<pubnumber>950R01013</pubnumber>
<title>Reproductive Toxicology Division's Peer Review, October 22-24, 2001</title>
<pages>355</pages>
<pubyear>2001</pubyear>
<provider>NEPIS</provider>
<access>online</access>
<origin>hardcopy</origin>
<author></author>
<publisher></publisher>
<subject></subject>
<abstract></abstract>
<operator>leidos</operator>
<scandate>01/13/17</scandate>
<type>single page tiff</type>
<keyword></keyword>
Reproductive Toxicology Division
National Health and Environmental Effects Research Laboratory
Office of Research and 
Development
Research Triangle Park, NC
Division Review
October 22-24, 2001
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UN,TED STATES environmental protection agency
I \ OFFICE OF RESEARCH AND DEVELOPMENT
National Health and Environmental Effects Research Laboratory
Reproductive Toxicology Division's Peer Review
October 22-24, 2001
Meeting Location:
NHEERL Building, Rm. 1405
2525 E. Hwy 54
Durham, NC 27713
Table of Contents
TAB
Table of Contents 1
Charge to Peer Reviewers 2
Agenda and List of Peer Reviewers 3
Schedule and Assignments 4
NHEERL Overview 5
RTD Overview 6
RTD GPRA Overview 7
Theme. Atrazine - Cooper 8
Theme: Critical Windows - Darney 9
Theme: DW/Dev - Hunter 10
Theme: DW/repro - Klinefelter 11
Theme: Dev Repro - Gray 12
Theme: Receptors - Abbott 13
Theme: Susceptible Populations - Rogers 14
Appendix A: 1997 Peer Review 15
Appendix B: Biosketches 16
Appendix C: Publications 17
Appendix D: Trainees 18
Appendix E: Seminars 19
Paper for Notes 20
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REPRODUCTIVE TOXICOLOGY DIVISION REVIEW
October 22-24,2001
CHARGE TO THE PEER REVIEW PANEL
Enclosed are background materials to be used in preparation for your on-site review of EPA's
Reproductive Toxicology Division (RTD). Please note that the review panel is not an advisory
committee Therefore, the objective is not to come to a consensus regarding the Division's program,
rather, the report should represent a summation of individual critiques, opinions, and viewpoints
OVERALL GOAL
The primary goal is to conduct a scientific peer review of the Reproductive Toxicology Division
to provide the Laboratory and Divisional senior management with guidance for the planning
and implementation of research and the allocation of resources over the next five years. The
review shall be conducted to evaluate 1) the nature and quality of the science within the
Division, and 2) the impact of the Division's science on Agency needs and problems. Each of
these two dimensions should be assessed relative to the research, advisory, and leadership roles
of the Laboratory. Specific questions relative to these roles are outlined on page 6.
RESPONSIBILITIES
Panel members are encouraged to bring their own laptop computers for use during the sessions. If
you cannot, we will have laptops available. We prefer you to use WordPerfect 9, but we have
converters for other programs. Printers and photocopiers also will be available
PANEL CHAIR:
Dr Mattison will serve as the Editor-in-Chief for the report and will draft the Executive
Summary
ALL PANEL MEMBERS (see attached List of Reviewers):
Each panel member has been assigned the role of primary or secondary author for the various
research areas of the Division's program. The proposed writing assignments may be found in the
notebook If you have objections to or concerns about these proposed assignments, we will be happy
to discuss them and try to accommodate your needs. Specified assignments do not preclude
reviewers from evaluating other research areas, in fact, such efforts are encouraged, especially when
disciplines cross research areas.
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What should be written prior to the on-site visit
Primary authors:
Primary authors, listed below, are asked to bring a written critique of their assigned research
area, based upon the materials provided in this notebook, to the on-site meeting (A recommended
format for your critiques is included on page 5, and the table on page 6 will assist you in preparing
these critiques.) A hard copy of your initial critiques should be given to the Peer Review Coordinator
during the Executive Session on October 21 Copies of these critiques will be distributed to all panel
members for discussion during the site visit.
Executive Summary Dr. Mattison
Drinking Water - Reproductive Effects Dr. Schrader
Drinking Water - Developmental Toxicity Dr. Holson
Critical Windows Dr. Terranova
Receptor-mediated Toxicity .... Dr. Hines
Chlorotnazmes Dr. Eldridge
EDCs Dr. Stocco
Susceptibility Dr. Hansen
Progress Since Last Review Dr. Mattison
The critiques should provide an analysis of 1) the Division's research (i.e , the nature and
quality of the science and the impact of the Division's research on Agency problems), 2) the extent
to which the Division provides advice and assistance to EPA and the scientific community, and 3)
leadership exhibited by the Division. Significant accomplishments, strengths, and weaknesses
should be highlighted. Please note that reviewers are not asked to evaluate individual members of
the RTD staff
Secondary authors:
All panel members have secondary responsibilities to prepare brief written statements or
bullets on the research areas not primarily assigned to them, using the table on page 6 as a guide.
These comments will be given to the primary authors at the meeting for them to incorporate into the
overall review
What should be written on-site
Primary authors:
Primary authors should incorporate the comments of secondary authors into their critiques,
editing and amending as necessary. Comments regarding poster sessions should be included
Secondary authors:
Secondary authors should amend their initial comments based on observations made during
the site visit and give this information to the appropriate primary author.
Please be aware that we must have a draft review report before your departure on Wednesday.
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Prior to adjournment, the Panel shall present its key findings to the NHEERL senior staff, the
senior staff of RTD, and the Peer Review Coordinator during an exit interview.
Post-meeting obligations
The Panel Chair, with assistance (if needed) from the Peer Review Coordinator, will complete
the draft report and circulate it to all members of the panel for their approval A final Peer Review
Report will be presented by the Panel Chair to the Laboratory Director no later than December 3,
2001
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SCHEDULE
All panel members are urged to arrive Sunday, October 21, in time to attend a 7:30 PM meeting with
NHEERL's Laboratory Director, Dr. Larry Reiter, the Associate Director for Health, Dr. Hal Zenick,
the Director of the Research Planning and Coordination Staff (RPCS), Dr. Robert Dyer, and the Peer
Review Coordinator, Virginia Houk. The meeting will be held in the Meredith Meeting Room at the
Hawthorn Suites Hotel where you have room reservations.
Upon arrival, the preliminary evaluations prepared by each panel member will be collected by the Peer
Review Coordinator. The material will be duplicated and distributed to all panel members to facilitate
discussions and consolidate evaluations
On October 22-24, the Division's program will be presented through oral and poster presentations
Ample time has been set aside for the reviewers to confer with Division staff and each other These
interactions will allow reviewers to obtain more detailed information on the Division's program, to
query researchers, and to clarify outstanding issues.
A portion of October 24 will be devoted to preparing the draft peer review report. During this
writing session, primary reviewers will modify, as necessary, their preliminary critiques to reflect
observations made during the site visit and to incorporate input from secondary reviewers Time also
has been set aside on this day for the chair and panel members to summarize and discuss major
findings and recommendations with the NHEERL and RTD senior managers
The timeline can be summed up as follows-
• Approximately 6 weeks prior to review (mid-September): Panel members receive
review package from Peer Review Coordinator
• Prior to arrival at on-site meeting (September and October): Reviewers prepare
preliminary text of review
• Evening before review (October 21): Reviewers meet in Executive Session with
Laboratory Director, Associate Director, RPCS Director, and Peer Review
Coordinator
• On-site review (October 22-24): Division discusses program with reviewers
• Last half day (October 24): Reviewers modify preliminary evaluation as needed;
panel chair and members hold exit interview with the Laboratory Director, Associate
Director, RPCS Director, Division senior staff, and Peer Review Coordinator
• Within approximately one month following site visit (December 3): panel chair
and members edit document and present final peer review report to the Laboratory
Director
The Peer Review Coordinator, Virginia Houk, will work with you throughout the peer review
process. Questions concerning the review should go directly to her (phone. 919.541-2815, fax.
919.541-4324; e-mail: houk virginia@epa gov). We would ask that there be no contact between you
and the staff of the Reproductive Toxicology Division regarding the review, but you may contact the
Division's Program Analyst, Teresa Wall, at 919.541.3591 for information regarding local
arrangements (hotels, transportation).
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GUIDELINES AND FORMAT FOR PREPARING THE REVIEW REPORT
The following outline for the review report is suggested. Parties responsible for the different pieces
are included in parentheses.
Q Executive Summary (Chair, with input from individual reviewers)
Description and evaluation of overall program, along with specific panel
recommendations and suggestions
~ Research (each reviewer, as assigned)
Evaluation of research areas based upon the review notebook, poster sessions, oral
presentations, and discussions Analysis of the nature and quality of the science and
the impact of the Division's research on Agency problems (see table on page 6 for
guidance)
Sample format
RESEARCH AREA (THEME)
Nature and Quality of Science
• Research Description and Division's Approach to Problem
• Critique (assessment of strengths and weaknesses, suggestions for
improvement, evaluation of future directions)
• Summary and Recommendations (overall evaluative statement and
recommendations)
Impact on Agency Needs and Problems
Q Advice and Assistance (each reviewer; Chair compiles and summarizes)
Critique of Division's role in providing advice/assistance to the scientific community
and EPA (see table on page 6 for guidance)
G Leadership (each reviewer, Chair compiles and summarizes)
Critique of Division's leadership role m the scientific community and within the
Agency (see table on page 6 for guidance)
~ Resources and Facilities (each reviewer)
Brief assessment of adequacy, special attributes, or deficiencies of resources and
facilities (relevant to the conduct of research)
~ Progress Since Last Review (Chair, with input from individual reviewers)
Critique of Division's responsiveness to the last Division review, taking into
consideration the former panel's recommendations and the Division's Response to
Comments, and steps taken to improve the program.
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GUIDANCE FOR DIVISIONAL REVIEW
Research
Advice/Assistance
Leadership
Nature/Quality
of the Science
Division's approach to a given environmental problem
Examples
• has the Division identified major uncertainties and appropriate
research priorities7
• are approaches scientifically sound7
• in what ways has the Division advanced scientific understanding
of the problem7
• are future directions sound7
Division's tesoitrces (assume fixed numbers)
Examples
• are Division's resources effectively and strategically allocated
across problems (appropriate depth and breadth)7
• is the skill mix optimized for the scientific direction taken7
• does workforce maintain cutting-edge knowledge and skills7
Mechanisms and extent to which findings /products are
disseminated to scientific audience in timely fashion
Extent to which Division's assistance is
sought by or provided to the scientific
community
Examples
• do scientists serve on national/
international workgioups, symposia,
professional societies, publication boards7
• are they members of research review
boards (e g , study sections) for other
organizations7
• do they provide scientific or technical
guidance to local, state, tribal, and
international governments
Division's leadership role in the scientific
community (influence on agendas,
decisions, priorities of other researchers /
organizations)
Examples
• do scientists lead collaborative research
efforts at the national / international level7
• do they serve on advisory boards of other
major agencies / organizations7
• are they invited to chair major
committees7
• do they organize major conferences,
symposia7
• do they receive awards / honors for
scientific contributions7
Impact on
Agency Needs
and Problems
Division's responsiveness to Agency needs
Examples
• is research driven by Agency priorities7
• does the research address the critical issues within EPA's
mission7
• is the Agency using the Division's data / products7
• does the Agency adopt approaches or methods developed by the
Division7
• does the Division provide information necessary for EPA users
to meet statutory requirements or other policy needs7
• what problems has the Division solved for the Agency7
Balance between core and problem-driven research, as defined by
the NRC
Mechanisms and extent to which findings /products are
disseminated to Agency in timely fashion
Extent to which Division's advice /
technical support is sought by the Agency
(Program Offices, Regional Offices)
Examples
• does Division staff paiticipate on major
within-Agency workgroups7
• do Division scientists assist the Agency in
developing testing guidelines, interpreting
research advances, reviewing Program
Office or Regional documents7
Division's leadeislup tole in the Agency
(influence on research planning efforts,
decisions, and prion ties of the Agency)
Examples
• does Division staff lead research planning
and coordination elfoits across Divisions,
Agency Labs, and Offices7
• do scientists represent ORD/Agency on
woikshops or workgroups addressing major
risk assessment, public health, and
environmental issues7
• do they receive major Agency awards/
honors7
SUPPLEMENTAL QUESTIONS
Provide suggestions for improvement in any of the above areas
What can be said about the adequacy of the Division's resources and facilities for meeting its scientific commitments''
Given the available resources, are productivity and efficiency optimized within the Division7
Considering the review of four years ago, has the Division made satisfactory progress on the panel's recommendations7 Where do reviewers see room for improvement7
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R
TD Peer Review Agenda
Monday
October 22
Tuesday
October 23
Wednesday
October 24
9:00 - 9:15
Welcome and Introductions
Intro to Atrazine Theme
Intro to Abn. Repro. Devel Theme
9:15 - 9:30
Division Overview
Intro to Receptor Theme
Abn. Repro Devel Posters
Reviewed (6)
9:30 - 10:00
Atrazine and Receptor Posters
Reviewed (5 + 4)
Critical Windows and Suscept.
Pop. Posters Displayed (8)
10:00 - 10:15
Intro to Water - Repro
Theme
10:15- 12:00
Water - Repro Theme
Posters Reviewed (8)
Water - Devel. Posters
Displayed (6)
11:30 - 12:00
Q&A, Abn. Repro. Devel.
12:00 - 12:30
Q &A, Water - Repro
Q & A, Atrazine and Receptor
Division Impact Summary
12:30 -- 1:30
Lunch
Lunch
Lunch
1:30 - 1:45
Intro to Water - Devel
Theme
Intro to Critical Windows Theme
Committee Report Writing
1:45 - 2:00
Water - Devel. Posters
Reviewed (6)
Water - Repro Posters
Displayed (8)
Intro to Suscept. Pop. Theme
2:00 - 4:30
Critical Windows and Sucept.
Pop. Posters Reviewed (3 + 5)
Atrazine and Receptor Posters
Displayed (9)
4:30 - 5:00
Q & A, Water - Devel
Q &A, Critical Windows and
Suscept. Pops
Exit Interview
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RTD REVIEW
LIST OF REVIEWERS
CHAIR:
Dr. Donald Mattison
March of Dimes Birth Defects Foundation
1275 Mamaroneck Avenue
White Plains, NY 10605
ph- 914.997.4649
fx: 914.997.
dmattison @ modimes.orci
PANEL:
Dr. Charles Eldridge
Wake Forest University School of Medicine
Winston-Salem, N.C. 27157-1083
Overnight Delivery Address:
115 S. Chestnut Street, Winston-Salem, N.C. 27101
ph: 336.716.8570 (office), 716-8572 (lab)
fx: 336.716.8501
eldridge@wfubmc.edu
Dr. Deborah Hansen
US Food and Drug Administration
National Center for Toxicological Research
3900 NCTR Road
Jefferson, AR 72079
ph: 870.543.7480
fx: 870.543.7682
dhansen @ nctr.fda.gov
Dr. Ronald N. Hines
Birth Defects Research Center
Department of Pediatrics
Medical College of Wisconsin and
Children's Hospital of Wisconsin
8701 Watertown Plank Road
Milwaukee Wl 53226-4801
ph: 414.456.4322
fx: 414.456.6651
rhines@mcw.edu
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Dr. Joe F. Holson
WIL Research Laboratories, Inc.
1407 George Rd.
Ashland, OH 44805
ph: 419.289.8700
fx. 419.289.
ifholson@wilresearch com
Dr. Steven Schrader
National Institute for Occupational Safety and Health
4676 Columbia Parkway
Cincinnati, Ohio 45226
ph: 513.533.8210
fx. 513.533.8138
sms4@cdc gov
Dr. Douglas M. Stocco
Department of Cell Biology and Biochemistry
Texas Tech University Health Sciences Center
3601 4th Street
Lubbock, TX 79430
ph: 806.743.2505
fx: 806.743.2990
doug.stocco@ttmc ttuhsc edu
Dr. Paul Terranova
The University of Kansas Medical Center
Molecular and Integrative Physiology
3901 Rainbow Blvd.
Kansas City, KS 66160
ph: 913.588.7435
fx: 913.588.7180
oterrano@kumc.edu
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TENTATIVE REVIEWER ASSIGNMENTS
Please keep in mind that these are tentative assignments. If you have objections to or
concerns about your proposed assignments, please contact the Peer Review
Coordinator, Virginia Houk, at 919.541.2815, and she will make every attempt to
accommodate your needs. Note that the posters for a given session will remain on view
the entire day, so if you are unable to visit an assigned poster during the scheduled
poster session, you should have ample opportunity to view it at some other time during
the course of the day.
It is the responsibility of the primary reviewer to make certain that a poster is adequately
reviewed, but it is not necessarily the responsibility of the primary reviewer to conduct
the review. Therefore, if you are a primary reviewer who feels your workload is too
heavy for a given set of posters, please ask another panel member to review a specified
poster and report back to you.
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Primary and Secondary Scientific and Impact Reviewers
Group
Theme
Leader
Posters
Primary
Scientific
Reviewer
Secondary
Scientific
Reviewer
Impact Reviewers
A
Water - Repro
Klinefelter
8
Schrader
Stocco
Holson (2)
Terranova (4)
Mattison (8.2)
Eldrige (8.3)
B
Water - Devel.
Hunter
6
Holson
Hines
Holson (2)
C
Atrazine
Cooper
5
Eldridge
Terranova
Hansen (3)
Matison (8.2)
Eldriqe (8.3)
D
Abnormal
Repro Develop
Gray
6
Stocco
Eldridge
Hansen (3)
Mattison (8.2)
Eldridge (8.3)
E
Critical
Windows
Darney
3
Terranova
Schrader
Terranova (4)
F
Receptor
Mediated
Abbott
4
Hines
Hansen
Mattison (8.2)
Eldridge (8.3)
G
Suscept. Pops.
Rogers
5
Hansen
Holson
Hansen (3)
Terranova (4)
Mattison (8.2)
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CROSSWALK OF RESEARCH THEMES, GPRA GOALS, SPECIFIC AIMS, AND REVIEWER RESPONSIBILITIES
GROUP No THEME LEAD INVESTIGATOR OBJECTIVE DESCRIPTION RESEARCH RESEARCH STRATEGY S"°N?^Y
T> STRATEGY FOCUS SPECIFIC AIMS REV,EWER "VIEWER REVIEWERS
A
1
Reproductive effects ot
dimlung water contamnants
Gary Khnofeltei
Testing paradigm to Study mode of action underlying habacid
induced testicular tonoty
2 1 7
Drinking Water
Evaluation of health effects
associated with exposure to
DSPs
2 13 74 1
Sctrader
Stocco
Hobon
A
2
Reproductive effects of
tanking water coniamnants
Gary Klinofeltai
Testing paradigms to determine consequences of haJoaod
oxposute durmg reproductwe development
2 1 7
Drtnkmg Water
Assessments of risks to
sensitive populations (krts)
from drinkng water exposue
11 1 8 5 52
Schrader
Stocco
Hotson
A
3
Reproductive effects of
tanlung water contamnants
Gary Klinefelter and Jeff
Welch
Identifcation and validation of tomarfcers of hafoaod induced
reproductive health effects
62 1
HHRA
Us© ol data on toologcal
mochansms tn risk
assessment
3 1 32 33 35
Schrafler
Stocco
Mattison
A
4
Reproductive effects of
tanking water contamnants
Sally Darney
Epdemologic evaluation ol DBP exposures on semen quality
2 1 7
Drinking Water
Evaluation of health effects
associated with exposure to
08 Ps
1 5
Sct*ader
Stocco
Hobon
A
S
Reproductive effects of
(kinking water contamnants
David Dix and John
Rocketl
Genome analysis of testicular toxicity
434
Safe Comnvubes
Suscoptibdity
24
Terranova
Schrader
Terranova
A
6
Reproductive effects of
(kinking water contamnants
David Dtx and John
Rocketl
Monitorng human reproductive health and development through
gene expresson profiling
62 1
HHRA
Cumutaovoteggrogato
38
Schrader
Stocco
Matt son
A
7
Reproductive effects of
(Jinking wator contamnants
Jerome Goldman
Haloacetic abds as endccrme disrupting chomcafs7 An
investigation of (he impact of these dnntgng water disinfection by
products on ovarian steroidogenesis
83 1
EDCs
Endocrine divuptors effects
1 3
Sctrador
Stocco
EkJndgo
A
e
Reproductive effects of
dimlung water contamnants
Mike Narotsky
Evaluation of endocrne disruptive effects on pregnancy
maintenance (oflowoig D6P eipsoure
S3 1
EOCs
Endocrine disuptors effects
1 2
Schrader
Stocco
Etdndge
e
9
Developmental effects ol
dnrtung water contamnants
Mike Narotsky
Does exposure to dtsmfeciants/OBPs/contamtnanis produco
developmental toxoty In animal models ci vivo
2 1 7
Diirtang Wator
11 17 3 1
Hotson
Hnos
Hotson
0
10
Developmental effects of
cbrtung water coniamnants
Mike Narotsky
Do mixtures of by products formed by disinfection after
development and are there differences in the effects pioducod by
different disinfection trams'
2 1 7
DnrtOng Wator
1 9
Hobon
Hmos
Holson
0
11
Developmental effects of
drriunQ wator contamnants
James Andrews
Are the haloacetc acids direct acting toxcants9
21 7
Oriitong Watei
2 1
Hobon
Hates
Hotson
B
12
Developmental effects of
drinking wator contaminants
Sid Hut Of
What aie the mode and mechanisms of action for hatoaceUc aod
Induced dysmorphogenese'
2 1 7
Dnrtung Water
22
Hobon
Hmos
Hotson
B
13
Developmental effects of
dwkmg water contamnants
Swj Hirter
Whai are (he critcal target cell populations for haloaceu: acid
nduced dysmorphogenesis'
2 1 7
Drlrtqng Water
23 24
Hobon
Htnos
Hobon
8
14
Developmental offects of
(frmkng water contamnants
Phip Hartig
What are the mode- and mocharasm-of action response for
arserucal »wJuced dysmorphogenese'
2 1 7
AfiorK
Hobon
Hmes
Holson
C
15
Identifying she mode and
mechanism of action ol
cNorotnazmes on endocrine
function in the rat
Ralph Cooper
Determine the Mode and Mechanism of Action ol the
CWorotnazme Her bodes using m vivo and m vifro approaches
82 1
83 1
HHRA
Harmonization
1 2 3
Eldndge
Terranova
Mattlson
Ettadge
C
16
Idenufyrig the mode and
mechanism of action of
crtorotriazmos on endoenne
function in (he rat
Suzanno Fertion
Effect ol atrazne on mammary gland morphogenesis and areolar
differentiation
82 1
83 1
HHRA
Sensitive subpoputations
10 11
Ettldge
Terranova
Mattlson
EUndge
c
17
Identifying the mode and
mechanism of action of
cMorotnaznes on endocrine
function In the rat
Susan Laws
Effects of atrazine on pubertal development comparng parent
compounds and metabolites
83 1
EDC/HHRA
Focus Area 1 EDSP
8
Eltadoe
Terranova
Eltadge
c
18
Identifying the mode and
mechanism of acton of
chlorotnajmcs on endocrine
function n the rat
Tammy Sloker
Atrazvie exposure during development induces prostatita In
adulthood Is this a model for EDC induced prostate cancer?
83 1
82 1 324
EDC/HHRA
Focus Area 2 developmental
issues
78
Eldndge
Terranova
EldrMge
Mattison
Hansen
c
19
Identifying (he mode and
mechanism of action of
chtorotrtazros on endocrine
functon In the rat
Mika Narotsky
Effects ol atrazne and metabobles on onplantation and pregnancy
maintenance
83 1
82 l
EDC/HHRA
Focus Area 2
devetopmnental issues
S 6
Ettldge
Terranova
EBrtdge
Mattlson
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20
CeAiai and molecular
mechanisms of atmomai
reproducirve development
Earl Gray and Vclue
Wilson
Effects mechanisms and modes of action ot EDCs thai alter
reproductive development n vitro and in vivo
82 1
83 1
EOCs and Human
Health
Harmonizing risk
2 3 9 10 11 12
13
Stocco
Etdndge
Mads on
Eldridge
D
21
CeQutai and molecular
mocharesms of abnoima)
roproducitve dovetopment
Earl Gray and Joe
Ostby
Cumulatrve rtsk of expesmes to EOCs with simlar and dasuriar
mechantsms and modes of action
32 1
Safe Food
Cumulatrve risk
5
Stocco
Eldridge
Hansen
D
22
CoOiiai and motocuiar
mechanisms of abnormal
reproductive dovetopment
Vtchio Wilson Jott
Welch Phdhp Harttg
Genome and proteonomc basts for interspecies extrapolations
basod upon estrogen and androgen receptor structure and function
among arvnah
82 1
HHRA
Hafmom?mg risk
6 ft and b
Stocco
Ektridpo
Mattison
D
23
CeOuiar and moiecutar
moctianisms ol abnormal
ropfoCucWQ development
C Lambnght and G
Ar* ley
Assessement of the utibty of In vitro assays to screen
environmental mixtures for androgens and antiandrogenc ac&vmes
83 1
324
Safe Food / EDCs
Harmontzvtg nsV
5
Stocco
Eldndge
Eldridge
Hansen
0
24
CeOular and molecular
mechanisms ol abnormal
reproducirve dovolopmont
Christy Lamtvight Kathy
Bobseine Vickie Witson
Phillip Haftig
Scientific and technological support on in vitro assays for the
Agency s Endocrine Disrupter Screer*ng Program
83 1
32 1
Sate Food / EDCs
Assay deveiopmeni lot
screermg
1 4 7
Stocco
Eldndge
EWridge
Hansen
0
25
CeDular and molecular
mechanisms of abnormal
reproductive deveiopmeni
Earl Gray and Joe
Ostby
Scientific and technological support on the Hershberger and other
in vrvo assays for the Agency's Endocrine Disrupter Screening
Program
831
32 1
Safe Food t EDCs
Assay development for
screening
8 10 t2
Stocco
Eldridge
Eldndge
Hansen
E
26
Crncal windows (or gamete
toxicology
SaDy Darney
Application of more sensitive and specif* end potnts to
charactenze male reproductive effects of pesticides and toxic
substances n arwnal and hunan studjes
434
Safe Communities
Dovelop & validate methods
and mode's
13 4 5
Terranova
Schrader
Terranova
E
27
Critical windows for gamete
toxicology
Jeff wetch
Identify sensitive stages of gonad and gamete development with
respect to glutathione homeostasis and protective mechanisms
4 34
82 1
Sale Communities
Methods to evaluate special
sensitivities based on
developmental stage
789
Terranova
Schiader
Terranova
e
28
Critical windows for gamete
toxicology
Tammy Stoker
Critical windows of exposure for female reproductive risk Effects
of acute exposure to pesbcides on the hormonal control of
ovulation and subsequent pregnancy outcome (Mode of action ol
tturam and parallel stuefces with mofanate)
4 34
Safe ComrTVKties
Develop A vabdate methods
and models
10 11
Terranova
Schrader
Terranova
F
29
Recoptor and signal
transduction pathways In
developmental toxeity
Barbara Abbott
Harmonization of mechanisms and responses across animal
models and cultured human t&suesfceSs
82 1
HHRA
Harmonizaton
1 7 15
Hines
Hansen
Mattison
F
30
Receptor and s^jnal
transduction pathways in
developmental toxicity
Barbara Abbott
Study ol receptor mediated pathways to reveal potential
mechanisms of differentia] sensiuvify and responsiveness
8 2 1
HHRA
SuscepiMity
8 10 19 21
Hmes
Hansen
Mattison
F
31
Receptor and s^jnal
transduction pathways m
developmental toxcity
Suzanne Fenton
Environmental toxicant erocts on the developing prostate and
mammary gland Mechantstc data on signal transduction pathways
undertymg differential responsiveness and susceptibly
82 1
HHRA
Susceptbdtty
17
Hirfij
Hanson
Mans on
F
32
Receptor and senal
transduction pathways in
developmental toxoty
JeromoGotaman
TojncantHndu:ud effects upon vascular growth Impact of
endocrtne-dorupfing chemicals on pre-natal development and organ
integrity wilhln the reproductive system
831
EDC
Endocrinedsruptors effects
12 13 16 18
Hmos
Hanson
Eldndge
G
33
Mechanisms of developmental
toxicity and susceptiWity
Chnslau
Construction of a bdogcafty basod dose response model lor
developmental toaoty In the rat
82 1
Human Health
Haimomzation
4 1
Hansen
Hols on
Mattison
G
34
Mecharnsms ol developmental
toxicity and susceptbdity
Ned Cher no n
Postnatal sequelae of tow btrth weight ejects of maternal toxcity
induced wwgtu loss dutng gesiaoon on fetal growth and
susceptive populations
82 1
324 434
Human Health
Susceptibility
3567
Hansen
Ho Is on
Mattison
Hansen
Terranova
G
35
Mechanisms ol developmental
toxicity and susceptibility
Mitch Rosen
Molecular approaches towards understands mode of acbon In
developmental toxoty
82 1
Hirnan Health
Harmorezauon
89
Hansen
Hofton
Mattison
G
36
Mechanisms ol developmental
toxoty and suscepubiity
John Rogers and James
Andrews
Evaluation of the role ol folate In susceptibbty to developmental
toxicants
821
Hurtan Health
Suscepobiiiy
t
Hansen
Hotson
Mattison
G
37
Metfianisrre ol developmental
tonory and susceptibfcty
Chrts Lau and John
Rogers
The developmental toxnty of perftuorooctane siilonate mduding
studies on putative modes of action
4 34
2
Hansen
Hotson
Teuan&ira
image:
cn
|!
2 m
® m
S u
image:
INTRODUCTION
The U.S. Environmental Protection Agency (EPA) was established in 1970 in response to
growing concerns over polluted air, unclean rivers, unsafe drinking water, endangered species, and
careless waste disposal. EPA was given the responsibility for implementing a broad set of federal
environmental laws, which have contributed in the intervening years to significant improvements
in environmental quality. Currently, EPA has jurisdiction over more than a dozen statutes enacted
to protect public health and the environment (e.g, the Safe Drinking Water Act and the Clean Air
Act).
EPA is both a regulatory and a scientific agency; it is one of only a few federal organizations
that operates in this capacity The environmental laws that form the legal basis for the Agency's
regulatory acti vities also authorize its research efforts. It is this research that provides the foundation
for scientifically defensible environmental policies, programs, and regulations EPA research is
housed chiefly in the Office of Research and Development (ORD), which includes the National
Health and Environmental Effects Research Laboratory (NHEERL)
EPA's OFFICE OF RESEARCH AND DEVELOPMENT (ORD )
ORD is the principal research arm of EPA. Its role is to integrate science into environmental
decision-making. Unlike most of the rest of the Agency, ORD has no direct regulatory function;
rather, its responsibility is to inform the regulatory process Through the development of technical
information and scientific tools, ORD's research strengthens EPA's science base, providing the
Agency's Program Offices and Regional Offices with sound data for use in developing and
implementing tenable environmental policies and regulations Comprising five national Laboratories
and Centers across the country, ORD's broad scope encompasses both human health and ecology
ORD is organized around the principles of risk assessment and risk management. These
principles not only help shape and prioritize ORD's research agenda, they also are an inherent part
of its organizational structure. A diagram of risk assessment and its relationship to ORD's Labs and
Centers is depicted in Figure 1 on the next page. The paradigm in Figure la applies to human health
risk assessment, while the framework in Figure lb, which conceptually mirrors the health paradigm,
is used for ecological risk assessment Simply put, risk assessment is the process of evaluating the
nature, magnitude, and likelihood of an adverse effect following exposure to a stressor, such as
pollution or habitat loss For health risk assessment, the step-wise process involves hazard
identification, dose-response assessment, exposure assessment, and risk characterization. Ecological
risk assessment, on the other hand, involves problem formulation, exposure and effects analysis, and
risk characterization. Once risk has been characterized, that information, together with factors such
EPA's Mission
Protect human health and safeguard the natural
environment - air, water, land - upon which life depends.
1
image:
Figure 1. Risk Assessment and ORD
a. Health Paradigm
Risk Assessment Ri*k Management
SldUiluiy <911J Lc>fdl
Dose-Response
At(»tcment
Public health \ *oc,al
ConwfcrabgniV FdCtor*
v. W
H«7*in
Identification
Risk Management
Derision
r
Characterization
\
Economic
Farum
nisk
Management
Optiom
Exposure
Assessment
PvjIlllCdl
Consideration*
•*flapirt1 frtrr
Ati A.tc-ii ktiJ i Om
«y Lh«- Awf|L
A«i)Mnf ol
UllAMt 15)01
iiwvc and kjOypmert
m MiiA Arieii/nom
K»tws** Keuvcn
COtTK'l I9V4
b. Ecology
Framework
Ofk
CKar«ei*m«t*en
Ctt«r*ct*m«l»on
Eeologeal Eltoett
PROBLEM FORMULATION
RISK CHARACTERIZATION
ANALYSIS
RISK MANAGEMENT DECISIONS
c. Alignment of ORD Labs / Centers with Risk Paradigm
National Exposure
Research Laboratory
National Health and
Environmental Effects
Research Laboratory
National Center for
Environmental
Assessment
National Risk
Management
Research Laboratory
National Center for
Environmental
Research
Research to measure
characterize, and assess
exposures and to support
compliance with
environmental regulations
and policies
Research to identif)
hazards, formulate
the problem, and
characterize response
Risk characterization
and research on risk
assessment methods
Research and technology
transfer to prevent,
mitigate and control
pollution
Extramural program •
grants, fellowships, and
national centers of
excellence - to
complement ORD's
intramural program
9
image:
as economic considerations, is used to make decisions on how to manage risk Figure lc shows how
ORD's institutional structure is aligned to comport with the risk paradigm, with each ofORD'sLabs
and Centers focusing on a different aspect of risk assessment NHEERL's singular focus is upon
potential effects of environmental stressors. Our role is to help formulate environmental problems,
identify the hazards, and characterize adverse effects.
ORD cannot address every problem in the environmental arena. It must be selective when
deciding which problems to tackle. Some research is required by law; other research is initiated in
response to specific environmental exigencies or opportunities. Thus, research in ORD can be
broadly viewed from two perspectives' scientific and programmatic This is an important
distinction because it reflects the dual responsibility of ORD to advance environmental science
frontiers while remaining responsive to the program needs and priorities of the Agency ORD
research must be scientifically relevant, but it also must be responsive to those in the Agency tasked
with making regulatory and policy decisions.
The framework for organizing research within ORD (and NHEERL) is drawn from EPA's
Strategic Goals, summarized in the box to the right These Goals identify the overall environmental
results, such as cleaner air, that EPA is working to attain.
We use these goals to systematize the way in which we
plan and prioritize our research, report our research
findings and products, and budget our programs. EPA
has 10 national environmental goals, the first eight of
which involve research Each goal is linked to key
environmental statutes For example, Goal 2- Clean and
Safe Water is arrayed with the Clean Water Act and the
Safe Drinking Water Act Accordingly, research
performed under a particular goal supports the regulatory
actions mandated by the corresponding legislation.
Goal 8, Sound Science, deserves some
explanation due to its confusing nomenclature. This
category is where our core research falls (similar to basic
research, and described more fully on page 6). As
opposed to problem-driven research, which addresses
specific environmental problems, core research improves
our fundamental understanding of complex environmental and human health issues. The
information and tools gleaned from this research are the kind that can apply to a wide variety of
environmental problems, including prospective environmental hazards. An example of "Sound
Science" research is our study of the unique susceptibilities of infants and children to toxic
substances, the results of which are far-reaching and can be used to address age-related scientific
issues in many problem areas
All research performed in ORD (and, therefore, in NHEERL) is driven by one of these
strategic goals. For each of the goals, ORD's Labs and Centers have committed to reaching certain
milestones and delivering specific products within a given time period, thus providing a mechanism
for measuring tangible progress toward completion of long-term objectives. This explicit
EPA's Strategic Goals
1. Clean Air
2. Clean and Safe Water
3. Safe Food
4. Preventing Pollution and Reducing
Risk in Communities
5. Better Waste Management
6 Reducing Global Risks
7 Expansion of American's Right to
Know About their Environment
8. Sound Science
9 Greater Compliance with the Law
10. Effective Management
3
image:
accountability grew out of the Government Performance and Results Act (GPRA) passed by
Congress in 1993; consequently, these Agency goals are sometimes referred to as "GPRA Goals."
THE NATIONAL HEALTH AND ENVIRONMENTAL EFFECTS
RESEARCH LABORATORY (NHEERL)
Organizational Structure. NHEERL is the largest research organization in ORD,
employing over 700 federal employees at various facilities across the country. The current
organizational structure is diagramed in Figure 2 Based in Research Triangle Park, NC, NHEERL
has nine divisions that specialize in different facets of human health or ecology research. Our Health
Divisions are centrally located in Research Triangle Park and Chapel Hill, NC Our Ecology
Divisions are based in Gulf Breeze, FL; Duluth, MN, Corvallis, OR, and Narragansett, Rl, each
location representing a significant regional ecosystem (Gulf of Mexico, Great Lakes, Pacific Coast,
and Atlantic Seaboard, respectively) Table 1 on the next page lays out the research focus for each
NHEERL division.
Figure 2. Organizational Structure of NHEERL
and its Relationship to ORD
Neurotoxicology Division
Gulf Ecology Division
Human Studies Division
Expenmental
Toxicology Division
Atlantic Ecology
Division
Mid-Continent Ecology
Division
Research Planning and
Coordination Staff
Western Ecology
Division
Environmental
Carcinogenesis Division
Reproductive
Toxicology Division
Office of the
Associate Director
for Health
National Outreach.
Information, and
Technology Staff
Office of the
Associate Director
for Ecology
National Accountability
and Resource
Management Staff
Environmental Monitoring
and Assessment
Program
> ..National Health
'.J-*j''and
f-- Environmental^.
-Effects Research -
Laboratory. V-'*
.NHEERL
4
image:
Table 1. Overview of NHEERL's Health and Ecology Divisions
DIVISION
LOCATION
RESEARCH FOCUS
Atlantic Ecology
Division (AED)
Narragansctt Rl
Studies the environmental effects of anthropogenic stressors on marine, coastal and estuanne water quality with an emphasis on the coastal waters and
watersheds of the Atlantic seaboard Areas of research specialization include modeling cumulative effects of multiple stressors on coastal ecosystems, developing
methods for assessing the ecological effects of contaminated marine sediments analysing the role of hiogeochemical processes on effects and conducting
geographic-based ecological assessments for the Atlantic coast
Environmental
C arcinogenesis Division
(ECU)
Research Triangle Park
NC
Performs research to assess potential carcinogenicity of environmental chemicals The aim is to reduce uncertainty in cancer risk assessment models by
developing mcchanisnc data underlying chemical carcinogenesis for agents of environmental concern (including mixtures) This approach is enhanced by
developing and applying biomarkers of response for predicting cancer outcomes and hy incorporating information gained from structure-activity and molecular
modeling approaches
Experimental Toxicology
Division (ETD)
Research Triangle Park
NC
Performs research to determine the health effects of environmental pollutants and cause-and-effect relationships at pollutant concentrations that mimic those in
the environment Investigations center on the pulmonary and cardiovascular systems, the immune system and susceptibility to infectious allergic, and neoplastic
disease Focal point for pharmacokinetic studies to elucidate dose-response relationships for systems susceptible to pollutants
(mlf Ecology Division
((.ED)
fiulf Breeze FL
Assesses the ecological condition of estuaries coastal wellands coral reefs and other critical habitats of the (iuir of Mexico Determines cause(s)of affected and
declining systems predicts future risk to populations communities and ecosystems from aquatic stressors and supports the establishment of cnlena to protect
critical habitats
Human Studies Division
(USD)
Chapel Hill NC
Conducts an interdisciplinary program of clinical and epidemiologic research that provides critical data for health risk assessment Clinical studies determine the
pharmacokinetics dosimetry, and effects of pollutants in controlled exposure studies of healthy and susceptible individuals Epidemiologic studies evaluate the
relationship between real-world exposures and observed health effects in populations of intircst The program focuses on the effects of pollutants in air and water
on the pulmonary cardiovascular, and neurobehavioral systems
Mid-conlinenl Ecology
Division (MED)
Duluth. MN
tirosse lie Ml
(field station)
Develops methods for predicting and assessing the effects of anthropogenic stressors on freshwater ecological resources, including the Great Lakes and Great
Rivers Conducts cause-and-effect research on the effects of nutrients, clean sediments climate change, and toxic chemicals on lake stream, and wetland
ecosystems as well as aquatic life and wildlife communities and populations Wildlife and aquatiL life toxicology research conducted in collaboration with
human health-based research performed in NHEERL s health divisions establishes advanced animal and dose-response extrapolation models to support
integrated risk assessments
Ncurotoxicology
Division (NTD)
Research Triangle Park.
NC
Performs research to provide the scientific and technological means to predict the neurotoxicity of environmental agents in humans Human neurotoxic disease is
modeled in laboialory animals and data arc collected in animals to make predictions about possible neurotoxic risk Studies range from the molecular level to the
whole organism and include neurobehavioral neurochemical ncurophysiological and ncuroanatomical approaches Major emphasis on the study of sensitive
subpopulations and developmental neurotoxicity
Reproductive Toxicology
Division (RTD)
Research Triangle Park
NC
Performs research on the effects of environmental pollutants on reproduction and development IXvclops biological indices for assessing germ cell maturation,
embryonic development and adult reproductive capacity and endocrine status integrating information into biologically based dose-response models Major
research emphasis on assessing modes of action for endocrine disrupting chemicals and drinking water disinfection by-products in order to reduce uncertainties in
the risk assessment of associated adverse reproductive outcomes
Western Ecology
Division (WED)
Corvallis, OR
Newport OR
(field station)
Studies estuanne. terrestrial and watershed ecology with a focus on the Pacific Nonhwcst region Research emphasizes marine coastal and inland ecosystem
functions and response to stress Areas of specialization include ecological theory for spatial and temporal analysis of regional environmental data, developing
methods for assessing regional-scale condition of ecological resources and assessing the effects of changes in habitat and land use on terrestrial systems and
modeling of estuanne systems
5
image:
NHEERL's Mission. NHEERL is a problem-solving organization We are EPA's focal point for
research on the adverse effects of contaminants and environmental stressors on human health and
ecosystem vitality. Our mission is to:
• perform high quality effects-based research to identify, understand and solve current
and future environmental problems;
• provide leadership in addressing environmental issues; and
• provide scientific and technical assistance at the local, state, federal, and
international level.
These three elements, discussed separately below, interface squarely with the missions of EPA and
ORD.
Research. Research provides EPA with the necessary information and technologies for
detecting, abating, and avoiding environmental problems. NHEERL's approach to research, in
accordance with ORD, is founded on principles of risk assessment. Our research is designed, within
a risk assessment context, to answer scientific questions and reduce major uncertainties about the
effects produced by pollutants and human activities on health and the environment. As shown in
Figure 1, our research focuses on two components of the risk assessment paradigm: problem
identification/formulation (does the contaminant or stressor cause the adverse effect?) and dose-
response/ stressor-response analysis (what are the relationships between the contaminant or stressor
and the extent of injury, disease, or damage?).
Rather than characterize our research as
basic or applied, we use the terms core and
problem-driven. Core research (supportingEPA's
Strategic Goal 8: Sound Science) seeks to produce
a fundamental understanding of the key biological,
chemical, and physical processes that underlie
environmental systems, thus forging basic scientific
capabilities that can be applied to a wide range of
environmental problems. Core investigations
address questions common to many EPA programs,
and they provide the preparedness needed to confront unforeseen environmental problems.
Examples of this type of research include NHEERL's multidisciplinary efforts to improve human
health and ecological risk assessment (in which issues such as susceptibility and extrapolation of
response are addressed) and the Environmental Monitoring and Assessment Program (designed to
develop the science needed to describe the condition of our nation's ecological resources). Problem-
driven research (supporting EPA Goals 1-7), on the other hand, focuses on specific environmental
problems. Studies in these areas respond to explicit Agency needs and may be motivated by
regulatory requirements or court-ordered deadlines. This type of research is exemplified by our
Particulate Matter research program, in which the relationships between airborne particles and
increases in morbidity and mortality are being studied to address critically important human health
Core and problem-driven research are similar, but
not equivalent, to basic and applied research.
Their complementary nature enhances NHEERL's
ability to address diverse environmental issues
Scientists often pursue both types of research
simultaneously, and cross-fertilization is
encouraged.
6
image:
Figure 3. The Relationship between
Problem-Driven and Core Research
(adapted from NRC, 1997)
Problem-Driven
Research
Core Research
Identifying existing and
"emerging" Issues
Elucidation of
environmental
, processes
Development
of tools
Use risk assessment to
rank Issues and pinpoint
largest uncertainties
O)
FEEDBACK,
Collection
of data
Narrow EPA focus based
on mission needs and
acknowledgment of
what others are doing
Improve understanding
and reduce uncertainties
Select protects based on broad
applicability, relevance to EPA,
and scientific merit
FRAMEWORK FOR ENVIRONMENTAL RESEARCH
questions. Another example is our Aquatic Stressors Program, in which we are developing stressor-
response models to advance our understanding of the effects of nutrients, habitat alteration,
sedimentation, and toxic chemicals on aquatic ecosystems, as well as techniques to diagnose cause
of ecosystem impairment.
Of course, core and problem-driven research are not entirely separable. In fact, they are
highly complementary and interactive, each informing the other (see Figure 3). An example from
our program can illustrate this. As previously mentioned, one of the issues under our core research
program is the unique susceptibilities of infants and children to toxic chemicals how does age
influence health effects? This is a fundamental question in toxicology, and the results of this
research may be far-reaching, providing useful information to other studies For instance, under
Goal 2 Clean and Safe Water, we are studying the reproductive and developmental effects of
disinfection by-products found in drinking water. Because this is an age-related susceptibility issue,
the results from our core program readily feed into this program area, and vice versa. Thus,
susceptibility research takes place in both areas (under Goal 8 and Goal 2), but the target question
is different In our core program (Goal 8: Sound Science), the question relates to the rudimentary
principles of age-related differences; in Goal 2 (Clean and Safe Water), the question is how
disinfection by-products impact an organism during its developmental stages This blend of core and
7
image:
problem-driven research yields a robust research portfolio that couples a stable core effort with
research focused on the mission needs of the Agency.
NHEERL's current core and problem-driven research topics are listed in Table 2 on the next
page. (All of NHEERL's core research falls under Goal 8: Sound Science ) Relative emphasis in
each of these topics may change as ORD priorities shift, as new data surfaces, as court-ordered
deadlines are met, or as budgets grow and shrink However, substantial efforts are made by
NHEERL to build and maintain research programs that are both relevant to the scientific problem
and responsive to Agency needs The objective is to create an integrated and coherent program, not
a collection of disconnected projects. There are three principal types of research product:
• test methods for detecting and characterizing hazard (e.g., new bioassays or
ecological indicators),
• predictive models for understanding and predicting relationships between stressors
and response (e.g., biologically based dose-response models or computer models that
predict the effects of climate change), and
• data designed to fill information gaps and address limitations associated with risk
assessment (e.g., toxicity test results).
Table 2. NHEERL's Core and Problem-Driven Research Programs
CORE
PROBLEM-DRIVEN
Goal 8: Sound Science
Coal 1: Clean Air
Goal 4: Reducing Risk in Communities
~ Human Health Risk Assessment
~ Particulate Matter
» Human Health Effects and
• Harmonizing Cancer/Noncancer Risk Assmt
~ Air Toxics
Susceptible Subpopulations
• Cumulative/Aggregate Risk
~ Ecosystem Effects
• Susceptibility
Goal 2: Clean, Safe Water
~ Research to Improve Ecosystems
~ Drinking Water
Goal 6: Global Risks
Risk Assessment
~ Aquatic Stressors
~ Global Climate Change
~ EMAP
Goal 3: Safe Food
Goal 7: Right-to-Know
~ Endocrine Disruptors
~ Effects of Pesticides
~ Chemical Information Databases
Leadership. NHEERL provides vital leadership in the environmental research arena, and its
scientists are proactive in the scientific community at many levels. Within the Agency, we help shape
the research agenda by contributing to research planning and coordination exercises, and we
participate in the development of ORD Research Plans and Strategies. Our scientists represent the
Agency on workshops and task forces addressing major risk assessment, public health, and
environmental issues Outside EPA, we influence the direction and priorities of environmental
research worldwide. We steer collaborative research efforts at the national and international level,
we are members of international planning committees and research review panels, we serve on
advisory boards of other major agencies and organizations, and we serve as adjunct faculty members
at major universities across the nation.
Scientific and Technical Assistance. As part of our mission, NHEERL responds to diverse
requests for scientific advice and technical consultation, both within and outside EPA. We provide
8
image:
technical support to the Agency by advising EPA Program Offices and Regional Offices on scientific
matters, by participating on Agency workgroups, and by helping develop testing and risk assessment
guidelines. We bring our expertise to bear at the national and international level by organizing
scientific workgroups and symposia, and by serving in professional and scientific societies and on
publication boards. We provide guidance to local, state, tribal, and international governments and
other federal agencies, informing them on issues of environmental importance and enabling them to
implement more effective environmental programs. We work to establish partnerships with the
corporate, public, private, and educational sectors and assist them in setting and achieving
environmental goals. We provide technical training and developmental opportunities for the senior
scientist as well as the post doctoral candidate and the student. By sharing our skills and knowledge,
we enhance the ability of other organizations to protect public health and the environment, and we
serve as an important catalyst for scientific and technological progress.
PRIMER ON RESEARCH PLANNING
AND RESOURCE ALLOCATION WITHIN NHEERL
Certain functions and operations within NHEERL are expressly governed by federal law. For
example, explicit limitations and constraints are placed upon the way in which we obligate research
funds. In other cases, NHEERL sets its own policies and procedures. The information below is
designed to present the ways in which NHEERL operates with respect to two important
responsibilities: research planning and resource allocation.
Research Planning. EPA's research agenda is determined by means of a research planning
process involving every organizational level within the Agency. Long-term guidance for research
direction is provided by several sources, the most important being Strategic Plans. These Plans
focus on both organizational issues and research topics, and they help set the course for research
direction. EPA's Strategic Plan is broad in scope, articulating EPA's mission and its 10 national
environmental goals ("GPRA Goals") and offering a framework for planning and resource allocation.
ORD's Strategic Plan and NHEERL's complementary
Organizational Strategy, on the other hand, are specific to each
organization's own research role within the Agency, though
they naturally adhere to the principles and long-term objectives
contained in EPA's Strategic Plan.
ORD's first Strategic Plan, published in 1996, was
pivotal to the research planning process. It instituted a new
system for determining research priorities. The system,
founded on a risk-based approach to decision-making, uses the
risk paradigm to shape the research agenda. Using this nsk-
based process, ORD identified - and later updated - research
areas of potentially greatest risk to human health and the
environment (see box to right). The selection of these high-
priority research topics was conducted in partnership with
ORD's many stakeholders, including the external scientific community (EPA's Science Advisory
Board, the National Research Council, other government agencies, and the private sector) and the
High Priority Research Areas
~ Particulate Matter
~ Drinking Water
~ Water Quality
~ Global Change
~Ecological Risk
~ Human Health Risk
~ Endocrine Disruptors
~ Pollution Prevention and New
Technologies
9
image:
Agency's Program and Regional Offices and scientific staff.
Once identified, these high-pnonty areas became the strategic targets for ORD research. For
each of these topics (plus several other high-profile areas), ORD has developed or is in the process
of developing Research Strategies and Plans. Research Strategies frame the scientific questions
associated with the environmental issue and delineate research needs. (If effects-based research is
needed, NHEERL becomes involved and identifies the areas where it has the expertise and technical
capability to reduce scientific uncertainty ) Research Plans, in contrast, are more detailed, outlining
the research approaches to be applied to the problems Issue-specific ORD Multi-Year Plans
integrate research across ORD and relate research to the Strategic Goals of the Agency; they are
developed in the context of existing Research Strategies and Plans with input from all of ORD's Labs
and Centers. Finally, NHEERL is developing multi-year Implementation Plans that bring the
planning process to the operational level. They are being developed by a steering committee made
up of two representatives from each Division as well as representatives from appropriate EPA
Offices. NHEERL and its staff play a lead role in developing all of the above-described documents.
ORD s research strategies and plans are available on the Internet at
htip//www ena gov/ORD/WebPubs/final.
This description of our long-range planning process and the accompanying documents that
serve as blueprints for research is meant to illustrate the interconnectedness of EPA's environmental
goals, ORD s research priorities, and the course set by NHEERL to address these priorities. This
process establishes research direction for a 5- to 10-year period. We are held accountable for meeting
the commitments made under this exercise, and certain measures are put into place to gauge our
progress Table 3 shows more clearly how NHEERL's research is aligned with, and linked to, ORD
priorities and EPA goals.
Annual research planning also takes place within the Agency as part of the federally mandated
planning and budgeting process. Annual planning in ORD is driven in part by the multi-year
commitments laid out in the process described above. Specific research needs are identified based
on input from the Program and Regional Offices and ORD's scientific staff, and these needs are then
prioritized by Agency-wide teams (called Research Coordination Teams). Special attention is paid
to research required to fulfill a legislative mandate, court order, or Agency GPRA commitment;
priority setting also takes into consideration scientific feasibility, the status of ongoing research,
budgetary constraints, and ORD's ability to make a contribution relative to other research institutions
that may be working in the same area. The objective is to focus on environmental problems that pose
the greatest risks to people and the environment (using criteria such as seventy, permanence, scale),
on uncertainties in risk assessment that can be effectively reduced, and on areas that clearly help the
Agency fulfill its regulatory mandates. These research needs become the priorities for ORD and, in
turn, for NHEERL
Whi le the problems NHEERL is tasked to sol ve are defined by the above process, the research
agenda for solving these problems is determined by NHEERL and its staff (see the Implementation
Plans description above). NHEERL structures a coherent research program around the problem areas,
with the various divisions playing specified roles. Divisions are held accountable for implementing
research activities within the program and for addressing the priorities established through the ORD
planning process Divisional scientists identify the critical paths for research to resolve the key
10
image:
scientific questions and are often the first to raise new questions and recommend new methods for
problem-solving. Their suggestions and ideas are fed back into the planning process by several
means, the most common being discussions with the appropriate NHEERL Associate Director and
Assistant Laboratory Director (ALD) and through the steering committee process. (It is the ALDs
who, as members of the ORD Research Coordination Teams, are at the interface of ORD and
NHEERL planning.) Figure 4 is a simplified diagram of the inter-relationships that exist in research
planning
11
image:
TABLE 3. Alignment of NHEERL's Research
with ORD's Research Priorities and EPA's Strategic Goals
EPA Goal 8 sound science
—II
EPA Goal 7 right-to-know
EPA Goal 6 reducing global risks
EPA Goal 5 better waste management
EPA Goal 4 safe communities
EPA Goal 3 safe food
EPA Goal 2 clean and safe water
EPA Goal 1 clean air
1
2
3
4
5
6
7
8
ORD
RESEARCH PRIORITY
NHEERL
RESEARCH TOPIC
Particulate Matter*
Particulate Matter
•
o
Air Toxics
Air Toxics
•
o
Drinking Water*
Drinking Water
•
o
Water Quality*
Aquatic Stressors
•
o
o
c
Safe Food
Effects of Pesticides
•
o
o
Safe Communities
Health/Ecosystem Effects
•
o
Contaminated Sites
Contaminated Sites
o
•
o
Global Change*
Global Climate Change
•
o
Right-to-Know
Chemical Info Databases
o
Ecosystem Assessment*
Ecosystems Research
o
o
o
o
Human Health Risk
Assmt*
Human Health Research
o
o
o
o
o
Endocrine Disruptors*
Emerging Risks (EDCs)
o
o
o
o
o
*ORD's highest research priorities
• Directly contributes to meeting Strategic Goal
O Supports achievement of Strategic Goal
12
image:
Figure 4. Research Planning
)
>
^ ORD
NHEERL
A
DIVISION
A
SCIENTIST
^K£^aE5^^^S^^tI^^555t
Identify national environmental priorities and
determine strategic direction
Set national environmental goals
Establish research priorities to help achieve Agency Goals
Develop research framework across ORD Labs/Centers
~ Identify priority needs
Plan effects-based research to address EPA/ORD priorities
Coordinate research programs across health/eco divisions
Identify emerging issues
Implement research to address EPA/ORD/NHEERL priorities
Direct specialized research within Division
~ Identify key scientific
questions
~ Conduct research to address EPA/ORD/NHEERL/Division
^ priorities
~ Develop methodologies for solving problems
~ Identify new hypotheses
STAKEHOLDERS |
~other federal agencies j
1
~Congress \
~the Administration j
i
~ industry ?
1
~ academia ^
H
~ taxpayers *
2
~ EPA Program Offices J
£
~EPA Regions !
s
t
~ other federal research |
partners J
r
5
~ other ORD Labs and J
Centers ¦
~ research collaborators
13
image:
Resource Allocation. Each year, EPA develops a budget that defines the funding required
to accomplish its goals and objectives. EPA provides its funding targets to ORD, and ORD provides
a funding target to NHEERL When approved by the President and Congress, the enacted budget
serves as the blueprint for all Agency activities. NHEERL research planning is done assuming a flat
overall budget from year to year. However, when fluctuations occur, resource allocation is adjusted
accordingly. Although the funding priority of a research area within NHEERL may shift over time
as research is completed or as new problems emerge, NHEERL's overall resources have remained
more or less fixed in the recent past. This means that if expenditures (and research) are allowed to
grow in one area, spending (and research) in another area must shrink. It should also be noted that
a limit on NHEERL FTEs is imposed by EPA, and each Division is required to operate with an FTE
ceiling.
In addition to salaries and benefits, travel, and operating expenses (e.g., equipment,
equipment maintenance, supplies, training, etc.) NHEERL's resources include those required to
sustain our in-house research capabilities (generally referred to as "research support" or
"infrastructure") and those that augment or leverage our in-house efforts (referred to as "above
infrastructure"). Above-infrastructure resources become available to the Laboratory through the
ORD planning process as the result of initiatives for research in certain high-priority areas. In certain
cases, an internal competition is held within the Laboratory for these funds, which are then awarded
to Divisions whose research proposals are meritorious based on reviews for scientific excellence and
programmatic responsiveness. These above-infrastructure resources may be used to provide funding
for
• support contracts (used, for example, for technical support, analytical services,
information technology, and animal care);
• competitive cooperative agreements (used, for example, to train post-doctoral
candidates); and
• interagency agreements (used to fund collaborative research across federal agencies)
The management of certain support services, such as computer support and animal
procurement and care, is location-based: such services are centrally managed in Research Triangle
Park for our health divisions, while the ecology divisions manage these activities themselves due to
their geographically separate locations. However, most other resources are managed by the
Divisions.
It is important to realize that there are explicit limitations - mandated by law - on spending
and obligating funds. The law states that spending (by an^ agency) cannot exceed the amount
appropriated by Congress. This means that NHEERL cannot apply for grants from other agencies
or institutions, and we cannot increase our technical support staff through the use of external
resources. However, NHEERL scientists are encouraged to collaborate both within the Agency
(within Divisions, across Divisions, across Laboratories) and outside of EPA. In these cases,
external resources may become available. Examples include Interagency Agreements, which foster
research across federal agencies, and Cooperative Research and Development Agreements, which
14
image:
allow us to work with industry partners on issues of mutual interest.
It should be stated that NHEERL does not have its own extramural grants program. EPA
research grants are handled by the National Center for Environmental Research in ORD and are not
administered by NHEERL
REFERENCES
National Research Council (NRC), Committee on Research Opportunities and Priorities for EPA
Building a Foundation for Sound Environmental Decisions Washington, DC, National Academy
Press, 1997.
U.S. Environmental Protection Agency (EPA), Office of Research and Development (ORD),
Framework for Ecological Risk Assessment, Washington, DC, EPA/630/R-92/001, 1992
U S Environmental Protection Agency (EPA), Strategic Plan, Washington, DC, EPA/190/R-00/02,
September 2000, www epa gov/ocfopage/plan/plan htm.
U S Environmental Protection Agency (EPA), Office of Research and Development (ORD),
Strategic Plan, Washington, DC, EPA/600/R-01/003, January 2001, www epa go\7ord/sp/.
15
image:
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RTD Overview - September 12, 2001
OVERVIEW OF THE REPRODUCTIVE TOXICOLOGY DIVISION
October, 2001
I. INTRODUCTION
II. HISTORICAL PERSPECTIVE
III. PERSONNEL
IV. RESEARCH FUNDING
V. RESEARCH FACILITY
VI. RESEARCH MANAGEMENT
VII. SCIENTIFIC RECOGNITION/PRODUCTIVITY
VIII. FUTURE DIRECTIONS
1
image:
RTD Overview - September 12, 2001
I. INTRODUCTION
Nationally, approximately one out of every ten couples seeks medical assistance
in achieving pregnancy. Several recent publications suggest marked decline in sperm
counts and semen quality as well as increases in testicular and genital abnormalities and
testicular cancer over the last four decades. In addition, at least 30 percent of all
recognized human pregnancies fail to culminate in a live birth because of spontaneous
abortions, and an equal number of clinically undetected pregnancies are also believed to
be lost. Congenital malformations recognized at birth affect at least 3% of infants; this
incidence doubles when later-recognized deficits are included, and increase to 21% when
including low birth weights. Indeed, birth defects are the fifth leading cause of potential
years of life lost, and states spend more money on development disabilities (including
mental retardation) than any other category of chronic disease. Concern about potential
environmental etiologies has increased with the publication of the National Academy of
Sciences' report on "Pesticides in the Diets of Infants and Children," and the highly
publicized endocrine-disruptor hypothesis that environmental contaminants may have
altered reproductive development and function in a variety of species. Unfortunately, the
underlying causes of reproductive and developmental abnormalities remain largely
unknown.
As one of nine major research units of the National Health and Environmental
Effects Research Laboratory (NHEERL), the Reproductive Toxicology Division (RTD)
brings together an unparalleled combination of expertise in teratogenesis and male and
female reproductive toxicity to investigate the health effects of environmental contaminants
such as pesticides, air and drinking water pollutants, and toxic substances, singly or in
combination, on all stages of the cycle. In the early 1990s, the Division established the
overarching goals of: 1) developing more sensitive methods to detect the adverse effects
of environmental contaminants, 2) elucidating how toxicants interact with living organisms
so as to foster accurate interpretation of toxicological data, and 3) developing models to
permit better extrapolation of experimental data across species and developmental stages
so as to permit better characterization of potential risks to exposed subpopulations. These
goals have remained central to the research activities of RTD, although the precise nature
of the work has changed considerably over the past decade, as the era of developmental
biology has opened new understandings of how systems operate, and era of the human
genome has provided new tools that for the first time really enable the potential to
characterize mechanisms of action of environmental toxicants at the cellular and molecular
level.
Structurally, the research staff is organized within three branches: Gamete and Early
Embryo Biology (GEEBB; Sally Perreault Darney, Chief), which specializes in studying the
production and function of gametes as well as the subsequent development of the fertilized
egg to an embryo up to the stages of organogenesis; Developmental Biology (DBB; John
Rogers, chief),which focuses on the effects of environmental toxicants on embryonic and
2
image:
RTD Overview - September 12, 2001
fetal development expressed either immediately at birth or later on in postnatal
development; and Endocrinology (EB; Ralph Cooper, Chief), which targets how
environmental toxicants disrupt endocrine processes essential to the development and
maintenance of normal reproductive function.
Although the branches are divided by specialization for the purposes of
administrative function, the various research programs conducted by RTD investigators
typically require collaborations between branches as well as across divisions and therefore
we operate scientifically through the formation of cross-organizational research programs.
The foci of these research programs are determined primarily by the GPRA Goals
identified by the Agency to meet the legislatively mandated needs of the EPA Program
Offices. The GPRA Goals relevant to RTD consist of both media-specific objectives (e.g.,
Clean Water under Goal 2, Safe Food under Goal 3), and more fundamental aspects of
toxicology such as contained within Goal 8 that are targeted to improving the scientific
basis of risk assessment from which many media programs would benefit. Within the
GPRA context, the Division and its investigators have broad leeway to develop and test
specific initiated hypotheses about particular environmental sources of adverse
reproductive outcomes within the media programs and to address more fundamental
issues and cross-cutting issues within the core research program. The defined research
efforts then are carried out in the form of Research Themes and pilot studies (investigators
are allowed to devote up to 20% effort on activities not explicitly covered in the Division's
annual planning exercise that aligns the research effort with the needs of GPRA). Themes
comprise the major research thrusts of the Division and are highly coordinated, multiple-
investigator, multi-disciplinary priority programs to which the Division has committed long-
term (i.e., 3- to 5-year) support and which directly support major components of the ORD
Strategic Plan. Themes are intended to promote overall productivity, efficient use of
resources, and the relevance of research projects by applying cutting-edge research into
far-reaching, mission-relevant programs. RTD currently supports seven Themes, and
these form the basis upon which the material will be presented in this review. Details of
the research planning process are provided later in this section and information on the
Themes are contained in separate tabs in this volume.
II. HISTORICAL PERSPECTIVE
RTD traces its roots back to the late 1970s with the movement within EPA's Health
Effects Research Laboratory (HERL) toward disciplinary research divisions. In 1979, the
reproductive and developmental toxicology research program was elevated to branch
status, at which time it consisted of 5 principal investigators. By 1981, the discipline was
further elevated with the creation of the Perinatal and Reproductive Branches within the
Developmental Biology Division. A subsequent merger in 1986 with the Experimental
Biology Division (which investigated the health effects of non-ionizing radiation) created the
Developmental and Cellular Toxicology Division. Three years later, concurrent with the
consolidation of the drinking water program from Cincinnati to RTP, DCTD was renamed
3
image:
RTD Overview - September 12, 2001
the Developmental Toxicology Division, with reassignment to other divisions of staff from
the old non-ionizing component. In 1995, DTD experienced considerable further expansion
with the conversion of our large on-site level-of-effort contract (ManTech Environmental
Technologies, Inc.) to permanent federal positions. With the consolidation of HERL and
ecology laboratories into the National Health and Environmental Effects Research
Laboratory (NHEERL) later that year, the Division adopted its current three-branch
structure and became the Reproductive Toxicology Division to reflect its broader focus as
well as the discipline's international nomenclature. Shortly thereafter, RTD moved to its
present location in the newly constructed NHEERL Building (see Research Highlights
1991-1996, pg. 5). The current organization of NHEERL and RTD has remained
unchanged since 1995.
Throughout its history, RTD has balanced its research program to incorporate
methods development, data interpretation, extrapolation models and chemical-specific
research, although the relative effort devoted to each area has changed as a function of
the needs of the disciplines and the Program Offices. For example, in the 1980s, the
Division's developmental toxicity research effort was devoted to developing tests of
neonatal organ function and evaluating the concept of the adult toxicity/developmental
toxicity (A/D) ratio as an extrapolation tool. At the same time, research programs in
reproductive toxicology were directed primarily toward developing methods to study the
reproductive effects of pesticides. RTD developed an alternative reproductive test protocol
that was peer-reviewed by an international panel in 1995. In response to its
recommendations efforts to expand some test measures such as sperm motility were
initiated. RTD scientists played a major role in optimizing computer-aided sperm analysis
(CASA) technology for use with rat spermatozoa in testing and research. Much of this
effort was funded under EPA's Superfund Research Program, and has included a series
of workshops (sponsored by EPA and NIEHS) involving scientists from government,
industry and academia. These efforts contributed significantly to the content of the
harmonized multi-generational toxicity test guidelines released in 1997 by the Office of
Prevention, Pesticides, and Toxic Substances (OPPTS). Over the past four years the
largest change to our program has been the result of the introduction of new technologies
that for the first time are allowing the possibility to characterize modes of action on the
molecular level using genomic technologies. RTD was a leader in NHEERL in recognizing
the potential for this technology, as evidenced by the organization of a workshop in
January 1999 to educate the NHEERL RTP staff on the current state of the science in
genomics. From that workshop grew a users group that is still active in the RTP area, as
well as an internal NHEERL users group and the NHEERL Molecular Profiling Group that
is charged with advising the laboratory on directions it should take relative to genomics,
proteomics and bioinformatics.
4
image:
RTD Overview - September 12,2001
As the fields of developmental and
reproductive toxicology matured, RTD
has gradually shifted its efforts away from
methods development and more toward
questions of interpretation (i.e., defining
adversity, elucidating the maternal role in
developmental toxicity) and ultimately in
the development of predictive risk models
that form the basis for many of the current
programs. For example, to improve the
risk assessment process for non-cancer
endpoints, RTD scientists, in conjunction
with the National Center for
Environmental Assessment (NCEA) staff
completed a major effort to compare
traditional NOAEL-based risk estimates
with a variety of statistical models to
obtain bench-mark doses (BMD) for both
quantal and continuous response
measures. The BMD approach offers the
advantages of using more experimental
data, of producing consistent potency
calculations across studies and
endpoints, and of eliminating the need to
repeat studies that fail to demonstrate a
NOAEL. Nearly eight years later, this
methodology is now used when practical
in the RfD (i.e., Reference Dose of the
IRIS program) process.
Research Highlights 1991 - 1996
1991 Developmental toxicity of
methanol reported and work
begun on 5-FU BBDR model
1993 Publication of standard
methods for first generation
CASA in toxicology
1994 Evaluation of BMD models for
developmental toxicity risk
assessment
1994 Vinclozolin reported to be
environmental anti-androgen
1995 Move to NHEERL Building
1995 Publication of 5-FU BBDR
model
1995 Publication of p.p'-DDE as an
anti-androgen
1995 Prominent role in research
needs workshop for EDCs
1996 Atrazine effects on ovarian
function characterized
Also during the early 1990's, RTD made a long-term investment into large-scale
studies to improve risk assessment through the development of biologically based dose-
response (BBDR) models, initially using the prototype toxicant 5-fluorouracil. BBDR
models seek to reduce the uncertainty of risk assessment by identifying and characterizing
the cascade of events in the pathogenesis of the adverse health effects that follow toxicant
exposure, and then to link them through a mathematical function that allows estimation of
the toxicant's potential risk to exposed organisms. This effort was initiated under an ORD
initiative called "Research to Improve Health Risk Assessment" (RIHRA) and funded
through investigator-initiated responses to internal RFAs. In many ways, this was a
pioneering effort in non-cancer health risk assessment, and has spawned broader efforts
to incorporate mechanistic information into the risk assessment process in a more formal
manner.
5
image:
RTD Overview • September 12, 2001
Along the way we have provided chemical-specific information (often the key
regulatory data) on a variety of high priority Agency chemicals such as nitrofen, benomyl,
boric acid, dinocap, triphenyltin hydroxide, branched chain aliphatic acids, phenols,
methanol, dioxin, PCBs, vinclozolin, p,p'-DDE, Linuron, procymidone, fenitrothion,
diethylhexylphthalate, atrazine, and perflurooctanoic sulfonate and some of the findings
from these efforts have led to current research projects covered in this review. To support
the legislative mandates of the Office of Water, we were engaged in a long-term effort to
elucidate the developmental and reproductive effects of water disinfection byproducts (e.g.,
haloacetic acids) following development in 1993 of an internal disinfectant byproduct
research strategy. In the area of air toxics, RTD investigators explored the developmental
toxicity of methanol while it was being considered as an alternative fuel source, and,
through an international collaboration, evaluated the effects of brown coal emissions on
semen quality in the Czech Republic (resulting in one of the first demonstrations of an
effect of ambient air pollution on reproductive parameters). Beginning in the mid 1990s,
the Division significantly increased its effort to elucidate the role of environmental
estrogens and androgens in disrupting the reproductive development of exposed
organisms during critical periods of pre- and post-natal development. In this ongoing effort,
the Division has received international recognition for the pioneering demonstration of the
ability of endocrine-disruptive chemicals (EDCs) to effect adverse reproductive
development outcomes through anti-androgenic action, and a number of the staff have
played pivotal roles in the development of the scientific database and research strategy
related to concerns that environmental contaminants are operating in human populations
via endocrine disrupting modes of action. This area remains a high priority topic for RTD,
and it appears that as we learn more about important modes of action and critical life
stages of susceptibility it will remain an important area of years to come.
In order to increase communication with the outside community, we have over the
past few years taken a more active role in planning and holding mini-workshops of topical
interest to the RTD program. We have used the outputs to refine our research directions
and develop new collaborative efforts. One of the most significant of these grew out of the
1997 Peer Panel's recommendations that we convene a working group to review progress
and future directions in the 5-FU BBDR program. We held such a workshop in 1998
(Reference #134) and used the output to guide our current effort in that area. In 2000 we
started what is expected to be an active program of holding two-day mini-symposia on
topics of interest to various members of the RTD staff. About a dozen outside researchers
are invited to these workshops. The first of these dealt with the role of altered folate
metabolism in embryonic toxicity, and in the spring of 2001 we held a similar one on
pattern formation processes. Current plans are to hold two mini-symposia in 2002 focused
on the developing brain as a target of endocrine disrupting chemicals, and one on the
status of biomarkers of sperm function. We anticipate each of these will increase the
6
image:
RTD Overview - September 12, 2001
awareness of outside scientists to the
NHEERL program, but also help foster
development of collaborative efforts with
scientists around the US and fine-tune
our research program by providing very
up-to-date views on rapidly changing
scientific fronts. RTD also took the
pioneering step in June 2001 of inviting to
RTP representatives of its major clients in
Washington, DC for a two-day workshop
on "Emerging Issues in Developmental
and Reproductive Toxicology." At this
workshop, RTD presented its vision for
future research needs relative to our
scientific disciplines, and the client offices
were asked to do the same. From this
came a number of recommendations for
increased communication and sharing of
information and both sides left with a
feeling that such efforts should be
coordinated on an annual basis in order
that long-term bridges be built across the
organizational units. Collectively we
believe that these types of steps will
continue to improve the quality of our
scientific endeavors, while at the same
time allow the output to have influence on
public policy. Through these efforts, the
Division has gained a reputation for high-
quality, Agency-relevant research.
Indeed, one particularly noteworthy
indicator of the division's research
success is the number of Scientific and
Technological Achievement Awards
(STAA) bestowed on its scientists. The
STAA is an externally reviewed ORD
award program for peer-reviewed
publications of scientific excellence and
relevance to the Agency, and in the
period between 1990 and 1994 (the last
year for which data are available), RTD
scientists captured approximately one-
third of the awards given in the area of
human health research. In addition,
Research Highlights 1997 - 2001
1997
Low dose effects of dioxin on reproductive tract
development
Demonstration of predictive value of SP-22 as
fertility maker
Assistance in promulgation of new multi-
generation protocol in EPA
Stereoselectivity of valproate enantiomers
shown in embryo culture
Posteriorization of skeletal morphology by
methanol exposure
1998
Adenoviruses used to deliver genes to
neurulation stage mouse embryos
Molecular characterization of SP-22
Boric acid shown to cause deletions of specific
aspects of axial skeleton
1999
Rodent-Human sensitivity for dioxin- induced
alterations in palate differentiation
Mode of action of chlorotriazines on
neuroendocrine function reported
Lead laboratory status for OECD validation of
the Hershberger assay
Prostatitis in adults from developmental
exposure to estrogens seen
2000
State of science reviews of male and female
pubertal EDSTAC assays published
DEHP shown to inhibit testosterone synthesis in
fetal rat testes
Czech studies of air pollution on semen quality
completed
Container for use in semen collection in
epidemiology
2001
Patent issued to EPA for SP-22 as fertility
biomarker
Mathematical construct of 5-FU BBDR model
Mouse testes array developed
7
image:
RTD Overview - September 12, 2001
since 1993, the EPA has awarded RTD investigators one bronze and four gold medals.
III. PERSONNEL
RTD has a staffing level of 64 FTEs, with 61 on-board full-time employees as of
August 2001. The distribution of full-time employees across General Schedule grades is
depicted in Figure 1 and the organizational structure of Ph.D. level researchers and their
support staff is shown in Figure 2. We currently have one biostatistician working in the
area of bioinformatics on detail to
Figure 1. RTD Grade Scale
16 -
//I
14-
12-
10-
8-
6-
4 -
2-
o J
1 1
GS-S GS-7 GS-8 GS-9 GS-10GS-11 GS-12GS-13GS-14GS-15
us pending an official
reorganization. We are presently
recruiting for two technical level
positions to fill recent vacancies,
a QA officer, and one NHEERL
Post-doctoral fellow. Having
recruited two new principal
investigators in 2000 (one to
work in the area of
toxicogenomics, the other in
cellular mechanisms of altered
endocrine function), we are
pursuing a strategic staffing plan
that will ultimately reduce the
number of designated principal
investigators to an average of
about 5 in each branch, with
subsequent increase in technical
support. We expect that the demographics of the RTD workforce will allow us to transition
to this state over the next 3-5 years as a portion of the workforce approaches retirement
age. In addition to the federal workforce, there are 4 part-time and 1 full-time senior-
environmental-employment-program (SEEP) employees, 7 post-doctoral fellows (plus
about 5 current vacancies), 1 guest scientist, and about 10 pre-doctoral students. The pre-
and post-doctoral trainees are associated with training agreements we have with the local
Universities (Duke, UNC, NCSU, NCCU), but we are also authorized to carry 4 NHEERL
post-doctoral trainees, which are federal employees on term (not to exceed 4 year)
appointments. Until this year, NHEERL divisions managed their own workforce within the
allocated FTE ceiling. Thus, when a slot was opened by departure of a division staff
person, it was the prerogative of management to back fill that slot, or redirect to another
area. This provided great flexibility in terms of strategic workforce planning and was in
place for about 4 years. With the advent of a reduced workforce within EPA in the
upcoming fiscal year, the ability to manage the FTE ceiling at the local level has been
eliminated, and HQ or Laboratory approval is now needed to replace a departing staff
member from any division.
8
image:
RTD Overview - September 12, 2001
Figure 2. RTD Organizational Chart
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9
image:
RTD Overview - September 12, 2001
IV. RESEARCH FUNDING
The FTE and resource allocations that have supported RTD's activities since FY97
are depicted in Table 1. Our FTE allocation has been relatively stable over the past four
years. The overall increase from 61 to 64.5, resulted from decentralizing QA operations
in NHEERL and the transference of one FTE, and the allocation of four "NHEERL
Postdoctoral Trainee" slots to each division following a transference of these FTE slots.
Relative to GPRA, about three-quarters of RTDs personnel are directed towards issues
contained in Goal 8 (Sound Science) and 4 (Safe Communities), with smaller efforts
directed at Goal 2 (Clean Water, -13%), Goal 3 (FQPA, -8%) and Goal 1 (Clean Air,
-3%). More information on the GPRA Goals and relationship to the RTD research
program is provided in the next tabbed section (Goal Directed Research).
Table 1. Resource Allocations to RTD, FY97 to Date ($ in thousands)
TYPE
FY97
FY98
FY99
FY00
FY01
Supplies
$887.8
$918.3
$1082.6
$880.6
$1015.7
Extramural
$1945.9
$1082.5
$411.9
$1005.9
$1597.6
T ravel
$37.3
$31.5
$44.7
$41.3
$53.5
FTEs
61
62.5
62
64.5
64.5
As with the FTE allocation, there has been a relatively stable amount of funds for
purchasing supplies and small equipment items that are largely distributed from NHEERL
on the basis of the number of GS-12s in a division. The extramural funds, which support
professional service contracts, histopathology, chemical synthesis, microarray
development, quality assurance efforts, workshops, traineeships and cooperative
agreements with academic institutions, are derived in two ways: a portion (this year
approximately $450K) is defined as "research support" and is given to the divisions to
support activities such as QA, analytical chemistry support, pathology, web site
development, clerical support, and seminar programs; the bulk of the funds are distributed
by NHEERL through a competitive RFA-driven process that supports primarily the media
based programs and can be used for cooperative agreements with non-profit organizations
and for application to our training agreements with local universities to support placement
of pre- and postdoctoral trainees in our laboratory. Most of the above "research support"
funds in the extramural category are directed at issues related to Goal 2 (Safe Water). The
Goal 2 extramural activities support a total of three cooperative agreements on various
topics that augment our intramural efforts, and the specifics will be covered in the Themes
supporting this Goal. Over the past several years we have also been successful in RFAs
related to Children's Health, Arsenic Toxicity and Endocrine Disruptors. The other budget
10
image:
RTD Overview - September 12, 2001
line item is travel, and this has shown a relatively steady increase in amount as a result of
an ORD initiative. Not shown in this table are the funds used to procure and maintain
animals in the vivarium, the cost of which is borne by NHEERL. One other source not
displayed is that of the ORD Capital Equipment Committee, which awards funding for
equipment that costs in excess of $75,000 based upon scientific and programmatic needs.
Our confocal microscope and fluorescent imaging capabilities were acquired through this
process over the past several years.
V. RESEARCH FACILITIES
The 80,000 square-foot NHEERL facility contains approximately 17,000 square feet
of laboratory space comprised of 24 specialized laboratories and 9 shared or core facilities.
In addition, the building houses an AAALAC-approved animal facility comprising 23 animal
rooms and a surgical suite, 2 necropsy rooms, a cold room, and 2 darkrooms, as well as
37 offices, 40 cubicles for technical staff and students, a resource room/library, and 31
administrative offices for the national laboratory. With the completion of the new EPA
facility on the federal campus with NIEHS, RTD will become a stand-alone division, and
will pick up some additional office and support space when the NHEERL senior
management group relocates in mid-2002.
VI. RESEARCH MANAGEMENT:
Following a strategic planning retreat in 1994, RTD initiated a theme-based planning
process intended to concentrate research activities on a fewer number of high priority
topics that required the skills of more than a single investigator, and a relatively long term
(i.e., 3-5 years) commitment of resources. This process was fully implemented in FY96,
and has continued, with minor modifications, to serve us today.
In this planning process, Principal Investigators are encouraged to develop multi-
disciplinary research teams comprised of individuals within and external to the division, to
address high priority research areas. The identification of high priority areas is through
multiple sources, but principally through the ORD planning process addressing the GPRA
Goals. General guidelines are set forth by the Agency Administrator and translated into
ORD Research Strategies that outline the major scientific questions and research needs,
and finally, into local implementation plans such as used by NHEERL. Throughout this
process, Associate Laboratory Directors (ALDs) are responsible for ensuring the
programmatic relevance of the research for a particular Program Office or GPRA Goal,
while the line managers (Division Directors and Branch Chiefs) within the research
divisions are responsible for ensuring the overall scientific quality and timeliness of the
research. Ultimately, however, it is up to the Principal Investigators to propose research
activities based upon the guidance documents, discussions with key personnel, and input
from the general scientific community through attendance at seminars, journal clubs,
scientific meetings, and internal/external research planning activities. In order to maintain
current awareness of the science, RTD sponsors a monthly invited seminar program
11
image:
RTD Overview - September 12, 2001
(participants listed in an Appendix), and journal clubs within each branch. In addition, we
organize and sponsor mini-workshops each year on specialty topics to help researchers
keep abreast of the field and to meet leading researchers. In June of this year, we took
the additional step of inviting our clients from a number of Program Offices for a two day
"Emerging Issues" workshop in which we had discussions about the scientific front, as well
as the regulatory front. This effort proved extremely beneficial in getting the RTD staff to
know the key personnel in the Program Offices, and to begin to identify cross cutting issues
for which actions can be started. One follow-up meeting to that effort occurred in late
August when nine staff from OPPTS came to RTP for a one day workshop on scoping out
an Agency position and policy on the use of emerging toxicogenomics technologies in risk
assessment.
The theme-based planning process used within RTD (Figure 3) involves an annual
cycle in which groups of investigators submit to the RTD management team 5-10 page
proposals for a research program that includes an overall programmatic and scientific
justification, a series of specific research aims addressing the overall research topic, a list
of involved staff, and a proposed supply budget. In this proposal they are specifically
asked to address:
The issue being addressed
The research questions being asked
The research approach for answering the questions
A tentative timetable for anticipated progress
The expected products
The personnel involved
Anticipated budgetary needs
Once the submissions have been received, they are evaluated by the RTD
Management Committee consisting of the Division Director and Branch Chiefs who assess
the quality of the proposed science program, its relevance to Agency needs, the
anticipated resource requirements, previous and potential productivity of the unit, and
needs for quality assurance and quality control. During discussions between the Review
Committee and the Theme Leader, feedback is provided on the overall direction and the
value of each proposed specific aim. The plan is then revised, reviewed for consistency
with comments, and approved at certain staffing and funding levels. These events
generally take place between September and November of each year. Progress by the
Theme is reviewed in at least four ways: (1) monthly Work-in-Progress meetings allow the
team to present various aspects of the ongoing work to division members; (2) quarterly
meetings of theme leaders with the Management Committee to review significant progress
and obstacles; (3) mid-year performance reviews of the Theme Leaders, and (4) evaluation
of the preceding year's progress should the Theme be submitted for potential funding in
the next fiscal year.
12
image:
RTD Overview - September 12, 2001
Figure 3. Research Planning in RTD
Program
Office
Dialogue
State
of the
Science
Agency/ORD
Planning
Documents
NHEERL
Implementation
Teams
Divisional
Research Themes
(specific aims, FTEs, $$,
and milestones)
Committee
•relevance
•scientific merit
•feasibility
•resources
RTD Review
Theme Leader
Proposals
•significance
•specific aims
•rationale
•progress
•goals
•methods
•discussion
13
image:
RTD Overview - September 12, 2001
From a research management standpoint, we expect that this process will facilitate:
More efficient use of limited human, financial and capital resources
Increased multi-disciplinary interactions and collaborations amongst
RTD, NHEERL and extramural Pis (see Table 2)
Greater alignment between the research programs and Agency
needs by directing resources toward high-priority issues
Increased incorporation of bottom-up input into the research planning
process
Increased productivity as gauged by the total number of publications
Increased quality of the research products, as evidenced, for
example, by recognition of EPA Scientific and Technological
Achievement Awards
Greater impact of the completed research on problems facing the
Agency.
We believe that by developing this planning process ahead of the new efforts to
develop "program projects" within the most important GPRA issues we are in a good
position to maintain the programmatic relevance that RTD has traditionally achieved
The RTD FY01 Research Plan is being provided as an electronic copy on the
accompanying CD for those reviewers wishing greater detail on the nature and content of
the proposals. A few hardcopies will also be available at the review The Cross Walk
(Rosetta) Table that links themes to specific research aims to composite posters provides
a quick and efficient overall reference for understanding the relationship of these various
planning and implementation processes. This Table is included in the Tab that provides
the "Schedule and Review Assignments."
14
image:
R. overview - September 12, 2001
Theme
Table 2: Collaborators
NHEERL Laboratories
ORD
NIH
OUTSIDE
COLLABORATORS
AED
ECD
ETD
GED
HSD
MED
NTD
WED
NERL
NRMRL
NCEA
NIEHS
Atrazine
~
~
~
See below
Critical
Windows
~
~
See below
DW - Dev
~
~
~
~
~
~
~
See below
DW - Repro
~
~
~
~
~
~
~
See below
Dev Repro
~
~
~
~
~
~
~
~
See below
Receptors
~
~
See below
Susceptible
Populations
~
~
See below
Collaborations with Universities and Industry:
Atrazine: Dr Parikshit Das, Univ of North Carolina-Chapel Hill; Dr. Lee Tyrey, Duke Univ Medical Center, Dr Lee Robinette, NC State Univ , Dr Thomas Sanderson, Utrecht,
The Netherlands; Dr. Johnathan Lindzey, Univ. of Southern Florida
Critical Windows: Dr. Aida Cancel, FHI, Dr Donald Evenson, SDSU, Dr Jiri Rubes, VRI - CZ Repubic, Dr Wendie Robbins, UCLA, Dr. Deborah O'Brien, Univ of NC-Chapel
Hill; Dr Patricia Hoyer, Univ of Arizona, Dr Jodi Flaws, Univ. of Maryland.
Drinking Water - Development: Dr. Shelly Tyl, Research Triangle Institute, Dr Tom Sadler, Univ of NC at Chapel Hill
Drinking Water - Reproduction: Drs. Andrew Olshan and David Savitz at UNC-Chapel Hill, Susan Rothmann, Fertility Solutions, Inc , Wendie Robbins, Univ of California-Los
Angeles (UCLA), Drs Ken Roberts and David Hamilton, Univ of Minnesota; Dr Steven Sylvester, Univ of Washington at Vancouver, Dr Harry Moore, Univ. of Sheffield in
England, Dr Gustavo Doncel, Eastern Virginia Medical School; Dr Richard Oko, Queens Univ. in Ontario, Dr. Giovanni Bernardini, Univ of Varese, Italy; Drs. Rao
Veeramanchaneni and Heywood Sawyer, Colorado State Univ; Dr. Rochelle Tyl, Research Triangle Institute
Dev Repro: Drs P. Foster and N. Barlow, CUT; Dr. G. LeBlanc, North Carolina State Univ. (NCSU), Dr L. Guillette, Univ of Florida (UFL),
EM Wilson, Univ of NC-Chapel Hill; DNR Veeramachaneni, Colorado State Univ ; J. Brock (formerly of CDC), R Tyl, ResearchTrianglre Institute
Receptors: Dr David Lee, Univ of NC-Chapel Hill; Dr. Dianne Bofinger, State Univ of New York-Buffalo; Dr Jeff Peters, National Cancer Institute. National Institutes of Health;
Dr Oliver Hankinson, Univ of California-Los Angeles, Dr. Richard Peterson, Univ. of Wisconsin-Madison
Suscepitable Populations: Bruce Allen, ICF; Tom Sadler, UNC
15
image:
RTD Overview - September 12, 2001
VII. SCIENTIFIC RECOGNITION/PRODUCTIVITY
Since January, 1997, RTD investigators have published nearly 250 research papers
and book chapters (Table 3) in more than 50 different journals, including ones in Biology
of Reproduction, Critical Reviews in Toxicology, Development, Developmental Dynamics,
Endocrinology, Human Reproduction, J. Biological Chemistry, J Molecular Medicine, J
Pharmacology and Experimental Therapeutics and Molecular Endocrinology.
Table 3. Scientific Productivity, 1997-2001
Abstracts
Research
Papers
Chapters
1997
57
35
10
1998
73
38
12
1999
57
35
6
2000
80
37
8
2001
72
58
8
A full listing of journals with impact factors is provided in the Bibliography tab, together with
a listing of journal impact factors.
One important indicator of the success of an ORD/EPA research program is the
number of Scientific and Technological Achievement Awards (STAA) bestowed on its
scientists. The STAA Award is an externally reviewed ORD annual award program for
peer-reviewed publications of scientific excellence and relevance to the Agency. The
awards come in four categories - Honorable Mention, and Levels I to III, with Level I being
the highest. Competition for the awards is strong, and only a small fraction of nominated
papers receive recognition. In the years preceding the past review, RTD received
approximately one-third of all the STAA awards given in the area of human health. More
recently, we have received a Level II award in 1999, a Level I, a Level III and two
Honorable Mentions in 1997, and a one Level I and two Level III awards in 1996.
Several RTD scientists have been recognized by the EPA Honor Awards Program.
Sally Darney, Earl Gray, Robert Kavlock, and Gary Klinefelter received Bronze Medals in
1999 for Commendable Service developing the EPA Harmonized Reproductive and
Developmental Test Guidelines. Earl Gray also received a Gold Medal for Exceptional
Service for his work on the Reproductive Risk Assessment Guidelines Workgroup in 1997
16
image:
RTD Overview - September 12, 2001
and a second Bronze Medal in 1999 for his activities on the Endocrine Disruptor Screening
and Testing Advisory Committee. Ralph Cooper received a Bronze Medal in 1998 for his
participation on the ORD/RAF Workgroup on Environmental Endocrine Disruption. The
Atrazine team received the Office of Pesticides Programs Health Effects Division Team
Award for work related to the chlorotriazines in 2000 and an EDSP team award for
Exceptional/Outstanding ORD Technical Assistance to the Regions or Program Offices in
2001.
RTD researchers are also active participants in many scientific societies and
organizations and have won numerous awards and honors over the years. Members of
the Atrazine team have won the honor of Best Paper Published in the Reproductive &
Developmental Toxicology Speciality Section of Toxicological Sciences in both 1999 and
2000. Sid Hunter received the Teratology Society's Clark Fraser Young Investigator Award
in 1999. Gary Klinefelter has been the recipient of two Outstanding Original Research
Awards from the American Society of Andrology, and Sally Darney presented the Society's
Buckeye Lecture in 1998.
Two outstanding achievements bear particular mention. Gary Klinefelter holds a
distinctive honor as inventor of EPA's first biotechnology patent, "Method for Evaluating
Male Fertility" issued March 6, 2001 as U.S. patent (# 6,197,940) on the SP22 protein,
fragments and recombinants. Earl Gray was elected as an American Association for the
Advancement of Science (AAAS) Fellow in 1999.
Over the past decade, RTD has attained broadening national and international
recognition, as evidenced by key demographic indicators of staff impact provided in the
individual biosketches and in the tables that cover invitations to international meetings
(Table 4), journals that we have served as scientific reviewers (Table 5), and elected
offices held (Table 6). For example, RTD staff have served as the treasurer for the Society
of Reproduction (Sally Darney), president and treasurer for the Teratology Society (Robert
Kavlock and John Rogers, respectively), councilor and treasurer for the Andrology Society
(Gary Klinefelter and Sally Darney, respectively), presidents of the North Carolina Society
of Toxicology (Robert Kavlock and Barbara Abbott), and president, treasurer, and councilor
of the Reproductive and Developmental Toxicology Specialty Section of the SOT (Robert
Kavlock, John Rogers and Barbara Abbott, respectively). The Staff holds a total of 15
adjunct appointments at research universities (e.g., Duke, UNC, NCSU), and many of the
adjuncts actively participate in teaching and mentoring graduate students. RTD staff
currently hold 19 editorships on peer reviewed scientific journals, including Archives of
Environmental Contamination and Toxicology; Biology of Reproduction; Cell Biology and
Biophysics; Journal of Andrology; Journal of Applied Toxicology; Journal of Toxicology and
Environmental Health; Molecular Reproduction and Development; Teratology;
Teratogenesis, Carcinogenesis and Mutagenesis; Toxicological Sciences; and
Reproductive Toxicology. We also provide ad hoc reviews for a wide number of journals
(see Table 5).
