&EPA
United States
Environmental Prote.
Agency
National Health
and Environmental
Effects Research
Human Health Research
Implementation Plan
r
esearch and Development
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EPA 600/R-03/069
September 2003
National Health and Environmental Effects Research Laboratory
Human Health Research Implementation Plan
US Environmental Protection Agency
National Health and Environmental Effects Research Laboratory
Office of Research and Development
Research Triangle Park, NC 27711
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Foreword
The National Health and Environmental Effects Research Laboratory (NHEERL), as part of the
Environmental Protection Agency's Office of Research and Development (ORD), is responsible
for conducting research to improve the risk assessment of chemicals for potential effects on
human health. This research is intended to address key Agency problems in a timely and
responsive manner. To meet this responsibility, NHEERL is developing research
implementation plans to achieve the following objectives:
Optimizing responsiveness of research activities to Agency needs,
Sharpening the focus of research programs where needed,
Providing a forum for engagement of scientific staff on issues and approaches,
Focusing on multi-year planning explicitly linked to Agency performance goals,
and
Providing a mechanism for prioritizing research.
This approach builds on the ORD planning process that identifies and prioritizes research needs.
Current areas for research include protection of susceptible subpopulations, harmonization of
risk assessment approaches, and improving aggregate and cumulative risk assessment in support
of the Agency's Program and Regional Offices and legislative mandates, including the Safe
Water Drinking Water Act, Clear Air Act, Toxic Substances Control Act, and Federal Insecticide
Fungicide and Rodenticide Act.
This document identifies the scientific problems and research that will be conducted concerning
human health. The ultimate goal of this research is to develop scientifically valid approaches for
improving human health risk assessment. This document was developed by representatives from
NHEERL research divisions and peer-reviewed by scientists from other ORD laboratories and
centers and EPA Regional and Program Offices. This document was also reviewed by scientists
external to the Agency. This implementation plan is intended to reflect research that will be
conducted over the next several years. As progress is made in achieving the goals outlined in
this document, it will be updated to address new and remaining human health challenges.
t^U/rV^-c^. "xj^^^
Lawrence W. Reiter
Director
National Health and Environmental Effects Research Laboratory
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TABLE OF CONTENTS
Foreword ii
List of Figures v
Acknowledgments vi
NHEERL Human Health Research Executive Committee vii
Peer Review viii
Acronyms ix
Executive Summary E-l
Section 1 Introduction 1-1
Section 2 Research Approach 2-1
Section 3 Harmonization of Cancer and Non-Cancer Risk Assessment 3-1
3.1 Problem 3-1
3.2 Goals 3-2
3.3 Critical Path 3-3
3.4 Program Projects 3-4
3.5 Gap Analysis and Links to Other Multi-Year Plans 3-18
3.6 References 3-21
Section 4 Susceptible Subpopulations 4-1
4.1 Problem 4-1
4.2 Goals 4-1
4.3 Critical Path 4-2
4.4 Program Projects 4-3
4.5 Gap Analysis and Links to Other Multi-Year Plans 4-36
4.6 References 4-38
Section 5 Cumulative Risk 5-1
5.1 Problem 5-1
5.2 Goal 5-2
5.3 Critical Path 5-3
5.4 Program Project 5-3
5.5 Gap Analysis and Links to Other Multi-Year Plans 5-7
5.6 References 5-8
in
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Appendix A Research Allocation for Program Projects A-l
Appendix B Annual Performance Goals and Measures for Harmonization A-2
Appendix C Annual Performance Goals and Measures for Susceptible
Subpopulations A-4
Appendix D Annual Performance Goals and Measures for Cumulative Risk A-8
IV
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List of Figures
Page
Figure 1 Mandates for Human Health Research 2-2
Figure 2 The Source-to-Outcome Continuum 2-3
Figure 3 Critical Path for Harmonization Research 3-3
Figure 4 Critical Path for Research on Cell Signaling 3-6
Figure 5 Critical Path for Research on Luteinizing Hormone. 3-11
Figure 6 Critical Path for Research of P-450 and XMEs 3-16
Figure 7 Framework for Use of Mechanistic Data 3-19
Figure 8 Critical Path for Research on Susceptible Subpopulations 4-2
Figure 9 Critical Milestones for Research on Children's Health 4-5
Figure 10 Critical Path for Research on Windows of Vulnerability 4-11
Figure 11 Critical Milestones for Research on Developmental Environment 4-14
Figure 12 Critical Path for Research on Susceptibility Associated with Older Adults 4-20
Figure 13 Critical Path for Research on Asthma 4-25
Figure 14 Critical Path for Research on Oxidative Stress 4-29
Figure 15 Critical Path for Research on Genetic Polymorphisms 4-34
Figure 16 Critical Path for Research on Cumulative Risk 5-2
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Acknowledgments
Preparation of the NHEERL Human Health Implementation Plan was coordinated by the
NHEERL Human Health Research Executive Committee whose members are listed on the next
page. Research themes for the proposals described in this document were developed at a
scientist-to-scientist meeting held at Research Triangle Park in November 2001. Participants at
that meeting included scientists from NHEERL, as well as scientists from other ORD
laboratories and Centers which include:
National Center for Environmental Assessment (NCEA),
National Center for Environmental Research (NCER),
National Exposure Research Laboratory (NERL), and the
National Risk Management Research Laboratory (NRMRL).
Scientists from several Regional Offices, the Office of Air and Radiation, Office of Water, and
the Office of Prevention, Pesticides, and Toxic Substances were also in attendance. Based on
recommendations generated from the scientist-to-scientist meeting, research proposals were
generated and reviewed by the NHEERL Human Health Research Executive Committee, as well
as the following non-NHEERL scientists:
Rick Hertzberg (Region 4),
Kim Hoang (Region 9),
Marc Rigas (NERL), Chris Saint (NCER),
Jennifer Seed (Office of Toxic Substances),
Sophia Serda (Region 9),
Linda Sheldon (NERL),
Michel Stevens (NCEA),
Cynthia Sonich-Mullin (NCEA), and
Edward Washburn (Office of Science Policy).
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NHEERL Human Health Research Executive Committee
Environmental Carcinogenesis Division
R. Julian Preston
Andrew Kligerman
Experimental Toxicology Division
Linda S. Birnbaum
Hugh A. Barton (Chair)
Jane Ellen Simmons
Human Studies Division
John Vandenberg
Tony Huang
Neurotoxicology Division
Stephanie Padilla
Stan Bar one
Reproductive Toxicology Division
Robert Kavlock
David Dix
Research Planning and Coordination Staff
Hugh A. Til son
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PEER REVIEW
Dr. Vicki Dellarco, U. S. EPA Office of
Prevention, Pesticides, and Toxic
Substances, Washington, DC
Dr. George Daston, Proctor & Gamble,
Miami Valley Laboratories, Cincinnati, OH
Dr. Alan Ducatman, Chair, Department of
Community Medicine, West Virginia
University School of Medicine,
Morgantown, WV
Mr. Ravi Rao, U. S. EPA Region 4, Atlanta,
GA
Dr. Winona Victery, U. S. EPA Region 9,
San Francisco, CA
Dr. James Gibson, East Carolina University,
Brody School of Medicine, Department of
Pharmacology and Toxicology, Greenville,
NC
Dr. Richard B. Schlesinger, Department of
Biological Sciences, Pace University,
Pleasantville, NY
Dr. Melvin Andersen, CIIT-Centers for
Health Research, Research Triangle Park,
NC
Dr. Ed Carney, Technical Leader,
Developmental & Reproductive Toxicology,
The Dow Chemical Company, Midland, MI
Dr. Jay Goodman, Michigan State
University, Department of Pharmacology &
Toxicology, East Lansing, MI
Dr. Moiz Mumtaz, AT SDR, Division of
Toxicology, Atlanta, GA
Dr. Penelope Fenner-Crisp, Risk Sciences
Institute, ILSI, Washington, DC
Dr. William Slikker, National Center for
Toxicological Research, Division of
Neurotoxicology, Jefferson, AR
Dr. John Moore, Hollyhouse, Inc.,
Arlington, VA
Dr. Diana M. Wong, U. S. EPA Office of
Water, Washington, DC
Dennis Pagano, U. S. EPA Office of Air and
Radiation, Research Triangle Park, NC
Dr. George Lambert, Director, Clinical
Research Center, UMDNJ-Robert Wood
Johnson Medical School, Rutgers
University, Piscataway, NJ
Dr. Marion Ehrich, Virginia-Maryland
Regional College of Veterinary Medicine,
Department of Biomedical Sciences and
Pathobiology, Virginia Polytechnic Institute
and State University, Blacksburg, VA
Dr. Michael I. Luster, NIOSH, Chief,
Toxicology & Molecular Biology Branch,
Morgantown, WV
Dr. Elizabeth G. Marshall, Department of
Pediatrics, UMDNJ-RWJMS, New
Brunswick, NJ
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Acronyms
ADHD Attention Deficit Hyperactivity Disorder
AMD Age-Related Macular Degeneration
APG Annual Performance Goal
APM Annual Performance Measure
As Arsenic
BBDR Biologically Based Dose Response
CCL Contaminant Chemical List
CDC Centers for Disease Control and Prevention
ChE Cholinesterase or acetylcholinesterase
CNS Central Nervous System
COPD Chronic Obstructive Pulmonary Disease
CVD Cardiovascular Disease
CYP Cytochrome P-450
2,4-D 2,4-Dichlorophenoxy Acid
DEHP Diesel Exhaust Particles
DBF Disinfection By-Product
EBIFs Ergosterol Biosynthesis Inhibiting Fungicides
EDC Endocrine Disrupting Chemical
FIFRA Federal Insecticide, Fungicide and Rodenticide Act
FQPA Food Quality Protection Act
FSH Follicle Stimulating Hormone
GJC Gap Junction Communication
y-GCS y-Glutamyl Cysteine Synthetase/Ligase
GnRH Gonadotrophin-Releasing Hormone
GPRA Government Performance and Results Act
GST-O Glutathione-S-Transferase Omega
HAAs Haloacetic Acids
HAPs Hazardous Air Pollutants
HO-1 Heme Oxygenase-1
iAs Inorganic Arsenic
IgE Immunoglobulin E
ICC Interagency Coordinating Committee
IUGR Intrauterine Growth Retardation
LH Luteinizing Hormone
LTGs Long-Term Goal
MAPK Mitogen-activated protein kinase
MO A Mode of Action
MYPs Multi-Year Plans
NCEA National Center for Environmental Assessment
NCER National Center for Environmental Research
NCS National Children's Study
NE Noradrenergic
NERL National Exposure Research Laboratory
NHEERL National Health and Environmental Effects Research Laboratory
NIH National Institutes of Health
NTFs Neurotrophic Factors
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NOAEL
NOEL
NQO-1
NRC
NRF2
NRMRL
OCHP
OP
OPP
OPPTS
ORD
OW
PBPK models
PCBs
PCR
PD models
PK models
PKC
PM
POD
RfD
RPE
ROS
SAR
SH Rats
STAR
TSH
TSCA
US
WKY Rats
XMEs
No-Observed-Adverse-Effect-Level
No-Observed-Effect-Level
NADPH Quinone Oxidoreductase-1
National Research Council
Nuclear Factor Erythroid-Derived 2, Like 2
National Risk Management Research Laboratory
Office of Children's Health Protection
Organophosphate
Office of Pesticide Programs
Office of Prevention, Pesticides, and Toxic Substances
Office of Research and Development
Office of Water
Physiologically Based, Pharmacokinetic Models
Polychlorinated Biphenyls
Polymerase Chain Reaction
Pharmacodynamic Models
Pharmacokinetic models
Protein Kinase C
Particulate Matter
Point of Departure
Reference Dose
Retinal Pigmented Epithelium
Reactive Oxygen Species
Structure Activity Relationship
Spontaneous Hypertensive Rats
Science to Achieve Results
Thyroid Stimulating Hormone
Toxic Substances Control Act
United States
Normotensive Wi star-Kyoto Rats
Xenobiotic Metabolizing Enzymes
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Executive Summary
This document describes the
implementation plan for human health
research within the National Health and
Environmental Effects Research Laboratory
(NHEERL) for the next 8-10 years
beginning in FY02. Human health research
in the Office of Research and Development
(ORD) of the Environmental Protection
Agency (the Agency) is based on needs and
goals established by the Agency in response
to the Government Performance and Results
Act (GPRA). The human health research
described in this document aims to provide
broad, fundamental scientific information
that will improve understanding of problem-
driven issues arising from risk assessment in
the Agency's Program and Regional
Offices. Human health research supports
the identification of fundamental
mechanisms of toxicity and development of
methods and models to elucidate key
scientific uncertainties important to
understanding and predicting the effects of
environmental agents on human health.
This implementation plan on human
health research provides a detailed approach
that NHEERL will take to address scientific
uncertainties described in the ORD's Human
Health Research Strategy document, which
includes harmonizing the use of mechanistic
data in risk assessment, protecting the health
of susceptible subpopulations, and
determining risk of exposure to multiple
chemicals. This document also outlines in
detail how NHEERL's human health
research will meet the Annual Performance
Goals (APGs) and Annual Performance
Measures (APMs) described in ORD's
Human Health Research Multi-Year Plan.
NHEERL's human health program is
organized around program projects, which
are multi-investigator, multi-disciplinary
efforts designed to address major research
themes. Programs projects were developed
by teams and subject to internal and external
review.
Research on Harmonization of Cancer and
Non-Cancer Risk Assessments
The assessment of health risk from
exposures to environmental agents has
traditionally depended on whether the
response is a cancer or non-cancer health
effect. However, there is a growing
consensus that there is a need to develop a
consistent, flexible set of principles for
using and drawing inferences from scientific
information in risk assessment. NHEERL's
research in this area will focus on
understanding the biological events that
precede toxic or adverse effects and
identifying common or similar modes of
action across cancer and non-cancer
endpoints that could provide the basis for a
harmonized approach for risk assessment.
Mechanistic information is also crucial for
reducing or replacing uncertainty factors in
risk assessment, especially for interspecies
extrapolation and for linking dosimetry
models such as pharmacokinetic (PK)
models with empirical or pharmacodynamic
(PD) models for effects of chemicals having
similar or differing modes or mechanisms of
action. Three program projects were
identified: (1) research on cell signaling
pathways, (2) neuroendocrine mechanisms,
and (3) P-450 and xenobiotic metabolizing
enzymes.
Susceptible Subpopulations
The variability in responsiveness of
humans to environmental pollutants can be
associated with differences in biological
susceptibility arising from intrinsic factors
such as life stage and genetics or with
acquired factors such as disease.
Approaches to better identify and
E-l
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characterize susceptible subpopulations by
describing the biological basis underlying
differential responsiveness is an overarching
goal of NHEERL. Research on susceptible
subpopulations will focus on developing a
scientific understanding of the biological
basis for differing responsiveness of
subpopulations within the general
population. NHEERL research on
susceptible subpopulations will also address
the role of life stage, genetic background,
and preexisting disease on responsiveness to
environmental agents. Seven program
projects were identified: four focusing on
life stage issues, one on asthma as a disease,
and two on genetic components of
susceptibility.
Cumulative Risk
Cumulative risk assessment is a major
concern to many Agency Program and
Regional Offices. NHEERL's research on
cumulative risk will focus on providing data
that may be used in the development and
refinement of approaches to predict
interactions of chemicals at low dose
concentrations. Research in this area will
utilize empirical and mechanistic data and
models to develop strategies to predict the
effects of chemical mixtures. The program
project identified in this area will explore
the limits of the additivity assumption that is
the standard assumption in non-cancer risk
assessment guidelines in the Agency. This
research will also address mixtures of
chemicals with common or dissimilar modes
of action and will evaluate the effects of
these mixtures in repeated exposure
situations.
The research described in this NHEERL
Human Health Research Implementation
Plan not only involves interdisciplinary
teams within NHEERL, but in many cases
involves collaborations with scientists from
other ORD Laboratories and Centers or
Agency Program Offices. Each of the
following sections attempts to link research
in that program project to other Agency
problem-driven research areas (i.e., air
toxics, drinking water, pesticides, and toxic
substances), as well as providing a narrative
on the programmatic impact of the work and
describing collaborative efforts with other
Laboratories and Centers within ORD.
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Section 1
Introduction
Purpose and Scope
This document, the NHEERL Human
Health Research Implementation Plan.,
describes the framework and
implementation plan for human health
research within the National Health and
Environmental Effects Research Laboratory
(NHEERL) beginning in FY02. Human
health research in the Office of Research
and Development (ORD) of the
Environmental Protection Agency (the
Agency) is based on needs and goals
established by the Agency in response to the
Government Performance and Results Act
(GPRA). The human health research
described in this document is core research
that aims to provide broad, fundamental
scientific information that will improve
understanding of problem-driven human
health issues arising from risk assessment in
the Agency's Program and Regional
Offices. Core research focuses on
developing and applying the best available
science for addressing current and future
environmental hazards, as well as on
developing new approaches for protecting
human health. NHEERL's human health
research program supports the identification
of fundamental mechanisms and
development of methods and models to
elucidate key scientific uncertainties
important to understanding and predicting
the effects of environmental agents on
human health.
This implementation plan on human
health describes the approach that NHEERL
will take over the next 8-10 years to address
the scientific uncertainties outlined in
ORD's Human Health Research Strategy
document, which includes harmonized use
of mechanistic data in risk assessment,
protecting the health of susceptible
subpopulations, and determining risk of
exposure to multiple chemicals. This
document also outlines in detail how
NHEERL will meet the Annual Performance
Goals (APGs) and Annual Performance
Measures (APMs) described in ORD's
Human Health Research Multi-Year Plan.
Multi-Year Plans (MYPs) provide a
framework to integrate research across
ORD's Laboratories and Centers, a basis for
creating annual plans, and a context within
which to understand how decision-making
in annual planning influences the ability of
ORD to meet future goals and outcomes.
MYPs identify Long-Term Goals (LTGs)
that are to be addressed over an 8-10 year
period while APGs and APMs represent
milestones that must be accomplished in
order to meet the LTGs.
Process for Developing this
Implementation Plan
This implementation plan was developed
through an iterative and interactive process
leading to the development of program
projects, which are multi-investigator, multi-
disciplinary efforts designed to address the
three major themes outlined in the ORD's
Human Health Research Strategy. A
Steering Committee oversaw the
development of the program projects and
was composed of two representatives from
each health division (one manager, one
scientist) and representatives from other
ORD Laboratories/Centers and Regional
Offices. NHEERL representatives on the
Steering Committee served as the Executive
Committee. ORD' s Human Health
Research Strategy and the F Y02 Human
Health Research Multi-Year Plan provided
background information and guided
strategic thinking as the planning effort
progressed.
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A scientist-to-scientist meeting was held
in November, 2001, to fashion a limited
number of integrated program projects
responsive to the programmatic needs of the
Agency. This meeting was attended by
approximately eighty NHEERL scientists
and twenty scientists from other parts of the
Agency (i.e., ORD, Program and Regional
Offices). Following this meeting, sixteen
preproposals were submitted by research
teams in NHEERL and reviewed by the
Executive Committee. At least one review
per preproposal was provided by a non-
NHEERL scientist. The Executive
Committee then recommended that eleven
of the research teams develop full proposals;
these evolved into the program projects
described in this document.
The NHEERL Human Health Research
Implementation Plan represents a work-in-
progress which puts forward current
thinking on major issues and ways to
address them. As the research progresses, it
will become increasingly apparent which
ideas and approaches are leading
successfully toward their goals and which
may not be. The Executive Committee will
convene a meeting of the researchers
involved in this effort approximately every
two years to present results and discuss
future directions. It is expected that
program projects will evolve over time in
response to the success of the research effort
and identification of new directions of
significance to the Agency. Changes in
work covered by the program projects at the
Laboratory level will be linked to strategic
revisions of the ORD Human Health
Research Multi-Year Plan.
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Section 2
Research Approach
Context for Research
The Agency is charged with the
responsibility of protecting public health
and the environment. To fulfill this
mandate, the Agency uses the process of
human health risk assessment to identify and
characterize environmentally related human
health problems. Human health risk
assessment provides a qualitative and
quantitative characterization of the
relationship between environmental
exposures and effects observed in exposed
individuals. In 1983, the National Research
Council (NRC) described four primary steps
in the process of risk assessment: hazard
identification, dose-response assessment,
exposure assessment, and risk
characterization. Risk assessment is the
primary scientific input to the risk
management process, which involves the
recognition of a potential new risk and
development, selection and implementation
of Agency actions to address the risk. Risk
management often considers a wide variety
of other factors. The overall process of risk
assessment and risk management is often
called the risk assessment/risk management
paradigm.
Risk assessment has become an integral
component of environmental decision-
making under the statutes implemented by
the Agency. There are many uncertainties
associated with the risk assessment process
because of severe limitations in available
data and the complex interactions between
the sources and environmental
concentrations of contaminants, dose at the
target site, and response. These
uncertainties frequently result in the use of
default assumptions and uncertainty factors
in risk assessments. NHEERL's human
health research program is based on the
assumption that major uncertainties in risk
assessment can be reduced by understanding
the fundamental determinants of dose and
the basic biological changes that follow
exposure to a chemical.
Human Health Research Themes
Use of Mechanistic Data in Risk Assessment
Protect Susceptible Subpopulations
Assess Cumulative Risk
Based on input from Regional and
Program Office risk assessors, human health
research at NHEERL will focus on three
major themes as described in the ORD's
Human Health Research Strategy and the
Human Health Research Multi-Year Plan:
(1) the use of mechanistic data in risk
assessments, (2) protection of susceptible
subpopulations, and (3) assessment of
cumulative risk. Research on harmonizing
risk assessment approaches will lead to a
common set of principles and guidelines for
drawing inferences about risk from available
scientific information. The overall goal of
this research, which is described in Section
3 of this document, is that Program and
Regional Office risk assessors will use
mechanistic data in a harmonized manner
for risk assessments for all health endpoints.
NHEERL will also focus on developing a
scientific understanding of the biological
basis for differing responsiveness of
subpopulations within the general
population. Research on biological
susceptibility, which is described in Section
4, will focus on the role of intrinsic factors
such as life stage and genetic background
and extrinsic factors such as preexisting
disease on responsiveness to environmental
pollutants. NHEERL's human health
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Limits of
Additivity
Biological
Basis of
Susceptibility
Use of Mechanistic
Information in
Risk Assessment
Scientific
Foundation for Risk
Assessment
1
Clean
Air
1
Safe
Drinking
Water
r i
Clean
Water
TSCA
i
FIFRA
Figure 1 Mandates for Human Health Research
research program will also address the fact
that humans are exposed to mixtures of
pollutants from multiple sources. This
research will provide scientific support for
decisions concerning exposure to a multiple
pollutants by assessing additivity as the
default assumption describing interactions
between chemicals in mixtures (see Section
5 of this document).
Authorization and Mandate for Human
Health Research
One of the reasons for focusing on human
health research is that virtually all of the
Agency's major legislative mandates (those
which require the Agency to promulgate
regulations to protect the public health and
welfare from environmental contaminants)
require that the Agency develop human
health risk assessments. These laws (and
amendments) include the Clean Air Act; the
Safe Drinking Water Act; the Clean Water
Act; the Toxics Substances Control Act
(TSCA); the Federal Insecticide, Fungicide,
and Rodenticide Act (FIFRA); the
Resources Conservation and Recovery Act;
the Comprehensive Environmental
Response, Compensation, and Liability Act;
the Superfund Amendments Reauthorization
Act; and the Food Quality Protection Act
(FQPA). In addition, Congress enacted
legislation in 1988 which mandated that the
Agency undertake Research to Improve
Health Risk Assessment. NHEERL's
research to harmonize the use of
mechanistic data in risk assessment, protect
susceptible subpopulations, and assess
cumulative risk will contribute to building
the scientific framework for human health
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Environmental
Release
Fate/Transport
Models/Data
Environmental
Concentration
Exposure
Models/Data
Exposure
Concentrations
PBPK
Models/Data
Target Organ
Dose
BBDR
Models/Data
Early Biological
Effects
Systems
odels/Data
Figure 2 The Source-to-Outcome Continuum
Adverse Outcome
risk assessments required by various
legislative mandates (Figure 1). Of
particular relevance to the human health
research program is the that the FQPA
requires children's risks to pesticide
exposures be considered during the
tolerance setting process. FQPA requires
the development of scientific information
necessary to "...ensure that there is a
reasonable certainty that no harm will result
to infants and children from aggregate
exposure to the pesticide chemical
residue...." Thus, information on children's
exposure is required to (1) consider the
susceptibility of children to increased
exposure and (2) account for aggregate
exposures to pesticides from all sources,
including food, drinking water, and
applications of pesticides in homes, schools,
daycare centers, and other micro-
environments. Because of these FQPA
mandates, the results of NHEERL's research
on susceptible subpopulations will be
important for risk assessments by the Office
of Prevention, Pesticides, and Toxic
Substances (OPPTS). This Office is
required to establish allowable levels of
exposure to pesticides and toxic chemicals
and protect the most susceptible
subpopulations. The Safe Drinking Water
Act Amendments (1996) also requires
consideration of susceptible subpopulations,
including children, when setting standards
for chemicals and other contaminants in
drinking water. Section 408 of the Federal
Food, Drug, and Cosmetic Act, as amended
by FQPA, identified several research issues
related to the determination of risk of
exposures to pesticides, including aggregate
exposure and cumulative risk, susceptible
subpopulations, an additional factor of 10
margin of safety for children, and questions
related to extrapolation of data in risk
assessment.
Multidisciplinary Human Health
Research at ORD
Research on human health at NFtEERL
is viewed as part of a multidisciplinary
research program within ORD that addresses
linkages lying along a continuum from the
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source of an agent through exposure and project based on FY03 FTE projections can
dose to adverse effects or outcomes (Figure be found in Appendix A.
2). Research at NHEERL focuses on
developing physiologically based,
pharmacokinetic (PBPK) models to
determine target organ dose, biologically
based dose response (BBDR) models to
determine early biological effects in
pathways associated with toxic effects, and
models to predict adverse effects. NHEERL
human health research aims to reduce
uncertainties in the risk assessment process,
including extrapolation across species,
extrapolation from short- to long-term
lifetime exposures, and variability of
response within the human population. For
purposes of dose assessment, NHEERL
works with the National Exposure Research
Laboratory (NERL) to develop
pharmacokinetic (PK) models to estimate
internal dose metrics and establish the basis
for metabolic differences between species.
The National Center for Environmental
Assessment (NCEA) performs complex risk
assessments of national interest and
develops risk assessment methods,
databases, and tools based on results from
NHEERL and other laboratories. The
National Risk Management Research
Laboratory (NRMRL) focuses on providing
the most effective and useful risk
management options and increasing linkage
between risk assessment and risk
management efforts. Intramural research
conducted by NHEERL is complemented by
extramural research sponsored by the
Agency's National Center for
Environmental Research (NCER). Through
the Science to Achieve Results
(STAR) Program, NCER supports grants
that focus on specific research needs
consistent with the mission of the Agency.
Specific linkages between NHEERL
research on human health and other
Laboratories/Centers within ORD, as well as
with other research groups, is described in
greater detail in the following sections.
Resource allocation for each program
2-4
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Section 3
Harmonization of Cancer and Non-Cancer Risk Assessments
3.1 Problem
The assessment of health risks from
exposures to environmental agents has
traditionally depended on whether the
response is a cancer or non-cancer health
effect. However, the 1997 report Risk
Management in Regulatory Decision
Making from the Commission on Risk
Assessment and Risk Management
concluded that the simple dichotomy
between cancer and non-cancer risk
assessment is not fully supportable by
current scientific evidence because it results
in expressions of risk that are not directly
comparable and differ significantly in
defining maximal exposures considered to
have negligible risk. In addition, the
National Research Council's report on
Science and Judgment in Risk Assessment
(NRC, 1994) noted the importance of a risk
assessment approach that is less fragmented,
more consistent in application of similar
concepts, and more holistic than endpoint-
specific guidelines. The Agency's Risk
Assessment Forum has also been actively
involved in reexamining approaches to
cancer and non-cancer risk assessment by
sponsoring internal colloquia to encourage
input and discussion between scientists and
risk assessors. One conclusion from these
discussions is that there is a need to develop
a consistent, flexible set of principles and
guidelines for using and drawing inferences
from scientific information in risk
assessment.
One issue related to the disparity in
approaches to cancer and non-cancer risk
assessments is the limited understanding of
the mode (MOA) or mechanism of action of
compounds. This lack of knowledge has
largely been due to limitations in the
experimental designs, methods, and animal
models that have been used to support
hazard identification and dose-response
phases of human health risk assessments.
Therefore, understanding an agent's MO A is
key to more accurate prediction and
characterization of hazard and risk and is the
basis for developing harmonized risk
assessment approaches for all health
endpoints. At present, risk assessment
guidelines for cancer and non-cancer
endpoints differ with respect to the use of
MOA information. Guidelines for non-
cancer effects such as reproductive toxicity
(US EPA, 1996) and neurotoxicity (US
EPA, 1998) use mechanistic information
largely in a weight-of-evidence framework
to strengthen the evidence that a human
hazard could exist. For example,
mechanistic data could be used to support
findings from an epidemiological study or
results from animal toxicity studies.
Mechanistic data may also be used on a
case-by-case basis to reduce the magnitude
of uncertainty factors used to calculate the
Reference Dose (RfD), Reference
Concentration, or Benchmark Dose. For
example, if the same MOA could be
demonstrated to occur in humans and
animals, the uncertainty factor for animal-
to-human extrapolation could be reduced in
the risk assessment.
The use of MOA information is spelled
out in detail in the draft final Guidelines for
Carcinogen Risk Assessment (US EPA,
2003). As in the case of non-cancer
endpoints, MOA data can be used in a
weight-of-evidence framework to support
the evidence for carcinogenic hazard
potential based on epidemiological or
animal toxicity studies. On the other hand,
these guidelines note that MOA information
is key to addressing various default options
in the risk assessment. BBDRs using
5-1
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mechanistic information can be used to
relate dose and response on an agent-
specific basis and to extrapolate to lower
dose levels, if needed. MOA data can be
crucial for establishing the point of
departure (POD) for risk assessment, the
linearity or non-linearity of the dose
response relationship, and the biological
plausibility of a response. MOA data are
also important for determining key events
that are of increased concern to susceptible
subpopulations, such as children, and
extrapolation from animals to humans.
MOA information plays an important role in
developing PK models to estimate dose in
target tissues and helps define relative
sensitivity of various tissues to carcinogens.
Unlike risk assessment guidelines for non-
cancer endpoints, the draft Cancer
Guidelines contain a framework for the
analysis of carcinogenic MOA information,
including steps necessary to define the
postulated MOA, identification of key
events, and assessment of experimental
evidence supporting the postulated MOA.
There is a clear need to develop a
common approach for the use of MOA data
in risk assessment (Bogdnanffy et al., 2001).
In the context of this implementation plan,
harmonization refers to developing a
consistent, flexible set of principles for
using and drawing inferences from available
information on MOA to support risk
assessment. "Mode of action" is defined as
a series of key events starting with
interaction of an agent with a cell and
proceeding through functional and
anatomical changes to result in an adverse
effect. "Mechanism of action" implies a
more detailed understanding and description
of the key events, including the molecular
level. Mechanism of action often describes
how a chemical works at all levels of
biological organization. For the purposes of
this document, mode and mechanism of
action are used interchangeably in this
document. "Key events" are defined as
empirically derived precursor steps linking
the proximal event to the adverse outcome.
The sequence of key events leading to an
adverse effect at a given internal dose is also
known as the "toxicity pathway."
Toxicities arise from a range of factors that
may be specific to the chemical or chemical
class. The physiology of the organism and
other environmental factors may affect the
expression of toxicity. A framework for
harmonization of risk assessment
approaches must facilitate incorporation of
data that accounts for these factors.
3.2 Goals
The overarching goal of mechanistic
research at NHEERL is to help derive a
commonly accepted set of principles
defining how MOA information can be used
in risk assessment, particularly as it relates
to extrapolation in risk assessment. The
NHEERL human health research
implementation plan addresses how MOA
information can be used in a more consistent
way to improve dose-response assessment
for the following:
For various types of cancer and non-
cancer endpoints,
For chemicals producing different
toxicities by a similar MOA, and
For chemicals producing similar
toxicities by different MOAs.
This research also addresses how
mechanistic data can be used in risk
assessments to
Compare adverse outcome across
toxicities,
Select the POD and approaches for
low-dose extrapolation, and
Account for inter- and intra-species
differences in PK and PD capabilities
for all health endpoints.
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Develop
Framework
to Define
Mechanism
Develop
Methods to
Study
Mechanism
Identify
Common
Modes
of Action
Dose-Response
Studies
on Prototypic
Chemicals
Framework for the Use of Mechanistic Information in
Risk Assessment, Especially as it Relates to Animal to
Human Extrapolation
Figure 3 Critical Path for Harmonization Research
3.3 Critical Path
Consistent utilization of MO A
information in risk assessment involves
several critical steps, as outlined in Figure 3.
In order to achieve a more harmonized
approach for risk assessment based on the
use of MO A data, there must first be a
common framework for defining MOA.
Questions to be addressed include the
following:
How much evidence is needed to show
that a substance acts via a particular
MOA in animals and its applicability
to humans?
How to show that two toxic
manifestations caused by the same
substance were produced by different
MOAs?
How to show that a common effect
produced by two chemicals may have
been caused by two different MOAs?
What should be the dose-response
assessment approach for a chemical
that produces multiple toxic
manifestations, but through a similar
MOA?
It is also highly probable that a key
element of research on the use of MOA data
in risk assessments will be the application of
emerging technologies, especially in
molecular biology. Methods and
approaches based on proteomics and
genomics will likely prove crucial in testing
hypotheses concerning common key events
that lead to both cancer and non-cancer
effects. Of particular concern will be
approaches to quantify changes in gene and
protein expression in toxicological studies
and to interpret such changes in a risk
assessment context. This will determine the
extent to which these emerging technologies
could be used to inform mechanistic
questions in risk assessment. A research
program applying molecular biological
techniques with mathematical and computer
models for prediction of effect and
understanding MOA is being developed by
ORD and is described in the draft document
A Framework for a Computational
ToxicologyResearch Program in ORD.
Once a framework has been developed
to define MOA for use in risk assessment,
work will be done to identify possible
common modes of action for chemicals
producing cancer and non-cancer effects.
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Research will develop a clear understanding
of the biological changes that occur
following delivery of the active chemical
moiety to target sites and the relationship of
response to dose. Emphasis will be placed
on identifying possible common key events
(e.g., cell proliferation, receptor interaction,
response to injury or stress) for prototypic
chemicals that have both cancer and non-
cancer effects. The overall objective of this
research will be to demonstrate the
feasibility of determining potential common
precursor MO As for cancer and non-cancer
effects. This information will provide the
basis for subsequent research to characterize
precursor steps for several prototypic
chemicals. Issues that will be addressed by
this research include the following:
How MO A data at the low end of the
dose-response curve can inform
decisions about the most appropriate
risk assessment model,
How MOA data can inform decisions
about the presence or absence of a
threshold, and
How mechanistic information can be
used to select the POD.
APGs and APMs related to research on
the use of mechanistic data in risk
assessment in the ORD Human Health
Research Multi-Year Plan cross referenced
to each program project can be found in
Appendix B.
3.4 Program Projects
Research on harmonization of risk
assessment consists of three program
projects addressing MO As and their use in
risk assessment. Program project 1
addresses the hypothesis that exposure to
different classes of chemicals will disrupt a
common key event, i.e., mitogen-activated
protein kinase (MAPK), in multiple tissues
leading to a range of toxicities. Program
project 2 addresses the hypothesis that
disruption of luteinizing hormone (LH)
secretion serves as a common MOA for
altered fertility, reproductive disease, and
cancer of the reproductive system in animals
and humans. Program project 3 addresses
the hypothesis that modulation of
cytochrome P-450s and other xenobiotic
metabolizing enzymes (XMEs) will lead to
common MOAs for multiple toxicities. A
fourth program project "Environmental and
Genetic Interactions in Hypertensive Rats:
Oxidative Stress as a Common
Susceptibility Attribute for Non-Cancer
Risks" contains some mechanistic research
concerning oxidative stress as a common
MOA. However, the focus of this program
project is primarily on questions related to
differential responsiveness to chemical
exposure, i.e., susceptible subpopulations
(see Section 4 for details). These four
themes were selected because they involve
toxicity pathways associated with prominent
adverse health effects of high priority to risk
assessors (i.e., cancer, reproductive and
pulmonary toxicity, neurotoxicity).
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Program Project 1: Harmonization of Cancer and Non-Cancer Risk
Assessment: Disruption of Mitogen-Activated Protein (MAPK) Signaling as
a Common MOA for Environmental Toxicants
Objectives:
Use both in vitro and in vivo models to
demonstrate that diverse groups of
chemical compounds can disrupt MAPK
signaling in multiple tissues,
Examine the disruption of MAPK
signaling as a common MOA leading to
the expression of multiple adverse effects
using pharmacologic and molecular
manipulations of the MAPK signaling
cascade as well as environmental
toxicants, and
Demonstrate that changes in MAPK
signaling studied in vitro can be used to
extrapolate the MOA of chemicals in
vivo. Determine the degree of homology
of response between animals and humans.
Scientific Approach
The transmission of external signals to
the interior of the cell is a fundamental
process in the regulation of cellular
responses to physiological stimuli. Virtually
every reaction that a cell makes in response
to its environment is regulated by signaling
processes. These signaling pathways exist
in all cell types and determine the activity of
the cell ranging from quiescence to fully
stimulated. Cell signaling pathways are
known to be upstream mediators of a
number of basic biological processes
including cell cycling, proliferation,
migration, differentiation, plasticity,
inflammation, and cell death. Signal
transduction pathways are critical
intermediates, accepting and integrating
extracellular information with the nucleus to
affect gene transcription and invoke a
biological response. Thus, cell signaling
pathways have the potential to serve as early
and sensitive indicators of biological
perturbation (NRC, 2000). In addition,
understanding the signal transduction
pathways that underlie the biological
response to environmental and chemical
insult is critical to identifying the MOA
leading to adverse health effects.
Protein kinases are signaling molecules
that interact with other messenger systems
and form a highly structured network to
integrate cell functions in an organism.
These signaling pathways consist of proteins
grouped in "cascade" fashion which
typically originate in the cell surface and
reach into the cell cytosol and nucleus.
Each of the proteins has a structural and/or
catalytic function that fits in the overall
pathway to attenuate the specificity,
magnitude, and duration of the signal being
transmitted. The pathway to be studied in
this proposal, the MAPK cascade, contains
receptor kinases as well as serine/threonine,
tyrosine, and dual specificity kinases with
multiple regulatory domains that determine
the specificity of the interactions (Chang
and Karin, 2001).
A number of studies conducted by
NHEERL scientists and others have
demonstrated that environmental
contaminants can disrupt signaling
processes in target cell types in a variety of
organ systems, including the lung, the
nervous system, the reproductive organs,
and the developing fetus. Although these
compounds differ markedly in their
physicochemical properties, they appear to
share a common MOA in their ability to
damage cell signaling by interfering with or
enhancing signal initiation, transduction, or
termination. Physiologically inappropriate
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Project 1
Project 2
Projects
Project 4
Demonstrate that
Diverse Classes of
Chemicals affect
MAPK
Determine if Dis-
ruption of MAPK
Leads to Multiple
Adverse Effects
Show that Effects
in In Vitro Models
Can Be Seen In
Vivo
Demonstrate Applic-
ability Across
Species
Demonstrate Disruption of
MAPK Underlies Toxicity
of Diverse Groups of
Chemicals
Figure 4 Critical Path for Research on Cell Signaling
signaling appears to result in a broad range
of toxic responses, including apoptosis,
inflammation, metaplasia, hyperplasia,
hypertrophy, and neoplasia. Because
MAPKs are involved in these basic cellular
processes, it is proposed that disruption of
MAPK signaling may be a common MOA
that underlies the multiple adverse effects
observed in diverse tissues. The critical
path for research on cell signaling is
illustrated in Figure 4.
Project 1 - Evaluation of MAPK
Signaling after Exposure to
Environmental Toxicants. External
stimuli play a major role in regulating
complex intracellular processes such as gene
expression, cell proliferation, growth,
differentiation, and death. These stimuli
include cell-substrate adhesion, growth
factors, hormones, neurotransmitters, and
cytokines that stimulate a plethora of
receptors. Environmental toxicants can act
at many of these sites to perturb intracellular
signaling, and, depending on exposure
conditions, tissue, and life stage, can result
in wide variety of adverse effects. In order
to harmonize risk assessment approaches it
will be necessary to determine whether the
array of possible upstream targets of
toxicants have a common downstream
response. In a multitude of cell types,
activation of a variety of receptors (e.g., G-
protein coupled receptors, ion channels,
receptor tyrosine kinases and cytokine
receptors) trigger the MAPK cascades which
serve as central downstream integrators to
modulate the cellular response to external
stimuli. Research in this project will test the
hypothesis that exposure to different classes
of chemicals will disrupt MAPK signaling
in multiple tissue types. This work is
intended to determine whether effects on
diverse upstream targets will lead to changes
in a common downstream endpoint, MAPK,
and provides the basis for continued testing
of MAPK signaling as a common MOA.
The studies outlined in this project will
use several different in vitro and in vivo
models to understand the potential for
altered upstream signaling to lead to
changes in MAPK in embryonic tissue
(developmental toxicity), brain tissue
(neurotoxicity), pulmonary tissue (airway
disease), and skin epithelium (cancer).
Because several different model systems
will be used, the chemicals to be examined
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will vary. For each model system,
chemicals with known adverse effects will
be chosen based on existing data. Research
in project 1 will demonstrate that known
environmental toxicants which act on
diverse cellular targets in multiple tissues
ultimately lead to alterations in MAPK
signaling. Based on considerations of dose-
response and toxicity, chemicals and models
which show a change in MAPK signaling at
relevant doses will be chosen for further
study in project 2. Research in project 1
will include the following:
Effects of arsenic (As) on cell
signaling in primary human
keratinocytes and bladder epithelial
and endothelial cells,
Effects of particulate matter (PM) on
MAPK cascade components inhuman
airway epithelial cells,
Effects of toxicants on ligand-receptor
interactions and subsequent MAPK
signaling events in the developing
embryonic palate,
Effects of developmental toxicants that
alter protein kinase and MAPK
signaling in whole embryo cultures,
Effects of toxicants that perturb
electrical excitability, integrin-receptor
interactions or calcium regulation of
MAPK signaling events in neuronal
tissue, and
Use of flow cytometry and confocal
microscopy to detect MAPK signaling.
Project 2 - Disruption of MAPK Signaling
as a Common MOA Leading to
Expression of Multiple Adverse Effects.
The studies in project 1 are designed to
demonstrate that toxicant-induced effects on
a variety of upstream targets ultimately
converge to perturb MAPK signaling. The
critical question to be addressed next is
whether the observed changes in MAPK are
related to the expression of toxicity in
multiple tissues. It is clear that MAPK
signaling is an important mediator of critical
cellular functions including embryogenesis,
cell proliferation, cell differentiation, and
cell death. Thus, alterations in MAPK
signaling is expected to be part of the MOA
that leads to changes in critical cellular
functions which are ultimately expressed as
the adverse effect in the different tissues and
model systems described above. This work
will attempt to tie the changes in MAPK
signaling identified in project 1 to
consequences downstream from this critical
signaling event. Research covered in project
2 will include the following:
Studies on MAPK-induced disruption
of cell cycle progression leading to
augmented cell proliferation and
carcinogenesis by arsenicals,
Effects of PM metal s on MAPK
activation and subsequent gene
transcription,
Studies on toxicants that interfere with
ligand-receptor interactions to alter
critical signaling events in the
developing embryo,
Effects of disruption of MAPK
signaling and craniofacial
development by altering differentiation
of neural crest cells after exposure to
haloacetic acids (HAAs) in vivo and in
vitro,
Research to determine if disruption of
cell signaling is a MOA by which
developmental neurotoxicants alter
neuronal differentiation and
synaptogenesis,
Studies to determine if disruption of
cell signaling is a MOA by which
developmental neurotoxi cants interfere
with activity-dependent plasticity in
intact neural circuitry, and
Use flow cytometry and confocal
microscopy detection of MAPK
signaling to demonstrate that alteration
of MAPK affects basic cellular
processes, including proliferation and
apoptosis.
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Project 3 - Use of In Vitro Signaling Data
for Extrapolating the MOA of
Environmental Toxicants In Vivo. The
Agency depends on mechanistic
toxicological research to provide biological
plausibility to strengthen the knowledge
base used in risk assessments in a weight-of-
evidence context. However, much of the
molecular and biochemical toxicological
research is presently conducted using in
vitro models which vary in their ability to
approximate toxic responses in the whole
animal or human. Therefore, evaluation of
the relevance of in vitro mechanistic
findings, such as MAPK activation, to in
vivo toxicity would lead to increased
confidence in the use of mechanistic data in
support of regulatory decision-making. This
project will determine if changes in MAPK
signaling studied in vitro can be used to
extrapolate the MOA of chemical
compounds in vivo. Research in project 3
will:
Determine the effects of PM metals in
MAPK activation in vivo and in vitro,
Compare the responses to
developmental toxicants in embryonic
organ culture to those observed
following in vivo exposure,
Determine the role of integrin-
mediated initiation of MAPK signal
transduction in vivo in neurotoxic
effects in vitro and in vivo,
Conduct in vitro and in vivo effects of
bioaccumulative chemicals such as the
polychlorinated biphenyls (PCBs) on
MAPK signaling in the developing
nervous system, and
Compare cell signaling in the nervous
system in vitro and in vivo and
determine it relationship to cognitive
effects of environmental toxicants.