17
image:
In addition, RTD investigators are highly active in numerous internal and external
scientific forums, including the Teratology Society, the Society of Toxicology, the North
Carolina Chapter of the Society of Toxicology, the Triangle Consortium for Reproductive
Biology, the American Society of Andrology, and the Society for the Study of Reproduction.
We have participated in and organized a number of national and international workshops
and symposia, notably in the areas of the reproductive risks of disinfectant by-products and
of endocrine disruption. Finally, a number of RTD investigators serve as advisors,
committee members or reviewers for the EPA Program Offices, the World Health
Organization, the International Agency for Research on Cancer, the International
Programme on Chemical Safety, the National Research Council, the National Toxicology
Program, The National Institutes of Health, the National Institute of Environmental Health
Sciences, the NORA program of NIOSH, the NTP Center for Evaluation of Risks to Human
Reproduction, working groups of the International Life Sciences Institute (ILSI), the
Interagency Coordinating Committee on the Validation of Alternative Test Methods, and
the CUT Centers for Health Research. The individual biosketches of RTD principal
investigators in a separate section of the briefing book provide more detailed information
on advisory and technical assistance issues. A full division bibliography is provided in the
appendix, and important publications are reiterated in the description of each research
theme. Hard copies of any publications will be provided upon request.
18
image:
Table 4. International Meetings Attended by RTD Scientists,
1997-2001
2001
9™ International Congress of Toxicology, Brisbane, Australia
7th International Congress of Andrology, Montreal, Quebec, Canada
2nd International Conference on Male-Mediated Developmental Toxicity,
Montreal, Quebec, Canada
3rd International Workshop on Endocrine Disruptors, Tsukuba, Japan
2000
International Federation of Teratology Societies, Matsue, Japan
Satellite IFTS Symposium Reproductive and Developmental Effects of
Dioxin in Endocrine Disruptors, Hiroshima, Japan
International Workshop on Hormones & Endocrine Disruptors in Food &
Water, Copenhagen, Denmark
2nd International Symposium on Environmental Endocrine Disruptors, Tokyo,
Japan
WHO/IPCS Endocrine Disruptors Steering Group, London, UK
UNU International Symposium on Endocrine Disruptors Pollution in East
Asian Coastal Hydrosphere, Kuala Lumpur, Malaysia
1999
IPCS Steering Group on Endocrine Disruptors, Stockholm, Sweden
Joint US/EU Endocrine Disruptor Research Expert Panel Meeting,
Ispra, Italy
WHO/IARC Handbooks of Cancer Prevention on Retinoids, Lyon, France
"99 International Symposium on Environmental Monitoring & Analysis in
the East Asian Region, Tokyo, Japan
European Society for Pediatric Endocrinology, Stockholm, Sweden
Dioxin'99, Venice, Italy
1998
International Congress of Toxicology VIII Meeting, Paris, France
WHO/IPCS Steering Group Meeting on Endocrine Disruptors, Milan, Italy
IARC Monographs Working Group, Lyon, France
1997
WHO Meeting, Geneva, Switzerland
2nd EC/US Wksp on the Integration of Multiendpoint Mechanistic Info in
Risk Assessment, Ispra, Italy
SETAC/Europe/OECD/EC Expert Workshop on Endocrine Modulators and
Wildlife, Amsterdam, The Netherlands
19
image:
Table 5. Ad Hoc Journal Reviews by RTD Scientists, 1997-2001
American Journal of Physiology
Analytical Biochemistry
Andrologia
Archives of Environmental Health
Archives of Toxicology
Biochimica Biophysica Acta
Biological Trace Element Research
Biology of Reproduction
Journal of Agricultural and Food
Science
Journal of Andrology
Journal of Biological Chemistry
Journal of Bone and Mineral Research
Journal of Clinical Investigation
Journal of Clinical Pathology
Journal of Craniofacial Genetics and
Biomedical and Environmental Sciences Developmental Biology
Bio Techniques
Brain Research
Brain Research Bulletin
Cell Biochemistry and Biophysics
Cell Stress & Chaperones
Chemical Research in Toxicology
Communications in Clinical Cytometry
Comparative Biochemistry and
Physiology
Critical Reviews in Toxicology
Cytometry
Endocrine
Endocrinology
Environmental and Molecular
Carcinogenesis
Environmental Health Perspectives
Environmental Research
Journal of Experimental Cell Research
Journal of Experimental Zoology
Journal of Nutrition
Journal of Reproduction and Fertility
Journal of Toxicology and Environmental
Health
Life Sciences
Molecular Pharmacology
Molecular Reproduction and Development
Mutation Research
Neurotoxicology and Teratology
Neurotoxicology
Reproduction
Reproduction Fertility and Development
Reproductive Toxicology
Risk Analysis
Science
Environmental Science and Technology Teratology
Environmental Toxicology and
Chemistry
Epidemiology
Experimental Cell Research
Fertility and Sterility
Food and Chemical Toxicology and
Pharmacology
Free Radical Biology and Medicine
Fundamental and Applied Toxicology
Human Reproduction
International Journal of Andrology
The Anatomical Record
The Cleft Palate-Craniofacial Journal
The Science of the Total Environment
Toxicology
Toxicology and Applied Pharmacology
Toxicology and Industrial Health
Toxicology Letters
Toxicological Sciences
Zygote
20
image:
Table 6 Activities in Professional Societies
1997- Present
Abbott, B.
North Carolina Society of Toxicology
President-elect, 2002-03
Society of Toxicology / Reprod &
Dev Toxicol Specialty Section
Councilor, 2002-04
Darney, S
Society for the Study of Reproduction
Strategic Planning Committee, 1997
Finance Committee, 1998-01
Treasurer, 1997-00
Development Committee, 2001-
American Society of Andrology
Nominating Committee, 1997
Future Meetings Committee, 1999- present
Women in Andrology
President-elect, 2001
Program Committee Chair, 2003
Hunter, S.
Teratology Society
Membership Committee, 1998-00
Public Affairs Committee, 1997-98
Fraser Young Investigator Award Committee, 1999-00
Kavlock, R.
Society of Toxicology
Continuing Education Committee, 1997-00
Society of Toxicology / Reprod. &
Dev. Toxicol. Specialty Section
President, 1997
North Carolina Society of Toxicology
President, 1999-00
Teratology Society
Councilor, 1994-97
Strategic Planning Committee, 1996-99
Endocrine Disruptor Position Paper ad hoc
Committee, 1997-99
Vice-President and Scientific Program Chair, 1999-00
President, 2000-01
Klinefelter, G.
American Society of Andrology
Executive Council, 1999-present
Society for the Study of Reproduction
Program Committee, 2000-01
Narotsky, M
Teratology Society
Education Committee, 2001
Rogers, J.
Society of Toxicology / Reprod &
Dev. Toxicol. Specialty Section
Secretary/Treasurer, 1997-00
Teratology Society
T reasurer, 1998-present
Strategic Planning Committee, 1997-present
Stoker, T.
Triangle Consortium of Reproductive
Biology
Board Member, 2001
21
image:
VIII. FUTURE DIRECTIONS:
There are three main scientific concerns that will impact the future efforts of RTD:
(1) what are the important adverse outcomes related to developmental and reproductive
health in the human population, (2) what current efforts do we have underway that require
additional resources in order to make the desired impact; and (3) what significant aspects
of developmental and reproductive toxicology are lacking from our current program. Input
from the ORD and NHEERL planning processes, reports of national advisory bodies such
as the NAS, the mini-workshops we hold, and the opinions of this peer review panel will
all influence which, and to what extent, of these concerns will impact the current program.
Whatever directions we pursue must be consistent with our existing scientific capabilities,
or entail retraining of our existing staff, as we have a more pressing need to augment the
support staff to our researchers than to add new researchers.
Relative to public health outcomes, we are intrigued by the implications of the
Barker hypothesis regarding the developmental origins of some adult-onset diseases, and
feel this is an area of high priority to examine from the viewpoint of developing and testing
animal models. We know from recent studies that there are early critical periods for proper
development of the mammary gland and prostate, and thus there is an interesting
complement between what we are beginning to know about latent effects in animal models
versus what has been suggested to be the latent effects in the human population (which
seem to be more cardiovascular and endocrinologic in origin). While the effect drivers also
seem to vary in the two groups, they are commonly united by being mediated through
modifications of endocrine system maturation and function.
The Human Genome Project and the accompanying revolution in genomics will no
doubt yield vast stores of information that will have considerable impact on the
determination of genetic susceptibility factors. Already the CDC is collecting a repository
of biological tissues for significant numbers of cases and controls for the 30 leading birth
defects, with the expectation that this material will help elucidate which genes and
polymorphisms are related to heightened expression of abnormal phenotypes. On the
other hand, the greater use of genomics in our toxicology program will hopefully begin to
identify those key precursor events that regulate the expression of toxicity or the ability to
repair damage. This will naturally lead to the exploration of polymorphisms for those genes
in exposed populations to determine whether those with altered risks can be identified and
what are the possible risk management decisions that result from that knowledge. The use
of improved animal models in which pathways have been knocked out, knocked in,
programmed or humanized will aid in moving this type of research forward. This activity
is strongly supported by the recent NAS report on frontiers of developmental toxicity risk
assessments. Another application of genomics technology that will be explored is their
use in examining expression patterns of accessible tissues as surrogates for responses
of internal organs (an approach we term biomonitoring).
In addition to the genomics area, there is a clear need to expand work in the area
of proteomics, as evidenced by the work with SP22 and with altered phosphorylation
22
image:
products associated with the DBP induced toxicity. Better measures of sperm integrity are
needed that could be used in routine toxicological settings. We also feel a need to
increase our attention on the brain as a neuroendocrine target of steroid hormone agonists
and antagonists, as well as agents which influence development of dopaminergic neurons.
Biomarkers of semen quality and the brain as a neuroendocrine target are likely to be our
two mini-workshops that we will sponsor in FY02.
Other public-health outcome-related considerations that may influence our program
are events such as pre-term delivery and precocious puberty. Preterm delivery is a leading
cause of infant morbidity and mortality, has no appropriate small animal model, and little
understanding of potential environmental influences. The age of onset of puberty appears
to be decreasing; but the extent to which environmental factors contribute is unknown.
While our current focus lies heavily with toxicological studies, we would like to seek
stronger ties with the Human Studies Division in order, when appropriate, to increase our
involvement in clinical or epidemiological research.
In terms of regulatory needs, the most evident emerging issues relate to the
characterization of modes and mechanisms of action for use in determinations of
aggregate and cumulative exposures (when and how should chemicals be combined in a
comprehensive risk assessment); the impact of acute/burst exposures on reproductive
events, and the need for chemical-specific information to inform important risk assessment
issues such as for the perfluorinated alkyl sulfonates.
23
image:
O D
? o
1-
* X
>
image:
Goal Directed Research
GOAL DIRECTED RESEARCH WITHIN RTD
When considering Goal directed research, it is important to understand
both the issues that are pertinent within a particular Goal, as well the inter-
relationships of some issues across multiple Goals. Thus, the potential public
health impacts, for example, of drinking water disinfectant byproducts and of
endocrine disruptors are of concern to Goal 2 and 8.3, respectively, and issues
such as special susceptibilities (age, genotype, pre-disposing disease,
concurrent exposures), biomarkers and the effects of multiple exposures, are of
concern to almost every GPRA Goal. The alignment of a particular experimental
focus to a Goal is therefore at times subjective and dependent on factors such as
the current state of the science (more fundamental research needs being
covered in Goal 8 (Sound Science) and more applied type work covered in the
media-specific Goals) and the origin of the research questions (is the research
being conducted to understand a particular environmental contaminant, or is it
being done to understand some fundamental aspect of a biological process and
its regulatory controls). Research conducted within Goal 8 is considered the
core research program of ORD, and is intended to be longer term in duration, to
address problems of high complexity requiring multi-disciplinary approaches, and
to produce results that are of more generic utility to the protection of public
health. About 40% of the ORD and NHEERL research portfolio consists of core
research, with the other 60% being more media (Program Office) needs driven.
There is a naturally an overlap in core and non-core efforts, as illustrated in
following figure.
Figure 1. Relationship of Core Research to Media-Oriented Research
Sate Water
(DBPs)
83
82
Endocrine
Disruptors
Safe Foods
(FQPA)
Human Health
Risk
Assessment
Sale
Communities
(Test
Methods)
1
image:
Goal Directed Research
Research activities within the GPRA Goals are driven through the
development of ORD Research Strategies and related NHEERL Implementation
Committees. The ORD Strategies provide broad definition of the problems and
establish priorities and long-term objectives for the research program. On the
local level, NHEERL began about two years ago to use a Research Strategy
Implementation process involving one manager and one bench scientist per
relevant Division to guide the process of translating the ORD Strategies into
operational activities. The major goals of this process are to provide closer
alignment between the NHEERL research portfolio and the Strategies, and to
develop more "program project" type multidisciplinary research efforts that more
effectively tap the broad expertise available within the laboratory. Because of the
wide variation in status of ORD Research Strategies and NHEERL
Implementation Teams, the amount of guidance to the researchers working in the
different Goals is also variable (see Table 1). RTD chairs the first NHEERL
Implementation Team that was established (for Goal 8.3 - Endocrine Disruptors),
and participates actively in the Goal 2, 4 and 8.2 Implementation Teams.
Table 1. Current Status of GPRA Guidance Documents (F=finai, LD= late draft,
MD = mid draft, ED = early draft, NS = not started)
Goal
1
Goal
2
Goal
3
Goal
4
Goal
8.2
Goal
8.3
ORD Strategy
MD
F
NS
NS
LD
F
NHEERL Implementation Team
MD
MD
NS
ED
LD
F
As mentioned in the RTD Overview and shown again in Figure 2,
approximately three-quarters of RTDs intramural resources are directed towards
issues contained in Goals 8 (Sound Science, 45%) and 4 (Safe Communities,
29%), with smaller efforts directed at Goal 2 (Clean Water, -14%), Goal 3 (Safe
Food, -8%) and Goal 1 (Clean Air, -3%). These allocations are adjusted each
year to reflect changing emphasis within the ORD, NHEERL and RTD planning
processes, but with a few exceptions where we have moved to consolidate our
efforts into fewer areas (e.g., reducing activity in Goal 1 (Clear Air) and in the
Candidate Contaminant List of Goal 2), the proportional distribution across Goals
has been relatively consistent over the past 4 years. The limited resources
available from Goal 1 is used primarily to provide technical assistance on matters
of reproductive and developmental toxicity relative to the air program, although
some of our work on concentration by time effects on toxic response is of interest
to them from the viewpoint of burst exposures to air pollutants and potential
effects on critical windows of susceptibility such as the control of ovulation and
the development of the embryo. Because of the relatively small size of this
effort, it will not be highlighted in this review.
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Goal Directed Research
Fig. 2 FTE Allocation by Goal
Goal 1 Goal 2
Goal 4
29%
EGoal 1 BGoal 2 DGoal 3 DGoal 4 BGoal 8
Goal 2: Clean Water:
The major human health concerns in Goal 2 relevant to RTD are to
characterize the hazards and risks of key disinfectant byproducts. This need is
driven by a number of factors, including the plethora of DBPs that vary as a
function of source water and treatment technology, the need to set not only
regulatory standards for critical by-products but also to design appropriate
sampling strategies to monitor compliance, the findings that some by-products
are indeed toxic to the developing embryo and to the reproductive system, and
finally, evidence of adverse pregnancy outcomes in humans that correlate with
various water sources. Particular research priorities have also been
recommended by a number of private (e.g., AWWARF, CCC) and governmental
advisory bodies (e.g., FSTRAC), and such recommendations have been central
in development of our program.
Research addressing these Goal 2 needs is supported in two RTD
Themes: one lead by Dr. Sid Hunter that focuses on developmental outcomes,
and the other lead by Dr. Gary Klinefelter that focuses on reproductive effects.
Through both a considerable intramural program, and partnership with other
research organizations (NIEHS, Colorado State University), RTD research is
striving to help identify which of the myriad of DBPs display the greatest potential
to affect development and reproductive function, what modes of action are
involved in the toxicities, and what is the extent of biological plausibility for the
epidemiological findings. The research strategy involves utilization of whole
animal screening methods to prioritize additional studies, longer term whole
animal studies to understand life-stage specific sensitivities, and in vitro systems
(e.g., whole embryo culture, seminiferous tubule cultures and ovarian follicular
culture) to characterize modes of action. Selection of chemicals for screening is
made after consultation with the Office of Water regarding their legislative
priorities, surveillance data on the prevalence and magnitude of DBPs from
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Goal Directed Research
national surveys of finished waters, and from the growing body of toxicological
knowledge. The in vitro studies are also useful for exploring relationships
between target tissue concentrations and the duration of exposure necessary to
initiate toxicity. At present, the vast majority of research is on the haloacetic
acids, and in particular, the brominated acetic acids, as they appear to be the
most potent toxicants based upon the screening level assays. Indeed, based on
our laboratory findings, we have recently expanded the scope of effort to include
participation in an epidemiological study in order to characterize the potential of
DBPs rich in brominated species to affect semen quality. We have also initiated
a comprehensive multi-generational study of bromochloroacetic acid in response
to a stated need of OW. We anticipate that the research findings will be
informative in setting the Stage II DBP rule, both in terms of the selection of
MCLGs for key contaminants, but also for the sampling frequency necessary to
adequately ensure that excursions above the standard do not occur at a
frequency that could pose a public health problem for pregnancy outcomes.
Involvement of the staff in national efforts discussing the interpretation of the
current findings and recommending future directions is evidence of the
leadership being displayed by RTD relative to Goal 2.
Over the next few years, it is anticipated that greater attention will be paid
to exploring the shape of the dose-response curve through efforts linking
pharmacokinetic studies with mode of action information, and perhaps extending
into alternative animal models. The research is already starting to encompass
greater emphasis on the use of genomic and proteomic technologies to answer
the key research questions, and will also be analyzing the potential interactive
effects of exposure to multiple DBPs. As work with individual DBPs matures, we
will be moving to work more with mixtures, and already have projects started with
NTP to examine the impact of model mixtures of haloacetic acids that would be
expected to originate from areas of high and low bromide, respectively. Another
mixture study is underway within NHEERL to look at water concentrates from two
disinfectant processes. Interestingly, the core design for that multi-division
experiment is based on assessment of reproductive function. There is likely also
to be a gradual shift in emphasis away from DBPs to the broader issues of CCLs,
but for the foreseeable future, RTDs efforts are remain focused in areas where
we know there is human exposure, and where there are suggestions of public
health problems.
Goal 3: Safe Food
The central focus of research in Goal 3 is related to the implementation of
the Food Quality Protection Act of 1996. In addition to requiring development of
a screening and testing program for estrogenic and other endocrine disrupting
chemicals (an effort covered in Goal 8.3), this act directed the EPA to consider
grouping together chemicals with common 'modes of action' when evaluating the
potential joint exposure of food use chemicals. Another important component of
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Goal Directed Research
the Act was to direct additional concerns to the issue of risks to children of
exposure to pesticide, and in fact, directed the EPA to use an additional 10 fold
uncertainty factor when the database was insufficient to evaluate whether the
developing organism was not more susceptible to toxicant exposure that was the
adult.
Work in the Cellular and Molecular Mechanisms Theme under Earl Gray is
exploring the consequences of concurrent exposure of agents with similar
mechanisms of action (e.g., androgen receptor antagonists) as well as dissimilar
mechanisms action but likely common modes of action (e.g., interference with
steroid production and steroid action) on the development of the male
reproductive track in order to understand the degree to which combinations
exhibit or depart from conditions of dose additivity. This work will be directly
applicable to the question of how aggregate risk should be considered for
specific chemical classes under FQPA. The Pesticide Program has been
particularly interested in results from studies using combinations of vinclozolin
and procymidione, two fungicides that act as competitive androgen receptor
antagonists. Other work is exploring the responses to Linuron, a herbicide with
anti-androgen receptor activity and dibutylphthalate, a plasticizer recently shown
to inhibit testosterone biosynthesis in the late gestation fetal rat testes.
Experiments with these anti-androgens combined with TCDD, which inhibits male
reproductive tract development by an as yet ill-defined mechanism are also
under consideration. Within the Susceptible Subpopulation Theme under John
Rogers, efforts are underway to develop experimental models to determine the
latent manifestations of adverse health effects following developmental
exposures. Adult-onset diseases such as hypertension, stroke and Type II
diabetes have recently been associated with altered patterns of fetal and
neonatal growth in humans, and there is a critical need to understand the extent
of biological plausibility of the correlation and to determine the extent to which
such findings can be explored in animal models. The need for this research is
made all the more important by the fact that altered fetal growth is one of the
most common findings in standard Segment II type study designs.
Goal 4: Safe Communities
Research covered in Goal 4 has traditionally dealt with the development of
new test methods for hazard identification, but is currently migrating more toward
issues related to aspects of interpretation of test method data and of
development of biomarkers applicable for characterization of risks to children.
Because there is often times a need to understand the basic underlying biology
of a system before developing a test methods for use in regulatory toxicology,
Goal 4 research is often dependent on outputs of our core research program
covered in Goal 8.2 (Human Health Risk Assessment), these is often overlap in
the research conducted in those goals.
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Goal Directed Research
Within the Critical Windows Theme lead by Sally Darney, research efforts
are ongoing to define improved endpoints of semen quality for potential
application in reproductive toxicity tests. Previously this group optimized
protocols for use of CASA (Computer Assisted Semen Analysis) methods for use
in rodents, and helped introduce the need for measures of sperm motility in the
new multigeneration reproduction test. Recognizing that other aspects of sperm
function were needed to characterize reproductive hazard, more recent efforts
have been directed at exploring tests of DNA or genetic damage in sperm (e.g.,
FISH for aneuploidy, acridine orange staining for DNA damage), when fertility
studies implicate male-mediated effects. Collaborations with epidemiological
studies of semen quality have allowed several of these techniques to be used
both in the experimental setting as well as in populations. In the area of female
reproductive health, methods development work is underway to examine
follicular health using confocal laser microscopy to visualize oxidative stress and
cell death in ovarian tissue. The ability to readily image and identify damage to
developing follicles would cover a notable gap in the current reproductive testing
methods. These studies are combined with those centered on the role of GSH in
protecting the oocyte from oxidant induced damage, and take on a particular
concern for how the susceptibility of the oocyte varies as a function of stage of
the estrous cycle.
One of the main thrusts in Goal 4 are to begin to understand how the use
of genomic and proteomic approaches can advance our understanding of age-
related toxicities, and has centered on the testes as the primary target organ of
interest. This is a relatively new effort, but is one sure to increase in importance
and visibility as the techniques begin to demonstrate the ability to detect sensitive
signals of tissue response and help define the molecular mechanisms that will
not only reduce uncertainties in extrapolation across species, but also begin to
point to the key susceptibility genes for which polymorphisms should be sought in
the human populations. Ultimately, this will lead to identification of increased (or
decreased) risks to individuals, as opposed to the population-based risk
assessment that is currently employed.
Within the Susceptibility Theme, we are currently working with OPPTS to
evaluate the manifestations and mode of action for a chemical class (the
perfluoro alkyl sulfonates) that recently been shown to exert significant adverse
effects on pregnancy outcome at relatively low dose levels. The toxicity is
manifest by high incidences of neonatal morality in the absence of over
malformations, and data suggests that impairment of thyroid function is a
potential contributing factor. Because these chemicals are quite persistent in the
environment and because there are a relatively large number of congeners with
significant production volume, the Program Office is very concerned with
identifying the key precursor events and establishing some structure-activity
relationships for the class.
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Goal Directed Research
Goal 8 (Sound Science):
Goal 8 houses the core research efforts of NHEERL in terms of
developing the principles and practices upon which media-specific research, and
ultimately risk assessment, is conducted. There are two main elements relative
to RTD: the Human Health Risk Assessment Strategy (Goal 8.2) and Endocrine
Disruption (Goal 8.3).
There are three main focus areas for Goal 8.2 articulated with the ORD
Research Strategy for Human Health Risk Assessment- (1) Harmonization of
Cancer and Non-Cancer Risk Assessment (i.e., research to understand modes
and mechanisms of action across "-icities" and using information in a consistent
way in the risk assessment process to reduce potential uncertainties): (2)
Aggregate/Cumulative Risk (i.e., issues related to the fact that we are always
exposed by multiple routes to multiple stressors, and how these might interact in
a concurrent or sequential exposure situation), and (3) Susceptible
Subpopulations (with emphasis on the key biological factors such as life stage
and genetics that convey heightened or lessened risk to an individual once
exposed to toxicant).
Goal 8.3 is currently focused on issues related to developing testing
methods, identifying chemical classes of concern, characterizing the shape of
dose-response curves for various modes of action, aspects of cumulative risk,
and extrapolating findings across levels of biological organization and species for
chemicals that exert their toxicity via interference with the function of endocrine
systems. Similar to Goal 8.2, there are 3 focal areas for this research: (1)
Support of the FQPA mandated screening and testing program for EDCs; (2)
Developmental susceptibilities to EDC exposures; and (3) Interspecies
extrapolation of EDC effects.
Because of the inherent fundamental nature of much of research needs in
Goal 8, and the key role that the endocrine system plays in development and
reproduction, multiple RTD Themes are directed at least in part to addressing the
problems identified here. The cross Theme compendium of targeted research
for each of the Goal 8 focus areas is presented below.
Goal 8.2: The Human Health Risk Assessment Research
The HHRA Research Strategy is reaching the final stages of development,
and with the three primary focal areas note above, is likely to have impact on
future directions of the NHEERL core research program. Much of RTDs work is
already aligned with the new directions, but there is likely to be some changes in
the portfolio over the next few years as the Strategy begins to have more
concrete influence on the allocation of resources. Historically, cancer and non-
7
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Goal Directed Research
cancer risk assessment have employed diverse assumptions and processes
(e.g., threshold vs non-threshold, scaling factors for species extrapolation or an
uncertainty approach) and dose extrapolation models (linear or RfD based). With
the advent of greater ability to determine modes of action and key precursor
events in toxicity, it is becoming apparent that the scientific basis for separate
processes may not be valid, and that efforts are needed to replace traditional
uncertainty factors with more biologically justifiable extrapolation factors.
Success here is predicated on the ability to understand modes of action in terms
of dose re-response (are they dose-dependent, life-stage dependent, influenced
by genetics) and inter-species extrapolations (are they species-dependent, how
do rate-limiting steps vary across species).
A prime example of this work is contained in the Chlorotriazine Theme
lead by Ralph Cooper. Atrazine is a large production volume chlorotriazine
herbicide that has been observed to increase the incidence of mammary tumors
in the Sprague-Dawley rat in a standard lifetime feeding study. Additional
studies by the registrant, however, did not find similar responses in the Fischer
344 rat, or in the B6C3F mouse. Furthermore, they characterized the induction
of mammary tumors in the sensitive rat as one of an acceleration of normal aging
process in that strain. This lead to uncertainty in how to treat the species and
strain sensitivity in the risk assessment process, and what dose-extrapolation
model (linear or margin-of-safety) should be used by the Agency. In order to
clarify the mode of action for the tumorogenesis, the Chlorotriazine team began
exploring the neuroendocrine status of several rat strains following acute and
sub-chronic exposures to atrazine. They found that the key precursor event in
the early onset of mammary gland tumor induction revolved around the ability of
atrazine to attenuate the LH surge of cycling female rats. This attenuation, in
turn, was posited to augment the normal aging process of the hypothalamic-
pituitary-ovarian axis, which ultimately lead to a physiological state of enhanced
endogenous estrogen levels, and a more suitable milieu for induction of
mammary tumors. The lack of a tumor response in Fischer 344 rats and B6C3F
mice is consistent with a different aging process in those strains. Furthermore,
the blunting of the LH pulse, were it to occur in humans exposed to atrazine,
would also not be expected to accelerate the reproductive aging process and
would therefore not be expected to lead to mammary tumors. Hence, because
the mode of action is non-genotoxic, a margin of safety approach was deemed
appropriate for the cancer risk assessment. However, the mode of action does
present other questions as to the consequences of atrazine exposure in the
human, particularly for non-cancer endpoints. Additional work is now clarifying
potential outcomes in male and female rats during different life stages when LH,
and the related pituitary hormone prolactin, are known to play key roles in
development and physiology. Data from the newer studies indicate that adverse
effects on prostate gland development can occur following brief early neonatal
exposures, and have opened up the question of life stage as a critical risk factor
for exposure to atrazine. This particular effort is covered in Goal 8.3. An
additional element in this Theme is addressing the differential susceptibility of
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Goal Directed Research
developing animals to atrazine in terms of mammary gland development and is
attempting to delineate whether age-specific sensitivities are present.
Collectively, these components directly address issues pertinent to the
harmonization of cancer and non-cancer assessments, and to the
characterization of particular age classes that may be more or less susceptible to
particular modes of action.
The Drinking Water Theme under Gary Klinefelter is developing a sperm
biomarker (SP-22) that shows high potential to predict fertility and which can be
employed in multiple species. A US Patent for the use of SP-22 as a biomarker
of fertility was issued earlier this year. Additional efforts in this Theme are
developing and employing genomic approaches to study testes-specific effects of
DBPs so as to provide information on the shape of the dose-response function
over a broader range of exposures that heretofore possible with phenotypic
analyses. By developing homologous DNA microarrays for different species,
improved extrapolation of findings may result. One novel aspect of this effort is
the use of microarrays to probe the mRNA content of sperm in semen, thus
affording the possibility for direct animal to human correlations to be established.
The genomics effort is relatively new, but shows particular promise for a number
of our research programs in helping, for example, to identify the molecular
pathways impacted by a chemical exposure and how they responds in the low
dose region.
In the Susceptible Population Theme under John Rogers, a long-standing
effort has been developing the data base necessary to construct a biologically
based dose-response model (one in which the key intermediary steps in a
sequence leading to a toxic manifestation are described by mathematical
functions). This work began on the prototype teratogen, 5-FU, and has evolved
into a more generic model of folate utilization, with subsequent expansion into
the broader area of interactions of dietary folate levels and risk of chemically-
induced teratogenesis. Lessons learned from this effort have been extremely
valuable in understanding the hurdles that are faced in transiting from qualitative
to quantitative based risk assessments. In order to explore the advantages of
genomic technology for detecting early signals of developmental toxicity, another
part of this Theme is using microarrays in various experimental situations to look
for predictive changes in gene expression from teratogenic exposures. Another
element of this Theme has begun to examine animal models of in utero growth
retardation in order to establish a protocol under which to evaluate the biological
plausibility of the "Barker hypothesis" that suggest that many adult-onset
diseases of endocrine and cardiovascular systems have an origin in patterns of
fetal growth Given that fetal growth retardation is common finding in
developmental toxicity studies, the implications of the Barker hypothesis for study
design, hazard characterization and risk assessment will only grow in importance
as the human evidence supporting it increases.
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Goal Directed Research
The Signal Transduction Theme under the leadership of Barbara Abbott is
devoted to addressing avenues for collecting mode of action information from
developing tissue exposed to toxicants in order to reduce reliance on uncertainty
factors in dose extrapolation. Specifically these efforts are directed at the
assessment of key signal transduction pathways that regulate the development
of the palate, mammary gland and prostate gland. Because of the multiplicity of
pathways and interactive events, the research effort is moving rapidly from the
analysis of a few genes and gene products to using higher density microarrays
applied to cells isolated by laser microdissection. The approach also relies
extensively on the use of transgenic animals to evaluate responses in systems in
which one or more signal transduction pathways have been altered by genetic
manipulation.
Goal 8.3- Endocrine Disruption
The ORD Research Strategy for EDCs was one of the first such
documents to become formally peer reviewed and officially accepted by the
Agency, and with the resulting NHEERL EDC Implementation Team effort, has
guided the research directions for the Goal 8.3 program for approximately 4
years now.. As noted above, there are three main areas of interest: screening
and testing assays; developmental susceptibilities, and interspecies
extrapolations. While the bulk of the EDC research is contained within the
Cellular and Molecular Mechanisms of Abnormal Reproductive Development
Theme lead by Earl Gray, elements are also found in other Themes when the
effort was largely driven by concerns of the implications of an EDC mode of
action. A cross cutting issue for investigators in RTD engaged in EDC research
is the support for OPP as they move to implement the recommendations of the
EDSTAC committee. There is literally daily interactions related to technical
assistance for the program office, and this responsibility is shared amongst a
number of investigators.
Beyond the technical support lie research efforts to develop and
standardize a number of in vitro and in vivo assays in preparation for their
entering the ICCVAM validation process. Relative to the in vivo assays, we are
currently the lead laboratory within OECD for validation of the Hershberger Assay
for (anti)-androgenic potential, and for the in vitro efforts, we have recently
submitted for publication a manuscript describing two cells lines for
characterizing androgenic and anti-androgenic potential (other efforts continue to
develop an analogous cell line for assessment of ER function). Two other major
elements of the Cellular and Molecular Mechanisms Theme use a variety of in
vitro and in vivo approaches to determine the mode of action of an EDC at its
target site. Such efforts have been successful in the past in determining how
several anti-androgens (pp-DDE, vinclozolin, Linuron, and certain phthalate acid
esters alter male reproductive tract development.
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Qoal Directed Research
In the Chlorotriazine Theme, the identification of the LH surge as well as
suppression of prolactin release as key targets in the adult rat to atrazine
exposure has lead to studies to determine the biological impact of exposure at
other developmental stages. The observation of prostatitis in adult rats exposed
as neonates exemplifies the need to bear in the mind the possibility for qualitative
differences in response as a function of age at exposure, and that these effects
are not limited to the period of organogenesis. Using a similar rationale of
exploring the impact across life stages, another activity has examined effects on
pubertal development of both atrazine and its major metabolites, while another
has looked at implications on pregnancy maintenance. Collectively, these
studies are providing a comprehensive understanding of the life-stage specific
consequences of an identified mode of action, and have pointed to important
issues to be considered within the expanded risk assessment.
Within the Drinking Water Repro Theme, the full litter resorptions in rats
exposed to bromodichloromethane has been shown to be related to an alteration
in LH responsiveness, with implications for the extrapolation of these findings to
humans. Other studies in this theme have shown that dibromoacetic acid can
alter steroidogenesis in the preovulatory follicle, with the site of action prior to
P450 side chain cleavage, leading to questions about potential relevance to
observations of the persistent estrous cycling in older females, and alteration of
pubertal development in younger animals.
SUMMARY:
RTDs Theme-based planning system has facilitated our alignment with a
problem solving orientation, and we feel that we have been successful in
directing our resources toward priority research areas. While our research
programs have to a large extent preceded the codification of Agency needs
through the GPRA effort, the specific aims of each Theme have become more
focused in that last few years on producing information of use to our clients.
Table 2 provides a measure of overall productivity of the combined research
themes as a function of GPRA, while Table 3 delineates some of the major types
of outputs for RTD research in relation to the needs of the Agency. These
outputs range from providing technical assistance in terms of reviewing
protocols, data sets, risk assessments, and testing guidelines to probing aspects
of how combinations of chemicals commonly found together in the environment,
or which act by different modes of action within the same target tissue, exert
toxicity, to using modern molecular techniques to analyze the precursor events
that determine the shape of the dose-response relationship.
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Goal Directed Research
Table 2 Cumulative Publications, 1997-2001 by GPRA Goal
Goal 1
Clean Air
Goal 2
Clean
Water
Goal 3
FQPA
Goal 4
Safe Communities
Goal 8.2
Human
Health
Goal 8.3
EDCs
Misc.
9
21
8
36
74
77
10
From our Emerging Issues Workshop with the Program Offices last May, a
number of specific issues were identified that should form the basis of future
impacts of the research program.
1. Communication
2. Levels of Biological Organization
3. Screening/Testing Strategies
4. Life Cycle Outcomes
5. Susceptibility Factors
6. Cumulative Risk
7. Children's Health
8. Genomics/Proteomics
Increased communication of on-going research was a clear area of need,
and will be addressed in part by wider distribution of ReproTimes, the monthly
RTD newsletter. Using molecular to explore deeper levels of biological
organization to obtain more sensitive endpoints that could be used to develop
quantitative dose-response models that cover broader range of dose levels was
another overarching concept. In terms of screening and testing, there was
interest in the development of comprehensive, innovative and more flexible
schemes that include Toxicokinetic components. Life Cycle Outcomes,
Children's Health and Susceptibility Factors all consider unique aspects of the
developing organism and include the need to better understand exposures and
outcomes of concern, the scope and patterns of exposure, the genetics of
response, and the signals from the public health arena about fetal origins of
adult-onset diseases. The final area of need relates to the introduction of the
new technologies of genomics and proteomics that impact each of the other
aspects. Clearly this technology offers unparalleled opportunities, but is will also
require careful attention to standard scientific procedure, as there is need
demonstrate that the technologies are both robust and replicable. Once those
hurdles are cleared and the peer review material distributed, there should be time
to consider all the options and possibilities that relate to their use in vitro and in
vivo screening techniques suitable for enhancing one's career goal.
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Goal Directed Research
Table 3. Agency Impacts for Research Outputs
Research Area
1- Clean Air
2- Clean Water
3- Safe Food
4- Safe
Communities
8.2- Human
Health Risk
8.3 - EDCs
Biomarkers
~
~
~
~
Hazard ID Studies
~
~
Dose-Response
~
~
~
~
~
Mixtures
~
~
~
Methods Development
~
~
~
~
Model Development
~
~
~
~
Children's Health
~
~
~
~
~
Testing Guidelines
~
~
~
~
Technical Advice
~
~
~
~
Toxicoqenomics
~
~
~
~
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Identifying the Mode and Mechanism of Action of Chlorotriazine
on Endocrine Function in the Rat
Ralph L. Cooper, Theme Leader
I. Introduction:
The chlorotriazines (i.e., atrazine, simazine, cyanazine, propazine) constitute the largest
group of herbicides sold in the United States. Despite their extensive usage, little is known about
the possible human health effects and mechanism(s) of action of these compounds. A major
adverse health effect involving the use of these herbicides has been the observation that they
bring about the premature occurrence of mammary gland tumors in rats. Long-term feeding
studies indicated that chlorotriazines induce a premature ovarian senescence. As reproductive
aging in the female rat is the result of altered hypothalamic control of the pituitary, we
hypothesized that the chlorotriazine herbicides interfered with the pulsatile release of
gonadotropin releasing hormone and subsequently the ovulatory surge of luteinizing hormone
(LH). To test this hypothesis, several experiments were conducted to characterize the extent to
which the LH surge was modified by atrazine and to demonstrate that the central nervous system
(specifically the hypothalamus) was the target site for this effect. Thus, with extended dosing,
this mode of action of atrazine would disrupt the hormonal control of ovarian function and induce
a premature reproductive aging. As a result of this premature senescence, the altered endocrine
milieu present in the exposed rat (elevated estrogen, low progesterone and appreciable amounts
of prolactin) facilitates the development of mammary gland tumors. The chlorotriazines have
been under Special Review within the Office of Pesticide Programs and Toxic Substance
(OPPTS) since November, 1995 and were the topic of a recent U.S. EPA, OPPTS, Science
Advisory Board/Science Advisory Panel meeting (6/29/2000). The impact of the research
conducted by the members of this theme is evidenced by the fact that a major portion of the
Atrazine Revised Preliminary Human Health Assessment (www.epa.gov/pesticides/reregistration/
atrazine/) focused on studies published by the members of this theme and these studies played
a key role in the SAB/SAP decision that atrazine is an unlikely human carcinogen.
Importantly, our work characterizing the mode of action of the chlorotriazines (i.e., altered
hypothalamic and pituitary function) has allowed us to propose and test hypotheses addressing
the effects of these herbicides on other reproductive parameters during key periods of
organization (perinatal), development (pubertal), adult function (cyclicity, pregnancy, lactation)
and aging of the reproductive system in a number of rat strains. Again, these studies were
highlighted in the recent SAB/SAP meeting and generated significant concern about potential
developmental effects of these herbicides (including setting the NOAEL for acute effects and
consideration of an additional 10 X safety factor in accordance with the FQPA concern for
children's health). Finally, this research has taken on an added significance, in that many of the
protocols being developed to test chlorotriazine toxicity promise to enhance our ability to better
identify the range of reproductive toxicity of many other environmental compounds.
The scientific and regulatory questions addressed by the specific aims of this theme focus
on Goal 8 of GPRA, Human Health, including: (1) The harmonization of cancer and non-cancer
risk assessment-effects wherein an understanding of the common mode(s) of toxicity will aid in
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identifying potential hazards, in extrapolating from animals to humans, and in determining if
different toxicological outcomes (cancer vs. non-cancer) arise from perturbation of common
pathways. (2) The evaluation of sensitive subpopulations, in particular, identifying methods and
dose-repose characteristics of reproductive toxicants in the developing organism. (3) The issue
of endocrine disruption. Specifically, do environmentally relevant levels of endocrine-disruptive
chemicals induce adverse reproductive and/or developmental effects in humans and/or wildlife?
Do current test guidelines and risk assessment procedures adequately evaluate potential
endocrine-mediated effects?
II. Impact of Research:
Human Heath, Harmonizing Cancer ys. Non-Cancer effects (Poster 15). These studies are
aimed at identifying modes(s) of action and mechanistic pathway(s) that are conserved across
species, tissues and levels of biological organization and are sensitive to perturbation by
toxicants (chlorotriazines, in particular). Although regulation of the endocrine system is complex,
several of the signaling events that regulate its development and function appear to be well
conserved across species. The work evaluating the mode and mechanisms of action of atrazine
has provided critical and timely information concerning several of the questions associated with
the hamonization of the cancer vs. non-cancer effects of these herbicides.
As discussed above, a primary health concern for the chlorotriazines is that they decrease
the latency for the onset of mammary tumors in the SD rat in standard lifetime feeding studies.
Similar effects were not noted in the Fischer 344 rat, nor in the B6C3F mouse. More recently,
this premature development of mammary tumors was reported to be associated with an atrazine-
induced premature reproductive aging. This led to uncertainty in how to treat the species and
strain sensitivity in the risk assessment process, and what dose-extrapolation model (linear or
margin-of-safety) should be used by EPA. In our work on the mode of action for tumorigenesis,
we conducted studies to identify the effect(s) of atrazine on the neuroendocrine status of several
rat strains following acute and sub-chronic exposures. We found that the key precursor event
for the early onset of mammary gland tumors revolved around the ability of atrazine to attenuate
the ovulatory surge of LH. This attenuation, in turn, was posited to augment the normal aging
process within the hypothalamic-pituitary-ovarian axis and ultimately lead to a physiological state
of constant endogenous estrogen stimulation and elevated prolactin, all conditions that promote
the induction of mammary tumors. The lack of a tumor response in Fischer 344 rats and B6C3F
mice is consistent with a different pattern of reproductive aging, although it should be cautioned
that other strains of rats (e.g., Long Evans) have a pattern of reproductive aging similar to the
SD rat and thus would likely respond as the SD to atrazine exposure. Importantly, reproductive
aging in women is not driven by the changes within the CNS, but is due to the depletion of
follicles within the ovary. Thus, if a similar atrazine-induced suppression of the LH surge were
to occur in women it would not be expected to accelerate reproductive aging and would therefore
not be expected to facilitate the development of mammary tumors. Hence, because the mode
of action is non-genotoxic, a margin of safety approach was deemed appropriate for the cancer
risk assessment. However, identifying a neuroendocrine mode of action has raised several
other questions about the consequences of atrazine exposure in the human, particularly for non-
cancer endpoints. Thus, current studies have focused on two issues. The first is to better
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characterize the target tissue and cellular mechanism(s) involved in atrazine exposure (including
in vitro studies and a careful analysis of tissue dosimetry following exposure). The second issue
is to determine the potential effects of atrazine exposure during key developmental periods in
an effort to better define the toxicity of this compound and its metabolites. These studies are
discussed below.
Human Health, Sensitive Subpopulations (Poster 16): Goal 8 addresses concerns about
sensitive subpopulations, including the identification of (1) human variability in susceptibility to
pesticide exposure due to age, sex, pre-existing disease, health and nutritional status and
genetic predisposition; (2) the most vulnerable developmental/maturational periods of exposure,
endpoints of response, and critical modes of action and (3) method development to detect subtle
developmental functional deficits or latent effects manifested later in life. One effort addressing
these issues involves the development of a model for the evaluation of the effects of xenobiotic
exposure (primarily gestational) on mammary gland development. This approach examines
mammary gland development (through puberty) and then determines whether or not the
susceptibility of the offspring to mammary gland tumors is altered due to toxicant exposure. This
model is being evaluated using two endocrine disrupting compounds (EDCs). The studies
suggest that gestation days 15-19 represent a sensitive period of mammary epithelial
development during which toxicant exposure may have a lasting effect on the female offspring.
Pups exposed to dioxin or atrazine during this period lacked sufficient lateral branching and had
fewer proliferating lobules following puberty. Furthermore, not only did the exposed offspring
have underdeveloped mammary epithelium, but they produced less milk than controls in timed
suckling experiments. Toxicant-exposed animals also had terminal end buds (highly proliferative
tissue that is sensitive to carcinogens) present in the glands for an extended period of time.
Environmental compounds that lengthen the time that these structures are present in the
developing gland increase the susceptibility of the animal to carcinogen-induced tumors. It is
unknown whether or not these EDCs act directly at the level of the mammary gland or if they
have more indirect endocrine modes of action (via the brain). Importantly, both of these EDCs
decrease circulating prolactin in dosed dams. The dam normally secretes large quantities of
prolactin into the milk which, in turn, is critical for normal development in the pup and prolactin
is known to cause proliferation of mammary epithelium. Therefore, it may be that
underdeveloped CNS regulation (i.e., decreased dopamine) and hyperprolactinemia in the pup
could lead to altered mammary development and function in adulthood. Alternatively, a critical
period in mammary gland development could be affected by low prolactin levels in utero.
Further, it is not known if the brief exposure to these compounds during gestation is all that is
required to cause the long-term and detrimental effects on mammary gland development. Future
studies will investigate other environmental agents that alter the normal pattern of mammary
gland development, milk production (as assessed by pup mortality) and terminal end bud
differentiation (as it affects susceptibility to breast cancer).
Human Health, Endocrine Disruptors: Pubertal Assays: (Poster 17). In response to the
Congressional mandate to develop and initiate a screening program to identify chemicals that
disrupt endocrine function (Public Law 104-170 and 104-182, August 1996), the U.S. EPA has
established an Endocrine Disruptor Screening Program (EDSP)
(www.epa aov/scipolv/oscoendo/index.htm). The Tier 1 Screening (T1S) Battery is a major
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component of the EDSP and includes a series of in vivo and in vitro assays designed to detect
chemicals that alter the estrogen, androgen or thyroid systems in humans, fish and wildlife. Prior
to implementation, the assays included in the T1S must undergo a rigid validation process to
document that the tests are scientifically sound. The Office of Science Coordination Policy
(OSCP) within OPPTS is coordinating the validation of the T1S assays. The objective of many
of the research activities within the theme is to assist OSCP in this endeavor. During the past
two years, members of this theme, with other RTD scientists, have conducted major research
and advisory activities which directly support the development, standardization and validation of
several of the T1S mammalian in vivo assays. In particular, they have played a primary role in
developing and standardizing the assay for the Assessment of Pubertal Development and
Thyroid Function in Juvenile Rats. These assays are designed to identify the effects of EDCs
on pubertal development and thyroid function in intact male and female rats (refs 217 & 78).
Recently we showed that the male and female pubertal protocols can be used to detect
chemicals with modes of action that are not directly mediated through the estrogen, androgen
or thyroid systems. Specifically, atrazine, known to have effects on the CNS and the hormonal
control of gonadal function, was shown to be positive in the pubertal assays. In the female,
vaginal opening was delayed in a dose-dependent manner. Additional studies evaluating the
metabolites of atrazine on pubertal development are underway. Also, studies designed to
address issues of possible sex differences in sensitivity, dose selection procedures, and strain
differences within these protocols are planned. Associated with this work will be an evaluation
of the lower limits of detection for both the female and male assays and (2) an evaluation of
subtle reductions in body weight on pubertal development. In addition to defining the effects of
atrazine and its metabolites on pubertal development, these studies will help determine the
lower limit capabilities of the protocols, as well as demonstrate any effect that reduction in body
weight alone (<10%, such as that which might occur if the doses are selected based upon the
MTD) might play during pubertal development.
Prostatitis Model: (Poster 18). A primary question concerning EDCs is whether or not exposure,
during certain developmental periods, will predispose the organism to reproductive disorders
and/or diseases (especially cancer) in later life. The purpose of this work is to identify
appropriate models for examining the question of whether or not toxicant exposure during certain
developmental periods will predispose the organism to reproductive disorders and/or diseases
(especially cancer) in later life. Specifically, this effort involves the investigation of the effects of
toxicants, including atrazine, on maternal prolactin secretion and the eventual development of
prostatitis in the adult male offspring. The specific studies include an evaluation of compounds
on suckling-induced prolactin release in the dam and the possibility that reduced maternal
prolactin will lead to impaired development in the offspring's tuberoinfundibular dopaminergic
neurons (TIDA neurons, the neurons which regulate prolactin secretion) which, in turn, lead to
increased prolactin secretion prior to puberty. A related set of studies is based on our findings
that a xenobiotic-induced rise in serum prolactin prior to puberty in the male rat will lead to
prostate inflammation in the adult (ref 216). We are currently characterizing the timing of
treatment and the classes of chemicals that lead to prepubertal hyperprolactinemia and, in turn,
prostatitis in the adult. The hypothesis to be tested is that regardless of the class of compounds
tested or exposure regimens employed during development, lateral lobe prostatitis will be
observed only if there is a significant rise is serum prolactin prior to puberty. In addition to
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developing and standardizing a protocol for the evaluation of xenobiotics on the prostate, these
studies have an added utility in that they will provide information on the extent to which the
chlorotriazines alter reproduction in the male rat. Atrazine and related chlorotriazines are of
concern to the OPPTS and this endpoint may be particularly sensitive to these compounds (i.e.,
identifying a new low dose in mammals). Finally, in future studies we plan to evaluate a broad
range of compounds to determine the extent to which these key developmental processes can
be altered.
In humans, nonbacterial prostatitis with undefined etiology has become a significant
clinical problem and has been associated with discomfort and infertility. Most interestingly,
chronic inflammation, the associated increase in cell turnover and increased oxidative stress
have been recently linked to neoplastic transformation and development of carcinomas (De
Marzo et al., Am J Pathol 1999; 155(6): 1985-1992).
Pregnancy Maintenance: (Poster 19). Another area of research addressing this goal is that
directed at determining whether or not the endocrine support of pregnancy can be disrupted by
chlorotriazines. Since atrazine has been shown to affect the ovulatory LH surge, we
hypothesized that these chemicals would also disrupt pregnancy in the rat via altered LH
secretion during the LH-dependent period of gestation. As predicted, atrazine did indeed disrupt
pregnancy when administered during the LH-dependent period (gestation days 7-10).
Conversely, treatment after the LH-dependent period (days 11-15) did not. Furthermore, a
comparison of three rat strains' sensitivity to atrazine revealed that the F344 strain was the most
sensitive (i.e., females lost litters at lower doses) when compared to SD and Long-Evans rats.
We are currently determining the hormonal profiles in pregnant rats following exposure to
atrazine and its metabolites. Also, based on the time course of effects on key hormones, we will
attempt to rescue the pregnancy with exogenous hormones to confirm their role in causing
pregnancy loss. These data will contribute to the identification of the mode of action of
chlorotriazine-induced pregnancy loss and demonstrate, as in other studies, that atrazine alters
several reproductive parameters in strains other than the SD rat. The ability of atrazine to cause
pregnancy loss when administered during the LH-dependent period, but not thereafter, supports
the hypothesis that atrazine alters LH-mediated mechanism(s). Future studies aimed at defining
the hormonal profiles and pregnancy rescue with exogenous hormones may give further support
for this hypothesis.
III. Future Directions:
Our planned research represents a logical extension of the work that has been conducted
by this theme during the past 3-5 years. Specifically, we will continue to investigate the mode
of action of atrazine and the related chlorotriazines in an attempt to identify the cellular
mechanism(s) associated with the loss of neuroendocrine control of gonadal function. These
studies will involve both in vivo (i.e., tissue dosimetry studies, relationship between
neurotransmitter and endocrine changes) and in vitro work (i.e., PC12 and othercell lines). Such
information is vital for the eventual extrapolation of the effects identified in rodents to humans.
Additionally, in vivo and in vitro studies designed to investigate the cumulative effects of the
primary chlorotriazines and their metabolites will be completed. The issue of cumulative effects
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is of primary concern to Goal 3 and the FQPA. Thus, we will evaluate the effects of combined
exposure to the chlorotriazines and other pesticides that, like atrazine, alter the control of
hypothalmic-pituitary function (are they additive or more than additive)? Other in vivo studies
will focus on whether or not the LOAEL would be less if the developing animal is exposed
gestationally and lactationally.
The studies focusing on the development of models will continue testing these protocols
with other chemicals to determine their validity. Finally, our work with the standardization and
development of the EDSP protocols (both in vivo and in vitro) will continue according to the
schedule outlined by OSPC. Upon completion of this work, the protocols will be reviewed by an
EDSP Federal Advisory Committee. Members of the atrazine theme and other RTD scientists
will assist OPPTS by evaluating data from the validation studies, and preparing the final
protocols for the final SAP review.
IV. Intramural Support (FTEs)
Total of 10 FTEs. Participating staff: Deborah Best, Christine Davis, Dorothy Guidici, Janet
Ferrell, Suzanne Fenton, Susan Laws, W. Keith McElroy, Michael Narotsky, and Jerome
Goldman, EB, RTD; Tammy E. Stoker, GEEB, RTD; Tim Shafer, Thomas Ward and C. A.
Meacham, NTD.
V. External Collaborators:
Parikshit Das, Curriculum in Toxicology, University of North Carolina, Chapel Hill
Lee Tyrey, Department of Obstetrics and Gynecology, Duke University Medical Center and
Johnathan Lindzey, Department of Biology, University of South Florida, Tampa Florida
VI. Productivity
CNS and Pituitary as Target Site for Reproductive Toxicants.
43. Cooper, R.L. (1997). Neuroendocrine Control of Female Reproduction. In Female
Reproductive Toxicology, vol. 10 Comprehensive Toxicology (K. Boekelheide, R. E.
Chapin, P. B. Hoyer and C. Harris, Eds.), pp. 273-281. Elsevier Science, New York.
76. Goldman, J.M., Parrish, M.B., Cooper, R.L., and McElroy, W.K. (1997). Blockade of
ovulation in the rat by systemic and ovarian intrabursal administration of the fungicide
sodium dimethyldithiocarbamate. Reprod Toxicol. 11,185-190.
77. Goldman, J.M., Gray, L.E., and Cooper, R.L. (1998). Reproductive and developmental
toxicity of endocrine disrupting chemicals: Steroid and non-steroid mechanisms using
rodent models. In Principles and Processes for Evaluating Endocrine Disruption in Wildlife
(R. J. Kendall, R. L. Dickerson, W. A. Suk and J. P. Giesy, Eds.), pp. 311-333. SETAC
Press.
45. Cooper, R.L., Goldman, J.M., and Tyrey, L. (1998). The hypothalamus and pituitary as
targets for reproductive toxicants. In Reproductive and Developmental Toxicology (K.
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Korach, Ed.), pp. 195-210. Marcel Dekker, Inc., New York.
145. Lindzey, J., Wetsel, W.C., Couse, J.F., Stoker, T., Cooper, R., and Korach, K.S. (1998).
Effects of castration and chronic steroid treatments on hypothalamic gonadotropin-
releasing hormone content and pituitary gonadotropins in male wild-type and estrogen
receptor-alpha knockout mice. Endocrinology. 139,4092-4101.
149. Miller, D.B., All, S.F., O'Callaghan, J.P., and Laws, S.C. (1998). The impact of gender
and estrogen on striatal dopaminergic neurotoxicity. Ann N Y Acad Sci. 844,153-165.
22. Ben-Jonathan, N., Cooper, R.L , Foster, P., Hughes, C.L., Hoyer, P.B., Klotz, D., Kohn,
M., Lamb, D.J., and Stancel, G.M. (1999). An approach to the development of
quantitative models to assess the effects of exposure to environmentally relevant levels
of endocrine disruptors on homeostasis in adults. Environ Health Perspect. 107 Suppl
4, 605-611.
88 Gray, L.E., Jr., Ostby, J., Cooper, R.L., and Kelce, W.R. (1999). The estrogenic and
antiandrogenic pesticide methoxychlor alters the reproductive tract and behavior without
affecting pituitary size or LH and prolactin secretion in male rats. Toxicol Ind Health. 15,
37-47.
46. Cooper, R.L., Goldman, J.M., and Stoker, T.E. (1999). Neuroendocrine and reproductive
effects of contemporary-use pesticides. Toxicol Ind Health. 15, 26-36.
211. Shafer, T.J., Ward, T.R., Meacham, C A., and Cooper, R.L. (1999). Effects of the
chlorotriazine herbicide, cyanazine, on GABA(A) receptors in cortical tissue from rat brain.
Toxicology. 142, 57-68.
48. Cooper, R.L., Stoker, T.E., Tyrey, L., Goldman, J.M., and McElroy, W.K. (2000).
Atrazine disrupts the hypothalamic control of pituitary-ovarian function. Toxicol Sci. 53,
297-307.
58. Das, P.C., McElroy, W.K., and Cooper, R.L. (2000). Differential modulation of
catecholamines by chlorotriazine herbicides in pheochromocytoma (PC12) cells in vitro.
Toxicol Sci. 56, 324-331.
59. Das, P.C., McElroy, W.K., and Cooper, R.L. (2001). Alteration of catecholamines in
pheochromocytoma (PC12) cells in vitro by the metabolites of chlorotriazine herbicide.
Toxicol Sci. 59,127-137.
Pregnancy Maintenance
157. Narotsky, M.G., Brownie, C.F., and Kavlock, R.J. (1997). Critical period of carbon
tetrachloride-induced pregnancy loss in Fischer-344 rats, with insights into the detection
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of resorption sites by ammonium sulfide staining. Teratology. 56, 252-261.
158. Narotsky, M.G., Pegram, R.A., and Kavlock, R.J. (1997). Effect of dosing vehicle on the
developmental toxicity of bromodichloromethane and carbon tetrachloride in rats. Fund.
Appl Toxicol. 40, 30-36
161. Narotsky, M.G., Best, D.S., Guidici, D.L., and Cooper, R.L. (2001). Strain comparisons
of atrazine-induced pregnancy loss in the rat. Reprod Toxicol. 15, 61-69.
57. Cummings, A.M., Rhodes, B.E., and Cooper, R.L. (2000). Effect of atrazine on
implantation and early pregnancy in 4 strains of rats. Toxicol Sci. 58, 135-143.
53. Cummings, A.M. (1998). Toxicology of Early Pregnancy, Implantation, and Uterine
Function. In Reproductive and Developmental Toxicology (K Korach, Ed.), pp. 397-412.
Marcel Dekker, Inc, New York.
56. Cummings, A.M., and Laws, S.C. (2000). Assessment of estrogenicity by using the
delayed implanting rat model and examples. Reprod Toxicol. 14,111-117.
Mammary Gland Effects
72. Fenton, S.E., and Sheffield, L.G. (1997). Prolactin inhibits EGF-induced DNA synthesis
in mammary epithelium via early signaling mechanisms: possible involvement of protein
kinase C. Exp Cell Res. 236, 285-293.
26. Bocchinfuso, W.P., Lindzey, J.K., Hewitt, S.C., Clark, J.A., Myers, P.H., Cooper, R., and
Korach, K.S. (2000). Induction of mammary gland development in estrogen receptor-
alpha knockout mice. Endocrinology. 141,2982-2994.
73. Fenton, S.E., and Sheffield, L.G. (Accepted). Prolactin activation of JAK2 via a positive
feedback loop involving c-src and SHP2. J Biol Chem.
74. Fenton, S.E., and Sheffield, L.G. (Submitted). Prolactin-induced tyrosine
phosphorylation, activation and receptor association of focal adhesion kinase (FAK) in
mammary epithelial cells: Critical involvement of JAK2 and c-src activation.
Endocrinology.
Pubertal Effects and Prostatitis
214. Stoker, T.E., Robinette,C.L., and Cooper, R.L. (1999). Perinatal exposure to estrogenic
compounds and the subsequent effects on the prostate of the adult rat: evaluation of
inflammation in the ventral and lateral lobes. Reprod Toxicol. 13, 463-472.
215. Stoker, T.E., Robinette, C.L., and Cooper, R.L. (1999). Maternal exposure to atrazine
during lactation suppresses suckling-induced prolactin release and results in prostatitis
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in the adult offspring. Toxicol Sci. 52, 68-79.
216. Stoker, T.E., Robinette, C.L., Britt, B.H., Laws, S.C., and Cooper, R.L. (1999).
Prepubertal exposure to compounds that increase prolactin secretion in the male rat:
effects on the adult prostate. Biol Reprod. 61, 1636-1643.
138. Laws, S.C., Carey, S.A., Ferrell, J.M., Bodman, G.J., and Cooper, R.L. (2000).
Estrogenic activity of octylphenol, nonylphenol, bisphenol A and methoxychlor in rats.
Toxicol Sci. 54,154-167.
139. Laws, S.C., Ferrell, J.M., Stoker, T.E., Schmid, J., and Cooper, R.L. (2000). The effects
of atrazine on female Wistar rats: an evaluation of the protocol for assessing pubertal
development and thyroid function. Toxicol Sci. 58, 366-376.
78. Goldman, J.M., Laws, S.C., Balchak, S.K., Cooper, R.L., and Kavlock, R.J. (2000).
Endocrine-disrupting chemicals: prepubertal exposures and effects on sexual maturation
and thyroid activity in the female rat. A focus on the EDSTAC recommendations. Crit Rev
Toxicol. 30, 135-196.
217. Stoker, T.E., Parks, L.G., Gray, L.E., and Cooper, R.L. (2000). Endocrine-disrupting
chemicals: prepubertal exposures and effects on sexual maturation and thyroid function
in the male rat. A focus on the EDSTAC recommendations. Endocrine Disrupter
Screening and Testing Advisory Committee. Crit Rev Toxicol. 30, 197-252.
218. Stoker, T.E., Laws, S.C., Guidici, D.L., and Cooper, R.L. (2000). The effect of atrazine
on puberty in male Wistar rats: an evaluation in the protocol for the assessment of
pubertal development and thyroid function. Toxicol Sci. 58, 50-59.
219. Stoker, T.E., Goldman, J.M., and Cooper, R.L. (2001). Delayed ovulation and pregnancy
outcome: effect of environmental toxicants on the neuroendocrine control of the ovary.
Environ. Toxicol. Pharmacol. 9,117-129.
220. Stoker, T.E., Guidici, D.L., Laws, S.C., and Cooper, R.L. (Accepted). The effects of
atrazine metabolites on puberty and thyroid function in the male Wistar rat. Toxicol Sci.
Endocrine Disruptors
44. Cooper, R.L., and Kavlock, R.J. (1997). Endocrine disruptors and reproductive
development: a weight-of-evidence overview. J Endocrinol. 152,159-166.
82. Gray, L.E., Jr., Kelce, W.R., Wiese, T., Tyl, R., Gaido, K., Cook, J., Klinefelter, G.,
Desaulniers, D., Wilson, E., Zacharewski, T., Waller, C., Foster, P., Laskey, J., Reel, J.,
Giesy, J., Laws, S., McLachlan, J., Breslin, W., Cooper, R., Di Giulio, R., Johnson, R.,
Purdy, R., Mihaich, E., Safe, S., Colborn, T., and et al. (1997). Endocrine Screening
Methods Workshop report: detection of estrogenic and androgenic hormonal and
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antihormonal activity for chemicals that act via receptor or steroidogenic enzyme
mechanisms. Reprod Toxicol. 11,719-750.
136. Laws, S.C., Goldman, J.M., and Cooper, R.L. (1997). Assessing the endocrine disrupting
potential of environmental chemicals. In Biotechnology International (T. H. Connor and
C. F. Fox, Eds.), pp. 208-215. Universal Med. Press, San Francisco.
50. Crisp, T.M., Clegg, E.D., Cooper, R.L., Wood, W.P., Anderson, D.G., Baetcke, K.P.,
Hoffmann, J.L., Morrow, M.S., Rodier, D.J., Schaeffer, J.E., Touart, L.W., Zeeman, M.G.,
and Patel, Y.M. (1998). Environmental endocrine disruption: an effects assessment and
analysis. Environ Health Perspect. 106 Suppl 1, 11-56.
130. Lamb, J.C., Matthiessen, P., Balcomb, R., Bens, C., M., Cooper, R.L., Gorsuch, J.W.,
Peden-Adams, M., and Voit, E.O. (1998). Hazard Identification/Epidemiology. In
Principles and Processes for Evaluating Endocrine Disruption in Wildlife (R. J. Kendall,
R. L. Dickerson, W. A. Suk and J. P. Giesy, Eds.), pp. 17-37. SETAC Press, Pennsacola,
Fla.
235. Zacharewski, T., Campbell, P., Routledge, E., Cooper, R., Kime, D., Wenzel, A., and
Huet, M.-C. (1998). Current Approaches for the Use of in Vitro Tests in Identifying the
Hazards of Endocrine Modulating Chemicals to Wildlife, pp. 41-58. SETAC-
Europe/OECD/EC Expert Workshop on Endocrine Modulators and Wildlife: Assessment
and Testing, Europe.
47. Cooper, R.L., and Goldman, J.M. (1999). Vaginal Cytology. In An Evaluation and
Interpretation of Reproductive Endpoints for Human Health Risk Assessment (G. Daston
and C. Kimmel, Eds.), pp. 42-56. International Life Sciences Institute/Health &
Environmental Sciences Institute, Washington, DC.
86. Gray, L.E., Jr., Wolf, C., Lambright, C., Mann, P., Price, M., Cooper, R.L., and Ostby, J.
(1999). Administration of potentially antiandrogenic pesticides (procymidone, linuron,
iprodione, chlozolinate, p,p'-DDE, and ketoconazole) and toxic substances (dibutyl- and
diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual
differentiation produces diverse profiles of reproductive malformations in the male rat.
Toxicol Ind Health. 15, 94-118.
49. Cooper, R.L., and Kavlock, R.J. (In Press). Endocrine Disrupting Chemicals (EDCs):
Session Overview. Intl. J. Human Ecol. Risk Assess.
91. Gray, L.E., Jr., Ostby, J., Wilson, V., Lambright, C., Bobseine, K., Hartig, P., Hotchkiss,
A., Wolf, C., Furr, J., Price, M., Parks, L., Cooper, R.L., Stoker, T.E., Laws, S.C., Degitz,
S.J., Jensen, K.M., Kahl, M.D., Korte, J.J., Makynen, E.A., Tietge, J.E., and Ankley, G.
(In Press). Xenoendocrine disrupters - tiered screening and testing: Filling key data gaps.
Toxicol. Lett.
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VII. Activities Relevant to Stakeholders
Member of the ORD/RAF Workgroup on Environmental Endocrine Disruption: An Effects
Assessment and Analysis Document, wrote document (1997).
Participant in EPA/WWF/CMA "Duke" Workshop on Endocrine Screening Methods. Report
published in 1997.
Participant in the SETAC-EUROPE OECD Expert Workshop on Endocrine Modulators and
Wildlife. Veldhoven Holland. (1997), Report published in 1998
Participant in the ILSI meeting on the Evaluation and Interpretation of Reproductive Endpoints
for Human Health Risk Assessment. Washington DC, November, (1997). And presented paper
on Evaluation and Interpretation of Rodent Vaginal Cytology in Toxicology Studies.
Presented summary on the Mode and Mechanism of Action of Atrazine (Triazine induced
alterations in the hormonal control of reproduction). Seminar presented to Office of Prevention
Pesticides and Toxic Substances, Crystal City, Virginia, April, (1998).
Presented seminar on the Effect of Atrazine on Reproductive Function in the Female Rat.
Seminar presented to the Triangle Wide Endocrine Disruptor Discussion Group, RTP, NC, April,
(1998).
Presented seminar entitled: Endocrine Disruptors: Fact or Fantasy. Presentation to National
Center for Environmental Research and Quality Assurance. Crystal City, June, (1998).
Presented seminar entitled: Lactational exposure to atrazine suppresses suckling-induced
prolactin. Endocrine Disruptors Workgroup, NHEERL, RTP, NC Apr., (1998).
Risk assessment issues of the chlorotriazines. Mode of action and Hazard ID. Briefing held for
the OPP chlorotriazine team, OPPTS RTP, NC, May, (1999).
Participated in EPA/NIEHS sponsored workshop on Characterizing the Effects of Endocrine
Disruptors on Human Health at Environmental Exposure Levels, Raleigh, NC. Paper published
in 1999.
Participated and presented a Seminar entitled Gene expression during breast development at
the Application of DNA Microarrays to Toxicology, NHEERL Workshop, Durham, N.C. (1999).
Evaluation and Interpretation of Rodent Vaginal Cytology in Toxicology Studies. Seminar
presented at the 20th annual meeting of the American College of Toxicology, McLean, Va.,
November, (1999).
Presented report entitled: A summary of research related to the effects of atrazine on
reproductive function in the rat to OPPTS, U.S. EPA, Washington, D.C., 1999-2000.
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Participated in the development of background document on Atrazine: Mode of Action and
Developmental Studies for the revised preliminary Human Health Risk Assessment for
Reregistration Eligibility Decision with Health Effects Division, OPPTS, U.S. EPA, Washington,
D.C. (2000).
Presentation to the FIFRA Scientific Advisory Panel on Hazard and Dose-Response Assessment
and Characterization of Atrazine June, Washington, D.C. (2000).
Participated in meeting held by the British Toxicology Society Annual Meeting and presented
paper entitled: Neuroendocrine and reproductive effects of pesticides. York University, Great
Britain, March (2000).
Presented seminar entitled: Prepubertal prolactin and prostate inflammation to the Breast and
Prostate Faculty, NIEHS, July. (2000).
Participant in the EPA Priority-Setting Workshop for the Endocrine Disruptor Screening, Durham,
NC. 2000.
Member Bisphenol A Panel for the EPA/NIEHS Low-dose workshop on endocrine disruptors.
RTP, NC. 2000., Report published 2001.
Presented seminar entitled: Atrazine and its metabolites alter puberty in the male rat. Endocrine
Disruptors Workgroup, RTP, N.C. Aug. (2000).
Chair, Session on: Determining indicators of exposure and effects for endocrine disrupting
chemicals (EDCs). 5th NHEERL Symposium, RTP, NC. (2000). Report in press.
Prepared document entitled Endocrine-disrupting chemicals: prepubertal exposures and effects
on sexual maturation and thyroid function in the male rat. A focus on the EDSTAC
recommendations. Endocrine Disruptor Screening and Testing Advisory Committee. (2000).
Prepared document entitled: Endocrine-disrupting chemicals: prepubertal exposures and effects
on sexual maturation and thyroid activity in the female rat. A focus on the EDSTAC
recommendations (2000).
Participant at the Joint US/EU Endocrine Disruptor Research Expert Panel Meeting, Ispra, Italy
2000.
Developed protocols and reviewed data from contract laboratories for the Male and Female
Pubertal Assays for the Office of Science Coordination and Policy, OPPTS (2000-present).
Prepared report entitled: Study Summary and Review: Assessment of pubertal development and
thyroid function in juvenile male and female rats. Study conducted by Therlmmune Research
Corporation, for OPPTS, U.S. EPA, Washington DC (2000).
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Chair, NHEERL Endocrine Disruptor Research Implementation Committee (2000-present).
Member, ORD Air Toxics Research Strategy Committee, (2000-present).
Member, NHEERL Air Toxics Research Implementation Committee (2000-present).
Member, NHEERL SP2K (Goal 3) Committee, 2000.
Developed protocols, trained contract personnel and reviewed data from contract laboratories
for the EDSP estrogen receptor assay for the Office of Science Coordination and Policy, OPPTS
(2000-present).
Presented seminar entitled: Mammary Tumors and Altered Reproductive Function Following
Atrazine in the Rat. University of Zurich, Zurich Switzerland (2000).
Member and Chair, Special Emphasis Panel Study Section, Conference Grant Applications (R-
13) National Institute of Environmental Health Sciences (2000-2001).
Participated and presented paper at the US/Japan International Workshop on Endocrine
Disrupting Chemicals and Their Toxicological Evaluation, Tsukuba, Japan Feb/Mar 2001.
Participated in workshop and presented paper entitled: Identifying EDCs with estrogen activity
at the ORD/Regional Workshop on Endocrine Disruptors, Region 4, Atlanta, Ga (2001).
Participated in workshop and presented paper entitled: Male and female pubertal assays.
ORD/Regional Workshop on Endocrine Disruptors, Region 4, Atlanta, Ga (2001).
US/Japan International Workshop on Endocrine Disrupting Chemicals and Their Toxicological
Evaluation, Tsukuba, Japan Feb/Mar 2001.
Co-Chair, RTD/Program Office and Region Workshop on Emerging Issues in Reproductive Tox.
June 2001.
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Poster 15. Mode and Mechanism of Action of the Chlorotriazine Herbicides in the Male
and Female Rat. RL Cooper1, TE Stoker1, JM Goldman1, T Shafer2, T Ward2, LL Tyrey3, J
Lindzey4 and P Das5. 1 EB, RTD,2 NTD,3 Dept Ob-Gyn, Duke Univ. Med Ctr.,4 Univ. South
Florida, and 5Toxicology Program, UNC
Atrazine and Simazine have been shown to cause an earlier onset of mammary tumors
in Sprague-Dawley rats following long-term dietary exposure (400 ppm or approximately 22.5
mg/kg/day). A fundamental question concerning this effect was whether or not the tumors were
induced by an action on the mammmary gland tissue itself or as a consequence of
chlorotriazine-induced hormonal changes that create an endocrine environment that is conducive
for mammary tumor growth. As the majority of studies rule out a cancer mechanism for the
development of mammary gland tumors, a primary focus of our laboratory has been to identify
the cascade of endocrine events that lead to the development of mammary tumors. Our studies
show that the primary mode of action of theses herbicides involves a disruption of the
hypothalamic control of anterior pituitary function. Specifically, atrazine inhibits the pulsatile
release of gonadotropin releasing hormone (GnRH) and subsequently LH release. A similar
decrease in prolactin (prl) secretion has also been identified. Studies using exogenous GnRH
stimulation and rats bearing ectopic pituitaries indicated that atrazine does not alter hormone by
a direct action on the pituitary. This finding was confirmed by ex-vivo and in vitro pituitary
perifusion studies in which the hormone release was not altered by atrazine. We conducted a
number of in vivo and in vitro studies in an attempt to determine the primary mechanism(s)
involved in the disruption of the hormonal control of these reproductive processes. We found
that atrazine increases dopamine (DA) and decreases norepinephrine (NE) in the hypothalamus.
These effects would be consistent with the inhibition of the GnRH pulses, decreased LH and
prolactin secretion. Using undifferentiated pheochromocytoma (PC12) cells, which constitutively
synthesize DA and NE, we have shown that the chlorotriazines and their metabolites alter
catecholamine metabolism suggesting that, in vivo, hypothalamic neurons represent one set of
target cells. However, we also have evidence that the chlorotriazines may interfere with
gamma-amino acid (GABA) neurotransmission by binding to the benzodiazepam recognition
sites on the GABA-a receptor and altering chloride ion flux. We are also evaluating the potential
involvement of aromatase in the male and female, as changes in this enzyme may represent
another cellular target.
Current or planned studies include a closer evaluation the chlorotriazine-induced changes
in DA, NE and GABA neurotransmission following in vivo exposure, further in vitro evaluation of
the cellular site of action of the chlorotriazines and their metabolites using cell lines that have
specific functional GABA receptors, an evaluation of the effects of atrazine and its metabolites
using GT1-7 neurons (which constitutively synthesize GnRH) and an examination of the
cumulative effects of parent chlorotriazines and the chlorinated metabolites using a variety of
endpoints.
Pertinent References: 43, 45, 46, 48, 58, 59, 21118.
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Poster 16. Effect of Atrazine on Mammary Gland Morphogenesis and Alveolar Differentiation.
SE Fenton, G Youngblood, J Rayner, and C Davis. Endocrinology Branch, RTD, NHEERL, ORD, U.S.
EPA.
Our studies address issues of importance to the Human Health Risk Assessment Research
Strategy (8.2.1, Sensitive Subpopulations), and focus specifically on identifying common mode(s) and
mechanism(s) underlying defects in mammary gland development or function, and susceptibility of
the pregnant or lactating dam and fetus to low or environmentally relevant levels of endocrine
disrupting compounds. Atrazine is a herbicide widely used to control weeds and grasses in farm crops
such as corn, sugar cane and sorghum. Continuous exposure to dietary atrazine causes an early
onset of mammary tumors in Sprague-Dawley (SD) rats. Our current research has determined that
exposure to atrazine during pregnancy decreased Long Evans (LE) rat pup weaning weight, increased
mortality rates and caused a delayed or runted development of the mammary epithelium in both the
dam and female offspring. The decreased body weights and increased mortality rates are correlated
with poor mothering behavior and a significant decrease in milk production in the exposed dam. Our
recent studies implement the use of a mixture of atrazine (25%), hydroxyatrazine (20%),
diaminochlorotriazine (35%), deethyl atrazine (15%) and deisopropyl atrazine (5%) that is 100-, 1000-
and 10,000-fold that reported in the literature for soil/ground water (1 X=0.873 ug/kg/d) to evaluate the
effect of low dose mixtures on adverse health outcomes in the offspring later in life. When the mixture
was administered to the pregnant dam for 5 days (GD15-19), delays in epithelial development were
observed in mammary gland whole mounts fromthe female pups (1000X), and an increased incidence
of prostatitis and lipomatous masses were visually detected in exposed male offspring (100X). We are
currently using cross-fostering as a means to clarify the mode of action for the effects of atrazine on
mammary development (unthrifty offspring can't consume enough milk, dam produces less milk, or
imprinting effect in fetus).
In an attempt to define the LOEL/NOEL for atrazine in the exposed fetus for delayed mammary
gland development, we have exposed time-pregnant LE and SD dams to 12.5, 25, and 50 mg/kg
atrazine twice daily on GD 15-19. Half of the offspring were given the chemical carcinogen DMBA on
postnatal day 45. Preliminary data suggest that atrazine exposure early in life could lead to an
increased number of mammary tumors of both LE and SD rats. We are also evaluating a large
number of non-cancer endpoints in our DMBA tumor studies for comparison with the tumor data to
determine whether or not the fetal risk of mammary tumors to a burst-exposure to atrazine is
endocrinological^ based or not. Comparisons between DMBA-treated and non-treated siblings will
help us identify any long-term impact of gestational exposures to atrazine. Related studies are
underway to identify early biomarkers of effect in the tissue from these exposed animals, using real-
time PCR and cDNA microarray analyses. Future studies will investigate atrazine and metabolite-
induced mammary developmental delays in Fischer 344 rats to confirm whether or not effects are
consistent across strains. Also, a lactating rat mammary epithelial cell culture model (HC11) will be
used to evaluate the mammary gland as a direct target site for atrazine and its metabolites (alone or
as a mixture). An effect of atrazine at this level would be significant as the rat and human mammary
tissue are developmentally similar, even though their ovarian cyclicity is not. Future work will identify
endpoints in the mammary gland to be evaluated in order to determine whether or not endocrine
disrupting compounds have the potential to alter the tissue development following a brief gestational
exposure. Ultimately we wish to provide a protocol for the evaluation of xenobiotic-induced effects on
mammary tissue development and mammary tumorigenesis for use in testing a variety of chemicals.
°ertinent References: None (new project)
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Poster 17. Effects of Atrazine and its Metabolites on Pubertal Development and Thyroid
Function in Male and Female Rats. SC Laws, TE Stoker, JM Ferrell, D Guidici, and RL Cooper.
EB, RTD, NHEERL, ORD, U.S. EPA, RTP, NC.
Based on our work demonstrating the effects of atrazine on the Central Nervous System (CNS;
and the hormonal control of gonadal function, we hypothesized that this chlorotriazine herbicide would
alter pubertal development in the male and female rat. To test this assumption, we exposed
developing rats to atrazine and it metabolites using the Protocols for the Assessment of Pubertal
Development and Thyroid Function in Juvenile Male and Female Rats. These protocols are integral
components of the Tier 1 Screening Battery for EPA's Endocrine Disruptor Screening Program
(EDSP). Atrazine delayed the onset of puberty in male and female rats. While the LOAEL (50 mg/kg)
for delayed puberty in the female was higher than that previously reported to cause mammary tumors
after prolonged treatment (i.e., 25 mg/kg/day), the LOAEL for the male was distinctively lower (12.5
mg/kg), indicating that, in both sexes, the effects of atrazine can be detected with a shorter treatment
period when this protocol is employed. Although necropsy body weights were reduced in both males
and females in the highest dose group, the inclusion of pair-fed controls (e.g., received the same
amount of food each day as consumed by a mate in the highest atrazine group) demonstrated that the
reduction in body weight was not solely responsible for the delay in puberty. Comparable studies using
metabolites at doses equimolar to that of atrazine demonstrated that all of the chlorinated metabolites
also delayed pubertal development with LOAELs in the same range of that of atrazine. These data
documented the potential cumulative risks associated with exposure to the chlorotriazines.
In contrast, higher doses of hydroxyatrazine, a metabolite without the chlorine group, were
required to delay the onset of puberty in both males and females, suggesting the structural
requirement for the chlorine group for the effect on puberty. Throughout all the studies with atrazine
and its metabolites, the LOAELs for delayed puberty in the males were consistently lower than that
observed for the females. Whether or not this was due to the longer exposure period prior to the onsc
of puberty in the males, or truly a difference in sensitivity to exposure between the males and females
will be further evaluated. Another major outcome of these studies was the observation that serum
estrogens were elevated in male rats following exposure to the highest dose of atrazine. This
observation, in conjunction with a recent report of induced aromatase activity and CYP 19 mRNA in
human adrenocortical carcinoma cells following in vitro exposure to atrazine, simazine and propazine
(Sanderson et al., 2000), supported the hypothesis that this chemical may alter aromatase activity in
the pubertal male rat. Studies have been initiated within our laboratory to test the hypothesis that
altered aromatase activity in the brain, testes and possibly the adipose tissue may be another mode
of action for atrazine. In addition, future studies are planned to address issues of dose selection
procedures, strain differences, and the lower limits of detection for the protocols. The research
described here addresses questions detailed in Goal 8 of GPRA, Human Health, endocrine disruptors,
and directly supports the efforts of the Office of Science Coordination Policy to develop and implement
the Agency's Endocrine Disruptor Screening Program.
Pertinent References: 78, 139, 217, 218, 220
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Poster 18. Early Toxicant Exposure and Alterations of Prolactin Secretion as a Key Event in
Prostate Inflammation in Adulthood TE Stoker, C Langdale, WK McElroy, RL Cooper.
Endocrinology Branch, NHEERL, ORD, U.S. EPA.
A primary question concerning Endocrine Disrupting Chemicals (EDCs) is whether or not
exposure, during certain developmental periods, will predispose the organism to reproductive
disorders and/or diseases (especially cancer) in later life. The primary hypothesis to be tested in this
series of studies is whether or not exposure to environmental agents, during certain key periods of late
gestational, early postnatal or prepubertal development, will increase the risk of prostate abnormalities
(inflammation or cancer) in adulthood. Embedded in this hypothesis is the assumption that some
environmental agents can disrupt normal brain-pituitary-gonadal development to the extent that the
prostate will display an increased risk of inflammation and dysplasia. One goal of this work is to
develop a rodent model for use in future testing and studying human prostate disease status. These
studies also provide an opportunity to determine the mode of action through which these changes are
induced, identify the specific mechanisms involved when the developing neuroendocrine system is
perturbed, and describe the specific lesions that occur in the brain, pituitary and reproductive tissues.
Treatment during certain sensitive periods of development may involve doses that are at or below
those known to alter reproductive function in adulthood, which may be in the range of human
environmental exposures. As we understand very little about the extent to which anomalies in the rat
reflect the potential for similar adverse changes in the human, the integrated approach offered in this
proposal will provide insight into the extent to which these processes may occur in similarly exposed
human populations.
In the first series of studies we investigated the relationship between prolactin and prostate
inflammation. With three different developmental exposure regimens and several environmental
compounds (atrazine, methoxychlor, bisphenol A), we were able to demonstrate that exposures which
result in increased pituitary prolactin secretion prior to puberty in the rat are correlated with lateral
prostate inflammation in the adult male rat. Of particular interest was the finding that lowering the
dam's prolactin during early lactation would result in a high incidence of prostatitis in the male
offspring. We are testing the hypothesis that a toxicant induced decrease in prolactin in the dam's milk
induces an alteration of the neurons responsible for controlling prolactin secretion in the pup
(tuberoinfundibular or TIDA neurons). In turn, this change leads to increased pituitary prolactin
secretion in the male offspring and eventually the development of prostatitis. Thus, we plan to examine
the effect of atrazine treatment to the dam on TIDA neuronal development. Once the basic technique
for examining TIDA neuronal development is established, we can broaden this research approach to
evaluate the effects of other EDCs on this critical neuronal population. The significance of this study
is that if we do find that TIDA neuron and prolactin regulation during development is modified, as
expected, we will have demonstrated that a non-steroidal, non-estrogenic/androgenic agent can alter
the organization of the neural tissue regulating adult reproductive function. In addition, once
developed and standardized, this model will provide a useful method to evaluate the effect of
environmental agents on neuroendocrine development and their potential to effect reproductive
function in adulthood.
Pertinent References: 48, 214, 215, 216
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Poster 19. Effects of Atrazine and Metabolites on Implantation and Pregnancy Maintenance.
MG Narotsky, AM Cummings, DS Best, RL Cooper, Endocrinology Branch, RTD, NHEERL, ORD, U.S.
EPA.
As part of an investigation of chlorotriazines' effects in nongravid female rats, RTD worker^
have demonstrated that the chlorotriazines alter both prolactin secretion and the ovulatory luteinizing
hormone (LH) surge via a neuroendocrine mechanism. Since prolactin and LH are both critical to
pregnancy maintenance in the rat, we hypothesized that the chlorotriazines would also impair
implantation (during the prolactin-dependent period) and disrupt the endocrinological maintenance
of pregnancy in mid-gestation (during the LH-dependent period). Since dramatic differences in strain
sensitivity to chlorotriazines have been shown for a variety of toxic responses, we compared several
strains for their sensitivity during both critical periods.
To evaluate its ability to impair implantation, atrazine was dosed once daily at the time of the
diurnal or nocturnal prolactin surge on GD 1-8; pregnancy status and hormones were evaluated on
GD 8 or 9. Four dose levels were tested to establish a dose response in the Holtzman, Sprague-
Dawley, Long Evans, and F344 strains. Implantation was significantly impaired, but only in the F344
strain.
Our investigative approach regarding effects on the LH-dependent period of pregnancy involves
several facets: 1) critical period, 2) strain comparisons, 3) hormonal profiles, 4) hormone
supplementation to rescue the pregnancy, and 5) establish dose response curves for atrazine
metabolites.
In assessing the critical period, we demonstrated that atrazine did indeed disrupt pregnancy
when administered during the LH-dependent period, but had no effect afterwards. We have show
that atrazine-induced pregnancy loss is associated with progressively decreasing levels of serui.
progesterone. These data strongly support an LH-mediated mechanism. To provide further evidence
for the target site of toxicity, we are currently determining the serum LH levels following atrazine
treatment. Also, to confirm the role of key hormones in causing pregnancy loss, we will attempt to
rescue atrazine-exposed pregnancies with exogenous progesterone, hCG (an LH agonist), and GnRH.
A comparison of three rat strains during the LH-dependent period revealed that the F344,
Sprague-Dawley, and Long Evans strains were similarly susceptible to pregnancy loss following a high
dose of atrazine, but only the F344 was sensitive at lower doses. In view of the F344 resistance to
atrazine-induced mammary tumors, the increased sensitivity of this strain to pregnancy disruption is
especially important to risk assessment and the issue of sensitive sub-populations.
In addition to atrazine, we have evaluated three chlorinated metabolites and one dechlorinated
plant metabolite for their ability to cause pregnancy loss in F344 rats. All four of these degradation
by-products disrupted pregnancy during the LH-dependent period. The relative potencies of these
metabolites provide insights the role of metabolism in atrazine toxicity and the structure-activity
relationships of this class of compounds. Furthermore, since these degradation by-products have all
been detected in water supplies or on food, these data provide valuable information about hazard-
identification and the potential for cumulative effects of the metabolites.
Pertinent References: 48, 57, 161
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CRITICAL WINDOWS FOR GAMETE TOXICOLOGY
Sally P. Darney, Theme Leader
I. Introduction:
The current EPA test protocol for reproductive toxicity (Health Effects Test
Guidelines OPPTS 870.3800 Reproduction and Fertility Effects) is designed to identify
reproductive hazards associated with continuous exposure to a given chemical of interest
over all phases of the reproductive cycle. This is achieved by dosing rats of both sexes
daily during adulthood and during breeding and pregnancy (parental generation) and
continuing exposures of the offspring (F1 generation) through development to adulthood,
breeding and pregnancy, and finally their offspring (F2 generation) through early
development to weaning, i.e., across 2-generations. In addition to the apical measures of
reproductive performance (fertility, fecundity and pregnancy outcomes), this protocol now
includes more specific measures of endocrine and gonadal function such as estrous
cyclicity in females and sperm measures in males (added to the test protocol in 1998).
The objective of these additional endpoints was to increase the sensitivity of the test by
picking up changes in reproductive function at a lower dose that might not impair fertility
but that would predict infertility at a higher dose. The existing test has certain limitations.
It does not necessarily identify the affected sex. It does not provide information about
cellular targets that would contribute to understanding the mechanism of action. Finally,
it does not reveal early, proximal responses, nor identify critical windows of exposure that
are important for understanding susceptibility to acute or intermittent exposures.
Under GPRA Goal 4, OPPTS has identified ongoing needs for research relevant
to testing: 1) to help interpret test data, especially those from the newly incorporated
endpoints, 2) to develop new and improved test methods, and 3) to develop protocols or
models to evaluate effects associated with other exposure conditions such as acute
exposures during critical periods of development or within biological cycles. Under GPRA
Goal 8, research is needed to improve the scientific basis of risk assessment in part by
elucidating mechanisms of toxicant action, and developing new methods to identify early
responses to low dose exposures.
This theme responds to all of these research needs in three focal areas selected
because they are related to critical windows for gonadal and gamete maturation and
function: 1) new and improved methods for evaluating altered sperm/semen quality in
rodents and humans after short-term or intermittent exposures; 2) models for
characterizing protective mechanisms induced by, and thresholds of response to, acute
oxidative stress; and 3) identification of cellular mechanisms responsible for pregnancy
failure after acute exposures during critical windows for the induction of ovulation.
Poster # 26 summarizes our work over the last three years on the "Application of
more sensitive and specific end points to characterize male reproductive effects of
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pesticides and toxic substances in animal and human studies." We are using
existing sperm samples derived from male reproductive toxicology studies conducted
within RTD to optimize approaches for using computer-aided sperm analysis (CASA). We
are defining "progressively motile" rat and mouse sperm in a reliable and objective manner
as is required for testing, and we are using CASA to identify specialized motility patterns
("hyperactivation") that are predictive of fertilizing ability and can be used as tools in
mechanistic studies. Since the capacities for progressive and hyperactivated motion
develop as sperm mature in the epididymis, measuring these capabilities is an excellent
way to identify adverse effects of toxicants during the critical window of sperm maturation.
In collaborative work we also helped characterize the sperm-specific glycolytic isozyme
(GAPD-S) in mouse and human sperm, reporting that it is localized to the sperm tail where
it plays a role in flagellar function and may be targeted by acute exposure to certain
toxicants such as alpha-chlorohydrin. We are also evaluating human semen from men
exposed episodically to high levels of air pollution in collaborative studies between EPA
and the Czech Ministry of the Environment. Here we are evaluating the usefulness of
CASA data, as well as the relative utility of several new tests for chromosome and DNA
integrity in sperm that may be predictive of male-mediated adverse pregnancy outcomes.
The focus is twofold: first on optimizing the methods and data analysis to address the
question at hand ("Does this particular exposure alter semen quality?"); and second, to
make recommendations concerning inclusion of these outcomes in future epidemiology
studies.
Poster #27 summarizes preliminary studies designed to "Identify sensitive stages
of gonad and gamete development with respect to glutathione homeostasis and
protective mechanisms." Previous work in our lab and by others has shown that
glutathione (GSH) plays critical roles in gamete development and function. Among these
roles is the protection of gametes from oxidative stress that may result from exposure to
reactive chemicals or their metabolites. We are currently developing baseline information
about GSH levels and the genes that regulate GSH production, metabolism and turnover
in developing gonads. Information about the ontogeny of GSH-associated gene profiles
during testis and ovarian development will be used to define and refine potential critical
windows of vulnerability for oxidative stress. Acute in vivo and in vitro exposure protocols
will be used in combination with known reactive chemicals to confirm critical windows. We
are also developing methods to visualize acute oxidative damage and its consequences
in ovaries and oocytes/zygotes. These include the use of specific fluorescent probes to
identify subcellular targets for oxidative damage in oocytes during oocyte maturation and
fertilization, and the use of innovative staining methods in combination with Confocal Laser
Scanning Microscopy (CLSM) to localize areas of oxidative damage and cell death in
ovarian tissue. In the long term, these studies are designed to provide important new
information regarding the existence of thresholds of effect for oxidative damage in the
gonads/gametes, and will contribute to the development of improved methods and test
systems for detecting acute oxidative damage in the gonads and mature gametes.