Project 4 - Extrapolation of Perturbations
in MAPK Signaling from Animals to
Humans. MAPK signaling cascades are
highly conserved across species, indicative
of the critical role these signaling pathways
play in cell function. Due to this high
degree of conservation, it is proposed that
toxicants which perturb these pathways in
one species will have qualitatively similar
effects in the same tissue of another species.
For the process of risk assessment, the
uncertainty associated with extrapolation of
data from animals to humans can be reduced
if it is demonstrated that the MOA is the
same in both species and if quantitative
differences can be accounted for through
data generated in tissues of humans and
animal models. Project 4 will determine if
effects of environmental toxicants on
MAPK in one species will be qualitatively
similar to those effects in other species. The
studies outlined in this project will use
several different in vitro models in which
comparable rodent and human tissue can be
obtained. These studies will also make use
of the information gained in projects 1 and 2
in order to focus on the upstream targets,
specific MAPK pathways, and critical
biological effects important to the
elaboration of pathogenesis for a particular
model. Research covered by this project
includes the following:
Research to determine if MAPK
signaling is perturbed by methylated
trivalent arsenicals in mouse and
human primary keratinocytes,
Research to determine the effects of
toxicants on MAPK signaling in
human and rodent embryonic palatal
cells, and
Research to determine if alterations in
cell signaling produced by
neurotoxicants in rodent neurons are
similar in human neurons.
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Impact
Cross-Agency Interactions
A major goal of ORD's research program
on human health is to develop a consistent,
flexible set of principles for determining risk
based on pertinent mechanistic data,
regardless of the nature of the toxicity (i.e.,
harmonize cancer and non-cancer risk
assessment approaches). There is an
emerging knowledge base which suggests
that the biochemical changes leading to
certain cancer and non-cancer health effects
are similar, and a more accurate prediction
and characterization of risk for cancer and
non-cancer endpoints can be based on
understanding a chemical's MO A. In order
to harmonize the risk assessment process for
cancer and non-cancer health effects,
information is needed regarding the
application of mechanistic data for
chemicals that produce different toxic
outcomes by a similar MOA. The goal of
this program project is to identify
intracellular signaling pathways critical to
the expression of carcinogenic and
noncarcinogenic effects following chemical
insult. This research will show that
disruption of critical cell signaling pathways
is a common MOA that underlies the
toxicity of diverse groups of chemicals, that
can be used as an in vitro predictor of the
mechanism of toxic response in vivo and
extrapolate from experimental animals to
humans. This program project will provide
an example of how data on common MOA
can be used for comparative risk assessment
across biological endpoints and also reduce
the reliance on uncertainty factors by
providing a common measure for
extrapolation. In addition, the results of
these studies may identify scientifically
defensible biomarkers of exposure and
effect.
As this program project progresses,
consultations with scientists from NCEA
will be held on a periodical basis in order to
develop principles that can be used to
formulate guidance documents on the
harmonized use of MOA data in risk
assessment.
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Program Project 2: Disruption of Luteinizing Hormone (LH) Secretion as
a Common MOA for Altered Fertility, Reproductive Disease, and Cancer of
the Reproductive System
Objectives
Characterize the range of environmental
agents that alter LH secretion,
Identify central nervous system (CNS)
mechanisms involved in this MOA,
Compare effects in animal models to
those in humans, and
Determine key events necessary for
altered reproductive function,
reproductive diseases and cancer of the
reproductive system in the rodent and
potentially the human.
Scientific Approach
One of the more difficult issues that
confront risk assessors is the interpretation
of toxicology data obtained in animal
species and applying them to human risk
assessment. A key to using such
information in risk assessment is the degree
of homology between humans and the test
species; that is, whether there exists a
common MOA. Within this context, the
research in this program project is intended
to derive a set of principles for application
to animal data pertaining to toxicants that
alter serum LH. This effort will focus on
how LH disruption, in both the test species
and humans, contributes to impaired fertility
and reproductive disease. These principles
can serve as a framework for evaluating the
effect of environmental chemicals that alter
the neuroendocrine control of LH.
Moreover, as a valuable tool for examining
test data, it will serve as a prototypical
approach for examining dysregulation in
other neuroendocrine axes of importance to
human health risk assessment (i.e., prolactin
and reproductive tumors;
thyroid-stimulating hormone [TSH] and
thyroid function; and adrenocorticotropin
hormone and adrenal function).
Many xenobiotics have been shown to
influence the regulation of LH. However,
the actual mechanism (e.g., receptor,
enzyme) may be different depending on the
toxicant. Recently, alterations in LH
secretion have been identified as the MOA
for altered reproductive function (i.e.,
delayed puberty in both sexes, altered
ovarian function in the adult female and
pregnancy loss), premature reproductive
aging, and mammary gland tumor
development in the female following
chlorotriazine treatment (Cooper et al.,
1998, 1999, 2000; Laws et al., 2000;
Narotsky et al., 2001; Stoker et al., 2002).
Similarly, exposure to selected xenobiotics
will produce changes in the secretion of LH
in the male resulting in decreased
testosterone; impaired fertility, or, as in the
case of linuron exposure, elevated basal LH
associated with an increased incidence of
Ley dig cell tumors. These effects of altered
LH secretion generally require a protracted
treatment before the adverse effect is
observed. In contrast, it is important to note
that all environmental compounds that
modify LH secretion do not necessarily
result in the same sequence of adverse
reproductive or cancer effects. Thus, all
toxicants that alter the LH surge (the MOA)
may not lead to the same adverse outcomes.
Based on these observations, the
fundamental hypothesis to be examined
herein is that, because the LH surge can be
modified by environmental agents that
interact with a variety of different CNS
mechanisms, the adverse outcomes observed
(in both rats and humans) will vary
depending on which CNS target site is
involved. Thus, a better understanding of
the dynamics involved in the disruption of
the LH surge will allow us to better predict
which adverse outcome(s) will emerge and
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Project 1
Project 2
Project 3
Project 4
Identify Range of
Chemicals that
Modify Regulation
ofLH
Identify Mechanisms
Involved in
Altering LH
Characterize Adverse
Cancer and Non-
Cancer Effects
Mediated by LH
Study Effects of
Cumulative
Exposures
Demonstrate How LH Disruption
Impairs Fertility and Reproductive
Health in Humans and Animals
Figure 5 Critical Path for Research on Luteinizing Hormone
thus the relevance of each to potential
human reproductive disorders can be better
predicted. These studies will attempt to link
the key cellular events responsible for
changes in LH and adverse outcome in
animals and humans, as summarized in
Figure 5.
Project 1 - Identification of the Range of
Chemicals that Modify the Regulation of
LH. The purpose of this project is to
determine the range of chemicals that may
influence the secretion of LH. Once a
chemical has tested positive in this
evaluation, the dose- and time-
response associated with oral dosing will be
characterized using well-established
techniques. These studies will provide
important data describing the range of
environmental chemicals that alter LH
secretion, as well as the baseline
information necessary for at least two of the
projects described below. To date, several
groups of environmental compounds have
been shown to disrupt LH secretion in the
female rat; however, the universe of
chemicals that may possess such effects
remains to be determined. To identify
compounds for evaluation and testing, two
different approaches have typically been
used. First, those agents reported to affect
any of the hypothalamic targets known to
regulate gonadotrophin-releasing hormone
(GnRH) and LH secretion would be
predicted to affect LH in an ovariectomized,
estrogen-primed rat model. Second, those
compounds known to affect the estrogen or
androgen receptor would test positive in
assays using the intact female or male
respectively. Either one or both models can
be used to fully characterize the effect of the
compound on LH. A third approach has
been to identify compounds that, in vivo, are
reported to cause adverse reproductive
effects suggestive of LH disruption (i.e.,
altered cycling in the female rat, blockade of
the LH surge, Leydig cell hyperplasia in the
male).
Project 2 - Identify the Mechanisms
Involved in Altering LH. The regulation
of the LH surge is under the control of a
variety of neurotransmitters and
neuropeptides. Of particular interest is the
fact that several xenobiotics affect
noradrenergic and cholinergic
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neurotransmission because both these
neurotransmitters have been shown to
regulate the pulsatile release of GnRH and
subsequently LH secretion. Thus,
compounds that alter noradrenergic (NE)
synthesis (i.e., chlorotriazines [atrazine,
simazine, propazine] and dithiocarbamates
[thiram, metam sodium, disulfiram, carbon
disulfide]), block NE receptors (i.e.,
chlordimeform, amitraz, etc.) or
acetylcholine availability (i.e, carbamates)
have all been reported to alter both basal LH
secretion and the LH surge. Other
environmental agents have been reported to
alter the regulation of LH secretion, but the
mechanism(s) remains to be determined.
The purpose of project 2 is to develop a
database that documents the various
mechanisms through which the regulation of
LH secretion (both basal and pulsatile) may
be altered. Should a compound be shown to
alter the regulation of LH (project 1) by
modifying pituitary or circulating
concentrations, studies will be initiated to
determine the particular mechanism of
action underlying the observed effect. The
particular approach(es) employed will
depend upon published information about
the compound in question, including
available data about the compound's effects
on other physiological processes using
similar mechanisms of regulation and
structure-activity (SAR) data available for
other members of its chemical.
Project 3 - Characterization of Adverse
Effects on Reproductive Function,
Reproductive Diseases, and Cancers of
the Reproductive System following
Modification of LH Secretion. This
research will use two approaches. The first
approach addresses the hypothesis that
environmental chemicals that change the
timing or magnitude of LH release will alter
reproductive function in both males and
females. The purpose of these studies is to
characterize the extent to which compounds
that interfere with the regulation of LH alter
a number of reproductive processes and to
provide an approach that will form the basis
for comparison between adverse
reproductive outcomes in the rodent versus
human. The second approach will address
the hypothesis that long-term alterations in
the endocrine milieu will lead to
reproductive diseases and/or cancer. These
studies will be dependent upon data
obtained from studies described in the
approach described above. For example, in
cases where the chemical causes long term
elevation of LH, additional endpoints will be
included in the male and female pubertal
studies to evaluate changes in ovarian and
testicular histology. These studies will
evaluate endpoints that are most relevant to
potential human health concerns associated
with this MO A.
Project 4 - Cumulative Exposures to
Environmental Compounds which
Suppress LH. There is concern that
exposure to multiple chemicals may alter the
effect in the target organ in ways not
predicted or expected based on the
dose-response curves of the single
chemicals and an assumption with regard to
additivity (i.e, toxicity may be greater or
less than expected based on the single
chemical information and an assumption of
either dose addition or response addition).
In addition, there is concern that cumulative
exposure may cause toxicity in an
unexpected organ. The default assumption
for risk assessment of non-cancer endpoints
is that toxic chemicals with similar MO As
will combine to cause toxicity in a dose-
additive manner. However, there are
relatively few data supporting this
assumption, particularly for scenarios where
toxic chemicals with similar MOAs, but
different mechanisms will combine to cause
toxicity. This scenario warrants further
study. In this project, chemicals from
pesticide classes which are identified in
projects 1 and 2 as having the MO A in
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suppressing LH will be evaluated in a dose-
related manner. Once the MO A and
portions of the specific mechanism within
each general class are determined, low-dose
cumulative exposure experiments will be
designed. This project is linked directly
with research on Cumulative Risk (see
Section 5). All APMs for this project are
reported in this Section on harmonization of
the use of mechanistic data in risk
assessment because the primary focus of the
research is on identification and
characterization of the LH MO A.
Impact
LH secretion is considered to be an
important MOA for risk assessment, and
chemical specific data will address key
issues such as whether or not LH secretion
is modified following exposure, the dose
response, the pattern of change that occurs
(chronic or acute disruption), the adverse
outcomes that develop, and the mechanism
involved. This information is critical for
determining the potential health risks of
exposure to the chemical in humans. By
determining the key events involved in
toxicant-induced chemical changes in LH
secretion, a better understanding of the
homology between the rat and human will
evolve. This research will also provide
significant information concerning the
conditions underlying altered reproductive
function and the role they may have in the
development of tumors of the reproductive
system (i.e., harmonization of cancer and
non-cancer endpoints).
The work evaluating cumulative effects
included in the current proposal will also
address important questions concerning the
evaluation of mixtures. There is a
substantial degree of confusion within the
Agency concerning how to select chemicals
to be used in a cumulative risk assessments.
The approach to be taken in this program
project will be to link the cellular MOA
responsible for the change in LH and
adverse outcome. In these studies, the
working hypothesis will be that compounds
with similar mechanisms will show dose
additivity; whereas combined exposure to
compounds with different mechanisms
would be either additive and potentially
more than additive or less than additive.
Cross-Agency Interactions
The research efforts will involve ongoing
communications with OPPTS. The effect of
environmental agents on LH secretion and
the demonstrated role of altered LH
secretion in the development of adverse
reproductive and cancer outcomes was of
primary significance to the risk assessment
of the chlorotriazine pesticides by OPPTS.
These studies have also played a major role
in the selection of compounds and the
development of the cumulative risk
assessment of the chlorotriazines again in
close collaboration with personnel in
OPPTS.
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Program Project 3: Modulation of Cytochrome P-450s and Other
Xenobiotic Metabolizing Enzymes (XME) Leading to Common MOA for
Multiple Toxicities
Objectives
Determine if P-450 and XME modulation
is a common key event for multiple
adverse effects in adult males,
Determine if P-450 and XME is a
common key event for multiple adverse
effects in developmentally exposed males
and females, and
Develop computational toxicological
approaches to evaluate if alterations in P-
450 and/or XME are common key events
for cancer and non-cancer effects.
Scientific Approach
This program project directly addresses
the need to derive a commonly accepted set
of principles defining how MOA
information can be used in risk assessments,
particularly as it relates to extrapolation
issues. Evaluating whether chemicals have
a common MOA for their multiple toxicities
and the implications of that MOA for dose-
response assessment are two of the
fundamental problems in harmonization of
human health risk assessment approaches.
This project addresses both qualitative and
quantitative methods that could be
eventually applied to risk assessment
activities.
Principles concerning the use of MOA
data in risk assessment will be derived
experimentally by examining a class of
prototypic chemicals, the conazole
fungicides (US EPA, 1999). Conazoles are
used both as pesticides and pharmaceuticals;
therefore, both experimental animal and
human toxicity data are available. These
fungicides are used in crop protection to
control fungal infections on fruits,
vegetables and cereal crops, and for seed
treatment of cereal crops. Medically, they
are used to treat local and systemic fungal
infections. Conazoles are a class of
ergosterol biosynthesis inhibiting fungicides
(EBIFs) which inhibit 14-cc-sterol
demethylase (CYP51, lanosterol
14-cc-demethylase). They contain either a
1,2,4-triazole or imidazole moeity which
prevents enzymatic activity through
interaction with the heme iron of P-450s.
Because ergosterol is an essential
component of fungal membranes, the
inhibition of its biosynthesis leads to cell
death. Conazoles have also been developed
to selectively inhibit aromatase for the
treatment of breast and prostate cancer. In
vertebrate species, conazoles have complex
effects on the hepatic and non-hepatic
microsomal monooxygenase systems. They
can act as both inducers and inhibitors of
cytochrome P-450s depending on the tissue
and specific conazole considered (Morita et
al., 1990; Vinggaard et al., 2000).
Many conazoles are hepatotoxic and
hepatocarcinogenic in mice. Some also
induce thyroid follicular cell tumors in rats,
and they are generally non-genotoxic using
standardized test systems. Several
conazoles are also hepatotoxic in humans.
Both thyroid and liver cancer is thought to
be mediated by modulation of P-450 or
XMEs. Specifically, thyroid tumors are
thought to be a result of increased hepatic
metabolism and biliary excretion of
thyroxine as the glucuronide leading to
increases of TSH and overstimulation of the
thyroid (Hurley, 1998).
Hepatocarcinogenesis is thought to be a
result of increased P-450 levels leading to
oxidative stress, mitogenesis, and altered
foci development ultimately leading to
neoplasia.
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In the liver, conazoles affect the activity
and expression of a number of P-450s. For
example, the pesticide propiconazole
induces the activities of CYP1A1, CYP1A2,
CYP2B1/2, CYP2B6, and CYP3A4.
Additionally, propiconazole inhibits
CYP2C11, and in reproductive and other
tissues also inhibits CYP19 (aromatase).
The pharmaceutical ketoconazole also
induces several rat P-450s: CYP1A1;
CYP2B and CYP3A2. Furthermore,
ketoconazole inhibits the activities of
CYP1A1, CYP1A2, CYP2A6, CYP2C19,
CYP2D6, CYP2E1, and CYP3A4 in the
liver. In other tissues, ketoconazole inhibits
essentially all the steroidogenic P-450s.
Ketoconazole most potently inhibits CYP17,
which is critical for two enzymatic steps in
the synthesis of androgens in the testis.
Ketoconazole also significantly affects male
and female reproduction and is a potent
developmental toxicant due to disruption of
steroidogenesis.
Long-term regulation of steroidogenesis
is primarily at the level of transcriptional
regulation of the genes for the various
steroidogenic P-450 enzymes. The P-450
genes are regulated in tissue-specific,
developmentally programmed, and
hormonally regulated fashions. There are
substantial differences in the expression of
these genes and the activity of their gene
products among various mammals.
Consequently, attention must be paid to
significant species differences between
humans and the rodent models. In the testis,
LH modulates the production of testosterone
in the Leydig cells, while follicle
stimulating hormone (FSH) modulates
estradiol synthesis (testosterone conversion
by CYP19 [aromatase]) in the Sertoli cells.
Both LH and FSH are secreted by the
anterior pituitary, in response to the GnRH
from the hypothalamus. Thus, monitoring
LH and FSH will be critical in
understanding conazole effects on
steroidogenesis in vivo and subsequent
effects and responses to altered steroid
levels in the relevant reproductive,
endocrine, and neural tissues. In addition to
the sex steroids and the
gonadalhypothalamic-pituitary axis,
conazoles also affect glucocorticoid
metabolism and the adrenal gland, thyroid
metabolism and thyroid gland, and retinoid
metabolism significant to embryogenesis.
The critical path for this program project is
summarized in the Figure 6.
Project 1- Profiling of Toxic Effects, P-
450s, and Gene Expression in Multiple
Tissues Following Conazole Exposures in
Adult Male Rats and Mice The purpose
of this project is to evaluate the hypothesis
that P-450/XME modulation is a common
critical event that contributes to the MOA
for hepatocarcinogenesis, thyroid
carcinogenesis, reproductive toxicity, and
neurotoxicity of selected conazoles in adult
male rodents. It is hypothesized that the
expression of numerous genes related to P-
450 expression and function will be affected
by conazole exposure in the various tissues
being studied and that these altered
expression profiles will provide mechanistic
insights useful to identifying common
MO As. Research covered in this project
will include the following:
Establish dose-responses for pre-
clinical and toxic effects in the liver,
thyroid, testis and brain of young adult
male Sprague-Dawley rats and CD-I
mice following 14 daily doses by
gavage with various conazoles;
Delineate conazole effects on P-450
protein expression and activity in liver,
thyroid, testis, and brain and evaluate
tissue dosimetry in these and selected
other tissues; and
Profile gene expression of P-450s and
XMEs in testis, brain, thyroid, and
liver of mice exposed to conazoles.
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Project 1
Profile Non-Cancer Effects of Conazoles
in Multiple Tissues in Adults
Project 2
Profile Cancer Effects of
Conazoles in Thyroid and
Liver
Project 4
Integrate Toxicity
Information for
Application in
Risk Assessment
Project 3
Common Modes of Action
for Developmental Exposure
Demonstrate that Modulation of P-450s and
XMEs are Common Mode of Action for
Multiple Toxicities
Figure 6 Critical Path for Research on P-450 and XMEs
Project 2 - Adult Exposures Resulting in
Thyroid and Liver Cancer. This research
will determine the effects of conazole in
thyroid and liver of rats exposed during
adulthood and will consist of the following
studies:
Research on the MO As of conazoles as
both thyroid hormone disrupters or as
progenitors of toxic intermediates with
both MO As occurring through
conazole-induced enhanced
metabolism, and
Studies to examine the early events in
conazole-induced hepatic neoplasia
and relate these findings to P-450
induction to exclude genotoxic
intermediate involvement as a result of
conazole-induced P-450 metabolism.
Project 3- Determining Common MOAs
for Reproductive and Neural Toxicities
Following Developmental Exposure to
Conazoles. Developmental effects are
expected at various stages of growth as a
result of both gestational and postnatal
exposures to conazoles. It is possible that
differential responses could be observed
depending on age, gender, or physiological
status. The goal of this research is to
understand how common MOAs produce a
range of toxicities in developing tissues,
following exposure to conazole fungicides.
These studies will focus upon modulation of
cytochrome P-450s and other xenobiotic
metabolizing enzymes to identify common
MOAs, but will also assess broader effects
on gene expression. The general hypothesis
is that disruption of steroidogenesis by
conazoles in developing neural, endocrine,
and reproductive tissues will result in
adverse effects at lower doses than in adult
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animals. Furthermore, because this
common MOA is shared across various
tissues, mechanistic data resulting from P-
450 and gene expression profiling will be
useful in developing new approaches for
harmonizing risk assessments across various
toxicities. These approaches should be
especially relevant and applicable in
assessing the risk of environmental
exposures for children. Research covered
by this project will evaluate the following:
Effects of gestational exposure to
conazoles on gestational hormone
levels, gestation length, pregnancy
maintenance and pregnancy outcome;
Effects of gestational exposure to
conazoles on female fetal
development;
Effects of gestational and postnatal
exposure to conazoles on timing of
female puberty and adult reproductive
function;
Effects of gestational exposure to
conazoles on male fetal development;
Effects of gestational and postnatal
exposure to conazoles on timing of
male puberty and adult reproductive
function;
Effect of gestational and postnatal
exposure to conazoles on the
developing neurological system in
both male and female rats;
Tissue dosimetry of parent compound
(and potentially active metabolites) to
assist in interpretation of results of
toxicity studies; and
Selected cancer-related biomarkers in
rats developmentally exposed for 90
days for comparison with 90-day
exposures in young adults (project 2).
Project 4 - Computational Toxicology:
Integrating Toxic Effects, P-450
Modulation, SAR Analysis, and Gene
Expression pRofiling for Application to
Risk Assessments. The term computational
toxicology will be broadly employed in the
context of this project to encompass a
variety of techniques and approaches for
accessing and modeling existing and newly
generated data on the conazoles, their varied
biological responses, and the activities of
structurally and biologically related
chemicals. The ultimate aim is to derive
useful generalizations with respect to
chemical structure, gene expression patterns,
and biological activation mechanisms that
can inform and harmonize future evaluation
of conazoles as well as, perhaps, other
chemicals with P-450-mediated toxicities
and common MOA characteristics.
The operating hypothesis is that shared
structural features and physicochemical
properties, as well as common patterns of
gene expression, can serve as useful
organizational principles and potential
predictors of metabolic and biological
response. More specifically, characteristic
profiles of structural/biological response
could suggest appropriate chemical
analogues, narrow the consideration of
potential adverse effects and animal models,
and aid in the design of a rational testing
strategy for new chemicals. Research
covered by project 4 will involve the
following:
Provide insight into the structural basis
for the P-450-mediated toxicological
effects of conazoles and related
compounds from survey of
environmental and pharmaceutical
data sources and use of SAR models;
Use of computational informatic
approaches for analyzing gene
expression profiles of P-450-mediated
toxicities across multiple test systems
(chemicals, species, tissues, doses)
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towards the goal of harmonizing risk
assessments; and
Utilization of relational database
searching strategies to explore broader
associations between P-450 expression
and activity, gene expression profiles,
chemical structure, and biological
effects to provide guidance in future
assessments of conazole-like
chemicals and P-450-mediated
toxicities.
Impact
This program project directly addresses
the need to derive a commonly accepted set
of principles defining how MO A
information can be used in risk assessments,
particularly as it relates to extrapolation
issues. Evaluating whether chemicals have
a common MOA for their multiple toxicities
and the implications of that MOA for dose-
response assessment are two of the
fundamental challenges in harmonizing
human health risk assessment approaches.
This project addresses both qualitative and
quantitative methods that could eventually
be applied to risk assessment activities.
Cross-Agency Interactions
From its inception, this project has been
discussed and modified based on numerous
meetings with the Office of Pesticide
Programs (OPP). OPP has provided critical
insight into chemical selection, dosing and
bioassay endpoints, throughout the project's
development. We have already planned
periodic meetings with OPP during the
conduct of this project to keep them
informed about progress as well as to garner
advice on future studies.
3.5 Gap Analysis and Links to Other
Multi-Year Plans
NHEERL research on the harmonization
of risk assessment will help identify
principles concerning the use of mechanistic
data in cancer and non-cancer risk
assessment through the study of common
MO As and key biological events associated
with three specific toxicity pathways, i.e.,
cell signaling pathways, alterations of LH
secretion, and modulation of P-450 and
XMEs. Because these are only several of
the many toxicity pathways that would be of
relevance to risk assessors, this may seem to
be a limitation. However, the pathways
selected are relevant to a large number of
environmental pollutants. It should also be
noted that the mechanistic research
described in this implementation plan has
significant links to several ORD MYPs (e.g.,
Air Toxics, Drinking Water, Endocrine-
Disrupting Chemicals [EDCs]). Information
from all of these areas will contribute to the
overall goal of developing a framework for
the use of mechanistic data in risk
assessment (see Figure 7).
For example, research on EDCs focuses
on mechanisms of estrogenic, androgenic,
and thyroid-mediated effects. EDC research
will characterize the effects of exposure to
multiple EDCs in various combinations such
as those with similar and different MO As
and will determine the degree to which
effects of EDCs with defined MO As can be
extrapolated across chemical class. EDC
research to develop standardized protocols
for screening chemicals for their potential
endocrine-mediated effects is also based on
an understanding of potential mechanisms
of MO As underlying the tests. Thus,
research on EDCs will inform discussions
on the identification of potential common
modes of action and will provide supporting
data with which to develop a framework
concerning the use of mechanistic
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Endocrine
Disrupters
Harmonization
Research in
GoalS
Air Toxics/PM
Pesticides and
Toxic
Substances
Framework for the
Use of Mechanistic
Data in Risk
Assessment
Drinking Water/
DPBs/CCLs
Figure 7 Framework for Use of Mechanistic Data
information in risk assessment.
Furthermore, emerging technologies
involving the use of proteomic and genomic
techniques are used to develop sensitive
screens and tests for EDCs and will
complement harmonization research to
determine the utility of emerging
technologies.
In the area of drinking water, much of the
research on (As) involves elucidation of the
MO A of cancer and non-cancer effects and
the development of PK models based on
mechanistic studies. The results from these
studies will complement harmonization
research on other potential common MO As
with other chemicals (i.e., cell signaling,
oxidative stress, hormonally-mediated
effects). Harmonization research will also
benefit from work on the mechanisms of
carcinogenicity of priority disinfection by-
products (DBFs), an evaluation of the
scientific basis for common MO As of DBFs,
and their application to risk assessment.
Research to provide a sound scientific basis
for the development of Contaminant
Chemical List (CCL) #3 will also provide
mechanistic data on cancer and non-cancer
endpoints for selected CCL contaminants.
Finally, research to improve data and tools
for assessing and managing potential health
risks associated with drinking water
contaminants will involve a development of
a weight-of-evidence approach based on
common mechanisms and toxicity for
cumulative risk assessment for drinking
water contaminants. Thus, mechanistic data
from research on several drinking water
contaminants will provide significant input
into the development of a framework for the
use of mechanistic data in risk assessment.
Research on Air Toxics involves the
development of mechanistic data to support
risk assessment, including research to
estimate human health effects and aggregate
exposures to hazardous air pollutants
(HAPs), to extrapolate animal-to-human
data for selected HAPs, to determine the
shape at low doses of the dose response
curve, and to develop models for
characterizing and predicting toxicity of
selected air pollutants and mixtures based on
MOA information. Harmonization research
to develop a framework for the use of
mechanistic data in risk assessment will also
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benefit from mechanistic work to identify
the mechanisms of toxicity for PM
constituents and/or its sources. MOA
research on potential common MO As of
pesticides and toxic substances will further
the development of a framework for use of
mechanistic data in risk assessment.
Research on the use of mechanistic data
in risk assessment will also significantly
affect the other themes described in this
implementation plan, i.e., susceptible
subpopulatons and cumulative risk. For
example, risk assessment for cumulative risk
(see Section 5) is based on the risk of
combined toxic effects of chemicals with
similar or dissimilar MO As. Mechanistic
research to identify common MO As and to
develop emerging technologies will be
crucial to developing the tools needed to
assess cumulative risks associated with
exposure to multiple chemicals, mixtures,
and other factors across the Source-to-
Outcome Continuum. Research on common
MO As using prototypic chemicals to
provide the scientific basis for the use of
mechanistic data in risk assessment will also
identify key events in specific toxicity
pathways that can be used to develop
framework and protocols for assessing
cumulative exposures and risks and to
develop methods and measurement data that
will support models of cumulative
exposures, dose, and effects.
Much of the research on susceptible
subpopulations (see Section 4) focuses on
the biological basis for differential
responsiveness to chemical exposures. A
crucial factor that must be determined is
whether the MOA of a chemical is the same
in the susceptible subpopulation as in the
general population. For example, to
evaluate the risks from childhood exposure,
it would be important to know if PK factors
resulted in an increased concentration of the
active chemical at the target site or if key
events in the toxicity pathway are of
increased frequency or expressed at a higher
rate in the susceptible subpopulation. It is
also possible that infants or children may
have differential sensitivity because a
chemical acts on a developmental process
that is not present in the adult.
Harmonization research on potential
common MO As and identification of key
events in representative toxicity pathways
from research on harmonization will be
important in determining the variation in
susceptibility to environmental agents as a
result of health status, aging, and genetic
factors. This research will also evaluate the
relationship between adverse health
outcomes and exposure to environmental
agents in utero and during infancy and
childhood and will elucidate the role of
environmental agents in the induction and
exacerbation of preexisting diseases such as
asthma.
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3.6 References
Bogdanffy, M.S., G. Daston, E.M. Faustman, C.
A. Kimmel, G. A. Kimmel, J. Seed, and V. Vu.
Harmonization of cancer and non-cancer risk
assessment: proceedings of consensus-building
workshop. Toxicological Sciences 61:18-31
(2001).
Chang, L., and M. Karin. Mammalian MAP
kinase signaling cascades. Nature 410:36-40,
(2001).
Cooper, R.L., J. M. Goldman, and L. Tyrey.
The hypothalamus and pituitary as targets for
reproductive toxicants. In K. Korach (ed.),
Reproductive and Developmental Toxicology.
Dekker, New York, pp 195-210 (1998).
Cooper, R. L., J. M. Goldman, and T. E. Stoker.
Neuroendocrine and reproductive effects of
contemporary-use pesticides. Toxicology and
Industrial Health 15:26-36 (1999).
Cooper, R. L., T. E. Stoker, L. Tyrey, J. M.
Goldman, and W. K. McElroy. Atrazine
disrupts hypothalamic control of pituitary-
ovarian function. Toxicological Sciences
53:297-307(2000).
Hurley, P. M. Mode of carcinogenic action of
pesticides inducing thyroid follicular cell tumors
in rodents. Environmental Health Perspectives
437-445 (1998).
Laws, S. C., J. M. Ferrell, T. E. Stoker, J.
Schmid, and R. L. Cooper. The effect of
atrazine on puberty in female Wistar rats: an
evaluation in the protocol for the assessment of
pubertal development and thyroid function.
Toxicological Sciences 58(2):366-376 (2000).
Morita, K., T. Ono, and H. Shimakawa.
Inhibition of testerone biosynthesis in testicular
microsomes by various imidazole drugs. J.
Pharmacobio-Dyn 13:336-343 (1990).
Narotsky, M. G., D. S. Best, D. Guidici, and R.
L. Cooper. Strain comparisons of atrazine-
induced pregnancy loss in the rat. Reproductive
Toxicology 15:61-69 (2001).
National Research Council. Science and
Judgment in Risk Assessment. National
Academy Press, Washington, DC, 1994.
National Research Council. Scientific Frontiers
in Developmental Toxicology and Risk
Assessment, National Academy Press,
Washington, DC, 2000.
Stoker, T. E., D. L. Guidici, S. C. Laws, and R.
L. Cooper. The effects of atrazine metabolites
on puberty and thyroid function in the male
Wistar rat: an evaluation in the male pubertal
protocol. Toxicological Sciences 67:198-206
(2002).
U.S. Environmental Protection Agency.
Guidelines for Reproductive Toxicity Risk
Assessment. Federal Register 61:56274-56322
(1996).
U.S. Environmental Protection Agency.
Guidelines for Neurotoxicity Risk Assessment.
Federal Register 63:26926-26954 (1998).
U.S. Environmental Protection Agency.
Propioconzaole, Establishment of Time-Limited
Pesticide Tolerances. Federal Register
64(54):13080-13086(1999).
U.S. Environmental Protection Agency. Draft
Final Guidelines for Carcinogen Risk
Assessment. Risk Assessment Forum,
Washington, DC. EPA/630/P-03/001A. 2003.
Vinggaard, A.M., C. Hnida, R. Breinholt, and J.
C. Larsen. Screening of selected pesticides for
inhibition of cyp!9 aromatase activity in vitro.
Toxicology In Vitro 14:227-234 (2000).
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Section 4
Susceptible Subpopulations
4.1 Problem
The variability in responsiveness of
humans to environmental pollutants can be
associated with differences in biological
susceptibility. As discussed in the ORD
Human Health Research Strategy, variation in
susceptibility depends on intrinsic factors such
as life stage and genetic factors, as well as
acquired factors such as disease. With regard
to life stage, there are specific periods or
windows of vulnerability during development,
particularly during early gestation, but also
throughout pregnancy and early childhood
through adolescence when toxicants might
permanently alter the morphology and/or
function of an organ system. Children may
also be more vulnerable to specific
environmental pollutants because of
differences in absorption, metabolism, and
excretion. In addition, children's exposures to
environmental pollutants are often different
from those of adults because of different diets
and different activities (e.g., playing on floors
and in soil and mouthing of their hands, toys,
and other objects) that can bring them into
greater contact with environmental pollutants.
The effect of aging on response to
environmental exposures is another area of
uncertainty. Older adults respond differently
than younger adults to environmental
exposures because of age-related changes that
limit the body's ability to maintain
homeostasis and respond to injury. Research
at NHEERL will examine the effect of aging
on responses to environmental pollutants and
will develop predictive models that can be
incorporated into the risk assessment process.
The genetic factors that could predispose
human subpopulations to adverse effects from
exposure to pollutants include genetic
polymorphisms for metabolizing enzymes,
differing rates of DNA repair, and different
rates of compensation following toxic insult.
The main scientific question for this research
is whether such genetic differences
significantly influence risk at realistic, low
dose exposures. Information on gene-
pollutant interactions as a result of long-term
exposure to environmentally relevant
concentrations of pollutants will be explored.
Preexisting diseases may influence the
response to environmental toxicants by
altering xenobiotic metabolism or otherwise
altering the host's response in a synergistic,
additive, or antagonistic manner. ORD
research has shown, for example, that mice
challenged with influenza have increased
mortality from exposure to several
environmental agents including dioxin, ozone,
and ultraviolet radiation. NHEERL research
will develop animal models of disease having
a high incidence in the human population
(e.g., asthma) and will determine the effects of
the disease on the dose-response curves for
high priority environmental agents (e.g., air
pollutants).
4.2 Goals
The overarching goal of NHEERL research
on susceptible subpopulations is to identify
the biological basis underlying differential
responsiveness of susceptible subpopulations
of humans to pollutant exposure. This
implementation plan will address the
following issues:
Identifying the adverse effects of
susceptible subpopulations that are
qualitatively or quantitatively different
from effects in the larger population,
Determining the PK and PD basis for
differential responsiveness of
susceptible subpopulations to
environmental agents,
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Identify Potential
Subpopulations
Not Covered
By 10XUF
Develop
Analogous
Animal Models
Determine Bio-
logical Basis for
Sensitivity
Confirm
Hypotheses in
Human Models
Improve Scientific Basis of Risk Assessment
for Susceptible Subpopulations
Figure 8 Critical Path for Research on
Susceptible Subpopulations
Developing methods and models in
animals that can be used to predict
responses in human susceptible
Subpopulations,
Determining the relationship between
exposures earlier in life to adverse health
effects later in life,
Determine the influence of critical
periods of development on expression of
toxicity, and
Determining the quantitative
contribution of genetic polymorphisms
to responsiveness to environmental
agents.
4.3 Critical Path
Research to improve the scientific basis for
risk assessment of susceptible Subpopulations
will follow the steps outlined in Figure 8.
Research must first identify which
Subpopulations in the general population may
not be protected by current risk assessment
methodologies. For example, in the case of
non-cancer risk assessments, an uncertainty
factor of 10 is used by risk assessors to
account for variability within the general
population. Research has indicated that this
factor, under some circumstances, may not be
fully protective of children's health.
NHEERL research will continue to focus on
infants and children as a susceptible
subpopulation while exploring the relative
sensitivity of other Subpopulations, especially
older adults, those with preexisting diseases
such as asthma, and those with specific
genetic polymorphisms. Once a potentially
susceptible subpopulation has been identified,
steps will be taken to develop animal models
that are as analogous as possible to the human
situation. This step is important to ensure that
the results from animal studies can be
extrapolated to humans and will reduce
uncertainties in the risk assessment process.
Appropriate animal models will also be used
to investigate the underlying PK and PD
differences between the susceptible
subpopulation and the general population.
Results from animal studies will generate
hypotheses that can be tested in human
epidemiological studies, and results from
human observational studies will be examined
in animal models to help determine
susceptibility factors and underlying MO As.
NHEERL research on susceptible
Subpopulations will be consistent with the
APGs and APMs for Susceptible
Subpopulations identified in the ORD Human
Health Research Multi-Year Plan (Appendix
C).
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4.4 Program Projects
Four program projects address life stage
issues. Program project "Identifying and
Validating Biologic Indicators of
Susceptibility and Sensitivity among Children
to Assess Potential Risk of Adverse Health
Outcomes" is concerned with identifying why
children are differentially susceptible to
environmental pollutants. Program project
"Extrapolating Across Windows of
Vulnerability to Assess Children's Health
Risks Using Rodent Toxicity Data" focuses on
how exposure, dose, and effect information
can be incorporated into risk assessment
methods to account for interindividual
variability. Program project "Long-Term
Effects of the Developmental Environment"
addresses the long-term consequences of
perturbations during the in utero period and
various diseases later in life. Program project
"The Aged as a Susceptible Subpopulation"
focuses on the differential sensitivity of the
older adults to environmental exposures.
The program project "Environmental Risk
Factors for Asthma" focuses on environmental
influences that act directly as allergens to
induce asthma or agents that enhance either
the induction or exacerbation of the disease
via non-specific stimulation of immune or
inflammatory responses. Two program
projects address genetic components that may
predispose subpopulations to exposure to
environmental agents. The program project
"Oxidative Stress as a Common Susceptibility
Attribute for Non-Cancer Risks" focuses on
the role of underlying oxidative stress as a
common susceptibility factor guiding
response to pulmonary, cardiovascular, neural
and reproductive toxicants. Program project
"Genotype and Phenotype in the Metabolism,
Toxicity and Carcinogenicity of Arsenic"
focuses on genetic capacity of individuals to
metabolize inorganic arsenic (iAs) as a
primary determinant in susceptibility.
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Program Project 4: Identifying and Validating Biologic Indicators of
Susceptibility and Sensitivity among Children to Assess Potential Risk of
Adverse Health Outcomes Associated with Environmental Exposure
Objectives
Identify and validate comparable biologic
indicators in both test animals and humans
that can be used to investigate the risk of
environmental exposures associated with
cancer and functional impairments of the
nervous, immune, and reproductive
systems; and
Identify genetic and physiologic factors
that can modify the associations between
exposures and outcomes in a similar
fashion in both animal and humans.
Scientific Approach
There is substantial public health concern
that variation in the quality of the environment
causes adverse health outcomes in children.
Many causes of childhood mortality, disease,
and disabilities affecting quality of life are
plausibly associated with environmental
factors. In order to better inform policy and
risk assessment, multifaceted scientific studies
are needed to provide bioindicators which
help identify environmental factors that are
harmful, harmless, or helpful in terms of their
effects on children's health and development.
The development and validation of
biomarkers that are applicable across species
are vital to improve the precision of risk
estimates. Thus, the major unifying theme for
this program project is identifying and
validating biological indicators of
susceptibility and sensitivity to assess the
potential risk of adverse child health outcomes
associated with environmental exposures.
This program project will assess a broad
range of potential biomarkers. For example,
neurodevelopmental biomarkers will focus on
neurotrophic factors (NTFs),
neurotransmitters, proinflammatory cytokines,
and classical conditioned responses. Research
on immunological biomarkers will assess
antibodies, cytokine proteins, message
analyses, and phenotypic lymphocyte profiles.
Reproductive function will be assessed using
mRNA and protein expression analyses,
immunoglobulins, proteins and other markers.
Cancer risk will be examined by looking at
underlying mechanisms which might account
for inter-individual phenotypic variation in
response measured by a wide range of health
indicators. All projects in this program
project will focus on maximizing animal-to-
human extrapolation by identifying and
validating common biomarkers with a focus
on samples that could be obtained non-
invasively from human subjects. While
human studies will be generally observational
with respect to exposure assessment, animal in
vivo studies and in vitro studies allow for
direct manipulation of exposure and
evaluation of biological indicators that are
predictive of adverse effects. This interactive
and iterative process between animal and
human studies will allow for the anticipation
of the types of health effects that might be
expected based on specific exposures and
gives important clues about the underlying
MO As for complex disease-exposure
relationships.
The research in this program project is
linked to the National Children's Study
(NCS), a multi-agency, multi-disciplinary
longitudinal study designed to evaluate
children's health in the United States (US).
Under the Children's Health Act of 2000, the
Agency will work with other Federal
Agencies to plan and implement the NCS.
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Project 1
Project 2
Projects
Project 4
Develop Biomarkers
of Neurologic
Function
Develop Biomarkers
of Immune Function
Develop Biomarkers
of Reproductive
Health
Develop Biomarkers
of Cancer and
Cancer Precursors
Battery of Biological Indicators to
Assess Potential Risk of Adverse
Child Health Outcomes
Figure 9 Critical Milestones for Research
on Children's Health
Research at NHEERL is designed to
coordinate animal and human studies that
focus on endpoints where the burden of
illness, disability, or death is high for children
in the US and where an environmental factor
is implicated in the disease process. One of
the unique strengths of NHEERL is the ability
to coordinate human and epidemiological
studies with toxicological studies in test
animals. This research program will influence
the selection of biomarkers assessed in the
NCS. The critical steps to be followed in this
program project are summarized in Figure 9.
Project 1 - Biomarkers of Neurologic
Function in Children. Neurological and
developmental disorders in children are a
major public health concern. The etiology of
most mental retardation and many other
neurodevelopmental disorders is unknown.
Autism spectrum disorder may be increasing
in prevalence, and some neuropsychologic
conditions of childhood such as attention
deficit-hyperactivity disorder (ADHD) are
being diagnosed at an epidemic rate.
Environmental exposures to known or
suspected neurotoxicants have been
implicated in these disorders of children. In
order to better understand the mechanisms
that link exposures to neurodevelopmental
outcomes in children, biomarkers in test
animals that can be applied to human
populations will be developed. Effects that
are observed in human populations will be
more fully evaluated by characterizing dose-
response relationships using animal models
(Barone et al., 1998; Lassiter et al., 2002;
White et al., 2002).
One series of studies will attempt to
demonstrate the association and predictive
validity of early postnatal serum measures of
NTFs and neurotransmitters with chemical-
induced brain damage in rats with the goal of
validating comparable early postnatal serum
measures with autism spectrum disorders,
ADHD, Downs Syndrome, and cerebral palsy.
The hypothesis for this work is that alterations
in expression profiles of NTFs, neuropetides,
and cytokines are predictive for
developmental disorders and functional
impairments of the nervous systems. These
studies will:
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Evaluate the association and predictive
validity of early postnatal serum
measures of NTFs, cytokines, and
neurotransmitters with chemically-
induced brain damage in animal models;
Assess the feasibility of measuring early
postnatal serum measures of NTFs,
neurotransmitters, and cytokines in
human infants; and
Assess the feasibility of determining the
predictive validity of early postnatal
serum measures of NTFs,
neurotransmitters, and cytokines in
human infants with developmental
neurological disorders.
A second series of studies will focus on
developing analogous methods to evaluate
neurobehavioral function and animal models.
The hypothesis for this research is that
developmental disorders in humans are linked
to fundamental processes that are common to
humans and experimental animals and can be
quantified. These studies will:
Assess the feasibility of a field ready test
system based on classical conditioning
(e.g., eye blink conditioning) for use in
human infants;
Investigate other potential measures of
neurobehavior (e.g., signal detection,
reaction time) applicable in both humans
and experimental animals;
Establish the ontogeny of motor function
in an animal model ;
Determine whether early (prenatal,
postnatal and/or perinatal) exposure to
toxicants alters the ontogeny of motor
function in animals and humans;
Establish a conditioned-behavioral test
for quantifying susceptibility in animals
and humans; and
Determine whether early exposure to
toxicants alters awareness and sensitivity
to the consequences of behavior in
animals.