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Poster #28 summarizes our recent results and newly initiated studies designed to
address: "Critical windows of exposure for female reproductive risk: Effects of acute
exposure to pesticides on the hormonal control of ovulation and subsequent
pregnancy outcome." This project extends previous research in the Endocrinology
Branch that showed that a single exposure to certain pesticides can interfere with the
timing of ovulation and thereby have a negative impact on fertilization and pregnancy
outcome. Specifically, we are determining the cellular mode of action of this effect on the
oocytes and zygotes by examining specific subcellular organelles (such as cortical
granules) and features (such as polyspermy) using improved visualization made possible
with CLSM, a core resource in RTD. We are also testing related chemicals to determine
if they act in the same manner, and whether other chemicals that alter the timing of
ovulation may produce the same effects. The previous studies also showed that continued
treatment with some of these pesticides is without effect on pregnancy outcome. This
could be due to induction of metabolism or to compensatory mechanisms involved in the
control of GnRH. As mentioned above, current EPA reproductive toxicity test protocols are
based on a paradigm that involves prolonged exposure to the compound of interest prior
to assessing fertility (i.e., the multigeneration test), but this approach ignores the possibility
that single exposures can impair reproductive performance. Thus, the current studies
identify a critical window not covered by the multigeneration tests, and demonstrate the
development of tolerance with extended exposures. Therefore, they challenge the validity
of the current testing paradigm.
II. Impact of the Research with respect to GPRA goals.
The research in this theme is responsive primarily to GPRA Goal 4, Safe
Communities, Human Health Effects, and Sensitive Subpopulations, with some of the
more exploratory aspects being responsive to GPRA Goal 8, Sound Science, Human
Health Research.
In the areas of human health effects, ORD's Goal 4 program specifies that research
is needed in order for OPPTS to comply with the mandates of TSCA and FIFRA to
evaluate existing test guidelines and to develop new and improved test methods. Also,
research is needed to develop new models for evaluating effects occurring under different
exposure conditions (such as critical developmental stages) and for using mechanistic
information in predicting risk.
Pertinent to this theme, optimization of CASA methods for semen analysis in
rodents and humans will assist OPPTS in reviewing and interpreting data provided through
the multigeneration reproductive test guidelines, and in interpreting mechanistic and
epidemiology data from the literature so that it can be used with confidence for risk
assessments. Additionally, this research will generate recommendations regarding the use
of preferred new tests for detecting DNA or genetic damage in sperm when fertility studies
implicate male-mediated development effects (see poster # 26).
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This aspect of the theme is more mature than the others, and the impact has
already been felt. Since the last peer review, we have continued to serve as leaders in the
field of sperm motion analysis by providing guidance to industry and academic laboratories
on the use of CASA to assess rodent sperm in toxicology testing and research. In original
research reports, invited reviews, workshops, and continuing education courses, we have
resolved issues of sample preparation and CASA instrument use and settings. We have
also developed new statistical analysis models, and we continue to contribute insights
regarding data interpretation. Such guidance is being applied to ensure that the data
collected in test and research protocols are valid, and to provide OPPTS the tools for
interpreting these data when they are submitted under the newly harmonized test
protocols. Thus, this research is designed to provide immediate results that are directly
applicable to the development of test guidelines. For example, we recently published
guidance on the use of CASA for setting an objective definition of progressive sperm
motion that industry can use when complying with the requirement for "the percentage of
progressively motile sperm" as specified by the OPPTS harmonized guidelines 870.3800,
Reproduction and Fertility Effects.
We are also evaluating the utility of new tests (developed by basic scientists in
academia) for the genetic integrity of human sperm (chromosome and DNA damage) that
are predictive not only of infertility but, perhaps more importantly, of abnormal pregnancy
outcomes. We have shown that it is feasible to include these tests in human field studies
and are currently evaluating the merits of these tests with respect to their specificity, ease
of use, and correlation with other semen measures. We recently participated in the
Second International Conference on Male-Mediated Developmental Toxicity and are
contributing a workshop report that summarizes the advantages and disadvantages of
each of these new methods and identifies future research needs in this area. Thus we
should be able to make recommendations regarding the inclusion of such tests in future
human reproductive health studies (applicable to the needs of OPPTS and other EPA
program offices).
The field of female reproductive toxicology is less mature, and our work in this
arena is also at an earlier stage of development. Our project designed to reveal the
cellular mechanisms through which delayed ovulation results in abnormal pregnancy
outcome (poster #28) will provide a model for extrapolating such effects to other
chemicals, and will provide insights regarding the need for specialized tests to detect such
effects as they may occur only during specific critical windows of the ovarian cycle. This
work has direct relevance to developing new models for reproductive toxicity testing, since
current multigeneration tests would likely miss such an effect for two reasons: first,
because dosing for multigeneration tests is usually done in the morning, but afternoon
dosing is required to "see" this effect; and, second, with continued dosing tolerance
develops and the effect disappears. Thus, such chemicals may come up negative in a
multigeneration study, but actually pose a risk to humans exposed intermittently at
sufficiently high levels. The animal model is critical since it would be almost impossible
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to detect such an intermittent effect in humans, given the difficulties involved in conducting
prospective fertility studies in humans.
The application of CLSM to visualize oxidative stress and cell death in ovarian
tissue, and to examine specific subcellular organelles in oocytes and zygotes, is highly
innovative and uses the latest advances in microscopy (poster # 27). These studies
should provide new tools for evaluating ovarian/oocyte function after acute exposures, and
allow us to identify critical windows for exposures during the ovarian cycle.
The ORD GPRA Goal 2 research plan also specifies the need for research to
evaluate special sensitivities of subpopulations defined in terms of age, genetic factors
and health status. Studies in this theme are establishing the baseline profiles of GSH-
system genes in developing gonads (poster # 27). We are starting with probes available
for northern analysis of a selected family of genes and will broaden this approach by
screening for gene profile changes using gene microarrays and other technologies as
these are developed for the rat. Our preliminary studies will provide the basis for
identification of critical windows of gonad development and for generating hypotheses
regarding susceptibility, within these windows, to oxidative stress that can be tested in
future work (see poster # 27). Because these studies should reveal information about
thresholds that depend on the effectiveness of protective mechanisms, and about oxidative
stress as a mechanism for adverse reproductive effects, this work is also responsive to
GPRA Goal 8, Sound Science (Harmonizing Cancer and Non-Cancer Risk Assessments).
NHEERL is currently developing a research strategy for Goal 4 after 2002 and an
associated Goal 4 Implementation Plan. In general the framework for this plan will expand
the NHEERL Goal 4 research program beyond individual test method development toward
providing tools and models for data interpretation in support of risk assessment. This will
include increasing emphasis on detection of early changes in response to toxicant
exposure that are predictive of adverse effects at a higher dose or after longer exposure
and that provide insights into the mechanisms of toxicant action. We see our research on
mechanisms of altered oocyte function (after delayed ovulation, and after oxidative stress)
as contributing to new reproductive risk assessment models. Our preliminary gene
profiling effort Should provide the basis for detecting early responses, understanding
thresholds of response and accompanying protective mechanisms, and ultimately
identifying subpopulations with elevated susceptibility for gamete-mediated adverse
reproductive effects.
III. Future Directions (next 3-5 years)
Continued research on measuring sperm motion in support of OPPTS test protocol
implementation will be limited to evaluating new CASA technologies as they evolve, and
to interpreting such data in combination with other outcomes. We will continue to optimize
methods for in vitro fertilization in rats with the aim of understanding the relationship
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between sperm hyperactivation and other biochemical changes in sperm that are
associated with fertility. The latter will help us refine specific tests for sperm function that
may be triggered by alterations in the sperm measures currently required in the OPPTS
multigeneration tests. Regarding tests for sperm DNA/chromosome damage, we expect
to continue to evaluate the utility of specific tests with respect to their value in a given
context. That is, should such tests be required on a routine basis, or added only when
existing test data or information on chemical structure indicate the potential for genetic risk
to sperm?
We have shown that the oocyte is vulnerable to delays in ovulatory signaling that
can be induced by exposures to specific pesticides during a critical window of the ovarian
cycle (via disruption of the pre-ovulatory surge of gonadotropins on the afternoon of
proestrus). We expect to determine whether this effect is generalizable to other pesticides
that act at different steps or target other cellular processes in ovulation induction but
produce the same general outcome (ovulatory delay). If we find that other chemicals
operate via the same mode of action, then it would be informative to examine this response
from a cumulative exposure perspective (i.e., exposure to two or more chemicals at the
same time). This project may lead to recommendations for new test paradigms, and
provide the impetus for OPPTS to expand their testing requirements to ensure protection
of cycling (adult) females.
We expect that the baseline information we are deriving on gene expression in
developing testes and ovaries will be used in future studies to define critical windows of
exposure, and to detect early changes that are predictive of adverse effects at higher dose
within the window. We anticipate using mouse models with loss-of-function GSH-system
gene knockouts in future studies designed to test hypotheses regarding vulnerability to
and thresholds for oxidative injury. These results should assist OPPTS in using
mechanistic information in risk assessment, and in developing unifying strategies for both
cancer and non-cancer risk assessments (bridging to longer term research in response to
Goal 8 objectives).
IV. Staffing Level
Currently a total effort equivalent to 4.25 FTEs is assigned to this theme, consisting
of partial efforts of the following staff: Sally Perreault Darney (theme leader), Tammy
Stoker, Jeffrey Welch, and Robert Zucker, with critical technical support provided by
Susan Jeffay, Lillian Strader, Dot Guidici, and Randy Barbee.
V. Significant External Collaborations
Sherry Selevan, ORD/NCEA - epidemiology support for Czech semen studies.
Jiri Rubes, VRI, Brno, Czech Republic - director of Czech semen studies and tests of
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sperm chromosome abnormalities.
Don Evenson, SDSU - tests of sperm DNA damage (Czech semen studies).
Wendie Robbins, UCLA - tests of sperm chromosome abnormalities (Czech semen
studies).
Aida Cancel, prior postdoctoral fellow - CASA and sperm hyperactivation; sperm function
tests.
David Dunson, NIEHS - Statistical models for interpretation of CASA data.
Deborah O'Brien, UNC - Characterization of sperm enzyme, GAPD-S.
Patricia Hoyer, U. Arizona and Jodi Flaws, U. Maryland - Model exposures for ovarian
toxicity detected by CLSM.
VI. Productivity:
SPERM MOTILITY
Cancel, A., Lobdell, Mendola, P. and Perreault, S.D. (2000). Objective evaluation of rat
sperm hyperactivation using computer-assisted sperm analysis (CASA). Hum.
Reprod. 75:1322-1328.
Perreault, S.D. and Cancel, A. (2001). Significance of incorporating measures of sperm
production and function into rat toxicology studies. Reproduction 121: 207-216.
Perreault, S.D. (2001). Smart use of computer-aided sperm analysis (CASA) to
characterize sperm motion. In: B. Robaire and B. Hinton (eds.) The Epididymis,
Kluwer Academic/Plenum Publishers, New York, in press.
Dunson, D.B., Weinberg, C.R., Perreault, S.D. and Chapin, R.E. (1999). Summarizing the
motion of self-propelled cells: Applications to sperm motility. Biometrics, 55:537-
543.
Dunson, D.B. and Perreault, S.D. (2001). Factor analytic models of clustered multivariate
data with informative censoring. Biometrics 57:302-308.
Bunch, D.O., Welch, J.E., Magyar, P.L., Eddy, E.M. and O'Brien, D.A. (1998).
Glyceraldehyde 3-phosphate dehydrogenase-S protein distribution during mouse
spermatogenesis. Biol. Reprod. 58:834-841.
Welch, J.E., Brown, P.L., O'Brien, D.A., Magyar, P.L., Bunch, D.O., Mori, C. and Eddy,
E.M. (2000). Human Glyceraldehyde 3-phosphate dehydrogenase-2 gene is
expressed specifically in spermatogenic cells. J. Androl. 21:328-338.
Selevan, S.G., Borkovec, L., Slott, V.L., Zudova, Z., Rubes, J., Evenson, D.P. and
Perreault, S.D. (2000). Semen quality and reproductive health of young Czech
men exposed to seasonal air pollution. Environ. Health Persp. 108:887-894.
Perreault, S.D., Selevan, S.G., Zudova, D., Zudova, Z., Evenson, D.P. and Rubes, J.
(2001). Male reproductive health studies in the Teplice Program: Air pollution and
semen quality in young Czech men. In: R. Sram (ed.) Teplice Program: Impact of
Air Pollution on Human Health, Academia, Prague, Czech Republic, pp. 145-156.
Selevan, S.G., Perreault, S.D. and Rubes, J. (2001). Epidemiologic aspects of semen
studies in Teplice and Prachatice, Czech Republic. In: R. Sram (ed.) Teplice
Program: Impact of Air Pollution on Human Health, Academia, Prague, Czech
Republic, pp 157-166.
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Slott, V.L., Jeffay, S.C., Dyer, C.J., Barbee, R.R. and Perreault, S.D. (1997). Sperm
motion predicts fertility in male hamsters treated with alpha-chlorohydrin. J.
Andrology,'\8:708-7'\ 6.
Perreault, S.D. (1997). The mature spermatozoon as a target for reproductive toxicants.
In: I.G. Sipes, C.A. McQueen and A. J. Gandolfi (eds.) Comprehensive Toxicology,
Vol. 10. Elsevier Science LTD, Oxford, 165-179.
Perreault, S.D. (1998). Gamete Toxicology: The Impact of New Technologies. In: K.
Korach, ed. Reproductive and Developmental Toxicology, Marcel Dekker, Inc., New
York, 635-654.
ESHRE Andrology Special Interest Group (S. Perreault, contributor) (1998). Guidelines
on the application of CASA technology in the analysis of spermatozoa. Human
Reprod. 13:142-145.
International Life Sciences Institute (1999) An Evaluation and Interpretation of
Reproductive Endpoints for Human Health Risk Assessment. Eds: G. Daston, C
Kimmel; Co-editors- B. Astroff, S. Perreault Darney, WD Faber et al. ILSI Press,
Washington DC
Wyrobek, A.J., Schrader, S.M., Perreault, S.D., Fenster, L., Huszar, G., Katz, D.F., Osorio,
A.M., Sublet, V. and Evenson, D. (1997). Assessment of reproductive disorders
and birth defects in communities near hazardous chemical sites III. Guidelines
for field studies of male reproductive disorders. Reprod. Toxicol., 11:243-259
SPERM - CHROMOSOME/DNA DAMAGE
Rubes, J., Lowe, Xiu, Moore, D., Perreault, S.D., Slott, V., Evenson, D., Selevan, S. and
Wyrobek, A.J. (1998). Smoking cigarettes is associated with increased sperm
disomy in teenage men. Fert. Steril. 70, 715-723.
Robbins, W.A., Rubes, J., Selevan, S.G. and Perreault, S.D. (1999). Air pollution and
sperm aneuploidy in healthy young men. Environ. Epidemiol. Toxicol. 7:125-131:
Perreault, S.D., Rubes, J., Robbins, W.A., Evenson, D.P. and Selevan, S.G. (2000).
Evaluation of aneuploidy and DNA damage in human spermatozoa: Applications in
field studies. Andrologia 32.247-254.
Ong, T.D., Xun, L., Perreault, S.D. and Robbins, W.A. Aneuploidy and chromosome
breakage in swim-up versus unprocessed semen from 20 healthy men. J. Androl.,
under revision.
Rubes, J., Vozdova, M., Robbins, W.A., Rezacova, O., Perreault, S.D., and Wyrobek, A.J.
Stable variants of sperm aneuploidy among health men show associations between
germinal and somatic aneuploidy. Amer. J. Hum. Genet., submitted.
Evenson, D.P., Jost, L.K., Perreault, S.D., Selevan, S.G. and Rubes, J. (2001).
Application of the sperm chromatin structure assay to the Teplice Program semen
studies: a new method for evaluating sperm nuclear chromatin damage. In: R.
Sram (ed.) Teplice Program: Impact of Air Pollution on Human Health, Academia,
Prague, Czech Republic, pp. 167-180.
Rubes, J., Vozdova, M., Selevan, S.G., Robbins, W.A. and Perreault, S.D. (2001). Impact
of air pollution on sperm aneuploidy. In: R. Sram (ed.) Teplice Program: Impact of
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Air Pollution on Human Health, Academia, Prague, Czech Republic, pp. 181-191.
OVARY. OOCYTES and GSH HOMEOSTASIS:
Perreault, S.D. and Goldman, J.M. (1997). Ovulation, Oocyte Maturation and Oocyte
Function. In: I.G. Sipes, C.A. McQueen and A. J. Gandolfi (eds.) Comprehensive
Toxicology, Vol. 10. Elsevier Science LTD, Oxford, 305-316.
Zuelke, K.A., Jones, D.P. and Perreault, S.D. (1997). Glutathione oxidation is associated
with altered microtubule function and disrupted fertilization in mature hamster
oocytes. Biol. Reprod. 57/1413-1419.
Zucker, RM Kershavih A, Price OT, Goldman J. (2000). Confocal laser scanning
microscopy of rat follicle development. J of Histochemistry and Cytochemistry. 48:
781-791.
Stoker, T.E., Goldman, J.M., and Cooper, R.L. (2001) Delayed ovulation and pregnancy
outcome: effect of environmental toxicants on the neuroendocrine control of the
ovary. Environmental Toxicology and Pharmacology 9(3):117-129.
Cooper, R.L., Goldman, J.M., and Stoker, T.E. (1999). Neuroendocrine and reproductive
effects of contemporary-use pesticides. Toxicol Ind Health. 15:26-36.
Note: Abstracts on visualization of cell death by CLSM in ovarian pieces were presented
at: Gender Differences in Reproductive Biology and Toxicology (Tuscon, AZ, 2000), two
microscopy meetings (2000-1), and SOT (2001). Abstracts on the GSH and ovulatory
delay projects were presented at the SSR (2000) meeting. Manuscripts based on these
presentations are currently in preparation.
ACTIVITIES RELEVANT TO STAKEHOLDERS (S. Darney):
1. Participated in US EPA Harmonized Developmental and Two-Generation Reproductive
Toxicity Test Guidelines Workgroup (1997-98)
2. Instructor: "CASA Applications in Reproductive Toxicology," Campus Course on
CASA, European Society of Human Reproduction and Embryology," San Miniato,
Italy (1997).
3. Instructor, Postgraduate Course on Effects of Toxicant Exposure on Sperm and Oocyte
Quality. American Society for Reproductive Medicine, Cincinnati, OH, (1997).
4. Instructor, "Biomarkers of developmental toxicity of paternal origin," Precongress
Course in Andrology and Male Reproductive Toxicology, 14th Annual Meeting of the
European Society of Human Reproduction and Embryology, Gothenborg, Sweden
(1998).
5. Instructor, "Improved methods for assessing sperm function and interpreting the
results," SOT Continuing Education course on Evaluation of Male Reproductive
Toxicity, New Orleans (1999).
6. Organizing Committee: Second International Conference on Male-Mediated
Developmental Toxicity, Montreal, 2001.
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Poster 26. Application of More Sensitive and Specific End Points to Characterize
Male Reproductive Effects of Pesticides and Toxic Substances in Animal and Human
Studies. SP Darney, J Welch, S Jeffay, L Strader, and R Barbee, in collaboration with A
Cancel (FHI), S Selevan (EPA/ORD/NCEA), J Rubes (VRI, CZ), Wendie Robbins (UCLA),
D Evenson (SDSU) and D Dunson, NIEHS.
Under GPRA Goal 4, research is needed to develop new and improved tests for
reproductive toxicity testing, new models for interpretation of test data for risk assessment,
and approaches for identifying mechanisms of toxicity to facilitate interspecies risk
extrapolation. This poster summarizes our work designed to enhance the scope and
usefulness of semen analysis as a tool for detecting adverse male reproductive effects,
especially those that occur during the critical windows for sperm maturation. The rationale
for this work is strengthened by the fact that semen can be obtained from humans as well
as test species. We concentrated on two aspects of semen analysis: first, the application
of new computer vision techology to evaluate sperm motion as a predictor of fertility in test
species and humans; and, second, exploration of new methods for assessing the genetic
integrity of the sperm as a predictor for its ability to support normal embryonic development
after fertilization.
During this review period we upgraded our Computer-aided semen analysis (CASA)
system to permit acquisition of digital images, developed an objective definition of
"progressively motile sperm" based on statistical distributions of dual CASA outcomes, and
made recommendations for using this approach in EPA test protocols. Likewise, we
developed an objective definition of "hyperactivated" sperm (for use in mechanistic, in vitro
studies) based on statistical distributions of two different CASA outcomes (in collaboration
with Dr. Cancel), developed several novel approaches for the statistical analysis of CASA
data (in collaboration with Dr. Dunson), and analyzed CASA data obtained in a human
semen study to evaluate its relationship with other semen outcomes (in collaboration with
Dr. Rubes). This work will assist OPPTS in interpreting sperm motion data in test species
and humans. Changes in sperm energy metabolism are also critical for sperm
function/motility, and have been linked to sperm capacitation and hyperactivation. The
sperm-specific glycolytic enzyme, glyceraldehye 3-phosphate dehydrogenase (GAPD-S,)
has been implicated as a key enzyme in the acquisition of hyperactivation and as a
potential target for known reproductive toxicants (a-chlorohydrin, ornidazole). Therefore,
we also completed a molecular characterization of this enzyme in mouse, rat, and human.
The high degree of conservation of this enzyme among species suggests that it provides
a common mechanism of modulating sperm motility, and that gene profiles designed for
use in future reproductive toxicology studies (see poster # 27) should include GAPD-S.
During the past five years, new methods have been developed for evaluating sperm
aneuploidy and chromosome breakage (fluorescence in situ hybridization or FISH, with
chromosome-specific probes), and DNA damage (sperm chromatin structure assay or
SCSA; sperm COMET; and, sperm TUNEL). We have applied these assays to the same
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human semen samples mentioned above (in collaboration with experts in each method),
to demonstrate that: episodic exposure to high levels of air pollution is associated with
elevations in sperm aneuploidy and abnormal chromatin structure; cigarette smoking is a
potential risk factor for sperm aneuploidy; and both of these measures are relatively
consistent within individuals but vary between individuals. We are currently assessing the
relative correlations among SCSA, TUNEL and COMET in order to make
recommendations concerning their use in future epidemiology studies. We are also
evaluating semen quality and these genetic markers with respect to the metabolic
genotype of the semen donor, to determine whether genotype may reveal a genetic
susceptibilities for adverse semen outcomes.
Pertinent References: 34, 35, 67, 68, 70, 71, 151, 167, 168, 170, 171, 172, 173, 182,
203, 204, 208, 209, 212, 229, 234
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Poster 27. Identification of sensitive stages of gonad and gamete development with
respect to glutathione homeostasis and other protective mechanisms. JE Welch,
RR Barbee, RM Zucker, S Jeffay, and SP Darney. Gamete and Early Embryo Biology
Branch, Reproductive Biology Division, ORD, NHEERL, US EPA, RTP, NC.
Research needs specified by OPPTS include the development of tests and models
for evaluating risks associated with short term exposures when they coincide with specific
stages of development or biologic cycles (responsive to GPRA Goal 4). Our hypothesis
is that developing gonads in juveniles and differentiating gametes in adults may exhibit
increased susceptibility to reactive chemicals based on the relative effectiveness of
endogenous protective mechanisms. This hypothesis is based on previous observations
that glutathione (GSH) levels are relatively high in these tissues. In order to identify
potentially susceptible critical windows of gonadal and gamete development, we have
begun to examine the degree of protection from oxidative stress afforded by
glutathione(GSH) homeostasis at the cellular/molecular levels. Preliminary efforts in this
new project are directed at both sexes, and can be grouped into three focal areas: 1)
Obtaining baseline information about the normal regulation of testicular gene expression
in the GSH synthetic pathway, 2) Modulation of oocyte meiotic spindle formation during
oocyte maturation and fertilization by toxicants that target GSH, and 3) Development of
methods needed to visualize cell death in situ in ovarian tissue by whole mount imaging
using Confocal Light Scanning Microscopy (CLSM).
Previous studies have shown that GSH levels are high in the testis, rivaling the
concentrations found in the liver and kidney. However, our preliminary work has shown
that the transcriptional activity of the three genes involved in GSH synthesis (glutamate-
cysteine ligase regulatory light subunitand catalytic heavy subunit genes and glutathione
synthetase gene) is unexpectedly low in adult testis. Interestingly, transcriptional activity
of these genes was found to be much higher in the juvenile testis. A gradual decline in
transcription seen during testis maturation suggests that the increase in GSH levels during
development acts to suppress transcription through feedback inhibition. If so, then acute
oxidative stress that depletes GSH should up-regulate these genes, and such a response
could be an early bioindicator of adverse effects of such an exposure on organ function.
Our next step is to test this assumption.
In the female, our previous work showed that there is a burst of GSH synthesis in
the oocytes shortly before ovulation (subsequent to the pre-ovulatory LH surge) resulting
in very high concentrations of GSH in mature oocytes. We have shown that disruption of
glutathione homeostasis at this time adversely impacts the formation of the male
pronucleus (via inhibition of sperm chromatin decondensation) and the formation and
maintenance of the meiotic spindle and chromatin organization in maturing oocytes and
early embryos. For example, acute exposure to reactive model chemicals which deplete
GSH (e.g., acrylamide, diamide) within this brief but critical window of time, results in
abnormal zygote formation at fertilization and subsequent early pregnancy loss. We are
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currently determining if we can generalize this effect to other toxicants such as organic
peroxides and whether the preimplantation embryo dies by apoptosis.
CSLM imaging techniques are being developed for use in whole ovaries, to allow
visualization of toxicant-induced cell death. Lysotracker staining for acidification
associated with cell death has been shown to correlate closely with TUNEL staining of
apoptotic cells in the ovary. This novel technique has allowed us to image folliculogenesis
in adult ovaries and to identify normal follicles undergoing atresia. Preliminary results
have also shown it is possible to detect abnormal follicles by increased Lysotracker
staining after toxicant exposure. These methods will make it possible to examine effects
of oxidative stress on ovarian development and on developing oocytes in situ, and together
with molecular probes developed for the testis studies above, to extend the work on gene
induction to the ovary.
Our initial results provide evidence for critical windows in gonad and gamete
development during which GSH homeostasis is essential. Ongoing work will determine
the extent to which GSH homeostasis can be altered in these cells tissues before causing
an adverse effect. This information is relevant to understanding thresholds for non-cancer
risk assessment (i.e., is responsive to GPRA goal 8, as well as 4). Further work in both
sexes will use both CLSM and DNA array profiling of GSH pathway-related genes to
develop sensitive screening methods for patterns of gene expression predictive of a
protective response at low dose or of adverse effects at higher dose.
Pertinent References: 166, 168, 243, 244
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Poster 28. Critical Windows of Exposure for Female Reproductive Risk: Effects of
Acute Exposure to Pesticides on the Hormonal Control of Ovulation and Subsequent
Pregnancy Outcome. TE Stoker, R Zucker, S Jeffay, RL Cooper, S Darney. Gamete
and Early Embryo Biology Branch, NHEERL, ORD, U.S. EPA.
In GPRA Goal 4, EPA is charged with preventing pollution and reducing risk in the
community, specifically to promote healthy, safe communities. Because EPA must
regulate the manufacture and use of pesticides and other chemicals, the research
conducted in this area must serve to examine the adverse human health effects that can
result from exposure to pesticides and develop protocols and models that allow us to
predict human health effects. The primary focus of this research is to examine the effect
of environmental agents on the neuroendocrine control of ovulation, an event that shares
considerable homology in the rat and human. We first sought to determine if xenobiotic
exposure during certain critical periods of the ovarian cycle would disrupt neuroendocrine
control of ovulation. Second, once an effect was identified, we examined the
consequences of such brief exposures on the viability of the oocyte, embryo development
and pregnancy outcome. In parallel studies, we evaluated the mode and mechanism of
action of several pesticides to further establish a basis for comparison to the human. A
related issue in these studies is the development of tolerance following a more extended
dosing regimen (i.e., 21 days). Previously, we showed that acute exposures to pesticides
such as chlordimeform or amitraz can disrupt the CNS control of the ovulatory surge of
LH on vaginal proestrus. Once the LH surge is blocked, ovulation is delayed for 24 hours.
Subsequently we tested the dithiocarbamate fungicide, thiram, in this protocol.
Dithiocarbamates are known to suppress dopamine-p-hydroxylase, the enzyme necessary
for norepinephrine synthesis in the CNS and a key neurotransmitter involved in the control
of LH secretion. Thiram delayed the LH surge and ovulation following relatively low doses.
Although a normal complement of oocytes was released, there was a significant reduction
in litter size. Since acute exposure is a "real-life" scenario and since there is a significant
degree of homology in the regulation of ovarian function in the female rat and human, we
continued our investigations during this review period, focusing on evaluating the
mechanisms and sequence of events following such xenobiotic-induced disruption of the
LH surge, ovulation and fertility. Thus far, we have shown that a 24 h thiram-induced
delay in ovulation significantly increased the number of dispermic zygotes. Such triploid
zygotes may develop and implant in the uterus, but would not develop to term. Thus,
polyspermy may account for at least some of the embryo/fetal loss seen previously We
have now begun to examine delayed fertilized and non-fertilized oocytes by confocal
microscopy to determine if there are alterations in cortical granule distribution, which may
explain the failure of such delayed oocytes to mount a normal block to polyspermy. In a
related study, we are examining the effects of a related chemical, molinate, which is a
thiocarbamate herbicide, on the LH surge and ovulation. In agreement with our
hypothesis, an acute dose of molinate also suppressed the LH surge and delayed
ovulation for 24 hours. This effect of molinate on LH has not been shown previously. In
vivo protocols are being used to verify the target tissue (brain, pituitary or gonad or any
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combination) and possible cellular mechanism of action of this herbicide. Planned studies
will examine whether tolerance develops with subchronic dosing of this herbicide, as it did
with thiram, and relate the significance of such observations to the current method of
assessing reproductive hazards in the female.
Pertinent References: 46, 219
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Assessments of Developmental Effects Attributed to
Drinking Water Contaminants
Sid Hunter, Theme Leader
I. Introduction
This Theme's research is designed to better understand the potential adverse health
effects produced by consumption of public drinking water. Our studies have focused on
disinfectants (D), disinfection by-products (DBPs), candidate chemical list (CCLs) and other
contaminants (such as arsenicals) found in drinking water. We are engaged in determining if
there is a hazard associated with exposure to these chemicals, establishing dose-response
relationships for toxicity as well as mode-and mechamsm-of-action studies.
The research being conducted under this theme focuses on addressing the public
health concerns that there are reproductive and developmental toxicants present in drinking
water that are formed by the disinfection process. Under GPRA Goal 2, Safe Water, there is a
major emphasis to protect public health by reducing the uncertainties and risks associated with
drinking water and assessing if there are susceptibilities to toxicity. Although disinfection of the
public's drinking water is among this century's greatest public health benefits, serious
questions have been raised regarding the safety of the disinfectants used and by-products
(DBPs) formed during the disinfection process. This concern has been raised by the results of
several epidemiology studies that conclude that there is an association between consumption
of drinking water with the highest levels of DBPs and an increase in adverse pregnancy
outcomes (spontaneous abortions, increased neural tube (anencephaly) and heart defects,
and reduced neonatal weight). In contrast, other epidemiology studies show no adverse effects
associated with drinking disinfected water. Thus, one critical element of our research is to
determine if there are potential developmental effects produced by disinfectants and DBPs.
Specifically, our research addresses these questions:
A. Does exposure to drinking water contaminants alter development in animal models in
vivo? In this project we have evaluated the developmental consequences of exposing
pregnant mice or rats to D/DBPs/CCLs. We have specifically filled data gaps and added
second species studies when not available in the extant literature. This work will be presented
in Poster 9.
B. Is there toxicity and are there differences in the toxicities produced by D/DBPs
formed by different disinfection processes? This project represents a collaboration of
investigators from 4 EPA Laboratories. We will evaluate the potential adverse effects produced
by exposure to drinking water concentrates from chlorinated and ozone/chlorinated source
water, using in vivo and in vitro test models. Bromide will be added to the source water to
mimic that present in naturally occurring high bromide source water. The disinfection and
concentration are being performed in EPA's National Risk Management Research Laboratory
(NRMRL). Scientists at the EPA's National Exposure Research Laboratory (NERL) analyze the
concentrates for presence and levels of D/DBPs and the stability of these compounds during
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exposure, NHEERL provides the assessment of toxicity and we closely interact with an NCEA
risk assessment group. This work will be presented in Poster 10.
C. Does exposure to the DBPs haloacetic acids directly alter rodent embryonic
development? The haloacetic acids (HAAs) are one class of DBP formed during water
processing. For several of the HAAs, administration to pregnant rodents produces
malformations and developmental toxicity. These studies have compared the relative potency
of the 9 bromo-, chloro- and bromochloro-haloacetic acids using the whole embryo culture
model system and established a QSAR for the induction of neural tube closure defects. We
have compared the sensitivity of mouse and rat conceptuses and compared the relative
potency of 4 HAA metabolites to the parent molecules. We have evaluated the interaction of 3
HAAs and the relationship between length of embryonic exposure to HAAs and induction of
dysmorphogenesis. These studies will provide us with a better understanding of the potential
of these compounds to produce teratogenesis and determine the proximate toxicant. This work
is present in Poster 11.
D. What are the mode- and mechanism-of-action responsible for HAA-induced
dysmorphogenesis? These studies are designed to better describe the cellular and molecular
events leading to HAA-induced dysmorphogenesis. We have evaluated the induction of cell
death and perturbation of the cell cycle by three disubstituted-HAAs. We have established that
pharmacological signal transduction inhibitors produce dysmorphogenesis, and several of the
HAAs perturb embryonic kinase activity and, using proteomic tools, we are describing the
changes in phosphorylation induced by exposure to these xenobiotics. This work is presented
in Poster 12.
E. What are the critical target cell populations for induction of HAA-induced
dysmorphogenesis? The time-dependent induction of heart defects in combination with the
specific outflow tract dysmorphology produced by administration of dichloroacetic acid strongly
suggests that the neural crest cells are the critical target cell population for induction of these
defects. We are assessing the effects of direct exposure of primary cultures of neural crest
cells to the HAAs. We are assessing migration, proliferation, induction of cell death and
perturbation in differentiation induced by the HAAs and compared those effects to
pharmacological kinase inhibitors. This work is presented in Poster 13.
F. What are the mode- and mechanisms-of-action responsible for arsenical-induced
dysmorphogenesis? Arsenicals are present at relatively high concentrations in drinking water
in several parts of the United States and in many regions of the world. Since the teratogenicity
of arsenicals is so well established in the literature, our project has focused on comparing the
toxicity of different forms of arsenicals in vitro, determining that reactive oxygen species (ROS)
play a critical role in the induction of dysmorphogenesis and assessing the pathogenesis
leading to defects. In order to assess the consequences of perturbation in gene expression we
developed a new technique for gene delivery and have used that technique to evaluate the
role of specific gene perturbations in gene expression in arsenical-induced dysmorphogenesis
and genetic susceptibility to these defects. This project has no further studies planned pending
publication of studies by other laboratories regarding metabolism and disposition. This work is
presented in Poster 14.
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II. Impact of Research.
Our first specific aim addresses the hypothesis that chemicals found in drinking water
produce adverse developmental effects. To address this hypothesis we employ in vivo models
to assess the potential for D/DBPs or CCLs to produce adverse developmental effects. Our
studies have included an evaluation of prioritized chemicals using standard teratological
assessments to fill data gaps and provide data on the response of a second species.
Specifically, we have employed in vivo Tier 1 screening studies, e.g. Chernoff-Kavlock
assessments, and Tier 2, definitive studies using classical Segment II study design. These
studies are designed to provide hazard identification and dose response information to add to
the general knowledge regarding the teratogenic potential of chemicals found in drinking water.
In addition to our intramural work, we have been involved in collaborative studies with
NTP/NIEHS to assess the potential reproductive and developmental effects of D/DBPs and a
mixture of haloacetates (HAAs). For these studies, we prioritized a group of ten D/DBPs that
were evaluated using the NTP 35 Day Study Design. These studies are now complete. The
NTP is now planning to perform a modified NTP 35-day study of a HAA mixture. This study will
compare the toxicity of HAA mixtures formed in low and high bromide source water and test
the hypothesis that regional differences in adverse pregnancy outcomes reported in
epidemiology studies are the result of differences in the mixture of DBPs formed because of
differences in the bromide content of the source waters. We have also established a
cooperative agreement with Research Triangle Institute to perform a multigeneration study of
the effects of bromochloroacetic acid. Although this study includes an evaluation of
developmental endpoints, the focus of this project is on the reproductive system and its
maturation. Therefore, this project will be discussed in the Assessments of Reproductive
Effects Attributed to Drinking Water Contaminants Theme. We also have collaborated with Dr.
Tom Sadler (UNC-CH) in an evaluation of the potential adverse effects of DBPs in an animal
model of folate deficiency. Studies by Dr. James Andrews are being performed in-house to
evaluate this potential interaction in rats. These studies will be presented in the Mechanisms of
Susceptibility and Toxicity Theme. In summary, these studies assess the potential
developmental effects of exposure to xenobiotics found in drinking water and support the
Agency's goal to Evaluate the Health Effects Associated with Exposure to DBPs.
Within the rubric of Evaluating the Health Effects Associated with Exposure to DBPs are
important questions regarding the relative risks associated with alternate disinfection
processes that could be utilized for drinking water. Since these disinfection processes use
different disinfectants, the types and concentrations of by-products formed will be different.
This leads to the hypothesis that drinking water produced by alternate disinfection processes
will have different toxic characteristics because of the D/DBPs mixtures produced. To test this
hypothesis we are involved in a collaborative project with other Laboratories of the EPA to
determine if there are adverse developmental and reproductive effects associated with drinking
water formed by chlorination and ozone/chlorination processes. For these studies surface
water will be collected, spiked with bromide to represent a region with a high bromide level and
disinfected using chlorination and ozone/chlorination by the NRMRL. Concentrates will be
prepared by reverse osmosis. Concentrates will be analyzed for known D/DBPs using
standard techniques. Unknowns will be determined using LC/MS by the NERL. The
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concentrates will be shipped to us (NHEERL Laboratory) where animals will be exposed to the
concentrates prior to and throughout gestation and lactation. The F1 neonates will be used for
evaluation of developmental effects, development and function of the reproductive system, as
well as other endpoints (neurotoxicity, immunotoxicity and carcinogenicity) by other NHEERL
scientists. We are working closely with the NCEA risk assessment group to best use these
data for protection of public health. This research project is represented in our second specific
aim.
In addition to our work in vivo to better characterize the effects of exposure to D/DBPs,
our third specific aim utilizes the in vitro whole embryo culture to compare the relative potency
across a class of DBPs, compare toxicity of parent and metabolites, compare sensitivity of
rodent (rat vs. mouse) conceptuses and assess the effects produced by combinations of
chemicals within a class. These data can then be used to set priorities for in vivo evaluations,
establish QSARs for induction of dysmorphogenesis, and preliminary information to determine
the proximate toxicant(s). Our studies began with a comparison of the 9 bromo-, chloro- and
bromochloro-acetates since only limited in vivo data were available. Using these results, in
collaboration with another EPA investigator, a QSAR model was established for the induction
of neural tube defects by the HAAs. We next assessed the effects of combinations of three
HAA and demonstrated an additive interaction of induction of dysmorphogenesis. These
studies provide an important insight into the potential direct toxicity of the HAA that can aid in
the risk assessment of this family of DBPs.
Since our in vivo results indicate that there are dramatic differences in the response of
mice and rats to HAA-induced teratogenesis, we compared the direct effects of 3 HAAs in rat
and mouse conceptuses in whole embryo culture. These studies indicate that the embryonic
response to HAA exposure is similar between the species. One explanation for the different
responses in vivo could be differences in the metabolism and elimination of the proximate
toxicant. We next used the Whole Embryo Culture (WEC) system to compare the relative
potencies of 4 HAA metabolites to the parent molecules. These studies suggest that the
proximate toxicant depends upon which HAA is being evaluated. For DBA and BCA the parent
compound is likely to be the proximate toxicant since they are far more potent than the
metabolites. In contrast, DCA is much less potent than the metabolites, suggesting that the
metabolites may be the proximate toxicants. These studies have important implications for
species extrapolations because of different rates of metabolism and provide critical information
regarding the direct toxicity and proximate toxicant(s) for the risk assessment of the HAAs.
In addition to our research focused on addressing hazard ID and dose-response
relationships, our fourth specific aim is designed to determine the modes and mechanisms of
action for DBPs. These studies have focused on understanding the toxicity of the haloacetic
acids because they are teratogenic in vivo and dysmorphogenic in vitro. Our mode of action
studies have evaluated the induction of cell death and perturbation of the cell cycle by the
HAAs. These studies show some similarities as well as differences among the 3 HAAs tested.
These differences suggest that the mechanisms responsible for dysmorphogenesis may be
different for individual yet structurally related HAAs. Alternatively, there may common
mechanisms for these acids, but differences in reactivity or transport/availability within the
embryo are responsible for the different effects observed. Our mechanistic studies have
focused on an assessment of signal transduction pathways as targets for HAA-induced
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craniofacial and heart defects. Based on the effects of the HAAs in adult tissues, we tested the
hypothesis that perturbation of signal transduction pathways is responsible for the induction of
dysmorphogenesis. We characterized the dysmorphogenic effects of several pharmacological
signal transduction pathway inhibitors and compared the induction of cell death and
perturbation of cell cycle to that induced by the HAAs. Since there was no information
regarding kinase activity in embryos at this stage of gestation, we measure PKC activity and
showed that some HAAs inhibit PKC in embryos. We have employed proteomic tools to
determine if exposure to the HAAs or kinase inhibitors changes the pattern of embryonic
protein phosphorylation. Our hypothesis is that because of the integration of signal
transduction pathways we will learn more about the effects of these agents by looking at the
entire pattern and not solely focusing on specific pathways. We have used Western analysis to
look specifically at phosphotyrosine containing proteins and have shown that the HAAs alter
their levels. Efforts are underway to identify two major phosphotyrosine containing proteins.
These studies support the hypothesis that exposure to the HAAs perturbs embryonic signal
transduction, and the defects produced by the kinase inhibitors suggest that these alterations
may be responsible for HAA-induced dysmorphogenesis. Our mode and mechanism of action
studies have suggested that the effects induced by dichloroacetic acid, dibromoacetic acid,
and bromochloroacetic acid are not the same. There are quantitative and qualitative
differences in the effects produced by these agents suggesting that the mechanisms of action
are not the same.
Our next specific aim asks questions regarding which embryonic cells are susceptible to
HAA-induced effects and contribute to the observed heart and craniofacial dysmorphogenesis.
Specifically, studies are underway to determine if neural crest cells are the critical targets. The
teratogenic effects produced by HAA administration in vivo (such as microphthalmia and heart
outflow tract defects) and the craniofacial dysmorphogenesis produced in vitro suggest that the
neural crest cells are a critical target cell population for HAA-induced defects. Studies to
evaluate the direct effects of the HAAs on neural crest development in primary culture are
underway. We have established the technique to explant regions of the embryonic
neuroepithelium that will allow neural crest cells (NCC) to develop and migrate into culture. We
have exposed the crest cells to the HAAs and pharmacological signal transduction inhibitors to
assess the effects on NCC development. These studies demonstrate that the HAAs can inhibit
migration and induce cell death in this population. Based on our observations in the whole
embryo, we plan to test the hypothesis that perturbations in signal transduction pathways are
responsible for HAA-induced crest cell effects.
In addition to our projects on the adverse effects of the DBPs we are also interested in
the mechanisms responsible for the teratogenic effects of arsenicals. These projects have
focused on determining the proximate toxicants, mode and mechanisms of action and genetic
susceptibilities to arsenical-induced dysmorphogenesis. Our studies of arsenicals have
compared the toxicities of arsenite (Aslll), arsenate (AsV), and the mono- and dimethyl-
arsenate metabolites and their combinations in vitro. We have shown that generation of
reactive oxygen species (ROS) is a critical event in the induction of craniofacial
dysmorphogenesis and described the time-course for arsenical perturbation of the cell cycle
and induction of apoptosis in vitro. Studies were performed in order to determine if ROS were
responsible for these cellular effects. To better understand the role of perturbation of gene
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expression as a mechanism leading to teratogenesis, to better understand the implications of
gene array data, and to provide insights into the genetic basis for susceptibility to arsenical-
induced dysmorphogenesis we have used molecular tools (such as antisense oligonucleotides)
to decrease gene expression and developed adenoviral tools to overexpress selected genes.
These studies demonstrated that induction of the heat shock 70-1 and 70-3 genes is critical to
prevention of malformations by decreasing susceptibility to the dysmorphogenesis. Reports
from another laboratory indicate that exposure to arsenate in vivo resulted in an increase in
bcl2 and p53 expression (among other changes) leading the authors to conclude that the
mechanism for induction of defects was perturbation of the cell cycle. We tested the
hypothesis that overexpression of p53 was dysmorphogenic using adenoviral vectors. We
demonstrated that p53 overexpression was not dysmorphogenic, but dramatically increased
the susceptibility of the embryo to arsenical-induced defects in vitro. These studies aid in the
assessment of risks to sensitive populations from drinking water exposure by determining
mode and mechanisms of action that will reduce the uncertainties associated with an
assessment of risk to the developing embryo. Utilization of mechanistic data for regulatory
decisions will require a more in-depth understanding of the basic processes of development.
Additionally, we have used novel techniques to better understand the relationship between
altered gene expression and dysmorphogenesis and the relationship between genetic
alterations and susceptibility to developmental toxicants. These studies will begin our efforts to
better understand data generated from DNA gene arrays as well as issues related to
differences in susceptibility to toxicants within the human population.
111. Future Directions of the Theme
With regard to an assessment of the potential developmental and reproductive effects of
D/DBPs, we have filled many data gaps over the last several years. Representative chemicals
from each of the major groups of known DBPs have been assessed. It is important to note that
only 40-50% of the formed DBPs have been identified, leaving as many unknown chemicals as
are already known. In recent publications, EPA scientists have identified more than 700
compounds previously unknown as DBPs (including nitro containing and iodinated
compounds). As the concentrations of these DBPs are determined, the Office of Water will
continue to need additional data on the potential toxicity of these agents. Additionally, our
research has focused on chemicals produced during chlorination. As the drinking water
industry considers switching to alternate or new disinfection processes (such as
ozonation/chloramination) new sets of DBPs will be produced. For example, ozonation tends to
produce much higher levels of halogenated aldehydes and alcohols than formed during
chlorination. Thus, we anticipate the continued need to provide descriptive data to prioritize
chemicals for definitive studies or possibly add second species analysis when necessary.
The "4-Lab" study has only progressed to a palatability study for the disinfection
concentrates. We are currently re-evaluating the process used in preparation of test material.
An alternative proposal, being evaluated by the NRML Engineers, is a concentration of the
source water to produce a high level of naturally occurring organic material followed by
disinfection to generate the desired D/DBP concentrates. This process has been used by other
investigators and has the added benefit of producing much higher concentratios of D/DBPs
than reverse osmosis of disinfected water. It has the added benefit of being amenable to many
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different disinfection processes. Thus, if preparation of D/DBP concentrates is no longer the
determining factor in how much water concentrates can be produced and which disinfection
processes can be employed, we anticipate that this project may be enlarged beyond the
original scope. Additionally, we are considering the feasibility of comparing the concentrates
from each disinfection process from source water with high, medium and low bromide
concentrations.
Our mechanistic studies of the HAAs have suggested that the effects induced by
dichloroacetic acid (DCA), dibromoacetic acid (DBA), and bromochloroacetic acid (BCA) are
not the same. There are quantitative and qualitative differences in the effects produced by
these agents suggesting that the mechanisms of action are not the same. We have begun to
employ proteomic tools to assess what proteins are phosphorylated in embryos exposed to the
DCA, DBA or BCA and used Western analysis to determine what phosphotyrosine-containing
proteins are present and if exposure to the HAAs changes their phosphorylation. We are
planning to have several proteins sequenced, and this should direct us to ask questions
regarding their function and relationship to induction of dysmorphogenesis. We have recently
begun an analysis of gene expression in the cranial region of HAA treated embryos using gene
arrays. Our preliminary analysis of the data indicates that DCA, DBA and BCA produce some
similar changes in gene expression. However, there are many more changes that are unique
to each chemical. This again suggests that the mechanisms responsible for dysmorphogenesis
are unique for these agents. Further studies to assess time and concentration dependent
alteration in specific genes and the potential dysmorphogenic effects of altered gene
expression (using adenoviral delivery of genes or antisense) are to be conducted in the future.
Our research on the adverse effects of arsenicals has been halted pending results of a
study comparing the metabolism and distribution of arsenic following oral and peritoneal routes
of administration. (It is our understanding that this study is being performed by another
laboratory). These are critical data because recent studies have clearly demonstrated that oral
administration of arsenic does not produce teratogenesis, in contrast to the high rates of
defects produced by the IP or IV routes. This suggests that environmental exposure to arsenic
in drinking water would not be expected to produce abnormalities. Once the distribution data
are published there may be additional studies, but we have none planned at this time.
IV. FTEs
Participating Staff: James Andrews, Debbie Best, Maria Blanton, Mary Cardon, Neil Chernoff,
Phillip Hartig, Sid Hunter, Clint Kawanishi, Leonard Mole, Mike Nartosky, Harriette Nichols,
Ellen Rogers, Mitch Rosen.
V. External collaborations that are critical to the successful execution of this research:
NTP/NIEHS/NIH for performing studies to assess the effects of exposure on the
reproductive and development effects in rodent models.
Other Laboratories within EPA: NRMRL, NERL, NCEA. Our collaboration is associated
with the "4-lab" study to assess the health effects produced by exposure to D/DBP
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concentrates formed by different disinfection processes. NRMRL does the disinfection and
prepares the concentrates, NERL performs the analytic chemistry to determine what is in the
water, and NCEA serves to coordinate and provide risk assessment context to this project.
Dr. Tom Sadler, UNC-CH Department of Cell and Developmental Biology, is performing
experiments on the interaction of folate deficiency and DBP-induced defects in mouse.
VI. Productivity
Our theme has published or submitted 15 Journal Articles and Book Chapters since January,
1997. We have been invited to present 19 presentations (including Seminars and Symposia
Talks) and organized 1 symposium at the Teratology Society Meeting.
Our publications are (numbered from the Division Bibliography)
204. Tabacova, S., Hunter, E.S , III., and Balabaeva, L. (1997). Potential role for oxidative
damage in developmental toxicity of arsenic. In Arsenic: Exposure and Health Effects (R.L.
Abernathy, Calderon and W.R. Chappell, Eds.), pp. 135-144. Chapman and Hall, New York.
221. Zucker, R.M., Hunter, S., and Rogers, J.M. (1998). Confocal laser scanning microscopy
of apoptosis in organogenesis-stage mouse embryos. Cytometry. 33, 348-354.
76. Hartig, P.C., and Hunter, E.S., III. (1998). Gene delivery to the neurulating embryo during
culture. Teratology. 58,103-112.
209. Ward, K.W., Rogers, E.H., and Hunter, E.S., III. (1998). Dysmorphogenic effects of a
specific protein kinase C inhibitor during neurulation. Reprod Toxicol. 12, 525-534.
222. Zucker, R.M., Hunter, E.S., III, and Rogers, J.M. (1999). Apoptosis and morphology in
mouse embryos by confocal laser scanning microscopy. Methods. 18, 473-480.
81. Hunter, E.S., III, and Hartig, P. (2000). Transient modulation of gene expression in the
neurulation staged mouse embryo. Ann N Y Acad Sci. 919,278-283.
210. Ward, K.W., Rogers, E.H., and Hunter, E.S., III. (2000). Comparative pathogenesis of
haloacetic acid and protein kinase inhibitor embryotoxicity in mouse whole embryo culture.
Toxicol Sci. 53, 118-126.
38. Chernoff, N., Hunter, E.S., Hall, L.L., Rosen, M.B., Brownie, C.F., Malarkey, D., Marr, M.,
and Herkovits, J. (In Press). Lack of teratogenicity of microcystin-LR in the mouse and toad.
J. Appl. Toxicol.
108. Klinefelter, G.R., Hunter, E.S., III., and Narotsky, M.G. (2001). Reproductive and
developmental toxicity associated with disinfection by-products of drinking water. International
Life Sciences Institute, pp. 309-324. ILSI Press, Washington, DC.
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82. Hunter, E.S., III., and Dix, D.J. (In Press) Heat shock proteins Hsp70-1 and Hsp70-3 are
necessary and sufficient to prevent arsenite induced dysmorphology in mouse embryos.
Molecular Reproduction and Development.
16. Andrews, J.E., Nichols, H.P., Schmid, J.E., Hunter III, E.S., and Klinefelter, G.R.
(Submitted). Developmental toxicity of mixtures: The water disinfection by-products dichloro-,
dibromo- and bromochloroacetic acid in embryo culture. Teratology.
83. Hunter, E.S., III., Cardon, M.C., and Hartig, P.C. (Submitted). p53 Overexpression
Increases Susceptibility Of Mouse Embryos To Chemical-Induced Malformations. Tox Sci.
84. Hunter, E.S., III., Cardon, M.C., Zucker, R.M., Elstein, K., and Hartig, P.C. (Submitted).
Pathogenic effects of arsenicals in neurulation staged mouse conceptuses in vitro. Teratology.
196. Smith, J.B., Hartig, P.C., Blanton, M.R., Sulik, K.K., and Hunter, E.S., III. (Submitted).
Amelioration of ethanol-induced malformations by adenoviral Cu,Zn-SOD and Mn-SOD
Overexpression in Vitro. Alcohol Exp. Clin. Res.
205. Tabacova, S., Harris, C., and Hunter, E.S., III. (Submitted). Developmental Toxicity of
Arsenic: Evidence for a Role of Oxidative Stress. Reprod Toxicol.
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Poster 9. Does exposure to drinking water contaminants produce adverse
developmental effects in rodent models in vivo? MG Narotsky, N Chernoff, EH Rogers,
DS Best, ES Hunter III.
Several epidemiology studies have raised concerns of an association between exposure
to DBPs and adverse pregnancy outcomes; however, a large number of DBPs have not been
evaluated for potential reproductive and developmental toxicity. In an effort to identify DBPs
that pose a developmental hazard, and to establish dose response relationships, we have
performed standard in vivo toxicity studies on a number of DBPs. These studies include
intramural work as well as extramural collaborations with NTP/NIEHS. Our goal is to provide
information about chemicals when no other data exist (fill data gaps), to assess toxicity in a
second species, and to address specific questions regarding effect or window of susceptibility.
Our intramural efforts have focused on tier-1 screening/priority setting studies using the
Chernoff/Kavlock study design. This protocol provides an effective screen for adverse effects
on prenatal development as well as on postnatal growth and viability. In these rodent studies,
pregnant animals are separated into 4-8 dose groups and chemicals are administered
throughout organogenesis. The dams are allowed to deliver their litters, and the pups are
counted, weighed, and examined on postnatal days 1 and 6. To further characterize effects of
xenobiotics on prenatal development, we have also used a standard Segment II study design,
incorporating detailed examinations of gross external, soft-tissue, and skeletal morphology of
full-term fetuses. Important findings from these studies include malformations of the tail, eye,
and urinary tract following exposure to dibromoacetic acid. In addition, reduced pup weights
and delayed parturition have been associated with exposure to several trihalomethanes and
halogenated acetic acids.
We used the Chernoff/Kavlock results and other criteria to select 10 D/DBP chemicals
for nomination to the NTP for assessment of reproductive and developmental effects. These
studies have been completed using the NTP 35-day study design which assesses female and
male reproductive endpoints, and the effects on development with exposure throughout major
organogenesis and exposure for 2 weeks prior to mating and throughout gestation. Results
from these studies (available at http://ntp-server.niehs.nih.gov/htdocs/pub-TT.html) indicate
that bromate may have some adverse effects on male reproduction; this finding led to a
continuous-breeding reproductive study currently being performed by the NTP. Also,
bromochloroacetic acid (BCA) produced pre- and post-implantation embryo loss, decreased
neonatal weight at term, altered postnatal growth, and increased postnatal mortality. We have
designed several studies to follow-up on these observations in-house.
In addition to D/DBPs, we have also evaluated microcystins for their developmentally
toxic potential. At maternally toxic doses there are no adverse effects on pregnancy outcome.
In summary, through the use of several study designs, we are filling specific data gaps, and
providing second-species data regarding the developmental toxicity of drinking water
contaminants. These data are valuable to the risk assessment of these chemicals and, thus,
will have substantial impact on the Agency's mission.
Pertinent References: 38, 126
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Poster 10. Toxicological and Chemical Evaluation of Alternative Disinfection Treatment
Scenarios. MG Narotsky, ES Hunter III, GR Klinefelter, JM Goldman, DS Best, EH Rogers, A
Murr, JE Simmons (Experimental Toxicology Division), L Teuschler (NCEA), R Miltner
(NRMRL), T Speth (NRMRL), G Rice (NCEA), K Schenck (NRMRL), S Richardson (NERL).
Epidemiological studies, as well as toxicological studies with laboratory animals, have
raised concerns regarding possible adverse health effects of disinfectant by-products (DBPs)
found in drinking water. The concentrations of DBPs are influenced by the choice of
disinfection process (e.g., chlorination vs ozonation) as well as source water characteristics
(e.g., pH, total organic carbon, bromide content). Disinfected water has been found to
contain more than 300 compounds; however, it should be noted that many DBPs remain
unidentified. For chlorination, the known DBPs might account for less than 50% of the mass
of total organic halide. Therefore, we plan to evaluate the toxicity of the complex mixtures
produced by different disinfection processes in order to better characterize the potential health
risks of drinking disinfected water.
Briefly, this project will entail four main steps: 1) Disinfect untreated water using either
chlorine ("chlorination") or an ozone-chlorine system ("ozonation"). 2) Concentrate these
waters approximately 100 fold; to the extent possible, volatile DBPs lost in the concentration
process will be spiked back into the concentrate. 3) Chemically characterize the concentrated
waters. 4) Evaluate the in vivo and in vitro toxicity of the concentrated waters, with a focus on
reproductive and developmental endpoints, but including other important endpoints to the
maximum extent possible.
To minimize logistical complications when preparing and analyzing the water, and
conducting the main study, a preliminary study, i.e., a "trial run," was conducted. For the
preliminary in vivo assessment, we conducted a Chernoff/Kavlock assay to screen the
concentrated waters for developmental toxicity. Pregnant rats were exposed to either boiled
distilled water, 100X chlorinated water, or 100X ozonated water during the period of major
organogenesis (gestation days 6-15) and their pups were examined for growth and viability on
postnatal days 1 and 6. No adverse effects of either disinfection scenario were found. The
day-6 pups will be further examined for skeletal alterations.
For the main study, we will conduct a multi-generational study with an emphasis on
reproductive and developmental endpoints. To maximize resources, however, collaborators
will also assess mutagenicity, carcinogenicity, immunotoxicity, hepatic and renal toxicity,
neurotoxicity, and toxicokinetics.
This project will have a tremendous impact on EPA's risk assessment of disinfected
water as it addresses public health concerns related to DBP exposure that cannot be
addressed directly from toxicologic studies of individual DBPs or simple DBP mixtures.
Furthermore, this effort addresses both a regulatory and a research need for the toxicological
assessment of real-world complex mixtures.
Pertinent References: None (new project)
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Poster 11. Does exposure to the disinfection by-products (DBPs) haloacetic acids
directly alter rodent embryonic development? JE Andrews, HP Nichols, EH Rogers, MR
Blanton, ES Hunter, A Richard
The reaction of disinfectants (such as chlorine) with natural organic matter (humic and
fulvic acids, i.e. dirt) produces a large amount of halogenated byproducts. The haloacetic acids
(HAAs) are one class of DBP formed during water disinfection. Chlorinated, brominated and
bromochloro-acetic acids are present in drinking water. This group of chemicals is regulated so
that the maximum concentration of 5 HAAs (TCA+DCA+MCA+DBA+MBA) present in water do
not exceed an annual average of 80 ^g/liter. These agents are measured by water treatment
facilities quarterly.
For several of the HAAs (e.g. DCA and TCA), administration to pregnant rodents
produces malformations and developmental toxicity in vivo. Since in vivo studies have not
been completed on all of the chemical family members, we used the rodent whole embryo
culture system to assess dysmorphogenesis, calculate benchmark concentrations and then
compare the relative potency of the 9 haloacetic acids. All of these agents produced
dysmorphogenesis, but the concentration necessary to alter development (e.g. benchmark
concentration) ranged from 5 (iM to 2 mM. In general, brominated forms were more potent
than chlorinated, and bromochloroacetic acids were similar to the brominated forms. We used
the whole embryo culture model system and established a QSAR for the induction of neural
tube closure defects. We have compared the sensitivity of rat and mouse conceptuses, and
these studies indicate that there is a similar sensitivity to the agents. However, mouse
conceptuses were more sensitive to the induction of neural tube closure defects than rats. We
have assessed the potency of 4 HAA metabolites (glycolic acid, glyoxylic acid, oxalic acid and
formate) relative to the parent molecules. These studies indicate that these metabolites are
less toxic than many of the HAAs, but much more toxic than DCA. This suggests that the
parent molecule is the proximate toxicant for many of the HAAs (such as BCA and DBA), but
that the metabolites may be the proximate toxicants for other HAAs (such as DCA). We have
also compared the relationship between length of exposure to the HAAs (DBA, BCA and DCA)
and the induction of malformations. These studies suggest that the reactivity of these agents
varies widely. Malformations were induced by BCA after a 1-hour exposure, but a 6-hour
exposure was needed with DCA to alter development. These studies further suggest that DCA
is not the proximate toxicant in vivo, but that BCA and DBA may be the active toxicant. We
have evaluated the interaction of 3 HAAs (DCA, DBA and BCA) for induction of
dysmorphogenesis and demonstrated an additive interaction between them. Once studies are
available that describe rodent and human metabolism and disposition of these agents, our
data will provide a rational basis for comparing their potential and relative potency.
Pertinent References: 16
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Poster 12. What are the mode- and mechanism-of-action responsible for HAA induced
dysmorphogenesis? JE Andrews, HP Nichols, MR Blanton, EH Rogers, ES Hunter, K Ward,
B Mundy
These studies are designed to better describe the cellular and molecular events leading
to HAA-induced dysmorphogenesis. We have evaluated the induction of cell death and
perturbation of the cell cycle by three disubstituted-HAAs. We have established that
perturbation of signal transduction pathways leads to dysmorphogenesis, and using proteomic
tools we are describing the changes in phosphorylation induced by exposure to
pharmacological kinase inhibitors and the HAAs.
The haloacetic acids (HAAs) are one group of disinfection byproducts (DBPs) formed
and found in drinking water. We have evaluated the developmental effects of the
administration of many of these agents in vivo and the entire family of brominatol, chlorinated
and bromochloro-acetic acids in vitro. Utilizing the in vitro data and the induction of neural
tube defects, a quantitative structure activity relationship was established. As a class of DBPs,
these agents consistently induce structural defects, are neurotoxic, and are carcinogenic.
Thus, we are interested in the potential mechanisms responsible for their effects. In vivo and in
vitro these chemicals produce craniofacial defects and heart dysmorphogenesis suggesting
that the neural crest may be a target cell population (these studies are described in abstract
30). Dichloroacetic acid (DCA) has been shown to inhibit the activity of pyruvate kinase, which
regulates the activity of pyruvate dehydrogenase in adult tissues. Additionally it has been
reported to increase PKA activity and decrease MAP kinase activity in adult liver. Based on
these studies we proposed that DCA, and other HAAs, may induce dysmorphogenesis by
perturbation of signal transduction pathways in the embryo. Because we don't know which
pathways are crucial for morphogenesis in neurulation staged mouse conceptuses we
evaluated the dysmorphogenic effects of pharmacological inhibitors of PKC, MAP kinase, PKG
and PKA. Bisindolmaliemide 1 (PKC inhibitor), PD98059 (MAP kinase inhibitor) and
staurosporine (a broad spectrum kinase inhibitor) produced high rates of dysmorphogenesis.
We next measured the relative activity of classic, atypical, and novel activity in the neurulation
staged embryo and showed that only 20% of the PKC activity was classic. We found that the
disubstituted HAAs (DCA, dibromoacetic acid (DBA) and bromochloroacetic acid (BCA))
altered PKC activity by different amounts (DCA>DBA>BCA). Because we know we are likely to
alter the activity of several kinases, we have established 2-dimensional gel electrophoresis in
our laboratory, labeled embryos with 33P-phosphate to assess what proteins were
phosphorylated and the effects of exposure to these HAAs. We have employed 2, 6 and 24
hour exposures and are in the process of evaluating these gels. Since antibodies are available
to recognize phosphotyrosine-containing proteins, we have used Western analysis to
specifically ask if these proteins are perturbed by exposure to the HAAs. After 6 and 24 hours
of exposure we have found that a 184 kDa PT protein is increased in DCA-treated embryos but
not in DBA or BCA-exposed embryos; we are in the process of identifying these proteins. Our
2-hour exposure data suggest a similar pattern of change, but are only preliminary at this point.
These data suggest that one or more tyrosine kinase is either upregulated following exposure
or that there is an inhibition of a phosphotyrosine phosphatase. Because inhibition of
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phosphatase activity by reactive oxygen species has been reported, we are interested in the
potential role of ROS in DCA-induced defects.
Pertinent References: 226, 227
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Poster 13. What are the critical target cell populations of Haloacetic Acid (HAA)-induced
dysmorphogenesis? JE Andrews, HP Nichols, ES Hunter
Administration of dichloroacetic acid (DCA) produces microphthalmia, heart outflow tract
defects and other perturbations of morphogenesis. Interestingly, the sensitive period for
induction of heart defects by DCA are gestational day 9-10 or day 12 in the rat. Because day
9-10 is a critical period for early neural crest development and neural crest cells are critically
involved in development and septation of the truncoconal heart tube, taken together these
studies suggests that neural crest cells are a critical target for DCA-induced defects.
Similarities in the effects produced by the HAAs in embryo culture, suggest that the crest cells
may be a target for all of the chemicals in this family.
To address the hypothesis that neural crest cells are sensitive to haloacetic acids
(HAAs), we began studies to directly expose primary cultures of neural crest cells to HAAs to
assess their early development. These studies have been performed using two separate study
designs. In the first design rat explants were grown in culture for 24 hours, photographed to
determine the extent of neural crest cell migration from the explant, exposed to HAAs for an
addition 24 hours, and then photographed to determine the extent of cell migration during the
culture period. An assessment of the induction of cell death was also performed using a dye
exclusion protocol. These studies demonstrate a concentration-dependent perturbation in
migration and induction of cell death. The second study design focuses on the earliest period
of crest cell morphogenesis; mouse neural fold explants (the source of the neural crest cells)
are exposed to the HAAs prior to neural crest cell emigration. These studies suggest that the
epithelial-mesenchymal transformation and/or emigration can be perturbed by the HAAs. As in
the previous study design effects on migration and induction of cell death are also observed.
Although these studies clearly demonstrate the adverse effects of the HAAs on neural crest
cells, they do not answer questions regarding the specificity of malformations. That is to say, if
the effect of the HAAs on the crest cells were on the general migration or induced widespread
cell death, then we would expect malformations in all neural crest derived tissues (such as
micrognathia), not specific microphthalmia and heart outflow tract defects. Thus, our focus has
turned to designing experiments to better understand the ramifications of early exposure of the
neural crest cells to xenobiotics and their ultimate differentiation. It is clear from studies of
endothelian antagonists that perturbation on mouse day 8 can lead to adverse consequences
in neural crest specification and/or differentiation without effects on migration and induction of
cell death. In future studies of differentiation, we have considered using a v-myc immortalized
mouse neural crest cell line to evaluate the influence of HAAs on growth factor-induced neural
crest cell differentiation. These MONC-1 cells are multipotent, and differentiation into
melanocytes, glia and peripheral neurons has been established. These studies would need to
be repeated in primary cultures of neural crest cells in order to investigate the potential for
early exposure to perturb differentiation.
The time-dependent induction of heart defects combined with the specific outflow tract
dysmorphology produced by administration of dichloroacetic acid strongly suggests that the
neural crest cells are the critical target cell population for induction of these defects. We are
assessing the effects of direct exposure of primary cultures of neural crest cells to the HAAs.
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We are assessing migration, proliferation, induction of cell death and perturbation in
differentiation induced by the HAAs and comparing those effects to effects of pharmacologicaf
kinase inhibitors.
Pertinent References: None (new project)
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Poster 14. What are the mode- and mechanisms-of-action responsible for arsenical-
induced dysmorphogenesis? ES Hunter, EH Rogers, MR Blanton, PC Hartig, MC Cardon, L
Gefrides, RM Zucker, KH Elstein, ML Mole, C Harris, S Tabacova, DJ Dix, F Copeland
Arsenicals are present at relatively high concentrations in drinking water in several parts
of the United States and in many regions of the world. Because the teratogenicity of arsenicals
is so well established in the literature, our project has focused on understanding the modes
and mechanisms of action responsible for arsenical-induced defects. We began our studies by
comparing the toxicity of arsenite, arsenate, mono-and di-methylarsenate and combinations of
these agents. We've confirmed that arsenite is far more potent than arsenate, and that
inorganic are more potent than organic arsenicals. There is a very high level of toxicity
produced by combinations of arsenite and arsenate, suggesting that in vivo this combination
may be an important mediator of effect. We have described the time course for induction of cell
death, by arsenical exposure and shown that there is a wave of death that peaks between 12-
18 hours of exposure. In embryos exposed to arsenic a G1 cell cycle block precedes induction
of cell death and this effect is different than that reported for many adult cells. We have
continued our experiments that have demonstrated that reactive oxygen species (ROS) are
critical mediators of arsenite-induced toxicity and have evaluated the relationship between
exposure to known ROS generators and perturbation of the cell cycle and induction of cell
death. We have also shown that administration of an antioxidant will ameliorate arsenite, but
not arsenate, induced defects in CD-1 mice in vivo following IP administration. These studies
confirm and extend our previous observations regarding the role of ROS in dysmorphogenesis.
In order to determine how exposure to arsenicals increases ROS, we assessed the
content of reduced glutathione (GSH), glutathione peroxidase (GPX) activity, and catalase
activity in embryos at several time points after exposure. These studies show that GSH is
rapidly depleted and that GPX activity parallels GSH content in arsenite-exposed embryos. As
embryonic GSH levels recover they increase to higher levels than in control embryos. GPX
activity continues to parallel GSH content and increases to >150% of control activity. At the
same time, catalase activity decreases to 30% of control activity. These changes are not
observed in arsenate-exposed embryos. Our working hypothesis is that the accumulation of
ROS in arsenite treated embryos results from a depletion of GSH, caused by direct binding of
GSH to arsenite, combined with a direct effect on mitochondrial metabolism, leading to a
decrease in antioxidative capability and increased release or production of ROS. Despite the
recovery of GSH, presumably through increased synthesis, and GPX activity, oxidative
damage has occurred and is sufficient to inactivate proteins including catalase. We also
demonstrated that an irreversible catalase inhibitor produces malformations in vitro. However,
the pattern of cell death and cell cycle perturbation is different than that produced by arsenite
indicating that inhibition of catalase by arsenicals is not the primary mechanism responsible for
dysmorphogenesis.
We have also been interested in the molecular effects of arsenicals in embryos and the
relationship between changes in genes and sensitivity to arsenical-induced dysmorphogenesis.
One well characterized effect of arsenical exposure is an induction of the heat shock response.
Although correlated with a decreased sensitivity to developmental toxicants, the direct role of
HSP70-1 and 70-3 had not been tested. Utilizing antisense oligonucleotides directed against
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these genes, we used Western analysis to demonstrate a decreased heat shock response and
observed an increased sensitivity to the dysmorphogenic effects of arsenite in embryo culture.
Additionally, an expression cassette of HSP70-1 decreased the sensitivity of embryos to
arsenite, confirming that heat shock proteins 70-1 and 70-3 were necessary and sufficient to
ameliorate arsenite-induced defects.
Rick Finnell's lab has published two papers on the perturbation of gene expression by
arsenate administration in vivo. Using gene arrays with selected genes, they reported
increased expression of p53, bcl2, and weel, and indicated that these changes indicated that
perturbation of the cell cycle was responsible for the defects. To directly test the hypothesis
that overexpression of p53 was associated with dysmorphogenesis, we developed a gene
delivery tool that delivers high levels of gene expression in the neurulation staged mouse
embryo in vitro. Adenoviral constructs of wild-type p53 expressed p53 in the embryo without an
induction of defects. However, expression of this gene dramatically increased sensitivity to
arsenite compared to when embryos expressed GFP or mutant p53.
These studies have described the effects of arsenicals and pathogenesis resulting from
exposure in vitro, and to a lesser extent, in vivo. We have shown that ROS are a critical
mediator of arsenite induced effects in vitro and in vivo, but not of arsenate-induced effects.
We have also developed a working hypothesis for how exposure to arsenite increases ROS in
the embryo. These studies will aid in the risk assessment of arsenicals because they provide
mode and mechanisms of action data that can be directly compared to those associated with
the carcinogenic effects.
The critical question to be answered in the area of arsenical developmental toxicity
relates to the metabolism and distribution of arsenicals following oral and IV/IP administration.
Oral administration of a maternally toxic dose of arsenicals does not produce teratogenesis,
while IV/IP routes produce high rates. One reason for this difference may be disposition. Our
understanding is that this research is being conducted by another laboratory. Based on the
outcome of this study we will consider further research when this question has been answered.
Pertinent References: 94, 99, 100, 101, 102, 213, 221, 222
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Assessments of Reproductive Effects Attributed to
Drinking Water Contaminants
Gary R. Klinefelter, Theme Leader
I. OVERVIEW
While the disinfection of public water supplies has been among the most successful
of all public health interventions in decreasing disease, the process has generated a
number of biologically-active, treatment-related chemicals in finished drinking water.
Common classes of chemicals among these disinfection by-products (DBPs) are
haloacids and trihalomethanes. To date, several epidemiology studies have identified a
positive association between levels of trihalomethanes in drinking water or drinking water
source (i.e. bottle water vs tap) and adverse reproductive outcomes. These adverse
outcomes include: increased incidence of spontaneous abortions, still births, and birth
defects (i.e. neural tube, cardiac, cleft palate). In a comprehensive attempt to comply with
the mission of ORD and provide data with which to reduce uncertainty and better assess
human health risk, several years ago we (RTD) formed a research team, which by virtue
of collective expertise, encompasses all stages of reproduction including toxicologic
assessments of the pregnant and non-pregnant female, as well as the adult and
developing male, spans facets of reproductive biology from gametogenesis through
fertilization and pregnancy maintenance, and comprises scientific subdisciplines ranging
from endocrinology to proteomics and genomics. This research embodies several
fundamental issues pivotal in today's assessment of human health risk, namely: hazard
identification with dose-response characterization, mode of action research incorporating
proteomics/genomics and biomarker development, target organ dosimetry, and a
significant extramural effort to expand on our recent findings regarding the potential of
DBPs to elicit lower dose effects when administered throughout reproductive development.
Over the years we have continuously responded to GPRA goals in both a
programmatic and regulatory fashion. For example, when the Office of Water (OW)
wanted us to fill DBP data gaps in the male and female, we implemented a tiered testing
strategy through an interagency agreement with the National Toxicology Program (NTP).
When the National Center for Exposure Assessment (NCEA) asked for DBP data with
drinking water as a route of administration and with exposure during reproductive
development, we responded with a developmental reproductive study of dibromoacetic
acid (DBA) administered in the drinking water. In anticipation of a need for interspecies
extrapolation we have recently completed the experimental phase of a Cooperative
Agreement with Colorado State University. This study paralleled our intramural
developmental reproductive study of DBA in the rat, but was performed in the rabbit, and
used a lower dose response range. That DBA effects reproduction in both the male and
female rabbit at these lower doses suggests that species with a longer period of
reproductive development (naturally including human) are more susceptible to DBP insult,
i.e. will elicit effects at lower exposures. This, together with our data supporting the notion
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that it is the duration not the window of the exposure, that is critical for manifestation of
pubertal delays and Sertoli cell only seminiferous tubules, is likely to impact decision
making on maximum tolerated contaminant levels for DBPs at some point in the future.
Finally, funding has been approved for an extension to an ongoing epidemiology study to
include a male cohort, (i.e. semen parameters including the SP22 biomarker) and fertility
measures (e.g. time to pregnancy). The potential value of this novel biomarker is likely to
extend beyond epidemiology studies focused on drinking water contaminants. Indeed,
commercial suitors are being sought to license SP22-based technologies (fertility
diagnostic, contraception, therapeutic enhancement) as patents issue.
Based on a recent Federal Advisory Committee recommendation, confidence in
public health safety associated with the Stage 2 M/DBP rule which sets limits on maximum
contaminant level goals (MCLGs) for DBPs is questionable, as there are still significant
uncertainties regarding the reproductive health risk in humans exposed to DBPs. To
elaborate on these uncertainties, we are in the process of organizing a Workshop and a
Symposium to be held at the Annual Meeting of the Society of Toxicology in spring 2002
entitled "Is there a reproductive risk associated with exposure to disinfection by-products
of drinking water?" and "Defining the cellular and molecular mechanisms of toxicant action
in the testis" (respectively). In addition, several important new manuscripts are presently
in various stages of completion. In a recent meeting consisting of representatives of
AWWARF, the M/DBP Council, OW, Stakeholders, and NHEERL research leaders it was
made clear that FACA recommendations for increased data on reproductive and
developmental outcomes associated with DBP exposures are being taken very seriously.
Indeed, RTD's research on reproductive and developmental outcomes has fueled this
need, and additional high profile research needs (i.e. multi-year project descriptions) have
been laid out and prioritized. The bottom line is that our data have raised serious doubt
about the adequacy of the existing 80 and 60 ug/L placeholders for maximum tolerated
levels of TTHMs and HAAs in finished drinking water. The following will now summarize
our efforts within each GPRA goal relevant to the research conducted within this theme.
Within each goal discussion, an effort is made to highlight the critical path our research
follows as it impacts the risk assessment process.
II. IMPACT OF RESEARCH
A. GOAL 2 -Safe Drinking Water
Evaluation of health effects associated with exposure to disinfection by-products
(relevant to Posters # 1 and 4)
As mandated by the 1996 Safe Drinking Water Amendment EPA research is being
conducted to better identify hazard and characterize dose-responsivity of reproductive
effects elicited by exposure to priority disinfection by-products and identify the mode of
action associated with these effects. The NTP has now screened 10 priority disinfection
by-products using their 35 day screen (i.e. hazard ID) but in general the sensitivity of
endpoints and duration of exposure have been insufficient. Research within RTD has
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been driven by the need to establish biological plausibility for observed effects, and in part
this is accomplished by a quantitative estimate of risk based on adequate dose-response
data (see Theme-Specific Aims 1.1, 3.7, 4.1). That we within RTD have observed
comparable alterations in spermatogenesis and fertility for structurally-related haloacetic
acids at relatively low experimental exposures has been fueling interest in the potential
human health risk associated with environmentally-relevant exposures Accordingly,
epidemiology studies are now being planned that will incorporate seminal and fertility
endpoints. To enhance subject recruitment and increase participation rates, a semen
collection unit is being validated for field use (see Theme- Specific Aim 1.5). This
container will allow for private, at-home collection and shipping of semen from men
recruited for study. In addition to obtaining routine measures of semen quality (semen
volume, sperm concentration, and sperm morphology), we plan to include measurement
of a new sperm biomarker, SP22 (see below).
Collectively, the animal studies to date indicate that the disubstituted haloacid,
bromocloroacetic acid, is more potent on an equimolar basis than either dichloroacetic
acid or dibromoacetic acid. Generating data on mode of action is also pivotal to
establishing biological plausibility. To this end RTD has a significant effort (see Theme-
Specific Aims 1.2, 1.3, 2.1). For example, in vivo/ in vitro data based on target organ
dosimetry and proteomics demonstrates that dibromoacetic acid inhibits protein synthesis
in stage-isolated seminiferous tubules, suggesting several specific proteins as new
candidate biomarkers. Moreover, these proteins might serve to elucidate the mode of
action of haloacetic acids within the testis (and perhaps other tissues). Likewise, the
manner in which DBPs to disrupt pregnancy maintenance and ovarian steroidogenesis is
crucial to our understanding of how these chemicals may increase the risk of the
spontaneous abortions and stillbirths as have been observed in epidemiology studies.
Assessment of risks to sensitive populations (Kids) from drinking water
exposure (relevant to Poster # 2)
As discussed in the December 2000 Report to Congress, assessment of risk to
sensitive subpopulations is now a fundamental consideration in drinking water regulation.
In this regard, we have recently launched significant intramural and extramural efforts
designed to determine whether priority DBPs administered via drinking water do indeed
perturb reproductive development, and if so, is perturbation exacerbated (so as to reduce
the LOAEL) in species with a longer period of reproduction development (i.e. species more
akin to human).
Data demonstrate that at least one haloacid, dibromoacetic acid, does indeed
produce body weight-independent pubertal delays in both male and female rats.
Moreover, the data demonstrate that it is the duration of exposure rather than the window
of exposure that is critical (see Theme- Specific Aim 1.1). This has obvious implications
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for chronic human exposures. By virtue of a Cooperative Agreement with Colorado State
University (see Theme- Specific Aim 5.1), a comparable study was performed using the
rabbit as animal model to examine whether species with a longer period of reproductive
development are more sensitive. Indeed, a much lower lowest observed adverse effect
level (LOAEL) was derived for alterations in mating ability and reduced fertility in the
rabbit. In fact, if the LOAEL observed in this study and an uncertainty factor of 100 rather
than 1000 were used (since comparable effects have now been observed across species),
a maximum contaminant level goal (MCLG) of 3.5 ug/ L would be calculated; DBA
occurrence typically ranges between 2 and 20ug/L. This research now prompts the
question of whether the LOAEL in human males might be appreciably lower, and if so, the
margin of safety provided by the existing 80 ug/L limits on total haloacids might be
insufficient. Interestingly, developmental exposure of female rabbits to a relatively low
dose of DBA resulted in a significant reduction in primordial follicle populations. The
significance of these findings with regard to early-onset menopause, i.e. reproductive
senescence, awaits clarification.
Based on our recent results demonstrating that haloacid exposure delays puberty,
assessments of reproductive development have been the driver in the design of an
upcoming NHEERL 4-lab study (see Theme- Specific Aim 1.6) designed to test relevant
mixtures of disinfection by-products produced by chlorination versus ozonation of water.
Finally, a multigenerational study of bromochloroacetic acid is now underway at the
Research Triangle Institute (see Theme- Specific Aim 5.2). Each of these studies will
incorporate various sensitive reproductive measures (i.e. in utero insemination to assess
fertility, pubertal indices, endocrine assessments, histopathology, proteomics) and involve
collaboration from 3 RTD investigators. Thus, four significant and separate efforts,
including one utilizing a relevant mixture exposure, are now underway to examine the
reproductive consequence of exposure to DBPs throughout reproductive development.
As stated earlier, preliminary findings from these studies are already driving decsions on
future research needs.
B. GOAL 4 - Safe Communities
Sensitive Subpopulations (relevant to Poster # 5)
One of the fundamental features requisite to assessing risk in children is
development of methods for rapid, early identification of uniquely susceptible molecules
of effect (both genes and proteins). Specifically, we wish to identify genes in developing,
aging, or genetically susceptible individuals that are uniquely susceptible to toxicant
exposure. As such our research theme now comprises a significant genomic and
proteomic effort (see Theme- Specific aims 2.1 - proteomic and 2.3- genomics). Knockout
mice have been used as a model to determine whether specific stress responsive genes
(i.e. heat shock genes) influence testicular toxicity induced by haloacetic acids. Data
have been derived from studies involving normal mice suggesting that some of the
testicular genes altered by exposure to bromocloroacetic acid, are implicated with signal
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transduction and cell adhesion. The sensitivity of specific genes, i.e. dose-response, is
now being characterized. This work is being linked with the proteomic work in the rat
mentioned above. Ideally, once the seminiferous tubule proteins compromised by
haloacid exposure are identified, at least some will match genes identified by genomic
array technology. In this manner not only will our mode of action / biomarker indications
be enhanced, but the value of the array technology to target functional genes will also be
demonstrated. Our proteomic strategy not only involves identification of target proteins
following both in vivo and in vitro exposures (after target organ dosimetry), but also
subsequent use of in vitro models to examine the concentration and time-dependent
nature of diminutions. In this manner proteins, once identified, can be evaluated from a
"target 1 > target 2" perspective, thereby facilitating a working hypothesis on mode of
action. Importantly, comparable proteomic and genomic efforts are underway within RTD
under the DW-Development theme. In this manner, target molecules (genes and/or
proteins) in one system can be applied to or examined in another system (e.g SP22 is
being evaluated in embryos via collaboration in RTD as well as collaboration in Varese,
Italy). This exchange of target molecules will result in an efficient use of resources,
comprehensive assessments of mode of action(s) across target tissues, and verification
of biological plausibility across tissues and species.
C. GOAL 8 - Human Health Risk Assessment
Data on biological mechanisms in risk assessment (relevant to Posters # 3 and 6)
The development and use of novel biomarkers is a critical component of improved
risk assessment as ideally these biomarkers lend themselves to use in human studies and
human risk extrapolations. To this end, we have invested considerable research effort
over the past decade to identify a novel protein which is predictive of fertility (see Theme-
Specific Aims 3.1-3.5). This research was cited 2 years ago by EPA's Assistant
Administrator as one of the three cutting edge scientific areas in the Agency. Since this
time the EPA has received a patent on SP22, EPA's first patent in the biotechnology
arena. Levels of SP22 in detergent extracts of sperm have now been correlated with
fertility in rats exposed to both dibromoacetic acid and bromocloroacetic acid. In fact, the
diminutions in SP22 and fertility have set the LOAEL for bromochloroacetic acid. SP22
is being/will be evaluated in our extramural efforts (see Theme- Specific Aims 5.1 and 5.2).
It is already quite clear that at the lowest dose which compromised fertility in the rabbit,
SP22 as well as other proteins are diminished in rabbit sperm extracts. Collectively, these
data establish a link (and biological plausibility) between low dose effects in rats and
rabbits.
While completely new to reproductive biology it is clear that SP22 plays a pivotal
role in fertility and efforts will continue to elucidate the molecular role of this protein.
SP22 is now being considered for incorporation as a seminal endpoint in new
epidemiology studies based on the fact that: 1) it is localized over the equatorial segment
of sperm from all species studied (i.e. rat, hamster, rabbit, bull, and human), and 2)
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antibodies to SP22 have now been shown to inhibit fertilization in three species (rat,
hamster, and human). To incorporate SP22 into the epidemiology studies mentioned
above, we will develop an enzyme-linked immunosorbant assay. Based on recent western
blot data obtained from human patients presenting at the fertility clinic, SP22 levels are
indeed diminished in idiopathic infertile men. Whether SP22 levels on human sperm and
(or) exposure to disinfection by-products of drinking water are linked to the putative decline
in semen quality in men remains to be determined.
While SP22 research provides a classical example of the value of proteomics, a
considerable effort is underway to realize similar potential in the genomics arena. It is
likely that gene expression analysis will aid in the early diagnosis of adverse
environmental exposures, as changes in gene expression typically precede clinical
manifestations of toxicity. Microarrays are currently being developed for human, rat and
mouse. These arrays contain many homologs between the three species and will
therefore be useful for cross-species comparisons. In addition to looking for susceptibility
genes (genomic biomarkers) of male infertility, studies will also be carried out on sperm
RNA of human male subjects who have suffered occupational exposures to environmental
chemicals to determine if gene expression profiles in the subjects' sperm can be related
to exposure.
Endocrine disruptors: effects (relevant to Posters # 7 and 8)
While disinfection by-products of drinking water have not been formally classified
as endocrine disruptors, the emerging data strongly suggest certain by-products at least
be considered as such. Multiple studies now indicate that trihalomethanes and haloacetic
acids disrupt the endocrine axis at different points (see Theme- Specific Aim 1.2). First,
trihalomethane (bromodichloromethane) exposure in Fischer 344 rats results in full litter
resorption when exposure occurs during the LH-dependent period of pregnancy
maintenance. We now have data demonstrating that serum LH is decreased during this
period accounting for reduced progesterone maintenance of pregnancy. While interesting
and definitely endocrine disruptive, it is doubtful that these findings are relevant to
spontaneous abortions in women. Another possibility that remains to be explored is
whether exposure to bromodichloromethane also results in reduced LH responsivity. If the
ovary is less able to produce progesterone in response to an LH signal, the human
placenta might be less able to produce progesterone in response to hCG and human
relevance would be indicated. Ideally, we will explore the nature of these effects in a
species in which pregnancy maintenance is comparable to that in humans, i.e. dependent
on placental progesterone production. In this manner, we will establish biological
plausibility by common mode of action.
The second demonstration of endocrine disruption by disinfection by-products was
provided by recent data (see Theme- Specific Aim 1.3) demonstrating that the preovulatory
follicles from the rat produce less progesterone when challenged with hCG in the presence
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of toxicologically relevant concentrations of dibromoacetic acid (i.e. concentrations
comparable to those found in serum following in vivo exposure). At lower exposures a
reduction in the primordial follicle population has been observed in rabbits (see Theme-
Specific Aim 5.1) exposed throughout reproductive development. More recently, it has
been shown that steroidogenic inhibition occurs prior to P450 side chain cleavage enzyme;
Steroid Acute Regulatory Protein (StAR) and the plasma membrane signaling cascade are
implicated. How these results relate to the observations of persistent estrous in cycling
adult females exposed to dibromoacetic acid, and delayed puberty (i.e. vaginal opening)
in young females exposed to the same haloacid remains unknown. However, that the
disubstituted haloacids compromise spermatogenesis and alter mating behavior and
fertility prompts similar studies in the male as these processes are known to be androgen-
dependent. Once the mode of endocrine disruption is established, it will be critical to
determine whether observed proteomic / genomic alterations are related.
III. Future Directions of the Theme
In the future, we hope to formally bridge exposures (i.e. chemical(s), route, species)
and issues (i.e. mode of action, biological plausibility driven by quantitative risk across
species) into an even more comprehensive theme research plan. In this regard, 1) we
have already drafted plans for inclusion of more sophisticated reproductive endpoints (e.g.
serum testosterone in boys, increased assessment of time to pregnancy, serum hCG,
SP22 on sperm, early menopause) in longitudinal cohorts studies; 2) we intend to
emphasize the value of interspecies extrapolation to establish biological plausibility and
mode of action by proposing additional studies in guinea pigs, rabbits, and dogs; 3) we
intend to develop high density gene microarrays for rat, mouse, and human, and to
improve linkage with our rat proteomic effort; 4) we have designed relevant mixture
studies to examine the potential for "effective" DBPs to behave in an additive or synergistic
fashion when exposure spans reproductive development; and 5) we need to consider
aggregate exposure designs whereby DBP exposure occurs via dermal and inhalation
routes in addition to drinking water. While the need for continued high quality data on the
reproductive consequences of exposure to existing DBPs is quite clear, it seems obvious
that emerging DBPs (those of increasing occurrence using new water treatment designs),
future CCLs (including pesticides/ herbicides/ pharmaceutical by-products), sensitive
subpopulations including the elderly, and consideration of disinfection by-products as
endocrine disruptors represent important new cross-cutting issues for drinking water
research.
IV. Intramural Support (FTEs)
Total of 12 FTEs. Participating staff: Juan Suarez, Naomi Roberts, Mike Narotsky, Debora
Best, Jerome Goldman, Ashley Murr, Lillian Strader, Sally Perreault-Darney, Susan Jeffay,
Rebecca Morris, John Rockett, David Dix, Jeff Welch, Robert Zucker, Clint Kawanishi, and
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Leonard Mole.
V. Extramural Collaborations
Poster # 4 (Theme- Specific Aim 1.5) includes collaboration with Drs. Andrew Olshan and
David Savitz at UNC; Dr. Susan Rothmann, Fertility Solutions, Inc.; Dr. Wendie Robbins,
UCLA; Dr. Sherry Selevan, US EPA/ORD/NCEA; and Dr. Pauline Mendola,
USEPA/ORD/NHEERL/HSD. Specific Aim 1.6 represents an upcoming collaboration with
other NHEERL divisions.
Poster # 3 (Theme- Specific aim 3.5) involves a collaboration with Dr. Ken Roberts and
Dr. David Hamilton at the University of Minnesota as well as collaborations with Dr. Steven
Sylvester at the University of Washington at Vancouver, Dr. Harry Moore at the University
of Sheffield in England, Dr. Gustavo Doncel at the Eastern Virginia Medical School, Dr.
Richard Oko at Queens University in Ontario, and Dr. Giovanni Bernardini at the University
in Varese, Italy.
Poster # 2 (Theme- Specific Aim 5.1) represents an existing collaboration with Dr. Rao
Veeramachaneni and Dr. Heywood Sawyer at Colorado State University, and Specific Aim
5.2 represents an existing collaboration with Rochelle Tyl at Research Triangle Institute.
VI. Productivity (since 1997)
A. Manuscripts and Relevant Review Articles
127. Klinefelter, G.R., Welch, J.E., Perreault, S.D. Moore, H.D., Zucker, R.M., Suarez,
J.D., Roberts, N.L., Bobseine, K., and Jeffay, S. Localization of the sperm protein
SP22 and inhibition of fertility in vivo and in vitro. J. Androl., 2001 (In press).