Project 2 - Biomarkers of Immune
Function, Developing Animal Models that
Predict Immune Function in Human
Children. Xenobiotic exposure during
critical windows of immune system
development can cause alterations in function
that persist into adulthood, and possibly for
life (Weisglas-Kuperus et al., 2000).
Evidence obtained in experimental animals
strongly suggests that the developing immune
system is much more susceptible than that of
the adult. This project will test the hypothesis
that results of standard clinical immunological
assays (e.g., antibody responses, cytokine
protein and message analysis, and flow
cytometric phenotyping of lymphocyte
subsets) are useful predictors of changes in
host immunocompetence and provide detailed
information on the specific response to
antigens or vaccines. Included in the
definition of xenobiotics are environmental
microorganisms and infectious agents; thus,
the influence of early-life infection on
immunocompetence (both positive and
negative) will be evaluated. The results may
provide useful markers for increased
susceptibility to infectious disease and may
predict the loss of homeostatic control that
may lead to the development of immune
suppression, allergy, or autoimmune disease.
Many disorders of childhood have an immune
function component and some, e.g., asthma,
are increasing and suspected to have
environmental antecedents. The hypothesis
for this work is that xenobiotic exposure
during critical windows of immune system
development will alter immunological
function that persist into adulthood and
possibly for life. Research in project 2 will
Evaluate the effects of developmental
exposure to pesticides and assess the
predictive value of the animal model for
responses of children exposed to
environmental chemicals,
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Assess the effects of developmental
exposure to endocrine-active xenobiotics
on immune function in animals, and
Determine MO A of rodent
developmental immunotoxicants as a
means to identify candidate markers that
reflect possible effects in exposed
humans.
Project 3 - Biomarkers Related to
Reproductive Health Risks. Environmental
exposures during preconception and early
pregnancy can affect fertility, outcome of
pregnancy, and the health and development of
children from the pregnancy. Two approaches
will be used to evaluate reproductive health
risks. One uses samples obtained with
minimal invasiveness (e.g., hair, milk, and
blood) that may be accessible for collection
from human subjects and compares the results
between effects on surrogate and target-tissue
(e.g., gonads) markers of exposure and effect.
The second approach looks at the placenta to
help identify useful biomarkers related to the
interface between the outside environment of
the mother and the developing fetus. Both
approaches are important for predicting the
eventual health of the offspring. In addition to
biomarkers that may be useful to predict the
offspring's eventual reproductive capacity,
biomarkers that predict reproductive success
in the parents also have predictive value for
child health outcomes. Parents with
suboptimal fertility (such as those with a long
time-to-pregnancy) are at higher risk for
adverse pregnancy outcomes including low
birth weight and preterm delivery. The
biomarkers developed in this project may
have value in measuring children's risk
associated with their parents reproductive
capacity as well as predicting the child's
reproductive health. The hypothesis for this
project is that gene expression profiling of
reproductive tissues can reveal genetic
biomarkers of exposure and effect for
reproductive toxicants. Studies included in
project 3 are as follows:
Evaluating the best methods to collect,
store and transport surrogate tissues
(blood, hair follicles, uroepithelial cells,
and semen) from humans and rodent
models such that sufficient quantities of
good quality RNA can be extracted for
gene expression analysis or
identification purposes, respectively;
Confirming that historical, archived
placental specimens have sufficient
macromolecular integrity for utilization
in mechanistic/biomarker studies;
Exposing fetal, juvenile, and adult rats
and mice to effective doses of known
and suspected reproductive toxicants
(i.e., disrupters of steroidogenesis such
as conazole fungicides) and using gene
expression analysis of target tissues
(testis, prostate, ovary, uterus) to
identify genetic biomarkers;
Determining whether gene or protein
expression profiling in surrogate tissues
can provide reliable biomarkers for
exposure to reproductive toxicants (e.g.,
conazoles, PCBs, chloroatrazines) in
adult humans; and
Determining whether gene expression
profiling in surrogate tissues from
children (blood, hair follicles,
uroepithelial cells) is possible using the
same methods and genes identified in
the fetal/juvenile rodents and adult
humans.
Project 4- Biomarkers of Cancer and
Cancer Precursors: Identification of
Genetic Susceptibility, Biomarkers of Gene
Expression, and Oxidant Status. Most
suspected associations between environmental
exposures and adverse health outcomes for
children are likely to have a component of
gene-environment interaction or variations in
susceptibility. These variations in
susceptibility make it difficult to assess risk in
the general population because we are unable
to effectively identify any at-risk subgroups.
Consequently, the heterogeneous risk pool
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dilutes risk estimates and drives them towards
the null. The aims of this project are designed
to enhance our ability to determine the
important genetic factors involved in gene-
environment interactions and to assess
potential co-factors such as reactive oxygen
species (ROS) and antioxidant status and how
they relate to disease. The capacity ability to
measure similar processes in animal and
human studies will advance our ability to
determine which genetic and physiologic
factors are most important in assessing
children's environmental health risk.
Research in project 4 will:
Evaluate whether dose, individual and
age-specific (adult vs children)
differences in sensitivity to chemicals
and mixtures can be assessed in human
blood, plasma, and/or individual blood
cells following in vitro treatment to
environmental agents;
Examine various housekeeping genes
and their functional products, such as
those associated with DNA repair, cell
cycle control and apoptosis, to determine
if they respond differently to chemical
stressors in a manner that is dependent
on the life stage;
Assess the utility of breast milk
epithelium and blood cells for
determining the contribution of genetic
variability (expression profiles and
polymorphisms) on an individual's
sensitivity to environmental toxicants;
Evaluate the extent to which plasma
concentrations of antioxidants
(ascorbate, urate, tocopherols and
glutathione) are cofactors that might
modulate risk factors in the initiation of
diseases in children;
Validate use of human blood ex vivo in
the evaluation of dose, genetic, and age-
specific factors that affect inter- and
intra-individual phenotypic disease
measurements; and
Develop reliable assays to measure
endogenous and exogenous constituents
of breast milk that are implicated in
altering health status of women or their
breast-fed children.
Impact
Under the Children's Health Act of 2000,
the Agency is working together with other
federal partners to plan and implement the
NCS. Within ORD, the importance of
research on children's environmental health is
a long-standing priority with critical
implications for risk assessment. This
program project will demonstrate scientific
leadership in children's environmental health
research by coordinating animal and human
studies that focus on endpoints where the
burden of illness, disability or death is high
for children in the US and where an
environmental factor is implicated in the
disease process. One of the unique strengths
of this implementation plan is the ability to
coordinate human and epidemiologic studies
with toxicology studies in test animals. This
research program can influence the selection
of biomarkers assessed in the NCS, which will
be the major study of children's
environmental health of this generation.
Cross-Agency Interactions
This program project benefits from
substantial interaction and coordination with
Agency efforts in planning the NCS. In
cooperation with several other federal
agencies, under the leadership of the director
of the National Institute for Child Health and
Human Development, the Agency has a strong
role in developing this major child
environmental health study. The Agency
representatives to the Interagency
Coordinating Committee (ICC) of the NCS
are supportive of this program project and
maintain contact with the investigators to
insure that the biomarkers evaluated and
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validated will be of optimum utility.
Members of the ICC come from across the
National Institutes of Health (NIH), Centers
for Disease Control and Prevention (CDC),
and the Agency. The Agency representatives
are from NHEERL, NERL, and NCEA. This
proposed research plan has been reviewed by
members of the ICC and the lines of open
communication are insured by the NCS web-
based portal (a copy of this research proposal
and updates are maintained on the portal for
ready access by study planners). The study
planners also interact regularly with NCER
regarding extramural funding for NCS
projects. This program project is also
integrated with a series of Agency-funded
pilot studies for the NCS which brings
together investigators from across ORD to
support method development for the NCS.
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Program Project 5: Extrapolating Across Windows of Vulnerability to Assess
Children's Health Risks Using Rodent Toxicity Data
Objectives
Focus modeling and laboratory research
around selected case studies including:
volatile organics (including methanol),
organophosphate (OP) pesticides, and
conazoles;
Obtain physiological, biochemical, and
anatomical parameters for rodents and
humans for selected windows of
vulnerability;
Quantify metabolism, and perhaps other
PK processes such as absorption in humans
and rodents for the selected windows of
vulnerability with in vitro methods;
Develop physiologically based PBPK
models and generate rodent PK data for the
case study chemicals during the windows
of vulnerability in rodents and humans;
Develop human exposure models during
the windows of vulnerability to link to the
PBPK models; and
Delineate the magnitude of age-related
differences in sensitivity to pesticides and
elucidate mechanisms (kinetic and/or
dynamic) underlying this difference in
sensitivity.
Scientific Approach
There are windows of vulnerability that
exist during various life stages during which
exposure to environmental chemicals may
result in permanent damage to biological
systems. These windows may exist at various
stages of gestation, as well as postnatally,
during infancy, childhood, or adolescence.
One major source of uncertainty and potential
source of error in the assessment of the health
risk of children derives from developmental
events occurring in different time periods for
different species. This is particularly apparent
for events that occur in the early postnatal
(lactational) period for rodents and in utero
for humans. Thus, lipophilic chemicals such
as PCBs, which disrupt thyroid hormones,
cause developmental hearing toxicity in
rodents during the lactational period due to
massive mobilization from the mother's fat
reserves to her milk during lactation. This
same effect would not likely occur following
postnatal exposure in humans because hearing
development occurs in utero. On the other
hand, the situation would be reversed if a
chemical were preferentially accumulated in
the fetus. Clearly, extrapolations between
animals and humans will be dependent upon
the developmental processes at risk. Because
animal toxicity data will continue to play an
important role in the assessment of a
chemical's potential to affect children's
health, there is a need to develop a systematic
approach to assess the relevance of animal
toxicity data occurring during different
developmental periods, both prenatal and
postnatal, to humans and to extrapolate the
response from animals to humans. It will be
necessary to first identify critical periods of
developmental processes in controls before
beginning studies using very specific
chemicals with known MO As. The critical
steps for research in this program project are
summarized in Figure 10.
Project 1 - Literature Review. The main
objective of this effort is develop a framework
that includes exposure scenarios and PK
processes associated with different windows
of vulnerability to assess children's health risk
from animal toxicity data. The following
issues will be addressed by the literature
review:
Physiological parameters for absorption,
distribution, metabolism and elimination
of selected environmental chemicals will
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PROJECT 1
Develop Framework
of Exposure and PK
Processes for
Different Windows
PROJECT 2
Delineate Magnitude
of Age-Related
Difference to
Insecticides
PROJECT 3
Determine
Developmental PK
for Fungicides
1
Establish Relevance of Animal
Toxicity Data from Developmental
Studies to Humans
J
Figure 10 Critical Path for Research on
Windows of Vulnerability
be obtained for several animal species
in vitro PK parameters will be determined
in order to:
- identify the enzymes involved
in chemical metabolism and to quantify
their expression in adults and children
when that variance may contribute to
susceptibility to toxic injury and
- develop chemical-specific in vitro-
derived measures of tissue distribution
of parent chemical and toxicologically-
active metabolites.
Develop PBPK models and rodent PK
information for various life stages.
Methanol was selected due to ongoing
risk assessment activities in NCEA.
Conazoles were selected in conjunction
with the developmental studies planned
for program project 3.
In collaboration with other ORD
Laboratories/Centers, develop exposure
models.
Project 2 - Determine Age-Related PD
Changes. The objective of project 2 is to
delineate the magnitude of age-related
differences in sensitivity to pesticides and
elucidate mechanisms (kinetic and/or
dynamic) underlying this differential
sensitivity. The approach has involved
systematic comparisons of the dose-response
and time-course of pesticide effects in rats of
different ages, followed by development and
testing of hypotheses concerning the
mechanisms for these differences. Behavioral
and neurochemical (cholinesterase [ChE]
inhibition) evaluations have been used to
characterize the acute dose-related effects at
the time of peak effect of several
anticholinesterase pesticides. One outcome of
prior research in this area has been to
emphasize the effect of kinetic differences in
detoxification pathways and to suggest that
the age-related differences in kinetics can
predict overt toxicity. Research will continue
in this area to characterize the absorption,
distribution, metabolism, and excretion of
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ChE-inhibiting pesticides in young and adult
animal models.
Project 3 - Evaluate Developmental PK of a
Class of Prototypic Pesticides. This project
will evaluate developmental PK information
on selected conazoles associated with
developmental toxicities. It serves as a case
study (as described in project 1 above) and is
a component of the harmonization proposal
addressing modulation of P-450s and other
xenobiotic metabolizing enzymes using
conazoles as a case study (see Section 4). The
PK factors evaluated will be useful both for
better understanding extrapolations across age
groups and across species. The following
studies will be performed.
Determination of the ability of selected
conazoles to transfer to mother's milk in
rats, and
Comparison of tissue dosimetry of
selected conazoles in pregnant and non-
pregnant females, as well as assessing
the tissue dose to the fetus.
Impact
This program project will identify
approaches to address PK and PD issues
related to extrapolation across windows of
vulnerability in multiple species. This will
provide options for risk assessment or for
additional research to improve those risk
assessments. This research will also define
for developmental endpoints and chemical
classes what risk assessment approaches are
feasible addressing windows of vulnerability
and what additional work is required to reach
a systematic set of guidelines for addressing
these issues of interspecies extrapolation for
exposure and dosimetry. Finally, the
development of computer simulation models
that can be parameterized for different species
and developmental time periods will be useful
for future development of guidelines dealing
with PK and PD issues across different life
stages.
Cross-Agency Interactions
This program project involves major
collaborative efforts between NHEERL,
NCEA, and NERL arising, in part, from
internal funding. The selection of case studies
and the implementation of the research is
being done jointly.
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Program Project 6: Long-Term Effects of the Developmental Environment
Objectives
Develop animal models to evaluate effects
of developmental perturbation on adverse
health outcomes during adulthood,
Assess key health outcomes during
adulthood following developmental
perturbation,
Investigate mechanism(s) of adverse health
effects in adults following developmental
perturbation, and
Provide input into interpretation of low
birth weight data from Agency testing
guideline studies.
Scientific Approach
Clinical and epidemiological studies have
shown significant correlations between
conditions during development and various
diseases later in life. In humans, low birth
weight has been used as a surrogate for
adverse developmental conditions, but the
specific conditions affecting birth weight are
usually unknown. Human studies demonstrate
inverse correlations between birth weight and
risk of later disease, and these correlations
hold even within the range of normal birth
weight. Adult-onset diseases with which birth
weight has been inversely correlated include
hypertension, coronary heart disease, diabetes,
obesity, schizophrenia, and early onset
chronic renal failure (Godfrey and Barker,
2001). There is also evidence that, for female
fetuses, the in utero environment can
significantly affect pregnancy outcome in
adulthood. Studies have shown that reduced
infant birth weight and other adverse effects
are more closely correlated with the mother's
weight at her birth (i.e., maternal intrauterine
environment) than with her infant's prenatal
environment (Rhind et al., 2001). While birth
weight has been used as a convenient and
available marker of developmental conditions
in humans, it is important to make clear that
adverse developmental conditions do not
always affect birth weight although such
adverse conditions may still have long term
effects.
The most common adverse effect seen in
standard rodent developmental toxicology
studies is reduced fetal weight at term.
Current Agency guidelines for interpretation
of developmental toxicity studies state that
fetal weight is an adverse outcome on par with
death and malformation, the other adverse
outcomes detectable in fetuses at term.
However, the interpretation of reduced fetal
weight is a contentious subject, especially
when fetal weight is affected only at
maternally toxic exposures. Current study
designs do not examine the long term health
of offspring exposed during development (in
utero and/or postnatally). The utilization of
animal models will help to define the causal
factors and identify long-term effects under
controlled experimental conditions that are
impossible to duplicate in epidemiological
studies. Current developmental toxicity tests
do not address this important issue and may
be missing important adverse effects highly
relevant to the human condition. Maternal
undernutrition during pregnancy in rats has
been shown to cause hypertension, diabetes,
and other metabolic derangements, as well as
adverse neurological sequelae in offspring.
This program project will examine the long
term health effects of toxic exposures during
development. The overall goal of this
program project is to determine the conditions
under which developmental perturbations will
lead to adverse health outcomes at adulthood.
There is evidence that some environmental
contaminants may alter developmental
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Project 1 Effects on PN
Growth, Cardiovascular,
Renal, Glucose Metabolism
Project 2 Effects on PN
Immune Function
Project 3 Identify
Placental Biomarkers
of Persistent Effects
i
i
Project 4 Effects on
Herbicides on Adult
Adverse Outcomes
Project 5 Imprinting
of Key Metabolic
Pathways and Adverse
Outcomes
Project 6 Intereenerational
Effects on Female
Reproduction
i
Project 7 Molecular
Pathways Involved in
Embryo/Fetal Programming
Project 8 Effects on
Development of
Allergy
1
Determine Conditions Under Which Perturbations
During Development Produce Permanent Adverse Effects
Figure 11 Critical Milestones for Research
on Developmental Environment
programming in a manner that does not
necessarily result in malformations but affects
function later in life. The best-studied
examples of this are environmental agents
with endocrine activity, and these agents
typically affect the reproductive tract or
reproductive function. Effects of these agents
may not become apparent until puberty, or in
some cases, may only hasten the onset of
reproductive senescence much later in life.
Latent health effects due to developmental
exposures to other classes of agents have
received scant attention, but there are
examples of neurological and
neurodegenerative effects of developmental
exposures. There is evidence that in utero
exposure to PCBs leads to altered thyroid
function and learning disabilities later in life.
The mechanisms underlying these effects are
poorly understood, but well-designed studies
and new technologies such as gene expression
profiling should begin to reveal clues to the
developmental pathways involved. Studies
using this technology will be designed and
incorporated into this program project as
results emerge from the more descriptive
animal studies. Critical milestones associated
with this program project are summarized in
Figure 11.
Project 1 - Effects of Developmental
Toxicant Exposure or Undernutrition on
Adult Physiology. Significant decrements in
fetal weight are the most common adverse
outcome of rodent developmental toxicity
bioassays in which pregnant animals are
exposed to xenobiotics. Maternal
undernutrition has been studied extensively in
rodents and is an established method of
inducing lower birth weight in offspring
without exposure to toxic chemicals. It was
observed over 35 years ago that poor nutrition
during pregnancy led irreversibly to reduced
cell number in many tissues. It has since been
established that maternal protein restriction
affects islet cells and insulin-sensitive tissues
such as the liver, muscle, adipocytes, kidney
and brain in the offspring. Maternal
undernutrition also programs offspring for
hypertension, and this can occur even when
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the undernutrition is only transient during
early gestation. Hypertension has also been
noted as early as 4 weeks of age in rodent
models of fetal programming, and this
hypertension persists throughout life
(Langley-Evans et al., 1998). Among major
organ systems, the kidney appears to be
targeted specifically by maternal
undernutrition, leading to decreased kidney-
to-body weight ratio and fewer nephrons in
the kidney at birth (Merlet-Benichou et al.,
1994). Prenatal malnutrition resulting in low
birth weight pups has also been associated
with behavioral abnormalities that persist into
adulthood. Cognitive deficits have been
reported in a variety of animal models using
both behavioral assessments of learning and
memory and neurophysiological indices of
synaptic plasticity (Kehoe et al., 2001). The
hippocampus subserves memory function in
humans, nonhuman primates, and rodents and
is also a target for glucocorticoids. Neonatal
stress induced by isolation induces changes in
transmitter function and neuroendocrine
activation and contribute to abnormal
behavioral and neurochemical responsiveness
later in life. Studies in project 1 will expose
pregnant rats to toxicants or undernutrition
during various developmental windows and
offspring will be observed during their entire
life span. This research will:
Demonstrate that physiologic and
metabolic development of an organism
is shaped in part by its developmental
environment, and adverse developmental
conditions can produce permanent and
deleterious physiological changes which
may become apparent later in life.
Project 2- Long-Term Immune Function
Deficits Following Immune System
Perturbation During Development.
Perturbation of immune system development
by various stressors has been documented to
result in functional decrements that may
possibly persist for life. For example,
perinatal and/or early postnatal exposure to
chemicals such as dioxin, organotins, and
organochlorine pesticides has been
demonstrated to suppress immune system
function later in life (Smialowicz et al., 2001).
These results indicate that the developing
immune system is more susceptible to
perturbation by chemicals and pesticides than
is the adult immune system and that these
alterations may persist into adulthood, and
possibly for life. Research in project 2 will
further investigate the conditions under which
in utero and/or early postnatal stressors lead to
permanent and deleterious alterations of the
immune system at any stage of post-weaning
life. This research will:
Demonstrate that results of standard
immunotoxicological assays (e.g.,
antibody responses, T cell-mediated
responses, cytokine protein and message
analysis, and flow cytometric phenotype
analysis of lymphocyte subsets) in the
rat are useful predictors of long-term
changes in immunocompetence that may
occur in humans.
Project 3 - Placental Tissue as a Potential
Source of Biomarkers Associated with
Intrauterine Growth Retardation (IUGR)
and Long-term Health Effects. There is
substantial evidence in the scientific literature
linking exposure to air pollution, cigarette
smoke and other agents to adverse pregnancy
outcomes including low birth weight,
intrauterine growth retardation (IUGR),
eclampsia, hypertension, premature delivery,
and effects on maternal health (Schardein,
2000). Many of these responses are
associated with physiological and
morphological changes in the placenta that
affect function, e.g., delivery of nutrients and
oxygen to the conceptus. Thus, the placenta
can be a source of biomarkers that relate to
exposure as well as to health effects. For
example, changes in gene expression, enzyme
induction/activity, and vascularization, among
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other endpoints, can be detected/monitored in
placental tissues from at-risk pregnancies and
compared with normal term placenta. Project
3 will develop a rodent model to evaluate the
effects of low birth weight on future health
and responses to toxicants. This research will
evaluate the role of altered placental
morphology, function, and gene expression in
the production of intrauterine growth deficits
and the long term health effects associated
with IUGR. This research will:
Provide a basis for comparing animal
and human responses to toxicants for the
potential to impact intrauterine growth
and development.
This project will also complement the
research proposed under program project 4
"Identifying and Validating Biologic
Indicators of Susceptibility and Sensitivity
among Children to Assess Potential Risk of
Adverse Health Outcomes Associated with
Environmental Exposures." In that program
project, human placental specimens will be
obtained and examined with genomic,
enzymatic, protein expression assays, and
proteomic analyses. The identification of
biomarkers that occur in both species would
facilitate laboratory study of toxicants
implicated in IUGR and having long-term
health consequences. Research in project 3
will:
Demonstrate that exposure to toxicants
affects growth and function of the
placenta, contributing to adverse effects
during gestation that persist throughout
life and can predispose to disease and
disability, and
Compare human and rodent biomarkers
associated with IUGR and adverse
health outcomes.
Project 4 - Effects of High use Herbicides
on Breast and Prostate Development and
Life-Time Disease Risk in Humans and
Rodents Following Gestational or
Lactational Exposures. The overall
objective of this project is to determine the
effects of gestational exposure to
chlorophenoxy and chlorotriazine herbicides
on breast and prostate development and to
compare their adverse health risk outcomes in
the rodent and human. Historically, 2,4-
dichlorophenoxyacetic acid (2,4-D) and
atrazine have been the top two herbicides used
in US agriculture by acre. Although the two
families of compounds differ in potential
MO As and metabolic clearance, they have
both been shown to have adverse health
outcomes following fetal or neonatal animal
exposures and little effect in adult animals.
These studies will:
Compare post-exposure health outcomes
in animal models with epidemiological
data in high chlorophenoxy and
chlorotriazine herbicide use counties
within the US.
Previous studies have shown that late
gestation exposure to atrazine caused impaired
mammary gland development in dams and
their female offspring, increased incidence of
prostatitis and lipomas in male offspring
(Fenton et al., 2002), and left the female
offspring with enhanced sensitivity to the
effects of a chemical carcinogen,
dimethylbenz[a]anthracene. Another study
found that atrazine, via modulation of
prolactin levels during early postnatal life,
increased the incidence and severity of
prostatitis in male offspring. Atrazine and its
individual metabolites induced adverse
reproductive tissue effects if delivered around
the time of puberty. However, there is little
information concerning the effects of 2,4-D.
Studies in project 4 will:
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Provide data on breast and prostate
developmental outcomes and cancer and
non-cancer endpoints following
exposure to chlorotriazine and
chlorophenoxy herbicides during periods
critical to target tissue or brain
development,
Provide dose-response information for
atrazine and its metabolite mixtures and
chlorophenoxy herbicide exposures
during pregnancy and lactation in rats,
and
Characterize adult risk to these
compounds following childhood
exposures in rodent models and in
humans.
Project 5 - Long-Term Metabolic System
Perturbations after Developmental
Exposures. Drug metabolizing systems are
known to be present to varying extent in
tissues of the embryo and fetus and can
influence the response of the conceptus to
toxic chemicals (Miller et al., 1996). Less
understood is how the ontogenetic profiles of
drug metabolizing enzymes are influenced by
exposure to toxicants and how toxicant
exposures during ontogeny of drug
metabolizing systems might affect their
expression and inducibility during adulthood.
Permanent alterations in enzyme expression
and/or inducibility due to the developmental
environment could be a basis for changes in
susceptibility to toxic exposures in later life.
Alterations in some of these metabolizing
systems could also adversely affect
metabolism of endogenous substrates
including steroid synthesis. Research in
project 5 will:
Demonstrate that during pre- and
postnatal development there are critical
metabolic pathways which may be
imprinted by the developmental
environment, leading to permanent
changes in their expression and/or
inducibility.
Project 6 - Intergenerational Effects on
Female Reproduction There is evidence
that, for female fetuses, the in utero
environment can significantly affect
pregnancy outcome later in life. Studies have
shown that reduced infant birth weight and
other adverse effects are more closely
correlated with the mother's weight at birth
(i.e., maternal intrauterine environment) than
with her infant's prenatal environment. There
is a body of epidemiological data that
indicates a correlation between the perinatal
environment of a woman and her chances of
having an adverse pregnancy outcome. There
is concern about the relationship of the health
of mothers and the health of infants. Research
suggests that the incidence of abnormal
pregnancy outcomes including stillbirths in
women within lower social classes might be
associated with childhood stunting brought on
by a poor environment. It has also been
shown that the incidence of neural tube
defects, stillbirths, and low weight babies is
high in women born during periods of general
malnutrition. Research in this project will:
Examine the effects of developmental
exposures on adult female fertility,
pregnancy maintenance and response to
developmental toxicants and
Determine whether a female's in utero
environment affects her ability to
maintain a normal pregnancy and
delivery in adulthood.
Project 7 - Gene-Expression Profiling for
the Elucidation of Developmental Pathways
Involved in Embryo/Fetal Programming.
New technologies for rapidly screening for
changes in gene expression offer exciting new
avenues for the elucidation of pathways of
developmental toxicity. There are a limited
number of conserved signaling pathways
involved in the process of development, and
the functions of these pathways are becoming
better understood. Because the biology
underlying embryo/fetal programming is
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poorly understood, gene expression profiling
may be the best approach for beginning to
understand the process of embryo/fetal
programming. Gene expression microarrays
have already been used to examine
mechanisms of teratogenesis, but there are no
established approaches for carrying out such
work. These studies will be initiated only
after adverse effects have been established in
previous projects. Gene expression profiles
will be assessed using microarray technology
and real-time quantitative polymerase chain
reaction (PCR). The specific developmental
times and tissues/structures to be studied will
depend on the nature of the effects observed in
the descriptive studies. This research will:
Demonstrate that embryo/fetal
programming involves changes in gene
expression leading to permanent
alterations in postnatal and adult
physiology, and these changes in gene
expression can be detected at early
stages using expression microarray
technology.
Project 8 - The Effects of Low Birth Weight
on the Development of Allergy. Research in
this project will determine if low birth weight
resulting from either maternal undernutrition
or in utero toxic exposure plays a role in
allergy development. Respiratory and oral
(food) allergies are thought to develop in early
life in a genetically predisposed population.
This project will:
Determine if low birth weight affects
allergy development by increasing
susceptibility to sensitization, and/or by
enhancing the severity of allergic
responses.
Impact
This project has the potential to change the
basic study designs for assessing
developmental toxicity and to reveal latent
toxic effects not seen before. If the results of
this study demonstrate long-term morbidity
associated with prenatal and/or postnatal
exposures, it may be necessary to extend
existing study designs or create new designs
to test for such effects. Perhaps some types of
morbidity associated with developmental
exposures are general in nature, while others
are chemical-specific. We may find that no
increase in disease is associated with
exposures that also affect fetal or early
postnatal growth, in which case we may not
need additional tests; however, such results
would clearly raise the significance of affects
on fetal weight or postnatal growth seen using
current study designs. These studies thus have
the potential to bring significant
improvements to the risk assessment process.
Cross-Agency Interactions
This project is being carried out in
collaboration with Dr. C. Kimmel of NCEA,
who will provide a critical risk assessment
perspective to the design, interpretation, and
application of these studies.
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Program Project 7: Susceptible Populations: Susceptibility Associated with
Older Adults
Objectives
Determine the characteristics that define
older adults as a susceptible subpopulation,
Determine PK and PD basis for differential
responses of older adults, and
Determine how susceptibility in older
adults varies across systems/tissues.
Scientific Approach
By the year 2030, one in every five
Americans will be older than 65 years, nearly
double the current population. Given the
burgeoning of this subpopulation, it is critical
for planning for the care of the older adults to
determine whether environmental exposure to
toxic agents can result in an age-related
accelerated decline in function or increase in
diseases of aging such as cancer. Current
testing guidelines do not include toxicological
assessments focused on older adults.
Research on aged human and animal models
will improve the scientific underpinning of
risk assessment decisions for the aged as a
susceptible population by moving risk
assessment toward more biologically-based
decisions about uncertainty factors.
The goal of this project is to understand
whether responses to environmental insults
differ in the aged compared to the young adult
population. Research designed to reach this
goal will generate data that will identify and
prioritize those functions and mechanisms that
most lead to age-related decline. By doing so,
these studies will address the uncertainty of
whether toxicity data collected in young
adults provides a sufficient margin of safety to
protect against effects that may result from
toxic exposure to an aged individual. Older
adults comprise a subpopulation that may
have special susceptibilities to
toxicant-induced dysfunction or degeneration
due to the critical characteristics of their
life-stage. Understanding the biological basis
for a differential response in older adults can
provide the rationale for decisions on how to
appropriately incorporate the differential
sensitivity of older adults into an
environmental risk assessment framework.
The candidate mechanisms driving age-
related susceptibility that will be considered
include changes in PK, age-related alterations
in cell signaling, deposition and accumulation
of harmful metabolic by-products, natural
decline in reserve capacity, cumulative gene
replication error-induced alterations, changes
in processes specific to the cell cycle, DNA
repair, cellular homeostasis, and response to
oxidative stress.
This program project includes research in
two of the major areas of concern to older
adults, i.e. cancer and degeneration in the
central nervous system (CNS). The project on
carcinogenesis will bring to bear the many
aspects of initiation, promotion, repair
processes, and homeostasis on the issue of the
interaction between aging and toxicant-
exposure leading to tumor production. The
studies on neurodegeneration concentrate on
age- and toxicant-induced changes in response
and repair mechanisms and function in the
CNS and visual system. Work on response
and repair mechanisms in the CNS will be
compared to similar work in other tissue
systems in the cancer project to see if age-
related changes in repair and homeostatic
mechanisms are similar in these different
tissue types. This will allow us to prioritize
particular processes in specific systems.
Figure 12 illustrates the critical path for this
program project.
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Characterize Biological
Differences
Between Young Adult
and Older Animals
Determine Age-Related
Responses to
Environmental Insult
Evaluate Mechanisms of
Susceptibility to Environmental
Insults
Framework to Inform Risk Assessment Decisions Concerning Aging
Figure 12 Critical Path for Research on
Susceptibility Associated with Older Adults
Project 1 - Mechanisms of Susceptibility to
Loss of Neural Function in Older Adults.
This project is designed to investigate age-
related changes in the CNS and sensory
systems at multiple levels. Studies on
response and repair mechanisms in the CNS
will focus on differences in age- and toxicant-
induced gene and protein expression in
different brain regions to begin to understand
the different processes that are associated with
functional losses in these brain areas. The
ultimate goal of these studies is to be able to
construct a model of the chain of events
leading from alterations at the molecular level
to alterations at the functional level. To
address the broad range of age-related
alterations that adversely influence the ability
to respond to toxic insult, we propose to
measure changes in gene and protein
expression for protective, repair, and plasticity
mechanisms in the brain as a function of the
interactions of age and toxic exposure. As a
focused example of the work linking changes
at the molecular and cellular levels to changes
in functional and morphological endpoints,
studies of age and toxicant-induced effects on
the retinal degeneration and visual function
will be examined. The studies on ocular
toxicity address the concern that pesticides
may be an environmental risk factor,
particularly to aging populations already
susceptible to retinal degeneration.
Normal aging processes result in changes
in physiological systems that could contribute
to increased susceptibility to neurotoxic
exposures in older adults. The studies of gene
and protein expression will focus on the
critical pathways involved in toxic response
and detoxification/repair pathways. That is,
pathways involved in ROS generation and
elimination, proteins involved in maintenance
and repair, and genes that have wide ranging
function in regulation of cell processes such as
transcription factors or homeobox genes and
signal transduction pathways. Monitoring
global patterns of gene expression allows one
to examine multiple physiological
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mechanisms simultaneously, gain insight into
interactions between mechanisms, prioritize
them in order of importance, and to associate
them with functional alterations. Because the
CNS shares with the other organs an overall
decline in cellular and genetic repair and
homeostatic processes with aging, we hope to
determine if mechanisms that are affected by
aging in the brain and nervous system play a
similar role in age-induced susceptibility in
other systems and for other endpoints (e.g.,
cancer, non-cancer).
A second set of studies, incorporating some
facets of the gene and protein expression work
described above, will focus on effects in the
visual system in an aging model. The aging
visual system is susceptible to ocular toxicity
because of a number of factors. Anatomical
examination of retinas taken from individuals
free of ocular disease show an age-related loss
of rod photoreceptors and ganglion cells.
Along with these losses is a lifelong
accumulation of non-degradable metabolic
by-products in and under the retinal
pigmented epithelium (RPE). In addition,
older individuals represent a growing
population uniquely susceptible to loss of
vision due to age-related macular
degeneration (AMD). AMD is the leading
cause of vision loss in individuals older than
65 years; the prevalence of AMD is estimated
to increase from approximately 1.6% before
age 64 to 28% eleven or more years later. The
pathogenesis of AMD is still largely
unknown, and it is not clear whether
environmental factors may push an individual
from the already compromised state due to
aging to a state of overt pathology. Broadly-
defined chemical exposure, for example, has
been identified as a risk factor in AMD.
Retinal degeneration has been reported in
humans exposed to OP chemicals and in
animal models of pesticide exposure and has
been linked to both aging and fungicide
exposure in epidemiological studies. In
addition, there is growing evidence that
inflammatory processes play a role in the
pathogenesis of age-related macular
degeneration and other important diseases of
older adults. The proposed studies examine
retinal physiology and a second messenger
(signal transduction) pathway in the retina
known to be affected by OPs in an older
animal model, the interactions of light and
environmental toxicants in models of retinal
degeneration, and a human population
exposed to pesticides and showing signs of
retinal degeneration. Increased age is a strong
co-factor. Research in project 1 will:
Assess qualitative and quantitative
differences in response to toxicant
exposure between young and the aged in
tissue culture, animal, and human
models for neurodegenerative, protective
or repair and plasticity mechanisms at
functional, cellular, and molecular
levels. This work aims to identify
mechanisms of susceptibility, targeted
sites of neurotoxicant action, and the
resiliency of the CNS;
Correlate age-related alterations with
functional endpoints, including brain
morphometrics / pathology, function,
and behavior;
Evaluate whether effects observed
involve changes in the PK of the
chemicals studied; and
Compare cell and molecular differences
found with those determined in other
tissues or resulting in other endpoints
(e.g., cancer versus non-cancer).
Project 2 - Mechanisms of Susceptibility to
Cancer in Older Adults This project is
designed to investigate age-related changes in
the body that lead to increased occurrence of
cancer. Age is the strongest factor associated
with the incidence of cancer in humans.
While the delay between an environmental
insult and the appearance of neoplastic growth
may explain some of this circumstance,
research is needed to determine what makes
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older individuals preferentially susceptible to
the occurrence of cancer. It is possible that
environmental agents have greater effect in
older individuals. There is general agreement
that cumulative damage caused by oxidative
stress is the fundamental cause of aging.
Additional changes occur in the body when
hormone production and maintenance are
altered beginning at 40 to 50 years of age.
Both of these processes, age-related changes
in response to oxidative stress and changes in
hormone levels, will be studied in this project
because they may be critical to the enhanced
cancer susceptibility of the older individual.
Factors mediating increased susceptibility
may be present at all stages of carcinogenesis.
The older population may be more susceptible
to cancer initiation because of age-related
alterations in DNA damage, detection and
repair-fundamental processes potentially
altered by some environmental agents.
Damage in or inappropriate expression of
specific genes controlling cell proliferation or
other functions critical to maintenance of cell
homeostasis can lead to uncontrolled cell
growth resulting in cancer. Cancer promotion
processes may also be affected. These include
processes associated with chromosome
stability, DNA methylation, suppressor gene
activity, signal transduction processes and
spindle assembly, all components in the web
of promotion processes that can lead to
reduced growth control in cells of various
tissues. Disruption of a homeostatic process,
gap junction communication (GJC), which
exhibits tumor suppressor activity, can also
lead to increased cell proliferation and cancer
formation.
Activities of some hormones are also
known to influence the occurrence of cancer.
It is possible that changing hormonal levels in
older individuals affect the mechanisms of
cancer initiation and promotion by
environmental agents. For example, the
serum levels of melatonin, a known oncostatic
agent, decrease as a function of age in humans
beginning at 40 to 50 years of age. GJC will
be one process studied because it has been
shown to be augmented by physiological
concentrations of melatonin. In addition, the
occurrence of DNA damage will be studied
because melatonin has been shown to be one
of the most potent free radical scavengers in
the body.
Chemicals selected for study will provide
experimental validation of principles as well
as have direct value in risk assessment
analysis. The chemicals include the
following: (1) As, which damages DNA,
alters GJC, and changes DNA methylation;
(2) 2,4-D, which alters GJC and causes liver
tumors in rodents; (3) chloral hydrate, which
alters GJC but has not been found to cause
cancer; (4) potassium bromate and bleomycin,
which damage DNA; (5) selected chemicals in
the conazole class, which includes
Pharmaceuticals and pesticides; and (6)
dimethyl benzathracene and N-nitroso-N-
methylurea, which cause DNA adducts either
directly or after metabolic activation, the latter
being influenced by melatonin. Ionizing
radiation may also be used because it causes
DNA damage that can lead to cancer in the
absence of metabolic conversion processes,
obviating PK concerns. As in project 1 of this
program project, much emphasis will be
placed on measuring age-related differences in
gene/protein expression and chemical toxicity
in order to evaluate susceptibilities of older
individuals.
These studies will generate directly
comparable data across diverse experimental
platforms (i.e., whole animal studies, animal
cell culture studies, and human cell culture
studies). This enhances the value of these
studies in that these data may provide critical
information needed for extrapolation for the
purposes of human health risk assessment.
Research in project 2 will:
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Establish animal and cellular models to
assess whether older animals are more
susceptible than younger adult animals
to the carcinogenic effects associated
with exposure to specific environmental
agents,
Evaluate the mechanisms of increased
susceptibility in older animals, and
Establish biomarkers of response to
estimate risk in humans.
Impact
ORD seeks to provide adequate protection
to all segments of the human population,
including susceptible subpopulations. In
response to a legislative mandate, research has
concentrated on effects during development or
in the very young. Numerous gaps in the
toxicological database still exist, however, for
older populations. This program project seeks
to examine whether there are special
susceptibilities associated with older adults
compared to the healthy young adult
population. Older adults comprise a
subpopulation that may have special
susceptibilities to toxicant-induced
dysfunction or degeneration due to the critical
characteristics of their life-stage. The very
young have been recognized as a population
of interest in the FQPA and the Agency has
recently promulgated an Initiative on Aging
that will help guide research in this area in the
future. Research on older adults that
incorporates age-specific data on biologically
effective dose and health effects will improve
the scientific underpinning of risk assessment
decisions by moving risk assessment toward
more biologically-based decisions about
uncertainty factors. These studies will address
the uncertainty of whether toxicity data
provides a sufficient margin of safety to
protect against effects that may result from
toxic exposure to an aged individual. As such,
it should help our understanding of how to
appropriately incorporate the differential
susceptibility of the aged into the current risk
assessment framework of the Agency.
Cross-Agency Interactions
We anticipate interactions with the
recently-launched Agency-wide Initiative on
Aging, coordinated by Dr. K. Sykes of the
Office of Children's Health Protection
(OCHP). Research on health effects of
environmental exposures to older adults has
already been identified in this initiative as one
of the critical elements. As this initiative
develops, we hope to see opportunities for
cross-agency interactions.
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Program Project 8: Environmental Risk Factors for Asthma
Objectives
Identify and rank mold allergens which
may be important in the induction of
allergic asthma, and
Assess the effect of air pollutant exposure
on the induction and exacerbation phases of
the disease.
Scientific Approach
More than 17 million people in the US had
asthma in 1998. The incidence of asthma has
doubled in the last 20 years; the largest
increase has been among children below the
age of 15 years of age (CDC, 1998). This
increase cannot be explained by changes in
diagnostic categorization or by alterations in
the gene pool and suggests a strong
association between environmental influences
and the incidence of disease. About 75% of
asthma is associated with allergy, and it
appears that the immune system can be
programmed to promote asthmatic responses
to certain antigens early in life. In nearly
100% of elementary school children with
asthma, allergens are the primary trigger for
asthma; and their disease is thought to result
from early exposure and sensitization to
common allergens in their indoor environment
(e.g., dust mite, cockroach, molds, animal
dander). While some of these allergens have
been studied extensively (e.g., dust mite and
cockroach), almost none of the mold allergens
have been characterized despite their
widespread distribution and potential
importance in the induction and exacerbation
of asthma. A recent National Academy of
Sciences Report concluded "There is
inadequate or insufficient evidence to
determine whether or not there is an
association between fungal exposure and the
development of asthma" (NAS, 2000).
Epidemiological and clinical studies have
also demonstrated that asthmatic responses
can be exacerbated by ambient combustion-
related products and by domestic and
occupational exposures to airborne chemicals.
While epidemiological studies have not
established a clear role in the induction of
asthma for air pollutant or pesticide
exposures, animal and clinical studies suggest
that ozone, nitrogen dioxide, residual oil fly
ash, and diesel exhaust particles can act as
adjuvants to enhance allergic sensitization.
In addition, recent studies have found that
children in inner cities or living closer to
major highways have more asthma than
children in rural and suburban centers or
living further from heavily trafficked roads.
Clinical and epidemiological studies have also
shown that children with asthma are
especially susceptible to the respiratory effects
of ambient combustion-related pollutants such
as sulfur dioxide and ozone.
The recent Agency's Asthma Research
Strategy (US EPA, 2002) describes research
needs related to susceptibility factors
contributing to asthma and risk assessment
issues such as dose and type of pollutant
which can increase the incidence or enhance
severity of allergic asthma. This NHEERL
Human Health Implementation Plan addresses
several classes of environmental pollutants
identified by the asthma research strategy
including bioaerosols (i.e., endotoxin, molds
and other allergens) and combustion-related
products formed from the burning of organic
fuels. The critical steps for this program
project are illustrated in Figure 13.
Project 1 - Fungal Antigens as Initiators of
Allergic Asthma. The hypothesis for this
research is that the structure and function of
the protein constituents of mold isolates
determines their allergenicity and can be used
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Project 1
Project 2
Remediation
(NERL, NRML)
Identify and
rank potency of
fungal allergens
Confirm potency
in naturally
exposed humans
Determine adjuvancy of
diesel & other pollutants
1
Quantitative model to
extrapolate animal to human
1
Assess health effects of
real-world exposures
1
Elucidate role of environmental
agents on induction and
exacerbation of allergic asthma
Figure 13 Critical Path for Research on Asthma
to determine the relative potency of different
mold components. This project will use a
mouse model to develop a cause-effect
relationship between exposure to fungal
extracts (and specific protein components) and
asthmatic responses; characterize the specific
protein allergens from three fungi
(Metarhizium anisopliae, Stachybotrys
chartamm, and Penicillium chrysogenum),
look for common structural characteristics,
and assess the potency of these allergens
relative to known human allergens. Studies in
the project will:
Identify those proteins in Stachybotrys
chartarum, and Penicillium
chrysogenum crude extracts that elicit an
allergic antibody response
(immunoglobulin E, IgE);
Confirm that the proteins identified as
IgE inducers are the relevant allergens in
the mouse disease model and determine
if these proteins are allergenic in
humans;
Characterize the physico-chemical
properties of the allergenic protein(s)
from the three fungi and compare to
other well-characterized protein
allergens such as dust mite allergens;
and
Determine the relative potency of the
fungal allergens compared to other
allergens such as dust mite, cockroach,
and alcalase.