97. Holmes M, Suarez JD, Roberts NL, Mole L, Murr AS, and Klinefelter GR.
Dibromoacetic acid, a prevalent disinfection by-product of drinking water
disinfection, compromises the synthesis of specific seminiferous tubule proteins
following both in vivo and in vitro exposures, J. Androl. 22:878-890, 2001.
126. Klinefelter, G.R., Hunter, E.S., and Narotsky, M. Reproductive and Developmental
Toxicity Associated with Disinfection By-Products of Drinking Water, In: Microbial
Pathogens and Disinfection By-Products of Drinking Water, ILSI Press, 309-333,
2001.
202. Royster, M.O., Lobdell, D. T., Mendola, P., Perreault, S.D., Selevan, S.A.,
Rothmann, S.G., and Robbins, W.A. (2000). Evaluation of a container for collection
and shipment of semen with potential uses in population-based, clinical and
occupational settings. J. Androl., 21:478-484
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18. Balchak, S.K., Hedge, J.M., Murr, A.S., Mole, M.L., Goldman, J.M. (2000).
Influence of the drinking water disinfection by-product dibromoacetic acid on rat
estrous cyclicity and ovarian follicular steroid release in vitro. Reproductive
Toxicology 14: 533-539.
79. Goldman, J.M., Murr, A.S. (Submitted, Toxicology). Alterations in ovarian follicular
progesterone secretion by the drinking water disinfection by-product dibromoacetic
acid: Examination of the potential site(s) of impact along the steroidogenic pathway.
125. Klinefelter, G.R. and Welch, J.E The Saga of a Male Fertility Protein (SP22).
Annual Review of Biomedical Sciences. 1:145-184, 1999.
228. Welch, J.E., Barbee, R.R., Roberts, N.L., Suarez, J.D., and Klinefelter G.R. SP22:
a novel fertility protein from a highly conserved gene family. J.Andrology 19:395-
393, 1998.
121. Klinefelter, G.R., Laskey, J.W., Ferrell, J., Suarez, J.D., Roberts, N.L. Discriminant
analysis indicates a single sperm protein (sp22) is predictive of fertility following
toxicant exposure. J. Andrology, 18:139-150,1997.
143. Under, R.E., Klinefelter, G.R., Strader, L.F., Veeramachaneni, D.N.R., Roberts,
N.L., Suarez, J.D. Histopathologic changes in the testis of rats exposed to
dibromoacetic acid. Reprod. Tox. 11:47-56,1997.
144. Under, R.E., Klinefelter, G.R., Strader, L.F., Suarez, J.D., and Roberts, N.L.
Spermatotoxicity of dichloroacetic acid. Reprod. Tox., 11:681-688, 1997.
190. Rockett JC, JC Luft, JB Garges, SA Krawetz, MR Hughes, DJ Dix (2001a).
Development of a 950-gene DNA array for examining gene expression patterns in
mouse testis. Genome Biol 2(4):research0014.1-0014.9.
http://genomebiology.eom/2001/2/4/research/0014/
188. Rockett JC, FL Mapp, JB Garges, JC Luft, C Mori, DJ Dix (2001 b). The effects of
hyperthermia on spermatogenesis, apoptosis, gene expression and fertility in adult
male mice. Biol Reprod 65, 229-239.
163. Ostermeier GC, DJ Dix, D Miller, P Khatri, SA Krawetz. What constitutes the normal
fertile male? New England Journal of Medicine (Submitted).
187. Rockett JC. Gene Expression Networks, in Encyclopaedia of the Human Genome,
Nature Publishing Group {In Press).
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179. Reid R, Dix DJ, Miller D, Krawetz SA (2001). Recovering filter-based microarray
data for pathways analysis using a multipoint alignment strategy. Biotechniques,
30(4);762-6, 768.
186. Rockett JC, DJ Dix. (2000). DNA Arrays: Technology, options and toxicological
applications. Xenobiotica, 30(2); 155-177.
184. Rockett, JC, DJ Dix (1999). Application of DNA arrays toToxicology. Environmental
Health Perspectives, 107(8):681-685.
Klinefelter GR. Method for evaluating and affecting male fertility. 2001; U.S. Patent
#6,197,940.
DNR Veeramachaneni (2000) Deteriorating trends in male reproduction: idiopathic or
environmental ? Animal Reproduction Science 60-61:121-130.
Activities Relevant to Stakeholders:
Klinefelter-
Organizing chair and speaker in workshop session entitled "Reproductive Risk Associated
with Exposure to Disinfection By-Products of Drinking Water", Society of Toxicology
(2002).
Served as Co-Chair in ILSI Workshop on Mechanistic Research of Disinfection Byproducts
(1998).
Presentation on Reproductive Risk Due to DBP Exposures at Health Effect Stakeholders
Meeting for the Stage 2 DBPR in Washington, DC (1999).
RTD representative on Steering Committee for NHEERL's Drinking Water Implementation
Plans (2000-present).
Presented an overview entitled "Reproductive and Developmental Toxicity Associated
with Disinfection By-Products of Drinking Water" at the 2nd International Conference on
The Safety of Water Disinfection: Balancing Chemical and Microbial Risks, in Miami, FL
(1999).
Served on select M-DBP Research Needs Workgroup sponsored by AWWARF and the
M/DBP council (2001).
Dix/Rockett-
Frontiers in Reproduction Symposium 2001, Cambridge, MA. "Expression Profiling to
Identify Genes Critical for Male Fertility."
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Poster 1. Testing Paradigm to Study Mode of Action Underlying Haloacid-lnduced
Testicular Toxicity. GR Klinefelter, M Holmes, JD Suarez, NL Roberts, ML Mole, and A
Murr. U.S. EPA, NHEERL, Reproductive Toxicology Division, RTP, NC.
To better determine reproductive health risk to humans exposed to low levels of
disinfection by-products, the toxicologist seeks to provide data that might elucidate a mode
of action for the most sensitive adverse outcomes observed in experimental animals. If
mode of action is determined, it is possible to determine biological feasibility in humans.
Dibromoacetic acid (DBA) is a byproduct of drinking water disinfection that alters
spermatogenesis in adult male rats. To identify a mechanism by which DBA alters
spermatogenesis, seminiferous tubules representing specific groups of spermatogenic
stages were exposed either in vivo or in vitro and structural and functional consequences
were evaluated.
Historically, seminiferous tubules from adult male rats have not been successfully
maintained in culture. Thus, a significant amount of preliminary work was done to optimize
the structural and functional maintenance of isolated tubules during overnight culture. For
this, isolated seminiferous tubules representing stages l-V, VI-VIII, and IX-XIV were
cultured overnight under conditions of reduced oxygen and temperature, i.e. 5% 02 and
26°C. For in vivo exposures, seminiferous tubules were recovered from animals which
received 250 mg/kg DBA via gavage for 5 days as it was previously established that initial
histological alterations occur after 5 days with this exposure. For in vitro exposures, 180
and 600/jM concentrations were tested; these concentrations bracketed the concentration
of DBA observed in the interstitial fluid of the testis following in vivo exposure. Protein
synthesis was evaluated by 35S-methionine labeling overnight and quantitative analysis
of radiolabeled proteins in mini, two dimensional (2D) SDS-PAGE gels. Radioinert
cultures were processed for light and electron microscopy. Morphological evaluation
indicated that all spermatogenic stages of the seminiferous tubules from control animals
were well maintained during the isolation and culture period. While no treatment-related
lesions were observed following the in vivo exposure, histological alterations were
observed at the lowest in vitro exposure. There was a significant diminution (P<0.05) in
the synthesis of four specific cytosolic proteins following both in vivo and in vitro
exposures. Diminution in these proteins was restricted to stages l-V and IX-XIV of
spermatogenesis suggesting that proteins involved in the early stages of spermiogenesis
are uniquely sensitive to DBA exposure. That both histology and protein synthesis were
affected in vitro by relevant target organ exposures indicates that DBA is capable of
altering spermatogenesis directly.
Genomic analysis of genes altered by BCA in vivo (see Poster # 5) suggest that
genes (and proteins) involved in cell adhesion and signaling may be compromised by
haloacid exposure. A major emphasis of our future work lies in the identification of the
proteins found to be compromised by DBA exposure. We believe this will allow us to
formulate a working hypothesis regarding the mode of action of haloacids in the testis, and
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perhaps other target tissues as well.
Pertinent References: 97, 126, 143, 144
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Poster 2. Testing Paradigms to Determine Consequences of Haloacid Exposure
During Reproductive Development. Gr Klinefelter1, R Veeramachaneni2, H Sawyer2,
LF Strader1, JD Suarez1, NL Roberts1, K Bodensteiner2, and C Moeller2. 1U.S. EPA,
NHEERL, RTD, RTP, NC. and 2Animal Reproduction and Biotechnology Lab, Colorado
State University, Fort Collins, CO.
To better assess reproductive health risk to humans exposed to low levels of
disinfection by-products, the toxicologist seeks to provide data to determine whether
certain periods of reproductive development are uniquely susceptible to insult, and
whether low effect levels can be modulated by a critical window of exposure or duration
of exposure. Once these data are available, a more informed decision can be made on
whether humans exposed during critical developmental periods or over long periods are
likely to be affected by low exposures. While numerous studies have now demonstrated
that haloacids alter reproduction in the adult male and female rat, studies were lacking to
determine the consequences of exposure to haloacids throughout reproductive
development. For this, we initiated separate studies involving rats and rabbits in which
dibromoacetic acid (DBA) was administered via drinking water from gestation day 15 until
adulthood. Subsequent to a dose-finding study in rats using 400,600, and 800 ppm DBA
which resulted in profound delays in vaginal opening and preputial separation, we
selected multiple exposure paradigms using 4, 40, and 400 ppm DBA. In study I,
exposure continued from GD15 through PND 98 and terminal assessments were made.
In study II, animals were exposed only from GD15 through weaning (PND21) with terminal
assessments made on PND98. In study III, animals were exposed from GD15 through
PND21 with terminal assessments on PND56 (i.e. puberty). In study IV, animals were
exposed from GD15 through PND56 and terminal assessments were made. In summary,
body weight independent delays in preputial separation and vaginal opening were
observed; delays were significant only in animals that were exposed continuously, i.e. from
GD21-acquisition. In contrast to these puberty indices, fertility in adult males was
decreased significantly at 400 ppm in animals that were exposed continuously as well as
animals in which exposure ceased at weaning. Thus, while pubertal alterations seem to
require continuous exposure, compromised sperm quality (i.e. fertility) can persist long
after exposure.
When rabbits were exposed to 1, 5, and 50 ppm (mg/kg) DBA from GD15 to
adulthood, significant alterations were observed in mating ability, fertility, and ovarian
histology. In general, males in the DBA groups showed reduced sexual interest and
ejaculation success. Males in the 50 mg group that did complete ejaculation took more
attempts (1.5 vs. 1 in control) and longer time (30 vs. 15 sec) to achieve ejaculation; two
had no sperm in their ejaculates at least once. The average conception rates for males
in the 0,1,5, and 50 mg group were 85,55,65 and 55%, respectively, and the conception
rate of each treatment group was significantly lower than that of the control group. The
male rats exposed similarly had significantly reduced fertility at 400 ppm (>50 mg/kg).
That sexual function and seminal quality (reflected by lowered fertility even at 1 mg/kg)
were more sensitive in rabbits compared to rats, may reflect longer exposure during
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reproductive development in the rabbit. Thus, the rabbit may be a more relevant model for
human reproductive health risk assessment as human reproductive development spans
many years. Finally, female rabbits in the DBA groups had reduced populations of
primordial follicles; numbers were significantly lower in both the 5 and 50 mg/kg groups.
In a parallel study of the effects of DBA in mice, Colorado State investigators found no
changes in the follicle populations. Again, this suggests that the rabbit is uniquely
sensitive, perhaps due to its longer period of reproductive development. Given the
possible implications these rabbit data regarding early reproductive senescence in
women, additional studies on the effects of haloacids in rabbits, or other species with a
longer period of reproductive development seem warranted.
Pertinent Reference:
Veeramachaneni, DNR. Deteriorating trends in male reproduction: idiopathic or
environmental? Animal Reproduction Science 60-61:121-130, 2000.
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Poster 3. Identification and Validation of Biomarkers of Haloacid-lnduced
Reproductive Health Effects. GR Klinefelter1, JE Welch1, S. Perreault-Darney1, RM
Zucker1, K Roberts2, D Hamilton2, H Moore3, G Doncel4, JD Suarez1, KL Bobseine1, RR
Barbee1, NL Roberts1, and SC Jeffay1. 1U.S. EPA, NHEERL, Reproductive Toxicology
Division, RTP, NC, departments of Urology and Cell Biology, University of Minnesota,
department of Molecular Biology and Biotechnology, University of Sheffield, England,
department of Obstetrics and Gynecology, Eastern Virginia Medical School.
While the toxicologist is continuously striving to establish biological feasibility for
effects observed in animal studies, there is an equal need to identify and validate
biomarkers for the biologically feasible effects. Such biomarkers have obvious utility not
only in streamlined, low dose animal tests, but also in epidemiological studies seeking to
establish or refute risk at environmental exposure levels. We previously found that levels
of a novel sperm protein (SP22) were highly correlated with reductions in fertility in
animals exposed to epididymal toxicants. More recently, we established that the same
association exists in animals exposed to testicular toxicants such as the haloacids (i.e.
bromochloracetic acid, dibromoacetic acid). Indeed, data for 131 animals exposed to
either epididymal toxicants or testicular toxicants yield a correlation of 0.83 for the
association between SP22 levels in detergent extracts of cauda epididymal sperm and the
fertility of these sperm as evaluated by in utero insemination. While these data suggest
that SP22 might be useful as a biomarker in both toxicologic and epidemiologic settings,
additional data were required to demonstrate that SP22 is expressed on sperm across
species, and that SP22 plays a pivotal role in the fertilization process.
For this, we raised polyclonal antiserum in sheep immunized with full-length
recombinant SP22 (rSP22) and monoclonal antibody (ascites) using hybridoma clones
from mice immunized with rSP22 and boosted with native SP22. Immunoblots indicated
that rSP22 Ig and ascites recognized denatured and native SP22, respectively.
Cytoplasm of pachytene spermatocytes and round spermatids immunostained with both
rSP22 Ig and ascites. In stage VIII, staining was concentrated along the spermatid head.
Isolated rete testis sperm revealed discrete staining at the base of the head; staining
migrated to the equatorial segment.of the head as sperm moved to the cauda
epididymidis. Ascites, rSP22 Ig, and rSP22 Fab each inhibited fertilization in vivo and
in vitro. For example, rSP22 Ig (1:50) added to cauda epididymal sperm 5 minutes prior
to in utero insemination decreased fertility from 74% to 34%, and rSP22 Ig (1:10) added
to sperm incubated overnight with eggs decreased fertilization from 81% to 39%. These
antibodies were capable of recognizing SP22 over the equatorial segment of all species
examined (rat, hamster, rabbit, bull, human). In vitro fertilization studies comparing zona-
free vs zona-intact hamster eggs revealed that the rSP22 Ig decreased fertilization of
zona-intact eggs in a concentration-dependent manner. That Ig only decreased
fertilization of zona-free eggs at the highest concentration tested suggests that SP22 plays
a primary role in fertilization at the level of the zona. Moreover, data using a human hemi-
zona assay indicates that Fab fragments of this antibody successfully inhibit binding of
human sperm to the zona. Finally, recent work suggests that SP22 expression (evaluated
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by Western blotting) is compromised in men with known or suspected infertility.
Thus, the collective data suggest that SP22 has promise as a useful (i.e. predictive)
biomarker of fertility in many applications. Linear epitope mapping of the 189 amino acid
SP22 sequence reveals at least three distinct sequences recognized by rSP22 Ig and one
sequence recognized by ascites. Current and future work is devoted to elucidating the role
of SP22 in sperm-egg interaction and expanding the data base on the relationship
between SP22 expression in humans and fertility (see Poster # ). To address molecular
function we will conduct additional in vitro fertilization studies to determine which, if any,
of the linear epitopes play a role in fertilization, determine 3-D structure of purified native
SP22 using x-ray crystallography, and identify protein-protein interactions using
immunoprecipitation, cross-linking, and yeast two-hybrid approaches.
Pertinent References: 127, 125, 228, 121
Klinefelter GR. Method for evaluating and affecting male fertility. 2001; U.S. Patent
#6,197,940.
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Poster 4. Epidemiologic Evaluation of DBP Exposures on Semen Quality. SD
Perreault, RA Morris, SC Jeffay, LF Strader, GE Klinefelter. U.S. EPA, NHEERL, RTD,
RTP, NC,(in collaboration with S Rothmann, Fertility Solutions, Inc.; W Robbins, UCLA;
S Selevan, US EPA/ORD/NCEA; P Mendola, USEPA/ORD/NHEERL/HSD, and A Olshan,
UNC).
Under GPRA Goal 2, NHEERL is charged with evaluating the health effects
associated with exposure to DBPs. The Office of Drinking Water (ODW), American Water
Works Association Research Foundation (AWWARF), and International Life Sciences
Institute (ILSI) have repeatedly called for epidemiology studies to evaluate reproductive
health effects of DBPs in men, since rodent toxicology studies have identified DBPs,
especially the HAAs, as male reproductive toxicants. Preliminary research conducted
during the past three years was undertaken to develop improved field methods for
surveying semen quality in populations of men exposed to varying levels of DBPs in their
municipal drinking water. Our first step was to develop a home-semen collection kit
whereby men could collect semen samples at their convenience in the privacy of their
homes, and then ship the samples to a common lab by overnight courier. In collaboration
with Dr. Susan Rothmann (Fertility Solutions, Inc.), such a system was designed and
tested to define the limits of sample preservation with respect to an array of semen
outcomes (including tests for chromosome and DNA damage). It was then field-tested in
collaboration with Dr. Pauline Mendola, HSD, and found to be satisfactory.
We now plan to use this kit in an epidemiology study, funded to begin in FY 2002
under a three-year cooperative agreement with UNC-Chapel Hill Department of
Epidemiology, Dr. Andrew Olshan, P.I. This study will be dove-tailed with a recently
funded (AWWARF) pregnancy outcome study in which 950 pregnant women will be
enrolled in each of three municipalities that differ with respect to DBPs in drinking water.
We will recruit the partners of a subset of these women, and survey their semen quality.
The municipalities were selected to provide data for men exposed to low levels of DBPs
(Nashville, TN), medium to high levels of chlorinated DBPs (Raleigh, NC), and high levels
of brominated DBPs (Galveston County, TX). Since brominated HAAs are more potent
male reproductive toxicants in animal studies, and we need to control for potential
geographic variability, men in Galveston will be over-sampled and stratified based on their
consumption of drinking water (exposure). During phase 1, which will begin in Raleigh this
fall, we will also develop and refine a test for measuring the sperm protein biomarker SP22
in human semen samples. The results of this study should demonstrate whether the
current MCLs are protecting male reproductive health, and if not, contribute to decisions
regarding changes in Maximum Contaminent Levels (MCL's) for DBPs in future ODW rule
makings.
Pertinent References: 202
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Poster 5. Genomic Analysis of Testicular Toxicity. DJ Dix, JC Rockett, JE Schmid,
LF Strader, DB Tully. U.S. EPA, NHEERL, RTD, RTP, NC.
In order to improve human health risk assessments, it will be necessary to
identify susceptible subpopulations (genetic or age dependent susceptibility),
characterize cumulative and aggregate risks, and harmonize approaches to non-
cancer and cancer risk assessments. We have developed a number of genomic tools
and approaches to generate data useful in addressing all three of these issues. First,
we have developed DNA microarrays and used them to identify toxicant mode-of-action
as indicated by effects on gene expression relevant to testis histopathology and
fertility. Second, we have used gene knockout (KO) mice as a model to determine
whether genetic susceptibility may influence toxic response.
After a series of experiments utilizing commercial human and mouse arrays
defined the testis transcriptome and identified gene expression changes linked to
fertility (Rockett et al., 2001b), we selected 950 mouse genes and created a custom
cDNA microarray focused on testicular gene expression (Rockett et al., 2001a). This
array has been used to characterize testicular gene expression following exposure of
mice to the water disinfectant by-product bromochloroacetic acid in a regimen that
induces subfertility at the higher doses (Luft et al., 2001c). We have also utilized
commercial arrays and our custom mouse testis array for analyzing the testicular
toxicity of cadmium (Liu et al., 2001). This type of toxicogenomic data has already
provided mechanistic data useful for identifying toxicant mode-of-action. In future
studies we plan to identify common changes in gene expression between chemicals,
tissues, strains and species in order to characterize cumulative and aggregate risks
and assist harmonized non-cancer and cancer risk assessments.
Two different lines of gene KO mice have been used to examine the significance
of HSP expression in mouse testis. First were the heat shock transcription factor 1
(Hsf1) KO mice, which have lost the ability for environmental stressors to induce the
expression of HSPs. In response to heat shock, there is diminished spermatocyte
apoptosis and limited recovery from the normally transient subfertility in Hsf1 KO mice
(Luft et al., 2001a). The linkage between disruption of the G2/M cell cycle transition
and cell death was further characterized in vitro using cell lines derived from the Hsf1
KO mice (Luft et al., 2001b). To pinpoint the specific function of the 70 kDa HSPs, we
created gene KO mice with deletions in the hsp70-1 and hsp70-3 genes. These mice
are currently being utilized in a number of toxicological studies at EPA and with
academic collaborators. Continuing a long-term research interest (Mori et al., 1999),
the functional significance of testis-specific Hsp70A2 and the association with fertility
status in humans has been reported (Huszar et al., 2001). All of these HSP projects
are focused on defining the role of HSPs in male fertility (Luft and Dix, 1999) and how
HSPs protect against environmental challenges to reproductive health. This work
might validate the use of HSPs as relevant biomarkers of exposure to toxicants, and
could also identify susceptible subpopulations.
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Pertinent References: 105, 146,147, 148, 156,189, 188
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Poster 6. Monitoring Human Reproductive Health And Development Through Gene
Expression Profiling. DJ Dix, JC Rockett, JE Schmid, DB Tully. U.S. EPA, NHEERL,
RTD, RTP, NC.
Through Goal 8.2.1 of NHEERL's Research Strategy, EPA aims to improve the
scientific foundation of human health risk assessment through the development of
methods for characterizing susceptible subpopulations and through examining the
potential utility of emerging technologies in harmonizing risk assessment and determining
health status Gene expression analysis may hold the key to early diagnosis of adverse
environmental exposures, as changes in gene expression always, with the possible
exception of necrosis, precede clinical manifestation of toxic effects. Inter-species
differences in response to toxicant exposure are a key area of investigation, as they may
provide evidence for conserved mechanisms or modes of toxicant action. In this respect,
we have developed a number of DNA arrays for use in examining the effect of toxicant
exposures on reproductive tissues.
Through a Cooperative Research And Development Agreement (CRADA) with a
commercial partner, we developed toxicology arrays for human, rat and mouse. These
arrays contain many homologs between the three species and are therefore useful for
cross-species comparisons. One way in which our allocation will be used is to look for
susceptibility genes (biomarkers) of male infertility by comparing the effects of male
toxicants on juvenile mouse exposure models. Studies will also be carried out on sperm
RNA of human male subjects who have suffered occupational exposures to pesticides and
heavy metals, to see if gene expression profiles in the subjects' sperm can be related to
adverse reproductive outcomes.
Furthermore, we are in the process of producing rat and mouse oligo-based DNA
arrays consisting of many thousands of genes each. Use of these arrays should provide
insight into both the mechanisms of action and the variability which exists amongst strains
and species, as well as providing leads on the basis of inherited and environmentally-
induced infertility.
In this manner, we are also currently searching for genetic markers of infertility
using commercially available arrays to examine gene expression in the testis of infertile
humans and mice with different histologies. By looking for similarities and differences in
gene expression within and amongst these species, we aim to identify key genes involved
in infertility and derive plausible hypotheses on the molecular mechanisms underlying the
infertile conditions.
We are exploring the feasibility of using gene expression changes in easily
accessible "surrogate" tissues to see if they mimic gene expression changes taking place
in target reproductive tissues. Our pilot study comparing gene expression profile changes
in the blood and uterus of female ovariectomized rats treated with estradiol shows that
there are many similar changes in gene expression. This suggests that this approach
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could be developed into a tool for biomonitoring if the gene changes can be linked to
developmental abnormalities or a toxic endpoint. The use of semen as a surrogate tissue
to characterize infertility problems in adults is also being examined. We have begun
characterization and comparison of the transcriptomes of normal adult human testis and
sperm. Such data may be helpful in identifying the nature of certain infertility disorders
using semen samples alone.
Children are viewed as being at higher risk from the effects of environmental
exposures, although little is actually known about the connection between childhood
exposures and adult reproductive health. As a model for examining the effects of the
stress genes that are typically regulated by toxicants, we used a male juvenile heat-shock
male mouse to look for long term affects of acute heat exposure. Our data indicate that
there may be long term affects on reproductive outcomes, and thus suggests that acute
exposures to other types of toxicants during juvenile development might produce lasting
adverse effects on reproductive function.
Pertinent References: 163, 179, 187, 184
Rockett JC and DJ Dix. Gene Expression Networks, in Encyclopaedia of the Human
Genome, Nature Publishing Group {In Press).
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Poster 7. Haloacetic Acids as Endocrine Disrupting Chemicals? An Investigation
of the Impact of these Drinking Water Disinfection By-products on Ovarian
Steroidogenesis. JM Goldman1AE Murr1 and S Balchak2. 1US EPA, NHEERL, RTD,
RTP, NC and 2UNC Curriculum in Toxicology, Chapel Hill, NC.
Previous studies have indicated that various by-products of drinking water disinfection
(DBPs) are able to adversely affect a number of reproductive endpoints and suggest
certain of these compounds may act as endocrine-disrupting chemicals (EDCs). For this
large group of compounds, the focus thus far has been on the haloacetic acids and
trihalomethanes. At increasing concentrations, a number of haloacetic acids have been
reported to cause abnormalites in sperm production and morphology, with declines in
testosterone present at higher dosages. Thus far, little data have been available
concerning effects in the female, particularly reproductive functions in the non-pregnant
female. Consequently, we have chosen to explore such potential alterations in response
to two model haloacetic acids- dibromoacetic (DBA) and bromochloroactic (BCA) acids.
This work was consistent with a laboratory-based progression within the risk assessment
framework: (1) hazard identification, (2) establishment of a dose-related effect, and (3)
identification of one (or more) mode(s) of action underlying the adverse effect.
Initial experiments focused on establishing whether or not the above two
haloacetic acids could perturb estrous cyclicity in the female rat, since this is a non-
invasive approach to evaluate general reproductive activity in animals that ovulate on a
regular, recurring basis. A 2 wk oral exposure was selected for use based upon earler
studies reporting alterations in the male rat. Daily examinations of vaginal epithelial
cells showed a dose-related disruption in cyclicity for both DBA (0-270 mg/kg) and BCA
(0-240 mg/kg). Because the cycle is under endocrine regulation, subsequent work
focused on a potential effect of these compounds on ovarian steroid production. Using
pre-ovulatory follicles maintained 24h in culture, it was observed that the 50 ng/ml DBA
in vitro (pH-adjusted) suppressed both the baseline and hCG-stimulated release of
progesterone (P4). Estradiol (E2) secretion was unaffected. This amount of DBA
represented the mean circulating concentration over 5h following oral exposure to a
dosage found to induce a reliable and persistent disruption in cyclicity. No effects on
P4 were seen following supplementation with pregnenolone (pregn), indicating that
follicular viability and the conversion of pregn to P4 were not affected.
Supplementation with 22(R)-hydroxycholesterol maintained P4 release, suggesting an
impact on steroidogenesis prior to the conversion of cholesterol to pregn by the P450
side chain cleavage enzyme and thereby implicating an alteration in intramitochondrial
cholesterol transport by steroidogenic acute regulatory protein.
Current work has focused on the added possibility that the effect on cyclicity
may be at least partially due to an alteration in E2 clearance and/or metabolism.
Groups of DBA-dosed ovariectomized females given estradiol implants had circulating
E2 concentrations at 72h post-implant that were up to 21/2-fold greater than controls,
depending on dose. An experiment is also being conducted to stimulate the hepatic
P450 enzyme-catalyzed hydroxylation of E2 to see if this can reverse the elevation and
address the possibility of a DBA-associated suicide inhibition. In summary, while these
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exposure levels are acknowledged to be above those concentrations typically present
in finished water, these data and those forthcoming should be of value to the Office of
Water in an overall consideration of the endocrine disruptive effects that these DBPs
may exert in vivo at lower dosages over longer periods of exposure.
Pertinent References: 18, 79
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Poster 8. Evaluation of Endocrine Disruptive Effects on Pregnancy Maintenance
Following DBP Exposure. MG Narotsky, DS Best, JM Goldman, AE Murr, R Pegram
(Experimental Toxicology Division), SR Bielmeier (UNC-Chapel Hill).
In a recent epidemiological study (Waller et al., 1998, Epidemiology 9:134-140),
consumption of high concentrations of trihalomethanes (THMs), particularly
bromodichloromethane (BDCM), were associated with an increased risk of spontaneous
abortions. We have shown that two of the brominated THMs cause pregnancy loss in
F344 rats. In view of the concerns raised by the epidemiological data, we are investigating
BDCM-induced pregnancy loss to determine its mode of action in rats. A further objective
is to address the question of whether this effect in rats is analogous to humans; more
specifically, would the mode of action explain the epidemiological findings? Our
experimental approach to this investigation involves several facets of experimentation: 1)
critical period, 2) hormonal profiles, 3) hormonal replacement to rescue the pregnancy, 4)
ex vivo evaluation of critical tissues, 5) metabolism and dosimetry, and 6) strain
comparisons.
In our early work, we demonstrated that the critical period for BDCM's effect is
during the luteinizing hormone (LH)-dependent period of pregnancy. During this period,
LH is required to maintain luteal secretion of progesterone, which, in turn, is required to
maintain pregnancy. In our evaluation of hormonal profiles, we demonstrated that
pregnancy loss is indeed associated with decreases in serum LH as well as progesterone.
These findings strongly support the hypothesis that BDCM's mode of action is mediated
by a disruption of LH secretion by the pituitary. Our attempts to rescue the BDCM-
exposed pregnancy with exogenous hormones provided further evidence of the pituitary
as a target site of action, both exogenous progesterone and hCG (an LH agonist) were
effective in rescuing exposed pregnancies. We also plan to attempt to rescue the
pregnancy with exogenous GnRH to help distinguish between the hypothalamus and the
pituitary as target sites of toxicity.
The data thus far strongly indicate that BDCM's effect in the rat is mediated by
altering pituitary LH secretion. This mode of action would not explain the epidemiological
findings as hCG (from the placenta), rather than LH, is required to maintain pregnancy at
the time of the spontaneous abortions. However, since hCG and LH bind to the same
luteal receptor, it is important to consider the hypothesis that BDCM may decrease luteal
responsiveness to LH/hCG, and thereby contribute to the disruption of pregnancy
maintenance. To test this hypothesis, we plan to evaluate rat corpora lutea ex vivo for
their ability to secrete progesterone following stimulation with hCG. Similar ex vivo
experiments are also planned to evaluate pituitary and hypothalamic responsiveness to
their respective stimuli.
In a collaborative effort with the Environmental Toxicology Division (ETD), we also
plan to assess BDCM's tissue distribution and the metabolic pathways that may contribute
to BDCM's effect on pregnancy. Finally, in contrast to the F344 strain, we have
25
image:
demonstrated that Sprague-Dawley rats are remarkably less sensitive to BDCM-induced
pregnancy loss. We plan to pursue this difference in strain sensitivity as a research tool
that may provide insights into susceptible sub-populations as well as BDCM's mode of
action.
Pertinent references: 23, 158
26
image:
(D _
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Cellular and Molecular Mechanisms of Abnormal Reproductive Development.
Earl Gray, Theme Leader
I. Introduction
As indicated in the figure below, our research integrates in vitro, ex vivo and in vivo
studies to provide mechanistic and dose response information for risk assessment on
Endocrine Disrupting Chemical (EDCs). Exposure to EDCs during development is of
concern because of the critical role that hormones play in differentiation and growth of the
reproductive system and other organ systems. EDC exposures have been shown to
cause population-and individual-level effects in some wildlife species and can induce
adverse developmental effects in humans and laboratory animals. Over the years, we
have examined the developmental effects of a variety of chemicals including
xenoestrogens, fetal germ cell toxicants, dioxin (2, 3, 7,8 TCDD), TCDD-like
polychlorinated biphenols (PCBS) and fungicides and toxic substances that inhibit
steroidogenesis. In the last few years our studies have focused primarily on
environmentally relevant chemicals that act as antiandrogens or androgens. These
chemicals include AR agonists and antagonists and chemicals that inhibit fetal
testosterone production. In addition to investigating the mechanisms of action of
environmentally relevant chemicals and providing the Agency with critical dose-response
information it is apparent that some of the assays that we have developed for these
purposes also are useful to the Agency as Tier 1 Screening (T1S) or Tier 2 Testing assays
in the Agency's Endocrine Disrupter Screening Program (EDSP).
In vitro
In vivo mechanistic
developmental
study of fetal
endocrinology
In vitro gene
with molecula
components
g5N& :ln VIVO
short-term dose
^response studying?
Integration of In vitro, ex vivo and in vivo studies to provide Mechanistic
and Dose Reponse information for Rish Assessment on EDCs
, In vivo
developmental
dose response
study
image:
Our research addresses specific problems in the ORD Endocrine Disruptors
Research Plan which indicated that research in this area was one of six high-priority
topics in the ORD Strategic Plan (USEPA, 1996a). The Agency recognized "the potential
scope of the problem, the possibility of serious effects on the health of populations, and
the persistence of some endocrine-disrupting agents in the environment". The ORD plan
identified 6 key questions considered important to the study of the effects of endocrine
disrupting chemicals. These question include:
1. What effects are occurring in exposed human and wildlife populations?
2. What are the chemical classes of interest, mechanism of action and their
potencies?
3. What are the dose-response characteristics in the low-dose region?
4. Do EPA testing guidelines adequately evaluate potential endocrine-mediated
effects?
5. What extrapolation tools are needed?
6. What are the effects of exposure to multiple EDCs and will a relative potency
approach be applicable?
Our research is addressing above questions 2, 3, 4 and 6.
More specifically, we are interested in:
What are the cellular and molecular mechanisms of EDC action? Currently studies are
focused on AR agonists and antagonists and inhibitors of steroid hormone
synthesis.
Are these mechanisms conserved among animals? We are examining or isolating steroid
hormone receptors from nonmammalian species.
What are the best in vitro and in vivo approaches to study these chemicals? We use both
in vitro, ex vivo and in vivo short-term and long-term assays to study EDCs.
How useful are these assays to the Agency for its Endocrine Screening and Testing
Program? We proposed the first "in vitro-in vivo screening battery" (Gray et al.,
1997), wrote the first in vitro and in vivo protocols (EDSTAC, 1998) and continue
to be involved in the EPA and Organization of Economic Cooperation and
Development (OECD) EDSP efforts.
What chemicals alter reproductive development and function? Over the years we have
studied EDCs and non-EDCs. Of the EDCs, we have studied estrogenic pesticides,
fungicides that inhibit steroidogenesis, antiandrogenic pesticides, phthalate esters,
and recently detected environmental androgens in samples of effluent from pulp
and paper mills and feed lots.
2
image:
What dosage levels or concentrations of the active metabolites are required to alter
endocrine action in vitro and in vivo? Where possible, we attempt to obtain data
on tissue levels of the active metabolites in vivo. We have done this to a limited
basis with vinclozolin, testosterone and a phthalate ester.
Do mixtures of these EDCs produce cumulative effects? We have initiated several
developmental reproductive toxicity studies with mixtures of antiandrogens. Effects
are cumulative, regardless of the mechanism of action.
How do the levels of these chemicals compare to those seen in some humans or wildlife?
Based in part upon our studies, it is apparent that some humans are exposed to
levels of DEHP, p,p' DDE and TCDD/PCBs (on a TEQ basis) that induce adverse
effects in the developing reproductive system of the rat. The Agency's new risk
assessment on vinclozolin, which cancelled or restricted several uses of this
fungicide, is based on our in utero and pubertal male rat dose response studies.
The ecological risk assessment on vinclozolin indicated that exposure levels for
some terrestrial vertebrates was of particular concern as some estimated
environmental concentrations could exceed NOELS by ten-fold.
This program also is responsive to issues identified in ORD Subobjectives on
Endocrine Disrupters and Human Health, under EPA's Science Goal 8. The focus of these
subobjectives is the development of a research program that will identify and evaluate
strategies to manage risks from exposures to endocrine disrupting chemicals capable of
inducing adverse reproductive and other effects in human and wildlife populations. In
addition, ORD Goal 3 is to facilitate the implementation of Food Quality Protection Act
(FQPA) 1996. In this regard, we are studying the effects of mixtures of EDCs, as FQPA
mandates that the Agency consider cumulative toxicity in it's risk assessments. These
projects also are covered in the NHEERL- EDC Research Implementation Plan (RIP),
Focus Areas 1 - 3.
II. Impact of Research: By Abstract.
Overview of Abstract # 20. Effects, mechanisms and modes of action of EDCs that alter
reproductive development: in vitro and in vivo approaches. Theme Aim #s
2, 3, 9, 10, 11, 12, 13.
EDC action can seriously alter reproductive development when administered during
critical, sensitive life stages. In utero, perinatal and pubertal exposures to EDCs can
produce severe effects on both male and female offspring, depending upon the mechanism
of action. Some effects are not obvious until late in life or are latent and not expressed
until after maturity or old age. The only protocols that can detect these types of effects are
long-term transgenerational or multigenerational studies because they are the only
protocols that 1) expose the animals during development and 2) evaluate the reproductive
3
image:
system in the offspring throughout life. Our studies typically dose the mother during
gestation and lactation and observe the development of male and female rat offspring
through maturity. The determination of the cellular and molecular mechanism of action of
an EDC is critical to understanding its potential to affect other species, including humans.
In order to elucidate mechanisms of action, we execute parallel in vitro or ex vivo studies
with EDCs or their active metabolites. In addition, we are attempting to obtain data on fetal
tissue levels of the active metabolites during the critical exposure period.
Over the last few years we have examined a variety of chemicals with different
mechanisms of endocrine action. We have studied estrogens (methoxychlor, zearalenone,
and chlordecone), inhibitors of steroidogenesis (ketoconazole, fenarimol (inhibits
aromatase)), germ cell toxicants (busulfan and benzidine-based diazo dyes), phthalate
esters (inhibit fetal testosterone production) and fungicides that are AR antagonists. We
are now beginning to study environmental chemicals with androgenic activity.
Over the last seven years, most of our work has been with environmental
antiandrogens. In 1994 we published the first study that demonstrated that a pesticide
altered male rat sexual differentiation by acting as an AR antagonist. This mechanism was
confirmed using in vitro AR binding and transcriptional activation assays, and by
examining AR-dependent ventral prostate gene expression (1997). In 1995 we were the
first to report in Nature that the p,p' DDE also was an AR antagonist. These studies have
now been extended to several other original observations on pesticides like procymidone
and linuron. Our work on linuron was the first to demonstrate that this herbicide induced
reproductive tract malformations, and points to weaknesses in former and current Agency
multigenerational test guidelines, as these malformations had been missed in several
published developmental toxicology and multigenerational tests. In addition to AR
antagonists, we, along with the laboratory of Dr P Foster at CUT, have been studying the
antiandrogenic effects of phthalate esters. We published the only report to date that
demonstrates that diethyl hexyl phthalate (DEHP) inhibits masculinization of the male rat
reproductive system by inhibiting fetal testosterone production. Currently, we are
attempting to increase our ability to examine the cellular and molecular events associated
with the effects of these chemicals on the fetal reproductive tract and testis. We also have
collected tissues from some of these studies for chemical residue or mRNA analyses.
For example, we have submitted fetal amniotic fluid and maternal urine samples from two
phthalate ester studies to the Center for Disease Control (CDC) for determination of MEHP
and MBP levels. We can then compare the dose to the fetus associated with adverse
developmental effects in the rat and we can compare MEHP and MBP levels to those
seen in humans.
In a risk assessment, the Agency considers many different exposure scenarios
besides intrauterine development. For example, EDC administration during puberty can
alter this developmental process. Estrogens accelerate pseudoprecocious puberty in the
female, while antiandrogens delay puberty in the male rat. In this regard, our pubertal
4
image:
studies on vinclozolin (1999) were the first demonstration that an antiandrogenic pesticide
could delay the onset of pubertal landmarks in the male rat. These data were used by
EPA to restrict uses of this chemical that were likely to result in childhood exposures to
vinclozolin at "levels of concern". We also found that linuron, di-n-butyl phthalate, diethyl
hexyl phthalate and p,p' DDE delay the onset of puberty in the male rat.
5
image:
Overview of Abstract # 21. Cumulative Risk of exposures to EDCs with similar and
dissimilar mechanisms and modes of action. Theme Aim # 5.
Humans and wildlife are never exposed to a single chemical at a time. For this
reason, the Agency is particularly interested in understanding how mixtures of chemicals
interact and if they produce cumulative effects. The FQPA of 1996 mandated that Agency
risk assessments on food use pesticides consider cumulative exposures to other
pesticides and toxic substances that act via the same mechanism of action. The Agency
also considers cumulative risk for chemicals that act by a similar modes of action, albeit
by a slightly different mechanism of action. Specifically, EPA has requested that we
develop data on cumulative effects of the pesticides vinclozolin, procymidone and
iprodione. We are determining if these chemicals interact in a predictable fashion in vivo
or in vitro? To address this issue we have initiated a program of research to determine
how 1) antiandrogens that act via identical mechanisms interact (two AR antagonists or
two phthalate esters), 2) antiandrogens act via different mechanisms of action (a phthalate
ester plus an AR antagonist) or 3) an androgen interacts with a pesticide with AR
antagonist activity. Initial studies have examined developmental reproductive toxicity in
the male rat, while other studies are examining cumulative effects on androgen-dependent
tissue weights, AR immunohistochemistry and mRNA expression levels. For chemicals
that act as AR antagonists, in vitro-mixtures also have been studied.
In general, we find that antiandrogenic EDCs produce cumulative effects when
coadministered at dosage levels that produce little effect by themselves. The responses
are "dose additive" but not always "effect additive" and resemble the example provided
at the right. With these dosing regimes, DBP and procymidone combined induce
hypospadias in 49% of the males, but they are not "effect additive", inducing only 1.5% and
0% individually. The individual dose response curves for DBP and procymidone indicate
that these are "dose additive" responses, not synergy.
Percentages of male rat offspring with reproductive
malformations. Dams were dose with procymidone at 50
mg/kg/d (Pro-60), dl-n-butyl phthalate at 500 mg/kg/d
(DBP-500) or a combination (Combo).
; Percent Hypospadias
Percent Vaginal Pouch
1
Con
Pro-60
DBP-600
Combo
Con
Pro-60
DBP-600
Combo
0
1.5
0
49
0
0
0
26 7
image:
Overview of Abstract # 22. Genomic and proteonomic basis for interspecies
extrapolations based upon Estrogen- and Androgen Receptor structure and function
among animals. Theme Aim #s 6 a and b
The Agency has a regulatory mandate to protect both human and ecosystem health.
Since steroid hormones and their receptors are highly conserved among vertebrates,
EDCs that act via ER or AR in one species potentially can affect other species via the
same mechanism. However, steroid receptors do display variations in amino acid
sequences. In general, the degree of variation depends upon the taxonomic distance
between the species. Several xenobiotics have been reported to display affinities for for
ER or AR in lower vertebrates that differ significantly from those reported for mammals.
If substantiated, significant species differences in steroid receptor affinity could have major
implications for interspecies extrapolation of effects of EDCs from one species to another.
The default assumption is that effects seen in one species can be extrapolated to another.
For example, the EDSP T1S includes only mammalian in vitro receptor-based assays. In
addition, wildlife are often viewed as "sentinel species". Estrogenic and androgenic
effects in wildlife are generally assumed to be of concern to other species, including
humans. In addition, effects from surrogate laboratory and non-laboratory species are
typically assumed to be relevant to the species of concern. The research described herein
is designed to determine how similar ER and AR (structure and function) are among
species from different classes (the approach and the current status for different species
are described in the figure below). Species were selected based upon several criteria.
1) Different affinities for ER or AR versus mammalian ER or AR have been reported. 2)
The species represents a different vertebrate class and has the potential to be studied
both in the laboratory and the field under natural and experimental conditions. 3) The
species has been adversely impacted by EDCs at the population level.
Quail, Reed Frog
V' Rana pipens, Daphnia
Alligator, Mud Snail
| Sequence ER or AR-
Fat Head Minnow
Express ER or AR
Rainbow trout ER
iPrepare cDNA Library ;
lsolate~ER or AR
Obtain animal Tissues
I . Rainbow trout AR
* -iT
1
jj. rtER versus hER
ER-orAR Function
Compare,. Function'
;; Across-Specles »;
image:
Overview of Abstract # 23. Assessment of the utility of in vitro assays to screen in
environmental mixtures for EDC activities. Theme Aim # 5
Although we usually use our in vitro AR assays to detect the activity of individual
chemicals or mixtures of chemicals, in the current project these assays are being used to
determine if effluent samples from the field display androgenic activity. In the first case,
masculinized female mosquitofish were described in rivers downstream from pulp and
paper mills over 30 years ago. Until now, however, the biological basis for this effect had
not been determined. Along with scientists from the University of Florida and EPA,
Midwest Ecology Division (MED) we recently reported that pulp mill effluent (PME) displays
androgenic activity. This was surprising because all other xenobiotic AR ligands have
displayed antiandrogenic activity. Chemical fractionation studies indicate that the
androgenic activity is not likely due to androstenedione in PME, in contrast to the reports
of another group. (Dark boxes indicate current research effort on PME).
In the second case, concern has arisen in the European Union about residues of
anabolic steroids in hormone-treated beef from the US. Europe has banned the use of
hormones for this purpose and refuses to import hormone-treated beef. These chemicals
are excreted in active forms and they persist in some field samples for long periods. In
collaboration with UFL and MED scientists, we determined that effluent from beef cattle
feed lots displayed androgenic activity. We have not yet determined if this activity results
from anabolic or natural steroids in the effluent samples. We believe that these methods
have application for other scientists in EPA within the Exposure and Monitoring
laboratories for assessing the presence of androgenic activity in drinking and waste water
samples. Discussions have been initiated in this regard. Several methods are in use to
screen water samples for estrogenic activity, but this is the first attempt to apply in vitro
assays for the detection of AR agonists and antagonists.
TIE Approach for Androgens in Pulp Mill Effluent
Observation of masculinzed female
mosquitofish in nvers contaminated
with Pulp Mill Efflent since 1970s
I Detei
I ^ dete
Rec<
Determine Bioconcentration and Magnification Factors and
determine Effects of Androgenic Mixture in other Species
3
Masculinize female fish with
Androgenic Mixture in lab
Reconstitute Androgenic Mixture "l
Identify Androgenic chemicals
Collect
contaminated
(PME) and
reference site
water
Fractionate active samples
Identfy Fractions with
Androgenic activity
Fractionate PME
AR agonist activity
displayed in vitro in
water contaminated
with PME
8
image:
Overview of Abstracts # 24 Scientific and Technological Support on in vitro assays for
the Agency's Endocrine Disrupter Screening program (Theme Aim #1,4,7) and # 25
Scientific and Technological Support on the Hershberger and other in vivo assays for the
Agency's Endocrine Disrupter Screening and Testing program (Theme Aim #s 8,10, 12)
In 1996, the USEPAwas
given a mandate to develop an
endocrine screening at testing
program. In 1998, the
Endocrine Disrupter Screening
and Testing Advisory
Committee (EDSTAC)
proposed a Tiered Screening
and Testing Strategy for
screening and testing
chemicals that act as or
antagonize estrogens or
androgens, or alter thyroid
(EAT) or hypothalamic-
pituitary-gonadal (HPG)
function. An example of the
screening and testing strategy,
including our recommendation
for a "repeat phase" to
eliminate false positives is
displayed at the right.
Our role in the EDSTAC-EDSP process is presented in detail in the abstracts
describing this work. Although the EDSTAC document presented T1S and T2T assay
protocols, research and scientific expertise are needed in this area because some of these
assays have not been rigorously standardized and validated for such purposes while in
other cases newer, more useful assays have been developed since we drafted the original
protocols for EDSTAC in 1998. For these reasons, we are conducting research
and providing scientific expertise to the Agency and the European Union (OECD) on the
development, standardization and validation of in vitro and in vivo assays for these
Prioritization and Screening programs. Many of these assays have been used in our
laboratories for decades for research purposes. This experience and expertise has
enabled us to facilitate the development of the Agency's EDC Screening and Testing
program and is unique in the field of reproductive toxicology. Several members of the
theme described herein, along with other members of the Endocrinology Branch under the
direction of Dr Ralph Cooper, have contributed significantly in this endeavor.
We have developed novel in vitro assays, conducted optimization experiments for
ER and AR binding and gene expression assays, provided on-site training and detailed
EDSP/EDSTAC Screening and Testing Program
(with our modifications - Repeat Phase)
Prioritization of 87,00 chemicals for
Screening
Negative
Positive
Repeat Phase
Repeat Positive
Responses in T1S
False +
Eliminated
before Testing
True +
Tier 2 Testing
Full Life Cycle Protocols
Dose response, adversity,
relevant route, etc
Tier 1 Screening
in vitro ER and AR
Steroidogenesis
Utertroplc assay
Hershberger assay
Pubertal Female Assay
9
image:
SOPS, planned standardization and validation experiments for contract laboratories,
analyzed data from these studies, and drafted reports and publications on several of these
assays. For the in vivo assays, we have developed assays, conducted optimization
experiments, provided on-site training and detailed SOPS, planned standardization and
validation experiments for contract laboratories and the OECD, analyzed data from these
studies, and drafted reports and publications on these assays.
III. Future Directions: BRIEF RESEARCH FUTURE PLANS.
Cumulative risk assessment / effects studies.
Complete ongoing mixture study with two phthalate esters.
Initiate studies using EDCs with estrogenic or AhR activities.
Initiate study of two dicarboximide fungicides to meet OPPTS needs.
Report on study of effects in male and female using an AR agonist plus an antagonist
Interspecies Extrapolation Issues.
Continue rtAR competitive binding studies and compare them to hAR affinities
Execute rtER competitive binding studies with bacilovirus expressed receptor protein.
Sequence fhER and AR and develop expression system for proteins to study functionality.
Probe cDNA libraries for steroid hormone receptors with emphasis on alligator and snail.
Identify environmental androgens in pulp mill effluent samples and other effluent samples.
Developmental Toxicology Studies.
Complete DEHP dose-response study and repeat, if warranted. Obtain fetal and maternal
tissue level data on active phthalate metabolites.
Examine fetal endocrinology after low-high dose exposure to AR agonists and antagonists
and inhibitors of fetal testosterone synthesis in vivo and in vitro.
Expand list of cellular and molecular endpoints evaluated in animals and tissues
especially including immunohistochemistry, rtPCR for key regulatory proteins and
DNA array effects of antiandrogens on target tissues.
Initiate developmental studies with environmental androgens including dose-response and
mechanistic studies.
In vitro, ex vivo and short-term in vivo mechanistic studies.
Determine mechanisms of action of phthalate esters on androgen-dependent tissues in
Hershberger and Pubertal Male rat assays.
Compare effects of different antiandrogens on in vivo gene expression using rtPCR,
northern blots, DNA arrays, immunohistochemistry and gravimetric methods.
Develop rtPCR methods for mRNA for proteins important to mammalian sexual
differentiation, and assess utility of more global, but potentially cruder analysis with
DNA arrays. Compare sensitivity of these methods for potential to address low-dose
issues for EDCs.
10
image:
EDSP ISSUES.
Hershberger Assay. As the Lead Laboratory
Analyze final Phase 1 OECD interlaboratory studies and draft report.
Design Phase 2 and present proposal to OECD VMG for approval.
Initiate Phase 2, analyze data and complete report and publish results after OECD
approval.
Publish background document on this Assay.
In Vitro Assay Development for T1S
Analyze contractor data on ER and AR binding assays, report to OPPTS.
Report our results on stable AR cell lines.
Report our results on Adenovirus transduced cell lines for AR
Continue development of ER stable cell line.
Examine utility of neonatal/fetal testis for assessment of steroidogenesis in vitro.
Conduct pubertal male and female rat and in-utero/lactational assays.
Work with OPPTS and contractor to design studies, standardize and validate these
assays analyze and interpret data from these studies.
IV. FTEs
RESOURCE BASE. FTEs AND STAFF
Staff FTE on Theme
Dr. LE Gray 1
Mr. J Ostby 1
Mr. J Furr 1
Ms. CWolf 1
Ms. C Lambright 1
Dr. V Wilson 1
Dr. P Hartig 0.5
Ms. M Cardon 0.5
Dr. J Welch 0.0
Dr. M Rosen 0.0
Dr. G Held 0.25
Ms. C Woods 0.25
Total 7.5 FTEs
Mr. M Price NCSU Undergraduate in Zoology on Training Coop.
Ms. M Gooding NCSU Predoctoral Trainee in Toxicology on Coop.
Dr. P Reynolds UNC Postdoctoral Student. Appointment anticipated
11/1/2001
Vacancy NCSU Coop. Postdoctoral Student
V. External collaborations.
Drs. L Guillette (UFL), EM Wilson (UNC), G LeBlanc (NCSU), DNR
Veeramachaneni (CSU), J Brock (formerly of CDC), P Foster and N Barlow (CUT), R Tyl
(RTI). EPA Staff. RTD: Members of the R Cooper Theme. B Abbott. MED scientists
including G Ankley, and T Henry (and their team members).
11
image:
VI. Theme References: by Abstract (note: some references are repeated as they
address more than one issue, reference numbers are taken from the Division list, those
without numbers were not on the Division list)
Abstract # 20. Effects, mechanisms and modes of action of EDCs that alter reproductive
development- in vitro and in vivo approaches. LE Gray, J Ostby, J Furr, C Wolf, L Parks,
C Lambright, V Wilson, G Held, C Woods, M Rosen, P Foster, N Barlow, R Tyl, EM
Wilson.
80. Gray LE Jr, Wolf C, Mann P and Ostby JS. In utero Exposure to Low Doses of
2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD) Alters Reproductive Development of
Female Long Evans Hooded Rat Offspring. Toxicol Appl Pharm 146:237-244,
1997.
81. Gray LE Jr, Ostby JS, Kelce WR. A dose-response analysis of the reproductive
effects of a single gestational dose of 2,3,7,8 tetrachloro-p- dioxin (TCDD) in Male
Long Evans Hooded rat offspring. Toxicol Appl Pharm 146:11-20, 1997.
82. Gray LE Jr, Kelce WR, Wiese T, Tyl R, Gaido K, Cook J, Klinefelter G, Desaulniers
D, Wilson E, Zacharewski T, et al. (33 authors). Endocrine Screening Methods
Workshop Report: Detection of Estrogenic and Androgenic Hormonal and
Antihormonal Activity for Chemicals that act via Receptor or Steroidogenic Enzyme
Mechanisms. Reproductive Toxicology 11 (4):719-750,1997.
83. Gray LE Jr. Chemically-induced alterations in female mammals. In Comprehensive
Toxicology (K. Boekelheide, R.E. Chapin, P.B. Hoyer and C. Harris, Eds) pp 329-
339. 1997. Elsevier Science, New York.
114. Kelce WR, Lambright CR, Gray LE Jr and Roberts KP. Vinclozolin and p,p' DDE
alter androgen-dependent gene expression: In vivo confirmation of an androgen-
receptor-mediated mechanism. Toxicol Appl Pharmacol 142:192-200, 1997.
115. Kelce WR and Gray LE Jr. Antiandrogens as environmental endocrine distrupters.
Health and Environment Digest 11:9-15, 1997.
175. Peterson RE, Cooke PS, Kelce WR and Gray LE Jr. Environmental Endocrine
Distrupters. Reproductive and Endocrine Toxicology: Male Reproductive
Toxicology. In: Comprehensive Toxicology. (Eds. G. Sipes, C. McQueen and J.
Gandolfi) pp. 181-191, 1997. Elsevier Science, New York.
17. Ankley G, Mahiach E, Stahl R, Tillitt D, Colborn T, McMaster S, Miller R, Bantle J,
12
image:
Campell P, Dickerson R, Fry M, Giesy J, LE Gray Jr, et al. Overview of a workshop
on screening methods for detecting potential (anti)estrogenic/androgenic chemicals
in wildlife. Environ Toxicol and Chemistry. 17:68-87, 1998.
84. Gray LE Jr, Ostby, Joseph, Wolf, Cynthia, Lambright, Christy, and Kelce, William.
The Value of Mechanistic Studies in Laboratory Animals for the Prediction of
Reproductive Effects in Wildlife: Endocrine effects on Mammalian Sexual
Differentiation. Environ Toxicology and Chemistry 17:109-118, 1998.
85. Gray, LE Jr and Ostby J. Effects of pesticides and toxic substances on behavioral
and morphological reproductive development: Endocrine versus nonendocrine
mechanisms. Toxicol Industrial Health 14:159-184, 1998.
61. Dickerson RL, Brouwer A, Gray LE Jr, Grothe DR, Peterson RE, Sheehan DM, Sills
C and Alfred Wiedow. Dose response relationships. In: Principles and Processes
for Evaluating Endocrine Disruption in Wildlife. (Eds Kendall, R., Dickerson, R.,
Suk, W and Giesy, J), pp. 1998. SETAC Press, Pensacola, FL.
116. Kelce WR, Gray LE, Wilson EM. Antiandrogens as environmental endocrine
disruptors. Reprod Fertil Dev, 10(1): 105-11. 1998.
117. Kelce WR and Gray LE Jr. Endocrine Disrupters: Effects of sex steroid hormone
receptors and sex development. Chapter 31 In: Handbook of Experimental
Pharmacology (Eds R Kavlockand G Daston) pp. 433-472,1998. Springer-Verlag,
Heidleberg.
24. Bigsby R, Chapin RE, Daston GP, Davis BJ, Gorski J, Gray LE, Howdeshell KL,
Zoeller RT, vom Saal FS. Evaluating the effects of endocrine disruptors on
endocrine function during development. Environ Health Perspect, 107(Suppl
4):613-8, 1999.
60. DeVito M, Biegel L, Brouwer A, Brown S, Brucker-Davis F, Cheek AO, Christensen
R, Colborn T, Cooke P, Crissman J, Crofton K, Doerge D, Gray E, Hauser P, Hurley
P, Kohn M, Lazar J, McMaster S, McClain M, McConnell E, Meier C, Miller R,
Tietge J, Tyl R. Screening methods for thyroid hormone disruptors. Environ Health
Perspect, 107(5):407-15. 1999.
86. Gray LE Jr, Wolf C, Lambright C, Mann P, Price M, Cooper RL, Ostby.
Administration of potentially antiandrogenic pesticides (procymidone, linuron,
iprodione, chlozolinate, p,p'-DDE, and ketoconazole) and toxic substances (dibutyl-
and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during
sexual differentiation produces diverse profiles of reproductive malformations in the
male rat. J Toxicol Ind Health 15 (1 -2):94-118,1999.
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87. Gray LE Jr, Ostby J, Monosson E, Kelce WR. Environmental antiandrogens: low
doses of the fungicide vinclozolin alter sexual differentiation of the male rat. Toxicol
Ind Health 15 (1-2):48-64, 1999.
88. Gray LE Jr, Ostby J, Cooper RL, Kelce WR. The estrogenic and antiandrogenic
pesticide methoxychlor alters the reproductive tract and behavior without affecting
pituitary size or LH and prolactin secretion in male rats.Toxicol Ind Health
15(1 -2):37-47, 1999.
152. Monosson E, Kelce WR, Lambright C, Ostby J, Gray LE Jr. Peripubertal exposure
to the antiandrogenic fungicide, vinclozolin, delays puberty, inhibits the
development of androgen-dependent tissues, and alters androgen receptor function
in the male rat. Toxicol Ind Health 15(1-2):65-79, 1999.
162. Ostby J, Kelce WR, Lambright C, Wolf CJ, Mann P, Gray LE Jr. The fungicide
procymidone alters sexual differentiation in the male rat by acting as an
androgen-receptor antagonist in vivo and in vitro. Toxicol Ind Health 15(1 -2):80-93,
1999.
231. Wolf CJ, Ostby JS, Gray LE. Gestational exposure to 2,,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD) severely alters reproductive function of female
hamster offspring. Toxicol Sci. 51(2):259-64,1999.
131. Lambright C, Ostby J, Bobseine K, Wilson V, Hotchkiss AK, Mann PC, Gray LE.
Cellular and molecular mechanisms of action of linuron: an antiandrogenic
herbicide that produces reproductive malformations in male rats. Toxicol Sci.
56(2):389-99, 2000.
232. Wolf CJ, LeBlanc GA, Ostby JS, Gray LE. Characterization of the period of
sensitivity of fetal male sexual development to vinclozolin. Toxicol Sci.
55(1 ):152-61, 2000.
164. Parks LG, Ostby JS, Lambright CR, Abbott BD, Klinefelter GR, Barlow NJ, Gray LE.
The plasticizer diethylhexyl phthalate induces malformations by decreasing fetal
synthesis during sexual differentiation in the male rat. Toxicol Sci. 58(2):339-49,
2000.
90. Gray LE Jr, Ostby J, Furr J, Wolf CJ, Lambright C, Parks L, Veeramachaneni DNR,
Wilson V, Price M, Hotchkiss A, Orlando E, Guillette L. Effects of environmental
antiandrogens on reproductive development in experimental animals. Human
Reprod Update. 7(3):248-64. 2001.
92. Gray LE Jr, Lambright C, Parks L, Tyl R, Orlando E, Guillette L, Wolf C, Seely J,
Chang TS, WilsonV, Hotchkiss A, Ostby J. (In press) Emerging issues related to
endocrine disrupting chemicals and environmental androgens and antiandrogens.
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In: Handbook of Environmental Chemistry: Endocrine distuptors. (M Metzler Ed).
2001.
Abstract # 21. Cumulative Risk of exposures to EDCs with similar and dissimilar
mechanisms and modes of action. LE Gray, J Ostby, J Furr, M Price, L Parks, A
Hotchkiss, C Wolf.
90. Gray LE Jr, Ostby J, Furr J, Wolf CJ, LambrightC, Parks L, Veeramachaneni DNR,
Wilson V, Price M, Hotchkiss A, Orlando E, Guillette L Effects of environmental
antiandrogens on reproductive development in experimental animals. Human
Reprod Update. 7(3):248-64. 2001.
92. Gray LE Jr, Lambright C, Parks L, Tyl R, Orlando E, Guillette L, Wolf C, Seely J,
Chang TS, WilsonV, Hotchkiss A, Ostby J. (In press) Emerging issues related to
endocrine disrupting chemicals and environmental androgens and antiandrogens.
In: Handbook of Environmental Chemistry: Endocrine Disruptors. M Metzler (ed).
2001.
Abstract # 22. Genomic and proteomic basis for interspecies extrapolations based upon
estrogen and androgen receptor structure and function among animals.
VS Wilson, GT Ankley, K Bobseine, M Cardon, MP Gooding, LE Gray, Jr, LJ Guillette, P
Hartig, G Held, J Korte, GA LeBlanc, J Welch, EM Wilson.
Abstract # 23. Assessment of the utility of in vitro assays to screen environmental
mixtures for EDC activities. LE Gray, J Ostby C Lambright, V Wilson, L Parks, G Ankley,
LJ Guillette, E Orlando, EM Wilson.
90. Gray LE Jr, Ostby J, Furr J, Wolf CJ, Lambright C, Parks L, Veeramachaneni DNR,
Wilson V, Price M, Hotchkiss A, Orlando E, Guillette L. Effects of environmental
antiandrogens on reproductive development in experimental animals. Human
Reprod Update. 7(3):248-64. 2001.
91. Gray LE Jr, Lambright C, Parks L, Tyl R, Orlando E, Guillette L, Wolf C, Seely J,
Chang TS, WilsonV, Hotchkiss A, Ostby J. (in press) Emerging Issues Related to
Endocrine Disrupting Chemicals and Environmental Androgens and Antiandrogens.
In: Handbook of Environmental Chemistry: Endocrine distuptors. (M Metzler, Ed).
2001.
165. Parks, LG, Lambright, C R, Orlando, EF, Guillette, LJ, Jr., Ankley, GT, and Gray,
LE, Jr.. Masculinization of female mosquitofish in Kraft mill effluent-contaminated
Fenholloway River is associated with androgen receptor agonist activity. Toxicol
Sci. 62,:257. 2001.
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Abstract # 24. Scientific and Technological Support on in vitro assays for the Agency's
Endocrine Disrupter Screening Program. V Wilson, P Hartig, C Lambright, K Bobseine,
M Cardon, S Laws, R Cooper, LE Gray.
82. Gray LE Jr, Kelce WR, Wiese T, Tyl R, Gaido K, Cook J, Klinefelter G, Desaulmers
D, Wilson E, Zacharewski T, et al. (33 authors). Endocrine screening methods
workshop report: Detection of estrogenic and androgenic hormonal and
antihormonal activity for chemicals that act via receptor or steroidogenic enzyme
mechanisms. Reprod Toxicol 11 (4):719-750, 1997.
162. Ostby J, Kelce WR, Lambright C, Wolf CJ, Mann P, Gray LE Jr. The fungicide
procymidone alters sexual differentiation in the male rat by acting as an
androgen-receptor antagonist in vivo and in vitro. Toxicol Ind Health 15(1 -2):80-93,
1999.
131 Lambright, C, Ostby, J, Bobseine, K, Wilson, V, Hotchkiss, AK, Mann, PC, and
Gray, LE, Jr. Cellular and molecular mechanisms of action of linuron: an
antiandrogenic herbicide that produces reproductive malformations in male rats.
Toxicol Sci. 56:389-399, 2000.
165. Parks, LG, Lambright, CR, Orlando, EF, Guillette, LJ, Jr, Ankley, GT, and Gray,
L.E, Jr. Masculinization of female mosquitofish in Kraft mill effluent-contaminated
Fenholloway River is associated with androgen receptor agonist activity. Toxicol
Sci. 62:257, 2001.
Abstract # 25. Scientific and Technological Support on in vivo assays for the Agency's
Endocrine Disrupter Screening and Testing Program. LE Gray, J Ostby, J Furr, R Cooper,
T Stoker, S Laws, J Goldman, R Tyl.
82. Gray LE Jr, Kelce WR, Wiese T, Tyl R, Gaido K, Cook J, Klinefelter G, Desaulmers
D, Wilson E, Zacharewski T, et al. (33 authors). Endocrine screening methods
workshop report: Detection of estrogenic and androgenic hormonal and
antihormonal activity for chemicals that act via receptor or steroidogenic enzyme
mechanisms. Reprod Toxicol 11 (4):719-750,1997.
86. Gray LE Jr, Wotf C, Lambright C, Mann P, Price M, Cooper RL, Ostby.
Administration of potentially antiandrogenic pesticides (procymidone, linuron,
iprodione, chlozolinate, p,p'-DDE, and ketoconazole) and toxic substances (dibutyl-
and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during
sexual differentiation produces diverse profiles of reproductive malformations in the
male rat. J Toxicol Ind Health 15 (1-2):94-118, 1999.
78. Goldman, J.M., Laws, S.C., Balchak, S.K., Cooper, R.L., and Kavlock, R.J.
Endocrine-disrupting chemicals: prepubertal exposures and effects on sexual
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maturation and thyroid activity in the female rat. A focus on the EDSTAC
recommendations. Crit Rev Toxicol. 30:135-196, 2000.
217. Stoker, TE, Parks, LG, Gray, LE, and Cooper, RL. Endocrine-disrupting
chemicals: prepubertal exposures and effects on sexual maturation and thyroid
function in the male rat. A focus on the EDSTAC recommendations. Endocrine
Disrupter Screening and Testing Advisory Committee. Crit Rev Toxicol. 30:197-
252, 2000.
VII. Activities relevant to the USEPA and other agencies. Theme members have
participated in the following activities that may have impact on Agency policies.
1996-1998 ORD Endocrine Disrupters Research Strategy Committee
1997 Chair: EPA/WWF/CMA "Duke" Workshop on Endocrine Screening Methods.
Report published in 1997.
1997 NCTR Site Visit Review Team/Consultant on "Estrogen Knowledge Base
Project".
1997- pres. Consultant to the US Department of Justice on several EDC issues.
1997-pres. Implement and organize the monthly RTP EDC Forum.
4/97 to 8/98 EDSTAC-Screening and Testing Workgroup Member.
1997 CMA/WWF/USEPA "Duke" Workshop Participant on Thyroid mediated
Toxicology.
1997 Participant at a SETAC Pellston Workshop on "Reproductive and
developmental effects of contaminants in Oviparous vertebrates", resulting
in a book.
1997 CMA/WWF/USEPA "KC" Workshop Participant on Detection of EDCs in
Wildlife 1997. EDSTAC presentation of "Screening and Testing Workgroup
Status Report" to the Plenary Committee.
1998 EDSTAC presentation of "Screening and Testing Workgroup Screening
Battery" to the Plenary Committee.
1998 4th Annual NHEERL, EPA Symposia presentation.
1998 USEPA/EDSTAC SAP/SAB Review: Presentation of T1S and HTPS at
OPPTS request.
1998 The First NIEHS Workshop Participant on "Low Dose Issues" which resulted
in a publication.
1998 NOAA / IWC Workshop on effects of contaminants in marine mammals.
1999 Toxicology Forum Panel Member and speaker.
1999 EPA-Endocrine Disruptor Screening Program Workgroup member which
prepared ORD Research Strategy Document.
1999-2001 Co-organizer and Speaker at USEPA, NHEERL Interspecies Extrapolation
Workshops.
2000-pres. NHEERL EDC Research Planning Implementation Committee which
prepared the NHEERL EDC Research Plan.
2000 Workshop on Effects of the Effects of Multiple Stressors on Reproduction
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in the Right Whale.
2000 OECD presentation of Hershberger Assay Standardized Proposal at
Validation and Management Group meeting and proposal and Phase 1
research plan for the Interlaboratory study, Paris.
2000 Denmark EDC Meeting on hormones in beef. Cochair and two presentations,
published presentations and discussions.
2000 SETAC Pellston Workshop on Interconnections of Ecosystem and Human
Health, Snow bird Utah. Book in preparation.
2000 The second NIEHS Low-Dose Workshop, The only data discussant for
Androgen Panel - data reanalysis and panel report on NIEHS web-site.
2000 Training contractor laboratory personnel in execution of AR binding assay
at request of OPPTS, EPA.
2000 Training technians from one contract and two industrial laboratories in
execution of in vivo Hershberger Assay following the OECD standardized
protocol.
2001 OECD presentation at Validation and Management Group meeting of Report
on Hershberger Assay Proposal and Phase 1 research from the
Interlaboratory study and presentation of a proposal for Phase 1b.
2001 NIES/EPA Meeting in Japan on EDCs. Speaker.
2001 Met with Japanese Pulp and Paper Mill representative at the request of
NHEERL/MED.
2001 Met with Japanese Congressional Delegation on EDC issues.
2001 EPA/ORD/Regional meeting on EDC Issues. Atlanta Speaker.
2001 RTD Delegate, Synergy Workgroup for allocation of awards for synergy
meetings, based upon submitted proposals.
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Poster 20. Effects, Mechanisms and Modes of Action of Edcs That Alter
Reproductive Development: in Vitro and in Vivo Approaches. LE Gray, J Ostby, J
Furr, C Wolf, L Parks, C Lambright, V Wilson, G Held, C Wood, M Rosen, P Foster, N
Barlow, R Tyl, EM Wilson.
Exposure to chemicals that alter steroid action or synthesis during critical
developmental periods can cause irreversible alterations of reproductive morphology and
function. Our studies are designed to address several factors including the period of
vulnerability, tissue dosimetry, and cellular and molecular mechanisms of action, which
ultimately will provide data for risk assessments on individual and mixtures of EDCs. Our
objectives include comparison of 1) dose-response relationships and 2) in vivo tissue
levels of the active toxicant. We have published several studies on AR-antagonists
(vinclozolin, procymidone) and other chemicals like the phthalates that act as
antiandrogens by other mechanisms, androgens and estrogens. In these studies,
pregnant animals are exposed during developmental stages and the reproductive system
of the male and female offspring assessed throughout lactation, puberty, mating and, on
occasion, old age. Studies have examined the effects of estrogens, androgens,
antiandrogens, AhR agonists (TCDD, PCBs), inhibitors of steroidogenesis, and fetal germ
cell toxicants on reproductive development in male and female offspring. Others studies
have examined the direct effects of exposure during puberty on maturation and
subsequent function of the reproductive system.
Mechanistic studies provide important information for understanding interspecies
extrapolation, windows of vulnerability, and dose-response relationships. In vitro,
mechanistic studies (AR binding and transcriptional activation, inhibition of 5 alpha
reductase, inhibition of steroidogenesis) are typically conducted in parallel with the in vivo
studies. In addition, we evaluate the endocrine system of a subsample of the litters during
fetal life. Included in such assessments are in vitro and ex vivo assays to evaluate fetal
T production, fetal tissue T levels, testicular steroidogenesis, fetal Leydig cell cultures,
immunohistochemistry for AR and EGF receptors and determination of receptor mRNA
levels by rtPCR. We have collected fetal reproductive tracts, amniotic fluid, carcass, and
placenta for analysis of testosterone to determine the effects of in utero treatment on this
endpoint. We also are incorporating a series of new methods to assess fetal testis
enzyme activity, fetal Sertoli cell products and analysis of specific genes/gene products
that are up- or down-regulated in the development of the urogenital system using cDNA
microarrays, northern and rtPCR analyses.
In some studies we are collaborating with MED, or CDC to obtain fetal and
maternal tissue level data on the concentrations of the active EDC metabolites at the
critical stage of development. We recently submitted fetal amniotic fluid and maternal
urine samples to CDC for determination of phthalate monoester levels that are associated
with malformations in male rat offspring.
Last year, an SAR profile for developmental toxicity of several phthalates was
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described and a mechanism of action purposed. DEHP, BBP, DBP, DINP all alter
reproductive development of the male offspring in an antiandrogenic manner. The first
three appear to be of equivalent potency, while DINP is 10-15 fold less potent. DEP, DMP
and DOTP were inactive. The adverse effects of DINP were replicated because industry
studies indicate that this phthalate had no adverse reproductive effects The FDA
reported that they were extremely concerned about DEHP levels in children on dialysis
and they also were concerned about exposures from breast milk.
Having completed the hazard identification, we have initiated dose response,
studies with DEHP and have found effects at doses below the reported NOAEL For the
DEHP dose response study, we are using longer-term dosing (option B2 of our ART
protocol, as described in Gray etal., 1988; Zenick et al., 1991). Dosing is initiated at GD
8 and continued through lactation. Near weaning, randomly selected males from each
litter are continued on treatment until they are necropsied after puberty. Other F1 males
are maintained through adult life and necropsied.
Our vinclozolin data, which established new NOAELs, have been used to restrict,
cancel, and/or change tolerance levels for this pesticide on August 22, 2001 (for both in
utero and pubertal exposures). A study by Wolf et al., (232) found that GD 16-17 was the
most sensitive period to the demasculinzing effects of vinclozolin. In contrast, Hotchkiss
et al., (in prep) found that the early postnatal stage of life was the only period of
development during which some sexually dimorphic behaviors could be demasculinized
by vinclozolin. Dr. Hotchkiss (in prep) also found that antiandrogenic effects of these
chemicals on neonatal anogenital distance and infantile areola/nipple numbers are
permanent and highly correlated with malformations in other tissues.
Our vinclozolin dose response data were recently the sole study selected for
reanalysis and review in the "Androgen-antiandrogen Panel" at the NTP/NIEHS Low Dose
Workshop (see NIEHS web site for report). They concurred with our assessment that the
low dose used in this study was a LOEL (3 125 mg/kg/d), not a NOEL and concluded that
the low dose effects were linear and did not display a threshold (the Agency's default
assumption), however, they suggested that nonmonotonic responses should not be
expected from antiandrogens. Several other studies on vinclozolin, procymidone, linuron,
and testosterone from this project were also included as supplemental studies that were
reviewed by the panel.
We recently submitted a dose-response study for publication on the effects of in
utero testosterone on sexual differentiation of the female offspring (Wolf et al., in press).
The results of this study are important because they demonstrate that very few, if any, of
the most sensitive androgen-responsive tissues are currently evaluated in the standard
multigenerational reproduction study.
In our studies we have found that several chemicals, reported to be without
teratogenic effect by industrial laboratories, are in fact positive causing epididymal
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agenesis and other malformations. It is evident that these laboratories are using protocols
that are inadequate, lacking sufficient sample sizes and an adequate assessment of the
appropriate endpoints. For example, perinatal maternal linuron-treatment, negative in a
teratology study and older multigenerational studies, causes malformations in over half the
males. Based on these industry studies, the developmental toxicity of linuron is not
properly addressed in the current EPA RED. (1995).
Pertinent References. 80-92, 114-118, 131, 152, 162, 164, 231, 232
also. Hotchkiss (in prep), Wolf et al., (in press)
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Poster 21. Cumulative Risk of exposures to EDCs with similar and dissimilar
mechanisms and modes of action. LE Gray, J Ostby, J Furr, M Price, L Parks, A
Hotchkiss, C Wolf.
The Food Quality Protection Act of 1996 mandated that the risk assessment
process consider combinations of chemicals that act via the same mechanism of action.
To this end, we have initiated studies to examine if mixtures of antiandrogens and
androgens act in an additive, antagonistic or synergistic manner. Our results support the
concept that risk assessments should consider the "cumulative" risk of the mixture as
opposed to examining risk on an individual chemical-by-chemical basis. While some of
combined effects of these toxicants may appear to be synergistic, we believe that these
are dose- but not effect-additive responses. Effects that tend to be linear in the low dose
region display responses that are obviously additive (i.e. 10+10 =20) (effect- and dose-
additive) while effects that typically display a threshold are dose-additive but not effect
additive (i.e. 0+1.5=49) It is clear from our results that toxicants that alter development
of the same reproductive tissues during the same critical period will produce cumulative
effects regardless of the mechanism of action.
Cumulative Effects of Vinclozolin (V) plus Procymidone (P).
The effects of graded doses of the AR antagonists P and V, ranging from 25 to 100
mg/kg/d individually or together, were evaluated in the Hershberger assay (castrate-
immature-testosterone-treated male rats for seven days). At low dosage levels the
mixtures of V plus P reduced ventral prostate and levator ani weights in an additive fashion
When the higher doses were combined the effects were less than additive because each
chemical completely inhibited the effects of testosterone by itself.
We also are examining the effects of a combination of V plus P on sexual
differentiation of the rat. The strategy employed in these developmental studies was to
select a dosing regime from past experience that would produce little or no effect when the
chemical was administered by itself on gestational days 14-18. In this study, only the P
plus V group displayed high incidences of malformations. The cumulative effects of this
mixture appeared "dose-additive" but not "effect-additive".
Cumulative Effects of an AR antagonist and an Inhibitor of Fetal T Synthesis.
These studies address the question, how do AR antagonists like P or linuron (L)
interact with phthalates like DBP (which are not AR ligands)? To address this question,
we have conducted two mixture studies using AR antagonists and an inhibitors of fetal
testosterone synthesis. In the first study, when P and di-n-butyl (DBP) were administered,
hypospadias was present in 1.5% and 0% of the male offspring in the P and DBP groups,
respectively, whereas 49% of the male offspring in the combination group (P plus DBP)
displayed this malformation. The incidence of males with a vaginal pouch displayed
similar additivity. In a second study, coadministration of L and benzyl butyl phthalate
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(BBP) also induced cumulative effects on the male reproductive system.
Cumulative Effects of Two Phthalates that Inhibit Fetal T Synthesis: DBP and BBP.
GD 14-18 administration of DBP or BBP at doses that induced minimal effects on
AGD, areolar retention and reproductive malformations produced cumulative effects on
these endpoints and on maternal growth and neonatal viability in a cumulative, "dose-
effect:" manner.
Antagonist interactions of the AR antagonist Vinclozolin and Testosterone (T),
administered during sexual differentiation.
When administered during sexual differentiation, V induces malformations of the
male reproductive tract, while TP-treatment masculinizes the female reproductive tract.
When V and TP are coadministered, V antagonizes the masculinzing effects of T on the
female rat, while T attenuates the effects of V on the male rat reproductive system.
Pertinent References: 90, 92
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Poster 22. Genomic and Proteomic Basis for Interspecies Extrapolations Based
upon Estrogen and Androgen Receptor Structure and Function among Animals.
VS Wilson, GT Ankley, K. Bobseine, M Cardon, MP Gooding, LE Gray, Jr, LJ Guillette,
P Hartig, G Held, J Korte, GA LeBlanc, J Welch, EM Wilson.
Most in vitro hazard assessments for the screening and identification of endocrine
disrupting compounds (EDCs), including those outlined in the EDSP Tier 1 Screening
(T1S) protocols, use mammalian steroid hormone receptors. There is uncertainty,
however, concerning differences that may exist in the binding affinities of toxicants for
steroid receptors from other species. For example, metabolites of the fungicide vinclozolin
have been shown to bind the androgen receptor from humans and rats, but the results of
binding assays in fish are contradictory. The goal of this work is to conduct an in-depth
comparison of androgen (AR) and estrogen receptor (ER) structure and function across
selected species. The cDNA sequences for the rainbow trout androgen (rtAR) and
estrogen (rtER) receptors were obtained from published sources. The rtAR has been
transfected into and the protein expressed in COS cells. Scatchard analysis with
[3H]R1881 has been completed and competitive binding assays are currently being
conducted in this system. Preliminary data indicates that the binding affinity of rtAR for
some chemicals appears to differ with some ligands from that of hAR. The cDNA
sequence for the rtER has been subcloned into a Baculovirus expression vector and semi-
purified. Competitive binding assays comparing the rtER to the hER are planned. Tissues
have been collected and cDNA libraries completed for alligator testis (Alligator
mississippenisis), fathead minnow viscera (Pimephales promelas), Northern Leopard frog
liver (Rana pipens), mud snail body (llyanassa obsoleta), and Daphnia magna. Tissues
have yet to be collected for Japanese quail and African reed frog. The ER and AR from
the fathead minnow have both been isolated and sequences confirmed. The full coding
region of the ER was isolated from the library using the rtER as a probe and full length AR
was isolated using a fathead AR fragment supplied by scientists from the US EPA lab in
Duluth. The alligator cDNA library is currently being screened for the ER by traditional
methods using the rtER as a probe and for the AR by rtPCR with primers designed from
the canary AR sequence. A fragment of the alligator AR of the expected length has been
isolated but the sequence has yet to be confirmed. If confirmed, this fragment will be used
to probe the alligator library for the full length AR. Initial low stringency probing of the mud
snail library with a published fragment from the whiptail lizard failed to isolate an AR
related sequence. The mud snail library is currently being screened both with the rtER
and, in separate experiments, with a probe targeting the most highly conserved region of
the DNA binding domain with the goal of isolation of any receptor within the steroid family.
As receptors are identified and sequenced, future studies will include detailed comparisons
of receptor sequences to known mammalian sequence information, development of
expressions systems for proteins to study functionality in comparison to mammalian
receptors, and development of additional in vitro assays that incorporate receptors from
these species. These investigations will help resolve some of the across-species
extrapolation issues associated with EDCs and, ultimately, may have a major impact on
future risk assessment protocols.
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Pertinent References: 90, 91
Poster 23. Assessment of the Utility of in vitro Assays to Screen Environmental
Mixtures for EDC Activities. LE Gray, J. Ostby C Lambright, V Wilson, L Parks, G Ankley,
LJ Guillette, E Orlando, EM Wilson
Female mosquitofish (Gambusia affinis holbrooki) downstream from Kraft paper mills
in Florida display masculinization of the anal fin, an androgen-dependent trait. The first
series of studies were designed to determine if water contaminated with pulp mill effluent
(PME) from the Fenholloway River, FL displayed androgenic activity in vitro and to relate
this activity to the reproductive status of female mosquitofish taken from this river. Eighty
percent of the female mosquitofish from the Fenholloway River were partially masculinized
while another 10% were completely masculinized based upon the number of segments in
the longest anal fin ray (18.0 ±0.4 versus 28.1 ±0.9 (p <0.001), in a control river versus
Fenholloway River, respectively). In a COS whole-cell binding assay, all three PME
samples displayed affinity for hAR (p < 0.001). In addition, PME induced androgen-
dependent gene expression in CV-1 cells (cotransfected with pCMV hAR and MMTV
luciferase reporter) which was inhibited by about 50% by coadministration of
hydroxyflutamide an AR antagonist. When CV-1 cells were transfected with human
glucocorticoid receptor (hGR) rather than hAR, PME failed to significantly induce MMTV-
luciferase expression. Further evidence of the androgenicity was observed using a COS
cell AR nuclear translocalization assay. PME bound hAR and induced translocalization
of AR into the nucleus. PME also displayed "testosterone-like" immunoreactivity in a
testosterone radioimmunoassay, whereas water from the reference sites did not. In
summary, water collected downstream of the Kraft mill on the Fenholloway River contains
unidentified androgenic substances whose presence is associated with masculinization
of female mosquitofish. Currently, PME samples are being fractionated in attempt to
identify the androgenic chemicals.
In a second project, we detected androgenic activity in feed-lot effluent from a feed
lot in Midwestern US. However, we have not yet determined if this activity arises from
natural or synthetic hormones. We also examined the potency of beta trenbolone for
androgenicity. Trenbolone, is an anabolic steroid used to promote growth in beef cattle
found in feed lot waste water and manure samples. Based upon observations of
reproductive alterations in fish in waters receiving feed-lot effluent, concern has arisen
about the presence and persistence of this hormonally active substance in effluent
reaching streams near the feed lots. In vitro, beta trenbolone was a full agonist about as
active as is dihydrotestosterone and this activity was fully inhibited by the antiandrogen
hydroxyflutamide. When examined in vivo in the Hershberger Assay, trenbolone displayed
selective androgenic receptor mediated activity (SARM), affecting some androgen -
dependent tissues much more than others in a manner suggesting that 5-alpha-reduction
inactivated rather than activated the parent compound. In summary, trenbolone is a potent
SARM. Further studies are in progress to determine 1) whether trenbolone is present in
feed-lot effluent in concentrations sufficient to induce effects and 2) to characterize the
ability of this chemical to alter vertebrate reproduction and development.
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Pertinent References: 90, 92 165
Poster 24. Scientific and Technological Support on in vitro assays for the Agency's
Endocrine Disrupter Screening program. V Wilson, P Hartig, C Lambright, K Bobseine,
M Cardon, S Laws, R Cooper, LE Gray
In response to the 1996 legislative mandate for an endocrine screening and testing
program, we are helping develop, standardize and validate relatively sensitive, robust and
relatively simple methods for in vitro screening of chemicals that affect estrogen, and
androgen function (Table below). The following types of assays are considered in this
project: ER alpha binding with the Pan Vera Assay, ER alpha ,and beta transcriptional
activation assays, AR binding assays and AR transcriptional activation assays (with
transient, transduced, and stable cells).
Androgen receptor binding assays. There are several in vitro binding assays
for the AR. These include using AR isolated from rat ventral prostate or COS whole cell
and a newer and unvalidated assay using a chimeric rat AR (Pan Vera). If some of the
newer AR binding assays could be standardized and validated then they could replace the
standard assay which uses AR from animal tissues (Kelce et al, 1994; 131, 162)
AR-dependent gene expression assays with transiently transfected or
transduced cells. Androgen-dependent gene expression can be assessed in vitro using
mammalian cells that are transiently transfected with a luciferase reporter gene and hAR.
The binding assays, cannot distinguish agonists from antagonists. Gene expression
assays using transiently transfected, or transduced cell, or stably transfected cell lines are
required to make this distinction. In our laboratory, we have used CV-1 cells that were
transiently cotransfected with MMTV-luc and hAR or hGR. We also have used Adenovirus
to transduce CV-1 cells with MMTV-luc and hAR or MDA-453 cells with MMTV-luc. (95,
131, 162, 165)
AR-dependent gene expression assays. K. Bobseine has developed two stable
cells lines to screen for androgenic and antiandrogenic chemicals. One of the assays
uses a cell line that has endogenous levels hAR (MDA-KB2) and has been stably
transfected with and androgen-responsive reporter gene while the other stable expresses
both hAR and reporter genes. These assays offer several advantages over the transiently
transfected cell lines (interassay variability, transfection of cells with genes not necessary).
These assays, and those above, have all been evaluated with a common suite of AR
agonists and antagonists. (230; Kemppainen, Bobseine, et al., 1999).
In vitro assessment of hER binding. The Fluorescence Polarization System (Pan
Vera) is used to assess binding of chemicals to ER a using the new ES2 kit. This is a
competitive binding assay using purified receptor, fluorescent ligand. We have
demonstrated that this assay provides reproducible data that are consistent with ER
26
image:
binding affinities collected from more established assays using about 20 chemicals
(including 17 beta-estradiol, estrone, ethinyl estradiol, genistein, DES, phenols,
DDT/DDEs). In contrast to the literature, we found that the ES1 kit was not useful and
recommend that it not be used and it was subsequently removed from the market.
Development of Cell lines that stably express hER alpha and/or beta for
screening EDCs. We are developing cells that stably express ER for research and
screening purposes. The ZR-75-1 and T47D cells are being evaluated with chemicals
that display known estrogenic activities. T47D breast cancer cell line has both ERs while
the ZR-75-1 parental cell line only has ER alpha Both estrogen responsive cell lines
demonstrate luciferase induction by E2 exposure while ICI consistently inhibits this
response.
Role of Endocrinology Branch Scientists in the Agency's EDSP in vitro assays for Tierl
Screening
Endocrinology
Branch/ Theme
Role
ER binding
Assays
AR binding
Assays
ER gene
expression
AR gene
expression
Steroid
Hormone
Synthesis
Assay
Originator
In progress
Three
Assays
Research to Optimize
protocol for EDSP or
OECD
Yes
Yes
Yes
Yes
Formerly
an active
effort
Drafted Standardized
protocol for EDSP or
OECD
Yes
Yes
Not Yet
Not Yet
Assisted in design of
validation program
Yes
Yes
Not Yet
Onsite and/or offsite
training of
participating labs
Yes
Yes
Analyzed data from
validation program
Yes
Yes
Published literature
review(s) of protocol
Two
submitted
Drafted EDSTAC
protocol for 1998
Yes
Yes
Yes
Yes
Yes
Purpose of Assay: to
Detect
ER ligands
AR ligands
ER agonist
and
antagonist
AR agonist
and
antagonist
Inhibitors
of steroid
hormone
svnthesis
Pertinent References: 95, 131, 162, 165, 230
27
image:
Poster 25. Scientific and Technological Support on in vivo Assays for the Agency's
Endocrine Disrupter Screening and Testing Program. LE Gray, J Ostby, J Furr, R
Cooper, T Stoker, S Laws, J Goldman, R Tyl.
We have been using short-term in vivo assays for research purposes since the late
1970s. Some of these in vivo assays have been designated for consideration in the
Agency's EDSP T1S program (Table below). In the early 1980s we developed the pubertal
male and female rat assays for screening chemicals for reproductive effects (former
"Repro Screen" Task). We first used the pubertal male assay to assess the effects of di-n-
butyl phthalate and the female assay to evaluate the estrogenic pesticides chlordecone
and methoxychlor (86, 88, 152, Gray et al., 1988 a,b, 1989).
In 1996, we led a workshop on EDCs which preceded the SDWA and FQPA and
EDSTAC. This multi stakeholder workshop proposed an in vitro, in vivo screening
strategy for estrogens and androgens that included all of the assays in the EDSTAC/EDSP
Screening Battery (82). As a member of the Screening and Testing Workgroup of
EDSTAC, we helped develop the T1S and T2T batteries and drafted the research
protocols for many of the in vivo screening assays (pubertal male and female assays, the
Hershberger assay, the uterotropic assay using intact juvenile or adult ovariectomized
female rats) and an Alternative Mammalian Reproduction Test contained in the EDSTAC
Final Report).
In 1999, we were designated as the lead laboratory by OECD and the Agency for
the Hershberger assay. In this role, we have conducted several studies in the laboratory
with weakly active chemicals to define the sensitivity of the assay and optimized the
protocol. In Feb 2000, we proposed a draft protocol, and a strategy for standardization
and validation of this assay to the OECD Validation and Management Group (Gray et al.,
2000). In the OECD Hershberger assay, castrate-immature male rats are treated orally
with androgenic chemicals for ten days and androgen-dependent tissues are evaluated
(ventral prostate, seminal vesicles, levator ani plus bulbocavernosus muscles, glans penis
and Cowper's Glands). This protocol is now in Phase 1 evaluation by OECD. Phase 1
includes two interlaboratory studies using a standardized protocol. For training purposes,
we developed a detailed set of photographs with necropsy procedures that was distributed
to the 17 participating laboratories. Three of these labs also received training on this
assay in our laboratory in Spring 2000. In Phase la, 17 laboratories were able to generate
reproducible dose-response data for sc administered testosterone propionate using the
OECD protocol. A similar study is ongoing with the antiandrogen flutamide. In addition,
we analyzed the data from the interlaboratory study and drafted a report of Phase 1 a in
Dec 2000. Currently, we are analyzing the data and preparing a final report of Phases
1a and 1b (due Oct 15, 2001) and are designing Phase 2 for presentation to the OECD
V.G. Phase 2 will involve a multi laboratory study of several different chemicals with
varying potencies and mechanisms of action.