Project 2 - Effect of Xenobiotic Exposure
on the Incidence and Severity of Allergic
Asthma. The hypothesis for this research is
that endpoints reflecting pollutant-enhanced
severity of allergic asthma seen in
experimental animals can be successfully
modeled in vitro (cell culture systems) and
that quantitative relationships exist between
animal and human cell responsiveness.
Research in this project will develop
laboratory-based assays which can predict the
ability of various pollutants to enhance the
induction or exacerbate the severity of allergic
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asthma, in a dose dependent manner. This
approach will be tested using a prototype
pollutant (diesel exhaust particles [DEHP]) in
in vitro, in vivo (animal and controlled human
studies), and epidemiological studies. The
validated paradigm will be used to test the
relative potency of a variety of different air
pollutants and materials of interest. Research
in this project will:
Compare the genetic expression and
release of pro-inflammatory
chemokines, receptors, and other
immunoregulatory molecules in healthy
and allergic airway epithelial cells and
macrophages from humans and rodents
after incubation with diesel exhaust;
Correlate in vitro findings with in vivo
markers of disease (altered gene
expression and cytokine output, immune
function and patho-physiology) in rat
and mouse models of allergic lung
disease;
Compare qualitative and quantitative
aspects of animal data with clinical
results from human studies investigating
the effect of DEHP exposure on non-
asthmatic and asthmatic lung responses;
Utilize in vivo animal techniques and in
vitro animal and human assays to screen
chemicals and obtain relative potencies
for air pollution particles and gases of
interest;
Develop methodology for preparing,
storing, and transporting clinical
samples for subsequent immunological
analysis; and
Assess the prevalence of biological
markers of exposure to DEHP and
biological markers of immunological
function across intra-urban gradients of
combustion-related products and air
toxics among school-age children.
Impact
The results from this program project will
greatly contribute to the understanding of
environmental risk factors for asthma and will
address several key Agency needs including
species extrapolation and quantitative risk
assessment, identification of susceptible
subpopulations, aspects of children's health,
as well as assessing the health effects of air
pollutants (both indoor and ambient) (ORD,
1999). This work will also contribute to the
identification of biomarkers of both exposure
and effect that will be subsequently used in
the NCS (see program project 4 in this
Section).
Cross-Agency Interactions
The first project has a strong inter-
laboratory collaboration between NHEERL
scientists and NERL engineers and biologists
in Cincinnati. In the second project, NRMRL
engineers are developing a diesel generation
and exposure system for use in the in vivo
studies performed by NHEERL scientists.
The work from both these projects will be
transmitted to the Office of Air and Radiation,
as well as to risk assessors in NCEA.
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Program Project 9: Environmental and Genetic Interactions in Hypertensive
Rats: Oxidative Stress as a Common Susceptibility Attribute for Non-Cancer
Risks
Objectives
Determine if oxidative stress is a common
susceptibility attribute for a variety of
environmental insults, including air
pollutants, pesticides, and herbicides;
Evaluate potential mechanisms and
biological pathways responsible for
susceptibility to toxicants in the presence of
preexisting oxidative stress conditions; and
Determine genetic contribution to oxidative
stress and susceptibility to toxicants.
Scientific Approach
Individuals with preexisting disease
conditions are likely more susceptible to
environmental exposures, and often the
uncertainty factor employed in risk
assessment may not be adequate to protect
these individuals. Although diseases such as
congestive heart failure, atherosclerosis,
Parkinson's and Alzheimer's diseases,
diabetes, infertility, and cancer have diverse
etiologies, they have one common element,
namely, the presence of oxidative stress
(Forsberg et al., 2001). Genetic susceptibility
to oxidative stress has been proposed in
individuals with diseases. Compensatory
antioxidant mechanisms are activated in
response to injury and the injury in healthy
individuals is followed by a normal tissue
repair. However, in genetically predisposed
individuals with chronic preexisting diseases,
these compensatory mechanisms are defective
either due to the presence of genetic
polymorphisms or a presence of disease;
therefore, these individuals are at increased
risk of morbidity and mortality from
environmental exposures.
One of the significant compensatory
antioxidant mechanisms involves regulation
of glutathione and a number of phase II
metabolism enzymes. Synthesis of
glutathione is regulated by y-glutamyl
cysteine synthetase/ligase (y-GCS). The gene
expression is under the control of nuclear
factor erythroid-derived 2, like 2, (NRF2)
nuclear translocation and binding to
antioxidant response element consensus
sequence present in phase II metabolism
enzymes. This protective transcriptional
pathway is induced by a variety of toxicants
which result in induction of y-GCS,
glutathione S-transferase Al, heme
oxygenase-1 (HO-1), NADPH quinone
oxidoreductase-1 (NQO-1), superoxide
dismutase-1, thioredoxin, catalase, and uridine
diphosphoglycosyl transferase Ia6. Recently,
it has been shown that NKF2 knock-out mice
showed increased toxicity to a variety of
toxicants. It has also been shown that a
sensitive (C57BL/6J) mice strain is more
susceptible to oxygen-induced pulmonary
damage than the C3H/HeJ strain, and that the
difference in sensitivity appears to be
associated with mutation of NKF2 promoter
(T-»C, bp-336). This mutation is predicted to
add an Sp-1 transcription factor binding site in
the C57BL/6J mouse strain compared to
C3H/HeJ strain. A recent study also reported
a single nucleotide polymorphism at -588 in
human y-GCS which decreases expression of
this enzyme and levels of plasma glutathione
in myocardial infarction patients. Thus, these
mutations are likely candidate single
nucleotide polymorphisms associated with
susceptibility to oxidative stress in animals
and humans.
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To understand the mechanism of
susceptibility, and the role of underlying
genetic oxidative stress as a common
susceptibility attribute, relevant animal
models exhibiting genetic predisposition to
oxidative stress can be employed. This
proposal involves the use of genetically
predisposed Spontaneously Hypertensive (SH)
rats as a model for increased oxidative stress
and compare to healthy normotensive Wistar
Kyoto (WKY) rats. SH rats are genetically
predisposed to increased oxidative stress and
have compromised compensatory ability to
increase tissue antioxidants in response to
mild to moderate tissue damage (expected
from environmental exposures). The
hypothesis underlying this program project is
that a rat model exhibiting phenotypic
predisposition to oxidative stress (associated
with hypertension/cardiovascular disease
[CVD]) will show measurable susceptibility to
a variety of insults affecting different organ
systems. The research done under this
program project will determine susceptibility
of SH versus WKY rats to known toxicants
that affect pulmonary, cardiovascular,
neuronal, ocular, and reproductive systems, as
well as investigate the roles for oxidative
stress in the exacerbation of the toxic
response. This will establish a base for
reducing uncertainty factors for individual
populations who are at increased risk of
environmentally induced disease because of
underlying genetic predisposition to oxidative
stress.
All individual projects listed below will
plan studies in three phases. In the first phase,
a dose-response characterization will be done
with acute exposure scenarios to determine
the level of susceptibility in SH versus WKY
rats in several organ systems using
appropriate organ specific toxicants. To
understand the role of oxidative stress and the
mechanism of increased susceptibility, the
second series of studies will involve
manipulating the oxidative stress of SH rats
using antioxidant treatment singly or as a
mixture (i.e., tempol, lazaroid, nacysteiline,
ascorbate, glutathione). Such agents will be
administered prior and/or during their
exposure to specific toxicants mentioned
under individual projects. The antioxidant
regimen that is most successful in reducing
systemic oxidative stress will be given
consideration for the purpose of this proposal.
Antioxidants and biomarkers of oxidative
stress will be evaluated in all projects in
addition to determining organ specific toxicity
markers. Tissues harvested from these studies
will also be employed to investigate oxidative
stress-related signal transduction (MAP
kinases and protein kinase C [PKC]-mediated
signaling) and gene expression using
comprehensive commercial rat gene arrays.
In the third phase of studies, tissues from SH
and WKY rats from selected studies will be
saved for isolation of DNA in order to screen
for possible polymorphism in select gene
regions (specifically genes forNRF2, NQO-1,
HO-1, and y-GCS) based on historical
evidence of polymorphisms in susceptible
humans and animals. These studies will
establish the mechanistic basis for genetic
susceptibility to oxidative stress, and its
implication in risk assessment. The
polymorphisms will also be evaluated in
patients with CVD and Chronic Obstructive
Pulmonary Disease (COPD) which will
confirm the role of oxidative stress in human
susceptibility and establish a genetic link
between animal and human susceptibility
traits. The critical milestones for research in
this program project are summarized in Figure
14.
Project 1 - Genetic Predisposition to
Hypertension-Associated Oxidative Stress
and Increased Cardiopulmonary
Responsiveness to Ozone and Zinc. This
research addresses the hypothesis that SH rats
will have exacerbated pulmonary injury and
cardiovascular effects from exposure to ozone
and zinc. Furthermore, this exacerbated
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Project 1
Relationship Between
Oxidative Stress and
Pulmonary?Cardio-
vascular Toxicity
Project 2
Relationship Between
Oxidative Stress and
Neurotoxicity
Project 3
Relationship Between
Oxidative Stress
and Ocular Toxicity
Project 4
Relationship Between
Oxidative Stress and
Reproductive Toxicity
Project 5
Determination of
Promoter Mutation
Frequency in Humans
Establish that Oxidative Stress is a Host Susceptibility
Factor Contributing to Exacerbated Toxicity for a
Variety of Toxicants in Diverse Organ Systems
Figure 14 Critical Milestones for Research
on Oxidative Stress
response will be diminished in SH rats when
pretreated with antioxidants due to diminished
oxidant-mediated cell signaling and activation
of inflammatory mediators. Ozone has been
selected based on its specific oxidant action
and its importance as one of the six criteria air
pollutants, while zinc is selected based on its
likely cardiac effects and its potential role as
one of the causative constituent of ambient
PM. Research in Project 1 will:
Determine relative susceptibilities of SH
and WKY rats to cardiovascular and
pulmonary effects of ozone and zinc
sulfate;
Use antioxidant treatment to establish
putative mechanism of action for
oxidative stress in cardiovascular and
pulmonary effects of air pollutants;
Assess cardiovascular function using
radiotelemetry prior to, during, and after
exposure to air pollutants; and
Determine comprehensive gene
expression profile of the lung and the
heart and measure oxidative stress cell
signaling and specific nuclear factors
activation.
Project 2 - Susceptibility of Spontaneously
Hypertensive Rats to the Neurotoxic
Effects of Pesticides: the Role of Oxidative
Stress. Oxidative stress is a condition
affecting tissues and cellular functions in a
number of disease states. In healthy
organisms, ROS which mediate oxidative
stress, are generated during normal
mitochondrial bioenergetics and are
scavenged by various enzymatic and non-
enzymatic antioxidants (e.g., glutathione,
catalase, xanthine oxidase, ascorbic acid, uric
acid). However, if the individual has genetic
errors which amplify the formation or reduce
the scavenging of ROS, this compensatory
response is reduced, resulting in a heightened
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state of oxidative stress to cells and tissues.
Clinical and experimental evidence indicates
that oxidative stress underlies the
neurodegenerative condition of Parkinsons
Disease. This disease is thought to be a
multifactorial condition involving a genetic
predisposition to errors in energy metabolism
(i.e., mitochondrial complex I), environmental
exposure to chemicals that target energy
metabolism (e.g., certain pesticides) and a
high susceptibility of mesencephalic
dopaminergic neurons and glia to oxidative
stress and energy depletion. Project 2 will
examine the interplay of such factors (i.e.,
genetics, chemical exposure,
neurodegenerative diseases) in SH rats, a
strain which has a genetic predisposition to
oxidative stress. The first phase of this study
will compare behavioral and neurochemical
endpoints of oxidative stress in response to
ChE-inhibiting pesticides (carbamates, OPs)
and paraquat. Endpoints that are
representative of oxidative stress will be
measured. Brains taken from representative
animals showing both behavioral (i.e.,
reduction in locomotor activity) and
neurochemical changes will be examined for
neuropathological changes. In vitro
experiments will examine the effects of
complex I inhibitors (e.g., paraquat, rotenone)
on mesencephalic glia and neurons. The
effects of antioxidant pretreatment on
pesticide-induced behavioral and
neurochemical changes will then be examined
in whole animals, their brain tissues, and CNS
target cells in vitro. It is hypothesized that
because of their condition of chronic stress
and reduced ability to scavenge ROS, SH rats
will show an increased susceptibility to the
neurotoxicity associated with selected
pesticides. Research in this project will:
Compare behavioral, neurochemical, and
neuropathological endpoints of oxidative
stress in response to various pesticides in
animals;
Describe the morphological and
neurochemical response of PD target
neurons and microglia to complex I
inhibiting pesticides;
Determine activation of PKC and MAP
kinase signaling and gene expression
patterns in response to pesticide
exposures; and
Determine the protective effects of anti-
oxidant treatment on pesticide-induced
neurotoxicity.
Project 3 - Ocular Toxicity and Oxidative
Stress. Visible light, the primary stimulus for
the visual system, is also its primary toxicant.
Many xenobiotic compounds absorb light in
the ultraviolet or visible wavelengths and
produce ROS (singlet oxygen) in response.
This project will investigate whether the
mixture of light-induced damage is a
mechanism for the generation of ocular
toxicity of the retina and the lens. The
hypothesis for this project is that xenobiotic
chromophores will absorb light and produce
damage through oxidative stress in ocular
structures and this damage will be exacerbated
in animal models deficient in protective
mechanisms due to either inherited
characteristics, e.g., as in SH rats, or due to
dietary deficiencies in antioxidants.
Antioxidant supplementation would likely
reduce damage. Research in this project will:
Use physiological, morphological and
biomicroscopic endpoints to assay
damage to the retina and lens following
exposure to insecticides and fungicides;
and
Determine levels of ROS in specific
ocular tissue to establish connection
between oxidative stress and retinal
damage.
Project 4 - Role of Oxidative Stress in the
Reproductive Function of Males and
Females. Epidemiological studies have
shown that certain subpopulations of people
have lower blood levels of glutathione and
may be more prone to oxidative injury in
somatic cells. Research in this project will
address the possibility that such individuals
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may be at increased risk of adverse
reproductive effects associated with the
induction of oxidative stress in gonadal
tissues. The hypothesis for this research is
that SH rats will have a diminished testicular
response to oxidative stress as compared to
the WKY strain and that lower amounts of
glutathione and decreased induction of
glutathione synthetic enzymes may be found
in the challenged SH animals. Differences in
levels of NRF2 would suggest a role for this
transcription factor in the altered response.
Sperm motility may also be affected in these
animals due to increased membrane lipid
peroxidation in the presence of diminished
glutathione levels. These animals will also
serve as positive and negative controls for
subsequent experiments (DNA array analysis).
The hypothesis for this research is that
diminished response to oxidative stress may
exacerbate the effects of known testicular
toxicants such as aery 1 amide and
thiocarbamate pesticides such as thiram or
molinate. Finally, the effects of
environmental toxicants on zygote formation
in SH and WKY rats will be tested. This
research will:
Determine if the SH rat is significantly
more susceptible to toxicant-induced
oxidative stress/antioxidant levels than
normal rats,
Determine oxidative stress-induced
responsive gene expression pattern in
testis following exposure to toxicants in
WKY and SH rats; and
Determine the biomarkers and the role
of oxidative stress in susceptibility to
toxicants.
Project 5 - Oxidative Stress in CVD and
COPD Patients and Possible NRF2
Promoter Mutation. Lower levels of
antioxidants and systemic oxidative stress
have long been considered risk factors
associated with CVD, COPD, and other
chronic diseases. The presence of genetic
mutations in antioxidant compensatory genes
and their role in pathobiologic mechanisms of
disease development are not well understood.
To determine if NKF2-mediated
compensatory antioxidant mechanisms are
involved in predisposition to oxidative stress,
selected gene mutations (specifically genes for
NRF2, NQO-1, HO-1, and y-GCS) that are
known to exist in humans and animals will be
evaluated in SH rats and humans with CVD
and COPD. This study will allow us to
establish the genetic basis to oxidative stress
and consideration of genetics in relevancy of
animal model to human diseases and will
Correlate the detection of increased
mutation frequency in NRF2 promoter
and other related genes with that of the
severity of disease in humans and its
incidence in SH rats.
Impact
Currently an uncertainty factor of 10 is
employed in risk analyses in order to account
for differences in susceptibility due to host
preexisting conditions and genetics.
However, recent epidemiological evidence
demonstrates that in many instances
susceptibility may lead to increased mortality
or disease such that an uncertainty factor of 10
may not be adequate. There is often
incomplete knowledge regarding the
existence, extent, and characteristics of
individual susceptibility about who is
susceptible and to what extent. Identifying
the mechanisms that underlie susceptibility to
many environmental toxicants that play a role
in the development or exacerbation of disease
leading to death is important in both basic risk
assessment and reducing the uncertainty.
Research in this program project will also
utilize a relevant animal model to determine
increased susceptibility to a variety of toxic
insults and organ systems due to underlying
oxidative stress/disease. Knowledge of the
mode or mechanism of action for specific
agents will reduce uncertainties in their risk
assessment. Mechanistic work generating in
this program project may also lead to the
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identification of new biomarkers of toxicity,
as well as susceptibility to toxic insult.
This research will also lead to the
development and use of technologies to
evaluate gene expression profiles to
understand patterns of pathological processes
and identify potential gene polymorphisms to
better define susceptibility. These
technologies will lead to a greater
understanding of the key events associated
with adverse effects. Thus, the data generated
from the gene expression profiling studies will
be useful to the emerging Computational
Toxicology research program.
Cross-Agency Interactions
As results are produced, collaborations with
NCEA and Program Offices will be
developed.
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Program Project 10: Genotype and Phenotype in the Metabolism, Toxicity
and Carcinogenicity of Arsenic (As)
Objectives
Identify genetic polymorphisms for
enzymes that metabolize As,
Examine the regulation of the gene and the
role of nutritional status in its regulation,
Examine the function of arsenate reductase
as a critical factor in metabolism of As, and
Examine As-protein interactions to gain
insight into the mode of toxicity for this
metalloid.
Scientific Approach
It is known that genetic polymorphisms and
mutations can greatly affect human
susceptibility to mutation and/or cancer (e.g.,
fast and slow acetylators of aromatic amines
and bladder cancer, xeroderma pigmentosum,
Cockayne's syndrome and
trichothiodystrophy). Genetic polymorphisms
and mutations may significantly affect the
outcome of exposures to chemical stressors,
such as As. Given the relatively high
frequency of polymorphisms in the human
population, this proposal will address the
potential effect of specific polymorphisms on
responses to environmental exposures.
This research will study the role of genetic
factors in the expression of toxicity by
investigating a prototypic compound having
environmental relevance, i.e., As. There is a
consensus that understanding variability in the
metabolism of iAs in humans is critical to
understanding the response of humans to
chronic exposure to iAs (Lin et al., 2002;
National Research Council, 1999). Inter-
individual variation in absolute and relative
amounts of methylarsenic and dimethylarsenic
in urine has been attributed to differences in
ethnicity, age, sex, nutritional status, and
extant disease. In humans chronically
exposed to iAs in occupational and
environmental settings, considerable inter-
individual variation has been found in the
occurrence of specific signs and symptoms of
As intoxication. For example, only a small
fraction of individuals chronically exposed to
iAs in drinking water develop blackfoot
disease, a progressive occlusive disorder of
the peripheral vasculature. A role for
metabolic capacity in determination of
susceptibility is supported by studies in
chronically-exposed populations that find
absolute or relative increases in the amount of
metabolites of As in urine are associated with
increased occurrences of As -induced skin
lesions.
This program project will determine
whether the inter-individual variation in
response is related to differences in the
capacity to metabolize iAs, if these
differences in metabolic capacity are primarily
determined by the kinetic properties of the
enzymes that catalyze the reduction of
pentavalent As and the methylation of
arsenicals, if the kinetic properties of these
enzymes are determined by their primary
sequence (hence, by the genotypes for these
proteins), and whether control of the
expression of these genes may be an important
in determination of the capacity for
metabolism of As. As a corollary to these
postulates, this research will determine if there
is a linkage between the genotypes for these
enzymes and the disease susceptibility
phenotype which is manifested by the capacity
of cells or organisms to metabolize iAs. It is
likely that polymorphisms in the genes which
encode these proteins determine their catalytic
activities and that the transcriptional
regulation of these genes affects their
expression. If susceptibility to the adverse
effects of chronic exposure to iAs is related to
capacity to produce methylated metabolites,
then genotypes for these critical enzymes are
highly relevant to predicting the hazard
associated with chronic exposure to this
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Identify Genetic
Polymorphisms
Important for
Metabolism
Develop Necessary
Molecular Methods
for Screening
Human Cells
Determine if Genes
Influence Sensitivity
In Vitro
Demonstrate
Susceptibility
and Sensitivity
In Vivo
Demonstrate Genetic Susceptibility
and Sensitivity in Humans and
Animals
Figure 15 Critical Path for Research on
Genetic Polymorphisms
metalloid. Studies of genotypic-phenotypic
relations in human cells and in individuals are
important steps in estimating the role of
interindividual variation in determination of
susceptibility. The critical steps for research
on genetic polymorphisms are summarized in
Figure 15.
Project 1 - Genotype-Phenotype
Correlations for As Methyltransferase in
Humans. Research in this project will
examine the relation between the genotype of
Cytl9, a gene encoding the human As
methyltransferase and the phenotype for the
capacity to methylate iAs in human cells and
in individuals chronically exposed to iAs. The
hypothesis for the proposed research is that
the genotype of Cytl9 affects the phenotype
for As metabolism as exemplified by the
pattern and amounts of iAs and metabolites
produced by cultured human cells or present
in blood or excreted in urine by chronically
exposed individuals. The specific aims for
this study are (a) to examine in cultured
primary human hepatocytes, the relation
between the Cytl9 genotype and the
phenotype for the metabolism of As, as
reflected by the amounts of iA and
metabolites in cells and media; (b) to examine
genotypic variation in Cytl9 using DNA
isolated from peripheral blood of a number of
human volunteers; and (c) to examine the
relation between the Cytl9 genotype and the
phenotype for the metabolism of As as
reflected by the amounts of iAs and
metabolites in blood and urine from humans
chronically exposed to iAs in drinking water.
Research in project 1 will:
Examine the relationship between Cytl9
genotype and phenotype in cultured
primary human hepatocytes from 20 to
30 donors,
Examine the genotypes for Cytl9 using
DNA from a larger number of human
donors; and
Study genotype and phenotype in
individuals who are chronically exposed
to iAs.