Recently, in depth, background review papers on the pubertal male and female
28
image:
assays (78, 217) were published in Critical Reviews in Toxicology and our laboratory is
now working on a similar manuscript for the Hershberger Assay with over 250 references,
to date. In summary, we continue to provide scientific leadership on the in vivo assays
now in the EDSP program.
We also are actively involved in helping the Agency develop an enhanced
muttigenerational assay for T2 Testing. In the late 1980s to early 1990s we developed
an Alternative Reproduction Test for assessment of the reproductive toxicity of chemicals
administered across generations (Gray et al., 1988; Zenick et al., 1991). As a result of
our recommendation, vaginal opening and preputial separation, landmarks of puberty in
the female and male rat, respectively, were first added to the Agency's Developmental
Neurotoxicology and the 1996 Multigenerational Reproduction Test Guidelines. For the
EDSP program, we have helped develop a contract (wrote the protocol, selected the
chemicals and dosage levels) to examine the limitations of the current multigenerational
study to determine how it can be improved to test EDCs. Here, we have reviewed the
literature and published summaries of EDC effects in multigenerational studies (86,90,92)
and provided text and references to the contractor for the background document.
Pertinent References: 78, 82, 86, 88, 90, 92, 152, 217
29
image:
Role of Endocrinology Branch and Theme Scientists in the Agency's EDSP in vivo assays for T1S and T2T
Endocrinology
Branch/Theme
Role
Hersh-
berger
Assay
Pubertal Male Rat
Assay
Pubertal
Female Rat
Assay
Rat
Uteropic
Assay
In utero-
lactational
Assay
"One Genera-
tion" Testing
Protocol
Assay
Originator
Yes
Repro
Screen
1980
Yes
Repro Screen
1980
Yes,
EDSP
2001
Yes
ART ARMT 1991;
1998
Research to Optimize protocol for EDSP
or OECD
Yes
Yes
Yes
Yes
Drafted Standardized protocol for EDSP
or OECD
Yes
Yes
Yes
Yes
Yes
Assisted in design of validation program
Yes
Yes
Yes
Yes
Yes
Onsite and/or offsite training of
participating labs
Yes
Analyzed data from validation program
Yes
Yes
Yes
Not Yet
Not Yet
Drafted reports of validation program
Yes
Published literature review(s) of protocol
Not Yet
Yes
Yes
Yes
Drafted EDSTAC protocol for 1998
Yes
Yes
Yes
Yes
Yes
Yes
Published Interlaboratory Validation
Study
In prep.
Purpose of Assay: to Detect
Arili-
androgen
ana
Androgen
HPG axis AR
ligand
Synthesis
inhibitors
Thyroid
HPG axis
ER ligand
Synthesis
inhibitors
Thyroid
ER ligands
Estrogen
Androgen
Thyroid
HPG
Estrogen
Androgen
Thyroid
HPG
NOAELs
30
image:
CO
® ®
1 o
J> (D
S-
si W
image:
Receptor and Signal Transduction Pathways in Developmental Toxicity
Barbara Abbott, Theme Leader
I. Introduction
This research theme addresses the problems inherent in extrapolation of toxicity
data across species. In order to achieve an improvement in the application of laboratory
animal data to human health risk assessment, this project explores the potential for
mechanistic models to correlate toxicological outcomes with pathways of response that are
conserved across species. These goals also encompass the potential to identify signal
transduction pathways and receptor-mediated mechanisms that contribute to differential
responsiveness and susceptibility The need for improved harmonization and
extrapolation is identified under GPRA Goal 8, Sound Science, Research for Human
Health Risk Assessment.
The research of this theme centers on receptor-mediated pathways of response,
including growth factor and steroid hormone pathways which are critical to reproductive
function and embryonic development. The overall approaches include development of in
vitro models, correlation of responses with target tissue exposure level, evaluating the
differential sensitivities and mechanisms of response between multiple target
tissues/organs, and determining the profiles of response across dose and time for multiple
gene and protein targets in these receptor pathways. Specifically, recent and current
research efforts center on the following:
A. Improve the linkage between exposure, target tissue level, mechanistically-linked
molecular responses and gross morphological and cellular outcomes. (Poster #29)
This project developed and evaluated in vitro models using laboratory animals
(mouse and/or rat) for comparison with cultured cells/tissues from human donors
(embryonic palatal organ culture, uterine endometrial cells). The goals were to estimate
dose and evaluate biomarkers of response that would enable comparison of sensitivities
and mechanisms of response across species and between laboratory animal and human
tissues/cells. The model compound utilized in the studies was 2,3,7,8-tetrachlorodibenzo-
p-dioxin (TCDD). The research evaluating responsiveness of cultured human cells/tissues
was completed and comparisons with the animal vivo and vitro models, relationships
between dose and response, and mechanistic pathways of response were reported in
peer-reviewed manuscripts. The responses of cultured human endometrial cells were
related to clinical observations and reports of endometrial responses in the non-human
primate models. The cleft palate research models established a means of comparing
tissue exposure levels across in vivo and in vitro models and allowed evaluation of the
sensitivity of human embryonic cells relative to the rodent models
1
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B. Evaluating critical receptor-mediated pathways for potential to provide insight
into differential responsiveness and susceptibility. (Posters 30 & 31)
Studies of the responses of highly conserved, receptor-mediated signal transduction
pathways are underway to evaluate the differential responses of reproductive and
embryonic target tissues. Aspects of this project that have been completed include a study
evaluating onset of auto-regulation of glucocorticoid pathway in the rat fetus. The
acquisition of competence of regulation of the stress hormone pathway in early
fetal/neonatal development has implications for susceptibility of this life stage to toxicants
that affect and/or activate that pathway. Research was also completed to address the
issue of whether the aryl hydrocarbon receptor (AHR) was required for TCDD-induced
teratogenic responses. Current research pursues the critical role of the epidermal growth
factor receptor (EGF-R) signal transduction pathway in reproductive and developmental
toxicity. Ongoing studies reveal the importance of this pathway in maturation and function
of the mammary gland and in the responses of the mammary gland to toxicants. The
pathway is also relevant to mechanisms of induction of cleft palate and hydronephrosis in
the embryo and studies reveal the potential for specific patterns of hgand expression
during development to be related to target organ specificity and sensitivity. Further
investigations will compare the expression of the ligands during development across target
tissues and in response to a variety of toxicants. A survey of signal transduction pathway
gene expression using gene array methodology and quantitative real-time Polymerase
Chain Reaction (PCR) amplification of relevant/responsive genes has begun.
C. Mechanisms of response for endocrine disrupting compounds: vascularization
and differential sensitivity of target tissues. (Poster 32).
This project focuses on improving the understanding of the impact of endocrine
disrupting compounds on human health, through increased knowledge of the mechanisms
and pathways of response in specific tissues. Methoxychlor, an environmental estrogen,
provides an opportunity to evaluate the vascularization response to estrogenic
compounds, as unlike estrogen which stimulates both uterus and pituitary, this agent fails
to stimulate vascularization of the pituitary gland. Vasculogenesis and angiogenesis are
regulated by receptor-mediated pathways and this project evaluates expression of genes
critical to the vascularization response, comparing the responsive and non-responsive
target organs. One study recently completed under this goal described the importance of
the vasculogenesis pathway in placental development and the involvement of the aryl
hydrocarbon receptor binding partner and hypoxia-related genes in regulation of
angiogenesis. Endocrine disrupting compounds that stimulate or inhibit this pathway have
the potential to impact fertility and embryo/fetal survival through effects on reproductive
organ development and function at all life stages.
II. Impact of Research:
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image:
These research projects are dedicated to fulfilling the mandates of GPRA Goal 8,
Sound Science, Objectives 8.2 Research for Human Health Risk Assessment and 8.3
Emerging Risks Issues. These GPRA goals delineate the need to reduce the reliance on
default assumptions in Human Heath Risk Assessment. To attain this objective will require
substantial involvement of the research teams to develop new models and tools. This
theme engages the challenge of these objectives on several levels in its projects.
Poster 29. This series of studies was directed toward harmonization of mechanisms
and responses between animal models and cultured human tissues/cells (Poster 29 and
Theme aims 1 [FYOO], 7 & 15). This work is aligned with goal 8.2.1 to develop data to
reduce uncertainties in risk assessment, supporting the ORD "Strategy for Research on
Environmental Risks to Children." This harmonization effort to improve the linkage
between exposure, target tissue level and molecular mechanisms of response developed
in vitro models for mouse and human embryonic palatal development and correlated the
levels of exposure in the target tissue in vivo and in culture with mechanistically relevant
molecular responses. The data supported analysis of differential sensitivity across species
based on mode-of-action in the embryonic target tissue A cell culture model evaluated
human uterine endometrial responses to the environmental toxicant TCDD and related
those molecular endpoints to clinical status (endometriosis). Similarly, in vitro models
were developed to evaluate the hormonal responsiveness of rat and mouse pituitary and
adrenal glands, and to examine the role of growth factors in producing hydronephrosis in
the developing urinary tract of the dioxin-exposed mouse embryo.
Posters 30 & 31. GPRA Goal 8.2.1 also addresses issues of susceptibility of
specific population groups including children and the research area specifically calls for
fundamental research to improve the scientific foundation for risk assessment. This theme
participates in those efforts (Theme Aims 8, 10 [FYOO], 17, 19 & 21) by developing
mechanistic models and tools to address risks to susceptible populations by evaluating
critical mechanistic pathways that have the potential to provide insight into differential
responsiveness. Models to address sensitivity compare the differential responses
between specific tissues as well as across species. Hypotheses include questioning what
makes one tissue a target and not another, what renders one tissue more sensitive, what
differences in the pathways of response can be detected and will those differences
account for differential responses or levels of sensitivity. Studies that address these
issues are highly relevant to the extrapolation across species as well as to those rendering
specific sub-populations more or less sensitive to environmental exposures.
The aims of the theme that engage these goals include evaluation of the role of
signal transduction pathways in mediation of toxic responses. Completed projects in this
theme (Aims 8, 10, 21) examined the importance of the AH receptor pathway in
developmental and reproductive toxicity, and the potential for delayed GR auto-regulation
to participate in increased susceptibility of the rodent fetus to toxicants acting through the
stress hormone pathway. Ongoing research (Aims 17 & 19) examines the importance of
4
image:
receptor-mediated growth factor pathways in reproductive and developmental toxicity.
Signal transduction pathways are closely related across species and most are highly
conserved and are generally essential for growth and development of the embryo as well
as maintenance and function of mature tissues/organs. Effects of environmental agents
on these pathways in one species may be highly predictive of responses in other
organisms. The EGF pathway is one such signal transduction cascade and the project
includes evaluation of the importance of this pathway on prostate and mammary gland
development, lactogenic function, and the role of the ligands of the EGF receptor in
teratogenic responses of the urogenital tract and cleft palate. The application of
transgenic models and inclusion of sensitive genomics techniques will provide mechanistic
data underlying differential responsiveness of the target tissues and dependence on
specific patterns of gene expression to mediate responses.
Poster 32. As a component of goal 8.3.1 Emerging Risks, the research (Theme
aims 12,13,16, & 18) focuses on improving understanding of the modes and mechanisms
of action of Endocrine Disrupting Compounds (EDC's) which have potential impact on
human health. The Research Strategy in this area calls for research to examine the
mechanisms through which endocrine disrupting chemicals produce their effects. A
component of this theme (Aims 12, 16, 18) evaluated effects of EDC's on developing
reproductive tract, placenta, pituitary, and uterus. Research was completed on the
endocrine responses to PCBs; reproductive tract malformations in developing rat vaginal
tract exposed to TCDD; and significance of the vasculogenesis/angiogenesis pathways
in embryonic survival and placentation. The study of the environmental estrogen
methoxychlor is ongoing (Aim 13) and addresses issues of responsiveness and target
specificity through involvement of the vasculogenesis pathway gene cascade. Endocrine
disruption of the pathway would be expected to impact fertility and embryo survival. This
aim continues to examine the signal transduction and endocrine pathways that regulate
the vascularization of the pituitary and the uterus. Vascularization is susceptible to
regulation by estrogens, and the pituitary and uterus respond to estrogenic chemicals with
increased vascularity. The atypical response of the pituitary to the environmental estrogen
methoxychlor provides a model for study of the estrogenic regulation of genes involved in
angiogenesis.
To summarize, this Research Theme provides increased understanding of the
involvement of receptor mediated pathways in reproductive and developmental toxicity and
can provide a basis for comparisons across species and to evaluate the potential for these
pathways to mediate similar responses in the human. This research as a whole addresses
the impact of toxicants on receptor pathways which are essential for normal embryo/fetal
development and reproductive function. A science-based risk-management strategy relies
on in-depth understanding of the biological pathways through which environmental
toxicants produce their biological effects. This level of knowledge provides a foundation
for interspecies comparisons, evaluation of the potential for low-dose responses, and
identification of population groups or individuals that may have greater susceptibility due
5
image:
to gene expression profiles associated with responsiveness.
III. Future Directions of the Theme:
The EDC component of this research is in initial stages and ongoing work includes
characterization of changes in expression of vascular growth factors in the pituitary and
uterus that accompany exposure to the estrogenic pesticide methoxychlor and which may
underlie the differential effects on tissue growth seen in the two tissues.
The importance of signal transduction pathways in mammary gland and the
developing embryo will be further characterized with the knockout mouse models and with
genomics approaches to study gene expression profiles. The strength of DNA array
technology will be utilized to generate profiles of responses in both mammary and
embryonic palatal tissues. Arrays for signal transduction genes and regulatory genes
important in development will be surveyed for effects of toxicants. These profiles are
expected to be informative regarding not only specific signal transduction pathways, but
the potential for association of gene profiles with mode-of-action. The studies of
responses of mammary gland will include prenatal exposure and postnatal maturation, as
well as effects on mammary maturation and function during puberty, pregnancy, and
lactation. The induction of cleft palate provides well characterized models (in vivo and in
vitro) in which to profile alterations in gene expression in response to developmental
toxicants and it will be of interest to challenge the developing embryo with several cleft
palate teratogens. The profiles of gene expression will be compared for teratogens that
each have differing underlying morphological basis for induction of the clefts.
The studies that specifically address the importance of the EGF-R pathway provide
an additional approach to mechanistic questions that impact extrapolation as well as
identification of sensitive populations. Human subpopulations expressing specific isoforms
of the TGF-alpha gene (an EGF-R ligand) have been associated with increased incidence
of cleft palate. Further, the EGF-R partner erb-B2, or neu, has been implicated in
metastatic cancers of the breast. The research project of this theme that examined the
responses of mammary gland revealed increased sensitivity to toxicants in knockouts with
compromised EGF pathways. There should be concern regarding the potential for
increased sensitivity in individuals with specific variants of critical genes. The potential
interaction of environmental exposures and specific genotypic variants presents an
extremely difficult research area to address in the laboratory. The signal transduction and
receptor models which are in development and under study show potential for examination
of at least some of the major issues in this area.
IV. Resources:
Total FTE: 5. Participating Staff: Barbara Abbott, Angela Buckalew, Suzanne
Fenton, Jerome Goldman, Gary Held, Christopher Lau, Ashley Shearin-Murr,
Carmen Wood, Robert Zucker.
6
image:
V. External Collaborators: The completed project to evaluate human endometrial tissue
responses to environmental toxicants was in collaboration with Dr. Dianne Bofinger
(SUNY). The project to evaluate the importance of the EGF signal transduction pathway
utilizes transgenic mice with inactivation of the EGF, TGF-alpha, EGF + TGF, and wild
type background strain mice which were provided through Dr. David Lee's laboratory (UNC
Chapel Hill). Characterizing the requirement for AH receptor for teratogenic responses
was in collaboration with Dr. Jeff Peters (NIH). Evaluation of the vasculogenesis pathway
in placental development was facilitated by use of knockout mice for AHR nuclear
translocator protein, which were characterized in collaboration with Dr. Oliver Hankinson
(UCLA). Recently a collaboration was initiated with Dr. Richard Peterson, University of
Wisconsin at Madison, to study the role of EGF signal transduction in responsiveness of
the developing prostate gland to environmental toxicants.
VI. Productivity:
1 Abbott, B.D. (1997) Developmental toxicity of dioxin: Searching for the cellular
and molecular basis of morphological responses. In Handbook of Experimental
Pharmacology: Section III: Pathogenesis and Mechanisms of Drug Toxicity in
Development (R. Kavlock and G. Daston, Eds.), pp. 407-433. Springer-Verlag,
New York.
2. Abbott, B.D., Probst, M.R., Perdew, G.H., and Buckalew, A.R. (1998). AH
receptor, ARNT, glucocorticoid receptor, EGF receptor, EGF, TGF alpha, TGF
beta 1, TGF beta 2, and TGF beta 3 expression in human embryonic palate, and
effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Teratology. 58, 30-43.
3. Abbott, B.D., and Birnbaum, L.S. (1998). Dioxins and teratogenesis. In Molecular
Biology of the Toxic Response (A. Puga and K. Wallace, Eds.), pp. 439-447.
Taylor and Francis, Washington, D.C.
4. Abbott, B.D., Held, G.A., Wood, C.R., Buckalew, A.R., Brown, J.G., and Schmid,
J. (1999). AhR, ARNT, and CYP1A1 mRNA quantitation in cultured human
embryonic palates exposed to TCDD and comparison with mouse palate in vivo
and in culture. Toxicol Sci 47, 62-75.
5. Abbott, B.D., Schmid, J.E., Brown, J.G., Wood, C.R., White, R.D., Buckalew,
A.R., and Held, G.A. (1999). RT-PCR quantification of AHR, ARNT, GR, and
CYP1A1 mRNA in craniofacial tissues of embryonic mice exposed to 2,3,7,8-
tetrachlorodibenzo-p-dioxin and hydrocortisone. Toxicol Sci. 47, 76-85.
6. Abbott, B.D., Schmid, J.E., Pitt, J.A., Buckalew, A.R., Wood, C.R., Held, G.A.,
and Diliberto, J.J. (1999). Adverse reproductive outcomes in the transgenic Ah
receptor-deficient mouse. Toxicol Appl Pharmacol. 155,62-70.
7
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7. Abbott, B.D., and Buckalew, A.R. (2000). Placental defects in ARNT-knockout
conceptus correlate with localized decreases in VEGF-R2, Ang-1, and Tie-2.
DevDyn. 219,526-538.
8. Abbott, B.D. (2000). Palatal Dysmorphogenesis: Palate Organ Culture. In
Methods in Molecular Biology: Developmental Biology Protocols, Vol. II (R. S.
Tuan and C. W. Lo, Eds.), pp. 189-195. Humana Press, Inc, Totowa, NJ.
9. Abbott, B.D. (2000). Insights from AhR and ARNT gene knockout studies
regarding responses to TCDD and regulation of normal embryonic development.
Congenital Anomalies Supplement. 40, s88-s93.
10. Abbott, B.D. (In Press). Laboratory Animal Models for the Study of Oral Clefts. In
Cleft Lip and Palate. From Origin to Treatment. (D. F. Wyszynski, Ed.), Oxford
University Press, New York.
11. Bryant, P.L., Clark, G.C., Probst, M.R., and Abbott, B.D. (1997). Effects of
TCDD on Ah receptor, ARNT, EGF, and TGF-alpha expression in embryonic
mouse urinary tract. Teratology. 55, 326-337.
12. Bryant, P.L., Reid, L.M., Schmid, J.E., Buckalew, A.R., and Abbott, B.D. (2001).
Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on fetal mouse urinary
tract epithelium in vitro. Toxicology. 162,23-34.
13. Bryant, P.L., Schmid, J.E., Fenton, S.E., Buckalew, A.R., and Abbott, B.D.
(2001). Teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice
lacking the expression of EGF and/or TGF-alpha. Toxicol Sci. 62,103-114.
14. Bryant, P.L., Reid, L.M., Diala, E.S., Buckalew, A.R., and Abbott, B.D. (In
Press). An in vitro model for murine ureteric epithelial cells. In Vitro Cellular &
Developmental Biology.
15. Ghosh, B., Wood, C.R., Held, G.A., Abbott, B.D., and Lau, C. (2000).
Glucocorticoid receptor regulation in the rat embryo: a potential site for
developmental toxicity? Toxicol Appl Pharmacol. 164,221-229.
16. Held, G.A., and Abbott, B.D. (2000). Palatal dysmorphogenesis. Quantitative
RT-PCR. Methods Mol Biol. 136,203-217.
17. Hurst, C.H., Abbott, B.D., DeVito, M.J., and Birnbaum, L.S. (1998). 2,3,7,8-
Tetrachlorodibenzo-p-dioxin in pregnant Long Evans rats: disposition to
maternal and embryo/fetal tissues. Toxicol Sci. 45,129-136.
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18. Hurst, C., Abbott, B.D., and Birnbaum, L.S. (Submitted). 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD) disrupts early morphogenetic events that
form the lower reproductive tract in female rat fetuses. Toxicological Sciences.
19. Kozak, K.R., Abbott, B.D., and Hankinson, O. (1997). ARNT-deficient mice and
placental differentiation. Dev Biol. 191,297-305.
20. Peters, J.M., Narotsky, M.G., Elizondo, G., Fernandez-Salguero, P.M.,
Gonzalez, F.J., and Abbott, B.D. (1999). Amelioration of TCDD-induced
teratogenesis in aryl hydrocarbon receptor (AhR)-null mice. Toxicol Sci. 47, 86-
92.
21. Pitt, J.A., Buckalew, A.R., House, D.E., and Abbott, B.D. (2000).
Adrenocorticotropin (ACTH) and corticosterone secretion by perifused pituitary
and adrenal glands from rodents exposed to 2,3,7, 8- tetrachlorodibenzo-p-
dioxin (TCDD). Toxicology. 151,25-35.
22. Pitt, J.A., Feng, L., Abbott, B.D., Schmid, J., Batt, R.E., Costich, T.G., Koury,
S.T., and Bofinger, D.P. (2001). Expression of AhR and ARNT mRNA in
cultured human endometrial explants exposed to TCDD. Toxicol Sci. 62, 289-
298.
Activities Relevant to Stakeholders:
Activities relevant to stakeholders are reflectred in the Biographical sketches of
the participating principal investigors (refer to the Honors and Awards, Professional
Activities, and Invited presentations sections). The publications originating from this
Theme's research activites are listed above.
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Poster 29. Harmonization of mechanisms and responses across animal models
and cultured human tissues/cells. BD Abbott, A Buckalew, G Held, C Wood, J
Schmid, JA Pitt1, PL Bryant2, D Bofinger3. 1Curriculum in Toxicology, UNC; 2School of
Public Health, UNC; 3SUNY, Buffalo.
The Human Health Risk Assessment Research Strategy identifies a need for
research to improve the linkage between exposure and dose, to assist in determining
the critical exposure and biologically effective dose, and to provide mechanistic
information that is applicable across species. Research addressing these critical areas
has the potential to provide support for extrapolation of data from laboratory animal
models to humans and in addition to improve the risk assessment process through
mechanistically-based interpretations of the data. The Receptor and Signal
Transduction Pathways Theme conducted research (Aims 1, 7 and 15) to improve
linkage between exposure and target tissue level and to correlate exposure level with
responses at the gross morphological and the molecular, mechanistic level. This
project emphasized comparisons across species and harmonization between in vivo
and in vitro models. The models evaluated responses to the environmental
contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the fetal secondary palate
of mouse (in vivo and vitro models) and human (palatal organ culture), the mouse
urinary tract (in vivo and vitro models), the rat and mouse pituitary and adrenal glands
(perifusion model), and human endometrial tissues in organ culture. The teratogenicity
of TCDD was evaluated in the mouse model which exhibits cleft palate and
hydronephrosis. The etiology of the teratogenic responses was characterized at the
morphological and cellular level. Critical response pathways were identified and
expression profiles of mechanistically important mRNA and protein were established,
including the aryl hydrocarbon receptor (AhR) which binds TCDD, its signaling partner,
ARNT, the dioxin-regulated cytochrome, CYP1A1, and members of several growth
factor pathways. In vitro models were developed and the distribution of chemical was
assessed for vivo and vitro exposures. The integration of this dose/response data,
target tissue levels, expression profiles of key biomolecules, and morphological
responses across species permitted insight into the responses and sensitivity of the
human fetal tissues relative to the laboratory animal model. In collaboration with Dr.
Diane Bofinger, SUNY at Buffalo, the responsiveness of human endometrial tissues
was evaluated in an explant culture system and correlated with expression of the Ah
receptor, ARNT and the regulation of bio-marker cytochromes CYP1A1 and CYP1B1.
The gene expression and morphological responses of the biopsy specimens in culture
were correlated with donor age, proliferative or secretory stage of the tissue, hormonal
responsiveness in culture (exposure to estradiol and/or progesterone), and the
presence of endometriosis in the donors. In a similar cross-species study, hormonal
responsiveness of pituitary and adrenal tissue was compared following in vivo
exposure to TCDD and using a perifusion model to evaluate the similarities and/or
differences between species and sexes (rat vs mouse, male vs pregnant female).
Studies of the mouse urinary tract also utilized an in vitro approach to characterize
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dose/response and growth factor involvement in that target tissue. The results
indicated that the epidermal growth factor (EGF) receptor pathway was involved in
responses of the urinary tract. As this is a key signal transduction pathway required for
regulation of cell proliferation and differentiation, further study of the involvement of
these proteins is underway (as presented in abstracts 20, 22) utilizing mice in which the
one or more of the ligands for EGF receptor is inactivated in knockout models.
Manuscripts specific to these efforts were recently completed and reported as peer-
reviewed journal articles.
Pertinent References: 1, 2, 3, 4, 5, 8, 10, 30, 31, 32, 33, 97, 177, 178
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Poster 30. Study of receptor-mediated pathways to reveal potential mechanisms
of differential sensitivity and responsiveness. BD Abbott, SE Fenton, CS Lau, R
Hanson, A Buckalew, G Held, C Wood, M Narotsky, J Schmid, J Diliberto1, J Peters2, B
Ghosh3. 1ETD,NHEERL, 2NIH, 3Curriculum in Toxicology, UNC
The Human Health Risk Assessment Strategy identifies a need for basic
research to reduce uncertainties and improve extrapolation by providing mechanistic
data specifically addressing determining factors that underlie differential
responsiveness and susceptibility to environmental toxicants. The research conducted
in this theme focuses on evaluation of chemical factors that potentially contribute to
differential responsiveness in gene expression or signal transduction pathways
deemed to be common across species and that are critical to embryo/fetal
development. Several of our research initiatives (Aims 8, 21, & 10) were recently
completed and these include study of the onset of auto-regulation of the glucocorticoid
pathway as a factor influencing susceptibility. Receptor auto-regulation is an integral
function for maintenance of the hypothalamic-pituitary-adrenal axis and alteration in the
timing of onset of competence of this system during development has the potential to
permanently alter functions and contribute to increased risk. Mapping onset of this
function defined a period of vulnerability and such information is relevant to improved
understanding of critical windows of exposure for children's health as well as lasting
effects in subsequent life stages. Studies in this thematic program also included
evaluation of the genes critical to the mechanism of response to dioxin. The
significance of the aryl hydrocarbon receptor (AhR) in mediating the teratogenic
responses to TCDD and in reproductive performance in mice was demonstrated in a
knockout mouse model lacking expression of AhR. Mice without expression of AhR
exhibited poor survival during pregnancy and decreased survival of pups in utero.
during the lactation period and after weaning. AhR was shown to be required for
induction of the teratogenic responses. A major and on-going research effort (Aim19)
in this thematic area involves defining the critical role of the epidermal growth factor
signal transduction pathway in developmental toxicity. Previous research
demonstrated altered regulation of the EGF receptor and its ligands in response to
several cleft palate and urinary tract teratogens. Current projects utilize knockout
animals in which one or more of these ligands is not expressed to evaluate responses
to teratogens and to link specific patterns of gene expression to target tissue selectivity
and sensitivity. There is a potentially broad applicability of these investigations as
signal transduction pathways such as EGF regulate growth and differentiation in a wide
range of organ systems and are important regulators of development in all mammalian
species. Future efforts in this research area compare gene expression profiles from
additional teratogens with the goal of detecting patterns associated with induction of
cleft palate. This effort will expand the signal transduction pathways evaluated through
surveys of gene expression via DNA array, quantification of gene expression with real
time PCR, localization and semi-quantification of protein expression with
immunohistochemical methods. Manuscripts specific to these efforts were recently
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completed and reported as peer-reviewed journal articles.
Pertinent References: 6, 9, 76, 130, 175
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Poster 31. Environmental toxicant effects on the developing prostate and
mammary gland: Mechanistic data on signal transduction pathways underlying
differential responsiveness and susceptibility. SE Fenton1, PL Bryant2, A
Buckalew2, and BD Abbott2. 1 Endocrinology and 2 Developmental Biology Branches,
Reproductive Toxicology Division, NHEERL, ORD, U.S. EPA. TM Lin and RE Peterson,
School of Pharmacy, University of Wisconsin, Madison, Wl.
A research area of the Human Health Risk Assessment Strategy addresses the
need to identify common modes of action of a chemical which may affect an individuals
susceptibility to toxicant exposure (Goal 8; 8.2.1). Definition of the mechanistic order of
events within the mode of action may allow identification of steps that are similar
between species, a sensitive subpopulation, or an early biomarker of exposure. Our
recent studies have uncovered a "critical period" in rat and mouse fetal mammary gland
development during which exposure to TCDD causes a persistent delay in mammary
epithelial growth and differentiation. Other environmental agents have been identified
that cause similar underdeveloped mammary glands following acute exposure. Many
hormones and growth factors are responsible for the normal maturation of the
mammary tissues, and these factors are comparable between rodents and humans.
However, the signaling pathway leading to the delayed mammary development
following gestational exposure to environmental agents, such as TCDD, is not known.
Studies in other laboratories have identified the epidermal growth factor receptor,
EGFR, as a integral cell membrane receptor that is altered upon gestational exposure
of male reproductive tract organs to TCDD. Further, this family of receptors (including
erbB2 or neu) is involved in metastatic breast cancer in humans. Therefore, we have
utilized a series of knockout mice lacking the ligands for the EGFR, as single or
double-null genotypes, to show that EGF and transforming growth factor-alpha (TGF-a)
are critical components in the TCDD-induced delay of mammary gland development.
Because there are 6 known ligands for the EGFR, their compensatory gene regulation
following a toxicant insult will be ascertained in future studies. Additionally, it will be
imperative to determine whether or not the wild-type and knockout animals lacking
these ligands (with or without a dose-response exposure to TCDD) are resistant to
chemical carcinogen-induced mammary tumors. If these studies prove that the EGFR
ligands are critical elements in normal mammary and breast tumor development, there
should be concern for increased sensitivity in individuals with specific variants of the
gene family members. Furthermore, recent collaborative studies with Drs. Richard
Peterson and Tien-Min Lin, University of Wl-Madison, preliminarily demonstrate that
prostatic tissue from male mouse fetuses exposed to TCDD on gestation day 12 show
specific lack of bud outgrowth and sensitivity/selectivity for this effect can be altered by
lack of expression of EGFR ligands. We will continue to use the EGFR ligand-null
mouse lines as a model to anticipate future environmental compounds that could
enhance a person's chances of suffering from breast or prostate cancer late in life
following an early life exposure. We believe that this knowledge will improve our ability
to assess risk in sensitive human subpopulations such as pregnant or breast-feeding
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mothers and their children, environmentally exposed wildlife such as seals and mink,
and other endangered species which already have diminishing populations.
Pertinent References: None (New start)
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Poster 32. Toxicant-induced effects upon vascular growth: Impact of endocrine-
disrupting chemicals on pre-natal development and organ integrity within the
reproductive system. J Goldman, B Abbott, A Murr, A Buckalew, R Zucker, C Lau, R
Hanson, G Held, C Wood, C Hurst1. 1Curriculum in Toxicology, UNC.
Much of the concern about the potential adverse health effects of endocrine-
disrupting compounds has been on the ability of these chemicals to affect early
development and later reproductive activity by compromising normal hormonal
regulatory functions. Our focus has been on identifying receptor-mediated mechanisms
through which environmental agents disrupt endocrine-mediated pathways. This effort
is consistent with the goals for Sound Science in the areas of Emerging Risks,
including the focus on Endocrine Disrupting Chemicals. Our research in this area
includes several completed Theme Aims (12, 16, and 18) which examined a range of
response pathways that are critical to normal embryonic development and reproductive
integrity. One critical activity in cell or tissue proliferation is the emergence of an
enlarged vascular network to supply cells with oxygen and nutrients and to take away
wastes. This outgrowth of new capillaries from pre-existing blood vessels, known as
angiogenesis, is an important natural process and underlies fetal and placental
development and normal proliferative events in the female uterus. Angiogenesis is a
receptor-mediated process with a complex cascade of regulatory steps, influenced by
exposure to estrogen, and also initiated by hypoxia. Genes which regulate
angiogenesis include the helix-loop-helix (HLH) family of regulatory proteins (which
includes the aryl hydrocarbon receptor (AhR), its partner protein ARNT, and the
hypoxic induction factor 1-alpha) Studies in this theme established that knockout of Ah
receptor nuclear translocator (ARNT) was lethal in early development due to abnormal
placental development. Evaluation of the mechanism involved revealed an important
link between this HLH peptide family and regulation of genes critical to vasculogenesis.
The impact of perturbation of this signaling cascade extends across all aspects of
reproduction and development A classic angiogenic promoter in the reproductive
system is estradiol, which will induce uterine growth and under extended exposures
cause tumorigenesis in a variety of tissues. A growing number of environmental agents
have estrogenic properties, including the capacity to induce angiogenic activity in
responsive tissues and impair normal reproductive activity, something which has been
of concern to the Agency and a focus of efforts within the Endocrine Disruptors
Screening Program. This endocrine-regulated angiogenic process is the focus of an
ongoing research effort (Aim 13). For this work, we have chosen to focus on the
expression of angiogenesis factors and markers of vascular growth (i.e., vascular
endothelial growth factor [VEGF], its receptor VEGF-R2, angiopoietin-1 [Ang-1] and -2
[Ang-2], the Ang-1 receptor Tie-2, and PECAM [platelet/endothelial cell adhesion
molecule]). The pesticide methoxychlor has been chosen for study (along with estradiol
as a positive control), since its primary metabolite is estrogenic, but unlike estradiol,
exhibits a differential effect on tissue growth within the uterus and pituitary. Pilot
semiquantitative evaluations of the expression of these angiogenic factors following
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methoxychlor exposure of ovariectomized rats underscored these prior observations
and further study is underway to delineate these effects. Since vascular growth is
critical to early development and affects the impact of estrogenic chemicals on
reproductive function, an assessment of the toxicant-associated alterations in these
angiogenic factors will contribute to an understanding of the mode(s) of action under a
variety of exposure paradigms Manuscripts specific to these efforts were recently
completed and reported as peer-reviewed journal articles
Pertinent References: 7,104,105
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Mechanisms of Toxicity and Susceptibility
John Rogers, Theme Leader
I. Introduction:
The "Mechanisms of Toxicity and Susceptibility" theme is new, having been started
just this year. Some of the research, however, carries forward from past research
programs on biologically based dose response models, methanol, and maternal nutrition
and toxicity. The focus of this theme is on developing mode-of-action approaches to
reduce uncertainty factors in extrapolation of animal developmental toxicity data, and
identifying and quantifying altered susceptibility to environmentally- induced disease
based on life-stage, nutrition, genetics or other factors Current study designs for
assessment of developmental toxicity do not consider these factors explicitly (with the
exception of requiring two species), but rather assume that any added risk they may carry
will be protected against by default uncertainty factors. This theme will examine specific
modes of action of a variety of developmental toxicants and interactions with specific
maternal conditions. Thus, our work tests the assumption that default uncertainty factors
protect individuals with conditions that may predispose them to increased risk of adverse
pregnancy outcome from environmental exposures.
To make real improvements in risk assessments for developmental toxicity, we are
striving to take advantage of the latest technologies and emerging knowledge in
developmental biology and toxicology, using these to better understand adverse effects
of developmental exposures. Recent major advances include elucidation of the genetic
control of development, new technologies to broadly evaluate patterns of gene and protein
expression, the importance of folate in the prevention of birth defects, and potential long-
term effects of the in utero environment. We have chosen a set of studies designed to
improve our ability to detect and extrapolate adverse effects in animal models to the
human situation. We have attempted to leverage these studies to address different goals
within the theme. For example, we are using maternal/embryonal folate deficiency in both
our BBDR studies of tomudex (a folate analog) and to study the potential role of folate in
conferring heightened developmental susceptibility to diverse toxicants. We have chosen
to pursue the mode of action of perfluorooctane sulfonate (PFOS) because of its
environmental pervasiveness and Agency relevance, as well as the advantage of having
identified putative modes of action. We will also use PFOS as one of the compounds in
our studies of the long-term effects of in utero toxicity, as well as for our gene array studies
to examine changes in patterns of embryonal gene expression as a means to identify
potential modes of action.
Specifically, our current research addresses these questions, which will be the
topics for the posters we will present:
How can mode of action/mechanistic data be used to reduce uncertainty factors and
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improve our ability to predict biological responses? (#33) In this project we have
developed a BBDR model for the developmental toxicity of the prototype teratogen 5-
fluorouracil (a thymidylate synthetase [TS] inhibitor), tested the model using another TS
inhibitor (tomudex), and are extending the model by examining the effects of varying
embryonal folate supply
Are there long-term effects of in utero exposures against which our current tests and
risk assessments may not be adequately protective? (#34) Recent studies suggest
that birth weight is inversely correlated with life-long morbidity in humans, but the biology
underlying this correlation is poorly understood. Does this relationship hold for toxic
effects on fetal growth in our animal models?
What are the critical physiologic and mechanistic factors that contribute to adverse
developmental outcome? (#35) By using candidate gene and gene array approaches to
evaluate toxicant-induced alterations in gene expression, we hope to identify critical
pathways of toxicity that can be assessed earlier and with greater sensitivity than
conventional endpoints.
Does reduced maternal folate status or embryonal folate availability increase
susceptibility to developmental toxicants? (#36) Folate supplementation is known to
reduce birth defects in humans. Pregnant rats rendered folate deficient or embryos grown
in folate-depleted sera will be exposed to tomudex (a toxic folate analog) as well as
diverse environmental toxicants to evaluate their susceptibility compared to folate-replete
animals.
What is the mode of action for the developmental toxicity of perfluorooctane
sulfonate (PFOS)? (#37) PFOS is a wide-spread environmental contaminant that
produces neonatal mortality in experimental animals. Putative modes of action include
inhibition of cholesterol synthesis and inhibition of thyroid function. We hope to use our
experience in BBDR modeling to guide our new multi-divisional studies on the
developmental toxicity of PFOS.
II. Impact of the Research:
The practical application of the results of several of the Specific Aims in this theme
could be relatively direct and short-term. If we demonstrate that in utero exposures that
induce little or no toxicity in term fetuses bear consequences later in life (Specific Aims 3
and 5), this could be an impetus for development of tests to specifically evaluate such
effects. Such results would also reinforce the Agency position that effects on fetal weight
are of equal importance to other adverse developmental outcomes following prenatal
exposures. More sensitive tests for functional deficits in offspring of exposed mothers are
still needed. The large-litter bioassay (Specific Aim 6) being explored in this theme may
provide a basis for testing for postnatal functional performance in an integrated manner.
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Our studies on the developmental toxicity of PFOS will aid the Agency in regulatory
considerations around a large number of commercial products related to this compound.
Other components of this theme have longer-term, yet no less clear benefit to
accomplishing the goals of GPRA and the Human Health Research Strategy. The
elucidation of the effects of altered maternal folate status on the expression of
developmental toxicity has obvious public health ramifications, but also serves to exemplify
the importance of considering the diverse factors that contribute to increased susceptibility
of some factions of our population. While such factors are often cited, they are rarely
studied. Understanding common modes of action for developmental toxicants will better
inform the risk assessment process, and may also help to point to potential compound
class-specific factors which may increase susceptibility. Having quantitative predictive
models based on model of action will allow much-improved risk assessments for
susceptible populations compared to the present situation.
33. Construction of a biologically based dose response model for developmental
toxicity in the rat. (Lau, et. al.)
The Human Health Research Strategy outlines research aimed at improving the
scientific foundation of human health risk assessment, and our research addresses the
"Effects" component (i.e., the dose = effect part of the continuum) of this element of the
Strategy. Specific objectives in the Strategy include identifying common modes of action
and developing mechanistic models of chemical effects, and developing mechanistic
models to study susceptible populations.
Work under Specific Aim 4 of this theme, and its predecessors, has developed a
mathematical model of the cascade of events between exposure and adverse
developmental outcome, using the prototype teratogen 5-fluorouracil, which alters
nucleotide pools. This effort has recently been extended by examining another teratogen
putatively affecting the same pathway, and by combining this exposure with perturbed
folate availability (see also Specific Aim 1), which can also affect nucleotide synthesis.
Perturbations of nucleotide synthesis and one-carbon metabolism can play a role in both
adverse developmental outcome and carcinogenesis, so this research may also move us
closer to harmonization of risk assessments for cancer and noncancer endpoints.
Interestingly, our modeling of the developmental toxicity of 5-FU suggests that this may be
a non-threshold response, more akin to assumptions for cancer endpoints than for
noncancer endpoints. Further, folate status may be a strong determinant of increased
susceptibility to both birth defects and cancer.
While it is widely held that mode of action information and BBDR models of toxicity
will inform the risk assessment process for developmental toxicity as well as other toxic
effects in humans, the ways in which such data will actually be used in the risk assessment
process remain to be enumerated. The most direct application of such information is the
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determination of whether the toxic pathway elucidated in an experimental animal species
is present in humans. This can be at the qualitative level (which speaks to the relevance
of the test species data), or may be extended to a quantitative comparison of the pathway
(i.e., a metabolic rate) in the animal model and the human, which could be used in a PBPK
model to predict target dose. The "effect" part of the BBDR model could include
determining dose response relationships for mechanistic endpoints in the cascade of
events leading to toxicty (i.e., enzyme activities, macromolecule syntheses, cell
proliferation rates, growth). These data could be used to posit particular dose response
models as being most appropriate for a given exposure and effect, and may allow
quantitative measures of effect at dosages below those eliciting the adverse outcome of
interest. However, it follows that repair capacity of effects on mechanistic endpoints will
have to be considered as well.
The data we have published to date on our 5-FU work demonstrate some of these
principals, and provides a model of a small piece of embryonal development that will be
useful for a variety of agents that might effect nucleotide synthesis or one-carbon
metabolism. While 5-FU is a prototype, our results demonstrate that mechanistic data
(here, TS activity and nucleotide pool imbalance) can be used to predict toxic oucome and
to choose an apropriate dose response model.
34. Postnatal sequelae of low birth weight; effects of maternal toxicity-induced
weight loss during gestation on fetal growth; and susceptible populations (Chernoff,
et. al.).
Life-long morbidity associated with a suboptimal in utero environment has been
demonstrated for humans and posited for experimental animals, but there are no extant
animal models for these effects. We seek to determine whether in utero insults including
toxic exposure or nutritional deprivation, which may only produce subtle toxicity in the
fetus, are associated with permanent effects later in life or in subsequent generations.
Elucidation of such effects could demonstrate a need for new testing paradigms or
alterations in existing tests for effects of prenatal exposures.
Specific Aim 6 of this theme seeks to develop a method for detecting subtle
functional effects in offspring of exposed animals. In this approach, neonates are stressed
by being placed in large artificial litters, forcing them to compete for limited resources.
While tests for specific organ function have been developed, our approach could produce
a holistic test of the vitality of the neonate, potentially demonstrating effects that would
otherwise not be apparent using current protocols. Specific Aims 2 and 4 are directed at
developing mechanistic models, and Specific Aim 1 examines the role of nutritional status
in increasing sensitivity, and all of the work under this theme is directed at the special
sensitivity of the developing organism. Specific Aims 3 and 5 examine unique endpoints
of toxicity following developmental exposure (long-term and intergenerational effects of in
utero exposure).
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Under GPRA Goal 3, Safe Food, there are major concerns about potentially
vulnerable developmental/maturational periods; which health endpoints are of greatest
concern; whether exposures experienced by children, as opposed to adults, produce
qualitatively different effects; and whether our current risk assessments adequately
protect children and other sensitive subpopulations from unreasonable risk.
All of the work in this theme is directed at determining the most vulnerable developmental
periods for specific effects and/or specific insults. Specific Aims 3,5,6, and 7 address the
special sensitivity of the developing organism, and the possibility of the induction of effects
different from those seen in adults.
35. Molecular approaches towards understanding mode of a ction in developmental
toxicity (Rosen, et. al.)
The Human Health Research Strategy posits that new technologies can be expected
to further our understanding of modes of action of environmental chemicals, and in this theme
we are exploring the use of gene array technology to elucidate critical pathways of
developmental toxicity. Gene expression analyses are carried out using commercially available
arrays following developmental toxicant exposure, and suites of genes whose expression is
changed are determined. This approach is expected to serve an hypothesis-generating function
that will lead to elucidation of important models of action for developmental toxicants.
The major obstacle in using mode of action data for environmental chemicals is that such
data rarely exist and are exceedingly hard to generate in the laboratory, particularly for
developmental toxicity given the complexity of embryo/fetal development. Use of gene array
technology (Specific Aim 8) represents a potentially powerful new approach based on the
premise that early steps in the cascade of events leading to toxicity will include or induce
changes in patterns of gene expression. The critical step of generating hypotheses about
putative modes of action is currently quite difficult for agents of unknown biochemical
mechanisms, such as is the case for the majority of environmental contaminants. Gene arrays
hold the promise, once they are developed using better understood toxicants, of providing at
least the first step in elucidating the etiology of environmentally-induced developmental
pathology.
36. Evaluation of the role of folate in susceptibility to developmental toxicants (Andrews,
et. al.)
Current studies (Specific Aim 1) examine the role of maternal folate status in response
to developmental toxicants. The first step in this process is to determine dietary folate levels
that are low (minimal for maintenance of pregnancy), adequate, and supplemental. Female rats
raised on these diets will then be exposed during pregnancy to tomudex (a folate analog used
here as a positive control/proof of concept) or diverse environmental compounds in a dose-
response fashion, and the effect of maternal folate status on dose response will be determined.
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While any number of dietary insufficiencies or other maternal factors could be hypothesized to
influence susceptibility to developmental toxicants, and could be chosen for study. Materns
folate status has been well-characterized and the positive effects of folate supplementation on
pregnancy outcome demonstrated in humans. Results of our animal studies will determine how
maternal folate status affects response to toxic exposures during pregnancy, providing at least
a first step in and an example of considering such factors in risk assessment and risk
management.
37. The developmental toxicity of perfiuorooctane sulfonate (PFOS), including studies on
putative modes of action (Rogers, et. al.).
PFOS is a ubiquitous and persistent environmental contaminant that causes
developmental toxicity in rodents and rabbits While this has been adequately demonstrated
by the registrant, the risk to humans remains unknown Our work will focus on elucidating the
mode of action for the neonatal mortality induced by maternal exposure to PFOS during
pregnancy, with the potential for expanding into development of a BBDR model for this
chemical. Information on model of action or development! of a BBDR model will improve our
ability assess the potential developmental risk to humans. PFOS and related chemicals are one
class of persistent environmental contaminant that is targeted for research in the Human Health
Research Strategy.
III. Future Directions:
Many of the Specific Aims and studies discussed above are relatively new and will likely
extend over a 3-5 year time frame. The studies of the relationship between maternal folate
status and expression of developmental toxicity (Specific Aim 1) will continue for at least the
next three years, and will include studies of the interactions of maternal folate status with the
developmental toxicity of diverse toxicants Studies on the long-term effects of the in utero
environment and intergenerational effects (Specific Aims 3 and 5) are just getting started and
will also require multiple years to complete, since we will have to study gestationally-exposed
offspring throughout their lives. We have made rapid progress in our studies of the
developmental toxicity of PFOS (Specific Aim 2), yet it remains difficult to predict where these
studies will lead us. Ideally, we will elucidate a mode or modes of action for PFOS that will be
amenable to incorporation into a BBDR model. The work on gene array technology (Specific
Aim 8) is in its infancy and will hopefully grow in scope and effort over the next 3-5 years,
attempting to identify patterns of changes in gene expression associated with specific modes
of action, chemical classes, and/or adverse outcomes. The project on developing BBDR models
for developmental toxicity (Specific Aim 4) was started a decade ago, and continues on a more
modest scale. The hope is to be able to apply at least parts of the models developed in that
study to our work on folate (e.g., evaluation of changes in nucleotide pools), and to expand the
model where necessary. Ideally, the experience gained in our work with 5-FU and tomudex will
provide guiding principals for building BBDR models for other critical pathways of
developmental toxicity as well, perhaps including effects of PFOS on cholesterol synthesis
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and/or maternal/fetal thyroid status.
IV. Staff (FTEs):
Total FTE: 7.0. Participating Staff: Barbara Abbott, Angela Buckalew, James Andrews, Harriette
Nichols, Brenda Barbee, Neil Chernoff, Chris Lau, Julie Thibodeaux, Brian Grey, Leonard Mole,
Mitch Rosen, Robert Zucker.
V. External Collaborators:
Current collaborations include NHEERL's Mid-Continent Ecology Division, The 3M Company,
and Dr. John Geisey (Mich State U.) (Specific Aim 2), Dr. Tom Sadler (UNC, Specific Aim 1).
Specific Aims 3 and 5 (Long-term and intergenerational effects) will benefit greatly from
additional collaborators from other health divisions, as a battery of physiological tests will need
to be developed for life-long assessments of these animals. Discussions are underway to
develop these crucial collaborations.
VI. Productivity:
Since this theme is in its first year, the publications listed below represent antecedents leading
up to formation of the theme.
Poster #33: Construction of a biologically based dose-response model for developmental
toxicity in the rat (Lau et al.)
69. Elstein, K.H., Mole, M.L., Setzer, R.W., Zucker, R.M., Kavlock, R.J., Rogers, J.M.,
and Lau, C. (1997). Nucleoside-mediated mitigation of 5-fluorouracil-induced toxicity
in synchronized murine erythroleukemic cells. Toxicol Appl Pharmacol. 146, 29-39A
75. Ghosh, B., Wood, C.R., Held, G.A., Abbott, B.D., and Lau, C. (2000). Glucocorticoid
receptor regulation in the rat embryo, a potential site for developmental toxicity?
Toxicol Appl Pharmacol. 164, 221 -229.
132. Lau, C., and Setzer, R.W. (1999). Biologically based risk assessment models for
developmental toxicity. In Developmental Biology Protocols (R. S. Tuan and C. W.
Lo, Eds.), pp. 271-281. Humana Press, Totowa, NJ.
133. Lau, C., Andersen, M.E., Crawford-Brown, D.J, Kavlock, R.J., Kimmel, C.A.,
Knudsen, T.B., Muneoka, K., Rogers, J.M., Setzer, R.W., Smith, G., and Tyl, R.
(2000). Evaluation of biologically based dose-response modeling for developmental
toxicity: a workshop report. Regul Toxicol Pharmacol. 31,190-199.
134. Lau, C., Mole, M.L., Copeland, M F., Rogers, J.M., Kavlock, R.J., Shuey, D.L.,
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Cameron, A.M., Ellis, D.H., Logsdon, T.R., Merriman, J., and Setzer, R.W. (2001).
Toward a biologically based dose-response model for developmental toxicity of 5
fluorouracil in the rat: acquisition of experimental data. Toxicol Sci. 59, 37-48.
135. Lau, C., Narotsky, M.G., Lui, D., Best, D.S., Setzer, R.W., Mann, P.C., Wubah, J.A.,
and Knudsen, T.B. (submitted). Exposure-disease continuum for 2-Chloro-2'-
deoxyadenosine (2-CdA), a prototype teratogen. 2. Induction of lumbar hernia in the
rat and species comparison for the teratogenic responses. Teratology.
150. Mole, M.L., Hunter, D.L., Gao, P., and Lau, C. (1998). Sample preparation and high-
performance liquid chromatographic analysis of deoxyribonucleoside triphosphates in
individual rat embryos. Anal Biochem. 259,245-252.
210. Setzer, R.W., Lau, C„ Mole, M.L., Copeland, M.F., Rogers, J.M., and Kavlock, R.J.
(2001). Toward a biologically based dose-response model for developmental toxicity
of 5-fluorouracil in the rat: a mathematical construct. Toxicol Sci. 59, 49-58.
233. Wubah, J.A., Setzer, R.W., Lau, C., and Knudsen, T.B. (2001). Exposure-disease
continuum for 2-chloro-2'-deoxyadenosine (2CdA), a prototype ocular teratogen. 1.
Dose-response analysis. Teratology. 64,154-169.
Poster #35: Molecular approaches towards understanding mode of action in developmental
toxicity. (Rosen et al.)
28. Branch, S., Francis, B.M., Rosen, M.B., Brownie, C.F., Held, G.A., and Chernoff, N.
(1998). Differentially expressed genes associated with 5-Aza-2'-deoxycytidine-
induced hindlimb defects in the Swiss Webster mouse. J Biochem Mol Toxicol. 12,
135-141.
199. Rosen, M., and Chernoff, N. (Submitted). 5-aza-2'-deoxycytidine-induced cytotoxicity
and long bone defects in the murine limb. Teratology.
8
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Poster 33. Construction of a biologically based dose-response model for
developmental toxicity in the rat. C Lau, RW Setzer, MG Narotsky, ML Mole, RM Zucker,
R Hanson, D Lui, D Best, OT Price, RJ Kavlock, and JM Rogers. RTD, *ETD, NHEERL, US
EPA, RTP, NC.
Estimation of a reference dose is a common practice for risk assessment at regulatory
agencies such as the EPA. One drawback of this practice is the degree of uncertainty
associated with low-dose and cross-species extrapolation of data obtained from laboratory
animal studies. Biologically based dose-response modeling is an emerging approach to
improve human health risk assessment by reducing these uncertainty factors These
models are designed to profile the pharmacokinetic and pharmacodynamic characteristics of
a chemical, and to relate the immediate cellular responses elicited to a cascade of aberrant
biological actions that leads to detectable adverse outcomes. For assessment of
developmental toxicity, these models should incorporate an integrated sequence of events
ranging from exposure of the chemical to pregnant dams, to molecular interactions within the
embryonal cells, interference with organogenesis, and ultimately defective anatomical
structures and/or deficient organ functions Our laboratory has attempted to construct such
a model using the chemotherapeutic agents 5-fluorouracil (5-FU) and Tomudex (TMD) as
prototype developmental toxicants. A single injection of 5-FU (doses ranging from 1 mg/kg
to 40 mg/kg) or TMD (20-60 mg/kg) was given to pregnant Sprague-Dawley rats on
gestational day 14, and various biochemical parameters were measured. 5-FU had a short
half-life in maternal serum and direct incorporation of 5-FU into embryonic nucleic acids was
low. However, activity of thymidyiate synthetase (TS) and deoxyribonucleotide (dNTP) pools
in the whole embryos, cell cycle profiles of embryonic tissues, total embryonic DNA content
and fetal weights were profoundly altered by 5-FU in a dose-dependent manner. Frank
terata (limb defects and cleft palate being the most prominent features) were noted at doses
above 25 mg/kg. Confocal microscopy revealed that a dose-dependent increase of cell
death in the limb buds paralleled the extent of digital malformation. The relationship
between dNTP pool imbalance and incidence of anatomical defects as well as fetal weight
deficits were evaluated by mathematical simulation. These models indicated different
associative patterns between dNTP alterations and the two endpoints of toxicity, perhaps
reflecting different underlying mechanisms. TMD is an anti-folate drug that inhibits TS
activity by competing with the natural co-substrate of the enzyme. Hence, a single exposure
to TMD led to profound imbalance of dNTP pools in the rat embryos, that was accompanied
by alterations of cell cycle progression in the limb buds, excessive cell death and an
increased incidence of digital malformation. The effects of TMD were remarkably similar to
those elicited by 5-FU and support the proposed teratogenic pathway in our BBDR model.
Further, maternal dietary folate deficiency exacerbated the toxicity of TMD. These findings
thus demonstrate the feasibility to construct a BBDR model for development toxicity and
illustrate their potential usefulness in low dose extrapolation for risk assessment. This
BBDR model may also be useful in assessing the role of folate in normal development and
developmental susceptibilities conferred by folate deficiency.
9
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Pertinent References: 69, 75, 132-135, 150, 210, 233
10
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Poster 34. Postnatal sequelae of low birth weight; effects of maternal toxicity-induced
weight loss during gestation on fetal growth; and susceptible populations. N
Chernoff, B Gersh, E Rogers, MB Rosen, ES Hunter III, C Lau, LL Hall, P Hartig, and JM
Rogers. RTD and ETD, NHEERL, ORD.
Fetal weight reduction is the most common effect at the LOAEL in Segment II
studies, but the postnatal consequences of low birth weight (LBW) have not been
adequately explored. Further, Segment II studies often show reduced maternal weight gain,
yet there is evidence that this effect may result from diminished food intake. Our studies
examine both of these issues. To evaluate postnatal growth of LBW pups, we used three
compounds that induce LBW. Results indicate no delay of parturition in treated dams, and
pup weights at birth have the same relationships across doses as those obtained the
following morning. LBW is correlated with lower postnatal viability, increased anomalies, and
reduced postnatal growth across different treatments. The growth of LBW pups without
external anomalies does not reach control levels by weaning. Studies using large, cross-
fostered litters of 16 pups derived from dams treated with one of four teratogens show that
litters of this size are undernourished and grow more slowly than normal-size litters. The
16-pup litters comprise either 8 treated LBW pups and 8 control pups or 16 controls of
matched weight. Regardless of treatment, growth of LBW pups from treated dams does not
differ from LBW controls. Studies to separate agent-induced from reduced food intake-
induced LBW used pups derived from gestationally food-restricted dams. This LBW, that we
assume is due to intra-uterine growth retardation (IUGR), was related to the extent of
maternal food deprivation. After birth, litters of 16 or 8 pups were formed from both control
and LBW pups, thus representing combined pre- and postnatal undernutrition; prenatal
undernutrition; postnatal undernutrition; and normal nutrition. Higher pup losses and
reduced growth occurred in the LBW groups (at both 8 and 16 pups/litter). Currently, we are
breeding females from all groups to examine fertility and pregnancy outcome, to address the
important issue of inter-generational effects. This concept developed from epidemiology
studies indicating that LBW and adverse outcomes are more closely correlated with the
mothers' perinatal environment than that during the pregnancy itself. Treatment-induced
reduction in maternal food intake may be the cause of IUGR rather than compound-induced
toxicity. While this will not affect the NOAEL, interpreting such an effect to be compound-
induced fetal toxicity may prove to be misleading. Paired feeding studies are underway to
address the relationship(s) of maternal food intake during gestation and toxicant-induced
fetal growth retardation. Inter-generational effects have been demonstrated in
epidemiological studies, but many questions remain unanswered. An animal model for this
phenomenon would allow us to address this phenomenon under controlled conditions.
Topics include the degree of "repair" possible with optimal postnatal nutrition and the
susceptibility of LBW (IUGR) offspring exposed to xenobiotics later in life - the question of
"susceptible populations." A series of tests will be done to evaluate critical organ functions
at various life stages of affected pups. The above studies address three issues of potential
importance to the EPA: the effects of different degrees of LBW on postnatal growth and
development; the relationship of reduced gestational maternal food and water intake to fetal
11
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growth and viability, and the life-long effects of IUGR and in utero toxic exposures.
Pertenient References: None (new project)
12
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Poster 35. Molecular approaches towards understanding mode of action in
developmental toxicity. MB Rosen, KC Brannen, W Harrouk, CA Kimmel, G Kimmel, ES
Hunter III, JM Rogers, and N Chernoff. RTD, NHEERL, RTP, NC and NCEA, Washington,
DC.
Basic assumptions concerning dose and species extrapolations are necessary when
making risk assessment decisions. Our goal is to reduce and improve these assumptions
by expanding our basic understanding of transcriptional changes associated with abnormal
development. One approach is to study how a toxicant might alter the expression of specific
candidate genes. As an example, we recently investigated the role of the endothelin
pathway in craniofacial development. Endothelin-1 (ET-1) binding was chemically disrupted
on gestation day 8 using an endothelin receptor antagonist. The subsequent expression of
a number of developmental genes was then examined. Of particular interest was an acute
down-regulation of Pitxl in the first arch mesenchyme on gestation day 10 as determined by
in situ hybridization. This is of potential importance since targeted mutations of Pitxl cause
similar defects to those resulting from either compound exposure or mutations of ET-1 or the
ET-1 receptor. These are the first data linking Pitxl to the ET-1 pathway. The use of gene
array technology is another approach we are using to help identify yet unrecognized genes
of significance or important genetic pathways. At present we are using this technology to
study the relationship between heat stress and boric acid exposure since both toxicants
cause similar anomalies of the axial skeleton and may therefore affect common
developmental pathways. This work is currently in the initial stages of data analysis.
Preliminary analysis indicates that genes involved in DNA damage repair, apoptosis, and
heat shock may be affected; however, experiments are still ongoing and data analysis is not
complete. A second gene array project currently underway relates to issues of drinking
water safety. Data have been collected from mouse embryos cultured for 6 hrs at
teratogenic concentrations of various haloacetic acids. It is too soon to report on the results
of these studies since they are in early stages of data analysis as well. In the future, we are
interested in using array technology to identify genes which may be of interest for continued
study. Array experiments could then be followed-up with more refined studies using real
time PCR, in situ hybridization, Northern analysis, etc., in which dose and time after
exposure are included as experimental variables. In the long term, we expect that
elucidation of mode-of-action for developmental toxicants will allow more biogically-based
risk assessments than are currently possible.
Pertinent References: 28,199
13
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Poster 36. Evaluation of the role of folate in susceptibility to developmental toxicants
J.E. Andrews, C. Lau, E.S. Hunter III, B. Grey, J.R. Thibodeaux, H. Nichols, R. Hanson, I
Chernoff, and J.M. Rogers. DBB, RTD, NHEERL, ORD.
Deficiencies of essential nutrients during pregnancy can result in birth defects or
other adverse pregnancy outcomes, and toxicant/nutrient interactions are well-known. In
terms of its roles in human pregnancy and development, folic acid is among the most
important and best studied essential nutrients, including studies demonstrating that folate
supplementation during pregnancy reduces the incidence of birth defects. Mechanism(s)
underlying the protective effect of folate have not been identified, but do not appear to be a
simple correction of deficiency. Little is known about the impact of marginal or low folate
status on pregnancy or susceptibility to developmental toxicants. We are examining the
relationship of maternal folate status or embryonal folate availability and susceptibility to
developmental toxicants both in vivo and in vitro. Research in our previous theme on
methanol demonstrated in mice that low or marginal maternal dietary folate intake
exacerbated the teratogenicity of methanol, which is detoxified in part via a folic-acid
dependent pathway. In our current theme, we have demonstrated that the developmental
toxicity of Tomudex, a thymidylate synthetase inhibitor and structural analogue of folate, is
exacerbated in rats by reduced maternal dietary folate intake. Tomudex, which is also being
used to test the BBDR model we are developing, causes embryonal nucleotide pool
disturbances and affects fetal growth and limb development when administered to pregnant
rats on gestation day 14. We have also demonstrated that supplementation of media with
folinic acid can completely abolish the toxicity of Tomudex to rat embryos grown in vitr
during organogenesis. While there is clear biological plausibility to the hypotheses tht.
folate deficiency will exacerbate the developmental toxicity of methanol (metabolized via
folate) or Tomudex (a folate analog), we are also evaluating the effects of altered folate
status on susceptibility to a broad range of developmental toxicants without such clear links
to folate. The hypothesis here is that if folate is a limiting substrate for normal development
and/or repair, then susceptibility may be increased to diverse toxicants with different modes
of action. We are currently studying the developmental toxicity of several drinking water
disinfection byproducts, including dichloroacetic acid, dibromoacetic acid and
bromochloroacetic acid. Preliminary data suggest that the toxicity of these chemicals is
exacerbated when embryos are cultured in sera from deficient donor rats, and that some,
though not all, of this toxicity is ameliorated by addition of folinic acid to the media. We
have also started studies testing the effect of maternal folate status on the developmental
toxicity of dichloroacetic acid in vivo, but data have not yet been analyzed. The plan for the
near future is to examine dose-response relationships for a number of known rodent
teratogens under conditions of varying maternal folate status. Women of child-bearing age
frequently have marginal dietary folate intake, although this may be improving with
education and supplementation of foodstuffs. Whether inadequate folate intake or status
renders mothers more susceptible to environmentally-induced adverse pregnancy outcomes
is important to understand, and would allow more informed and comprehensive assessments
of risk for such outcomes.
14
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Pertinent References: None (new project)
15
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Poster 37. The developmental toxicity of perfluorooctane sulfonate, including studies
on putative modes of action. JM Rogers, B Grey, JR Thibodeaux, R Grasty1, R Hanson,
Chernoff, and C Lau, RTD, NHEERL, ORD, U.S. EPA, and 'Curriculum in Toxicology,
University of North Carolina, Chapel Hill, NC.
Perfluorooctane sulfonate (PFOS) is a breakdown product of Scotchgard® and other
commercial products, is a widespread, persistent environmental contaminant, and has a
long biological half-life. Studies in several species have demonstrated developmental
susceptibility to PFOS, with neonatal mortality being the most sensitive endpoint of toxicity.
Thus, EPA is currently reviewing this compound. We have carried out studies in Sprague
Dawley rats and CD-1 mice to better define the dose response for both teratogenicity and
postnatal mortality, the most sensitive developmental stage(s), and potential modes of
action. Based on orally administered maternal dosage throughout gestation (gd 2-20 in rats
or 2-17 in mice), rats were more sensitive than mice to PFOS-induced neonatal mortality.
Maternal dosages of 5 mg/kg/day and above resulted in virtually 100% mortality in rats,
while dosages of 15 mg/kg/day in the mouse were uniformly lethal. All pups were born alive,
but neonates became moribund and died in hours to days, depending on maternal dosage.
PFOS did not affect fetal viability. Teratogenicity, including cleft palate and skeletal defects,
and reduced fetal weight occurred at higher dosages than did neonatal mortality. When
pregnant rats were dosed with PFOS at 25 mg/kg/day for different 4-consecutive-day
periods during gestation, the incidence of neonatal death increased with increasingly later
dosing, reaching nearly 100% with dosing from gd 17-20. Single-day dosing with 50 mg/kg
on either gd 17 or gd 20 resulted in 60-80% neonatal mortality, with gd 20 dosing beir
slightly more toxic. Maternal serum and fetal tissue levels of PFOS following the variou
exposure regimens are being evaluated. Preliminary examination of lungs from affected
neonates revealed evidence of hemorrhage and possibly immaturity. Assays of maternal
serum revealed a marked reduction of thyroid hormones, and to a lesser extent, cholesterol
and triglycerides. Hence, perinatal hypothyroidism may be a contributing factor in PFOS
developmental toxicity. The enzyme HMG-CoA reductase is known to be inhibited by PFOS,
so impaired cholesterol metabolism is another putative mode of action. Because of its
widespread environmental occurrence and persistence, it is important to understand the
developmental toxicity of PFOS. Elucidation of the mode of action for the neonatal mortality
induced by PFOS is critical for extrapolation of these animal data to predict potential human
risk.
Pertinent References: None (new project)
16
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1997 Peer Review Contents
Pages
Peer Review Comments dated 8/13-15/97 1-18
Divisional Peer Review Response dated 9/18/98 19-29
Update to 1998 Response to 1997 Peer Review 29-35
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PEER REVIEW OF
U.S. ENVIRONMENTAL PROTECTION AGENCY'S
Reproductive Toxicology Division
August 13-15, 1997
Research Triangle Park, North Carolina
image:
TABLE OF CONTENTS
OVERVIEW 1
DYSMORPHOLOG Y 2
GAMETE TOXICOLOGY 5
DBPa 7
ENDOCRINE DISRUPTORS 1 10
ENDOCRINE DIRUPTORS II 11
PEER REVIEW PANEL 16
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1
OVERVIEW
The scientists of this division have a long history of important contributions to the scientific
fields and disciplines tha they represent. The following comments are made to suggest that with
some changes in overall planning and allocation of resources, the group can be even more
influential and productive in the future. The vision for the future is not thoroughly laid out to
clearly identify areas of priority research and the basis for their selection. Priorities should be
defined relative to needs of the regulatory offices of the EPA and the current status of the
respective fields of science. Currently, some areas of research, such as use of subtractive
hybridization technology, seem to be methods in search of a need, rather than a priority need for
which the method is the most relevant approach. Confocal microscopy is another example of a
modem technology driving a research effort, research being driven by a tool rather than a
well-defined pionty.
Two factors should be considered that would likely improve productivity of the division. The
range of productivity of scientists extends from very high to little evidence of productivity.
Some individuals scattered throughout the division do not appear to contribute to publications in
peer-reviewed journals, the only real measure of productivity emphasized in this review. This is
particularly noticeable and critical at the higher GS levels. The second contributing factor may be
the tendency for people to be spread too thin, lacking focus on narrowly defined areas where
they could make a well structured contribution.
When an organizational unit of a laboratory is responsible for a wide range of research, as is the
case with the Reproductive Toxicology Division, allocation of resources to various components
of the laboratory is a major challenge. This is especially true within the federal government
where practices are often inconsistent with rapid changes in work-force needs determined by
rapidly changing science and regulatoory needs. Nonetheless, there appears to be some imbalance
in the allocation of people and resources in the opinion of the review team. Management should
review the possibility of allocating a larger share of the resources to the area of gametogenesis.
Also, while TCDD is an interesting chemical by which to explore the biological basis for
toxicological findings, the review team felt that there was too much emphasis on TCDD within
the division. Less emphasis on TCDD and more on research in gametogenesis would result in a
division with a more balanced allocation of resources relative to the apparent goals of the
division. Also, emphasis should be continued to develop biologically based dose response
models because of the likely value to support sound regulatory decisions in the future.
The last recommendation relates to a problem that is not unique to the Reproductive Toxicology
Division or to NHEERL. The review team recommends greater emphasis on bridging of scientific
activities within NHEERL and with scientists outside of the EPA. Division scientists would
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2
benefit from more collaboration and communicaiton with investigators in other research labs
where there is commonality of research questions and approaches, and with scientists in other
disciplines where new findings have a direct bearing on reproductive and developmental
toxicology research. In the context of research planning, the review team recommends more
communication between NHEERL division directors to enhance the overall planning process of
NHEERL.
DYSMORPHOLOGY
Goal:
This focal area deals with the abnormal morphology in embryos resulting from toxicant exposure.
A number of different research paradigms have been utilized to address this issue: methanol
toxicology, skeletal defects induced by toxicants, arsenite effects, 5-fluorouracil action, and
nutrient involvement in teratogenicity. The studies in this area are largely phenomenological,
characterized in large measure by morphological endpoints.
Critique:
While efforts are underway to identify mechanistic causes of morphological aberrations, most ^
evidence is, at best, preliminary. In addition, some of the studies resemble "fishing expeditions .
For example, a number of possible genes have been screened to see if expression patterns are
altered by boric acid treatment (in conjunction with rib defects) or by 2,-ueoxy-5-azacyti<Jine (in
conjunction with hindlimb phocomelia); but the results thus far are largely negative. Further, the
importance of the methanol data comes into question considering the waning interest in methanol as
a toxicant. It is not clear how modeling the 5-FU effects will contribute to a general understanding
of toxicant action. While 5-FU inevitably works through altered pynmidine nucleotide pools, is
there any precedence to presume that a reasonable number of other toxicants act in a similar
capacity*?
The core facility has the capabilities to contribute considerably to a number of different research
projects. Sensitive nucleotide assays have been utilized in the 5-fluorouracil modeling project, and
confocal microscopy has been used to visualize the ability of different toxicants to induce
apoptosis. However, these faciliues appear to be underutilized, and more extensive use could be
made of them.
One study for which there seems to be a good comprehensive approach to the problem is that of
asenic induction of apoptosis. The investigators have used flow cytometry as well as confocal
microscopy to characterize cell cycle status and apoptosis. In addition, a variety of molecular
techniques are being used, including loss- and gain-of-function models to investigate the
involvement of P53, P21, and Bcl2. They will also attempt to overexpress different antioxidant
molecules to counteract presumed mediation of toxicant effects by reactive oxygen species. Tnjs
marriage of morphological, cell biological and molecular biological approaches is to be applauded.
The panelists generally felt that the mission of the Pattern Formation Focal Area was poorly
delineated. Projects included within this Focal Area included those that investigated:
o A specific chemical/toxicant
o The perturbation of a particular developmental process.
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3
Not only were the projects quite diverse, but also they were not well integrated The reasoning for
the selection of each research topic was not transparent to the reviewers. Since the overarching
mission of the Reproductive Toxicology Division (RTD) appears to be embedded in the
Biologically Based Dose Response (BBDR) Framework, one possible approach is to reorganize
the research missions of this Focal Area to fit under that Framework. Such a reorganization would
help'to tie the projects together and would provide a less uncertain concept of the mission of this
Focal Area relative to the overarching RTD mission. The emphasis would be on Tilling in the
black boxes" and the biological relevance, i.e. the target dose (parent compound or metabolite), the
target molecule(s), target interactions, and not on mathematically modeling the data for each
chemical. Comments are offered below for each of the individual areas within this theme:
McThanolIhcmc
The results generated to date are comprehensive and solid; they should be completed and
published The Panel recognizes that methanol is no longer a priority chemical for the Agency. In
the future, expenments in which methanol is used as a model chemical should fit under other
themes. For instance, it appears that the results of these projects could fit well under the pattern
formation or apoptosis themes. The future directions described, however, extend beyond the
bounds of these other themes and do not appear to be highly focused. Recognizing the decreased
priority of this theme for the Agency, the planned experiments should be redesigned to fit clearly
under the appropriate themes.
Pattern Formation Theme
Given that changes in rib number are of regulatory importance because varying rib number is
frequently the oily significant morphological finding in developmental toxicity tests, the projects
that investigate this issue have clear relevance. Early results suggest an approach to investigate an
important toxicological problem through the study of families of patterning genes. However, there
is a plethora of data on patterning genes being generated in many laboratories throughout the
world. The question arises as to whether the changes in patterning gene expression observed in
this laboratory are an endpoint that will be of value. That is to say, while the results may be of
importance to mechanistic developmental toxicology research, will such information be of
assistance in target identification on a routine basis? Beyond the bounds of the current projects, i(
is not clear that the results will lead to an assay for hazard identification or assist in risk
assessment; the relevance to Agency interests is less obvious.
Considerable effort has been devoted to the development of the subtraction hybridization method.
This has been an ambitious effort. The technique, however, is a descriptive, labor intensive
method which identifies changes between control and treated tissues but is unable to distinguish
cause from effect. Its very complicated and time consuming methodology makes it unlikely that it
will be useful as a routine assay for hazard identification. Consequently, its immediate relevance to
Agency goals is not clear.
The technique for manipulation of gene expression is in preliminary stages of development This
technique may hold promise for defining the target of developmental toxicants. The methodology is
in its infancy and requires extensive further development. Providing the technique meets its
expectations, its application should be in the delineation of the target molecule(s); this fits well into
the BBDR Framework. A challenge that will remain will be to translate the method into an in vivo
system.
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4
Aooptosis Theme
This theme is important to the field of developmental toxicology. It is critical that the controlling
and modulating events of this phenomenon during organogenesis be discerned. Experiments on
the roles of bcl-2, p53, and p21 should contribute significantly in this respect. It is important that
experiments to study / assess Apoptosis not be limited to one technique (confocal microscopy).
Nevertheless, the unifocal microscopy techniques being developed are state of the ait and are likely
to yield novel and important quantitative information concerning the relevance of the phenomenon
to the generation of developmentally toxic effects. By developing this technique, the lab is doing
breakthrough methods development This core lab clearly has the potential to contribute
significantly to a number of themes I branches.
This theme could be enhanced with additional projects that study apoptosis in vivo, as well as the
integration of experiments using other agents already studied at RTD (such as S-FU). Does
nucleotide imbalance lead to apoptosis? What is the role of apoptosis in 5-FU embryotoxicity? If
such a role is identified, then confocal microscopy could be usenil in helping to quantitate the
rescue work under the 5-FU theme.
5-FU BBDR Theme
The "biological aspects" of this theme are really the overarching paradigm of the RTD. The
selection of S-FU was useful in helping to build the BBDR Framework, but now thai the
framework is completed, it needs to be expanded by including other chemicals. Much time, effort
and money have been expended in developing the FU-BBDR story. The true impact of the
exercise, however, has not been the results produced, but rather the concept developed. The
bioloyv that is important to understand in each step of the BBDR Framework should be readily
translatable to other toxicants end to other themes. The likelihood that mathematical aspects of the
BBDR model will come to fruition in the near term is remote. The conceptual thinking concerning
the biology, however, is extremely relevant to the RTD mission and could be the overarching
mission of the division. This review committee felt that in "filling in the biology" for 5-FU in the
BBDR framework, the theme has become too focused on 5-FU. Yet the level of information with
respect to the biology still appeared to be insufficient to justify a major effort in mathematical
modeling. The Committee recommends that more consultation be sought with respect to the focus
on mathematical modeling.
Maternal Metabolism
This IRP project fits well under the target dose (parent compound / metabolite / stereoisomer)
segment of the BBDR Framework. The project is focused, well-designed and its results are clear
cut. The project answered a question that was germane to the methanol project The methodology
should be useful to other projects and could be readily transferred to other laboratories. Although
we recognize the independent nature of an IRP, this area could be more integrated into other
themes. For example, a further enhancement would be to attempt to include gene manipulation
methods in order to study the role of specific metabolizing enzymes in the activation / detoxification
of chemicals of interest.
Summary and Conclusions:
The contributions by some of the Pis have been significant and have been recognized both within
and outside the Agency. Nevertheless, some of the projects were not too clearly related to the
mission of the Agency.
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5
A number of Pis in this focal area have made a significant impact at the national / international
levels in terms of participation in scientific societies, workshops and symposia, while others have
bad minimal impact The net result is that it appears that this focal area may be overstaffed for its
level of productivity.
GAMETE TOXICOLOGY
Goal:
This Focal Area has been divided into two main sections: 1) Sperm regulation and function and 2)
Protective mechanisms in gametes and early embryos. The first deals with methods of assessing
sperm morphology and motility patterns, carbohydrate metabolism, and the potential biomarker
SP-22 in response to different toxic insults as measures of sperm health and viability. The second
concerns protective means by which oocytes and embryos respond to toxicant exposure and
includes measuring a series of heat shock proteins and glutathione homeostasis.
Critique:
High quality research is being done in the protective mechanisms portion of this focal area.
Although the amount of research is heavily distributed toward the male system, the work with
oocytes is timely and important. One important segment of these studies is the analysis of
glutathione accumulation and its involvement in sperm nuclear condensation following fertilization.
Through assays and modulation of glutathione levels, the investigators have convincingly
demonstrated the importance of glutathione homeostasis in spindle formation md pronuclear
formation. Of more relevance to the interests of the EPA are subsequent experiments that
examined the actions of different toxicants on chromatin and spindle integrity in mature oocytes
and the importance of GSH. Thai depletion of GSH in mature oocytes exacerbates the negative
actions of toxicants on these parameters provides farther support for a vital role for QSH in
maintaining normal oocyte fertilizability. Important future studies are planned to better define the
protective function of GSH in oocyte physiology by depletion of oocyte glutathione levels both in
vivo and in vitro and monitoring the redox potential by a sensitive HPLC assay. Using these
procedures, the investigators will be in a position to test the efficacy of the GSH protection system
on the detrimental effects of a variety of reproductive toxicants.
The phase of oocyte maturation that these experiments have focused on is principally at a late stage,
just after fertilization, as they reach metaphase Q. This is a rational starling point, however other
phases of oocyte development should also be considered as possible targets for toxicant action.
It is important to note that the oocyte remains arrested at the late prophase I stage of meiosis
throughout the period of oocyte and follicle development; during this time the chromatin remains in
a decondensed slate and is presumably susceptible to environmental insults. The possibility that
oocytes in this condition sustain damage at both the cytoplasmic and nuclear / genetic levels from
toxicant exposure would appear to be a fertile target for research. Moreover, techniques are
available for in vitro culture of developing follicles, enabling the testing of toxicant effects in the
growing oocyte. It is certainly conceivable that growing oocytes exposed to toxicants could
sustain damage that may not necessarily be manifested immediately but appear later at
postimplantation or postnatal stages of development.
While there may be detrimental effects of toxicants acting directly on the germ cells, the
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6
participation of somatic cells in toxic action should be also considered. Since ovarian follicle cells
play an intimate role in oocyte development, they may be an important positive influence in the
protective potential of the oocyte against oxidative insult They might also serve to mediate the
negative actions of certain toxins on the oocyte, since numerous agents are unable to be taken up
directly by the oocyte but can accumulate in the oocyte, if first taken up by the follicle cells and
transferred lo the oocyte by a patent gap junctional pathway.
While the bulk of the glutathione work has been carried out in oocytes, a recent sttidy has been
conducted in males that examined the actions of aciylamide, a GSH-depleung agent, on sperm
function The suggested relationship between GSH depletion and anomalous sperm behavior
indicates that glutathione homeostasis may be of universal importance in gametes to combat
oxidative insult Future work along these lines will be important in establishing this relationship.
In addition, parallel studies involving heat shock protein-mediated protection from toxicants has
been largely limited to male gametes. It would be relevant to expand this work to the female, as
different protective systems might be effective against different toxicants.
Are the projects that have been selected in the field of gamete toxicology the most appropriate ones
to assess environmental toxicants and to increase our knowledge about potential biomarxers in
gamete reproductive toxicology? In general, the themes and IRPs that nave been identified are
those that are needed and they will continue to need further research. However, the decision has
been made to focus primarily on the later phases of gamete maturation, and to place little emphasis
on the effects of toxicants on the gametes while in the gonads. This may have been a wise uuual
choice much less is known about the effects of toxicants on the latter phases of gamete
maturation. However, there is clearly a need for this branch to expand in the direction of
developing expertise in identifying how environmental toxicants act on gametes in the gonads.
IndfTJ* the studies on epididymal toxicity have been instrumental in establishing this tissue as a
target for reproductive toxicants. A concern within this particular IRP is the apparently oversold
interpretation of sp22 as a marker for the fertilizing ability of spermatozoa. The proposed studies to
better characterize sp22 and other proteins are appropriate. A complication that the investigators
may want to consider in planning and interpreting their efferent duct ligation studies is that there are
direct consequences to the epididymis of withholding components of the testicular fluid, ix.,
efferent duct ligation does more than simply block the entry of sperm into the epididymis.
The studies on CAS A have pioneered the validation of this method for toxicological studies in
rodents. The significance of changes in parameters of sperm motility and extrapolation to humans
remains to be established. However, the scientific community is now in a posiuon to assess tne
importance of this biomarker thanks to the work of this group,
The theme on stress proteins is an important one. The proposed studies with the novel HSP70-2
are exciting and appropriate to a better understanding of how stress proteins could mediate
xenobiotic action. The knockout studies should help establish the biological significance of testis
specific HSPs.
The IRP on sperm energetics has potential interest. However, there has been a plethora ofstudies
on enzymes in intermediary metabolism that have an isoform that is specific to me tesus. These
studies have not shed much light, either on testicular function or on the action of xenobiotics.on
either the testis or on spermatozoa. It may be appropriate to reconsider the value / focus of this
IRP.
image:
Summary and Conclusions:
One of the concerns relating to this foe a] area is to assure that the recently appointed Pis are
encouraged to become autonomous, independent scientists while continuing to work in a team
environment. In deciding on future appointments, we believe that it will be important to identify
individuals whose research objectives are to understand, at the molecular level, how environmental
agents interact with gonads. To that end, the DNA analysis / molecular biology core should
provide up to date effective support for these and other individuals working in this focal area.
In order to optimize the effectiveness of the Pis in this focal area as. well as in the others, more
extensive technical support should be made available, so that Pis do not have to do routine lab
bench work once they have established the requisite methods for the questions that they are asking.
With respect to the relative role of this Branch within the RTP, and to the outstanding output by its
senior members, we feel that it is inappropriate that the total manpower in this branch *
approximately half of that in each of the other two branches. Thane are crucial areas in the field or
gamete toxicology which need to be covered and which are not covered because of lack of
appropriate manpower.
The contribution made by this branch to the reproductive toxicity testing guidelines has been
pivotal in introducing elements of gamete biology. The role of EPA in this area has certainly Been
fostered by the activities of the members of this branch.
A strong leadership posiuon at the national and international scale has been made by the Branch
Chief. This individual continues to bring a high level of visibility to the EPA. The other
investigator in this branch is also gaining national recognition for his solid, novel wore, wewiy
appointed Pis have promising futures but have yet to prove themselves at the national and
international levels. Little visibility can be seen in the DNA / molecular biology core area.
DBPs
Goal:
The RTD strategy in this area was comprised of four main elements: 1) hazardidentification
studies, 2) mode of action studies, 3) bfomarker development studies, and 4) development of
biologically based dose-response models. The Focal Area is reviewed below on the basis of those
four areas.
Critique;
pnTflrri Identification Studies
These studies were performed as a mix of extramural and intramural I^sea^"
have identified 34 compounds of environmental concern in six categories: 1)
halogenated acids. 3) halonitrile and aldehydes, 4) ketones, 5) miscellaneous, and 6) disinfectants.
There are 3 research areas: The investigators have completed or initialed screening of 13
compounds in Developmental Toxicology and have plans for 4 more. Eleven Sc^^ st"J"
have been initiated or completed. In the area of male reproductive toxiCoJogy lS st^ haveban
initiated and five are planned. In female reproductive toxicology, four stupes have been imtiatcd
and six are planned. The studies are distributed among all six categories of chemicals listed above,
and there is at least one study in each research area within each category of chemicals.
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8
Some of the work is supported extramurally and it is not clear to the reviewers that the RTD plays a
significant role in the design, performance or interpretation of the studies.
DBPs, a family of structurally related compounds, lend themselves to QS AR. We would
recommend this approach 10 get at prediction of risks rather than test each of the 34 components for
each of the end points of concern. The objective of this focal area should be to determine whether
or not disinfected drinking water is safe
The reviewers have major concerns regarding the brevity of the dosing period for reproductive
toxicants (i.e., 35 d, 14 d), especially for male reproductive studies. Dosing should extend
through at least one spermatogenic cycle and epididyroal transit time for the species being studied.
This concern is amplified when short exposure periods are used to test mixtures of compounds for
toxicity. The concern is thai all components of the mixture will be judged negative if the mixture is
negative after an inappropriately short exposure period (see additional comment under the
biologically based models area, below).
Mode of Action Studies
Studies in this area ranged from basic reproductive and developmental biology to cell and
molecular biology and have identified promising leads in defining modes of action of toxicants.
However, effects on fetuses, pups, ana pregnant females, as well as on parameters of the male
reproductive system, have been seen only at relatively high doses, i.e., effects were seen only at
doses of hundreds of mg/kg/day.
Efforts to elucidate the mode of action of haloacetic acid-induced neural tube and heart defects are
interesting and exciting. Nevertheless, given the effort expended to study these high-dose effects,
the reviewers question the place of these studies in a laboratory with a mission-orientation toward
providing information about environmentally relevant doses. It was perceived thai this type of
effort is appropriate within the 20% effort allocated to each investigator for personal research
initiatives.
Biomarker Development Studies
The most prominent work in this area is that resulting in the identification and partial
characterization of SP22, a rat epididyma] sperm protein. This protein is found on rat sperm
beginning in the rete testis, is prominent on normal cauda sperm, and has been found to decrease in
sperm membrane preparations as sperm fertility decreases. The protein has been sequenced and
found to have high homology with DJI, a human nuclear (presumptive) protein. Both the cellular
localization and the function of the protein are unknown and are appropriate for study within the
RTD if the protein is found to be a true biomarker for toxin exposure, rather than a general marker
for infertility. The reviewers are concerned that while SP22 has been highlighted by the RTD as a
biomarker for toxin-induccd infertility, it remains unclear that this protein is, in fact, relevant for
purposes of toxicology. Appropriate control studies (e.g., cryptorchidism, heat exposure,
ischemia) have not been performed to demonstrate the molecule's specificity to toxin-induced
infertility; therefore, advancing this molecule as a biomarker for toxin-induccd infertility appears
premature.
The potential of SP22 as a general marker for infertility, or its use in a potential contraceptive
development strategy, while very interesting, appear to the reviewers to be outside the mission of
the RTD.
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9
The reviewers are seriously concerned about the proposal to use human testicular tissues for in
vitro studies of staged seminiferous tubules This proposal does not reflect a knowledge of the
literature regarding (he microanatomy of spermatogenesis or the physical characteristics of human
testicular tissue. Additionally, available human tissues are typically reproductively compromised.
Development of Biologically Based Dose-Response fBBDRI Models
The investigators in this focal area appear to view the BBDR model as a framework for research
initiatives to fill in the "black boxes" between administered dose and adverse outcome. The
reviewers concur with this approach. The first "black box" of the framework is target dose. It is
obvious that all of the compounds of concern undergo metabolism, but it is not obvious whether
the parent compound or the metabolite is the proximate toxin. This concern is compounded in
exposures to mixtures of toxins Parent compounds may affect (e.g. inhibit, stimulate) their own
metabolism or the metabolism of other compounds of the mixture. We concur that a PBPK model
is appropriate here; however, the pharmacokinetics may be very different at high doses than at low
environmentally relevant doses. The reviewers repeat a concern that although the agency is
concerned with the reproductive effects of DPB's, the effects shown only occur at very high,
potentially irrelevant doses. Perhaps long-term, low dose exposure to the most potent DPB's in
the drinking water would establish whether there is, in fact, a reproductive risk at concentrations to
which humans and wildlife would be exposed.
Summary and Conclusions:
Personnel Contributions to the Agency
The qgency has asked the RTD for information on reproductive effects of DPB's and the division
has responded The four components of the research strategy are reasonable and the chemicals
under examination arc basically mandated by what is found in disinfected drinking water. The
extramural and intramural testing programs have successfully identified the most potent drinking
water contaminants of the DBP class and have identified the most sensitive end points for these
contaminants. The reviewers do question whether continued study of the high-dose effects
mentioned previously contribute to the agency's mission of identifying reproductive risks of
DPB's when the compounds are present in environmentally relevant doses. The reviewers
recognize and applaud the recognition from within the EPA that several investigators within the
division have received for their research.
National Scientific Leadership
The national scientific leadership provided by investigators in the areas of mechanisms of
epididymal toxicology and of teratogenicity is evidenced by the investigators' scientific
productivity, their quality of research, and the recognition they have received from outside the
agency. The reviewers are concerned, however, that too many of the division's investigators
evidence low productivity and absence of participation in the nauonal science scene.
The reviewers applaud the proposed investigations of DBP's involving nonpregnant, cycling
females, pregnant females, and males, and the use of rabbit, rat and mouse species. While the
future directions provided to the reviewers appeared complete and in detail, the reviewers could
have assessed better their real merit if the future plans had been more focused and presented with
some sense of priority. It is recommended that the exact list of future directions provided by the
reviewers be prioritized and retained for presentation to the next review panel for the RTD. In this
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10
way, that review panel will know what the focal group set out to do and will be able to assess how
much progress has been made.
ENDOCRINE DISRUPTORS I
Goal:
This focus group concentrates its efforts on receptor-mediated (i.e., nuclear steroid receptors)
mechanisms for alterations in development, investigating such compounds as TCDD and PCBs.
cda rccluw effects mediated by the Ah/ARNT system as well as those proposed to act via the
ER, AR, and Tk receptors. The emerging area of endocrine disrupting compounds is
controversial in the areas of applicability of animal models and in vitro assays to potential effects
on humans, U-shaped dose-response cuives, and lack of discernible threshold effects. In
response to the potential complexity of biological responses, a multilevel approach including
investigation of developmental as well as reproductive effects on exposed individuals, or their
progeny, is being carried out. The breadth of the research projects, experimental approaches, and
techniques are necessary and appropriate to answer the questions posed.
Critique:
Strengths
o Strong, productive investigators
o Well-denned model systems and relevant goals
o Antiandrogen work ground-breaking
o Human and mouse palatal cultures notable, potential comparisons powerful
o Demonstrated leadership within and outside agency on EDC questions
Weaknesses
o Evidence linking reproductive problems with toxin-mediated effects on stress axis is absent
(and does not warrant major effort)
o Although anti-thyroid effects of toxins are of interest, thyroid-based effects on learning are
unlikely to provide a useful screening mechanism and break no new ground on thyroid
hormone action
Several significant scientific findings on potential antiandrogenic activities were presented, as well
as important information on the presence or lack of estrogenic effects and on the synergistic
responses of specific compounds. This group of studies, taken together, has the highest current
scientific impact. The findings that agents such as thephthalates, procymidone, vinclozolin, and
p,p' DDE are antiandrogenic followed up initial anginal observations and the work of otters and
significantly expanded the understanding of both mechanism of action and potential risks of riwcg
compounds.
Important collaborations with outside investigators have been made, and parallels to the Tfm
mouse on development and other effects have been established in some cases, and should be
continued to be drawn. EPA researchers have used both in vivo and in vitro models (Endocrine
dismptors II) to assess biological actions and biochemical properties. At present, in animal
systems have not been confirmed with regard to controversial suggestions that human sperm
quality is declining; however, significant biochemical and cellular effects demonstrated in these
studies may be causes for potential concern. Data on potential estrogenic and antiestrogenic
compounds have in general agreed with the consensus of the emerging literature. Importantly, the
synergism originally reported by others (and recently withdrawn) between environmental estrogens
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11
was carefully examined and not substantiated, although additivity was found in some cases.
Advantage should be taken of the ERKO (ERx form) knockout mouse to examine some of the
cr>rf®inu 05 rccePtor versus non-ER mechanisms of action. Also, potential roles for
ERB, which binds both antiestrogens and environmental estrogens at much higher affinity and is
contained in brain, ovary, testis, and epididymis should be considered. This is particularly
important when there is a failure to observe effects on ERx or PR, as documented here and in
Endocrine Disruptors n, and when considering screening methods.
Morphological changes induced in the embryo by TCDD, as well as effects on growth factor and
receptor levels were documented Interesting model systems, including the rodent and human
palatal cultures, were established, and although species specific differences exist, many assay
measures were correlated with subsequent morphological effects Disparity in doses between
species will be significant in evaluating effects on humans, or in negating the usefulness of a
particular assay (such as the CYPIA1 knockout gene) to assess human risk. Experimental results
on the Ah receptor and its cellular association with ARNT are extremely interesting; although
dissociation of localization is at present unclear, it may suggest additional transcription
partners for these molecules. Data on glucocorticoid and GR actions, as well as induction of
specific growth factors, are intriguing but not well developed, and in some reports the type of
experiments are outlined, but the bottom line of experimental results is lacking. Additional studies
with the Ah knockout mouse as proposed should be useful.
Chemicals which affect the pituitaiy-adrenal axis, although potentially important, do not appear ai
this point to result in appreciable effects on the reproductive axis. Knowledge of basic endocrine
mechanisms and strongly developed rationales for these studies are lacking. The link between Ah
SJ?0111 mothers and loss of pups is transient, the link to "stress" is transient and no connection
to GR-mediated mechanisms has been made. A major effort on this axis with regard to endocrine
disruption is not warranted.
Data on the thyroid hormone system are similarly intriguing, but require additional information for
validation or for assessment of continuing studies (i.e., reversibility with T3. effects on TSH).
Thyroid hormone effects on the brain and learning are well-known, and decreases in fetal exposure
to T3 may indeed affect learning processes if untreated. Neurosensory deafness has frf*n
associated with some types of thyroid hormone resistance (TR mutations), but likely involves more
than lowering of T3 levels. The original finding that these toxins have antithyroid activity is of
interest and has potential importance. However, these long-term labor-intensive assays will be of
little use as a screen, and are unlikely to provide much information on thyroid hormone action. A
more general thrust to examine effects of maternal toxin exposure on both pre- and post-natal
development is, however, of great relevance and importance. The tadpole bioassay and TR
binding screening in ED II are of more immediate use for Agency impact purposes.
Data from this group of investigators have been used to support recommendations from an outside
panel of reviewers to use anogenital distance in the risk assessment for vinclozolin, which will
likely result in a new lower NOAEL, and a restriction on its use in Canada. Data on this
compound's induction of Leydig cell tumors also had significant impact in evaluating cancer risk.
Promcymidone was also reviewed. TCDD studies, particularly those documenting effects on the
fetus at concentrations three orders of magnitude lower than on the adult, have been incorporated
into the Agency's risk assessment. Dr. Gray has participated in several risk assessment and test
guideline committees.
image:
12
Summary and Conclusions:
In addition to comments above, mechanistic studies to compare in vivo and in vitro models, to use
Ah receptor knockout animals for mechanistic effects, and to examine synergy between various
compounds have bera proposed. Proposed studies on synergy at this point are not well defined,
and could take considerable resources and effort Furthermore, synergistic responses should be
examined not so much in compounds postulated to act through identical mechanisms (i.e.,
estrogens or antiandrogens alone) but also by those which should act independently, For example,
the emerging literature on synergistic interactions between steroids and growth factors, acting on
the steroid receptors themselves and thus on their responsive genes and on independently regulated
processes, is a critically important concept in endocrine cancer and likely to be similarly important
in development. This group, along with ED II, must also define / identify the number and type of
assays needed to evaluate estrogenic and anuandrogenic compounds in the environment.
ENDOCRINE DISRUPTORS II
Goal:
Endocrine Disruptors D provides research on improving methods for the assessment of
reproductive toxicity of endocrine disrupting chemicals. This group provides continuity with the
theme of Endocrine Disruptors I and broadens the approach by developing and improving in vitro
and in vivo models and tests for screening potential endocrine disruptors that are reproductive
toxicants, especially estrogenic and androgenic compounds.
The projects presented in this focal area are:
(1) Environmental anti androgen research focused on identifying and characterizing
molecular mechanisms of action of vinclozolin metabolites, the methoxychlor metobolite, HFTE,
and octylphenol. In addition, this project developed strategies to verify that the anuandrogenic
mechanisms observed in vitro were also operational in vivo.
(2) In vitro assays for environmental estrogens that tested a synergy hypothesis, estrogenic
activity of several compounds using a ER regulated reporter gene in MCF-7 ceils, and structure
activity relationships.
(3) In vitro and in vivo tests for evaluating estrogen and progesterone receptor function.
The in vitro studies identified the relative binding affinity of environmental chemicals for ER and
PR. The in vitro studies compared and evaluated relative estrogenic activity of chemicals using
different in vivo exposures (dose-time response and mechanism of action).
(4) The developmental toxicity of chlorotriazines is being investigated in a ret model that
measures the suppression of suckling induced prolactin release, delayed vaginal opening and
induced prostatitis.
(5) Single burst exposure to toxicants during the female cycle disrupts CNS control of
ovulation by inhibition of LH secretion.
(6) The development of tolerance of females to repeated dosing, when injection of toxicant
at a specific time point in the female cycle induces an effect (delay of ovulation).
(7) The ovary as a target for noradrcnergenic neurotoxicants that reveal direct exposure of
the ovary to the toxicant may be related to blockage of follicular rupture.
(8) An early pregnancy protocol is used to evaluate chemicals, and a sensitive and specific
bioassay for estrogenic properties of chemicals using a hypophysectomized, delayed implantation
rat model is proposed. In addition, a rat model of endometriosis and the effects of TCDD on that
model are being investigated. Ishikawa cells, the human uterine cell line, are being used to
determine effects of chemicals on cellular and molecular processes.
(9) Halocarbon induced pregnancy loss.
image:
13
Critique:
Overall, the research is broadly based with a wide range of topics, a variety of expertise and
numerous experimental approaches A strength is'the testing of multiple paradigms. For example*
there is a disparity between compounds —TCDD accumulation may be important in demonstrating
an effect after a prolonged period whereas CCU and atrazine, each alone, have critical windows of
sensitivity for effects on pregnancy and in the female cycle, respectively. This also emphasizes the
caution needed in developing single assays for reproductive dysfunction
The research on environmental anti androgens is solid and significant Considerable effort has
been expended on short-term screening strategies of estrogenic and androgenic compounds; such
studies should also be useful for screening of antiestrogenic and antiandrogenic compounds. Hie
expanding literature has demonstrated that the cellular complement of cefaclors and corepressors,
as well as receptor isoforms, is critically important in defining cell-specific responses to steroids,
and that such responses should, in fact, be expected on more complex biological promoters and
responses. Furthermore, specific nonbiological estrogens can combine with the ER to form a
complex which activates gene sequences which cannot be activated by the biological estrogen
bound to ER. Such data, in part, explain the disparity of results between mammalian cell-based
and yeast-based screening systems. Thus, the use of CV-1 cells may be valuable to determine
relative binding affinities, but would be insufficient to assess biological steroid effects. However,
a combination approach, using perhaps breast (the suggested MCF7 cells), and prostate cell lines,
might be more biologically relevant. At the very least, it is recommended that assays be conducted
on the binding of compounds to the ERp isoform, which is found at high levels in prostate, testis,
ovary and brain (with fairly low levels of ERq ) and in very low levels in uterus and breast While
the ERphas similar gene (so far) induction effects, and binds to natural estrogens in a similar way,
it has been found to have dramatically different binding capabilities for antiestrogens and
environmental estrogens, with several orders of magnitude higher affinity for these compounds
than ER. Given its cellular distribution in cell types of great interest to this group and this
differential ligand binding characteristics, it should be included in- similar screens. As mentioned
previously, the data on the synergy of potential environmental estrogens were not supported by
data from the EPA (and other groups), and have since been withdrawn. Careful studies by EPA
scientists played an important role in this effort
In project 2 above, it was observed that xeno-estrogens such as p.p'DDT, methoxyclor and others
inauced estrogen dose responses similar to in vitro reports by others who also used mammalian
cells. Although these data are positive, the lack of ERp in this system is a shortcoming; ERqER^
effects should be distinguished in order to provide a more complete investigation.
The estrogen synergy studies were found to be negative even though some of the xenoestrogens
produced additive results. Some compounds (dicofol) were found to have no effect on MCF-7
proliferation yet the reporter gene was activated in the MVLN cells. Other compounds (bisphenol
A) showed consistency in estrogenic proliferation of MCF-7, reporter gene activity and in vivo
estrogenic responses. The variation in consistency between compounds in the various assays is
inexplicable and confusing. The rationale for the use of all of these assays for high throughput is
questionable, given the variations and the likely need for in vivo testing regardless of the outcome
of the in vitro testing. One cannot overlook the positive responses as strong indicators; however,
the lack of response in vivo and in cdl based in vitro assays is problematic for interpretation.
In project 3 above, competitive binding studies using ER and PR, various hormones, drugs and
image:
1 4
environmental chemicals were conducted. Chlordeccme, o, pDDT and nonylphenol inhibited ER
and PR binding with similar affinities. In vivo studies revealed estrogenic acuviues of some test
compounds at extremely high doses; the time between dosing and killing (and route of
administration) was critical for detecting changes in uterine weight The likelihood of achieving
these levels from environmental exposure is highly unlikely, although a solid point of these studies
is that binding to BR + PR can occur. However, the involvement of ER alone or in combination
with other factors in disruption of cycles after chemical treatment in vivo is questionable; other
mechanisms may be involved. The further complication of metabolism and clearance of the
chemicals tested weakens the in vitro model.
In the 4th project above, atrazine was found to suppress prolactin in suckling mothers on postnatal
days 1-4 and delay vaginal opening in female offspring depnved of prolactin from the mothers
milk. The male offspring of the atrazine treated mothers showed a higher incidence of prostatitis.
Both the delayed vaginal opening and prostatitis correlated with hyperprolactinemia. Although
effects of decreased prolactin on postnatal days 1 -5 had been shown previously, this is the first
demonstration of an environmentally relevant chemical having this effect. The future studies
proposed seemed reasonable and will provide further evidence for a role of prolactin in these
In the 5th and 6th projects above, it has been shown that a single appropriately timed injection of
the toxicant atrazine disrupts the female cycle whereas repeated dosing of the same toxicant induce
an acclimation (or tolerance). The importance of these results is obvious and they indicate Bie neea
to use more than one paradigm. Additional studies have tracked the mechanism of cycle disruption
and provided strong evidence using classical neuroendocrine methods that noradrenergic
transmission is disrupted by atrazine, leading to inhibition of the LH surge Tpe mechanism ol
tolerance (after repeated dosing) is unknown. However, it will be a subject of numerous fiiture
u>;th >h,» nhiM^ives nf extending the scooe of the problem to other compounds and
events.
assessing wneiner meiaooiism ui— -- . •
central description of additional studies to assess the effects of the ovulatory delay ra toe quality
of the oocyte, the preimplantation embryo and functionality of offspring were provided. The role
of the ER and AR in atrazine action was lacking;this could tie it to the antiandrogen studies but no
evidence was provided A more cellular model does not appear forthcoming as the mechanism 01
GnRh pulsatility is poorly defined and evidence for its control by norepinephrine is lacking, in
.... * > ¦ • « ¦ 4a lu >«•« >a1
addition, multiple neural pathways are likely to be involved.
¥_ .l. -».l —nvini Kqc t>M>n nrrmnsed to serve as a direct target for atrazine. This concept
chemical provided to the ovary may also far exceed the level reacmng tne ovary ««cr
administration or by gavage. Thus, a hypophysectomized
may be more appropriate (this would also reduce the chance of adrenal involvement).
•n.. o.i j.ou ivirh eft,en* nf chemicals on uterine function that then lead to adverse
done to determine mechanism oi acuon. More vigorous mui».uiai
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15
needed to provide insight into these mechanisms.
In the endometriatic models, TCDD and methoxychlor, each aJone, were found to enhance the
growth of the lesions. These are gross observations that lack any biochemical mechanisms other
than the receptor involvement. The proposed studies on calcium channel blockers and the role of
the adrenals in this response are not supported by theoretical underpinning.
The hypophysectomized delayed implanting rat model is interesting but has been described by
others as a model for testing estrogenic (and antiestrogenic) chemicals. The research will identify
other chemicals thai have estrogenic effects. The future direction of this model will apparently only
serve as a screen.
The studies on Ishikawa cells are too preliminary for thorough evaluation and the proposed studies
appear weak. Failure to establish the cell line screening method prevented assessment of human in
vitro models.
In the 9th project (last project), various halocarbons were administered at specific time points
during pregnancy. The most interesting compound tested was CC1< which when given on days
6-7 of gestation induced resorption by inhibiting LH and progesterone secretion. Reversal of the
deleterious effects was accomplished by LH or progesterone or GnRh by replacement therapy.
Thus, the target for the action of CCU appears to be the hypothalamic pituitary axis. These studies
provide an additional example of the use of classical reproductive endocrine techniques to
determine the endocrine mechanism responsible for a chemical induced pregnancy termination. In
depth studies are unlikely to be forthcoming.
Summary and Conclusions;
Overall, the most exciting and productive studies are die molecular mechanisms of androgen
(anti-androgen) action. These studies use solid and state-of-the-ait techniques that provide keen
insights and unique hypotheses. A moderate level of enthusiasm is given to the neuroendocrine
studies of atrazine and its direct effects on ovarian function, although the major weakness is the
classical nature of the studies and lack of rigorous in depth mechanistic approaches. The weakest
studies include the haJocarbOn and uterine targets for chemical induced pregnancy loss
endometriosis, and the in vitro testing of estrogenicity.
Although this theme was split from ENDO I, there appears to be little cross talk between projects.
Although there is some indication of interaction, it is minimal. There is no synergy in this group.
The reasons for this are unclear but it may be related to the disjointed nature of the projects. There
was little indication of extrapolation of this information from animals to humans.
Some of the weaker projects have appeared to reach the point of diminishing returns. The lack of
specific preplanned future directions, with sufficient detail to allow a thorough evaluation, is also a
shortcoming.
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16
RTD PEER REVIEW
August 13-15, 1997
Dr B.A. Scbwetz, Director, NCTR, 3900 NCTR Road, Jefferson, Arkansas. 72079*
Dr. John M. DeSesso, Mitre Technical Systems, 7525 Colshire Drive, McLane, VA 22102-3481
Dr. Steve Downs, Marquette University, Biology Dept., 2514 N. 64th St, Wauwatosa, W1
53213
Dr.Barbara Hales, McGill University, Dept Of Pharmacology and Therapeutics, Mclntyre
Medical Sciences Bldg., 3655 Drummond St, Montreal, Quebec, H3G IY6 rnnadn
Dr. Bernard Robaire, McOill University, Dept Of Phannacolocy, Rm 115,3655 Drummond
Street, Montreal, Quebec, H3G 1Y6, rnrmda
Dr. Margaret Shupnik, University of Virginia, Health Sciences Center, Dept Of Medicine, Box
578 HSC, Charlottesville, VA 22908
Dr. Paul Terranova, University of Kansas Medical Center, Center for Reproductive Sciences,
3901 Rainbow Blvd., Kansas City, ICS 66160
Dr. Teny Turner, DepL of Urology, Box 442, University of Virginia School of Medicine
Charlottesville, VA 22908
Dr. Rochelle Tyl, Research Triangle Insitute, 3040 Cornwall is Road, Research Triangle Park,
NC 27709-2194
* Chair
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1 7
OVERVIEW
The Reproductive Toxicology program at the RTP site of the EPA consists of five focal groups
including dysmoiphogenesis, gamete toxicology, disinfection byproducts, and endocrine
disrupters I and II. Together, the RTP groups address potential actions of toxic substances on the
reproductive system ranging from dramatic effects on morphology and sexual development of
offspring to more subtle changes in reproductive capacity of exposed individuals and multiple
generation studies of their progeny. Given previous examples of and information on
PCBs, DDT and DES, this approach is both appropriate and warranted.
In an effort to develop appropriate and accurate predictive tools, a mixture of both in vivo and in
vitro assays either have been established or are in development. Since many rapid ceil culture
based assays may not adequately reflect all possible physiological responses, a major question in
this field is the acceptance of a particular assay and its predictive ability. It may not be possible to
eliminate whole animal studies for many observed effects, particularly those on development or
multigenerational effects. Assays suggested by this group have concentrated on specific
mammalian tissues. These include mouse and human palatal cultures, which appear to be accurate
reflections cf dysmorphology effects, and several mammalian clonal cell lines (MCF7, COS) to
investigate sex steroid effects [discussed below] As many of these steroid effects are documented
to be cell-specific, some of these assays may need to be expanded or altered to address emerging
issues. The development of models to correlate in vivo with in vitro effects is important, and
should be continued.
A basic problem to be addressed by all workers in this area, is the relevance or veracity of
anecdotal clinical reports, or the ability to extrapolate animal models to the human condition.
Finally, an important issue, which is being addressed, is the basic difference between toxic and
endocrine dose approaches, and the fact that the large doses needed for some toxic effects may not
correlate with biological effects seen at lower doses; efforts to correlate effective doses with actual
tissue accumulation are underway or have been performed.
While the program in general appears to have a strong mix of classical and cutting-edge
technologies, the approach is not homogeneous across focal groups. When appropriate, transgenic
and molecular approaches should be encouraged because of their power and sensitivity. Of
necessity, many of the programs have been established in a response to previous observations by
others, and due to their very nature would not be expected to break new scientific ground. That is,
it is their purpose to carefully test a perceived problem or danger at the level of biological effect to
provide data to judge the relative risk. However, even given this situation, some important
advances in knowledge have resulted, including data on antiandrogenic properties of some
pesticides, and data on ARNT/Ah receptor localization and expression. The topics pursued by
these investigators have become, m the past 2-3 years, of great interest to the general public as well
as the scientific community; this is reflected by recent increases in the invitations to national and
international meetings. Prior to this time, it appears that many investigators here were somewhat
isolated, but this situation seems to have greatly improved, at least for some. In some emerging
fields, such as endocrine disniptors and gamete toxicology, EPA scientists have the opportunity to
be leaders. In order for this to materialize, these investigators have to be involved in the endocrine
as well as the toxicology communities, by attending those meetings, by publishing in high quality
and well-cited peer-review journals outside the realm of narrow disciplines. There is variability in
the success or completion rate of projects among and within the focus groups, as well as strong
directions for future work. For example, there does not seem to be a strong rationale to continue
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18
studies on mechanisms of methanol and arsenic toxicity, and a search for affected kinases in the
developing embryo may not result in valuable new information. Studies on prolactogen and
placental lactogen on egg sac and embryo development are also not well developed or rauonalued,
and do not appear to be tightly related to the overall goals of the dysmorphogenesis group.
In all programs, it is difficult to evaluate resources and budget, as no spreadsheets were given.
Further, although core facilities were referred to, they are not elaborated upon or defined, and
cannot be evaluated. From information given, the assumption is that all salaries are line items in an
overall budget, as well as equipment, travel, and animals costs (unclear from material provided);
therefore, budget allotments refer only to supplies for an overall focus group and in some cases to
an IRP. Based on that information and set of assumptions, and compared to other academic or
institute laboratories, the physical resources appear adequate to address the scientific problems and
to carry out the proposed studies. However, the rationale for the attribution of personnel and their
relative distribution among focal areas is elusive and in some cases does not appear lo be well
founded.
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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
National Health and Environmental Effects Research Laboratory
Reproductive Toxicology Division
Research Triangle Park, NC 27711
OFFICE OF
RESEARCH AND DEVELOPMENT
September 18, 1998
MEMORANDUM
SUBJECT: Divisional Peer Review
FROM: Robert J. Kavlock, Director
Reproductive Toxicology Division (MD-71)
THRU: Linda Birnbaum
Acting Associate Director for Health, NHEERL
TO: Hal Zenick
Acting Director, NHEERL
The Reproductive Toxicology Division (RTD) Program Review was held on August
13-15,1997, and a report by the panel was submitted to RTD in June 1998. This memo
comprises the official RTD response and actions related to the recommendations of the
peer review panel.
The report focused on the overall function of RTD, and the research functions
grouped into five main focal areas. The report noted that RTD has a long history of
important scientific contributions to the field of reproductive toxicology, but commented that
the group could become even more influential in the future should it undertake some
changes in the methods of overall research planning and allocation of resources. Two
factors in particular were deemed worthy of attention: the broad range of productivity
between investigators, and the tendency for some to be spread too thin. In terms of areas
of focus, the panel suggested that more resources should be devoted to the study of
gamete toxicology, and less attention should be devoted to research on dioxin. A final
general recommendation dealt with the need for broader scientific interactions within and
outside NHEERL. A synopses of the general- and focal-area specific comments provided
by the panel are provided in Appendix 1. In addition to responding to recommendations
of the peer panel, these changes also address some of the communication issues raised
by the Williamsburg process, and accountability issues raised by GPRA.
^osr-%
* ifh
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General Comments:
In FY98, RTD revamped the research planning process to more clearly articulate
the major goals and objectives of its research program. Each research theme or individual
research plan now contains a relevancy statement, a statement on the signification of the
effort, and a series of specific aims intended to be addressed over one-two year
increments. For each aim, there are defined resources and time lines for accomplishment.
The disaggregation of research efforts into specific aims has greatly facilitated transition
to the GPRA framework. Indeed a crosswalk between specific aims and GPRA research
areas was completed this year along with establishment of milestones through FY2000.
Allocation of FTE effort to GPRA Goals and Objectives has been made according to the
cross walk of the specific aims to GRPA. Thus, the accomplishments of specific aims are
tied directly to GPRA performance in terms of both level of effort and performance.
Progresses on the aims are reviewed semiannually, and formal decisions on continuations
and new starts made annually. The division is currently drafting a strategic research plan
to guide the direction of our work over the next 3-5 years; we feel this exercise will aide in
the critique that we need to establish a clearer vision and priorities for the research
program. We will also be actively promoting publication in a more diverse range of
scientific journals and in presenting findings outside the traditional toxicology meetings in
order to interact with a broader range of audiences.
Other reforms/staffing initiated include: further strengthening the functioning of the
research themes by instituting quarterly theme meetings, and monthly meetings of the
theme leaders. Recent topics in these meetings have included hiring strategies, research
planning, the response to the Peer Review, and recognition of team efforts under the new
PERFORMS system. For example, to increase input of Team Leaders in research
decisions and to address the issues of breadth vs. depth cited by the Peer Review panel,
this fiscal year RTD management worked with Team Leaders to develop guidance on
procedures for approving new theme (i.e., team-based research) proposals and for
evaluating existing themes (self assessments and management feedback). These two
activities are designed to enable RTD to incorporate emerging issues into its research
strategy and to respond to changing Agency needs, ensure that resources are being
directed toward the most productive and relevant research programs, provide ample
opportunities for all Pis to develop their research programs, and ensure that RTD achieves
the appropriate balance of breadth and depth in its research pursuits. Along these lines,
we will be de-emphasizing the use of individual research plans in FY99 and encourage
even stronger team efforts in the planning process. Individual creative research will still
be possible within the context of the 20% effort each investigator is allowed outside the
existing formal planning process. Strong consideration is being given to
reducing/consolidating the number of themes supported to create greater depth in the
program.
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All three branches now have active journal clubs to increase intra-divisional
communication of broader aspects of science, and a molecular biology course has
been established within the division to familiarize the technical staff with recent
advances in this area of science.
To ensure that our core facilities are effectively utilized, we have formed an
ad hoc team (Darney, Dix and Abbott) to address questions about the space
utilization and better define expectations for the investigators in charge of specific
labs. At present, the specialists in these laboratories contribute in their area of
expertise to the research in one or more themes. Thus, they are expected to
remain abreast of advances in their field, provide advice to RTD staff, and develop
new methods in support of RTD theme objectives. They are not expected to
develop an independent hypothesis-driven research program, but this does not
preclude them from conducting their own studies (and publishing the results) within
the context of the theme research plan. The committee will evaluate current and
projected needs in these areas, and make recommendations to RTD management
concerning how we can best prioritize the expertise available in these laboratories
to strengthen our theme-based research.
RTD has also developed retrospective budgetary summary tables that
detailed for each Principal Investigator the amount of annual funding (expense,
extramural, animal care, PC&B, and R&D) required to support his/her laboratory
along with a tally of the number of technical publications resulting from his/her
efforts over a two-year period. While it is hoped that this information will impart to
the Principal Investigators an appreciation for how much their research efforts cost
the taxpayer, these data also will be used as a cost/benefit index during the
retrospective theme review, and to subsequent external peer review panels.
In terms of personnel issues to help achieve a better balance in technical
support, several changes have been instituted. We converted one investigator
position (formerly occupied by a GS-13 who retired in 1/98) to a technical level
support function in the analytical chemistry laboratory and are reserving placement
of an additional technical position pending completion of the current hiring plans
(see below). To better utilize expertise, we detailed one senior level GS-15 position
to the Associate Director for Ecology to work on the declining amphibian and reptile
issue. We are also planning to switch representatives on the IACUC to better
balance the committee assignment load within RTD (more in the DBP Theme).
Finally, we have markedly boosted the recruitment of postdoctoral fellows to high
priority areas (detailed in the succeeding sections). The newly authorized
NHEERL Postdoctoral positions were particularly used to bolster areas in need of
additional resources and to bring more advanced molecular techniques into the
mode-of-action studies (e.g., see the Gamete Biology section). The technical level
position mentioned above that is being held in escrow is currently being used for
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less than permanent full time staff (e.g., the STEP program), as division
management evaluates the optimal use of that FTE. To increase staff's
understanding and appreciation of emerging technologies in the rapidly evolving
field of molecular biology, an area the Peer Panel felt strongly about, RTD, in
conjunction with ECD, has begun a series of internally taught introductory lectures
that have been well attended by both RTD and ECD staff. It is hoped that this
forum will encourage productive interactions among staff as RTD accelerates its
transition toward molecular approaches to elucidating mechanisms of normal and
toxicant-perturbed development. We are also contracting to provide technical
writing training, both on an introductory level and on more professional aspects to
assist the staff in communicating their research to the outside community.
For new staff positions, we have modified our plans to recruit a theoretical
modeler of pattern formation into a more experimentally oriented position, and
conducted national searches for this position and the one in signal transduction.
As of September 27, we are pleased to say that Dr. Suzanne Fenton will be joining
RTD to pursue toxicant effects on membrane functions. Dr. Fenton received her
Ph.D in Endocrinology-Reproductive Physiology (with a minor in Statistics) from the
University of Wisconsin, Madison in 1993 and completed a postdoctoral research
fellowship at the Lineberger Comprehensive Cancer Center, University of North
Carolina. Her research has focused on the molecular regulation of signal
transduction factors involved in development (e.g., mammary gland) and ovarian
function (gamete physiology) and has extensive experience with a number of
sophisticated techniques that will help evaluate the target sites and mechanisms
through which reproductive and developmental toxicants disrupt function. Although
still in the planning stages, Dr. Fenton will be involved with research evaluating the
effect of the chlorotriazines (mammary gland and prostate development) as well as
collaborating and serving as a resource for themes. We are also in final
negotiations with our top candidate for the pattern formation slot and hope to have
that filled within the next month. Each of these positions has an associated
technical support slot. Of final note, Dr. Kelce has left the Agency, and plans are
being initiated now for a national recruitment to replace that function in the
Endocrinology Branch.
With the completion of the comparative embryonic sensitivity studies of
dioxin-induced cleft palate, we have reduced the resources devoted to dioxin-
mediated toxicity, as encouraged by the panel (see Dysmorphology section for more
details).
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Dysmorphology.
The review noted that some of the Pis in this unit have made significant
contributions and have been recognized both within and outside the Agency. We agree
with the comment that components of this area are largely phenomenological,
characterized by morphological endpoints, but this represents the current status of the field
in the area of genetic control of development. Gene expression in terms of temporal and
spatial patterns is in the process of being determined for many of the genes of potential
interest to prenatal toxicology. Most of the genes studied to date have, in fact, turned out
to be transcription factors rather than structural genes, and the linkages among them or
how they actually produce a nascent structure is far from understood. Our pioneering
experience with developing a mechanistically based dose-response model for 5-FU, and
efforts to elucidate molecular correlates of altered skeletal patterning provide ample
demonstrations of the current limitations in the field. Nevertheless, we are taking several
steps to address the overall criticisms of this focal area.
We have convened a small workshop of about ten experts to specifically review
progress and potential future directions for the 5-FU project and the expectations
for BBDR modeling in risk assessment. This group was chaired by a member of the
Peer Panel (Tyl), and consisted of modelers (Portier, NIEHS; Andersen, ICF Kaiser;
Crawford-Brown, UNC), risk assessors (Kimmel, NCEA; Jarabek, NCEA),
developmental biologists (Muneoka, Tulane; Knudsen, Thomas Jefferson
University), developmental toxicologists (Tyl, RTI) and pharmacologists (Smith,
Glaxo-Welcome). It met on September 9-10, 1998, and we expect to have a
written report to a series of questions poised to the panel by the end of October.
Given the interesting discussions of the workshop, we anticipate that the report
should be published in a journal. We expect the outcome of the discussions will
help guide our program in this area over the next several years.
We have undertaken a top to bottom review of the research directions of the
Developmental Biology Branch. This effort is identifying those particular questions
that the research program should be addressing, and formulating research projects
to address them. The BBDR framework will likely provide the overarching
organization of the effort, but specific targeting of a project is still under discussion.
Input of the EPA Risk Assessment and Program Offices are being sought as part
of this project to bolster the relevance of the research to the function of the Agency.
The outcome of the effort is expected to be incorporated into our FY99 planning
process.
As noted above, the recruiting action for a new investigator in this area was
significantly modified, and a final selection is imminent. It is expected that this
position will greatly strengthen our efforts in identifying the role of pattern formation
genes in dysmorphogenesis caused by environmental toxicants. We hope to
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eventually rely on the new EPA-sponsored, Triangle-wide modeling consortium
currently being put together (a member of the BBDR review team is involved in that
effort via UNC) to bring what we feel is the requisite integration of the biological
information into a more formal mathematically-based construct.
For methanol, our previous efforts are being brought to publication, and
future efforts will likely be melded into the newly emerging research plan within the
DBB.
Gamete Toxicology:
The review was quite favorable with regard to the two main elements presented
under gamete toxicology: 1) Sperm regulation and function, and 2) Protective mechanisms
in gametes and early embryos. In general, the established investigators were commended
for their contributions to EPA test guidelines in the area of gamete considerations, and for
validating the use of CASA analysis for rodent toxicology studies. General
recommendations included expanding efforts in evaluating earlier stages of
gametogenesis, and in applying molecular biology to understand mechanisms of toxicant
action in the gametes and gonads. Accordingly, increased manpower in gamete toxicology
was recommended, with emphasis on molecular expertise. In the area of sperm
regulation, the report commended the establishment of the epididymis as a target for
reproductive toxicants. While cautioning against "overselling" SP-22 as a biomarker for
fertilizing ability before its utility is demonstrated, the report was supportive of the plan to
better characterize this and other proteins. On the other hand, the report questioned the
ultimate value (not the quality) of the sperm energetics project as a priority research area
for RTD. The report also affirms the relevance of research being conducted in the
protective mechanisms theme on the importance of glutathione and stress proteins in
oocytes and seminiferous tubules, respectively, and encourages extension of this
approach to characterize protective mechanisms in both male and female gametes and
gonads.
Since the review, progress on characterizing SP-22 has been extensive and
the results address many of the concerns of the reviewers. In particular, using an
affinity-purified antibody, SP-22 has been localized to the equatorial region of the
acrosome of rat, bull and human sperm. Moreover, the levels of SP22 on bull
sperm, determined by quantitative FITC labeling, appear to correlate with fertility
in this species as they do in the rat. Finally, the SP-22 gene has been cloned, and
using a testes cDNA library, two testes-specific transcipts coding for SP-22 have
been identified. The increased pace of the research was made partially possible
by a de-emphasis in the sperm-energetics work over the past year. These results
have appeared as a "Breakthrough" paper appearing in the July/August issue of J.
Andrology, and are being used to support worldwide patent protection for the
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invention. Furthermore, several additional candidate biomarkers of sperm or
testicular function have been identified that will be pursued in the future.
Localization of these proteins in seminiferous tubules, as well as mature gametes,
is being defined using confocal microscopy, and this effort will be expanded by the
addition of a postdoctoral fellow in the coming year. Another postdoctoral fellow will
join in the molecular biological studies of SP-22 this winter from divisional
infrastructure funds.
In response to these recommendations regarding the need for attention to
early stages of gametogenesis, a junior level investigator has been detailed to the
Gamete Biology group from the Endocrinology Branch. Tammy Stoker, who
recently received her Ph.D., will work underthe direction of Sally Darney in the area
of oocyte growth and development. This achieves two important objectives-
increased resources for evaluating early stages of gametogenesis, and career
development for a new investigator. Our initial plan is to convene an internal
working group to prioritize research needs in this area relevant to the ORD strategic
plan. Input will also be sought from several outside experts in folliculogenesis and
ovarian toxicology through the RTD seminar program with the goal of forming an
external advisory committee as we consider additional research needs in the area
of environmentally-mediated impacts on gametogenesis.
The HSP project will be expanded and strengthened by focusing the efforts
of an NHEERL postdoc. In addition, and partly in response to the review
recommendations, expertise in molecular biology is being added to the gamete
toxicology group in the form of a second NHEERL postdoc. This individual will
focus on the use of DNA arrays to evaluated altered gene expression in response
to testicular insult. We are very excited and optimistic about this approach since
it encompasses all components of the gamete toxicology program. Thus, we expect
to be able to monitor genes for HSPs, oxidative stress (GSH system), and sperm
biomarkers including SP-22. This effort will be tied, in part, to an increased
emphasis on children's health. We plan to choose a "positive control" for the gene
array project that affects testis function when given in the postnatal, pre-pubertal
exposure window. Dr. Dix is RTD's point person for the children's health initiative,
and his understanding of this issue will help guide this effort.
The above plan should also help to integrate the junior investigators in the
GEEBB into high priority, theme-based research projects, and at the same time
encourage their development as autonomous investigators (another concern of the
reviewers).
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Disinfectant By Products:
The RTD strategy in this important programmatic research element is to identify
hazards, conduct mode-of-action studies, develop biomarkers for potential field
application, and build BBDR models as necessitated by the data. The review panel
expressed its concerns regarding the use of relatively high dose levels in the toxicology
studies and the lack of apparent input into the studies being conducted with the NTP while
applauding the movement into potential effects on the non-pregnant female.
In terms of the dose levels used in the studies, we wish to point out that the
first function of the RTD DBP program is that of hazard identification that almost by
necessity focuses on high dose level effects. Once an effect is identified in this
component of the program, the dose-response relationship is further examined, and
mode-of-action studies initiated as deemed necessary and appropriate. Indeed, our
work with the brominated haloacetic acids has lead to the determination of
exposure scenarios that interfere with spermatogenesis that are well below those
causing other significant adverse health effects in experimental models. How these
levels compare to real world exposures may still indicate a broad margin of
exposure, but this can only be determined by experimental observations. However,
we do recognize the importance of studying long-term low-level exposures which
may be more reflective of the human situation, and we have taken steps to build
collaborative efforts with both CSU and RTI to pursue such exposure paradigms.
The latter project in particular will focus on the effects of a multi-generational
exposure of the rat to chronic, low levels to one of the most potent DBP-related
reproductive toxicants identified to date (bromochloroacetic acid). Due to several
recent epidemiological studies reporting altered reproductive outcomes following
consumption of disinfected drinking water, we feel it is all the more imperative to
provide careful and thorough assessments of the dose-response relationships for
these chemicals, regardless of the dose level where adverse effects are observed.
One postdoctoral fellow, and one predoctoral fellow have been recruited to conduct
research on potential effects of DBPs on female reproductive physiology.
In terms of our relationship with the NTP, the panel was concerned about the
selection of the experimental protocols and the extent of involvement of the RTD
staff in the effort. We wish to offer some clarification. Relative to the study
protocols, we have nominated, and NTP has accepted, a list of DBPs (based on
both absolute concentrations and prevalence of detection) for testing using
standard NTP protocols. These protocols have gone through their peer-review
process and are scientifically defensible. We recognize that the protocols may only
detect the more active of the DBPs, but this is exactly the function of that
component of our research program we seek. Relative to the RTD involvement in
the NTP studies, we are actively consulted on each phase of the effort, from the
selection of dose levels, through to the review and acceptance of the final contract
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report. Our investigators review each research report derived from this interaction,
and provide feedback to the NTP project officer on the quality of the study and its
interpretation. Therefore, we feel that we are active collaborators in this effort, and
have opportunities at many levels to provide input and oversight.
Therefore, we feel that we are continuing to provide the Agency with the
necessary data for priority DBPs. As we progress in this research area, we will
undoubtedly be shifting from an emphasis on DBPs to others based on the new
contaminant list of the Safe Drinking Water Protection Act.
Endocrine Disruptors I and II:
This research area was perceived as composed of strong, productive investigators,
with well-defined model systems and relevant goals. The research on environmental
androgens was considered particularly solid and significant A noted strength of the effort
was the ability to use multiple experimental paradigms ranging from molecular biological
studies on receptor-ligand activation to classical neuroendocrine methods, suggestions
were made in area of synergisms, and extent of effort placed on dioxin, pituitary-adrenal
interactions, and thyroid gland function.
Our work with development of screening assays for endocrine disruptors has
continued with the development of a permanently transfected cell line containing
the molecular machinery for evaluating androgen receptor activation. A similar
effort is being considered for the estrogen receptor(s), pending resolution of the
EDSTAC screening battery and the recruitment of a replacement for Kelce. We are
remaining active in the Screening and Validation Workgroup that will begin the
implementation process for acceptance of the EDSTAC Tier 1 Screening Battery.
In this regard, we have contributed the mammalian screening protocols and
developed a list of chemicals for use in validating the HPTS. While the peer
review encouraged work on TR binding and the tadpole assay, we feel that these
areas, particularly the latter, are better suited for MED/NHEERL and we have
recently assisted in the training of an NRC fellow from MED in the cellular-based
assays of steroid receptor activation.
Future plans in the area of anti-androgens include the recruitment of an
NHEERL Fellow (Parks) to work in the characterization of genetic alterations in
exposed developing reproductive tracts. This will be a collaborative effort of the
Abbott and Gray laboratories.
With regard to the perceived over-emphasis on TCDD, a considerable
amount of research has been brought to conclusion in the last year, and three
manuscripts are being published on the findings. A small effort is continuing in
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investigating the roles of AhR and ARNT as transcription factors important to some
developmental events such as angiogenesis and vasculogenesis. Further effort
in this area is under evaluation as part of the overall planning exercise being
conducted in DBB.
Although we agree that evidence directly linking chemically-induced
disruption of the adult/neonatal hypothalamic-pituitary-adrenal axis and
developmental toxicity is lacking, we feel a small effort is still justified in this area
of endocrine toxicology, as it is known that stress in general can alter
developmental processes (both structural changes to the axial skeleton and
functional changes in reproductive behaviors).
The work on the hypothyroid effects of PCB exposure is being concluded,
and attention is being directed at perturbations in glucocorticoid receptor functions,
which appears to be down-regulated in the developing brain following PCB
exposure.
The effort devoted to adrenergic control of ovulation is being brought to a
conclusion, and the focus shifted to that of female reproductive effects of DBPs (see
above).
The atrazine effort is moving from a descriptive site-of-action approach to a
more molecular approach involving whether GnRH neural cell lines are targets,
whether atrazine effects dopamine beta hydroxylase activity in PC12 cells, and
characterizing the effects on the GABA receptor using both PC12 cells and an
insulin secreting cell that contain all GABAa receptor variants. A new postdoctoral
fellow has been recruited to assist in this effort. Relative to the comment regarding
the need to examine the effects of atrazine on the ER and AR, these studies had
previously been conducted by ourselves (and reported at the SOT in 1996) and
others and found to be negative, although we do plan to evaluate any potential
effects of the chlorotriazines (and their metabolites) on phosphorylation of steroid
receptors as well as steroid biosynthesis in the near future.
SUMMARY: RTD appreciates the recommendations of the Peer Panel, and has taken a
number of steps over the past 12 months to address the major issues. Specifically, we
have revamped our research planning process to have a more focused and accountable
basis, we have aligned the research aims with the GPRA Goals and Objectives for ORD
and NHEERL, we have modified our hiring plan for principal investigators and are in the
midst of adding two sought after areas of expertise (signal transduction and molecular
morphogenesis), we have added a number of postdoctoral positions to areas considered
in need of more resources (primarily gamete biology), we have increased specific training
to the staff in the areas of molecular biology and technical writing, formed journal clubs
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within the branches, and opened up new avenues of communication within the division to
facilitate the interface of the research to the mission of the Agency, we are encouraging
broader selection of journals for submission of manuscripts and meetings to attend that will
increase our connections to the scientific community, we have undertaken a
comprehensive review of the research effort in BBDR models and in developmental
biology, and we have begun to de-emphasize some particular chemical-specific work as
recommended by the Panel. We fully anticipate that these changes will put us in a
stronger position to defend our program in the next cycle of the peer review.
cc: RTD Staff
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Update on the 1998 Response
to the 1997 Peer Review
September 12, 2001
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General Comments:
The following response is structured according to the outline of both the Peer
Review Report and our subsequent Response. The 1997 review of RTD raised
several general issues: the need to improve the process by which future research
areas are selected and laid out, the broad range of productivity among
investigators; the tendency to be spread too thin in certain areas; and the need for
greater interactions with the broader scientific community. In addition, the Panel
suggested that greater attention should be given to research in the area of gamete
toxicology, although no specific guidance on important research needs or gaps was
provided other than the need to examine gametes while in the gonads.
Research Planning: As noted in our 1998 response, we have taken, and
continue to take, a number of steps to strengthen our theme-based research
planning process, and to align our research activities with the goals established by
GPRA. Our research planning process continues to evolve and to have greater
impact and influence, and we feel we have made considerable progress in this area
over the past 4 years. The process provides the scientific staff with an opportunity
to articulate how they perceive their research can have impact on the Agency, and
to receive feedback from management on the alignment and scientific goals. Many
of the items detailed below provide direct evidence of the steps taken and the
outcomes witnessed that we think demonstrate the utility of our planning process.
By improving our planning process, we also feel that we have reduced the
tendency to have a technology-driven, as opposed to problem-driven, research
program, but we realize that this is always a struggle when new and enabling
technologies such as genomics are made available to biological scientists.
Research Productivity Relative to issue of variable productivity, the
importance of communicating our research in the peer-reviewed literature has been
continually emphasized within the Division, and the extent to which this aspect has
been addressed is best judged by evaluating the performance of the research
themes and the related investigators. There is an ever-increasing tension between
attempting to cover the scope of the problems before us, and the gradually
tightening restraint of FTE slots available to conduct the work. The challenge is
made greater by the generally low staff turnover, and the need to maintain a basal
level of administrative support to the operations. The net consequence of changes
to the workforce is that technical support to investigators is at a premium, and
without adequate technical support, productivity is likely to be weak in some areas
RTD management has continued to examine placement of resources, and to try
and maintain critical mass in the highest impact areas relative to the Aqency's
mission.
While we have had increased access to post-doctoral trainee slots (both
through the intramural ORD Post-doctoral Fellows Program as well as the
extramural training agreements with UNC, NCSU and Duke University), we, like
other institutions across the country, have faced significant challenges in recruiting
qualified applicants for these positions. Partially for this reason, we have recently
become more dependent on the use of pre-doctoral trainees in our workforce mix.
31
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Determination of where to place the trainees, be they pre-doctoral or post-doctoral
fellows, is done on a competitive basis within the Division, with proposals sought
from investigators, and judged by RTD management based upon programmatic
relevance, scientific quality and track record of the investigator. This helps place
more resources in the areas of highest priority and productivity. We have also
offered specific in-house training courses to the technical staff to help with their
career development and broadening of their job skills. These have included a
lengthy course on molecular biology, and a shorter one on writing skills. Currently,
we are nearing the end of an employee-initiated and organized 13-week program
on statistical techniques. We have also availed ourselves of the opportunities
available at institutions around RTP and a number of staff have taken courses from
the nearby academic institutions.
Like with trainee allocations, we have focused utilization of permanent
vacancies to bolster the more productive and promising areas. As noted in the
1998 Response, we hired Dr. Sue Fenton to develop a program on mammary gland
development with the Endocrinology Branch, and assigned one permanent FTE as
support. This helped provide critical expertise in the area of signal transduction,
which we did, and continue to, consider a key area that we need to work in (witness
the recent report of the NAS on New Frontiers in Developmental Toxicology Risk
Assessment). We were not able to fill the other recruitment we were pursuing at
that time, a molecular developmental biologist working on pattern formation, and
have since decided not to increase activities in that area with additional outside
recruits. More recently, we hired Dr. John Rockett to work in the area of
toxicogenomics within the Gamete and Early Embryo Development Branch, and Dr.
Vickie Wilson in the Endocrinology Branch to work on steroid-receptor assays
suitable for characterization of endocrine disruptor mechanisms of action. Dr.
Wilson was assigned an existing FTE support position from within EB, while Dr.
Rockett does not currently have full time technical support (he is the highest priority
for the next available FTE). The long-term strategic goal for RTD's workforce is to
gradually approach a situation with approximately 5 principal investigators in each
branch, with subsequent increased technical support. Given the demographics of
the division, this is expected to take about 3-5 years to accomplish as retirements
and departures accumulate, and this challenge is likely to be higher than we
imagined if the management of FTE ceiling is not maintained on the division level,
as it was for much of the late 1990s through this past year. Importantly, we feel our
core competencies are now adequate to address our mission and do not anticipate
recruiting additional principal investigators unless the FTE ceiling were to
significantly increase, or that we experience an unanticipated loss of one of the
current key investigators.
Increasing Efforts in Gamete Toxicology: The effort in gamete cell biology
has been fortified in several ways. In addition to building the division's emerging
genomics effort with the GEEEB (see the section below under Gamete Toxicology),
we have also arranged to have Dr. Tammy Stoker, who received her PhD under Dr.
Ralph Cooper in the EB, to have post-doctoral training in the laboratory of Dr. Sally
Darney, Branch Chief for GEEEB. In aggregate, we believe we have provided
additional mass to the gamete program as suggested by the peer review.
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Broadening Scientific Circles: In terms of strengthening scientific
interactions, several of our themes (notably the Chlorotriazines under Ralph
Cooper, Mechanisms of Abnormal Reproductive Development under Earl Gray,
Critical Windows under Sally Darney, Signal Transduction under Barbara Abbott,
and Reproductive Effects of DBPs under Gary Klinefelter) have developed wide
research networks, and the researchers are sought out for collaborations with a
number of academic, industrial and governmental laboratories. At the same time,
NHEERL has been moving towards a model of more multi-disciplinary research
efforts among the intramural investigators, and as a result, stronger efforts are now
apparent in the areas of EDCs, Disinfectant Byproducts, and Human Health Risk
Assessment, where research implementation teams are now guiding the
development of programs to address needs identified in those research strategies.
Another example of increased intra-Laboratory interactions is our involvement in
the NHEERL Synergy Program. This scientist-lead structure provides travel funds
to bring together geographically dispersed members of NHEERL to formulate
research on common issues. RTD scientists have played a lead role in one of the
main Synergy projects, that of interspecies extrapolation of EDC-related effects.
This group meets annually, alternating between the Duluth Division and RTD, and
a number of productive collaborations have resulted from the effort.
Interactions and communications have also been addressed within the
Agency, as RTD recently hosted an "Emerging Issues" workshop at which we
invited our clients with the Program Offices to come to the laboratory and engage
in discussions on future research directions that RTD should consider assisting
them in their duties. A number of action items arose from that highly successful
forum. About four years ago we also instituted a monthly divisional newsletter, the
ReproTimes, that has since been emulated by many other NHEERL divisions. This
provides a forum for other divisions and outside organizations to keep up to date
on our activities. Finally, we continue to have monthly journal clubs within each
Branch, and a monthly program of internal Work-in-Progress seminars and invited
outside seminar speakers. One element we have instituted recently is a round table
discussion between our trainees and the external speakers that has proven very
beneficial to the trainees in understanding options and considerations for future
employment.
Breadth of the Research Program: The final general issue concerned the
potential to be stretched too thin. While we do have broad demands to address
within the GPRA Goals, we have moved to reduce efforts in Air Toxics and CCLs,
and to build up efforts addressing EDCs under Goal 8.3 and harmonization and
susceptibility issues under Goal 8.2. A strong research program in Goal 8 will
provide generic benefits to the more media-oriented programs in terms of novel
approaches to risk assessment, while at the same time the problems tend to be at
a level that required greater commitment of time and resources - hence the need
to bolster the effort. We have also built up resources used to study the effects of
DBPs on developmental and reproductive processes under Goal 2, as this is the
large area of uncertainty relative to public health consequences. At the same time,
we have eliminated the possibility of investigators submitting and having funded
research efforts outside the overarching theme areas (they are still allowed to
33
image:
spend up to 20% effort on research of their choosing), and we have worked to
reduce the number of supported themes from nine down to seven. Collectively
these steps have helped provide a stronger focus on fewer research issues.
Specific Areas:
Dysmorphology: As a follow-up recommendation to evaluate directions for
the BBDR (Biologically Based Dose-Response Modeling) effort with 5-FU, we held
a mini-workshop (see Lau et al, 1999) to assess the project from a scientific and
programmatic direction. From that discussion, the program began work on the
broader context of nucleotide pool alterations and involvement of folates in
embryonic toxicity and conducted confirmatory experiments with tomudex. A
subsequent mini-conference on the role of folates on embryonic health held in 1999
provided further impetus for the program to move in the direction of defining the
extent to which folate levels can modulate the response to a number of
environmental stressors, and that program is now developing additional research
concepts as it migrates towards an effort targeted on defining susceptible
populations relative to folate status. Another new area of research involves
determining the long-term consequence of altered fetal growth on the postnatal
organism, partially as experimental facet to the "Barker Hypothesis" on human
growth, and partly because altered fetal growth is the most common finding in
standard developmental toxicity assays. A final segment of the program is now
studying the manifestation and mode of action of a class of chemicals called
fluorinated alkyl sulfonates, which have recently been shown to be ubiquitous in the
environment, and which induce a high level of postnatal death at relatively low
concentrations levels following prenatal exposure. All three of these components
represent new directions for the group, and employ more mechanistically-oriented
approaches than the more classical descriptive activities of the past. To make
these shifts, we have eliminated efforts in subtractive hybridization, and the
molecular basis of altered rib formation, but we still feel that this latter area is an
important data gap that impacts the Agency's ability to conduct informed risk
assessments. While we no longer have a methanol research program, it is
interesting to note that in early October, the Center for Evaluation of Risk to Human
Reproduction of the NIEHS will be conducting an Expert Panel evaluation on the
developmental and reproductive risks of methanol, and the research conducted by
RTD in the 1990s will play a prominent role in the deliberations.
Gamete Toxicology. The Review Panel was generally complementary of this
research area, but cautioned about "overselling" the work on SP22 as a sperm
biomarker In order to establish its validity, we have assigned more resources to
clarify its molecular biology and function. We are pleased to report that our efforts
to understand the utility of SP22 continue to show progress. Indeed we received
a US patent on the application early this year. Details of the progress are available
in the thematic write-up for Reproductive Effects of DBP where this work is housed.
We have also continued to pursue the role of protective mechanisms on gamete
development, with focus on anti-oxidants and stress proteins. The addition of
expertise in toxicogenomics to this effort has opened up a number of new research
34
image:
areas detailed in the Critical Windows Theme. A workshop we organized in early
1998 on genomics helped raise the knowledge level of this emerging technology
in NHEERL and helped spawn the creation of the Triangle Array User's Group
hosted by CUT and the NHEERL Molecular Profiling Users Group hosted by RTD.
In addition, we are playing leadership roles both within the laboratory by our
involvement in the NHEERL Genomics Steering Committee, and within the Agency
by bringing together involved parties for discussion on the nature of the EPA
approach to genomics. We have also been able to add a MS level statistician to
the group who is developing her expertise in bioinformatics in order to help with the
volume of data that this area generates. The Critical Windows Theme contains
several elements that demonstrate the direction we see this technology taking us
in the future.
The work on sperm energetics has been winding down, and the molecular
expertise applied to the SP22 effort with considerable success. It is important to
note that gamete program has the strongest links in the division with studies in
human population, as evidenced by the recently completed air pollution studies in
the Czech Republic, and the soon to be started study of semen quality in men from
three areas of the US with differing profiles of drinking water contaminants.
Disinfectant Byproducts: A primary concern expressed by the panel was the
need to conduct more long-term, low-dose exposure of DBPs during critical
developmental periods. We have addressed this concern by developing two
cooperative agreements, one with Colorado State University to look at the rabbit a
better model of postnatal human reproductive development and the other with
Research Triangle Institute to conduct a multi-generation study using drinking water
as the route of exposure. Both studies were guided in selection of DBPs by the
work conducted in RTD that has identified the critical developmental periods and
brominated haloacetic acids as the more potent contaminants. We have also
expanded our efforts at using drinking water as the route of administration in
studies involving pubertal animals that have shown some novel findings. Details
are provided in the Reproductive Toxicity of DBPs Theme. Our interaction with
NTP on individual DBPs has been completed and we are currently negotiating the
conduct of a mixture study involving the haloacetic acids. Finally, as noted under
Gamete Toxicology, we are addressing the human relevance of our findings of the
sensitivity of the male to brominated haloacids by developing a cooperative
agreement with the University of North Carolina to study semen quality in men from
three areas with three different DBP characteristics.
Endocrine Disruptors I and //: The Review Panel was generally very positive
regarding these efforts, and the efforts on anti-androgens, chlorotriazines and
endocrine screening batteries remain strong points in the division's research
portfolio both in terms of publications and impact. The chlorotriazine work recently
played an instrumental role in the Agency's decision to use a non-threshold model
for cancer risk assessment, and to begin to pay attention to developmental
exposures as a new critical period based on the identified mode of action in the
CNS. The interpretation was endorsed by the SAB, an external advisory board to
the Pesticide Program. In part, this success was made possible by the
35
image:
encouragement of the Review Panel to take on a more molecular and biochemical
approach to the problem, although understanding of the neurophysiological
consequences became paramount in developing the Agency's risk assessment
document on atrazine. Over the past two years we have been providing increasing
technical assistance to efforts to implement the EDSTAC recommendations, and an
award recently recognized the group for "Outstanding Technical Assistance to a
Program Office" by the Office of Research and Development. Details of these
efforts are provided in the Mechanisms of Abnormal Reproductive Development
theme.
Consistent with the recommendations of the review, we have continued to
reduce our efforts in characterizing the health effects of dioxin, although some of
this work continues in the Signal Transduction theme. We have largely moved out
of the work on the development of the pituitary-hypothalamic-adrenal axis and
related studies on PCBs in favor of the PFOS work being conducted in the
Susceptible Population Theme. Similarly, the control of ovulation work is now
focused on the potential effects of disinfectant by products in the WaterRepro
theme.
SUMMARY: RTD has continued to rely on the recommendations of the Peer Panel
in formulating its research program within the context of the needs of the Agency.
In addition, we have through time moved our research programs into closer
alignment with programmatic needs, and have phased out several areas that
although productive and of high quality, were not addressing the highest priority
areas. Our process for assigning pre- and post-doctoral trainee positions has
helped increase the mass of effort being applied to important questions, and we
have worked to have fewer work units addressing more focused problems. Several
of our recently completed studies in developmental and reproductive toxicants are
now used by outside parties as textbook examples of how research in these fields
should evolve. Our scientists play lead roles both in the Agency and in the
scientific community, as evidenced by the number of programs organized and
invitations extended. Our publication list demonstrates a broader range of journals
being used than before, and a wider array of collaborators, although much of the
fruits of these efforts have yet to appear in publications. Whiie we recognize there
remains room for improvement, we believe we are a stronger and more prominent
research unit than we were at the time of the last review; we also feel that we are
making significant progress in addressing the problems in developmental and
reproductive toxicology presented to us by the Agency.
36
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BIOGRAPHICAL SKETCH
°rovide the following information for the key personnel in the order listed on Form Page 2
NAME
POSITION TITLE
Barbara D. Abbott
Research Biologist
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training )
INSTITUTION AND LOCATION
DEGREE
(it applicable)
YEAR(s)
FIELD OF STUDY
NC State University, Raleigh, NC
Northeastern Illinois University, Chicago, IL
NC State University, Raleigh, NC
NC State University, Raleigh, NC
LRDT, NIEHS, Research Triangle Park, NC
NTP, NIEHS, Research Triangle Park, NC
M S
M S
Ph.D.
1962-1964
1977-1979
1980-1982
1982-1985
1985-1987
1987-1990
Preveterinary Medicine
Biology and Chemistry
Toxicology
Toxicology
NRSA Postdoctoral Fellow
IRTA Postdoctoral Fellow
— ^>ivwuni ^wuiiiwi i, ii%< i, >ii wiiivsi iviv^j ivui ui o(iwu<f ui • i « iwi u, ioi loo, oj ivj
honors Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications dunng the past three years and to representative earlier publications pertinent to this application If the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant
application)
Professional Experience:
1990 - Present: Research Biologist, Reproductive Toxicology Division, Health Effects Research Laboratory, US.
Environmental Protection Agency, Research Triangle Park, NC 27711
1979-1980 Research Assistant in Biochemistry, Northwestern University, Evanston, IL
1975-1978 Research Tech (Virology and Cell Culture, 1975-1976, Teratology, 1976-1978), G.D Searle Laboratories,
Skokie, IL
1963-1964. Undergraduate Research Program, NC State University, Raleigh, NC, in conjunction with NC Wildlife
& Conservation
rofessional Societies
Teratology Society
Society of Toxicology
N.C Branch of the Society of Toxicology
American Association for the Advancement of Science
Honors. Awards: (selected from last 5 years)
1999 Scientific and Technological Achievement Award (Level II) for four research publications addressing the
interspecies comparison of developmental toxicity for human and mouse embryonic tissue: correlation of tissue
dose and gene expression Awarded by US EPA.a
President NC Chapter Society of Toxicology, 2002-2003
Councilor, Reproductive and Developmental Toxicology Specialty Section, Society of Toxicology, 2002-2004
Professional Activities (selected from last 5 years)
Research Theme Leader for "Receptor-mediated Mechanisms of Developmental Toxicity"
Synergy Workgroup Committee, NHEERL, 1998-2001.
Human Health Risk Assessment Framework Colloquium II, Discussant, June 1998, Washington DC
Publications Committee, Teratology Society, 2000-2004
Risk Assessment Task Force, Society of Toxicology, 1999-2001
Education Committee, Teratology Society, 1998-2000
Invited Presentations (selected from last 5 years)
Symposium presentation "Angiogenesis gene downregulation in placentae of ARNT-deficient embryos", at the International
Federation of Teratology Societies Satelitte Symposium "Reproductive and Developmental Effects of Dioxms and
Endocrine Disrupters", July 12-17, 2000, in Hiroshima, Japan
"Abbott, Barbara D. "Responses of mouse and human palatal shelves to TCDD" Presented at Horizons in Developmental
and Molecular Toxicology Dioxinsand PCBs Assessing Health and Environmental Effects June 1-2, 2000, University
of Wisconsin-Madison, Madison, Wl
PHS 398 (Rev 4/98) (Form Page 6) Page FF
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image:
Teratology Society annual meeting, Continuing Education Course, invited Lecturer, "Vasculogenesis and Angiogenesis: A
critical gene pathway for embryonic development" Annual meeting of the Teratology Society,June, 2000
Invited speaker for Wiley-Liss Symposium, "Vasculogenesis and Angiogenesis Responses to teratogenic agents." anm
Teratology Society meeting, June, 2000.
"ARNT (0HLH-PAS) Gene Knockout Disrupts Placental Vasculogenesis", a presentation at the North Carolina Alliance for
Developmental Biology and Birth Defects Research, Inaugural meeting November 20,1998, N C Biotechnology Center,
Research Triangle Park, NC
Seminar presentation for the Department of Toxicology, North Carolina State University, Raleigh, NC. "Developmental
Toxicity of Dioxin", April 21, 1998
1997 Triangle Conference on Reproductive Biology, invited symposium on "Reproductive effects of AhR deficiency in
transgenic knockout mice", Jan 25,1997.
Publications: (selected from last 5 years)
Abbott, Barbara D. Laboratory Animal Models for the Study of Oral Clefts In. Cleft Lip and Palate. From Origin to
Treatment, Diego F Wyszynski (Editor), Oxford University Press, New York (2001)
Bryant, P LaMont, Judith E Schmid, Suzanne E. Fenton, Angela R Buckalew, and Barbara D. Abbott Teratogenicity of
2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) in mice lacking expression of EGF and/or TGF-a. Toxicol. Sciences 62:103-
114, 2001
Bryant, P LaMont, Lola M Reid, Judith E Schmid, Angela R Buckalew, and Barbara D. Abbott Effects of 2 3 7.8-
tetrachlorodibenzo-p-dioxin (TCDD) on embryonic mouse urinary tract epithelium in vitro Toxicology 162:23-34, 2001
Bryant, P LaMont, Lola M Reid, Edward S. Diala, Angela R Buckalew, and Barbara D. Abbott An in vitro model for murine
ureteric epithelial cells In Vitro Cellular & Developmental Biology", 37 (in press), 2001
Hurst, Christopher, Abbott, Barbara, and Linda Birnbaum 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) disrupts early
morphogenetic events that form the lower reproductive tract in female rat fetuses Toxicol Sci Submitted (2001)
Pitt, Jeff A., Feng, Li, Abbott, Barbara D., Schmid, Judy, Batt, Ronald E , Costich, Theodore G., and Diane P. Bofinger
(2001) Expression of AhR and ARNT mRNA in Cultured Human Endometrial Explants Exposed to TCDD. Toxicol
Sciences 62.289-298, 2001
Abbott, Barbara D and Angela R. Buckalew. Placental defects in ARNT-knockout conceptus correlate with localize
decreases in VEGF-R2, Ang-1, and Tie-2. Developmental Dynamics 219 526-538, 2000
Abbott, Barbara. Chapter 21. Palatal Dysmorphogenesis Palate Organ Culture In Methods in Molecular Biology,Vol. 136.
Developmental Biology Protocols, Volume II, eds R S TuanandC W Lo, Humana Press Inc., Totowa, NJ, pp195-201,
2000
Held, G.A and Abbott, Barbara-Chapter 22: Palatal Dysmorphogenesis Quantitative RT-PCR In Methods in Molecular
Biology,Vol 136 Developmental Biology Protocols, Volume II, eds R S TuanandC W Lo, Humana Press Inc.,
Totowa, NJ, pp203-217, 2000
Ghosh, Basavdutta, Wood, Carmen R , Held, Gary A , Abbott, Barbara D. and Chnstopher Lau Glucocorticoid receptor
regulation in the rat embryo A potential site for developmental toxicity7 Toxicol Appl Pharmacol 164:221-229,2000.
Pitt, Jeff A., Buckalew, Angela R., House, Denis E., and Barbara D. Abbott ACTH and Corticosterone Secretion by
Perifused Pituitary and Adrenal Glands From Rodents Exposed to TCDD Toxicology 151 25-35, 2000
Parks, Louise G , Joe S Ostby, Christy R. Lambright, Barbara D. Abbott, Gary Klinefelter, Norman J Barlow and L Earl
Gray, Jr The Plasticizer Diethylhexyl Phthalate induces malformations by decreasing fetal testosterone synthesis during
sexual differentiation in the male rat Toxicol. Sciences 58 339-349, 2000
Abbott,B. D., G A. Held, C R Wood, A.R. Buckalew, J G Brown, and J. Schmid AhR, ARNT, and CYP1A1 mRNA
quantitation in cultured human embryonic palates exposed to TCDD and comparison with mouse palate in vivo and in
culture Toxicological Sciences, 47 62-75,1999
Abbott.B. D., J Schmid, JG Brown, C R Wood, R D White, A R. Buckalew and GA Held RT-PCR quantification of
AHR, ARNT, GR, and CYP1A1 mRNA in craniofacial tissues of embryonic mice exposed to 2,3,7,8-tetrachlorodibenzo-p-
dioxin and hydrocortisone. Toxicological Sciences, 47 76-85, 1999
Peters, Jeffrey M , MichaeIG Narotsky, GuillermoElizondo, Pedro M Fernandez-Salguero, FrankJ Gonzales, and Barbara
D. Abbott Amelioration of TCDD-induced teratogenesis in aryl hydrocarbon receptor (AhR)-null mice Toxicology
Sciences, 47:86-92,1999
Abbott.B. D., A.R Buckalew, J J Diliberto, C. R. Wood, G Held, J Pitt, and J Schmid. Adverse reproductive outcomes
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FF
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in the transgenic Ah receptor-deficient mouse. Toxicol Appl Pharmacol 155 62-70,1999
Abbott, B.D. and Birnbaum, L S. Dioxins and teratogenesis. In. Molecular Biology of the Toxic Response, A. Puga and K.
Wallace eds. Taylor and Francis, Washington, DC Chapter 25, Dioxin and Teratogenesis, pp439-447, August, 1998.
Abbott, B.D., M R Probst, G H Perdew, and A.R Buckalew. AH receptor, ARNT, glucocorticoid receptor, EGF receptor,
EGF, TGFa, TGF01, TGF02, and TGFP3 expression in human embryonic palate and effects of 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD) Teratology 58.30-43,1998
Hurst, Christopher H , Barbara Abbott, Michael DeVito, and Linda S Birnbaum 2,3,7,8-tetrachlorodibenzo-p-dioxin in
pregnant Long Evans rats Disposition to maternal and embryo/fetal tissues Toxicological Sciences, 45 129-136,1998.
Kozak, Katherine R , Barbara Abbott and Oliver Hankinson ARNT deficient mice and placental differentiation. Dev Biol.
191:297-305, 1997.
Bryant, P.L, G C. Clark, M R Probst, and B. D. Abbott- Effects of TCDD on Ah receptor, ARNT, EGF, and TGFa
expression in embryonic mouse urinary tract Teratology, 55 326-337,1997
Abbott, B.D. Developmental Toxicity of dioxin Searching for the cellular and molecular basis of morphological responses
In Handbook of Experimental Pharmacology Section III Pathogenesis and Mechanisms of Drug Toxicity in Development
(R Kavlock and G Daston, Eds ), pp 407-433 Springer-Verlag, NY, 1997
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FF
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BIOGRAPHICAL SKETCH
"Vovide the following information lor the key personnel in the order listed on Form Page 2
hotocopy this page or lollow this lormat (or each person
NAME
POSITION TITLE
James E. Andrews
Research Toxicologist
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training )
INSTITUTION AND LOCATION
DEGREE
(if applicable)
YEAR(s)
FIELD OF STUDY
UNC Chapel Hill, NC
NC State University, Raleigh, NC
NC State University, Raleigh, NC
BS.
M.S
Ph D
1974
1983
1986
Chemistry and Zoology
Toxicology
Toxicology
RESEARCH AND/OR PROFESSIONAL EXPERIENCE Concluding with present position, list, in chronological orde.r previous employment, expenence, and
honors Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications during the past three years and to representative earlier publications pertinent to this application If the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant
application)
Professional Experience:
1987-1988 Research Toxicologist, Cell Biology Branch, DCTD, HERL, USEPA, RTP, NC
1980-1986 Research Toxicologist, Toxicology Branch, ITD, HERL, USEPA, RTP, NC
1975-1977 Chemist, Toxic Effects Branch, Environmental Toxicology Division, HERL, USEPA, RTP, NC.
1973-1975 Chemist, Toxic Effects Branch, Pesticides and Toxic Substances Effects Laboratory, USEPA, RTP, NC.
1971-1979 Research Chemist, Toxic Effects Branch, Environmental Toxicology Division, PTSEL, USEPA, RTP, NC.
Professional Societies
N.C. Chapter Society of Toxicology
merican Chemical Society
Society of Toxicology
Teratology Society
Professional Activities (selected from last 5 years)
Editorial Board of Reproductive Toxicology
Ad hoc reviewer for the following journals: Teratology, Toxicology Letters, Reproductive Toxicology
Fundamental and Applied Toxicology, Food and Chemical Toxicology and Pharmacology and Toxicology
Advisor for Guest Worker, Dr T.S. Roy, All India University, New Delhi, India, March-May, 1997
Publications: (selected from last 5 years)
J.E. Andrews, H.P. Nichols, J.E Schmid , E.S Hunter III, and G.R Klinefelter, Developmental toxicity of mixtures: The
water disinfection by-products Dichloro-, Dibromo- and Bromochloro Acetic Acid in embryo culture.Teratology (submitted).
Huang, V.S G A Held, J.E. Andrews and J.M Rogers, UC Methanol Incorporation into DNA and Proteins of
Organogenesis Stage Mouse Embryos in Vitro. Reproductive Toxicology 15, 429-435, 2001.
Roy, T.S., J.E. Andrews, F.J Seidler and T.A. Slotkin, Nicotine evokes cell death in embryonic rat brain during
neurulation J Pharmacol Exp. Ther., 287, 1136-1144, 1999.
Roy, T.S , J.E. Andrews, F.J Seidler and T.A. Slotkin, Chlorpyrifos elicits mitotic abnormalities and apoptosis in
neuroepithelium of cultured rat ebryos. Teratology 58, 62-68, 1998
Andrews, J.E., M Ebron-McCoy, J.E. Schmid, and D. Svendsgaard, Effects of combinations of methanol and formic acid
on rat embryos in culture. Teratology 58, 54-61, 1998.
Andrews, J.E., M Ebron-McCoy, U. Bojic, H Nau and R.J. Kavlock, Stereoselective Dysmorphogenicity of the Valproic
Acid analogue 2-N-Propyl-4 -Pentynoic Acid (4-yn-VPA) Cross-species evaluation in whole embryo culture. Teratology,
55, 1-5, 1997.
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BIOGRAPHICAL SKETCH
°rovide the following information for the key personnel in the order listed on Form Page 2
hotocopy this page or follow this format for each person
NAME
POSITION TITLE
Neil Chernoff
Scientist
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training )
INSTITUTION AND LOCATION
DEGREE
(if applicable)
YEAR(s)
FIELD OF STUDY
Brooklyn College
University of Miami
BS
Ph D
1959-63
1964-69
Biology
Zoology
— ¦ — — -JJ ...... f www >> 11^1, III will WIIWIV^IWCU UIUC,I piQVIVUS CIM^IUyi IICI It, CA^OIICIIUC, CU IU
honors Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications dunng the past three years and to representative earlier publications pertinent to this application If the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant
application) a
Professional Experience:
1969-1971 National Institute of Environmental Health Sciences - Staff Fellow
1971 -1980 Environmental Protection Agency - Scientist
1980-1981 Environmental Protection Agency - Chief, Developmental Biology Branch
1981-1987 Environmental Protection Agency - Director, Developmental Biology Division
1987-Present Environmental Protection Agency - Scientist
Professional Societies
Teratology Society
Society of Developmental Biology
Professional Activities (selected from last 5 years)
North Carolina State University, 1990-Present Adjunct Professor, Department of Toxicology.
Editorial/Grant Review Duties.
Member, Editorial Board of Teratogenesis, Carcinogenesis and Mutagenesis, 1982-Present
Member, Editorial Board of Journal of Applied Toxicology, 1985-Present
Member,Editorial Board of Toxicology Methods. New Approaches in Toxicology and Safety Assessment, 1990-Present.
Member, Editorial Board of Archives of Environmental Contamination and Toxicology, 1992-Present
1995-Present - Member, International Scientific Committee of the Instituto de Ciencias Ambientales y Salud, Buenos
Aires, Argentina
1997 - Member, EPA Workshop on Occurrence of Deformities in Populations of Frogs Found in The United States and
Canada.
1998 - Member, Reproductive Toxicology Epidemiological Research Findings Committee at the National Institute of
Environmental Health Sciences, EMF
Epidemiology RAPID Science Review Symposium, San Antonio, TX
Invited Presentations (selected from last 5 years)
1995 - Health Effects Institute, Harvard, Course on Health Effects of EMF Radiation "Reproductive Effects of Microwaves
in Animals"
1998 - Midwest Teratology Association, "Determination of the significance of supernumerary ribs in risk assessment-
molecular and postnatal functional strategies"
Publications: (selected from last 5 years)
anch, S , Francis, B M., Rosen, M.B , Brownie, C.F , Held, G.A., and Chernoff, N Differentially expressed genes
associated with 5-aza-2'- deoxycytidine-mduced hindlimb defects in the Swiss-Webster mouse J Biochem. Molecular
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Toxicology 12.135-141, 1998
Branch, S , Hall, L., Blackshear, P., and Chernoff, N Infectious dermatitis in a ball python (Python regius) colony. J.
Zoo and Wildlife Medicine, 29 461-464, 1998.
Francis, B M., Metcalf, R.L., Lewis, P.A , and Chernoff, N. Maternal and developmental toxicity of halogenated 4'-
mtrodiphenyl ethers in mice. 59 69-81 .Teratology, 1998
Branch, S., Chernoff, N„ Brownie, C.F., and Francis, B M..5-AZA-2'-deoxycytidine -induced dysmorphogenesis in the
rat. Teratogenesis, Carcinogenesis, and Mutagenesis, 19 329-338, 1999.
Hunt, J.H , O'Donnell, S., and Chernoff, N. Observations on two neotropical swarm-founding wasps, Agelaia yepocapa
and A panamaensis (Hymenoptera Vespidae) Annals of Ecology and Population Biology, 2000 (in press).
Chernoff, N , Hunter, E S. Hall, L.L., Rosen, M B Brownie, C F , Malarkey, D , Marr, M , and Herkovits, J Lack of
teratogenicity of microcystin-LR in the mouse and toad J Appl Tox (Submitted, 2001)
Rosen, M and Chernoff, N. 5-aza-2'-deoxycytidine-induced cytotoxicity and long bone defects in the murine limb
Teratology, 2001 (submitted)
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BIOGRAPHICAL SKETCH
NAME
POSITION TITLE
^alph L. Cooper
Chief, Endocrinology Branch
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training)
INSTITUTION AND LOCATION
DEGREE
(if applicable)
YEAR(S)
FIELD OF STUDY
Monmouth College, W Long Branch, NJ,
Rutgers University, Newark, NJ, 1973
Duke University, Durham, NC, 1973-1975
B A.,
Ph D
NIH Postdoc
1963-1968
1968-1973
1973-1976
Psychology
Psychobiology
Neuroscience Program
RESEARCH AND/OR PROFESSIONAL EXPERIENCE
Professional Experience:
Chief, Endocrinology Branch, RTD, NHEERL, ORD EPA 1995 - Present
Chief, Endocrinology/Gerontology Section, RTD, NHEERL, 1984-1995
Assistant Medical Research Professor, Medical Psychology Dec 1977-Aug 1984
Duke University Medical Center, Durham, NC
Research Associate, Dept Psych , Duke University Jan 1976-Dec 1977
Professional Societies
Sigma Xi Society for Neuroscience, North Carolina Chapter Endocrine Society
Honors, Awards: (selected from last 5 years)
1998 Bronze Medal ORD/RAF Workgroup on Environmental Endocrine Disruption. An Effects Assessment and
Analysis Document
J99 Best Paper Published in Reproductive & Developmental Toxicology Speciality Section, Toxicological Sciences
2000 Recipient, Office of Pesticides Programs Health Efects Division Team Award for work related to the chlorotriazines
2001 Recipient, EDSP team award for Exceptional/Outstanding ORD Technical Assistance to the Reqions or Proqram
Offices.
2001 Best Paper Published in Reproductive & Developmental Toxicology Speciality Section, Toxicological Sciences
Professional Activities (selected from last 5 years)
SETAC-EUROPE OECD Expert Workshop on Endocrine Modulators and Wildlife Veldhoven Holland (1997).
Joint US/EU Endocrine Disruptor Research Expert Panel Meeting, Ispra, Italy, 2000.
Member and Chair, Special Emphasis Panel, Conference Grant Applications (R-13) National Institute of Environmental
Health Sciences (2000-2001).
Chair, NHEERL Endocrine Disruptor Research Implementation Committee (2000-present)
Member, ORD Air Toxics Research Strategy Committee, 2000-present)
US/Japan International Workshop on Endocrine Disrupting Chemicals and Their Toxicological Evaluation, Tsukuba, Japan
Feb/Mar 2001
Co-Chair, RTD/Program Office and Region Workshop on Emerging Issues in Reproductive Tox June 2001
Adjunct Assistant Professor, Department of Psychology, Duke University Medical Center, Durham, N C.
Adjunct Assistant Medical Research Professor, Division of Medical Psychology, Duke University Medical Center 1984-
present
Adjunct Professor, Department of Anatomy, Physiological Sciences and Radiology, North Carolina State University
liege of Veterinary Medicine 1995-present
[PHS 398 (Rev 4/98) (Form Page 6) Page _
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Invited Presentations (selected from last 5 years)
Atrazine Research Meeting, Hilton Head, S. C., April, (1997)
ILSI meeting on Evaluation and Interpretation of Reproductive Endpoints for Human Health Risk Assessment
Washington DC, November, (1997).
Alterations in the hormonal control of reproduction. Office of Prevention Pesticides and Toxic Substances, Crystal City,
Va, April, (1998)
Endocrine Disruptors- National Center for Environmental Research and Quality Assurance. Crystal City, June, 1998
Triazine Expert Panel Meeting, Panama City, Florida, May, (1999).
20,h annual meeting of the American College of Toxicology, McLean, Va , November, (1999)
Presentation to the FIFRA Scientific Advisory Panel on Hazard and Dose-Response Assessment and Characterization of
atrazine June (2000).
Conference on Gender Differences in Toxicology. Tuscon Arizona (2000)
Mammary Tumors and Altered Reproduction Univerity of Zurich, Zurich Switzerland. (2000)
US/Japan International Workshop on Endocrine Disrupting Chemicals and Their Toxicological Evaluation, Tsukuba, Japan
Feb/Mar 2001
Publications: (last 5 years, selected from a total of more than 110 papers and 2 books)
Cooper, R.L. and Kavlock, R J Endocrine disruptors and reproductive development: A weight-of-evidence
overview. Journal of Endocrinology 152.159-166,1997.
Cooper, R.L (1997) Neuroendocrine Control of Female Reproduction. In. K Boekelheide, R.E. Chapin, P.B.
Hoyer and C Harris, (eds ). Female Reproductive Toxicology, vol. 10 Comprehensive Toxicology, Elsevier Science,
New York, pp 273-281.
Goldman, J.M., Parrish, M.B., Cooper, R.L., McElroy, W.K. Blockade of ovulation in the rat by systemic and
ovarian intrabursal administration of the fungicide sodium dimethyldithiocarbamate. Reproductive Toxicology. 11:185-
190, 1997.
Gray LE, Kelce WR, Wiese T, Tyl R, Gaido K, Cook J, Klinefelter G, Desaulniers D, Wilson E, Zacharewski
Waller C, Foster P, Laskey J, Reel J, Giesy J, Laws S, McLachlan J, BreslinW, Cooper R, Di Giulio R, Johnson R, Purdy
R, Mihaich E, Safe S, Colborn T. Endocrine Screening Methods Workshop report detection of estrogenic and androgenic
hormonal and antihormonal activity for chemicals that act via receptor or steroidogenic enzyme mechanisms.
Reproductive Toxicology Sep-Oct;11 (5).719-50. 1997
Lamb, J C , Matthiessen, P., Balcomb, R , Bens, C.M., Cooper, R.L. Gorsuch, J W., Peden-Adams, M.MN and
Voit, E.O (1998) Hazard Identification/Epidemiology. In: R.J. Kendall, R.L. Dickerson, W.A Suk and J.P. Giesy (eds.).
Principles and Processes for Evaluating Endocrine Disruption in Wildlife SETAC Press. Pp 17-37
Goldman, J.M., Gray, L.E and Cooper, R.L. (1998). Reproductive and developmental toxicity of endocrine
disrupting chemicals: Steroid and non-steroid mechanisms using rodent models In: R.J. Kendall, R.L. Dickerson, W A
Suk and J.P. Giesy (eds.). Principles and Processes for Evaluating Endocrine Disruption in Wildlife. SETAC Press
pp. 311-333.
Crisp, T.M., Clegg, E.D., Cooper, R.L., Wood, W P., Anderson, D.G., Baetcke, K.P. Hoffmann, J L , Morrow, M.S.,
Rodier, D.J., Schaeffer, J.E., Touart, L.W., Zeeman, M.G., Patel, Y.M Environmental Endocrine Disruption- An Effects
Assessment and Analysis, Environmental Health Perspectives 106' 11-56,1998
Cooper, R.L.„ Goldman, J M. and Tyrey, L (1998). The hypothalamus and pituitary as targets for reproductive
toxicants. In ed. By K. Korach, Reproductive and Developmental Toxicology. Dekker, New York, pp 195-210.
Lindzey, J., Wetsel, W.C., Couse, J.F., Stoker, T.E., Cooper, R.L., and Korach, K.S Effects of castration and chronic
steroid treatments on hypothalamic GnRH content and pituitary gonadotropins in male wild-type and estrogen-a receptor
knockout mice. Endocrinology, 139:4092-4101, 1998
Cooper, R.L., Goldman, J.M., Stoker, T.E. Neuroendocrine and reproductive effects of contemporary-use
pesticides. Toxicology and Industrial Health 15:26-36 (1999).
Gray, L.E , Ostby, J., Cooper, R.L., and Kelce, W. R The estrogenic and anitandrogenic pesticide methoxychlor alters the
reproductive tract and behavior without affecting pituitary size or LH and prolactin secretion in male rats. Toxicolgy e
Industrial Health. 15- 37-47, 1999
Gray, L. E , Wolf, C., Lambright, C, Ferrell, J., Mann, P., Price, M , Cooper, R.L and Ostby, J Administration of
fr>HS 398 (Rev 4/98) (Form Page 6) Page |
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image:
potentially antiandrogenic pesticides (procymidon, linuron, iprodione, chlozolinate, pp"-DDE and ketoconazole) and toxic
substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation
produces diverse profiles of reproductive malformations in the male rat Toxicolgy and Industrial Health 15" 94-108,
1999.
Ben-Jonathan, N., Cooper, R.L., Foster, P , Hughes, C Hoyer, P.B., Klotz, D., Kohn , M ., Lamb, D And
Stancel, G M. (1999). An approach to the development of models to quantitatively assess the effects of exposure to
environmentally relevant levels of endocrine disruptors Environmental Health Perspectives. 107 605-611
Stoker, T E , Robinette, C L. and Cooper, R.L. (1999) Maternal exposure to atrazine during lactation suppresses
suckling-induced prolactin release and results in prostatitis in the adult offspring Toxicological Sciences 52(1) 68-79.
Shafer, T J , Ward, T.R , Meacham, C A , and Cooper, R.L., (1999) Effects of the chlorotriazine herbicide,
cyanazine, on GABAa receptors in cortical tissue from rat brain Toxicology 142(1) 57-68
Cooper, R.L. and Goldman, J M (1999). Vaginal Cytology. In G Daston, C Kimmel (Eds ), An Evaluation and
Interpretation of Reproductive Endpoints for Human Health Risk Assessment International Life Sciences Institute/Health
& Environmental Sciences Institute. Washington pp 42-56
Stoker, T E., Robinette, C.L, Britt, B H. and Cooper, R.L. (1999) Prepubertal exposure to compounds that
increase prolactin in the male rat Effects on adult prostate Biology of Reproduction 61(6)1636-1643
Stoker, T.E., Robinette, C.L and Cooper, R.L. (1999) Perinatal exposure to estrogenic compounds and the
subsequent effects on the prostate of the adult rat Evaluation of inflammation in the ventral and lateral lobes.
Reproductive Toxicology 13 463-472
Laws, S.L , Cary, S A., Ferrell, J M , Bodman, G.J , and Cooper, R.L. (2000) Estrogenic activity of octylphenol,
nonylphenol, bisphenol A and methoxychlor in rats Toxicological Sciences 54-154-167.
Cooper, R.L., Stoker, T E , Tyrey, L , Goldman, J. M and McElroy, W K (2000) Atrazine disrupts hypothalamic
control of pituitary-ovarian function Toxicological Sciences 53-297-307.
Goldman, J M., Laws, S.C , Balchak, S K , Cooper, R.L. and Kavlock, R J (2000) Endocrine disrupting
chemicals Prepubertal exposures and effects on sexual maturation and thryroid activity in the female rat A review of the
EDSTAC recommendations Critical Reviews in Toxicology. 30'135-196.
Stoker, T E., Parks, L.G , Gray, L.E., and Cooper, R.L., (2000) Effects of endocrine disrupting chemicals on
puberty in the male rat: A review of the EDSTAC recommendations Critical Reviews in Toxicology 30.197-252
Bocchinfuso, W. P., Lindzey, J.K., Curtis-Hewitt, S. Clark, J.A., Myers, P.H., Cooper, R. L. and Korach, K. S
'2000). Induction of Mammary Gland Development in Estrogen Receptor-a Knockout Mice. Endocrinology 141: 2982-
Das, P.C , McElroy, W K. and Cooper, R.L. (2000). "Differential Modulation of Catecholamines by Chlorotriazine
Herbicides in Pheochromocytoma (PC12) cells in vitrcf Toxicological Sciences. 56 324-331.
Cummings, A. M., Rhodes, B.E , and Cooper, R.L. (2000). Effect of atrazine on implantation and early pregnancy in four
strains of rats Toxicological Sciences 58*135-143
Laws, S.C., Ferrell, J.M., Stoker, T E., Schmid, J., and Cooper, R.L. (2000) The effect of atrazine on puberty in
female wistar rats an evaluation in the protocol for the assessment of pubuertal development and thyroid function
Toxicological Sciences
Stoker, T.E., Laws, S C , Guidici, D., and Cooper, R.L. (2000) The effects of atrazine on puberty and thryroid
function in the male wistar rat- An evaluation of a protocol for the assessment of pubertal development and thyroid
function Toxicological Sciences 58. 50-59
Narotsky M G., Best, DS , Guidici, D. L., and Cooper, R.L. (2001) Strain comparisons of atrazine-induced
pregnancy loss in the rat Reproductive Toxicology 15 61-69
Das, P.C., McElroy, W.K. and Cooper, R.L. (2001) Alteration of catecholamines in pheochromocytoma (PC12)
cells in vitro by the metabolites of chlorotriazine herbicide. Toxicological Sciences 59- 127-137.
Stoker, T E , Goldman, J M., and Cooper, R.L., (2001) Delayed Ovulation and pregancy outcome- Effect of
environmental Toxicants on the neuroendocrine control of the ovary. Environmental Toxicology and Pharmacology 9
117-129
Putz, O., Schwartz, C B., Kim, S., LaBlanc, G.A., Cooper, R. L. and Prins, G.S. (In press) Neonatal low-and high
dose exposure to estradiol benzoate in the male rat I. Effects on the prostate gland. Biology of Reproduction.
Putz, O., Schwartz, C.B., LaBlanc, G A , Cooper, R. L and Prins, G S. (In press) Neonatal low-and high-dose
exposure to estradiol benzoate in the male rat. II Effects on male puberty and the reproductive tract Biology of
Reproduction.
Cooper, R.L., and Kavlock, R.J. (In press) Endocrine Disrupting Chemicals (EDCs) Journal of Human and
Ecological Risk Assessment.
^HS 398 (Rev 4/98) (Form Page 6) Page
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BIOGRAPHICAL SKETCH
Provide the following information tor the key personnel in the order listed on Form Page 2
hotocopy this page or follow this format for each person
NAME
POSITION TITLE
Audrey Mane Cummings
Research Biologist
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training)
INSTITUTION AND LOCATION
DEGREE
(if applicable)
YEAR(s)
FIELD OF STUDY
Cornell University, Ithaca, NY
Univ of Kansas, Lawrence,
Texas Tech Univ Health Sciences Center, Lubbock,
TX
B A
Ph.D.
Postdoctoral
Fellowship
1974
1982
1982-1985
Biological Sciences
Reproductive
Endocrinology
Biochemistry
RESEARCH AND/OR PROFESSIONAL EXPERIENCE Concluding with present position, list, in chronological orde.r previous employment, expenence, and
honors Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications dunng the past three years and to representative earlier publications pertinent to this application If the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant
application)
Professional Experience:
1977-1979. Research Assistant., Dept of Biochemistry, Univ. of Kansas, Lawrence, KS.
1977-1982 Research Asstistant, Dept. of Physiology and Cell Biology, Univ. of Kansas, Lawrence, KS.
1985-presenf Research Biologist, Endocrinology Branch, Reproductive Toxicology Division, NHEERL, ORD,
U.S E.P.A.
Professional Societies
Society for the Study of Reproduction, 1981 - present.
North Carolina Society of Toxicology, 1993 - present.
Society of Toxicology, 1994 - present.
Endocrine Society, 1984 - 1994.
Honors, Awards: (selected from last 5 years)
Team Award, Office of Pollution Prevention, Health Effects Division, U.E.P.A., 2000.
Professional Activities (selected from last 5 years)
Adjunct Professor, North Carolina State University, Department of Toxicology. October, 1996 - present
Awards Committee, Reproductive Toxicology Division.
360 Degree Feedback Advisory Group, NHEERL representative.
Work Improvement Team, RTD.
NHEERL Customer Service Workgroup.
.ditonal Boards Biology of Reproduction and Reproductive Toxicology
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Ad hoc Reviewer, Toxicology, Toxicology and Applied Pharmacology, and Journal of Reproduction and
Fertility 1985 - present.
Invited Presentations (selected from last 5 years)
Publications: (selected from last 5 years)
CUMMINGS, A.M. Methoxychlor as a model for environmental estrogens. CRC Critical Reviews in
Toxicology 27(4) 367-379, 1997.
CUMMINGS, A.M Toxicology of Early Pregnancy, Implantation, and Uterine Function In:
Reproductive and Developmental Toxicology (K. Korach, ed.) Marcel Dekker, Inc., New York, 1998,
pp. 397-412.
JOHNSON, K., CUMMINGS, A., and BIRNBAUM, L Promotion of endometriosis by polychlonnated
dibenzo-p-dioxins, dibenzofurans, and biphenyls Environmental Health Perspectives 105 (7).750-755,
1997.
CUMMINGS, A.M., HEDGE, J L., and LASKEY, J Ketoconazole impairs early pregnancy and the
decidual cell response via alterations in ovarian function. Fundamental and Applied Toxicology 40.238-
246,1997
CUMMINGS, A.M. AND HEDGE, J.M Dibromoacetic acid does not adversely affect early pregnancy
in rats. Reproductive Toxicology 12(4).445-448, 1998
CUMMINGS, A.M., HEDGE, J.M., and BIRNBAUM, L. Effect of prenatal exposure to TCDD on the
promotion of endometnotic tissue growth by TCDD in adult female rats and mice Toxicological
Sciences 52.45-49, 1999.
CUMMINGS, A.M. and LAWS, S C. Assessment of Estrogenicity Using the Delayed Implanting Rat
Model and Examples Reproductive Toxicology 14:111-117, 2000.
CUMMINGS, A.M., RHODES, B E., and COOPER, R.L Effects of Atrazme on Implantation and Early
Pregnancy in Four Strains of Rats. Toxicological Sciences 58 (1): 135-143, 2000
BIRNBAUM, L S. and CUMMINGS, A.M. Dioxins and Endometriosis' A Plausible Hypothesis.
Environmental Health Perspectives In press, 2001.
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BIOGRAPHICAL SKETCH
Provide the following information lor the key personnel in the order listed on Form Page 2
'hotocopy this page or lollow this format for each person
NAME
POSITION TITLE
Sally Perreault Darney
Chief, Gamete & Early Embryo Biology Branch
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and tnclude postdoctoral training )
INSTITUTION AND LOCATION
DEGREE
(if applicable)
YEAR(s)
FIELD OF STUDY
Newton College, Newton, MA
Brown University, Providence, Rl
University of Hawaii, Honolulu, HI
Johns Hopkins, Baltimore, MD
B A
MAT
Ph D
Postdoctoral
1967
1968
1980
1981-1984
Biology
Biomedical Sciences
Reproductive Biology
Reproductive Biology
Professional Experience:
1968-1977 Assistant/Associate Professor, Biology Dept, Community College of R. I., Warwick, Rl.
1984-1985 Research Biologist, Reproductive Toxicology Branch, Developmental Biology Div., HERL/ORD/US EPA.
1986-Present Supervisory Research Biologist, since 1995 Chief Gamete & Early Embryo Biology Branch/RTD/NHEERL
/ORD/US EPA
1991-Present Adjunct Professor, Department of Animal Science, NCSU, Raleigh, NC
Professional Societies
Society for the Study of Reproduction (SSR) American Society of Andrology (ASA)
N C. Society of Toxicology N.C. Consortium of Reproductive Biology
'onors. Awards (selected from last 5 years)
Juckeye lecture, American Society of Andrology, 1998
US EPA, 1997 Scientific and Technological Achievement Awards, honorable mention
US EPA, Bronze Metal for Commendable Service developing the EPA Harmonized Reproductive and Developmental Test
Guidelines, 1999
Professional Activities (selected from last 5 years)
Editorial Boards Journal of Andrology, 2001- 2004; Molecular Reproduction and Development, 1996-present; Journal of
Toxicology and Environmental Health, Part B, Critical Reviews, 2001-present.
Society offices/committees: SSR. Strategic Planning Committee, 1997, Finance Committee, 1998-2001; Treasurer, 1997-
2000, Development Committee, 2001-. ASA Nominating Committee, 1997, Future Meetings Committee, 1999-present,
President-elect of Women in Andrology, 2001, 2003 Program Committee Chair.
Organizing Committees Hazardous Substances and Male Reproductive Health, An International Conferenace, N.Y. Academy
of Medicine, New York, N Y (May 13-15,1998), Conference on Gender Differences and Reproductive Biology and Toxicology,
Tuscon, AZ (November 10-12,2000); Second International Conference on Male Mediate Development Abnormalities, Montreal,
Canada (June 19-21, 2001)
EPA and other Federal Committees- National Occupational Research Agenda subcommittee on Fertility and Pregnancy
Abnormalities, 1998-present, US EPA Harmonized Developmental and Two-Generation Reproductive Toxicity Test Guidelines
Workgroup, 1992-99, US EPA, NHEERL Technical Qualifications Review Board (Promotions) 2000-present;
Invited Presentations (selected from last 5 years)
"CASA applications in Reproductive Toxicology," Campus Course on CASA, European Society of Human Reproduction and
Embryology," San Miniato, Italy, 1997
"Fertilization Ignition of the first cell cycle and target for toxicants," Keynote Talk at Endocrine-Reproductive Physiology Annual
Symposium, U Wisconsin, Madison, Wl, 1997.
iffects of Toxicant Exposure on Sperm and Oocyte Quality," Postgraduate Course, American Society for Reproductive
Medicine, Cincinnati, OH, 1997
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"Fertilization. Ignition of the first cell cycle," Buckeye State-of-the-Art lecture at Annual Meeting of the American Society of
Andrology, Los Angeles, CA, 1998.)
"Animal Models" , International Conference on Hazardous Substances and Male Reproductive Health, N V Acad' ">f
Sciences, New York, N.Y. 1998
"Biomarkers of developmental toxicity of paternal origin," Precongress Course in Andrology and Male Reproductive Toxicology,
14th Annual Meeting of the European Society of Human Reproduction and Embryology, Gothenborg, Sweden, 1998.
"Improved methods for assessing sperm function and interpreting the results," SOT Continuing Education course on Evaluation
of Male Reproductive Toxicity, New Orleans, 1999.
'The oocyte as a target for reproductive toxicants," 5,h Internatn. Symposium of Andrology in the Nineties, Capetown, SA, 1999
"On the lookout for drug and environmentally induced male infertility" 5th International Symposium of Andrology in the Nineties,
Capetown, SA (March 25, 1999).
"Endocrine disruptors and male infertility- Fact or fiction7" Department of Urology and Andrology, U Pretoria Medical School,
Pretoria, SA. 1999.
"On the lookout for drug and environmentally induced male infertility" American Society of Andrology Annual Meeting, Louisville,
KT, 1999.
"In vitro reproductive toxicology. Sperm-egg interactions and early post fertilization events " Vision 20/20 Workshop TestSmart
A Humane and Effective Approach to SIDS Data. Fairfax, VA , 1999
"Fertilization- Ignition of the first cell cycle and target for toxicants" Japanese Testis Workshop, Tokyo, JP, 1999
"Sperm studies at EPA." Mini-symposium, West Japan Group on Endocnne Dismptors Issues Osaka, JP, 1999
"Assessment of toxicant-induced DNA damage in human sperm" Molecular Aspects of Male Reproductive Toxicology,
International Symposium, Giessen, Germany, 1999.
"Human Semen Studies, 1992-99" Conference on Teplice Program, Prachatice, Czech Republic, 2000.
"Altered semen quality as a biomarker of adverse environmental effects on male reproductive health in humans Studies in the
Czech Republic." Population Council, Center for Biomedical Research, New York, NY, 2000.
"Gender Differences in the Response of Mature Gametes to Reproductive Toxicants," Gender Differences in Reproductive
Biology and Toxicology, Tuscon, AZ, 2000.
"Gender Differences in the Response of Maturing Gametes to Reproductive Toxicants, Keynote address at Quebec r
Meeting on Reproductive Biology, Quebec, Canada, 2001.
"Air Pollution Effects on Semen Quality," 2001 Health Effects Annual Conference Air Pollution and Populations at Risk,
Washington, DC, 2001
"Failed Fertilization versus Early Embryo Loss," Second International Conference on Male-Mediated Developmental Toxicity,
Montreal, Quebec, Canada, 2001.
"Integrating Semen Analysis and Tests of Sperm Nuclear Integrity," March of Dimes Symposium on Male Mediated
Developmental Toxicity, Annual Meeting of the Teratology Society, Montreal, Canada, 2001
Publications: (selected from last 5 years)
Slott, V.L., Jeffay, S C , Dyer, C.J., Barbee, R.R. and Perreault, S.D. (1997). Sperrr. motion predicts fertility in male hamsters
treated with alpha-chlorohydrin. J. Andrology, 18 708-716.
Zuelke, K.A., Jones, D.P. and Perreault, S.D. (1997). Glutathione oxidation is associated with altered microtubule function and
disrupted fertilization in mature hamster oocytes Biol. Reprod. 57,1413-1419.
Wyrobek, A J , Schrader, S.M., Perreault, S.D., Fenster, L., Huszar, G., Katz, D.F., Osorio, A.M , Sublet, V and Evenson, D
(1997) Assessment of reproductive disorders and birth defects in communities near hazardous chemical sites III.
Guidelines for field studies of male reproductive disorders Reprod Toxicol., 11.243-259.
Perreault, S.D. (1997) The mature spermatozoon as a target for reproductive toxicants In- I.G. Sipes, C A McQueen and A
J Gandolfi (eds.) Comprehensive Toxicology, Vol. 10 Elsevier Science LTD, Oxford, 165-179.
Perreault, S.D. and Goldman, J M. (1997). Ovulation, Oocyte Maturation and Oocyte Function In. I.G. Sipes, C.A. McQueen
and A J Gandolfi (eds.) Comprehensive Toxicology, Vol. 10. Elsevier Science LTD, Oxford, 305-316.
Rubes, J., Lowe, Xiu, Moore, D., Perreault, S.D., Slott, V , Evenson, D., Selevan, S. and Wyrobek, A J (1998) Smoking
cigarettes is associated with increased sperm disomy in teenage men Fert. Stent 70, 715-723
Kempinas, W.D , Suarez, J.D., Roberts, N.L., Strader, L.F., Ferrell, J.M., Goldman, J.M., Narotsky, M.G , Perreault, S.D.,
Evenson, D.P , Ricker, D.D and Klinefelter, G.R. (1998) Fertility of rat epididymal sperm after chemically and sur
induced sympathectomy. Biol. Reprod. 59-897-904.
Perreault, S.D. (1998) Gamete Toxicology. The Impact of New Technologies. In- K Korach, ed Reproductive and
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Developmental Toxicology, Marcel Dekker, Inc., New York, 635-654.
Dunson, D.B , Weinberg, C.R , Perreault, S.D. and Chapin, R.E (1999) Summarizing the motion of self-propelled cells-
Applications to sperm motility. Biometrics, 55 537-543
bobbins, W A , Rubes, J., Selevan, S G and Perreault, S.D. (1999) Air pollution and sperm aneuploidy in healthy young men
Environ. Epidemiol. Toxicol V125-131.
Perreault, S.D. and Kirby, J.D. (1999) Internal fertilization in birds and mammals In E Knobil and J D Neill, eds,
Encyclopedia of Reproduction, Vol 2 Academic Press, NY, pp 856-866
Royster, M.O, Lobdell, D. T., Mendola, P , Perreault, S.D., Selevan, S A, Rothman, S G , and Robbins, W A (2000)
Evaluation of a container for collection and shipment of semen with potential uses in population-based, clinical and
occupational settings J Androl, 21:478-484.
Perreault, S.D., Rubes, J., Robbins, W.A., Evenson, D.P and Selevan, S G (2000) Evaluation of aneuploidy and DNA damage
in human spermatozoa Applications in field studies Andrologia 32:247-254
Cancel, A , Lobdell, Mendola, P and Perreault, S.D. (2000) Objective evaluation of rat sperm hyperactivation using computer-
assisted sperm analysis (CASA). Hum Reprod 15 1322-1328
Selevan, S G., Borkovec, L , Slott, V.L , Zudova, Z , Rubes, J , Evenson, D P and Perreault, S.D. (2000) Semen quality and
reproductive health of young Czech men exposed to seasonal air pollution Environ Health Persp 108 887-894
Lemasters, G K , Perreault, S.D., Hales, B F , Hatch, M , Hirshfield, A.N., Hughes C L , Kimmel, G.L., Lamb, J.C , Pryor, J.L.,
Rubin, C. and Seed, J G (2000) Workshop to identify critical windows of exposure for children's health Reproductive
health of children and adolescents. Work group summary Environ Health Persp. 108 (Suppl 3).505-509
Moline, J , Golden, A , Bar-Chama, M., Smith, E., Rauch, M E , Chapin, R E , Perreault, S.D., Schrader, S , Suk, W.A , and
Landrigan, PJ (2000) Exposure to hazardous substances and male reproductive health A research framework. Environ
Health Persp 108 803-813.
Dunson, D B and Perreault, S.D (2001) Factor analytic models of clustered multivariate data with informative censoring.
Biometncs 57.302-308
Perreault, S.D. and Cancel, A (2001) Significance of incorporating measures of sperm production and function into rat
toxicology studies. Reproduction 121 207-216
'obbins, W A., Witt, K.L., Haseman, J.K , Dunson, D.B , Troiani, L., Cohen, M S , Hamilton, C.D., Perreault, S.D., Libbus, B.,
Beyler, S A., Rayburn, D.J , Tedder, S T , Shelby, M.D., and Bishop, J.B Antiretroviral therapy effects on genetic and
morphologic end points in lymphocytes and sperm of men with human immunodeficiency virus infection J Infectious Dis
184.127-135
Klinefelter, G.R., Welch, J. E., Perreault, S.D., Moore, H D , Zucker, R.M., Suarez, J.D , Roberts, N L., Bobseine, K, and Jeffay,
S (2001) Localization of the sperm protein SP22 and inhibition of fertility in vivo and in vitro J Androl., in press.
Ong, T D., Xun, L , Perreault, S.D. and Robbins, W A Aneuploidy and chromosome breakage in swim-up versus unprocessed
semen from 20 healthy men J Androl, under revision..
Clegg, E D , Perreault, S.D. and Klinefelter, G R (2001). Assessment of male reproductive toxicity. In: A W Hayes (ed.)
Principles and Methods of Toxicology, 4rd Edition. Taylor and Francis, Philadelphia, pp 1262-1300
Perreault, S.D. (2001) Smart use of computer-aided sperm analysis (CASA) to characterize sperm motion In B Robaire and
B Hinton (eds ) The Epididymis, Kluwer Academic/Plenum Publishers, New York, in press
Perreault, S.D., Selevan, S G., Zudova, D , Zudova, Z., Evenson, D P. and Rubes, J (2001). Male reproductive health studies
in the Teplice Program' Air pollution and semen quality in young Czech men In R Sram (ed.) Teplice Program• Impact
of Air Pollution on Human Health, Academia, Prague, Czech Republic, pp 145-156
Selevan, S G , Perreault, S.D. and Rubes, J (2001) Epidemiologic aspects of semen studies in Teplice and Prachatice, Czech
Republic In- R Sram (ed.) Teplice Program- Impact of Air Pollution on Human Health, Academia, Prague, Czech Republic,
pp 157-166.
Evenson, D P , Jost, L.K., Perreault, S.D., Selevan, S G and Rubes, J. (2001) Application of the sperm chromatin structure
assay to the Teplice Program semen studies a new method for evaluating sperm nuclear chromatin damage In: R. Sram
(ed.) Teplice Program. Impact of Air Pollution on Human Health, Academia, Prague, Czech Republic, pp 167-180.
Rubes, J , Vozdova, M , Selevan, S G , Robbins, W.A. and Perreault, S.D. (2001) Impact of air pollution on sperm aneuploidy.
In R Sram (ed ) Teplice Program¦ Impact of Air Pollution on Human Health, Academia, Prague, Czech Republic, pp. 181 -
191
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BIOGRAPHICAL SKETCH
'Ovide the following information for the key personnel in the order listed on Form Page 2
NAME
POSITION TITLE
Pankshit C Das
Research Associate
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include
postdoctoral training )
INSTITUTION AND LOCATION
DEGREE
Of
applicable)
YEAR(s)
FIELD OF STUDY
Surendranath College, Calcutta Univ , WB, India
B S. (Hons)
1972-1976
Human Physiology(Hons)
Physics & Chemistry
Calcutta University, WB, India
M Sc.
1976-1978
Human Physiology
(Endocrinology)
Calcutta University, WB, India
Ph.D
1991
Physioloqv
RESEARCH AND/OR PROFESSIONAL EXPERIENCE Concluding with present position, list, in chronological orde.r previous employment, experience, and
honors Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications dunng the past three years and to representative earlier publications pertinent to this application If the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant application)
Professional Experience:
1998-Present Research Associate, Curr. In Toxicol, UNC-CH, NC & Endocrinology Branch, RTD, NHEERL, U.S. EPA,
RTP, NC
1996-1998: Research Associate, Deportment of Medicine, Division of Hematology/Oncology, Duke University Medical
Center, Durham, NC
1992-1996: Visiting Associate, Laboratory of Molecular Carcinogenesis, NIEHS, NIH, RTP, NC
191-1992: Research Associate, Department of Oncology, Howard University Cancer Center, Washington, DC
J89-1990: Research Associate, Department of Pharmacology, Howard University College of Medicine, Washington,
DC
1988-1989 Senior Research Fellow, Department of Food Technology & Biochemical Engineering, Jadavpur University,
Calcutta, WB
1982-1987: Research Fellow, Department of Physiology, Calcutta University College of Science & Technology, Calcutta,
WB
Professional Societies
Member (1994-Present)
Associate Member (1992-Present)
Life Member (From 1983)
American Society for Cell Biology
American Association for Cancer Research
Physiological Society of India
Professional Activities (selected from last 5 years)
Co-Chairperson Session on Free Communication on Endocrinology and Reproduction 3rd. Congress of Federation of Indian
Physiological Societies, November 24-26, 2000, Calcutta.
Invited Presentations (selected from last 5 years)
Mode and Mechanism of action of chlorotriazineherbicide mediated disruption of reproduction. Third Congress of Federation
of Indian Physiological Societies, November 24, 2000, Calcutta
Publications: (selected from last 5 years)
js P C, McElroy W K, and Cooper R L: Differential modulation of catecholamines by chlorotriazine herbicides in
pheochromocytoma (PC12) cells in vitro. Toxicological Sciences 56, 324-331, 2000.
PHS 398 (Rev 4/98) (Form Page 6) Page FF
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Das P C, McElroy W K, and Cooper R L Alteration of catecholamines in pheochromocytoma (PC12) cells in vitro by the
metabolites of chlorotriazine herbicide Toxicol. Sci. (USA) 59,127-137, 2001.
PHS 398 (Rev 4/98) (Form Page G) Page pp
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BIOGRAPHICAL SKETCH
'ovtde the following information tor the key personnel in the order listed on Form Page 2
NAME
POSITION TITLE
David J. Dix
Research Biologist
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training )
INSTITUTION AND LOCATION
DEGREE
YEAR(s)
FIELD OF STUDY
(il applicable)
University of Illinois Chicago, Chicago, IL
B.S
1985
Biological Sciences
Rush University, Chicago, IL
Ph.D
1990
Physiology
RESEARCH AND/OR PROFESSIONAL EXPERIENCE Concluding with present position, list, in chronological orde.r previous employment, expenence, and
honors Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications during the past three years and to representative earlier publications pertinent to this application If the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant
application)
Professional Experience:
1995-Present
1998-present
1992-1995
1990-1992
989-1990
Research Biologist, Gamete and Early Embryo Branch, Reproductive Toxicology Division,
National Health & Environmental Effects Research Laboratory, USEPA, RTP, NC
Adjunct Professor, Department of Biology, North Carolina Central University, Durham, NC
Intramural Research Fellow, Laboratory of Reproductive and Developmental Toxicology,
National Institute.of Environmental Health Sciences, RTP, NC.
Research Associate, Department of Biochemistry, NC State University, Raleigh, NC
Visiting Research Specialist,Department of Physiology and Biophysics, University of Illinois
Chicago, IL
Professional Societies
American Society for Cell Biology (1990-1995)
Society for Developmental Biology (1995-1997)
Teratology Society (1996-1999)
North Carolina Society for Toxicology (1998-present)
Society for the Study of Reproduction (1999-present)
Society of Toxicology (2001-present)
Honors. Awards: (selected from last 5 years)
1997 U.S. EPA Scientific and Technological Achievement Award for "Meiotic Mechanisms Dependent on a Unique
Heat Shock Protein".
1997 Quality Step Increase awarded by U.S EPA for high quality performance.
1998 Career tenure from U.S EPA
1999 Special Accomplishment Recognition Award from U.S EPA for organizing and reporting NHEERL workshop
"Application of DNA Microarrays to Toxicology" and forming the EPA Microarray Consortium (EPAMAC)
2000 Special Accomplishment Recognition Award from U.S. EPA for organizing NHEERL Array User's Group and
facilitating NHEERL's foray into array applications.
2001 Special Accomplishment Recognition Award from U.S. EPA for developing hsp70-1/hsp70-3 gene knockout
mice
2001 Special Accomplishment Recognition Award from U.S. EPA for leadership in launching toxicogenomics effort in
RTD
Professional Activities (selected from last 5 years)
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1996-1999 RTD research theme leader Protective Mechanisms in Reproductive Toxicology
1998-present RTD representative on issues of Children's Health
1998-present RTD lead on application of gene array technologies in toxicology Organizer of NHEERL Workshc,
Application of Microarraysto Toxicology, Jan 7-8,1999, scientists from government, industry and academia presented array
data and discuss future applications.
1998-present Organizer, EPA Microarray Consortium (EPAMAC).agroupof extramural scientists and EPA staff developing
mouse, rat and human gene microarrays and applications
1999-present Organizer of NHEERL Array User's Group, now the NHEERL Molecular Profiling Group, encompassing both
genomics and proteomics.
1999-2000 Member of NHEERL Strategic Plan 2000 committee
1999-present RTD representative on NHEERL Emerging Science Committee
1999-2003 Co-investigator on cooperative research and development agreement (CRADA) with Clontech Inc to develop
gene microarrays for toxicology applications
2000-present RTD representative on NHEERL Capital Equipment Committee
2000-present RTD representative on NHEERL Human Health Risk Assessment Research Implementation Team.
2001-present RTD representative on NHEERL Molecular Profiling Program
Steering Committee, to direct coordinated NHEERL efforts in genomics and proteomics
Non-EPA Service
2000 Member of scientific committee, Critical Assessment of Techniques for Microarray Data Analysis, conference held at
Duke University, Durham, NC
2001 Reviewer, applications responding to National Institute of Environmental Health Science RFA for establishing research
centers to cooperate with the National Center for Toxicogeno
Invited Presentations (selected from last 5 years)
Teratology Society, West Palm Beach, 1997 "Expression and function of Hsp70s during preimplantation
embryogenesis"
Laboratory of Reproductive and Developmental Toxicology, Natl Inst of Environmental Health Sciences, RTP, NC,
1997 "G2/M arrest and apoptosis in spermatocytes lacking HSP70-2 "
Program in Toxicology, North Carolina State University, Raleigh, NC, 1998. "Expression and function of Hsp70s during
spermatogenesis and embryogenesis."
Faculty of Toxicology, Texas A&M University, College Station, TX, 1998 "Expression and function of Hsp70s during
spermatogenesis and embryogenesis"
Center for Molecular Medicine and Genetics, Wayne State University, Detroit, Ml, 2000 "Application of DNA microarrays
to reproductive toxicology and development of a testis chip."
Center for Ecogenetics and Environmental Health, University of Washington, Seattle, WA, 2000 "Application of DNA
microarrays to reproductive toxicology and development of a testis chip"
North Carolina Society for Toxicology, Chapel Hill, NC, 2000 "Functional Genomic Analysis of Male Reproductive
Toxicity"
Gender Differences in Reproductive Biology and Toxicology Symposium, University of Arizona, Tucson, AZ, 2000.
"Application of DNA microarrays to reproductive toxicology and the development of a Testis Array."
Frontiers in Reproduction Symposium 2001, Cambridge, MA "Expression Profiling to Identify Genes Critical for Male
Fertility"
International
EU Workshop on Reproductive Toxicology In Vitro Germ Cell Developmental Toxicology, Granada, Spain, 1997,
"Protective mechanisms in germ cells, stress proteins in gametogenesis "
Japan National Institute of Environmental Science/U.S. EPA International Workshop on Endocrine Disrupters, Tsukuba,
Japan, 2001 "Biomonitoring the toxicogenomic response to EDCs in humans, laboratory species, and wildlife."
Toxicogenomics International Forum 2001, Nat'l Inst. Health Sciences, Tokyo, Japan. "Gene Expression Profiling to
Identify Mechanisms of Male Reproductive Toxicity "
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Publications: (selected from last 5 years)
")ix, D.J.: Hsp70 expression and function during gametogenesis Cell Stress & Chaperones, in press, 1997
Jix, D.J. Stress proteins in reproductive toxicology. Environmental Health Perspectives, 105(4) 436-438, 1997
Dix, DJ, JW Allen, BW Collins, P Poorman-Allen, C Mori, EH Goulding, BD Strong, EM Eddy HSP70-2 is required for
desynapsis of synaptonemal complexes during meiotic prophase in juvenile and adult mouse spermatocytes, submitted,
1997.
Hunter, ES, DJ Dix Heat shock protein 70-1 and 70-3 are necessary and sufficient to prevent arsenite-induced neural
tube defects submitted, 1997
Mori, C, N Nakamura, DJ Dix, M Fujioka, S Nakagawa, K Shiota, EM Eddy Morphological analysis of germ cell
apoptosis during postnatal testis development in normal and Hsp70-2 knockout mice Developmental Dynamics, 208"
125-136, 1997.
Zhu, D, DJ Dix, EM Eddy. HSP70-2 is required for CDC2 kinase activity in meiosis I of mouse spermatocytes
Development, in press, 1997
Dix DJ, JB Garges, RL Hong Inhibition of hsp70-1 and hsp70-3 expression disrupts preimplantation embryogenesis and
heightens embryo sensitivity to arsenic Mol Reprod Devel 51, 373-380, 1998
Mori C, JW Allen, DJ Dix, N Nakamura, M Fujioka, K Toshimori, EM Eddy Completion of meiosis is not required for
acrosome formation in hsp70-2 null mice. Biol Reprod, 61, 813-822, 1999
Rockett, JC, DJ Dix Application of DNA arrays to toxicology Environmental Health Perspectives, 107, 681-685, 1999
Huszar G, K Stone, D Dix, L Vigue. The putative creatine kinase m-isoform in human sperm is identified as the 70 kDa heat
shock protein HSPA2 Biol Reprod 63, 925-932, 2000
Rockett, JC, DJ Dix, DNA arrays, technology, options and toxicological applications Xenobiotica, 30, 155-177, 2000
Barnes J A, D J Dix, BW Collins, JC Luft, J W Allen Expression of inducible Hsp70 enhances the proliferation of MCF-7 breast
cancer cells and protects against the cytotoxic effects of hyperthermia Cell Stress & Chaperones, in press, 2001
Hunter ES III, DJ Dix. Heat Shock Proteins Hsp70-1 and Hsp70-3 are necessary and sufficient to prevent arsenite-induced
dysmorphology in mouse embryos Mol Reprod Devel, 59, 285-293, 2001.
u J, C Corton, DJ Dix, Y Liu, MP Waalkes, CD Klaassen. Genetic background but not metallothionem phenotype dictates
sensitivity to cadmium-induced testicular injury in mice. Toxicology and Applied Pharmacology, in press
Luft JC, JB Garges, IJ Benjamin, DJ Dix Heat shock factor 1 mediates heat-induced G2/M cell cycle arrest and prevents
cell death Cell Stress & Chaperones, in press, 2001
Reid R, DJ Dix, D Miller, SA Krawetz Recovering microarray data for Pathways analysis using a multipoint alignment
strategy Biotechmques 30, 762-768, 2001
Rockett JC, JC Luft, JB Garges, SA Krawetz, MR Hughes, DJ Dix Development of a 950-gene DNA array for examining
gene expression patterns in mouse testis Genome Biol2(4):research0014.1-0014 9, 2001
Rockett JC, FL Mapp, JB Garges, JC Luft, C Mori, DJ Dix The effects of hyperthermia on spermatogenesis, apoptosis, gene
expression and fertility in adult male mice Biol Reprod 65, 229-239, 2001.
Ostermeier GC, DJ Dix, D Miller, P Khatri, SA Krawetz What constitutes the normal fertile male. Submitted, 2001.
Luft JC, JC Rockett, JE Schmid, DB Tully, DJ Dix The reproductive effects and altered gene expression of juvenile and
adult male mice exposed to the water disinfectant byproduct bromochloroacetic acid Submitted, 2001.
Rockett JC, JB Garges, DJ Dix Long-term effects of juvenile testicular heat shock Submitted, 2001
Rockett JC, RJ Kavlock, C Lambright, L Parks, JE Schmid, V Wilson, DJ Dix Use of DNA arrays to monitor gene expression
in blood and uterus from Long-Evans rats exposed to 17-b-estradiol- a new approach to biomonitoring effects of endocrine
disrupting chemicals Submitted, 2001
Rockett JC, MS Ricci, P Patrizio, NB Hecht, JE Schmid, DJ Dix. Gene expression patterns associated with infertility in rodent
and human models Submitted, 2001
PHS 398 (Rev 4/98) (Form Page 6) Page
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BIOGRAPHICAL SKETCH
Yovide the following information for the key personnel in the order listed on Form Page 2
NAME
POSITION TITLE
Suzanne E. Fenton
Research Biologist
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training)
INSTITUTION AND LOCATION
DEGREE
(if applicable)
YEAR(s)
FIELD OF STUDY
University of Wisconsin, Madison, Wl
University of Wisconsin, Madison, Wl
University of Wisconsin, Madison, Wl
University of North Carolina, Chapel Hill, NC
BS
M.S
Ph D.
Post-doc
1984-88
1988-90
1990-93
1993-98
Dairy Science/Pre-Vet
Endocrinology/Repro Phys
Endocrinology/Repro Phys
Mammary gland
carcinogenesis
Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications dunng the past three years and to representative earlier publications pertinent to this application If the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant
application)
Professional Experience:
1992 Lecturer, University of Wisconsin-Madison (Reproduction in Farm Animals)
10/98-present Research Biologist, U.S EPA, Reproductive Toxicology Division, Endocrinology Branch
RTP, NC
Professional Societies
American Society for Cell Biology (1997)
American Association for Cancer Research (1997-8)
American Association for the Advancement of Science (AAAS, 1997-)
'C Society of Toxicology (1999-)
ociety of Toxicology, Full Member (2001-)
Women in Toxicology (2001-)
Honors. Awards: (selected from last 5 years)
1997 - 1998 National Cancer Institute NRSA grant recipient, University of North Carolina, Chapel Hill, NC
2000 - Present Office of Pesticides Programs Health Effects Division Team Award - Atrazine
Professional Activities (selected from last 5 years)
U.S EPA
1999-
1/00
2000-2002
2001-2003
1/99
8/99
Outside EPA
1999-
2001-
9/98
1999
1999
1/99
99
o/99
Member, Molecular Profiling Group, NHEERL (previously NHEERL Array User's Group)
CRADA #0176-99, Project Manager Development of Toxicology/Stress DNA Microarrays
Chairperson, Reproductive Toxicology Division Seminar Series
Member, RTD Performance and Recognition Committee
"Application of DNA Microarrays to Toxicology" Workshop, U S. EPA-sponsored
Synergy Workshop, U.S EPA Endocrine Disruptors and Dioxin workgroups
Member, Triangle Array User's Group
Member, Study Assembly of the Longitudinal Cohort Study of Environmental Effects on Child
Health and Development
"Cell Signaling Processes Underlying Toxicology Responses" Workshop, CUT, participant
Triangle Conference on Reproductive Biology, participant
North Carolina Society of Toxicology,member and participant
"Growth Factors, Cell Signaling and Cell Death" Conference, NIEHS, participant
Real-Time Quantitative PCR User's Group, NC Biotechnology Center, participant
GEMS Meeting "Regulatory Assessment of Toxicological Data", Marriott Hotel, RTP, participant
PHS 398 (Rev 4/98) (Form Page 6) Page FF
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8/99
"Biomarkers: Taking Stock" Workshop, The Friday Center, Chapel Hill, participant
9/01 Environmental Mutagen Society, "Breast Cancer & Environmental Mutagens", RTP, NC, participi.
Invited Presentations (selected from last 5 years)
1/99 Application of DNA Microarrays to Toxicology, NHEERL Workshop, U.S EPA. "Gene expression during breast
development", (oral presentation)
6/99 Mammary Gland Biology Gordon Conference, Henniker, NH. Poster presentation entitled, "Early Adverse Effects
of Prenatal Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in the Rat Mammary Gland".
6/99 Mammary Gland Biology Gordon Conference, Henniker, NH Poster presentation entitled, 'Targeted Inactivation
of the EGF and Amphiregulin Genes Reveals Distinct Roles for EGF Receptor Ligands in Mouse Mammary Gland
Development".
12/99 Triangle Endocrine Disruptors Group, U.S EPA. "Early adverse effects of prenatal exposure to
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the rat mammary gland", (oral presentation)
2/00 Prolactin Gordon Conference, Ventura, CA. Poster presentation entitled, "Prolactin-lnduced Tyrosine
Phosphorylation, Activation and Receptor Association of Focal Adhesion Kinase (FAK) in Mammary Epithelial Cells"
8/00 Dioxin 2000 Meeting, Monteray, CA "Adverse effects of TCDD on mammary gland development in Long Evans rats:
A two generational study" (oral presentation)
3/01 Society of Toxicology, San Francisco, CA "Environmental Toxicants and Disrupted Mammary Gland Development.
The window of susceptibility". In Breast Cancer Issues in Screening and Testing of Potential Human Carcinogens.
(oral presentation)
Publications: (selected from last 5 years)
Johnson, J.L., S.E. Fenton, and L.G Sheffield. 1996. Prolactin inhibits epidermal growth factor-induced Ras-MAPK
signaling
in mammary epithelial cells J. Biol. Chem. 271:21574-21578.
Fenton, S.E. and L.G. Sheffield. 1997 Prolactin inhibits EGF-induced DNA synthesis in mammary epithelium via early
signaling mechanisms Possible involvement of protein kinase C. Exp Cell. Res. 236 285-293.
Leutteke, N.C., T.H. Qiu, S.E. Fenton, K. L. Troyer, R. F Riedel, A Chang, and D.C. Lee 1999. Targeted inactivation of
the
EGF and amphiregulin genes reveals distinct roles for EGF receptor ligands in mouse mammary gland development.
Development 126:2739-2750
Bryant, P L , J.E. Schmid, S.E. Fenton, A.R. Buckalew, B.D. Abbott. 2001. Teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-
dioxin (TCDD) in mice lacking the expression of EGF and/or TGF Toxicol. Sci. 62103-114
Fenton, S.E. and L.G. Sheffield. 2001 Prolactin activation of JAK2 via a positive feedback loop involving c-src and SHP2
J Biol Chem (Accepted).
Fenton, S.E. and L G. Sheffield. Prolactin-induced tyrosine phosphorylation, activation and receptor association of focal
adhesion kinase (FAK) in mammary epithelial cells: Critical involvement of JAK2 and c-src activation. (Submitted to
Endocrinology in July 2001).
Fenton, S.E., J.T Hamm, L.S. Birnbaum, and G.L. Youngblood Persistent abnormalities in the rat mammary gland
following gestational and lactational exposure to 2,3,7,8 - tetrachlorodibenzo-p-dioxin (TCDD). (Submitted)
PHS 398 (Rev 4/98) (Form Page 6) Page
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BIOGRAPHICAL SKETCH
"•rovide the following information for ihe key personnel in the order listed on Form Page 2
notocopy this page or tollow this format for each person
NAME
POSITION TITLE
Jerome M. Goldman
Research Biologist
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training)
INSTITUTION AND LOCATION
DEGREE
(il applicable)
YEAR(s)
FIELD OF STUDY
University of Pittsburgh, Pittsburgh, PA
East Carolina University, Greenville, NC
York University, Toronto, Ontario, Canada
University of Waterloo, Waterloo, Ontario, Canada
Duke University, Durham, NC
U S EPA (National Research Council)
BS
M.A
M A
Ph.D
Post-doc
Post-doc
1967
1969
1974
1983
1983-1984
1984-1987
Psychology
Experimental Psychology
Psychobiology
Biopsychology
Neuroendocrinology
Reproductive Toxicology
— — — — ...... ^IWWV'M f/vwniwil! <11 WIIIWMWIW^IWUI w I UU ,1 ^lOVIVUd Ol I I IOI ll, U I 1I I w i Ul IU
honors Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications during the past three years and to representative earlier publications pertinent to this application If the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant
application)
Professional Experience:
1991 - present Research Biologist, Reproductive Toxicology Division, NHEERL, USEPA, RTP, NC
1987 - 1991 Research Scientist / Project Supervisor, ManTech Environmental Technology, RTP, NC
1970 - 1972 Instructor in Clinical Chemistry, U S. Army Medical Field Service School, Fort Sam Houston, San
Antonio, TX
Honors. Awards: (selected from last 5 years)
000 Shared ORD Award for Outstanding Technical Assistance to the EPA Office of Prevention, Pesticides and Toxic
Substances Recognition for Involvement during the Standardization and Validation of the Tier 1 Screening Battery
of the Endocrine Disruptors Screening Program
2000 Recognition by NHEERL Senior Management for serving on the NHEERL Strategic Plan 2000- Goal writing team
(1999-2000).
2000 Selection of Toxicoloaical Sciences Paper of the Year for: Cooper RL, Stoker TE, Tyrey L, Goldman JM and
McElroy, WK Atrazme disrupts the hypothalamic control of pituitary-ovarian function. Toxicological Sciences 53:
297-307 (2000)
Professional Activities (selected from last 5 years)
2001 Member, NHEERL Quality Assurance Workgroup.
2000 - present Project Officer, EPA Cooperative Agreement with Research Triangle Institute (RTP, NC) to
investigate the reproductive effects of the drinking water disinfection by-product,
bromochloroacetic acid
1999 - 2000 NHEERL Strategic Plan 2000. Goal writing team
1991-1999 NHEERL Institutional Animal Care and Use Committee (Member, Also- Head, Facilities
Subcommittee)
Invited Presentations (selected from last 5 years)
1999 Invited Participation in American Water Works Association Research Foundation, Drinking Water Research
Needs Expert Workshop, Leesburg, VA.
Publications: (selected from last 5 years)
"oldman, J.M., Murr, A S. Alterations in ovarian follicular progesterone secretion by the drinking water disinfection by-
product dibromoacetic acid Examination of the potential site(s) of impact along the steroidogenic pathway (Submitted)
Stoker, T.E , Goldman, J. M, Cooper, R L Delayed ovulation and pregnancy outcome Effect of environmental
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toxicants
on the neuroendocrine control of the ovary Environmental Toxicology and Pharmacology 9 117-129 (2001)
Balchak, S.K., Hedge, J.M., Murr, A.S., Mole, M.L , Goldman, J.M. Influence of the drinking water disinfection by-
product
dibromoacetic acid on rat estrous cyclicity and ovarian follicular steroid release in vitro Reproductive Toxicology 14.
533-539 (2000).
Zucker, R M., Keshaviah, A P, Price, O.T., Goldman, J.M. Confocal laser scanning of rat follicle development Journal
of
Histochemistry and Cytochemistry 48 781-791 (2000)
Goldman, J.M., Laws, S.C , Balchak, S.K , Cooper, R L , Kavlock, R.J Endocrine disrupting chemicals. Prepubertal
exposures and effects on sexual maturation and thyroid activity in the female rat. A focus on the EDSTAC
recommendations. Critical Reviews in Toxicology 30 135-196(2000)
Cooper, R.L , Stoker, T.E , Tyrey, L , Goldman, J.M., McElroy, W.K Atrazine disrupts the hypothalamic control of
pituitary-ovarian function Toxicological Sciences 53. 297-307 (2000)
Cooper, R L , Goldman, J.M., Stoker, T E Neuroendocrine and reproductive effects of contemporary use pesticides
Toxicology and Industrial Health 15 26-36 (1999)
Cooper, R L., Goldman, J.M. (1999) Vaginal cytology In G Daston and C.Kimmel (Eds ). An Evaluation and
Interpretation of Reproductive Endpoints for Human Health Risk Assessment I LSI Press Washington (1999) pp 42-
56
Cooper, R L , Goldman, J.M., Tyrey, L The hypothalamus and pituitary as targets for reproductive toxicants. In- K.S
Korach (Ed.), Reproductive and Developmental Toxicoloov Marcel Dekker New York (1998). pp 195-210
Goldman, J.M., Gray, L.E., Cooper, R.L. Reproductive and developmental toxicity of endocrine disrupting chemicals-
Steroid and non-steroid mechanisms using rodent models In R.J Kendall, R.L Dickerson, J.P. Giesy, W P. Suk
(Eds ), Principles and Processes for Evaluating Endocrine Disruption in Wildlife SET AC Press: Pensacola, FL (1998).
pp 311-333.
Kempinas, W.G., Suarez, J.D., Roberts, N.L , Strader, L , Ferrell, J., Goldman, J.M., Klinefelter, G R Rat epididymal
sperm quantity, quality and transit time after guanethidine-induced sympathectomy Biology of Reproduction 59' 890-
896 (1998)
Kempinas, W.G , Suarez, J D.t Roberts, N L , Strader, LF„ Ferrell, J , Goldman, J.M., Narotsky, M G„ Perreault, S D ,
Evenson, D.P., Ricker, D.D., Klinefelter, G R Fertility of rat epididymal sperm after chemically and surgically induced
sympathectomy Biology of Reproduction 59 897-904 (1998)
Goldman, J.M., Parrish, M B , Cooper, R L., McElroy, W K Blockade of ovulation in the rat by systemic and ovarian
intrabursal administration of the fungicide sodium dimethyldithiocarbamate Reproductive Toxicology 11:185-190
(1997)
Laws, S C , Goldman, J.M., Cooper, R L Assessing the endocrine disrupting potential of environmental chemicals In:
TH Connor, C F Fox (Eds ). Biotechnology International. Vol 1 Universal Med. Press San Francisco (1997) pp
208-215
Perreault, S D , Goldman, J.M. Ovulation, oocyte maturation and oocyte function In: P Hoyer (Ed ), Reproductive and
Endocrine Toxicology. Vol 10, Section II, Female Reproductive Toxicoloov (Comprehensive Toxicology. I.G Sipes,
CA McQueen, A J Gandolfi, eds -in-chief) Pergamon Press Oxford (1997) pp. 305-316.
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BIOGRAPHICAL SKETCH
Vovide the following information tor the key personnel in the order listed on Form Page 2
notocopy this page or follow this format for each person
name Dr Leon Earl Gray Jr
position title Research Biologist GS 15
INSTITUTION AND LOCATION
DEGREE
YEAR(s)
FIELD OF STUDY
(if applicable)
Cornell University, Ithaca New York
BS
1967
Biology
North Carolina State University
Ph D
1976
Zoology
Professional Experience:
1995-Present Team Leader of "Cellular and molecular mechanisms of abnormal reproductive development produced by
Endocrine Disrupting Chemicals (EDCs) administered during Critical Developmental Periods", Endocrinology Branch,
Reproductive Toxicology Division, NHEERL, USEPA
1986 - 1995. Section Chief, Developmental and Reproductive Toxicology Section, Reproductive Toxicology Branch
1979 - 1986 Research Biologist. Reproductive Toxicology Branch
1976 - 1979 Research Associate- Faculty of Duke University on IPA to Experimental Biology Division, HERL, EPA
Professional Societies
AAAS Fellow-1999, Society for the Study of Reproduction, Society for the Study of Toxicology Society for Environmental
Toxicology and Chemistry, Audubon Society, National Wildlife Federation, World Wildlife Federation, NC Wildlife Federation
Honors. Awards: (selected from last 5 years)
1997 USEPA Gold Medal for Exceptional Service Reproductive Risk Assessment Guidelines Workgroup.
1999 Elected as a AAAS Fellow for research on endocrine disrutping chemicals
1999 USEPA Bronze Medal for activities on the Endocrine Disruptor Screening and Testing Advisory Committee
1999 USEPA Bronze Medal for developing and harmonizing guidelines for develpmental/reproductive toxicity testing.
°000 ORD Award for Exceptional/Outstanding Technical Assistnace to the Program Office
)00 Honorable mention, for four papers in Toxicol Ind Hlth, 1999
STAA Award. Level I. Scientific and Technological Achievement Award (awarded in 1997, 13 total over EPA career) for
Three manuscripts, including "Persistent DDT metabolite p,p' DDE is a potent androgen receptor antagonist" in
Nature.Numerous cash awards
Professional Activities (selected from last 5 years)
ORD Endocrine Disrupters Research Strategy Committee, 1996-present
Chair EPA/WWF/CMA "Duke" Workshop on Endocrine Screening Methods Publixhed 1997
NCTR Site Visit Review Team/Consultant on "Estrogen Knowledge Base Project" '97
Consultant to the US Department of Justice on EDC issues. '97-present
EDSTAC-Screening and Testing Workgroup Member 4/97 to 8/98
EPA-Endocrine Disruptor Screening Program Workgroup member 2/99
Research Managment Group (RMG) Committee Member 1999
NHEERL EDC Research Planning Committee 2000 to present
Former Editorial Board of Journal of Toxicology and Environmental Health
Former Editorial Board of Biology of Reproduction
Ad Hoc Journal Reviews for Science, Neurotoxicology, Toxicology, Toxicology and Applied Pharmacology, Toxicological
Sciences, Fundamental and Applied Toxicology, Teratogenesis, Carcinogenesis and Mutagenesis, Biology of Reproduction,
Reproduction and Fertility, ReproductiveToxicology, Neurobehavioral Toxicology and Teratology, Environmental Toxicology
and Chemistry, etc
Advisor for an NHEERL Postdoctoral Trainee
Graduate and undergraduate research advisor for University of Florida, North Carolina State University and UNC students
under the USEPA/NCSU and UNC cooperative research grants for undergraduate, predoctoral and postdoctoral students
nvited Presentations (selected from last 2-3 years)
1 1999 Jan. Keystone Molecular Biology Symposia Cochair and Speaker
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2.
1999
Feb North Carolina State University Zoology Department Seminar
3.
1999
March SOT Continuing Education Course Organizer and Speaker
4
1999
April
Toxicology Forum Panel Member and speaker
5.
1999
Apri.
Teratogen Update Course Speaker at Mass General Hospital
6
1999
May
Speaker at CUT Open House
7
1999
Aug
Co-organizer and Speaker at USEPA, NHEERL Species Extrapolation Workshop
8
1999
Nov
Seminar for the School of Environmental Health, CSU
9
1999
Nov
Seminar for the School of Vet Med, Dept Physiolgy, CSU
10
1999
Nov
Invited SETAC EDC Symposia speaker
11
2000
Jan
OECD presentation of Hershberger Assay Proposal at VMG meeting, Paris
12.
2000
Feb
NYS Wadsworth Laboratory Seminar (invited)
13
2000
May
Denmark EDC Meeting Cochair and two presentations (invited)
14
2000
Spring
Duke University Seminar in Course on EDCs
15
2000
Summer
Society for Toxicologic Pathologists Continuing Ed Course on EDCs
16
2000
Summer
SETAC Pellston Workshop on Interconnections of Ecosystem and Human Health, Snow I
I Itah
17.
2000
Aug
LHaf l
Presentation of Hershberger Assay at SSR Annual meeting, Madison Wl
18.
2000
Sept
NCSU Toxicology Course Lecture
19
2000
Oct
SOCIETY FOR RISK ANAYLSIS MEETING, RAL, NC
20
2000
Cct
NIEHS Low-Dose Workshop, Data Discussant for Androgen Panel
21
2000
Oct
FALL FSTRAC MEETING, RTP NC
22
2001
Jan
Univeristy of Rochester Environmental Sciences Seminar
23
2001
Mar
SOT Meeting, Cochair and speaker Symposia on EDCs
24
2001
April
NIES/EPA Meeting in Japan on EDCs Speaker
25.
2001
May
EPA/ORD/Regional meeting on EDC Issues Atlanta Speaker
26
2001
July
ICT IX Symposia on EDCs, Brisbane AUS, Speaker
27
2001
July
SSR Annual Meeting Ottawa, Cochair Symposia on Effects in Males
Publications; (selected from last 5 years: Career total of over 100)
1. Gray LE Jr, Wolf C, Mann P and Ostby JS In utero Exposure to Low Doses of 2,3,7,8 Tetrachlorodibenzo-p-dioxin
(TCDD) Alters Reproductive Development of Female Long Evans Hooded Rat Offspring. Toxicol Appl Pharm 146.237-244,
1997
2 Gray LE Jr, Ostby JS, Kelce WR. A dose-response analysis of the reproductive effects of a single gestational dose of
2,3,7,8 tetrachloro-p- dioxin (TCDD) in Male Long Evans Hooded rat offspring Toxicol Appl Pharm 146:11-20, 1997
3 Gray LE Jr, Kelce WR, Wiese T, Tyl R, Gaido K, Cook J, Klinefelter G, Desaulniers D, Wilson E, Zacharewski T, et al.
(33 authors) Endocrine Screening Methods Workshop Report Detection of Estrogenic and Androgenic Hormonal and
Antihormonal Activity for Chemicals that act via Receptor or Steroidogenic Enzyme Mechanisms. Reproductive Toxicology
11(4) 719-750, 1997
4 Kelce WR, Lambright CR, Gray LE Jr and Roberts KP Vinclozolin and p,p' DDE alter androgen-dependent gene
expression In vivo confirmation of an androgen-receptor-mediated mechanism. Toxicol Appl Pharmacol 142, 192-200,
1997.
5. Ankley G, Mahiach E, Stahl R, Tillitt D, Colborn T, McMaster S, Miller R, Bantle J, Campell P, Dickerson R, Fry M, Giesy
J, LE Gray Jr, et al. Overview of a workshop on screening methods for detecting potential (antiestrogenic/androgenic
chemicals in wildlife. Environ Toxicol and Chemistry 17.68-87,1998
6 Gray LE Jr, Ostby, Joseph, Wolf, Cynthia, Lambright, Christy, and Kelce, William. The Value of Mechanistic Studies in
Laboratory Animals for the Prediction of Reproductive Effects in Wildlife. Endocrine effects on Mammalian Sexual
Differentiation Environ Toxicology and Chemistry 17.109-118, 1998.
7 Gray, LE Jr and Ostby J. Effects of pesticides and toxic substances on behavioral and morphological reproductive
development. Endocrine versus nonendocrine mechanisms Toxicol Industrial Health 14 159-184,1998.
8 Colborn T, et al Gray, LE Jr Consensus Statement from Erice "Wingspread" conference Italy, Toxicol Industi
Health 14 1-8, 1998
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9 GrayLEJr Xenoendocrine disrupters laboratory studies on male reproductive effects Tox Lett 28,102-103 331-5,1998.
10 KelceWR, Gray LE, Wilson EM Antiandrogens as environmental endocrine disruptors ReprodFertil Dev, 10(1).105-11.
,998
11 Gray LE Jr. Tlered screening and testing strategy for xenoestrogens and antiandrogens Toxicol Lett 28; 102-103.677-80,
1998
12. Gray LE Jr, Ostby J. Effects of pesticides and toxic substances on behavioral and morphological reproductive
development endocrine versus nonendocrine mechanisms. Toxicol Ind Health 14(1 -2)' 159-84, 1998.
13 DeVito M, Biegel L, Brouwer A, Brown S, Brucker-Davis F, Cheek AO, Christensen R, Colborn T, Cooke P, Crissman
J, Crofton K, Doerge D, Gray E, Hauser P, Hurley P, Kohn M, Lazar J, McMaster S, McClain M, McConnell E, Meier C, Miller
R, Tietge J, Tyl R Screening methods for thyroid hormone disruptors Environ Health Perspect, 107(5) 407-15.1999
14 Bigsby R, Chapin RE, Daston GP, Davis BJ, Gorski J, Gray LE, Howdeshell KL, Zoeller RT, vom Saal FS Evaluating
the effects of endocrine disruptors on endocrine function during development Environ Health Perspect, Aug, 107(## Suppl
4)613-8, 1999
15 Gray LE Jr, Wolf C, Lambright C, Mann P, Price M, Cooper RL, Ostby Administration of potentially antiandrogenic
pesticides (procymidone, linuron, iprodione, chlozolinate, p,p'-DDE, and ketoconazole) and toxic substances (dibutyl- and
diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles
of reproductive malformations in the male rat J Toxicol Ind Health 15(1-2)94-118, 1999
16 Ostby J, Kelce WR, Lambright C, Wolf CJ, Mann P, Gray LE Jr The fungicide procymidone alters sexual differentiation
in the male rat by acting as an androgen-receptor antagonist in vivo and in vitro Toxicol Ind Health 15(1-2)-80-93, 1999
17 Monosson E, Kelce WR, Lambright C, Ostby J, Gray LE Jr Peripubertal exposure to the antiandrogenic fungicide,
vinclozolin, delays puberty, inhibits the development of androgen-dependent tissues, and alters androgen receptor function
in the male rat Toxicol Ind Health 15(1 -2):65-79,1999.
18 Gray LE Jr, Ostby J, Monosson E, KelceWR Environmental antiandrogens low doses of the fungicide vinclozolin alter
sexual differentiation of the male rat. Toxicol Ind Health 15 (1-2) 48-64,1999
19 Gray LE Jr, Ostby J, Cooper RL, Kelce WR The estrogenic and antiandrogenic pesticide methoxychlor alters the
9productive tract and behavior without affecting pituitary size or LH and prolactin secretion in male rats Toxicol Ind Health
,5(1-2) 37-47, 1999
20 Wolf CJ, Ostby JS, Gray LE. Gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) severely alters
reproductive function of female hamster offspring Toxicol Sci 1999 Oct,51(2).259-64
21. Stoker TE, Parks LG, Gray LE, Cooper RL. Endocrine-disrupting chemicals prepubertal exposures and effects on sexual
maturation and thyroid function in the male rat. A focus on the EDSTAC recommendations Endocrine Disrupter Screening
and Testing Advisory Committee Crit Rev Toxicol 2000 Mar,30(2)197-252 Review
22 Wolf CJ, LeBlanc GA, Ostby JS, Gray LE Characterization of the period of sensitivity of fetal male sexual development
to vinclozolin Toxicol Sci 2000 May,55(1 ):152-61.
23 Lambright C, Ostby J, Bobseine K, Wilson V, Hotchkiss AK, Mann PC, Gray LE Cellular and molecular mechanisms
of action of linuron- an antiandrogenic herbicide that produces reproductive malformations in male rats. Toxicol Sci. 2000
Aug;56(2):389-99
24. Parks LG, Ostby JS, Lambright CR, Abbott BD, Klinefelter GR, Barlow NJ, Gray LE The plasticizer diethylhexyl phthalate
induces malformations by decreasing fetal synthesis during sexual differentiation in the male Rat Toxicol Sci 2000
Dec,58(2).339-49
25 Gray LE, Ostby J, Furr J, Price M, Veeramachaneni DN, Parks L. Perinatal exposure to the phthalates DEHP, BBP, and
DINP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male Rat Toxicol Sci 2000 Dec;58(2):350-65
26. Gray L E Jr, Ostby J , Furr J., Wolf C J , Lambright C, Parks L., Veeramachaneni D N R., Wilson V, Price M., Hotchkiss
A , Orlando E , Guillette L 2001 Effects of environmental antiandrogens on reproductive development in experimental
animals Human Reproduction Update May-Jun;7(3).248-64
27 Tamura H, Maness SC, Reischmann K, Dorman DC, Gray LE, Gaido KW 2001 Androgen receptor antagonism by the
organophosphate insecticide fenitrothion Toxicol Sci Mar,60(1) 56-62
28. Tamura H, Gaido KW, Dorman DC, Gray LE 2001 Reply Toxicol Sci Jul,62(1 )*183-4
?9 Parks LG, Lambright CS, Orlando EF, Guillette LJ Jr, Ankley GT, Gray LE Jr. 2001. Masculinization of Female
losquitofish in Kraft Mill Effluent-Contaminated Fenholloway River Water Is Associated with Androgen Receptor Agonist
Activity Toxicol Sci 62(2) 257-267.
PHS 398 (Rev 4/98) (Form Page 6) Page
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BIOGRAPHICAL SKETCH
Provide the following information tor the key personnel in the order listed on Form Page 2
'hotocopy this page or follow this format for each person
NAME
POSITION TITLE
Phillip C. Hartig
Research Biologist
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training)
INSTITUTION AND LOCATION
DEGREE
(if applicable)
YEAR(s)
FIELD OF STUDY
William Carey College, Hattiesburg, MS
Virginia Commonwealth Univ, Richmond, VA
Pennsylvania State Univ, Stale College, PA
Medical College of Virginia, Richmond, VA
BS
M.S
Ph D
1977
1980
1984
Biology
Biology/Virology
Genetics
Pathology/Virology
RESEARCH AND/OR PROFESSIONAL EXPERIENCE Concluding with present position, list, in chronological orde.r previous employment, experience, and
honors Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications dunng the past three years and to representative earlier publications pertinent to this application If the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant
application)
Professional Experience:
1994-Present
1994-1995
1986-1994
1984-1986
1978-1984
Professional Societies
merican Society for Gene Therapy American Society for Microbiology
American Society for Virology Chi Beta Phi
Teratology Society
Publications: (selected from last 5 years)
Gray, L.E. Jr, J Ostby, V. Wilson, C Lambright, K Bobseine, P. Hartig, A. Hotchkiss, C Wolf, J Furr, M Price, L
Parks, R. Cooper, T Stoker, S. Laws, S J Degitz, K. M Jensen, M D Kahl, J J Korte, E. A Makynen, J. E Tietge,
and G Ankley (cleared submitted) Xenoendocrine disrupters-tiered screening and testing. Filling key data gaps
Toxic Let
Hunter,III, E S, P. C. Hartig, E. H Rogers, and C Y Kawanishi (cleared submitted)
Pyran copolymer does not ameliorate all trans Retinoic acid-induced defects Terat. Carcin. Mut
Hunter,III, E S , P. C. Hartig, E H. Rogers, and C. Y. Kawanishi (cleared/submitted) Aspirin selectively ameliorates al
trans-retinoic acid-induced axial skeletal anteriorization in mice
Hunter, III, E S, M C Cardon, and P. C. Hartig (cleared submitted) P53 Over expression increases susceptibility of
mouse embryos to chemical-induced malformations Terat Carcin Mut.
Hartig, P. C , K L Bobseine, B. Brett, M. C. Cardon, C. Lambright, V.S Wilson, and L. E Gray, Jr (cleared submitted)
Development of two novel androgen-receptor (AR) assays using adenoviral transduction of MMTV-Luc reporter and/or
hAR for endocrine screening Toxicol Sci.
Hunter, III, E S , K Dam, L. A Gefrides, M C Cardon, R M Zucker, K Elstein, and P. C. Hartig . ( cleared)
Pathogenic effects of arsenicals in neurulation staged mouse conceptuses in vitro Teratology.
Smith, J B , P. C. Hartig, M R. Blanton K. K Sulik , and E S Hunter III. ( cleared/submitted) Amelioration of ethanol-
induced malformations by adenoviral Cu,Zn-SOD and Mn-SOD Overexpression in Vitro Alcohol Exp. Clin Res
Hunter,III, E. S., and P. C. Hartig 2000 Transient modulation of gene expression in the neurulation staged mouse
"rnbryo . Ann N Y Acad Sci. 919.278-83
,artig, P. C , and E S. Hunter, III 1998 Gene delivery to the neurulating embryo during culture. Teratology 58 103-112
Research Biologist
Research Scientist, ManTech Environmental Inc , RTP, NC
Project Scientist, ManTech Environmental Inc , RTP, NC
Instructor of Pathology, University of South Alabama, Mobile, AL
Instructor of Biology, Virginia Commonwealth University, Richmond VA
PHS 398 (Rev 4/98) (Form Page 6) Page FF
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image:
BIOGRAPHICAL SKETCH
Provide the following information tor the key personnel in the order listed on Form Page 2
Photocopy this page or follow this format for each person
NAME
POSITION TITLE
Gary A. Held
Research Microbiologist
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include
postdoctoral training)
INSTITUTION AND LOCATION
DEGREE
(if applicable)
YEAR(s)
FIELD OF STUDY
University of Illinois, Chicago, IL
University of Illinois, Chicago, IL
Kansas State Univ., Manhattan, KS
Postdoctoral Research Associate, University of Idaho,
Moscow, ID
BS,
MS
Ph D
1971
1976
1983
1984
Biology
Biology
Microbiology
Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and complete references
to all publications during the past three years and to representative earlier publications pertinent to this application If the list of publications in the last three years
exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant application)
Professional Experience:
1984- Current Position Research Microbiologist, USEPA, Research Triangle Park, NC
1983-1984 Postdoctoral Research Associate, University of Idaho, Moscow, ID
1976-1978 Research Assistant Protein and Carbohydrate Laboratory ABI, Manhattan KS
Professional Societies
American Association for the Advancement of Science
Publications: (selected from last 5 years)
Huang Ys, Held, G.A., Andrews J E , Rogers,J M , 14C methanol incorporation into DNA and proteins of organogenesis
stage mouse embroys in vitro Reprod Toxicol 2001 Jul-Aug; 15(4) 429-35
Held G,A, Abbott B, D Palatal dysmorphogenesis Quantitative RT-PCR Methods Mol Biol 2000;136:203-17.
Ghosh B, Wood C,R, Held G,A, Abbott B,D, Lau C Glucocorticoid receptor regulation in the rat embryo-a potential site
for developmental toxicity7 Toxicol Appl Pharmacol 2000 Apr 15;164(2)-221-9
Abbott BD, Schmid JE, Brown JG, Wood CR, White RD, Buckalew AR, Held GA.,RT-PCR quantification of AHR, ARNT,
GR, and CYP1A1 mRNA in craniofacial tissues of embryonic mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and
hydrocortisone Toxicol Set 1999 Jan,47(1) 76-85
Abbott BD, Held GA, Wood CR, Buckalew AR, Brown JG, Schmid J AhR, ARNT, and CYP1A1 mRNA quantitation in
cultured human embryonic palates exposed to TCDD and comparison with mouse palate in vivo and in culture
Toxicol Sci 1999 Jan,47(1).62-75.
Abbott BD, Schmid JE, Pitt JA, Buckalew AR, Wood CR, Held GA, Diliberto JJ Adverse reproductive outcomes in the
transgenic Ah receptor-deficient mouse. Toxicol Appl Pharmacol. 1999 Feb 15,155(1).62-70
Branch S, Francis BM, Rosen MB, Brownie CF, Held GA, Chernoff N Differentially expressed genes associated with 5-
Aza-2'-deoxycytidine-induced hindlimb defects in the Swiss Webster mouse. J Biochem Mol Toxicol 1998:12(3) 135-
41
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BIOGRAPHICAL SKETCH
'rovide the following information for the Key personnel in the order listed on Form Page 2
notocopy this page or follow this format for each person
NAME
POSITION TITLE
Sid Hunter
Toxicologist, DBB, RTD, NHEERL, ORD, US EPA
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training)
INSTITUTION AND LOCATION
DEGREE
(if applicable)
YEAR(s)
FIELD OF STUDY
Hampden Sydney College, Hampden-Sydney, VA
Old Dominion University, Norfolk, VA
University of North Carolina at Chapel Hill, Chapel Hill, NC
University of North Carolina at Chapel Hill, Chapel Hill, NC
BS
MS
PhD
Post-Doc
1980
1983
1986
1986
Chemistry
Biochemical Toxicology
Anatomy/Embryology
Embryology
RESEARCH AND/OR PROFESSIONAL EXPERIENCE Concluding with present position, list, in chronological orde.r previous employment, expenence, and
honors Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications dunng the past three years and to representative earlier publications pertinent to this application If the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant
application)
Professional Experience:
1993 - Present Toxicologist, Developmental Biology Branch. RTD, NHEERL, US EPA, RTP, NC
1998 - Present Adjunct Assistant Professor, Curriculum in Toxicology, UNC - Chapel Hill, NC
1990 - 1993 Toxicologist, Developmental and Reproductive Toxicology Group, Systems Toxicology Branch
National Toxicology Program, National Institute of Environmental Health Sciences, RTP,
NC
987 - 1990. Research Assistant Professor, Department of Cell Biology and Anatomy, UNC - Chapel Hill, NC
Professional Societies
Sigma Xi
Teratology Society
Honors. Awards: (selected from last 5 years)
1999 Clark Fraser Young Investigator Award, Teratology Society
Professional Activities (selected from last 5 years)
Current Teratology - Experimental Teratology and Developmental Toxicology Section Editor
Former Teratology Society - Membership Committee, Public Affairs Committee, and Fraser Young Invesitgator Award
Committee
2000 ICCVAM Review of FETAX, Research Triangle Park, NC
1999 Toxicology Excellence for Risk Assessment, Peer Review of Arsenic,
Invited Presentations (selected from last 5 years)
2001, 2002 Guest Lecture in Developmental Toxicology, College of Veterinary Medicine, NCSU, Raleigh, NC
1992-Present Guest Lecturer in Advanced Toxicology, Toxicology Curriculum, UNC-Chapel Hill, NC.
1989-1999 Guest Lecturer in Developmental Toxicology and Teratology, Department of Cell Biology and Anatomy,
UNC-Chapel Hill, NC.
February 1998 Departmental Seminar, Department of Toxicology, North Carolina State University, Raleigh, NC
March 1998 Spring Symposium, Molecular Substrates of Development Impact on Toxicology. Integrated Toxicology
Program, Duke University, Durham, NC.
March 1998 Continuing Education Course, Current Developmental Biology Relevance and Application for
Developmental Toxicology. Society of Toxicology, Seattle, WA
"ibruary 1997 Grand Rounds, Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College,
Philadelphia, PA.
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June 1997
February 1999
June 1999
Keystone,
Sept. 1999
October 1999
June 2000
June 2000
Nov 2000
January 2001
March 2001
Toxicology
May 2001
October 2001
April 2002
Continuing Education Course, Experimental Methods for Studying Mechanisms of Abnormal
Development. Teratology Society Meeting, FL.
Health Effects Stakeholders Meeting for the Stage 2 Disinfection Byproduct Rule, Washington, DC.
NIEHS Symposium, Oxidative stress and apoptosis in birth defects, Teratology Society Meeting,
CO.
NY Academy of Sciences Meeting, Toxicology for the Next Millennium, New York Academy of Sciences,
Arlie, VA.
Michigan State University, Environmental and Industrial Health, Toxicology Program, Ann Arbor, Ml
Drinking Water and Reproduction, Symposium Teratology Society, FL
Signal Transduction: Pathways to Growth, Differentiation, and Morphogenesis Continuing Education
Course, Teratology Society Meeting, FL.
Understanding the Developmental Effects of Haloacetic Acids, Federal-State Toxicology and Risk
Analysis Committee, Research Triangle Park, NC
Early Craniofacial Development Life among the signals, Triangle Consortium for Reproductive Biology,
Research Triangle Park, NC.
The role of apoptosis in developmental neurotoxicity and neurodegeneration in adults, Society of
Symposium, San Francisco, CA.
Genes and Gene Delivery for Diseases of Alcoholism, Symposium, Alcohol Research Center, UNC at
Chapel Hill, NC
Departmental Seminar, Department of Toxicology, North Carolina State University, Raleigh, NC
Drinking Water and Reproduction, Symposium. Society of Toxicology, Nashville, TN
Publications: (selected from last 5 years)
Tabacova, S., E.S. Hunter, III and L. Balabaeva (1997). Potential role for oxidative damage in developmental toxicity
arsenic IN Arsenic Exposure and Health Effects Abernathy, R L. Calderon and W R Chappell, Eds, Chapman and
Hall, NY, pp. 135-144
K W Ward, E.H. Rogers and E.S. Hunter, III (1998). Dysmorphogenic effects of a specific protein Kinase C inhibitor
during neurulation Reprod Toxicol., 12(5), 525-534.
Hartig, P C. and E.S. Hunter, III (1998) Gene Delivery to the neurulating embryo during culture Teratology. 58.103-
112.
R M. Zucker, E.S. Hunter. Ill and J Rogers (1998). Confocal laser scanning microscopy of embryo apoptosis.
Cytometry 33- 348-354.
G A Boorman, V. Dellarco, J K Dunnick, R.E Chapin, E.S. Hunter. III. F Hauchman, H. Gardner, M Cox, R C Sills
(1999). Drinking water disinfection byproducts. Review and approach to toxicity evaluation. Environmental Health
Perspectives, 107(Suppl 1). 207-217.
K.W Ward, E H Rogers and E.S. Hunter. Ill (2000). Comparative pathogenesis of haloacetic acid and protein kinase
inhibitor embryotoxicity in mouse whole embryo culture. Toxicol Sci 53, 118-26
E.S. Hunter. Ill and D.J. Dix (2001) Heat shock proteins Hsp70-1 and Hsp70-3 are necessary and sufficient to prevent
arsenite-induced dysmorphology in mouse embryos Molecular Reproduction and Development, In press
N. Chernoff, E.S. Hunter III. L L Hall, M B Rosen, C. F. Brownie, D Malarkey, M. Marr, J. Herkovits (2001) Lack of
Teratogenicity of Microcystin-LR in the Mouse and Frog Accepted
Tabacova, S., C. Harris and E.S. Hunter. Ill (2001). Developmental Toxicity of Arsenic: Evidence for a Role of Oxidative
Stress Reproductive Toxicology. Submitted
S. Degitz, J Rogers and E.S. Hunter. Ill (2001). Strain Differences In Response To Methanol In Mouse Whole Embryo
Culture. CD-1 Vs C57bl/6J Mice.. Toxicology Sciences Submitted
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Andrews, J.E., J E. Schmid, H P. Nichols, E.S. Hunter. Ill and G.R. Klinefelter (2001). Developmental Toxicity of
fixtures: The water disinfection by-products dichloro-, Dibromo- and bromochloro-Acetic acids in embryo culture
eratology Submitted
E.S. Hunter. III. M Cardon and P Hartig (2001) p53 Overexpression Increases Susceptibility Of Mouse Embryos To
Chemical-Induced Malformations. Submitted.
E.S. Hunter. III. M Cardon, R Zucker, K. Elstein, P. Hartig (2001) Pathogenic effects of arsenicals in neurulation staged
mouse conceptuses in vitro. Submitted
E. Sidney Hunter. III. Phillip Hartig, Ellen H Rogers and Clinton Y Kawanishi (2001) Pyran copolymer does not
ameliorate all-trans Retinoic acid-induced defects Cleared for submission
E. Sidney Hunter. III. Phillip Hartig, Ellen H. Rogers and Clinton Y Kawanishi (2001) Acetylsalicylic Acid does not
ameliorates all trans retinoic acid-induced axial skeletal anteriorization in mice Cleared for submission.
L A Gefrides, F Copeland, P Hartig, M Cardon and E S Hunter. Ill (2001Dysmorphogenic Effects of an Irreversible
Catalase Inhibitor in Mouse Embryos in vitro In Clearance Process
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BIOGRAPHICAL SKETCH
Vovide the following information for the key personnel in the order listed on Form Page 2
hotocopy this page or follow this format for each person
NAME
POSITION TITLE
Robert J. Kavlock
Dir, ReproductiveToxicology Division, NHEERL
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training)
INSTITUTION AND LOCATION
DEGREE
YEAR(s)
FIELD OF STUDY
(if applicable)
University of Miami, Coral Gables, FL
BS
1973
Biology
University of Miami, Coral Gables, FL
PhD
1977
Biology
RESEARCH AND/OR PROFESSIONAL EXPERIENCE Concluding with present position, list, in chronological orde.r previous employment, experience, and
honors Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications during the past three years and to representative earlier publications pertinent to this application If the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEEPTHREE PAGES (for a modular grant application)
Professional Experience:
1999-2000 Acting Associate Director for Health, NHEERL (June-January)
1981-1989 Chief, Perinatal Toxicology Branch, DTD, HERL, USEPA, RTP, NC
1979-1981- Research Biologist, Perinatal Toxicology Branch, DTD, HERL, USEPA, RTP, NC
1977-1979 Research Associate, Dept. of Biology, Univ. of Miami, Coral Gables, FL
Professional Societies
""ociety of Toxicology, Continuing Education Committee (1997-2000); Reproductive & Developmental Toxicology Specialty
,ection, (Secretary, 1989- 1991; Councilor, 1993-1994, President, 1997)
Teratology Society, Scientific Program Chair, 1988; Education Committee, 1989-1992, Public Affairs Committee, 1992-
1994, Councilor 1994-1997, Strategic Planning Committee (Organizing Committee Member), 1996-1999, Endocrine
Disruptor Position Paper ad hoc Committee, 1997-1999, Vice-President and Scientific Program Chair, 1999-2000;
President 2000-2001
North Carolina Society of Toxicity, President, 1999-2000
Honors, Awards: (selected from last 5 years)
U S EPA Bronze Medal, 1998 for efforts on Harmonized Reproductive Testing Guidelines
Professional Activities (selected from last 5 years)
President, Teratology Society, 2000-2001
Co-Organizer, Japanese NIES/US EPA Workshop on EDCs, Tokyo, February 2000
NHEERL Human Health Research Strategy Implementation Team, 2001-
American Chemistry Council Endocrine Implementation Planning Group (Consultant), 2000-
Chair, NTP Center for Evaluation of Risk to Human Reproduction Expert Panel on Phthalates, 1999-2000
Chair, NHEERL Emerging Science Committee, 1999-
Advisor, United Nations University's Program on Endocrine Disruptors in the Coastal Environment of Southeast Asia,
1999-
US/EU Workshop on Endocrine Disruptor Research Needs, Rapporteur, 1999
NIOSH Working Group to Develop Research Priorities for Reproductive Epidemiology, 1999
EPA Endocrine Disruptor Screening Program Implementation Steering Group, 1998-2000
'PCS/WHO Steering Group for International State-of-Science Assessment of Endocrine Disruptors, 1997-
IH ALTX-4 Study Section, Standing Member, 1997-2001
Chair, NHEERL Branch Chief Career Ladder Committee, 1997-1998
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Chair, ORD Endocrine Disruptor Research Strategy Committee, 1995-1998
WHO/IPCS Working Group on the International Harmonization of Risk Assessment Guidelines for Reproductive and
Developmental Toxicity, 1994
EPA Working Group on Harmonized Testing Guidelines for Reproductive and Developmental Toxicity, 1991-1999
Co-Chair, HERL(NHEERL) Technical Qualifications Board, 1989-1997
Invited Presentations (selected from last 5 years)
Neurotoxicology Division, NHEERL, Research Triangle Park, NC, January 1996
Duke University, Department of Pharmacology, Durham, NC, January 1996,
German Environmental Agency, April, 1996,
Swiss BUWAL, Berne, April 1996,
The University of Zurich, Zurich, Switzerland, April 1996,
European Teratology Society, Rome, Italy, September 1996,
Mid-Atlantic Society of Toxicology, October 1996,
Society of Environmental Toxicology and Chemistry, Washington, DC November 1996,
European Commission Workshop on Endocrine Disruptors, London, England, December 1996,
NIEHS/NTP Board of Scientific Councilors, December 1997, Zeneca, England, January 1997,
Toxicology Forum, Washington, DC, February 1997;
Mississippi State University, April 1997,
Second EC/US EPA Workshop on The Integration of Multi-endpoint Mechanistic Information in Risk Assessment, Ispra, Italy,
May 1997;
Teratology Society Continuing Education Committee, June 1997,
International Meeting on Boron, October 1997,
Minnesota Pollution Control Agency Endocrine Disruptor Workshop, October 1997,
Second International Symposium on the Health Effects of Boron and Its Compounds, October, 1997;
Endocrine Disruptor Workshop, Rome, Italy, November 1997,
UNU EDCs in the Coastal Environment Workshop, Tokyo, February, 1999,
Japanese Toxicology Society, Sapporo, Japan, July, 1999,
2nd International Conference on EDCs, Kobe, Japan, 1999;
UNU EDCs in the Coastal Environment Workshop, Kuala Lumpur, April, 2000;
European Teratology Society, Ferrara, Italy, September 2000,
NTP Board of Scientific Councilors, October 2000;
FDA/CDC Workshop on Post marketing Surveillance for Adverse Pregnancy Outcomes, Washington, December 2000,
3rd International Conference of EDCs, Yokohama, Japan, December 2000;
US/Japan Joint Conference on EDCs, Tsukuba, Japan, December 2001;
EPA ORD/Regional Workshop on EDCs, Atlanta, May, 2001
Publications: (selected from last 5 years)
Kavlock, RJ (In Press) Pesticides as endocrine-disrupting chemicals IN: Handbook of Pesticide Toxicology
Andersen, ME, Conolly, RB, Faustman, EM, Kavlock RJ, Portier, CJ, Sheehan, DM, Wier, PJ and Ziese, L.
Environmental Health Perspectives 107.Suppl 4-631-638.
Rogers, JM and RJ Kavlock (2001). Developmental toxicity. In: Casarett & Doull's Toxicology The Basic Science of
Poisons, 6h edition Curtis D Klaassen, editor McGraw-Hill, Inc., New York, NY, 301-331
Moorman, WJ, Ahlers, HW, Chapin, RE, Daston, GP, Foster, PM, Kavlock, RJ, Morawetz, JS, Schnorr, TM and
Shrader,
SM Prioritization of NTP reproductive toxicants for field studies Reproductive Toxicology 14(4) 293-301
Lau, C, Andersen, ME, Crawford-Brown, DJ, Kavlock, RJ, Kimmel, CA, Knudsen, TB, Muneoka, K, Rogers, JM, Setzer,
RW, Smith, G and Tyl, R (2000). Evaluation of biologically-based dose-response modeling for developmental toxicitv
a workhsop report Regul. Toxicol Pharmacol. 31 (2 Pt 1) 190-199.
Goldman, JM, Laws, SC, Balchak, SK, Cooper, RL and Kavlock, RJ (2000). Endocrine-disrupting chemicals:
prepubertal exposures and effects on sexual maturation and thyroid activity in the female rat. A focus on the ESTAC
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recommendations. Critical Reviews in Toxicology 30(2). 135-196
Jarlow, S, RJ Kavlock, JA Moore, SL Schantz DM Sheehan, DL Shuey, and JM Lary (1999) Teratology Society position
paper: The developmental toxicity of endocrine disruptors to humans Teratology 60(6) 365-375
Narotsky, MG, JE Schmid, JE Andrews, and RJ Kavlock (1999) Effects of boric acid on axial skeletal development in
rats Biological Trace Element Research 66, 373-394
Kavlock, RJ (1998). What's Happening to our Frogs? Environmental Health Perspectives, 106(12) 773-774.
Reiter, LW, C DeRosa, RJ Kavlock, G Lucier, MJ Mac, J Melillo, RL Melnicj, and T Sinks (1998) The U.S. Federal
framework for research on endocrine disruptors and an analysis of research programs supported during Fiscal Year
1996. Environmental Health Perspectives Vol 106(3)105-113.
Kavlock, RJ and GP Daston (1997) Handbook of Experimental Pharmacology, Vol. 124 Drug Toxicity in Embryonic
Development I. Advances in Understanding Mechanisms of Birth Defects Morphogenesis and Processes at Risks
(610 pgs) Springer-Verlang, Heidelberg, Germany, ISBN 3-540-61259-9
Kavlock, RJ and GP Daston (1997) Handbook of Experimental Pharmacology, Vol 124 Drug Toxicity in Embryonic
Development II Advances in Understanding Mechanisms of Birth Defects Mechanisistic Understanding of Human
Developmental Toxicants. (485 pgs) Springer-Verlag, Heidleberg, Germany, ISBN 3-540-61261-0
Cooper, RL and RJ Kavlock (1997) Endocrine disruptors and reproductive development A weight-of-evidence
overview Journal of Endocrinology 152.159-166
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BIOGRAPHICAL SKETCH
"Ovide the following information for the key personnel in the order listed on Form Page 2
xitocopy this page or follow this format for each person
NAME
Gary R Klinefelter
POSITION TITLE
Research Biologist
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training )
INSTITUTION AND LOCATION
DEGREE
(if applicable)
YEAR(s)
FIELD OF STUDY
Pennsylvania State University, University Park, PA
Pennsylvania State University, University Park, PA
University of Minnesota, Minneapolis, MN
Johns Hopkins School of Hygiene, Baltimore, MD
B.S.
M S.
Ph D
Post Doc
1976
1979
1984
1987
Animal Science
Dairy Science
Anatomy
Reproductive Biology
RESEARCH AND/OR PROFESSIONAL EXPERIENCE Concluding with present position, list, in chronological orde.r previous employment, experience, and
honors Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications dunng the past three years and to representative earlier publications pertinent to this application If the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant
application)
Professional Experience:
1988-1991 Research Scientist, Mantech Environmental, Inc., Research Triangle Park,NIC
1991-Present Research Biologist, U S Environmental Protection Agency, Research Triangle Park, NC
Professional Societies
Society of Toxicology
Society for the Study of Reproduction
American Society of Andrology
honors. Awards: (selected from last 5 years)
Recipient of two Outstanding Original Research Awards from the American Society of Andrology
Recipient of an unprecedented two Level I Scientific and Technological Achievement Awards from ORD.
SP22 research was cited in the EPA Assistant Adm inistrator's 1999 Congressional Report as one of the cutting edge research
areas in ORD.
EPA Bronze Metal for Commendable Service in 1999 for tireless and innovative work developing and harmonizing guidelines
for reproductive toxicity testing improving assessment of children's health risk.
Inventor of EPA's first biotechnology patent, "Method for Evaluating Male Fertility" issued March 6, 2001 as U.S. patent (#
6,197,940) on the SP22 protein and fragments and recombinants
Professional Activities (selected from last 5 years)
Journal of Andrology (Editorial Board, 1998 to present)
Biology of Reproduction (Editorial Board, 1995 to 2000)
Serving on the Executive Council of the American Society of Andrology (1999-present).
Serving on the Editorial Board of Toxicological Sciences (00- present).
Serving on the Program Committee for the meeting of the Society for the Study of Reproduction (2000-2001).
Adjunct Professor at University of North Carolina at Chapel Hill, Curriculum in Toxicology, (2000-present).
Invited Presentations (selected from last 5 years)
Symposium presentation at the 1998 Triangle Consortium for Reproductive Biology entitled "SP22, A Novel Sperm
Biomarker of Fertility".
Presentation entitled "Toxicology as a Tool for Physiology: The Saga of a Male Fertility Protein" at the Department of
nysiology, Colorado State University, 1998
resentation entitled "Assessments of Reproductive Risk Associated with Disinfection Byproducts of Drinking Water" at the
Department of Environmental Health, Colorado State University, 1998.
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Lecture entitled Toxicology of the Epididymis: From Unique Experimental Strategies to Novel Biomarkers" for a Continuing
Education Course "Evaluation of Male Reproductive Toxicity- Sperm Markers and Epididymal Mechanisms of Toxicity
sponsored by SOT, 1999.
Seminar entitled The Saga of a Male Fertility Protein Potential of SP22 as a Biomarker in Toxicology", presented
to the Environmental Carcinogenesis Division at NHEERL, EPA, 1999
Presented a lecture entitled 'Toxicology of the Male Excurrent Ducts and Accessory Sex Glands" to the Departments of
Pharmacology and Anatomy at the Institute de Biociencias in Botucatu, Brazil (1999)
Presented a lecture entitled 'Toxicology of the Epididymis From Unique Experimental Strategies to Novel
Biomarkers" in a Postgraduate Course at the 14th Annual Meeting of the Federation of Societies of Experimental
Biology held in Caxambu, Brazil (1999)
Presented a symposium lecture entitled Toxicology of the Epididymis- The Saga of a Male Fertility Protein" at
the 14lh Annual Meeting of the Federation of Societies of Experimental Biology held in Caxambu, Brazil (1999)
Presented a lecture entitled "Breakthrough in Andrology The Saga of Male Fertility Protein" to the Department
of Pharmacology, Experimental Endocrinology Division, at the Federal University of Sao Paulo in Sao Paulo,
Brazil (1999)
Presented seminar entitled "Saga of a Novel Male Fertility Protein (SP22)" at the Center for Recombinant Gamete
Contraceptive Vaccinogens, Department of Cell Biology, University of Virginia (2000)
Organizing chair and speaker in workshop session entitled "Reproductive Risk Associated with Exposure to
Disinfection By-Products of Drinking Water", Society of Toxicology (2002)
Publications: (selected from last 5 years)
Linder, R E., Klinefelter, G.R., Strader, L F., Veeramachaneni, D.N R., Roberts, N.L , Suarez, J.D. Histopathologic changes
in the testis of rats exposed to dibromoacetic acid Reprod. Tox. 11:47-56, 1997.
Klinefelter, G R., Laskey, J W., Ferrell, J., Suarez, J.D., Roberts, N.L. Discriminant analysis indicates a single
sperm protein (sp22) is predictive of fertility following toxicant exposure. J Andrology, 18 139-150, 1997
Ricker, D D., Crone, J.K, Chamness, S L., Klinefelter, G.R., Chang, T.S.K. Partial sympathetic denervation of the
epididymis permits fertilization but inhibits embryo development J. Andrology, 18:131-138, 1997
Linder, R.E , Klinefelter, G.R., Strader, L.F., Suarez, J D., and Roberts, N L Spermatotoxicity of dichloroacetic acid
Reprod. Tox , 11:681-688, 1997
Klinefelter, G R and Suarez, J.D. Toxicant-induced acceleration of epididymal sperm transit androgen-dependent proteins
may be involved Reprod Tox., 11:511-519,1997.
Gray, L E , Kelce, W.R., Weise, T., Tyl, R, Gaido, K., Cook, J , Klinefelter, G., Desaulniers, D., Wilson, E ,
Zacharewski, T., Waller, C., Foster, P., Lakey, J , Reel, J , Giesy, J., Laws, S., McLachlan, J , Breslin, W., Cooper,
R„ DiGiulio, R., Johnson, R„ Purdy, R , Mihaich, E , Safe, S., Sonnesnschein, C., Welshons, W„ Miller, R„ McMaster, S ,
and Colborn, T Endrocrine screening methods workshop report: detection of estrogenic and androgenic hormonal and
antihormonal activity for chemicals that act via receptor or steroidogenic enzyme
mechanisms. Reprod Tox 11.719-750,1997.
Klinefelter, G.R. Ethane Dimethane Sulphonate (EDS) A Prototypic Epididymal Toxicant In. Comprehensive
Toxicology, Volume 10, (Sipes I.G., McQueen C.A., Gandolfi A.J., eds), pp 151-164, Pergamon Press, 1997
Klinefelter, G R and Hess, R.A. Toxicology of the Male Excurrent Ducts and Accessory Glands In. Reproductive
and Developmental Toxicology, (Korach, K„ ed), pp 553-591, Marcel Dekker Inc., 1998.
Chen, Y.C., Bumck, D., Bahr, J.M , Klinefelter, G.R., and Hess, R.A Isolation and culture of epithelial cells from rat efferent
ductules and initial segment epididymidis Tissue and Cell 30.1-13,1998.
Welch, J E , Barbee, R R , Roberts, N.L , Suarez, J.D., and Klinefelter G.R. SP22: a novel fertility protein from a highly
conserved gene family. J. Andrology 19395-393,1998
Kempinas, W D , Suarez, J.D., Roberts, N.L., Strader, L.F., Ferrell, J.M., Goldman, J.M, and Klinefelter, G.R., Rat
epididymal sperm quantity, quality, and transit time after guanethidine-induced sympathectomy Biol. Reprod
59.890-896, 1998.
Kempinas, W D , Suarez, J.D., Roberts, N.L., Strader, L F„ Ferrell, J M., Goldman, J.M., Narotsky, M.G., Perreault,
S.D., and Klinefelter, G.R. Fertility of rat epididymal sperm after chemically and surgically-induced sympathectomy. Bit
Reprod 59 897-904, 1998.
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Walensky L.D., Dawson, T.M , Steiner, J P., Sabatini, D.M., Suarez, J D , Klinefelter, G.R., and Synder, S H , The
12 kD FK506 binding protein, FKBP12, is released in the male reproductive tract and stimulates sperm motility.
i\/lolecular Medicine, 4.502-514, 1998
Cook, J C., Foster, P M D., Hardisty, J F., Klinefelter, G R , Sharpe, R.M A review of Leydig cell hyperplasia in
rodents and the relevance man Critical Reviews in Toxicology, 29(2) 169-261,1999.
Klinefelter, G R and Welch, J E The Saga of a Male Fertility Protein (SP22). Annual Review of Biomedical Sciences.
1 *145-184, 1999
Akingbemi, B.T, Ge, R S , Klinefelter, G R , Gunsalus, G.L., and Hardy, M P A metabolite of methoxychlor, 2,2-
Bis (p-Hydroxyphenyl)-l,l ,1-Trichlorethane, reduces testosterone biosynthesis in rat Leydig cells through the
suppression of steady-state mRNA levels of the cholesterol side-chain cleavage enzyme Biol Reprod 62:571-578,
2000
Klinefelter, G R , Hunter, E S , and Narotsky, M. Reproductive and Developmental Toxicity Associated with
Disinfection By-Products of Drinking Water, International Life Sciences Institute (In Press), 2000
Parks, L G., Ostby, J.S , Lambright, C R., Abbott, B D , Klinefelter, G R , and Gray, L.E., Jr. The plasticizer diethylhexyl
phthalate induces reproductive malformations by decreasing fetal male rat testosterone synthesis Tox.
Sciences 58 339-349, 2000
Clegg, E D., Perreault, S D, and Klinefelter, G R Assessment of male reproductive toxicity. In Principles and
Methods of Toxicology, Volume 4E, (Hayes A Wallace, ed ), pp 1263-1300, Raven Press, 2000
Salva, A., Klinefelter, G R., and Hardy, M.P. Purification of rat Leydig cells increased yields after unit gravity
sedimentation of collagenase dispersed interstitial cells, J Androl., 2001, (In Press).
Modulation of rat Leydig cell steroidogenic function by di(2-ethylhexyl)phthalte. Akingbemi BT, Youker RT, Sottas
CM, Ge R, Katz E, Klinefelter GR, Zirkin BR, and Hardy MP Differential effects of DEHP on Leydig cell function
throughout reproductive development, Biol. Reprod , 2001, (In Press)
Holmes M, Suarez JD, Roberts NL, Mole L, Murr AS, and Klinefelter GR. Dibromoacetic acid, a prevalent disinfection by-
oroduct of drinking water disinfection, compromises the synthesis of specific seminiferous tubule
roteins following both in vivo and in vitro exposures, J. Androl., 2001, (In Press)
flomualdo, G S , Klinefelter, G.R , and Kempinas W. Postrotational exposure to gossypol results in epididymis-
specific effects throughout puberty and adulthood in rats, J Androl 2001, (In Press)
Klinefelter, G R , Welch, J.E., Perreault, S.D. Moore, H.D., Zucker, R M., Suarez, J.D., Roberts, N L , Bobseine,
K., and Jeffay, S Localization of the sperm protein SP22 and inhibition of fertility in vivo and in vitro. J. Androl.,
2001, (In Press)
Klinefelter, G.R. Actions of Toxicants on the Structure and Function of the Epididymis In The Epididymis
(Robaire B. and Hinton B, eds ), (In Press), 2001.
PHS 398 (Rev 4/98) (Form Page 6) Page
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BIOGRAPHICAL SKETCH
rovide the following information tor the key personnel in the order listed on Form Page 2
notocopy this page or follow this tormat tor each person
NAME
POSITION TITLE
Christopher S Lau
Pharmacologist
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training)
INSTITUTION AND LOCATION
DEGREE
YEAR(s)
FIELD OF STUDY
(it applicable)
Duke University
AB
1975
Chemistry and Zoology
Duke University
Ph D
1982
Pharmacology
RESEARCH AND/OR PROFESSIONAL EXPERIENCE Concluding with present position, list, in chronological orde.r previous employment, experience, and
honors Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications during the past three years and to representative earlier publications pertinent to this application If the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant
application)
Professional Experience:
1990-Present Pharmacologist, RTD, NHEERL, US EPA, RTP, NC
1984-1990 Research Scientist, NSI Technology Services, Environmental Sciences, RTP, NC
1982-1984 Research Associate, Dept of Anat, Medical College of Pennsylvania, Philadelphia, PA
Professional Societies
Society for Neuroscience.
Teratology Society.
Professional Activities (selected from last 5 years)
djunct Assistant Professor, Dept of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC
Team leader, "Biologically based dose response modeling" research team
Vice chairman, NHEERL Diversity Steering Group
Invited Presentations (selected from last 5 years)
1999 Teratology Society meeting discussion on Teratogen-induced alterations in developmental pathways leading to
dysmorphogenesis: What do we know and where do we go from here7"
Publications: (selected from last 5 years)
Lau, C., Narotsky, M.G , Lui, D., Best, D., Setzer, R.W., Mann, P.C , Wubah, J.A. and Knudsen, T.B. (2001) Exposure-
disease continuum for 2-chloro-2'-deoxyadenosine (2-CdA), a prototype teratogen 2. Induction of lumbar hernia in the rat
and species comparison for the teratogenic responses. Teratology, submitted
Carter, C.S., Lau, C. and Stanton, M.E. (2001) Developmental exposure to propylthiouracil (PTU) and cognitive development
in the rat Neurotoxicol. Teratol, in press.
Zahalka, E A., Ellis, D.H., Goldey, E.S., Stanton, M E. and Lau, C. (2001) Perinatal exposure to polychlorinated biphenyls
Aroclor 1016 or 1254 did not alter brain catecholamines nor delayed alternation performance in Long-Evans rats Brain Res
Bull., in press
Wubah, J.A., Setzer, R.W , Lau, C , Charlap, J.H. and Knudsen, T.B. (2001) Exposure-disease continuum for 2-chloro-2'-
deoxyadenosine (2CdA), a prototype ocular teratogen 1. Dose-response analysis. Teratology, in press
Lau, C., Mole, M.L., Copeland, F.M , Rogers, J M , Kavlock, R.J , Shuey, D L., Cameron, A.M., Ellis, D., Merriman, J. and
Setzer, R.W. (2001) Toward a biologically-based dose-response model for developmental toxicity of 5-fluorouracil in the rat
Acquisition of experimental data Toxicol. Sci. 59.37-48
Setzer, R W., Lau, C , Mole, M L., Copeland, F.M , Rogers, J.M and Kavlock, R.J. (2001) Toward a biologically-based
jse-response model for developmental toxicity of 5-fluorouracil in the rat A mathematical construct Toxicol Sci. 5949-58.
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Ghosh, B., Wood, C.R , Held, G A., Abbott, B.D. and Lau, C (2000) Glucocorticoid receptor regulation in th rat embryo: A
potential mechanism for developmental toxicity'' Toxicol Appl. Pharmacol 164 221-229
Lau, C., Andersen, M.E., Crawford-Brown, D J., Kavlock, R.J., Kimmel, C.A , Knudsen, T.B , Muneoka, K., Rogers, J.N.,
Setzer, R W , Smith, G And Tyl, R (2000) Evaluation of biologically based dose-response modeling for developmental
toxicity A workshop report Regul Toxicol Pharmacol 31 '190-199
Chiu, A , Chiu, N , Beaubier, N.T., Beaubier, J., Nalesnik, R , Singh, D , Hill, W R , Lau, C and Riebow, J (2000) Effects
and mechanisms of PCB ecotoxicity in food chains algae fish -*• seal polar bear. Environ Carcino Ecotox Revs
C18(2) 127-152.
Barr, M., DeSesso, J M , Lau, C., Osmond, C , Ozanne, S E , Sadler, T W , Simmons, R.A and Sonawane, B R (2000)
Workshop to identify critical windows of exposure for children's health Cardiovascular and endocrine work group summary
Environ Hlth. Perspect 108, Suppl 3 569-571.
Lau, C and Setzer, RW (1999) Biologically based risk assessment models for developmental toxicity In Developmental
Biology Protocols, vol I, eds. R.S TuanandCW Lo, Humana Press, pp 271-281
Sawin, S , Brodish, P., Carter, C.S., Stanton, M E and Lau, C. (1998) Development of cholinergic neurons in rat brain
regions dose-dependent effects of propylthiouracil-induced hypothyroidism Neurotoxics Teratol. 20:627-635
Mole, M L , Hunter, D.L , Gao, P and Lau, C (1998) Sample preparation and high performance liquid chromatographic
analysis of deoxyribonucleoside-triphosphates in individual rat embryos. Anal Biochem 259 245-252
Elstein, K H , Mole, M.L , Setzer, R W., Zucker, R M , Kavlock, R.J , Rogers, J M and Lau, C. (1997) Nucleoside-mediated
mitigation of 5-fluorouracil-induced toxicity in synchronized murine erythroleukemic cells Toxicol Appl Pharmacol.
146 29-39
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BIOGRAPHICAL SKETCH
Provide the following information for the key personnel in the order listed on Form Page 2
'hotocopy this page or follow this format (or each person
NAME
POSITION TITLE
Susan C Laws
Research Biologist
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training)
INSTITUTION AND LOCATION
DEGREE
(if applicable)
YEAR(s)
FIELD OF STUDY
Meredith College, Raleigh, NC
B.S.
1975
Biology
North Carolina State University, Raleigh, NC
M.S
1982
Physiology, Statistics
North Carolina State University, Raleigh, NC
Ph.D
1988
Biochemistry
RESEARCH AND/OR PROFESSIONAL EXPERIENCE Concluding with present position, list, in chronological orde.r previous employment, experience, and
honors Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications dunng the past three years and to representative earlier publications pertinent to this application If the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant
application)
Professional Experience:
1994 - Present Research Biologist, Endocrinology Branch, RTD, NHEERL, ORD, U S EPA
1975 - Sept. 1994 Research Biologist, Reproductive Toxicology Branch, DTD, ORD, U S EPA
Professional Societies
NC Society of Toxicology
Triangle Consortium of Reproductive Biology
Honors. Awards: (selected from last 5 years)
1999 Recipient, S Award for Contributions to the Agency's Endocrine Disruptor Screening Program
2000 Recipient, S Award for Contributions to the Agency's Endocrine Disruptor Screening Program
2000 Recipient, S Award for Contributions to the Agency's Special Review of Chlorotriazines
2000 Recipient, Team Award for Technical Assistance to the Office of Prevention, Pesticides and Toxic.Substances
(OPPTS)
2001 Recipient, Team Award for Exceptional/Outstanding ORD Technical Assistance to the Regions or Programs Offices
Professional Activities (selected from last 5 years)
Member, Triangle Consortium for Reproductive Biology, 1993 - Present
Member, RTD Performance Recognition Committee, NHEERL, 1998 - Dec 2000
Member, NHEERL-RTP Quality Assurance Committee, 1998- October 2000
Member, NHEERL SP2K (Goal 3) Committee, 2000
Member, Review Team, QA Contract for Instrumentation Validation, 2000
Member, RTD EDSP Team, 1999 - Present
Reviewer, NIEHS RTI Contract, 2000
Editorial Board, Journal of Toxicology and Environmental Health, 1998 - Present
Invited Presentations (selected from last 5 years)
Biological Actions of Estrogen: Genomic and Non-Genomic Mechanisms March 1999 Workshop. Endocrine Disruption
and Neurotoxicity- Why Toxicologists Should be Concerned About the Actions of Estrogenic Chemicals in the CNS 38th
Annual Meeting of the Society of Toxicology. New Orleans, LA.
"ffects of Atrazine on Female Reproductive Development. November 2000 Toxicology Department Seminar Series, N C.
jtate University, Raleigh, NC
PHS 398 (Rev 4/98) (Form Page 6) Page FF
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Identifying EDCs with Estrogenic Activity. May 2001. ORD/Regional Workshop on Endocrine Disruptors, Region 4, Atlanta,
GA.
Publications: (Of 38 publications (13 first-authored and 2 co-authored book chapters)
Gray LE Jr, Kelce WR, Wiese T, Tyl R, Gaido K, Cook J, Klinefelter G, Desaulmers D, Wilson Zacharewski T, Waller C,
Foster P, Laskey J, Reel J, Giesy J, Laws S, McLachlan J, Breslin W, Cooper R, Di Giulio R, Johnson R, Purdy R,
Mihaich E, Safe S, Colborn T. 1997. Endocrine Screening Methods Workshop report detection of estrogenic and
androgenic hormonal and antihormonal activity for chemicals that act via receptor or steroidogenic enzyme
mechanisms Reprod Toxicol 11 (5) 719-750.
Laws SC, Goldman JM, Cooper RL 1997. Assessing the endocrine disrupting potential of environmental chemicals.
Biotechnology International. 1:208-215
Miller DB, All SF, O'Callaghan JP, Laws SC. 1998 The impact of gender and estrogen on striatal dopaminergic
neurotoxicity. Ann N Y Acad Sci. 844 153-16
Stoker TE, Robinette CL, Britt BH, Laws SC, Cooper RL. 1999 Prepubertal exposure to compounds that increase
prolactin secretion in the male rat: effects on the adult prostate Biol Reprod 61(6) 1636-1643
Laws SC, Carey SA, Ferrell JM, Bodman GJ, Cooper RL 2000. Estrogenic activity of octylphenol, nonylphenol,
bisphenol A and methoxychlor in rats Toxicol Sci. 54(1) 154-1667
Goldman JM, Laws SC, Balchak SK, Cooper RL, Kavlock RJ 2000 Endocrine disrupting chemicals prepubertal
exposures and effects on sexual maturation and thyroid activity in the female rat A focus on the EDSTAC
recommendations Crit Rev. Toxicol. 30(2) 135-196
Cummings AM, Laws SC. 2000 Assessment of estrogenicity by using the delayed implanting rat model and examples.
Reprod Toxicol 14(2)111-117
Stoker TE, Laws SC, Guidici DL, Cooper RL 2000. The effect of atrazine on puberty in male wistar rats, an evaluation in
the protocol for the assessment of pubertal development and thyroid function Toxicol Sci 58(1):50-59.
Laws SC, Ferrell JM, Stoker TE, Schmid J, Cooper RL 2000 The effects of atrazine on female wistar rats* an
evaluation
of the protocol for assessing pubertal development and thyroid function. Toxicol Sci 58(2)'366-376
Gray LE, Jr., Ostby J, Wilson V, Lambright C, Bobseine K, Hartig P, Hotchkiss A, Wolf C, Furr J, Price M , Parks L,
Cooper R, Stoker T, Laws S, SJ Degitz , KM Jensen , MD Kahl, JJ Korte, EA Makynen, JE Tietge and G Ankley
2001 Xenoendorcrine Disrupters Tiered Screening and Testing- Filling Key Data Gaps Submitted Toxicological
Letters
Reports to Program Offices:
Cooper RL, Cummings AM, Laws SC, Narotsky M, and Stoker TE 2000. A summary of research related to the effects of
atrazine on reproductive function in the rat Report to Office of Prevention, Pesticides and Toxic Substances, US EPA,
Washington, DC
Laws SC, Stoker TE, Gray LE, Goldman J, Kavlock RJ, Cooper RL 2000. Study Summary and Review: Assessment of
Pubertal Development and Thyroid Function in Juvenile Male and Female Rats Study conducted by Therlmmune
Research Corporation Report to Office of Prevention, Pesticides and Toxic Substances, US EPA, Washington, DC
PHS 398 (Rev 4/98) (Form Page 6) Page FF
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BIOGRAPHICAL SKETCH
Drovide the following information tor the key personnel in the order listed on Form Page 2
hotocopy this page or lollow this (ormat (or each person
NAME
Edward J Massaro
POSITION TITLE
Senior Scientist, Senior Science Advisor
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training)
INSTITUTION AND LOCATION
DEGREE
(il applicable)
YEAR(s)
FIELD OF STUDY
Rutgers University , New Brunswick, Newark, NJ
B A
1955
Biology, Chemistry
University of Texas, Austin, TX
MA
1958
Biochemistry, Cell
Biology
University of Texas, Austin, TX
University of Texas Medical Branch, Galveston, TX
Ph D
1962
Anatomy, Biochemistry,
Physiology
RESEARCH AND/OR PROFESSIONAL EXPERIENCE Concluding with present position, list, in chronological orde.r previous employment, expenence, and
honors Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications dunng the past three years and to representative earlier publications pertinent to this application If the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant
application)
Professional Experience:
1983 - Present USEPA/NHEERL/RTD/DBB
1983 -1985 Director, Inhalation Toxicology Division, USEPA National Health and Environmental Effects
Laboratory, Research Triangle Park, NC
1982 -1986 Senior Research Associate, Institute for the Study of Human Development, The Pennsylvania
State University, University Park, PA
978 -1986 Director, Center for Air Environment Studies and Professor of Toxicology, Department of
Veterinary Science, The Pennsylvania State University, University Park, PA
1978 -1979 Director of Toxicology and Chemical Carcinogenesis, Mason Research Institute,
Worcester, MA 1975 - 1977 Professor, Biochemistry, SUNVAB
1973 - 1977 Director of Biological Research, New York State Nuclear Reactor Facility, SUNYAB
1972 - 1974 Interim Chairman, Department of Biochemistry, SUNYAB
1971 - 1975 Associate Professor, Biochemistry, SUNYAB
Professional Activities (selected from last 5 years)
It is to be noted that the Professional Activities noted below are accomplished predominately on my personal time.
Journal: Cell Biochemistry and Biophysics Humana Press, Totowa, NJ 1995-
Book Series Methods in Toxicology. CRC Press, Boca Raton, Fl 1995-
Book:
Handbook of Human Toxicology First Edition. CRC Press, Boca Raton, Fl 1992-
(Second Edition in preparation)
Handbook of Neurotoxicology (2 volumes) Humana Press, Totowa, NJ In press
Handbook of Copper Pharmacology and Toxicology Humana Press, Totowa, NJ In press
Editor
Boole.
Folate in Health and Disease Humana Press, Totowa, NJ (with John M Rogers). In press
Skeletal Development. Biochemical, Genetic, and Molecular Biologic Mechanisms,
with John M Rogers) Humana Press, Totowa, NJ In preparation
PHS 398 (Rev 4/98) (Form Page 6) Page
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Editorial Advisory Board'
Book Series:
Advances in Modern Nutrition. CRC Press, Boca Raton, FL 1993-.
Invited Presentations (selected from last 5 years)
It is to be noted that this Professional Activitiv was undertaken on my personal time
Invited Discussant: International Symposium on Programmed Cell Death, Shanghai, China, September 8-12, 1996
Publications: (selected from last 5 years)
Massaro, E. J. Tissue Uptake and Subcellular Distribution of Mercury In Handbook of Human Toxicology (Massaro,
E J., ed.). CRC Press, Boca Raton, FL. pp 285-301, 1997
PHS 398 (Rev 4/98) (Form Page 6) Page
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FF
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BIOGRAPHICAL SKETCH
"Vovide the following information for the key personnel in the order listed on Form Page 2
hotocopy this page or follow this format tor each person
NAME
POSITION TITLE
M Leonard Mole
Research Chemist
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training)
INSTITUTION AND LOCATION
DEGREE
(if applicable)
YEAR(s)
FIELD OF STUDY
Berry College, Rome, GA
Clemson University, Clemson, SC
Clemson University, Clemson, SC
Dept of Chemistry, University of Oklahoma, Norman, OK
A.B
MS
Ph D
Postdoc
1963
1966
1971
1971-1972
Chemistry & Math
Organic Chemistry
Organic Chemistry
Natural Products
Isolation/ID
honors Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications during the past three years and to representative earlier publications pertinent to this application If the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant
application)
Professional Experience:
1992-Present Research Chemist, Developmental Biology Branch, RTD.NHEERL, USEPA, RTP, NC
1987-1992 Research Chemist, Reproductive Toxicology Branch, DTD, HERL, USEPA, RTP, NC
1984-1987 Research Chemist, Perinatal Toxicology Branch, DBD, HERL, USEPA, RTP, NC
1979-1984 Research Chemist, Toxicology Branch, ITD, HERL, USEPA, RTP, NC.
1974-1979 Research Chemist, Biochemistry Branch, ETD, HERL, USEPA, RTP, NC.
ep. 1974 Scientific Collaborator, Organisch Chemisch Laboratorium, Der Rijksuniversiteit te Utrecht, Netherland.
.972 -1974 Investigator/Research Associate, Research Institute of Pharmaceutical Sciences, University of
Mississippi, Oxford, MS.
1971-1972 Visiting Asst Professor, Chemistry Dept., University of Oklahoma, Norman, OK
1964-1971 Grad Student/Teaching Asst, Chemistry Department, Clemson University, Clemson, SC
Summer 1965 Chemist, Deenng Milliken Research Corporation, Spartanburg, SC
1963-1964 Chemist, US Air Force Civil Service, WRAMA, Warner Robins, GA.
Professional Societies
American Chemical Society, Sigma Xi, N.C. Society of Toxicology, Kappa Psi Pharmaceutical Fraternity,
American Society of Pharmacognosy
Honors. Awards: (selected from last 5 years)
2000 Two EPA Special Act or Service Awards
1999 Two EPA Special Act or Service Awards
1999 EPA S-Award
1998 EPA Scientific and Technological Achievment Award,
1998 EPA Performance award (Honorable Mention)
Publications: (selected from last 5 years)
Holmes, M., Suarez, J.D , Roberts, N.L , Mole, M.L., Murr, A.S , and Klinefelter, G R.. Dibromoacetic Acid, a Prevalent
By-Product of Drinking Water Disinfection, Compromises the Synthesis of Specific Seminiferous Tubule Proteins
Following both In Vivo and In Vitro Exposures Submitted.
"<5tzer, R.W., Lau, C., Mole, M.L., Copeland, F.M , Rogers, J.M., and Kavlock, R.J.: Toward a biologically based dose-
;sponse model for developmental toxicity of 5-fluorouracil in the rat A mathematical construct. Toxicological
Science, 53 49-58, 2001.
PHS 398 (Rev 4/98) (Form Page 6) Page
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Lau, C , Mole, M.L., Copeland, M F., Rogers, J.M., Kavlock, R.J., Shuey, D.L., Cameron, A.M., Ellis, D H , Merriman, J.,
and Setzer, R.W • Toward a biologically based dose-response model for developmental toxicity of 5-fluorouracil in the
rat: Acquisition of experimental data. Toxicological Science, 59- 37-48, 2001
Balchak, S K., Hedge, J.M., Shearin, A.E., Mole, M.L., and Goldman, J M Influence of the drinking water disinfection
by-product dibromoacetic acid on rat estrous cyclicity and ovarian follicular steroid release in vitro Reproductive
Toxicology, 14" 533-539, 2000.
Mole, M.L., Hunter, D.L., Gao, P., Lau, C Sample preparation and high performance liquid chromatographic analysis of
deoxyribonucleoside triphosphates in individual rat embryos Analytical Biochemistry, 259" 245-252, 1998
Elstein, K H , Mole, M.L., Setzer, R.W., Zucker, R M., Kavlock, R.J., Rogers, J.M., and Lau, C.- Nucleoside-mediated
mitigation of 5-FU-induced toxicity in synchronized MELC Toxicology and Applied Pharamcology, 146 29-39, 1997.
Rogers, J.M and Mole, M.L.- Critical periods of sensitivity to the developmental toxicity of inhaled methanol in the CD-1
mouse. Teratology, £5 364-372, 1997
PHS 398 (Rev 4/98) (Form Page 6) Page
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image:
BIOGRAPHICAL SKETCH
"•rovide the following information for me key personnel in the order listed on Form Page 2
'hotocopy this page or follow this format lor each person
NAME
POSITION TITLE
Rebecca A. Morris
Biologist
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training)
INSTITUTION AND LOCATION
DEGREE
(it applicable)
YEAR(s)
FIELD OF STUDY
King College, Bristol, TN
Univ of S Carolina School of Medicine, Columbia, SC
U S EPA NHEERL, Raleigh, NC
BS
Ph.D.
Post Doc
1995
2000
Present
Molecular Biology
Biomedical Sciences
Reproductive Toxicology
RESEARCH AND/OR PROFESSIONAL EXPERIENCE Concluding with present position, list, in chronological orde.r previous employment experience, and
honors Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications during the past three years and to representative earlier publications pertinent to this application If the list ol
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEEDTHREE PAGES (for a modular grant application)
Professional Experience:
1995-2000 Graduate Fellow, University of South Carolina School of Medicine, Columbia, SC.
1996-2000 Medical Biochemistry Teaching Assistant, Columbia, SC
1997 Medical Microanatomy Laboratory Assistant, Columbia, SC
2001-Present U.S EPA NHEERL Postdoctoral Fellow, Reproductive Toxicology Division, RTP, NC
Professional Societies
American Society of Cell Biology
American Society of Andrology
ociety for the Study of Reproduction
dociety of Toxicology (North Carolina Chapter)
Honors. Awards: (selected from last 5 years)
Dean C Barnhart Excellence in Research Award (University of South Carolina School of Medicine, 1998)
Invited Presentations (selected from last 5 years)
Publications: (selected from last 5 years)
Kimple RJ, De Vries L, Tronchere H, Behe CI, Morris RA, Gist Farquhar M, Sidero\ski DP RGS12 and RGS14 GoLoco
motifs are G alpha(i) interaction sites with guanine nucleotide dissociation inhibitor activity Journal of
Biological Chemistry. 276(31) 29275-29281. 2001.
Lough J, Jackson M, Morris R, Moyer R. Bisulfite-induced cytosine deamination rates in E.coli SSB:DNA complexes.
Mutation Research.478 191-197,2001
Boockfor, F R , R.A. Morris, D.C. DeSimone, D.M. Hunt, and K.B Walsh. Sertoli cell expression of the cystic fibrosis
transmembrane conductance regulator American Journal of Physiology (Cell Physiology 43). C922-C930, 1998
PHS 398 (Rev 4/98) (Form Page 6) Page 1
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BIOGRAPHICAL SKETCH
Provide the following information tor the key personnel in the order listed on Form Page 2
'ftotocopy this page or follow this tormal tor each person
NAME
POSITION TITLE
Michael G Narotsky
Toxicologist
EDUCATION/TRAINING (Begm with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training)
INSTITUTION AND LOCATION
DEGREE
YEAR
FIELD OF STUDY
State University of New York at Buffalo
North Carolina State University, College of Veterinary
Medicine, Raleigh, NC
B.A
Ph.D
1978
1997
Biology & Psychology
Veterinary Medical Sciences
Major Pharmacology
Minor Toxicology
Professional Experience:
1979-1986 Senior Technician, Reproductive Toxicology, Ayerst Laboratories, Chazy, NY
1986-1987 Staff Scientist/Teratologist, Reproductive Health Division, Dynamac Corporation, Rockville, MD
1987-1994 Senior Scientist, Perinatal Toxicology, ManTech Environmental Technology, Inc , RTP, NC
1995- Present Toxicologist, Endocrinology Branch, Reproductive Toxicology Division, USEPA, RTP, NC
Professional Societies
Teratology Society
North Carolina Society of Toxicology
Triangle Consortium of Reproductive Biology
Honor Society of Phi Kappa Phi
Professional Activities (selected from last 5 years)
Adjunct Professor, University of North Carolina at Chapel Hill, School of Public Health
Air Toxics Research Strategy Implementation Team, EPA
Contaminant Candidate List Research Needs Team, EPA
Education Committee, Teratology Society - 2001
Ad hoc reviewer: Teratology, Reproductive Toxicology, Biomedical and Environmental Sciences
Invited Presentations (selected from last 5 years)
Effects of boric acid on the developing axial skeleton in the rat In. The Status of Current Knowledge on Biochemical,
Genetic, and Molecular Mechanisms of Skeletal Development Symposium/Workshop. Durham, NC (2001)
Effects of chlorotriazines and trihalomethaneson pregnancy maintenance in rats In Seminar Senes, Dept of Environmental
& Molecular Toxicology. North Carolina State University, Raleigh, NC (2001).
Effects of disinfection by-products on reproduction and development In Dnnking Water Disinfection By-Products Fall
Meeting of the North Carolina Chapter of the Society of Toxicology, Chapel Hill, NC (2001)
Trihalomethane-induced pregnancy loss in rats In Developmental Risks of Disinfectant By-Products Symposium, T eratology
Society, Palm Beach, FL (2000)
Publications (selected from last 5 years)
Narotsky, M.G., Best, D S , Guidici, D.L., and Cooper, R L (2001) Strain comparisons of atrazine-induced pregnancy loss
in the rat Reprod. Toxicol. 15.61-69
Bielmeier, SR, Best, D.S , Guidici, D.L., and Narotsky, M.G. (2001) Pregnancy loss in the rat caused by
bromodichloromethane Toxicol Sci 59.309-315
Klinefelter, G R., Hunter, E.S III, and Narotsky, M.G. (in press) Reproductive and developmental toxicity associated with
disinfection by-products of drinking water In The Safety of Water Disinfection: Balancing Chemical and Microbial Risks.
ILSI Press, Washington, DC
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Narotsky, M.G. and Rogers, J.M. (2000) Examination of the axial skeleton of fetal rodents Methods Mol Biol. 135.139-
150. In. Developmental Biology Protocols Ed. R Tuan. Vol 1, Chap 14 Humana Press, Totowa, NJ.
Peters, J.M., Narotsky, M.G., Elizondo, G., Fernandez-Salguero, P.M., Gonzalez, F.J., and Abbott, B A. (M
Amelioration of TCDD-induced teratogenesis in aryl hydrocarbon receptor (AhR)-null mice Toxicol. Sci 47.86-92.
Narotsky, M.G., Schmid, J.E., Andrews, J.E., and Kavlock, R.J (1998) Effects of boric acid on skeletal development in
rats Biol. Trace Element Res 66:373-394.
Kempinas, W.D., Suarez, J.D., Roberts, N.L., Strader, L.F , Ferrell, J , Goldman, J.M., Narotsky, M.G., Perreault, S.D.,
Evenson, D P , Ricker, D.D., and Klinefelter, G.R (1998) Fertility of rat epididymal sperm after chemically and surgically
induced sympathectomy Biol. Reprod 59(4) 897-904
Narotsky, M.G., Brownie, C.F., and Kavlock, R.J (1997) Critical period of carbon tetrachloride-induced pregnancy loss in
Fischer-344 rats, with insights into the detection of resorption sites by ammonium sulfide staining Teratology
56(4):252-261
Narotsky, M.G., Pegram, R A , and Kavlock, R.J (1997) Effect of dosing vehicle on the developmental toxicity of
bromodichloromethane and carbon tetrachloride in rats Fundam Appl Toxicol 40(1)30-36
PHS 398 (Rev 4/98) (Form Page 6) Page
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BIOGRAPHICAL SKETCH
"Yovide the following information for the key personnel in the order listed on Form Page 2
hotocopy this page or follow this format tor each person
NAME
POSITION TITLE
John C. Rockett
Research Biologist
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training )
INSTITUTION AND LOCATION
DEGREE
(it applicable)
YEAR(s)
FIELD OF STUDY
University of Warwick, Coventry, W. Midlands
University of Warwick, Coventry, W Midlands
B.S.(Hons)
Ph D
1987-1991
1991-1995
Microbiology & Microbial
Technology
Immunology of
Oesophageal Cancer
RESEARCH AND/OR PROFESSIONAL EXPERIENCE Concluding with present position, list, in chronological orde.r previous employment, expenence, and
honors Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications dunng the past three years and to representative earlier publications pertinent to this application II the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant application)
Professional Experience:
12/00 - Present Research Biologist, Gamete and Early Embryo Biology Branch, Reproductive Toxicology Division,
National Health and Environmental Effects Research Laboratory, USEPA, RTP, NC
8/98 -12/00 NHEERL Post-Doctoral Research Fellow, Gamete and Early Embryo Biology Branch, Reproductive
Toxicology Division, National Health and Environmental Effects Research Laboratory, USEPA,
RTP,NC
5/95 -7/98" Rhone-Poulenc Post-Doctoral Research Fellow, Molecular Toxicology Group, School of Biological
Sciences, University of Surrey, Guildford, Surrey, England.
rofessional Societies
Society of Toxicology (Application under consideration)
Science Advisory Board (2001 -present)
North Carolina Chapter of the Society of Toxicology (1999-present)
British Toxicology Society (1996 - 2000)
Institute of Biology (U.K.) (1989 - present) - C.Biol. and M.I Biol designate
Biochemical Society (U.K.) (1992-1995)
British Society for Immunology (1992-1995)
Professional Activities (selected from last 5 years)
Charter member of Fertility and Early Pregnancy Work Group of Children's Longitudinal Cohort Study
US EPA (NHEERL) representative on International Life Sciences Institute (ILSI) subcommittee on Application of Genomics
and Proteomics to Mechanism-Based Risk Assessment
RTD alternate on NHEERL Molecular Profiling Group Steering Committee (2001-present)
Ad Hoc grant application reviewer lor four grant awarding bodies
Ad Hoc reviewer for three journals
Session Chair, Atlantic Symposium on Computational Biology and Genome Information Systems & Technology, Duke
University, March 17, 2001, Durham, NC, USA.
Member of Scientific Committee for international meetings, Critical Assessment of Techniques for Microarray Data Analysis
(CAMDA 00 and 01), Duke University, Durham, NC.
Consultant for Front Line Strategic Management Consulting, Inc (Foster City, CA) in DNA Technology Concept Testing
Program, 2/7/00
Triangle Array Users Group (TAUG) member
NHEERL Molecular Profiling Group (NMPG) member
EPA Microarray Consortium (EPAMAC) member
'ebmaster for EPAMAC website (http.//www.epa.gov/nheerl/epamac/).
Invited Presentations (selected from last 5 years)
PHS 398 (Rev 4/98) (Form Page 6) Page
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John C. Rockett and David J. Dix. "Gene arrays for elucidating mechanistic data from models of male infertility and
chemical exposure in mice, rats and humans" Platform presentation at the symposium "Defining the cellular and
molecular mechanisms of toxicant action in the testis". Society of Toxicology 41s' Annual Meeting, March 17th-21
2002, Nashville, TN, USA
John C Rockett, Sigmund Degitz, Suzanne E Fenton, Leroy Folmar, Michael J. Hemmer, Joe E Tietge, and David J.
Dix "Use of DNA Arrays in Environmental Toxicology". Platform presentation at the 4'h Annual Lab-on-a-Chip and
Microarrays for Post-Genomic Applications meeting, January 14,h-16'h, 2002, Zurich, Switzerland.
John C. Rockett "DNA Arrays" Seminar at EPA Molecular Biology Course, USEPA, RTP, April 8th, 1999
John C Rockett. "Contract Services for Array Applications". Seminar at the Triangle Array Users Group Meeting,
CUT, RTP, May 1s', 1999
Publications: (selected from last 5 years)
John C. Rockett Genomic and Proteomic Techniques Applied to Reproductive Biology Genome Biology (accepted)
John C. Rockett. Chipping away at the mystery of drug responses The Pharmacogenomics Journal (accepted)
John C. Rockett and David J Dix. Gene Expression Networks, in Encyclopaedia of the Human Genome, Nature
Publishing Group (accepted)
John C Rockett, Faye L Mapp, J. Brian Garges, J Christopher Luft, Chisato Mori and David J. Dix (2001) The
effects of hyperthermia on gene expression, apoptosis, spermatogenesis and fertility in adult male mice Biology of
Reproduction, 65,229-239
John C. Rockett, J. Christopher Luft, J Brian Garges, Stephen A. Krawetz, Mark R Hughes, Kwan Hee Kim, Asa
Oudes, and David J. Dix (2001) Development of a 950-gene DNA array for examining gene expression patterns in
mouse testis Genome Biology 2(4) research0014.1-0014.9 fhttp //aenomebioloav.com/2001/2/4/research/0014)
JC Rockett, DJ Esdaile and GG Gibson (2000). Use of a suppression-PCR subtractive hybridisation method to
identify
gene species which demonstrate altered expression in male rat and guinea pig livers following 3-day exposure to [4-
chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid Toxicology, 144(1-3); 13-29
John C. Rockett and David J Dix. (2000). DNA Arrays. Technology, options and toxicological applications
Xenobiotica, 30(2);155-177. (A review article)
Rockett, John C and Dix, David J. (1999). U.S EPA workshop' Application of DNA arrays to Toxicology.
Environmental Health Perspectives, 107(8):681-685 (A meeting report)
John C. Rockett, David J Esdaile and G Gordon Gibson (1999). Differential gene expression in drug metabolism:
practicalities, problems and potential. Xenobiotica, 29(7).655-691. (A review article)
MC Murphy, CN Brookes, JC Rockett, C Chapman, JA Lovegrove, BJ Gould, JW Wright and CM Williams (1999).
The quantitaion of lipoprotein lipase mRNA in biopsies of human adipose tissue, using the polymerase chain reaction,
and the effect of increased consumption of n-3 polyunsaturated fatty acids European Journal of Clinical Nutrition
53 441-447
JC Rockett, DJ Esdaile and GG Gibson (1997). Molecular profiling of non-genotoxic carcinogenesis using differential
display reverse transcription polymerase chain reaction (ddRT-PCR,). European Journal of Drug Metabolism &
Pharmacokinetics 22(4).329-333.
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BIOGRAPHICAL SKETCH
Provide the following information for the key personnel in the order listed on Form Page 2
"hotocopy this page or follow this format tor each person
¦JAME
POSITION TITLE
John M Rogers, Ph.D
Chief, Developmental Biology Branch, NHEERL, EPA
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training)
INSTITUTION AND LOCATION
DEGREE
(if applicable)
YEAR(s)
FIELD OF STUDY
University of Miami, Coral Gables, FL
BS
1976
Biology
University of Miami, Coral Gables, FL
M S
1979
Biology
University of Miami, Coral Gables, FL
Ph.D.
1982
Biology
University of California, Davis, CA
Postdoctoral
1984
Nutrition
RESEARCH AND/OR PROFESSIONAL EXPERIENCE Concluding with present position, list, in chronological orde.r previous employment, expenence, and
honors Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications dunng the past three years and to representative earlier publications pertinent to this application If the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant
application)
Professional Experience:
Sept 1976-May 1982
May 1982 - July 1982
Nov 1984 - Dec 1988
Spring, 1987, Summer,
1990
Jan 1989-Mar. 1991
larch 1991 - Present
Professional Societies
Teratology Society, Society of Toxicology, Society for Developmental Biology, Society for Experimental Biology and
Medicine
1997 - 2000 Secretary/Treasurer, SOT Repro./Devel. Tox. Specialty Section
1998 - Present Treasurer, Teratology Society
Honors. Awards:
EPA Scientific and Technical Achievement Awards Agency-wide competition for best papers, Levels l-lll ~ Level I is
the highest EPA award for a published paper 1991 Level II, Level III; 1992 Level II; 1993 Level III (2); 1994 Level I,
1996, Level III; 1998, Honorable Mention
Professional Activities (last 5 years)
Editorial Board, J. Toxicol Environ. Health, 1992-99
Teratology Society Strategic Planning Committee, 1997-present
External Peer Reviewer (Final Reports), Health Effects Institute, Methanol Initiative Grants Program, 1998.
External Peer Reviewer, FDA Office of Women's Health 1998 Intramural Funding Program
Chair, SOT Continuing Education Course: "Current Developmental Biology Relevance and Application for
Developmental Toxicology " Seattle, WA, Society ol Toxicology, March 1, 1998
International Advisory Committee, International Conference on Environmental Contamination, Toxicology and Health,
Hong Kong Baptist University, Hong Kong, September 23-25, 1998
rganizer, North Carolina Alliance for Developmental Biology and Birth Defects Meeting, Research Triangle Park,
November 6, 1998.
Graduate Teaching Assistant, Biology Dept, University of Miami
Lecturer in Biology, Biology Dept, University of Miami
Research Biologist, Perinatal Toxicology Branch, Developmental Biology Division,
HERL, U.S. EPA
Visiting Assistant Professor, Department of
Zoology, North Carolina State University, Raleigh, NC
Chief, Experimental Teratology Section, Perinatal Toxicology Branch, Developmental
Toxicology Division, Health Effects Research Laboratory, U.S EPA
Chief, Developmental Biology Branch, Reproductive Toxicology Division, NHEERL, U S
EPA
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Member, Expert Registry, NTP Center for Evaluation of Risks to Human Reproduction 1998-present
Invited Participant, CDC/ATSDR Workshop on Children's Health Issues, Green Bay, Wl, August 3-4, 1999.
Organizer, Workshop and Symposium on Current Knowledge of Folate in Human Development, Durham, NC, Octobe'
17-19, 1999
Invited Participant, Chemical Manufacturer's Association Workshop on Reproductive and Developmental Toxicology
Research Needs, Arlington, VA, February 22-23, 2000
Member, Expert Panel, Review of FETAX (Frog Embryo Teratogenesis Assay -Xenopus), National Institute of
Environmental Health Sciences, Research Triangle Park, April-May, 2000i
Member, ILSI Health and Environmental Sciences Institute's Working Group on Interpretation of Skeletal Variations for
Human Health Risk Assessment 1999-present
Member, Center for the Evaluation of Risks to Human Reproduction, Panel on Methanol, 2000-present
Invited Presentations: (last 5 years)
Enzymes In' Experimental Approaches for Studying Mechanisms of Abnormal Development Continuing Education
Course, Teratology Society, Palm Beach, FL, June 21, 1997
Disruption of Embryonal Pattern Formation by Methanol, Department of Toxicology, North Carolina State University,
Raleigh, NC, January 27, '998
The Developmental Toxicity of Methanol Basic and Applied Research, Toxicology and Molecular Biology Branch,
National Institute of Occupational Safety and Health, Morgantown, WV, March 9, 1998
Elucidating Mechanisms of Developmental Toxicity for Model and Environmental Toxicants 5-Fluorouracil and
Methanol Department of Biological Sciences, Florida Institute of Technology, Melbourne, FL, April 23, 1998.
Epigenetics in Developmental Toxicology Toxicant-Induced Phenocopies and Developmental Gene Expression
Genotoxicity and Environmental Mutagenesis Society, Research Triangle Park, NC, May 1, 1998.
Developmental Control Genes Cross-Species Homology and Effects of Maternal Toxicant Exposure Office of Research
and Development, U.S. EPA, Washington, DC, August 12, 1998
Developmental Toxicology. Overview and Relevance to Children's Health Issues CDC/ATSDR Workshop on Children''
Health Issues, Green Bay, Wl, August 3-4, 1999.
Toxicant-Induced Fetal Skeletal Phenocopies and Associated Alterations in Gene Expression Midwest Teratology
Association Meeting, Lima, OH, May 13-14, 1999
Chemically-Induced Fetal Skeletal Phenocopies and Alterations in Developmental Gene Expression. NIH Research
Festival, Minisymposium on Craniofacial and Skeletal Biology and Disorders, Bethesda, MD, October 6th, 1999.
Mechanisms of Development and Thresholds of Response University Integrated Toxicology Program Fall Symposium,
Duke University, Durham, NC, Novembers, 1999
Development of Biologically Based Dose-Response Models for Developmental Toxicity Human Biology Models for
Environmental Health Effects Virtual Body Workshop, National Institute of Environmental Health Sciences,
Research Triangle Park, NC, June 22, 2000
Reproductive Hazards in the Workplace. Physicians Assistant Certificate Program in Occupational and Environmental
Medicine. Duke University Medical Center. July 20, 2000.
Spatiotemporal Patterns of Gene Expression Developmental Stage-Related Susceptibility Continuing Education course
entitled "Principles of Teratology. Application of New Technologies Towards Understanding Abnormal Development
2001 Teratology Society Annual Meeting, Montreal, Quebec, Canada. June 24, 2001.
Publications: (last 5 years)
Connelly, L E , and J.M. Rogers. (1997) Methanol causes posteriorization of cervical vertebrae in mice Teratology
55.138-144
Rogers, J.M., and M.L Mole (1997) Critical periods of sensitivity to the developmental toxicity of inhaled methanol in
the CD-1 mouse Teratology, 55 364-372
Elstein, K H., M L. Mole, R W. Setzer, R.M. Zucker, R J Kavlock, J.M. Rogers, and C.L. Lau (1997) Nucleoside-
mediated mitigation of 5-fluorouracil-induced toxicity in synchronized murine erythroleukemic cells. Toxicol. Appl.
Pharmacol. 146 29-39.
Keen, C L , M W Taubeneck, S. Zidenberg-Cherr, G P Daston, and J.M. Rogers (1998) Toxicant exposure and tract,
element metabolism in pregnancy Environ Toxicol Pharmacol 4 301-308.
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Zucker, R.M., S. Hunter, and J.M. Rogers (1998) Confocal laser scanning microscopy of apoptosis in organogenesis-
stage mouse embryos Cytometry 33:348-354
Tucker, R.M., S Hunter, and J.M. Rogers (1999) Apoptosis and morphology in mouse embryos by confocal laser
scanning microscopy. Methods 18'473-80
Jankowski-Hennig, M.A., M.S Clegg, G.P. Daston, J.M. Rogers, and C L. Keen (2000) Zinc-deficient rat embryos have
increased caspase 3-like activity and apoptosis Biochem Biophys Res Comm 271.250-256
Narotsky, M G., and J.M. Rogers (2000) Examination of the axial skeleton of fetal rodents. Methods Mol. Bio! 135139-
150
Zucker, R M., and J.M. Rogers (2000) Embryo/fetal topographical analysis by fluorescence microscopy and confocal
laser scanning microscopy. Methods Mol Biol 135.203-209
Zucker, R.M., E.S Hunter III, and J.M. Rogers (2000) Confocal laser scanning microscopy of morphology and apoptosis
in organogenesis-stage mouse embryos. Methods Mol Biol. 135 191-202
Lau, C , Andersen, M.E., Crawford-Brown, D J , Kavlock, R.J., Kimmel, C A , Knudsen, T.B , Muneoka, K„ Rogers,
J.M.., Setzer, R W., Smith, G , and Tyl, R (2000) Evaluation of biologically based dose response modeling for
developmental toxicity. A workshop report Regul Toxicol. Pharmacol. 31.190-199
Lau C, Mole ML, Copeland MF, Rogers JM, Kavlock RJ, Shuey DL, Cameron AM, Ellis DH, Logsdon TR, Mernman J,
Setzer RW. (2001) Toward a biologically based dose-response model for developmental toxicity of 5-fluorouracil in
the rat acquisition of experimental data Toxicol Sci 59.37-48.
Setzer R W , C Lau, M L Mole, M.F Copeland, J.M. Rogers, and R J Kavlock (2001) Toward a biologically based
dose-response model for developmental toxicity of 5-fluorouracil in the rat a mathematical construct Toxicol Sci.
59 49-58
Rogers, J.M., and R.J Kavlock (2001) Developmental toxicology. In: Casaretl and Doull's Toxicology. Sixth Ed.,C D
Klaassen, ed McGraw-Hill, NewYork, pp.351-386
Rogers, J.M., and E.S. Hunter, III (2001) Toxicological highlight. Redox redux A closer look at conceptal low molecular
weight thiols. Tox. Sci. 62:1-3.
Huang, Y.S., J.E Andrews, and J.M. Rogers (2001) uC-Methanol incorporation into DNA and specific proteins of
organogenesis stage mouse embryos in vitro. Reprod. Toxicol., In press.
i_eazer, T.M., and J.M. Rogers (2001) Role of the acute phase response and tumor necrosis factor alpha in the
developmental toxicity of lipopolysaccharide in the CD-1 mouse. In press, Reprod. Toxicol
Massaro, E.J., and J.M. Rogers (eds). (2002) Folate in Human Development. Humana Press, Boca Raton, FL (In
press).
PHS 398 (Rev 4/98) (Form Page 6) Page
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BIOGRAPHICAL SKETCH
Provide the following information tor the key personnel in the order listed on Form Page 2
'TQtocopy this page or tollow this tormat tor each person
NAME
POSITION TITLE
Tammy E. Stoker
Research Biologist
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training)
INSTITUTION AND LOCATION
DEGREE
(if applcabie)
YEAR(s)
FIELD OF STUDY
North Carolina State University, Raleigh, NC;
B.S.,
1983-1987
Zoology,Pre-Vet Medicine
North Carolina State University, Raleigh, NC;
MS ,
1990-1993
Toxicology
North Carolina State Unversity College of Veterinary Med
Ph.D
1995-1998
Pharmacology
Professional Experience:
1995-Present Research Biologist, Endocrinology Branch, RTD, NHEERL, ORD EPA
1988-1994 Senior Scientist, Section Supervisor, ManTech Environmental, RTP, NC
Professional Societies
NC Society of Toxicology
Triangle Consortium of Reproductive Biology (TCRB)
Society for the Study of Reproduction
Honors. Awards: (last 5 years)
1999 Best Paper Published by: Toxicological Sciences," Reproductive & Developmental Toxicology Speciality Section"
""000 Recipient, Office of Pesticides Programs Health Effects Division Team Award for work related to the mode of action
of the chlorotriazine herbicides
2001 Recipient, EDSP team award for Exceptional/Outstanding ORD Technical Assistance to the Regions or Program
Offices.
2000 Best Paper Published by Toxicological Sciences,"Reproductive & Developmental Toxicology Speciality Section"
Professional Activities (last 5 years)
External-
Atrazine Workshop Invited participant. Atrazine Research Meeting, Hilton Head, S C , Apr, (1997)
Adhoc reviewer for Biology of Reproduction (2000-present).
Adhoc reviewer for Toxicological Sciences (2000-present)
NIEHS/EPA Breast and Prostate Faculty Group (1999-present)
Triangle Consortium of Reproductive Biology (TCRB) Board Member (2001)
Internal-
Coordinator of RTD Seminar Series, (1995-2000).
Member of RTD EDSP Committee (1999-present)
Invited Presentations (last 5 years)
Atrazine and Prostate inflammation Atrazine Research Meeting, Hilton Head, S C , Apr, (1997)
Lactational expsure to atrazine suppresses suckling-induced prolactin Endocrine Disruptors Workgroup, Apr., (1998)
Neuroendocrine control of ovulation:effects of environmental toxicants Pembroke State University, Biology Dept.,
Pembroke, NC, Sept, (1998)
Alterations in developmental prolactin levels and subsequent prostate inflammation. North Carolina State Univ , Dept of
Toxicology, Raleigh, NC, Oct (1999).
auroendocrine and reproductive effects of pesticides. British Toxicology Society Annual Meeting, York University,
Great
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Britain, March (2000)
Prepubertal prolactin and prostate inflammation. Breast and Prostate Faculty, NIEHS, July (2000)
Atrazine and its metabolites alter puberty in the male rat Endocrine Disruptors Workgroup, Aug (2000).
Publications: (Of 22 manuscript publications (9 first-authored) and 2 coauthored book chapters
Lindzey, J., Wetsel, W.C , Couse, J.F , Stoker, T.E., Cooper, R L., and Korach, K S Effects of castration and chronic
steroid treatments on hypothalamic GnRH content and pituitary gonadotropins in male wild-type and estrogen-a
receptor knockout mice. Endocrinology, 139 4092-4101, 1998
Cooper, R.L., Goldman, J M , Stoker, T.E. Neuroendocrine and reproductive effects of contemporary-use pesticides
Toxicology and Industrial Health 15. 26-36 (1999)
Stoker, T.E., Robinette, C L and Cooper, R.L. (1999) Maternal exposure to atrazine during lactation suppresses
suckling-induced prolactin release and results in prostatitis in the adult offspring Toxicological Sciences
52(1).68-79
Stoker, T.E., Robinette, C L , Britt, B H and Cooper, R.L (1999) Prepubertal exposure to compounds that increase
prolactin in the male rat Effects on adult prostate. Biology of Reproduction 61(6)1636-1643
Stoker, T.E., Robinette, C L and Cooper, R.L (1999) Perinatal exposure to estrogenic compounds and the subsequent
effects on the prostate of the adult rat Evaluation of inflammation in the ventral and lateral lobes Reproductive
Toxicology 13 463-472
Cooper, R.L., Stoker, T.E., Tyrey, L , Goldman, J. M and McElroy, W K (2000) Atrazine disrupts hypothalamic control
of pituitary-ovarian function. Toxicological Sciences 53 297-307.
Stoker, T.E., Parks, L.G., Gray, L.E , and Cooper, R.L , (2000) Endocrine-disrupting chemicals Prepubertal exposures
and effects on sexual maturation and thyroid function in the male rat A focus on the EDSTAC recommendations.
Critical Reviews in Toxicology 30:197-252
Laws, S C , Ferrell, J.M., Stoker, T.E., Schmid, J., and Cooper, R.L (2000). The effect of atrazine on puberty in female
wistar rats, an evaluation in the protocol for the assessment of pubuertal development and thyroid function
Toxicological Sciences 58(2). 366-76
Stoker, T.E., Laws, S.C., Guidici, D., and Cooper, R.L. (2000) The effects of atrazine on puberty and thryroid function
in the male wistar rat An evaluation of a protocol for the assessment of pubertal development and thyroid function
Toxicological Sciences 58' 50-59
Stoker, T.E., Goldman, J.M , and Cooper, R.L. (2001) Delayed ovulation and pregnancy outcome- effect of
environmental toxicants on the neuroendocrine control of the ovary Environmental Toxicology and Pharmacology
9(3):117-129
Stoker, T.E., Guidici, D.L., Laws, S.C and Cooper, R.L. (Submitted, Aug. 2001) The Effects of Atrazine Metabolites on
Puberty and Thyroid Function in the Male Wistar Rat: An Evaluation in the Male Pubertal Protocol. Toxicological
Sciences.
Gray, L.E., Ostby, J., Wilson, V , Lambright, C., Bobseine, K., Hartig, P., Hotchkiss, A., Wolf, C., Furr, J., Price, M.,
Parks, L, Cooper, R., Stoker, T.E., Laws, S , Degitz, S.J., Jensen, K.M , Kahl, M.D., Korte, J.J., Makynen, E A , Tietge,
J.E. and Ankley, G T (In Press, 2001) Xenoendocrine disrupters-tiered screening and testing Filling key data gaps.
Toxicology Letters.
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image:
BIOGRAPHICAL SKETCH
°rovide the following information for the Key personnel in the order listed on Form Page 2
hotocopy this page or follow this format tor each person
NAME
POSITION TITLE
Douglas B. Tully
Biologist
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training )
INSTITUTION AND LOCATION
DEGREE
YEAR(s)
FIELD OF STUDY
(rf applicable)
North Carolina State University, Raleigh, NC
BS
1974
Biological Sciences
University of North Carolina, Chapel Hill, NC
PhD
1990
Biochemistry
RESEARCH AND/OR PROFESSIONAL EXPERIENCE Concluding with present position, list, in chronological order, previous employment, expenence, and
honors Include present membership on any Federal Government public advisory committee list, in chronological order, the titles, all authors, and
complete references to all publications during the past three years and to representalive earlier publications pertinent to this application If the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant
application)
Professional Experience:
2001 -present
2001-
2000-present
1999-2000
1994-1999
1990-1994
Biologist, Gamete and Early Embryo Biology Branch, Reproductive Toxicology Division, National
Health and Environmental Effects Research Laboratory, U S Environmental Protection Agency.
Guest Worker, Gamete and Early Embryo Biology Branch, Reproductive Toxicology Division,
National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection
Agency
Adjunct Assistant Professor, Department of Biology and Health Sciences, Meredith College,
Raleigh, NC.
Staff Assistant to the Director, NIEHS/NTP, Office of the Director, National Institute of
Environmental Health Sciences (NIEHS), Research Triangle Park, NC.
Biologist (Project Officer), Functional Toxicology Group, Environmental Toxicology Program,
NIEHS, Research Triangle Park, NC
Fellow in Medicine, Department of Physiology, University of North Carolina, Chapel Hill, NC.
Professional Societies
American Association for the Advancement of Science (1979-present)
American Society for Biochemistry and Molecular Biology (1990-present)
Endocrine Society (1991-present)
New York Academy of Science (1991-present)
North Carolina Chapter of the Society of Toxicology (1994-present)
Honors. Awards: (selected from last 5 years)
1994-1995 Intramural Research Training Award, National Institute of Environmental Health Sciences,
Research Triangle Park, NC
Professional Activities (selected from last 5 years)
1995-present Ad hoc reviewer, Biochimica et Biophysica Acta, Environmental Health Perspectives,
Mutagenesis
Invited Presentations (selected from last 5 years)
Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, US EPA. Aug 6,
2001 "Using gene expression to assess chemical toxicity"
^productive Toxicology Section, National Institute of Environmental Health Sciences, Feb 25, 1999. "New biomarkers
i toxicity for four high priority heavy metal pollutants"
Reproductive Toxicology Section, NIEHS, Feb 19, 1998 "Six high priority organochlorine pesticides, either singly or in
PHS 398 (Rev 4/98) (Form Page 6) Page
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FF
image:
combination are nonestrogenic in transfected HeLa cells"
Laboratory of Toxicology and Functional Toxicology Faculty, NIEHS Aug 7, 1997 Testing of heavy metal pollutants in
Xenometrix CAT-Tox(L) assays."
Fourth Annual Research Symposium of the Institute of Nutrition of the University of North Carolina, Oct 11,1993.
"Vitamin B6 modulation of steroid hormone regulated gene expression in human breast cancer cell lines"
Receptor Mechanisms Discussion Group, NIEHS, RTP, NC, Aug 3, 1993 "Role of vitamin B6 in glucocorticoid hormone
action"
Publications: (selected from last 5 years)
Tully, D. B., Jones, F and Tully, M R (2001) Donor milk: What's in it and what's not? J Hum Lact 17, 152-155.
Tully, D. B., Collins, B J , Overstreet, D J , Smith, C S., Dinse, G.E., Mumtaz, M M and Chapin, R. E. (2000) Effects
of arsenic, cadmium, chromium and lead on gene expression regulated by a battery of 13 different promoters in
recombinant HepG2 cells Toxicol, and Applied Pharmacol 168,79-90
Tully, D. B , Cox, V T , Mumtaz, M M., Davis, V. L and Chapin, R E (2000) Six high priority organochlorine
pesticides, either singly or in combination, are nonestrogenic in transfected HeLa cells. Reprod Toxicol 14, 95-102.
Shelby, M.D , Newbold, R R., Tully, D.B , Chae, K and Davis, V.L. (1996) Assessing environmental chemicals for
estrogenicity using a combination of in vitro and in vivo assays Environ. Health Perspec. 104, 1296-1300
Sewall, C H., Bell, D A , Clark, G. C., Tritscher, A M , Tully, D. B., Vanden Heuvel, J and Lucier, G W. (1995)
Induced gene transcription Implications for biomarkers Clin Chem. 41,1829-1834
Tully, D. B., Allgood, V E and Cidlowski, J A (1994) Modulation of steroid receptor mediated gene expression by
vitamin B6, FASEBJ. 8, 343-349.
Tully, D. B. and Cidlowski, J. A (1993) Protein-blotting procedures to evaluate interactions of steroid receptors with
DNA inWu, R (ed ) Methods in EnzymologylW, 535-551.
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BIOGRAPHICAL SKETCH
"rovide the following inlormation tor the key personnel in the order listed on Form Page 2
rcotocopy this page or follow this format lor each person
NAME
POSITION TITLE
Jeffrey E. Welch
Research Biologist
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training)
INSTITUTION AND LOCATION
DEGREE
YEAR(s)
FIELD OF STUDY
(if applicable)
University of North Carolina, Chapel Hill, NC
BS
1982
Zoology
University of North Carolina, Chapel Hill, NC
Ph D
1988
Cell Biology
RESEARCH AND/OR PROFESSIONAL EXPERIENCE Concluding with present position, list, in chronological order, previous employment, expenence, and
honors Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications dunng the past three years and to representative earlier publications pertinent to this application If the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant
application)
Professional Experience:
1995-Present Research Biologist, Gamete and Early Embryo Biology Branch, Reproductive Toxicology Division, US EPA,
Research Triangle Park, NC
1991-1994 Senior Staff Fellow, Gamete Biology Section, Laboratory of Reproductive and Developmental Toxicology,
NIEHS, Research Triangle Park, NC
1989-1991 Staff Fellow, Gamete Biology Section, Laboratory of Reproductive and Developmental Toxicology, NIEHS,
Research Triangle Park, NC
1988-1989. IRTA Fellow, Gamete Biology Section, Laboratory of Reproductive and Developmental Toxicology, NIEHS,
Research Triangle Park, NC.
1988 NIH Postdoctoral Fellow, Department of Cell Biology and Anatomy, School of Medicine, University of North
Carolina, Chapel Hill, NC
Professional Societies:
American Society for Cell Biology
Society for the Study of Reproduction
Triangle Consortium for Reproductive Biology
Professional Activities (selected from last 5 years)
US EPA Fuel and Fuel Additives Testing Review Panel
Human Health Risk Assessment/Susceptible Sub-populations Writing Team
US EPA Synergy Workshop Discussant, August 2000, Duluth, Minnesota
USDA Enhancing Animal Reproductive Efficiency Grant Review Panel
Biology of Reproduction Reviewer
Journal of Andrology Reviewer
Molecular Reproduction and Development Reviewer
Publications: (selected from last 5 years)
Klinefelter GR, Welch JE, Perreault SD, Moore HD, Zucker RM, Suarez JD, Roberts NL, Bobseine K, Jeffay S
Localization of the Sperm Protein SP22 and Inhibition of Fertility in vivo and in vitro J Androl (in press)
Welch JE, Brown PL, O'Brien DA, Magyar PL, Bunch DO, Mori C, Eddy EM. Human glyceraldehyde 3-phosphate
dehydrogenase-2 gene is expressed specifically in spermatogenic cells J Androl 21:328-338 (2000)
Klinefelter GR, Welch JE. The Saga of a Male Fertility Protein Ann Rev Biomed Sci 1 145-182 (1999)
Dremont RT, Macrae AD, Aparicio SA, Kendall HE, Welch JE, Lefkowitz RJ The GRK4 subfamily of G
protein-coupled receptor kinases. Alternative splicing, gene organization, and sequence conservation. J Biol Chem
274.29381-29389 (1999).
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Welch JE, Barbee RR, Roberts NL, Suarez JD, Klinefelter GR SP22 a novel fertility protein from a highly conserved gene
family J Androl 19:385-393 (1998)
Hagaman JR, Moyer JS, Bachman ES, Sibony M, Magyar PL, Welch JE, Smithies O, Krege JH, O'Brien DA
Angiotensin-converting enzyme and male fertility Proc Natl Acad Sci U S A 95:2552-2557. (1998)
Bunch DO, Welch JE, Magyar PL, Eddy EM, O'Brien DA. Glyceraldehyde 3-phosphate dehydrogenase-S protein distribution
during mouse spermatogenesis. Biol Reprod 58:834-841 (1998)
Mori C, Nakamura N, Welch JE, Gotoh H, Goulding EH, Fujioka M, Eddy EM Mouse spermatogenic cell-specific type 1
hexokinase (mHk1-s) transcripts are expressed by alternative splicing from the mHkl gene and the HK1-S protein is
localized mainly in the sperm tail Mol Reprod Dev 49.374-385 (1998)
Mori C, Nakamura N, Welch JE, Shiota K, Eddy EM. Testis-specific expression of mRNAs for a unique human type 1
hexokinase lacking the porin-binding domain. Mol Reprod Dev 44 14-22 (1996)
O'Brien DA, Welch JE, Goulding EH, Taylor AA Jr, Baba T, Hecht NB, Eddy EM. Boar proacrosin expressed in spermatids
of transgenic mice does not reach the acrosome and disrupts spermatogenesis Mol Reprod Dev 43.235-247 (1996).
PHS 398 (Rev 4/98) (Form Page 6) Page
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BIOGRAPHICAL SKETCH
°rovide the following information for the key personnel in the order listed on Form Page 2
'hotocopy this page or follow this format for each person
NAME
POSITION TITLE
Vickie S Wilson
Research Biologist
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training )
INSTITUTION AND LOCATION
DEGREE
(if applicable)
VEAR(s)
FIELD OF STUDY
Framingham State College, Framingham, MA
North Carolina State University, Raleigh, NC
US EPA/NCSU Post-Doc Fellowship, RTP, NC
B.S.
PhD
1992
1999
1999-2000
Food Science
Toxicology
Reproductive Toxicology
RESEARCH AND/OR PROFESSIONAL EXPERIENCE Concluding with present position, list, in chronological orde.r previous employment, experience, and honors
Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications dunng the past three years and to representative earlier publications pertinent to this application If the list of
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant application)
Professional Experience:
1989-1990
1991 - 1993
1993- 1994
1994-1995
Summer 1996
1996-1999
1999-2000
000 - Present
Assistant Research Scientist, GeneTrak Systems, Framingham, MA
Technical Assistant II, Framingham State College, Framingham, MA
Research Technician I, Duke University Med Center, Stedman Center for Nutritional Studies,
Durham, NC
Technician III, Good Mark Foods, Garner, NC
Research Assistant, North Carolina State University, Raleigh, NC
Research/Teaching Assistant, NC State University, Department of Toxicology, Raleigh, NC.
Post Doctoral Fellow, NCSU/USEPA Cooperative Training Agreement, U.S EPA, NHEERL, RTD,
Research Triangle Park, NC.
Research Biologist, U S EPA, NHEERL, RTD, EB, Research Triangle Park, NC.
Professional Societies
Society of Toxicology
NC Branch of the Society of Toxicology
American Association for the Advancement of Science
Society fo the Study of Reproduction
Triangle Consortium of Reproductive Biologists
Honors. Award(s): (selected from last 5 years)
2001 ORD Award for Exceptional/Outstanding ORD Technical Assistance to OPPTS during the standardization and
validation of the Tier 1 screening battery of the EDSP
Professional Activities (selected from last 5 years)
2000 - Present RTD delegate, Synergy Workgroup Committee, NHEERL
2000 Judge, Dept of ToxicologyGraduateStudentResearchSymposium.NorthCarolina State University,
Raleigh, NC
Invited Presentations (selected from last 5 years)
September 2001 North Carolina State University, Toxicology Seminar Series
November 2000 Inter-species steroid receptor extrapolation studies for endocrine disrupting chemicals 2151 Annual
Meeting of the Society of Environmental Toxicology and Chemistry, Nashville TN
August 2000 Use of in Vitro Assays for the Assessment of Endocrine Disrupting Chemicals Work-in Progress
Seminar, USEPA, Reproductive Toxicology Division
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August 2000 Progress in Cross Species Extrapolation Studies and the Use of In Vitro Assays to Assess the
Mechanisms of Action of Endocrine Disrupting Chemicals, Synergy Workgroup Meeting, USF
Duluth, Ml
February 2000 Testosterone metabolism as an endocrine biomarker University of North Carolina at Pembroke,
Pembroke, NC
February 1999 Xenobiotic induced alterations in testosterone biotransformation profiles: consequences and
significance U.S. Environmental Protection Agency, NHEERL, Reproductive Toxicology Division,
RTP, NC
Publications: (selected from last 5 years)
V. S. Wilson, K. Bobseine, C. R. LambrightJ_.E Gray, Jr (2001) A novel cell line, MDA-kb2, that stably expresses an
androgen and glucorticoid responsive reporter for the detection of hormone receptor agonists and antagonists Toxicol
Sci. Submitted.
Gray LE Jr, Ostby J, Wilson V, Lambright C„ Bobseine K , Hartig P, Hotchkiss A, Wolf C, Furr J, Price M, Parks L,
Cooper R , Stoker T , Laws S, SJ Degitz , KM Jensen , MD Kahl, JJ Korte, EA Makynen, JE Tietge and G Ankley
(2001) Xenoendocrine disrupters-tiered screening and testing Filling key data gaps Toxicol Letters In Press
Gray, L.E., Jr, J Ostby, J. Furr, M. Price, C.J. Wolf, C R Lambright, L G Parks, D.N.R. Veermachaneni, V.S. Wilson, A.
Hotchkiss, E Orlando, and L Guillette. (2001) Effects of environmental antiandrogens in experimental animals
Human Reproductive Update 7(3) 248-264
Gray, L. E., Jr, C Lambright, L Parks, R W. Tyl, E F Orlando, L J Guillette, C.J Wolf, J.C. Seely, T-Y Chang, V. S.
Wilson, A Hotchkiss, J.S Ostby (2000) Emerging issues related to endocrine disrupting chemicals and environmental
androgens and antiandrogens IN Handbook of Environmental Chemistry Endocrine Disruptors, Springer-Verlag In
press.
Lambright, C., J. Ostby, K. Bobseine, V. S. Wilson, A.K Hotchkiss, and L.E. Gray, Jr (2000) Cellular and molecular
mechanisms of action of Linuron. an antiandrogenic herbicide that produces reproductive malformations in male rats
Toxicol. Sci. 56. 389-399
Wilson, V. S. and G. A. LeBlanc (2000) The contribution of hepatic inactivation of testosterone to the lowering of serum
testosterone levels by ketoconazole. Toxocol. Sci May, 54(1)- 128-137
Wilson, V.S., J.B McLachlan, J.G Falls, and G.A. LeBlanc (1999). Alteration in sexually dimorphic testosterone
biotransformation profiles as a biomarker of chemically-induced androgen disruption Environ Health Perspec. May,
107(5): 377-384.
Wilson, V.S. and G.A LeBlanc (1998). Endosulfan modulates testosterone metabolism and elimination in female CD-1
mice Toxicol. Appl Pharmacol 148 158-168
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image:
BIOGRAPHICAL SKETCH
Drovtde the following information lor the key personnel in the order listed on Form Page 2
NAME
POSITION TITLE
Robert M. Zucker
Research Biologist
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training)
INSTITUTION AND LOCATION
DEGREE
(il applicable)
YEAR(s)
FIELD OF STUDY
University of California at Los Angeles (UCLA)
University of California at Los Angeles ( UCLA)
University of California at Los Angeles ( UCLA)
BS.
MS
Ph D
1965
1966
1970
Physics
BioPhysics & Nuclear Med
BioPhysics & Nuclear Med
RESEARCH AND/OR PROFESSIONAL EXPERIENCE Concluding with present position, list, in chronological orde.r previous employment, expenence, and
honors Include present membership on any Federal Government public advisory committee List, in chronological order, the titles, all authors, and
complete references to all publications dunng the past three years and to representative earlier publications pertinent to this application If the list ol
publications in the last three years exceeds two pages, select the most pertinent publications DO NOT EXCEED THREE PAGES (for a modular grant
application)
Professional Experience:
1995-Present USEPA NHEERL, RTD, Research Biologist
1985-1994 Senior Project Scientist, HERL, ManTech, Inc., USEPA, RTP, NC
1972-1984 Senior Scientist, Papanicolaou Cancer Research Institute, Miami, FL
1974-1984 Associate Professor, Dept of Medicine, Univ of Miami, Miami, FL
1970-1972 Postdoctoral Fellow, Max Planck Institute for Eiweiss and Lederforschung, Munich, Germany
Professional Societies
American Association for Advancement of Science
iternational Society of Analytical Cytology
oigma XI Research Society
American Society of Cell Biology
Microscopy Society of America
Honors. Awards: (selected from last 5 years)
USEPA Science and Technological Achievement Award, 1996.1997
Professional Activities (selected from last 5 years)
Member of Advisorary Board on Bioscope, Purdue University 1998-2002
Member of NSF rewiew committee on Core equipment support 1998
USEPA Science and Technological Achievement Award, 1996 1997
Review flow cytometry Atrazine literature for USEPA/OPP
ad hoc journal reviewer Cytometry
ad hoc journal reviewer-- miscellaneous journals
ad hoc grant reviewer for NSF
Designed and Implemented Training Course on Confocal Microscopy for USEPA Staff.
Designed and Implemented Training Course on Confocal Microscopy for Scinetists at Research and Clinical Cytometry
Imaging Symposium Washington DC 1998, 1999
Designed and Implemented training course on Flow Cytomety for USEPA Staff
Tutorial on Apoptosis at ISAC March 1998
Workshop on Flow Cytometry at USEPA March 14 2000
Tutorial on Confocal microscopy ISAC May 20 2000
Workshop on Confocal Microscopy ISAC May 23 2000
ivited Presentations (selected from last 5 years)
1M Zucker Evaluation of Confocal Microscopy System Performance with Applications to whole Organ/Orgainsm
apoptosis Microscopy and Microanalysis 2001, Long Beach California August 4-9 2001
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RM Zucker Mammalian Apoptosis in Whole Neonatal Ovaries, Embryos and Fetal Limbs using Contocal Microscopy.
Microscopy and Microanalysis 2001, Long Beach California August 4-9 2001
RM Zucker Tutorial on Confocal microscopy ISAC May 20 2000 Montpeiller France
RM Zucker Workshop on Confocal Microscopy ISAC May 23 2000 Montpeiller France
RM Zucker flow cytometry artifacts in print New yourk New Jersey Flow Cytometry Users Group Cold Spring Harbor
June 14 2000
RM Zucker Confocal Microscopy applications to reproduction East Carolina University June 1999
R M. Zucker K. Sulik and OT Price Confocal Laser Scanning Microscopy of Whole Mouse and Human Embryos-
Apoptosis and Morphology Microscopy and Microanalysis meeting '1999 Portland Oregon August 1999
R. M Zucker O T Price Optimization and the Evaluation of confocal system performance to quantify fluorescence
Microscopy and Microanalysis meeting 1999 Portland Oregon August 1999
Zucker, RM, Paul Robinson and Nick Terry : Confocal Microscopy Workshop Sixth Annual Clinical Cytometry and I
maging Symposium. Rockville Maryland April 11-13 1999
Zucker RM- Applications of confical microscopy to reproductive problems Wake Forest University July 1998
Zucker, RM, Paul Robinson and Nick Terry : Confocal Microscopy Current Applications and future directions' Fifth
Annual Clinical Cytometry and Imaging Symposium Rockville Maryland April 23-25 1998
Zucker, R M Paul Robinson and Nick Terry : Confocal Microscopy and image analysis workshop. Fifth Annual Clinical
Cytometry and Imaging Symposium. Rockville Maryland April 23 1998
Zucker, R M. ISAC PRESENTATION on APOPTOSIS in solid tissues :Embryos and ovaries ISAC Colorado springs
March 1998
Zucker, R M. Hunter E.S Haas A R Rogers J M. Confocal Laser Scanning microscopy of apoptosis in embryogenesis.
Teratology Society June 1997
Zucker, R M Flow cytometry and confocal microscopy Fine Particle workshop NHEERL SYMPOSIUM RTP June 1997
Zucker, R.M ROCKVILLE 1997 Fourth Annual Clinical Cytometry and Imaging Symposium Rockville Maryland April 23
1997
Zucker, R M. ROCKVILLE 1997 COURSE Fourth Annual Clinical Cytometry and Imaging Symposium Rockville
Maryland April 23 1997
Publications: (selected from last 5 years)
Klinefelter, G.R , Welch, J.E , Perreault, S.D. Moore, H.D., Zucker, R.M., Suarez, J.D., Roberts, N.L., Bobseine, K., and
Jeffay, S Localization of the sperm protein SP22 and inhibition of fertility in vivo and in vitro J. Androl., 2001 (In
press)
Zucker, R.M. Price OT Evaluation of confocal system performance Cytometry August 2001
Zucker, R.M. Price OT Statistical Evaluation of Confocal Microscopy Images Cytometry Augest 2001 2000
K K Sulik, D B. Dehart, C Johnson, S L Ellis, S-y Chen, W C Dunty and R M. Zucker Programmed cell death in
extraocular muscle tendon/sclera precursors submitted Molecular Vision Acepted July 2001
W C Dunty, Jr, S-yu Chen, R M. Zucker, D B. Dehart, and K K. Sulik Selective Vulnerability of Embryonic Cell
Populations to Ethanol-lnduced Apoptosis: Implications for Alcohol Related Birth Defects and Neurodevelopmental
DisorderH A.2001 Accepted July 2001 Alcoholism: Clinica and Experimental researchZucker, R.M. Kershavih A Price OT
Goldman J Confocal laser scanning microscopy of rat follicle development. J of Histochemistry and Cytochemistry.
48 781-791,2000
Kligerman AD, Doerr CL, Tennant AH, Zucker RM. Cytogenetic studies of three triazine herbicides Environmental
Mutagenesis 465 53-59 2000.
Zucker, R.M. Sid Hunter Rogers JM. Apoptosis and Morphology of Embryonic Structures in Mouse Embryos by
Confocal Laser Scanning Microscopy—METHODS A Companion to Methods of Enzymology ed. Anda Cornea 18"
473-480 1999
Zucker, RM, Rogers JM Embryonic/fetal Topographical Analysis by fluorescence Microscopy and confocal Laser
scanning microscopy In- Developmental Biology Protocols, vol II, eds. R.S. Tuan and C.W. Lo, Humana Press,
chapter 20 pp 203-209 1999
Zucker, R.M. Rogers JM Confocal Laser Scanning Microscopy of Morphology and apoptosis in Organogenesis stage
Mouse embryos.ln- Developmental Biology Protocols, vol. II, eds R.S. Tuan and C.W. Lo, Humana Press, chapter 19
pp 191-202 1999
Zucker, R.M. Price,OT -Practical confocal Microscopy and the Evaluation of system performance. METHODS a
companion to Methods of Enzymology ed. Anda Cornea 18.447-458 1999
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Zucker, R.M. Hunter S., Rogers JM Confocal Laser Scanning Microscopy of Apoptosis in Organogenesis-Stage Mouse
Embryos Cytometry 33:348-354 1998
clstein, K H , M.L Mole, R.W. Setzer, R.M. Zucker, R.J. Kavlock, J.M Rogers, and C Lau. (1997) Nucleoside-
ediated Mitigation of 5-Fluorouracil-induced Toxicity in Synchronized Murineg Erythroleukemic Cells. Tox. Appl
Pharmacol 146.29-39
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Impact Factors - RTD Publications
Impact Factor
Journal
(1999)
Analyt Biochem
2.146
Alcohol Exp Clin Res
2013
Andrologia
0.982
Ann NY Acad Sci
0.964
Annu Rev Biomed Sci
NL
Biochem Biophys Res Commun
3.161
Biol Reprod
3417
Biol Trace Elem Research
0.951
Biometrics
1 335
Biotechniques
1 749
Brain Res Bull
1.977
Cell Stress Chaperon
2.847
Chemosphere
1.255
Congenital Anomal Suppl
NL
Crit Rev Toxicol
4.906
Cytometry
2.843
Development
10.880
Develop Biol
6.049
Develop Dynamics
3.939
Endocrinology
5.365
Environ Carcinogen Ecotox Revs
0.538
Environ Epidem Toxicol
NL
Environ Health Perspect
2 469
Environ Toxicol Chem
2 462
Environ Toxicol Pharmacol
0.707
Exp Cell Res
3.256
Fertil Steril
2.955
Fundam Appl Toxicol
2.205
Genome Biol
NL
Health Environ Digest
NL
Human Reprod
3 003
Number of RTD publications
(1997-2001)
1
1
1
2
1
1
9
2
2
1
1
3
1
1
4
1
3
1
2
3
1
1
15
2
3
1
1
2
2
1
2
1
image:
Journal
Human Reprod Update
Int J Hum Ecol Risk Asses
In Vitro Cell Devel Biol
J Androl
J Biochem Molec Toxicol
J Biol Chem
J Endocrinol
J Histochem Cytochem
J Infect Dis
J Molec Med
J Pharmacol Exp Therap
J Zoo Wildlife Med
Methods
Molec Endocrinol
Molec Reprod Devel
Neurotoxicology
Neurotoxicol Teratol
New Engl J Med
Pharmacogenomics J
Proc Natl Acad Set USA
Regul Toxicol Pharmacol
Reproduction
Reprod Fertil Devel
Reprod Toxicol
Teratogen Carcinogen Mutagen
Teratology
Tissue Cell
Toxicol Appl Pharm
Toxicol Indust Health
Toxicol Lett
Toxicol Sci
Toxicology
Xenobiotica
Impact Factor
(1999)
2 297
NL
0.372
1.972
NL
7 666
2.580
2.675
4.842
3 748
3.300
0 322
1.679
7.566
2 658
1.282
1.822
28.857
4.640
10.260
1 530
NL
1.082
1.277
1.018
2.028
1.028
2.723
1.545
0 773
1.778
1.343
1-335
Number of RTD publications
996-2001)
2
17
1
15
1
7
7
2
32
4
_2_
Note. NL = impact factor not listed
2
image:
Publications 1997-2001
1 Abbott, B.D (1997) Developmental Toxicity of Dioxin Searching for the Cellular and Molecular
Basis of Morphological Responses. In Handbook of Experimental Pharmacology. Section III-
Pathogenesis and Mechanisms of Drug Toxicity in Development (R Kavlock and G. Daston,
Eds ), pp 407-433 Springer-Verlag, New York
2 Abbott, B.D, Probst, M.R , Perdew, G H., and Buckalew, A R. (1998). AH receptor, ARNT,
glucocorticoid receptor, EGF receptor, EGF, TGF alpha, TGF beta 1, TGF beta 2, and TGF beta 3
expression in human embryonic palate, and effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
Teratology 58, 30-43
3 Abbott, B.D., and Birnbaum, L.S (1998) Dioxins and teratogenesis In Molecular Biology of the
Toxic Response (A Puga and K Wallace, Eds ), pp 439-447 Taylor and Francis, Washington,
DC
4 Abbott, B D , Held, G A., Wood, C R , Buckalew, A R , Brown, J G , and Schmid, J (1999) AhR,
ARNT, and CYP1A1 mRNA quantitation in cultured human embryonic palates exposed to TCDD
and comparison with mouse palate in vivo and in culture Toxicol Sci 47, 62-75
5 Abbott, B.D., Schmid, J E., Brown, J G , Wood, C R , White, R D , Buckalew, A.R., and Held, G A
(1999). RT-PCR quantification of AHR, ARNT, GR, and CYP1A1 mRNA in craniofacial tissues of
embryonic mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and hydrocortisone Toxicol Sci
47, 76-85
6 Abbott, B.D , Schmid, J.E., Pitt, J A , Buckalew, A R„ Wood, C R , Held, G.A., and Diliberto, J J
(1999) Adverse reproductive outcomes in the transgenic Ah receptor-deficient mouse Toxicol
Appl Pharmacol. 155,62-70.
7. Abbott, B.D., and Buckalew, A.R (2000) Placental defects in ARNT-knockout conceptus
correlate with localized decreases in VEGF-R2, Ang-1, and Tie-2. Dev Dyn 219, 526-538.
8 Abbott, B D (2000) Palatal Dysmorphogenesis Palate Organ Culture In Methods in Molecular
Biology: Developmental Biology Protocols, Vol II (R S. Tuan and C W. Lo, Eds.), pp. 189-195.
Humana Press, Inc, Totowa, NJ.
9 Abbott, B.D. (2000) Insights from AhR and ARNT gene knockout studies regarding responses to
TCDD and regulation of normal embryonic development Congenital Anomalies Supplement 40,
s88-s93.
10 Abbott, B D (In Press) Laboratory Animal Models for the Study of Oral Clefts In Cleft Lip and
Palate From Origin to Treatment (D. F Wyszynski, Ed ), Oxford University Press, New York
11 Akingbemi, B T., Ge, RS„ Klinefelter, GR, Gunsalus, G.L., and Hardy, M.P. (2000). A
metabolite of methoxychlor, 2,2-bis(p-hydroxyphenyl)-1,1,1- trichloroethane, reduces testosterone
biosynthesis in rat Leydig cells through suppression of steady-state messenger ribonucleic acid
levels of the cholesterol side-chain cleavage enzyme Biol Reprod 62, 571 -578
12. Akingbemi, B.T., Youker, R T., Sottas, C.M., Ge, R., Katz, E , Klinefelter, G R., Zirkin, B R., and
Hardy, M.P. (In Press) Modulation of rat Leydig cell steroidogenic function by di(2-
ethylhexyl)phthalate Differential effects of DEHP on Leydig cell function throughout reproductive
development Biol. Reprod.
13 Anderson, M E., Conolly, R B , Faustman, E M., Kavlock, R J., Portier, C.J., Sheehan, D.M , Wier,
P.J., and Ziese, L. (1999). Quantitative mechanistically based dose-response modeling with
endocrine-active compounds Environ Health Perspect 107,631-638
3
image:
14 Andrews, J.E., Ebron-McCoy, M.T , Bojic, U., Nau, H., and Kavlock, R.J. (1997). Stereoselective
dysmorphogenicity of the enantiomers of the valproic acid analogue 2-N-propyl-4-pentynoic acid
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231 Wolf, C J , LeBlanc, G A , Ostby, J.S., and Gray, L.E., Jr. (2000) Characterization of the period
of sensitivity of fetal male sexual development to vinclozolin. Toxicol Sci. 55,152-161
232. Wubah, J.A , Setzer, R.W., Lau, C., and Knudsen, T.B. (2001). Exposure-disease continuum for
2-chloro-2-deoxyadenosine (2CdA), a prototype ocular teratogen 1. Dose-response analysis
Teratology 64, 154-169.
233. Wyrobek, A.J , Schrader, S.M., Perreault, S.D., Fenster, L„ Huszar, G„ Katz, D.F., Osorio, A.M.,
Sublet, V , and Evenson, D (1997). Assessment of reproductive disorders and birth defects in
communities near hazardous chemical sites III Guidelines for field studies of male reproductive
disorders. Reprod Toxicol. 11,243-259.
234 Zacharewski, T„ Campbell, P., Routledge, E., Cooper, R., Kime, D„ Wenzel, A , and Huet, M -C.
(1998). Current Approaches for the Use of in Vitro Tests in Identifying the Hazards of Endocrine
Modulating Chemicals to Wildlife pp 41-58. SETAC-Europe/OECD/EC Expert Workshop on
Endocrine Modulators and Wildlife: Assessment and Testing, Europe
235 Zahalka, E.A., Ellis, D.H., Goldey, E.S., Stanton, M.E, and Lau, C (In Press) Perinatal
exposure to poylchlorinated biphenyls Aroclor 1016 or 1254 did not alter brain catecholamines nor
delayed alternation performance in the Long Evans rats. Bra/n Res Bull
236. Zhu, D„ Dix, D.J , and Eddy, E.M. (1997) HSP70-2 is required for CDC2 kinase activity in
meiosis I of mouse spermatocytes. Development 124, 3007-3014.
237. Zucker, R M., Hunter, S., and Rogers, J.M. (1998). Confocal laser scanning microscopy of
apoptosis in organogenesis-stage mouse embryos. Cytometry 33, 348-354
238. Zucker, R.M., Hunter, E.S., III, and Rogers, J.M. (1999) Apoptosis and morphology in mouse
embryos by confocal laser scanning microscopy Methods 18, 473-480
239. Zucker, R.M., and Price, O T. (1999) Practical confocal microscopy and the evaluation of system
performance. Methods. 18, 447-458.
18
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241. Zucker, R.M., Hunter, E.S., III, and Rogers, J.M (2000) Confocal Laser Scanning Microscopy of
Morphology and Apoptosis in Organogenesis-Stage Mouse Embryos In Methods in Molecular
Biology (R S TuanandC. W Lo, Eds ), pp. 191-202 Humana Press, Totowa, NJ
242 Zucker, R.M , and Rogers, J M (2000) Embryo/Fetal Topographical Analysis by Fluorescence
Microscopy and Confocal Laser Scanning Microscopy In Methods in Molecular Biology (R S.
Tuan and C W Lo, Eds ), pp 203-209 Humana Press, Totowa, NJ
243 Zucker, R.M , Keshaviah, A P , Price, O T„ and Goldman, J M (2000) Confocal laser scanning
microscopy of rat follicle development J Histochem Cytochem 48, 781 -791
244 Zuelke, K A , Jones, D P , and Perreault, S.D (1997) Glutathione oxidation is associated with
altered microtubule function and disrupted fertilization in mature hamster oocytes Biol Reprod
57,1413-1419
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Reproductive Toxicology Division Trainees
1997- Present
Name
Trainee
Status
Mentor
Affiliatio
n
Training Period
Current Position
Balchak, Sharon
Post Doc
Goldman, S
UNC
Nov 98-Jun 99
Notre Dame
Bielmeier, Susan
Pre Doc
Narotsky, M
UNC
Mar 98 - Mar 02
Incumbent
Brannen, Kimberly
Pre Doc
Rogers, J.
UNC
Auq 97 - Auq 01
Univ Shefield
Bryant, Lamont
Pre Doc
Abbott, B
UNC
Sep 95 - Dec 00
Proctor & Gamble
Cancel, Aida
Post Doc
Darney, S
UNC
May 97 - Auq 99
Family Health Intl
Das, Parikshit
Post Doc
Cooper, R
UNC
Jul 98 - Oct 01
Incumbent
Degitz, Siqmund
Post Doc
Rogers, J
UNC
Sep 96 - Feb 98
MED/NHEERL
Gersch, Beniamin
Pre Doc
Chernoff, N
NCSU
Jan 00 - Dec 02
Incumbent
Ghosh, Basavdutta
Post Doc
Lau, C
NRC
Oct 96 - Auq 99
Bayer Corp
Goetz, Amber
Pre Doc
Dix, D
NCSU
Auq 01 - Auq 03
Incumbent
Grasty, Rayetta
Pre Doc
Rogers, J
UNC
Auq 01 - Auq 03
Incumbent
Goodinq, Meredith
Pre Doc
Wilson, V
NCSU
Mar 01 - Feb 02
Incumbent
Greiner, Sarah
Pre Doc
Fenton, S
Duke
Auq 99 - May 00
USGS
Holmes, Michael
Pre Doc
Klinefelter, G
NCCU
May 97 - May 01
Rockefeller Univ
Hotchkiss, Andrew
Pre Doc
Gray, E
NCSU
Jul 98 - Jun 01
Univ Ohio
Johnson, Cory
Pre Doc
Hunter, S
UNC
Auq 00 - Dec 02
Incumbent
Kaydos, Emily
Pre Doc
Klinefelter, G
NCSU
Auq 01 - Auq 03
Incumbent
Lanqdale, Christopher
Pre Doc
Cooper, R
NCSU
Jan 01 - Jan 03
Incumbent
Leazer, Tyra
Pre Doc
Roqers, J
UNC
Auq 93 - Feb 00
Univ. Kansas
Luft, Christopher
Post Doc
Dix, D.
EPA
Auq 98 - Mar 01
Artecel Sciences
Mapp, Faye
Pre Doc
Dix, D
NCCU
Feb 97 - May 98
St Judes
Modic, Walker
Pre Doc
Laws, S.
NCSU
Jan 01 - Dec 02
Incumbent
Morris, Rebecca
Post Doc
Darney, S.
EPA
Jan 01 - Jan 04
Incumbent
Parks, Louise
Post Doc
Gray, E
EPA
Oct 98 - Jun 01
DuPont
Pitt, Jeff
Post Doc
Abbott, J
UNC
Feb 97 - Apr 99
W.I.L. Res. Labs
Pooler, Melanie
Pre Doc
Fenton, S
NCCU
Auq 01 - Aug 03
Incumbent
Price, Matthew
Under Grad
Gray, E
NCSU
Mar 99 - Feb 02
Incumbent
Rayner, Jennifer
Pre Doc
Fenton, S
UNC
Jun 01 - Jun 03
Incumbent
Reynolds, Phillip
Post Doc
Gray, E
UNC
Oct 01 - Oct 04
Incumbent
Richards, Monique
Pre Doc
Abbott, B.
UNC
Auq 01 - Auq 02
Incumbent
Rockett, John
Post Doc
Dix, D
EPA
Auq 98 - Dec 00
RTD/NHEERL
Smith, Jan
Pre Doc
Hunter, S
UNC
Jan 99 - Jan 02
Incumbent
Tarka, Dana
Pre Doc
Roqers, J
UNC
Jun 98 - Auq 03
Incumbent
Toews, Erin
Under Grad
Gray, E.
UNC
May 99 - May 01
UNC
Thompson, Kary
Post Doc
Dix, D.
EPA
Jan 02 - Jan 05
Incumbent
Tully, Douglas
Post Doc
Dix, D
EPA
Aug 01 - Aug 04
Incumbent
Ward, Keith
Post Doc
Hunter, S
UNC
Jan 97 - Jul 97
Glaxo Smith Kline
Wiese, Tom
Post Doc
Gray, E
UNC
Sep 95 - Jul 98
Tulane Univ.
Wilson, Vickie
Post Doc
Gray, E
NCSU
Aug 99 - Dec 00
RTD/NHEERL
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Reproductive Toxicology Division Guest Workers
1997 - Present
Name
Research Focus
Affiliation
EPA Mentor
Brownie, Cecil
Effects of stress during
gestation on embryonic gene
expression
NCSU School of
Veterinary Medicine
Chemoff, N
Cancel, Aida
Evaluation of sperm function in
rats
Family Health
International
Darney, S.
Ghosh, Basavdutta
Endocrine-disruptive potential
of polychlorinated biphenyls
Self
Lau, C
Goetz, Amber
Training in molecular
toxicology and transgenics
Self
Dix, D
Hwang, Seong, Hee
Training in reproductive
toxicology and evaluation of
endocrine disruptinq chemicals
Self
Cooper, R.
Kempinsas, Wilma
Effects of chemically-induced
denervation on sperm quality
and fertility in adult rats
Universidade
Estradual Paulista,
Botucatu, SP, Brazil
Klinefelter, G
Kirpalani, Jaya
Training in cDNA cloning
techniques
Self
Welch, J
Moon, Hyun-Ju
Effects of Environmental
Toxicants on mammary gland
development
Korea Food and Drug
Administration
Fenton, S
Tully, Douglas
Training in molecular
toxicology and toxicoqenomics
Self
Dix, D.
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SEMINAR SERIES (1997 - 2001)
SPEAKER
DATE
TOPIC
Dr. Delores Lamb
Baylor College of Medicine
1/14/97
Assessment of Sperm Function in the Progesterone Receptor
Knock Out Mouse
Dr. Keith Ward
UNC - Curriculum in Toxicology
2/11/97
Haloacetic Acid - Induced Embryo Toxicity: Mechanisms and
Pathogenesis
Dr. Aida M. Cancel
PA State Univ
3/14/97
Osteropontin in the 55 kDa Fertility-Associated Protein in
Bovine Seminal Plasma
Dr. Richard Finnell
Texas A&M Univ, Coll of Vet Med
3/19/97
Genetic basis of Susceptibility to Environmentally-Induced Birth
Defects
Dr. Thomas Knudsen
Jefferson Medical College
3/20/97
Tumor Suppressors and the Embryonic mtDNA Genome
Dr. Roger Gorski
UCLA Medical School
4/17/97
Sexual Differentiation of the Brain: Some Unanswered
Questions
Dr. Barbara Hales
McGill University
5/15/97
In Vitro Approaches Towards Understanding Molecular
Mechanisms of Teratogenesis
Dr. Craig Nelson
Harvard University
5/22/97
Gene Expression in Normal Limb Development: Evolution of
the Tetrapod Limb
Dr. Harry Moore
University of Sheffield (UK)
6/5/97
Fertilizing Capacity and Survival of Epididymal Sperm
Dr. Jacques Picard
Universite Catholique De Louvain
7/11/97
Boric Acid and HOX Expression in Rats
Dr. Glen Van Der Kraak
University of Guelph (Canada)
8/21/97
Endocrine Disruptors in Fish Populations
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SPEAKER
DATE
TOPIC
Dr. David Anderson
California Institute of Technology
11/20/97
Neural Crest Cell Migration
Dr. Ken Korach
NIEHS
12/4/97
Molecular and Physiological Phenotypes in Estrogen Knockout
Mice
Dr. Gary Ankley
Mid-Continent Ecology Div., NHEERL
1/28/98
Effects of Methoprene and Ultraviolet Light on Survival and
Development of the Northern Leopard Frog, Rana pipiens
Dr. Patricia Rodier
Univ. of Rochester Medical Center
2/4/98
Phenotype Predicts the Genotype of Autism
Dr. Matthew Hardy
The Population Council
2/19/98
The Regulation of Leydig Cell Steroidogenesis
Dr. Robert Chapin
NIEHS
3/24/98
Stertoli-Late-Spermatid Adhesions: Toxicant-Induced
Separation Anxiety
Dr. Adele Turzillo
Colorado State University
4/6/98
Physiological Consequences of Estrogen Exposure in the
Anterior Pituitary Gland
Dr, Insong J. Lee
NIH, NIAAA
4/9/98
Molecular Characterization of Fetal Alcohol Syndrome Using
mRNA Differential Display
Dr. James A. Coffman
California Institute of Technology
4/21/98
Spatial Regulation of Gene Expression in the Sea Urchin
Embryo
Dr. Char Farin
North Carolina State Univ.
4/16/98
Transcriptional Regulation of bovine Oocyte Maturation
Dr. H. Scott Stadler
University of Utah
5/15/98
Aberrant Cranial Neural Crest Migration & Apoptosis in Hoxa-1
Mutants
Dr. Joseph Sebastian
North Carolina State Univ.
5/21/98
Estrogen Action and EGF-Receptor Signaling in Mammary
Gland Development
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SPEAKER
DATE
TOPIC
Dr. Rocky S. Tuan
Thomas Jefferson Univ.
6/11/98
Molelcular and Cellular Mechanisms of Developmental
Skeletogenesis: Genes, Cells and environmental Perturbations
Dr. Gail Prins
Univ. of Illinois at Chicago
9/16/98
Developmental Estrogenization of the Prostate
Dr. Kenneth Setchell
Children's Hospital Medical Center
9/24/98
Phytoestrogens and Early Exposures and Implications of
Hormone Dependent Diseases
Dr. Lonnie Russell
Southern Illinois University
10/15/98
Spermatogonia! Transplantation
Dr. Susan Bryant
University of California - Irvine
11/19/98
Cell Cycle Length, Gene Expression and Pattern Formation in
Development
Dr. Michael Evgen'ev
Russian Academy of Sciences
12/3/98
Comparison of Heat Shock Response in Ecologically Different
Populations Including Humans
Dr. Norman Hecht
University of Pennsylvania
1/6/99
Post-Transcriptional Regulation of Male Germ Cell
Differentiation
Dr. Pat Hoyer
University of Arizona
2/25/99
Ovarian Toxicity of 4-Vinylcyclohexene Diepoxide
Dr. Douglas J. Crawford-Brown
Univ. of North Carolina - Chapel Hill
3/11/99
Uniting Mechanistic Models at the Biochemical, In Vitro, Animal
and Human Levels of Organization: A Case Study from Radon
Dr. Peter G. Wells
University of Toronto
4/29/99
Reactive Oxygen Species and Oxidative Damage in Chemical
Teratogenesis
Dr. Blanch Capel
Duke University
5/20/99
SRY and the Testis: Pathways of Organogenesis
Dr. Dona Chikaraishi
Duke University
5/17/99
Role of Norepinephrine in Embryonic Survival
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SPEAKER
DATE
TOPIC
Dr. Jim Simpkins
University of Florida
8/19/99
Neuroprotective Effects of Estradiol and Novel Estrogens
Dr. Kim Boekelheide
Brown University
10/19/99
Testicular Toxicity and Response to Injury
Dr. Michael E. McClure
NIEHS
11/9/99
Research on Reproduction in the Millennium of the Environment
Dr. Michael E. Burczynski
Univ. of Pennsylvania Sch. of Med.
12/8/99
Roles and Regulation of Human Aldo Keto Reductases in
Xenobiotic Metabolism and Oxidative Stress
Dr. Alex Merrick
NIEHS
2/24/00
Proteomics in a Gene Expression Center: Applications to
Environmental Toxicology
Dr. Rochelle Tyl
RTI
3/9/00
Research Needs in Developmental and Reproductive
Toxicology
Dr. Lou Guillette
University of Florida
4/6/00
Sexual Dimorphisms: Effects of EDC's in the Alligator
Dr. Marion Miller
Univ. of California - Davis
4/20/00
Mechanistic Data to Model Human Risk of Molinate-lnduced
Male Reproductive Toxicity
Dr. Kipak Mahato
NIEHS
6/1/00
Elucidating the Role of Estrogen Receptor-alpha in
Spermatogenesis
Dr. Jill James
Nat. Center for Toxicological Res.
5/31/00
Gene-Nutrient Interactions in the Maternal Risk of Down
Syndrome
Dr. Tyrone B. Hayes
Univ. of California - Berkeley
6/15/00
African Reedfrogs, Soft Shell Turtles, and Kingsnakes: Non-
traditional Models Filling in the Blanks in the Endocrine
Disruotor Story
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SPEAKER
DATE
TOPIC
Dr. Bradley Thatcher
Ciphergen Biosystems, Inc.
6/12/00
The Use of Protein Chip Arrays and SELDI for Proteomic and
Toxicological
Dr. D. Stewart Irvine
Center for Reproductive Biology (UK)
6/8/00
Detection of Oxidative Damage in the Sperm Genome
Dr. Kurt Zuelke
Victorial Institute of Animal Sci (Aust)
7/12/00
Transgenice Modification of Cow's Milk for Value-Added
Processing
Dr. Bruce Lessey
Univ. of North Carolina - Chapel Hill
7/27/00
Use of Biomarkers to Study Implantation Defects in Women with
Hyperandrogenism
Mr. David Choiniere
Arcturus Engineering, Inc.
10/17/00
The Capture and Analysis of Pure Cell Populations from Tissue
Sections Using Laser Capture Microdissection
Dr. Mary Ann Handel
University of Tennessee
11/2/00
From Prophase to Division Phase: Meiosis in Spermatocytes of
the Laboratory Mouse
Dr. Mitch Eddy
NIEHS
12/13/00
HSP70 Chaperones in Spermatogenesis
Dr. Jodi Flaws
University of Maryland
1/18/01
The Effects of Environmental Chemicals on the Female
Reproductive System
Dr. Patricia Wainwright
University of Waterloo (Canada)
2/15/01
Role of Nutrition in Development Studies of Brain and
Behavioral Development in Rodents
Dr. Christopher J. Bowman
University of Florida
3/8/01
Teleost Gene Regulation by Estradiol and Estrogen Mimics
Dr. Steve Krawetz
Wayne State University
3/14/01
Analysis of Sperm mRNA using Gene Microarrays
Dr. Nira-Ben Jonathan
University of Cincinnati
3/15/01
Xenoestrogens and the Neuroendocrine-Reproductive Axis
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SPEAKER
DATE
TOPIC
Prof. David J.P. Barker
Univ. of Southampton (UK)
4/2/01
Developmental Plasticity: The Biological Origins of Heart
Disease
Prof. David J. P. Barker
Univ. of Southampton (UK)
4/3/01
Human Growth During the Industrial Revolution: The Social
Origins of Coronary Heart Disease
Dr. Edwin Lepart
Brigham Young University
5/8/01
Detection of Brain Aromatase Activity in Rodents
Dr. Tiaan deJager
University of Laval (Canada)
5/10/01
Semen Quality in Men Exposed to High Levels of DDT and
PCBs in Mexico
Dr. Terry Brown
Johns Hopkins University
5/17/01
Age-dependent and Lobe-specific Prostatic Hyperplasia in the
Brown Norway Rat
Dr. Cynthia Brame
University of Virginia
6/14/01
Protein Identification Using Mass Spectroscopy
Dr. Jim Stevens
Syngenta/Wake Forest Univ.
7/10/01
P-glycoprotein: Does its absence in the neonatal rat pup reduce
the value of this model for pesticide testing in the
developmental-neurotoxicity, postnatal lactational studies, and
the 2-generation reproduction studies?
Dr. Steve Sylvester
Washington State Univ. - Vancouver
7/19/01
Toward the Mechanism of Bromochloroacetate Toxicity in the
Testis
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