Project 2 - Genetic Susceptibility and
Sensitivity to As Exposures. This project
will address the hypothesis that cells, animals,
or individuals who might show
polymorphisms at the gene for glutathione
transferase omega (GST-O) will be
differentially susceptible to the genotoxic and
hence carcinogenic effects of As. Research in
this project will:
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Determine if polymorphism in GST-O
exists and if it may be responsible for
differential sensitivity to arsenic
genotoxicity.
Project 3 - Altered Phenotype-Binding
Studies of Arsenicals and Purified Proteins
or Synthetic Peptide Amino Acid Sequences
of Importance in As Metabolism Reductase
and Cytl9) or Mechanism of Action. This
research address the hypothesis that there are
polymorphisms in As metabolism and
mechanism of action (Kitchin, 2001). Genetic
susceptibility to arsenicals will be revealed by
the binding of arsenicals to certain amino acid
sequences of proteins that are important in As
metabolism or mechanism(s) of action. If
there is a polymorphism in human Cyt 19,
there will be an altered amino acid sequence
that can be studied via binding experiments.
If there is a genetic deletion or substitution of
key sulfhydryl(s) required for the binding of
the arsenical would be altered. These studies
will:
Evaluate altered binding affinity and/or
amount of binding of arsenicals to
polymorphic animal or human proteins.
Project 4 - Interactions of Dietary Folate
Deficiency and As: Evaluations of Gene
Expression Changes in Association with
Enhanced DNA Fragility and Possible
Alterations in Methylation Patterns. As
toxicity may be enhanced in individuals with
folic acid (folate) deficiency. As this vitamin
is needed for normal DNA synthesis and
methylation, its deficiency can result in effects
which interact with those of As via different
pathways. Folate deficiency can result in
uracil misincorporation and increased DNA
fragility, an effect which could be increased
by As inhibition of repair enzymes. This
deficiency may also lead to a depletion of S-
adenosylmethionine required for methylation
to result in DNA hypomethylation and
possibly decreased As detoxification. These
studies will:
Use a mouse model to evaluate global
changes in gene expression associated
with folate deficiency, As exposure, and
most important, folate deficiency plus
As exposure.
Project 5 - Transcriptional Control To
study the transcriptional control of Cytl9 (As
[III] methyltransferase) and the expression
pattern of Cyt 19 in various human tissues.
Cyt 19 has been recently identified as the
methyltransferase that catalyzes the formation
of methylated and dimethylated As.
Methylated arsenicals that contain As(III) are
more potent than other forms of arsenicals in
regards to genotoxicity (Mass et al., 2001) and
inhibition of various enzyme activities. Our
hypothesis is that the polymorphisms in the
transcriptional activity of Cyt 19 contributes
to human susceptibility to mutation and/or
cancer caused by As. By understanding the
transcriptional control of this gene, we are in
better position to understand the human
susceptibility to As. Methylation of As is a
critical feature of its metabolism, therefore,
characterization of Cyt 19 gene and its
expression pattern will improve our
understanding of the mechanism of As as a
toxin and a carcinogen. This research will:
Delineate the regions and factors that
control the expression of the Cyt 19
gene,
Study the tissue specificity of this gene
by looking at the presence or absence of
the expression factors in different
tissues, and
Study the expression of this gene in
different populations.
Cross-Agency Interactions
We have ongoing collaborations with
NERL-Cincinnati to develop and validate new
analytical methods that are used in our work
on genetic polymorphisms of As metabolism.
We also have a ongoing collaboration with
Region 10 to examine metabolism of As in
individuals ingesting a variety of marine
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organisms. This work may include studies of
genetic polymorphisms of As metabolism.
Impact
Although most attention has focused on the
potential health effects of chronic ingestion of
As-contaminated drinking water, substantial
exposure (and risk) occurs from other sources.
As is a component of particulate material
generated by fuel combustion, is introduced
into the environment by the use of As-
containing pesticides, and occurs in complex
organic species (e.g., arsenobetaine,
arsenosugars) in many foods. Hence, data
from the proposed research on the control of
the metabolism and toxicity of As will
contribute to risk assessments for chronic
exposure to As from water, air, and food.
Because exposure to As occurs from each of
these media, the availability of new data on
the metabolism and effects of As will improve
the risk assessment for this element and assist
the Agency in its mission to protect public
health.
In general terms, the proposed research will
emphasize three distinct, but related, aspects
of the health effects of As. Understanding the
role of genetic susceptibility in the control of
As metabolism will benefit the risk
assessment process by identifying
subpopulations of individuals who are
genetically predisposed to greater
susceptibility to the toxic or carcinogenic
effects of chronic As exposure. Second,
experimental evidence may be obtained that
show unusual sensitivity to As. Third, the
factors that are identified in the proposed
research as modifiers of the metabolism and
toxicity of As can be incorporated into
quantitative models which describe the
systemic and cellular metabolism of As and its
MOA as a toxin and carcinogen.
This approach would extend current
modeling efforts that examine the fate of other
forms of As (i.e., total As or total methylated
As) and improve the utility of these models in
risk assessment. Taken together, the proposed
research effort examining the genotypic and
phenotypic control of metabolism, toxicity,
and carcinogenicity of As will contribute to
the Agency's periodic reevaluation of risk
assessments for this element in water, air,
pesticides, and other media.
4.5 Gap Analysis and Links to Other Multi-
Year Plans
NHEERL's implementation plan with
regard to susceptible subpopulations focuses
on life stage, genetic polymorphisms, and
asthma. NHEERL research on children is
consistent with research needs outlined the
Agency's Strategy for Research on
Environmental Risks to Children (US EPA,
2000). NHEERL research on older adults
responds to the Administrator's interest and
concern about the environmental health of
older Americans. Research described in the
program project on Aging has been included
in the recent inventory of Agency intramural
and extramural research projects organized by
the OCHP. Research related to older adults is
envisioned to increase over the next few years.
NHEERL research on genetic
polymorphisms deals primarily with a limited
number of possible genes associated with
differential sensitivity to the prototypic
chemical used in these studies, i.e., As. There
is considerable human and animal data
available on As, and it is clear that there is a
subpopulation of people that respond
differently to this metal. As indicated in the
program project on genotype and phenotype in
the metabolism, toxicity, and carcinogenicity
of As, there are several testable hypotheses
concerning potential gene-chemical
interaction that should provide basic
information needed to develop strategies for
addressing this issue with other environmental
agents from other chemical classes. Research
on a non-cancer class of chemicals, such as
the CHE-inhibiting pesticides, would provide
useful complementary information with
regard to gene/chemical interactions. Results
4-36
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from a cancer and non-cancer agent could be
used to help develop risk assessment
strategies that include potential gene/chemical
interactions in susceptible subpopulations.
NHEERL research with regard to
preexisting disease focuses on asthma and is
consistent with research priorities outlined in
the Asthma Research Strategy (US EPA,
2002). Other diseases such as autoimmune
deficiency diseases, cardiopulmonary
diseases, diabetes, and neurodegenerative
disorders such as Parkins disease could
potentially interact adversely with exposure to
environmental pollutants. NHEERL does not
plan to conduct extensive research on these
diseases at the present time, although
compelling epidemiological research
suggesting an interaction between a chemical
class and a disease could generate hypotheses
that could be tested in animal models.
Research on susceptible subpopulations is
also linked to several other MYPs. In the case
of Drinking Water, NHEERL research will
contribute to other studies to identify the risk
of birth defects due to drinking water
contaminants, provide fundamental
information on the basis of inter-individual
variation in As metabolism. For Air Toxics
and PM, NHEERL research will help identify
the role of specific polymorphisms in the
metabolism and repair genes and the relative
increase or decrease in tumorigenic risk
associated with exposure to air pollutants,
identify parameters for models describing the
deposition of fine and coarse mode particles in
older adults and moderate asthmatics, provide
basic research on the effects of concentrated
air particles in asthmatic humans and animals,
provide data for a report on the acute
respiratory effects of PM and co-pollutants in
asthmatic children, help describe the effects of
metals in animal model of allergic asthma,
and provide basic data to help describe the PK
of oxygenate mixes in young and older adult
populations. For EDCs, NHEERL research on
susceptible subpopulations will contribute to
the evaluation of toxicant-induced alterations
in mammalian reproductive development as a
function of critical window of exposure,
provide basis information for research on the
influence of life stage on the sensitivity and
reproducibility of estrogen cDNA
macroarrays, help evaluate critical windows
of exposure for alterations in mammary gland
development and lactational function, and
characterize the effects of atrazine delivered
during critical windows of development.
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4.6 References
Barone, S., N. Haykal-Coates, D. K. Parran, and
H.A. Tilson. Gestational exposure to methyl
mercury alters the developmental pattern of trk-
like immunoreactivity in the rat brain and results
in cortical dysmorphology. Dev Brain Res
109(1), 13-31(1998).
Centers for Disease Control and Prevention.
Forecasted state-specific estimates of self-
reported asthma prevalence-United States,
1998. MMWR 47:1022-1025 (1998).
www. cdc. gov/epo/mmwr/preview/mmwrhtml/O
0055803 .ht.
Fenton, S. E., and C.C. Davis. Atrazine increase
dimethylbenz{a}anthracene-induced mammary
tumor incidence in Long Evans offspring
exposed in utero. Toxicol Sci 66, 185 (2002).
Forsberg, L., U. de Faire, and R. Morgenstern.
Oxidative stress, human genetic variation, and
disease. Arch Biochem Biophys 389:84-93
(2001).
Godfrey, K.M. and D.J. Barker. Fetal
programming and adult health. Public Health
Nutr 4:611-624 (2001).
Kehoe, P. K. Mallinson, J. Bronzino, and C.M.
McCormick. Effects of prenatal protein
malnutrition and neonatal stress on CNS
responsiveness. Brain Res Dev 132(1):23-31
(2001).
Kitchin, K.T. Recent advances in arsenic
carcinogenesis: modes of action, animal model
systems and methylated arsenic metabolites.
Toxicol Appl Pharmacol 172:249-261 (2001).
Langley-Evans, S.C., D.S. Gardner, and S.J.M.
Welham. Intrauterine programming of
cardiovascular disease by maternal nutritional
status. Nutrition 14:39-47 (1998).
Lassiter, T.L., L.D. White, S. Padilla, and S.
Barone. Gestational exposure to chlorypyrifos:
qualitative and quantitative neuropathological
changes in the fetal neocortex. Society of
Toxicology 2002 Abstract, Nashville, TN
(2002).
Lin, S., Q. Shi, F.B. Nix, M. Styblo, M.A. Beck,
K.M. Herbin-Davis, L.L. Hall, J.B. Simeonsson,
and D.J. Thomas. J Biol Chem 211:10795-
10803 (2001).
Mass, M.J., A. Tennant, B. Roop, W.R. Cullen,
M. Styblo, D.J. Thomas, and A. Kligerman.
Methylated trivalent arsenic species are
genotoxic. Chem Res Toxicol 14:355-361
(2001).
Merlet-Benichou, C., T. Gilbert, M. Muffat-Joly,
M. Lelievre-Pegorier, and B. Leroy. Intrauterine
growth retardation leads to a permanent nephron
deficit in the rat. Pediatr Nephrol 8:175-80
(1994).
Miller, M.S., M.R. Juchau, P.P. Guengerich,
D.W. Nebert, and J.L. Raucy. Drug metabolic
enzymes in developmental toxicology. Fundam
Appl Toxicol 34:165-75 (1996).
U.S. Environmental Protection Agency, Office
of Research and Development. Draft Strategy
for Research on Environmental Risks to
Children. 92pp. (1999).
http://www.epa.gov/ncea/pdfs/Draft21 .PDF
National Academy of Sciences. Clearing the
Air: Asthma and Indoor Air Exposures.
National Academy Press, Washington, DC.
(2000).
National Research Council. Arsenic in Drinking
Water. National Academy Press, Washington,
DC. (1999).
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Rhind, S.M., M.T. Rae, andN. Brooks. Effects
of nutrition and environmental factors on the
fetal programming of the reproductive axis.
Reproduction 122:205-214. (2001).
Schardein, J.S. Chemically Induced Birth
Defects. 3rd Ed., Marcel Dekker, New York.
(2000).
Smialowicz, R.J., W.C. Williams, C.B.
Copeland, M.W. Harris, D. Overstreet, G.J.
Davis, and R.E. Chapin. Effect of
perinatal/juvenile heptachlor exposure on adult
immune and reproductive system function in
rats. Toxicol Set 61:164-175 (2001).
U.S. Environmental Protection Agency, Office
of Research and Development. Strategy for
Research on Environmental Risks to Children.
Washington, DC. EPA/600/R-00/068. 2000.
http://cfpub.epa.gov/ncea/cfm/recordisplav.cfm?
deid=20068
U.S. Environmental Protection Agency, Office
of Research and Development. Asthma
Research Strategy. Washington, DC.
EPA/600/R-01/061. 2002.
http://cfpub.epa.gov/ncea/cfm/recordisplav.cfm?
deid=54825
Weisglas-Kuperus, N., S. Patandin, G. Berbers,
T. Sas, P. Mulder, P.J.J. Sauer, and H.
Hooijkaas. Immunological effects of back-
ground exposure to polychlorinated biphenyls
and dioxins in Dutch preschool children: an
exploratory study of health effects from
"normal" environmental exposure. Environ
Health Perspec 108:1203-1207 (2000).
White, L.D., T.L. Lassier, K.P. Das, and S.
Barone. Prenatal exposure to chlorpyrifos alters
neurotrophin immunoreactivity and apoptosis in
rat brain. Society of Toxicology 2002 Abstract,
Nashville, TN (2002).
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Section 5
Cumulative Risk
5.1 Problem
Cumulative risk is of interest to a wide
variety of Agency's Program Offices. For
example, the FQPA directs the OPP to include
in its assessments of pesticide safety the
risk(s) associated with the cumulative effects
of chemicals that have a common MOA. The
Office of Water (OW) is concerned with risks
from mixtures of DBFs and balancing any
risks associated with DBFs, as single
chemicals or mixtures, against the risks
associated with microbial agents in water.
Recently, CCL chemicals have become a
concern of OW, leading to considerations of
cumulative risk for CCL chemicals as well as
mixtures containing DBFs and CCLs. The Air
Office is concerned with mixtures of criteria
air pollutants and volatile organic compounds.
The Waste Program is concerned with
mixtures of chemicals from the same or
different chemical classes that are present in
the air, water and soil in and surrounding
waste sites. In addition to these
chemical-specific concerns, the Agency is
concerned with communities that may either
have elevated exposure to chemicals or may
be subject to stressors such as poverty, lack of
access to medical care, or inadequate
nutrition.
NHEERL's research will focus on
providing data that may be used in
development and refinement of risk
assessment approaches, particularly
addressing principles that may underlie
interactions of chemicals at low dose levels
(Teuschler et al., 2002). This research will
utilize empirical and mechanistic data and
models described below, as well as results
derived from work on harmonization (see
Section 4), to develop strategies to predict the
effects of chemical mixtures. Either or both
bottom-up (component-based) and top-down
(whole mixture) approaches may prove useful
for development of mechanistic data and
predictive models.
The vast majority of risk assessments for
chemical mixtures rely on component-based
approaches. This raises a concern with
cumulative exposure because exposure to
multiple chemicals may interact in ways not
predicted or expected based on the
dose-response curves of the single chemicals
and on an assumption with regard to additivity
(i.e., toxicity may be greater or less than
expected based on the single chemical
information and an assumption of either dose-
addition or response-addition). There is also a
minor concern with cumulative exposure that
toxicity may be seen in an unexpected organ.
As dose-additivity for non-cancer health
effects is a default assumption on which many
Program Offices rely, including OPPTS for
OP pesticides, a major goal of this program
project is to examine those circumstances
under which dose-additivity is a reasonable
assumption. Included in this effort is the
development of efficient experimental designs
and statistical methods for mixtures more
complex than 2-3 chemicals.
Another very important research issue is
what happens when there are components of a
mixture which have different MOAs or
multiple mechanisms. As response-addition is
the risk assessment default assumption for
mixtures of chemicals with diverse MOAs,
another major research goal of the program is
the examination of the potential for additivity
of mixtures of chemicals with different
MOAs. Included in this research will be
comparisons of the appropriateness and
predictive value of response-addition versus
dose-addition for various mixtures.
Another important issue for either
component-based or whole mixture
approaches is whether the quality, e.g., nature
(greater than-, less then-, or additive) or
5-1
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Mechanism or
Mode of
Action
Project 1
Test Dose-Additivity for
Agents with Common
Mode of Action
Project 2
Test Response-Additivity
for Agents with
Dissimilar Modes
of Action
Project 3
Test Additivity
Assumptions following
Repeated Exposure
Framework to Predict Interactive Effects of Pollutants in Mixtures
Figure 16 Critical Path for Research on Cumulative Risk
magnitude, of the interactions of chemicals
change as the dose level changes (Simmons,
1995) or as a function of repeated exposure.
Data are needed to describe the dependence of
interaction magnitude on mixing ration, total
mixture dose and on component fractions.
NHEERL research on cumulative risk will
explore the influence of dose and exposure
scenario on interactive toxicity. Historically,
most mixtures research has focused on short-
term exposure to higher portions of the dose-
response curve where such situations as
saturation kinetics might influence the
interactive outcome. In contrast to this
historical situation, NHEERL research will
include a targeted focus on the low end of the
dose-response range and on repeated
exposures.
NHEERL research will also address the
problem of determining a threshold response
for the endpoint(s) of interest at
environmentally relevant doses. These may
include doses at or below the No-Observed-
Effect-Level (NOEL) or No-Observed-
Adverse-Effect-Level (NOAEL) of the single
chemicals and doses representing either levels
found in the environment or at or near the
RfD. Doses will be selected to be as close to
environmentally relevant levels as permitted
by the limits on experimental sensitivity due
to the number of animals used. In vitro work
will make assumptions regarding the
extrapolation of experimental doses to the
target site concentration in vivo. The upper
end of the dose-response curves will include
effects which are determined to be adverse for
the endpoint in question. It should be noted
that experiments utilizing the mixture ray
design explicitly measure responses over a
range of effect levels. Effects will not,
however, be characterized at dosages which
produce overt toxicity such as animal
lethality. The critical path for this research is
illustrated in Figure 16.
5.2 Goal
The overarching goal of NHEERL research
on cumulative risk is to provide the scientific
basis for predicting interactive effects of
pollutants in mixtures and the most
5-2
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appropriate approaches for combining effects
and risks from pollutant mixtures.
5.3 Critical Path
Figure 16 outlines the critical steps that will
be taken for NHEERL research on cumulative
risk. First, either mechanistic data on selected
environmental pollutants or assumptions
about MOA will be used to design subsequent
research on mixtures (see Section 3). A
second step will be to study interactions
among chemicals with a known or assumed
common MOA to determine if the interactions
among them are dose-additive. A next step
will be to determine if interactions among
chemicals with dissimilar MOAs are
response-additive. The last step will be to
investigate whether the nature (i.e., additive,
greater than additive) of the interact!on(s) and
their magnitude, if non-additive, that result
from acute exposure are different from those
that result from repeated exposure. Research
in this program project will be consistent with
APGs and APMs in the Cumulative Risk
Section of the ORD Human Health Research
Multi-Year Plan (Appendix D).
5.4 Program Project
Only one program project "Exploring the
Limits of Additivity" was developed. It is
concerned with the assumption of addivity use
in risk assessment of chemical mixtures.
5-3
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Program Project 11: Exploring the Limits of Additivity
Objectives
Test the assumption of dose-additivity for
chemicals with a similar MO As,
Test the assumption of response-additivity
for chemicals with dissimilar MOAs, and
Determine if the assumption of dose- and
response-additivity changes following
repeated exposure.
Scientific Approach
The Agency has published several
documents describing methods to perform
health risk assessments of chemical mixtures
(US EPA, 1986; 1990, 2001) in which three
approaches (i.e, actual mixture, similar
mixture or component-based) to quantifying
health risk for a chemical mixture are
recommended depending on the type of data
available to the risk assessor. The preferred
approach is to utilize data on the complex
mixture itself, but such data are often not
available. The second approach, which is
recommended when data limitations prohibit
the use of the first approach, uses data on a
"sufficiently similar mixture or a group of
similar mixtures." As these data are also very
scarce, the overwhelming majority of
mixtures risk assessments are conducted with
component-based approaches. Thus, this
research will focus on component-based
approaches to provide information useful to
how mixtures risk assessment are actually
done in the present and in the future. In
addition to this component-based focus,
several research projects will take a "mixture
as the whole" approach.
The overall approach of project 1 of this
program project will be to test the assumption
of dose-additivity based on the evaluation of
several classes of chemicals, including OPs,
carbamates, pyrethrins, DBFs, persistent
organic pollutants, and azoarene-contaminated
water effluent. Project 2 will use the same
overall approach to determine if the default
assumption of response additivity holds for
chemicals having dissimilar MOAs. Project 3
will focus on predicting effects of exposure to
selected pesticides following repeated
exposure.
Project 1 - Develop and Apply Methods to
Examine Whether the Assumption of Dose-
Additivity Holds for Mixtures Comprised
of Two or More Chemicals Acting via a
Common MO A. This project will use
relatively low exposure levels for the
component chemicals in the mixtures. Many
earlier interaction studies have explored the
consequences of high dose combinations, i.e.,
LD50s. Instead, these mixture studies will use
low doses, e.g., levels that do not produce
overt toxicity and approach more
environmentally relevant levels. In addition,
attention will be paid to the ratios of
chemicals present in the mixture because the
proportion of individual chemicals may
influence the nature of the interaction.
Research in this project will focus on
determining whether an interaction between
pesticides in a mixture can be assessed by
their actions at specific biomolecular targets,
i.e., ion-gated or second messenger-linked
neurotransmitter receptors, various enzymes,
cellular macromolecules, DNA, proteins, or
even specific moieties of a chemical structure.
This research will address the hypothesis that
compounds that interact with a common
biomolecular target (after adjustment for
potency and PK differences) will behave in a
dose-additive manner. Specific issues to be
addressed by these studies include the
following:
Development of molecular and
mathematical models for the interactions
of pesticides with specific bio-molecular
targets,
5-4
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Assessment of interactions of pyrethroid
pesticides on voltage-gated Na+ channel
function and membrane excitability,
Assessment of interactions between OP
and carbamate pesticides in rats
Assessment of interactions of mixtures
of multiple (3-5) OP pesticides in adult
and pre-weanling rats, and
Assessment of the interaction of
multiple carbamate pesticides in adult
rats.
Research in this project will also use
diagnostic genotoxicity assays to examine low
dose additivity assumptions for mixtures of
compounds that act via a common MOA.
This research will examine the following null
hypothesis: regardless of the genotoxic
MO As and the specific type of xenobiotic
metabolism, mixtures of genotoxic
compounds that act by a common MOA, when
applied at low doses, do not depart from the
linear dose-additivity assumption. This
research will:
Compare the low dose-response of
environmental mixtures and their
corresponding "totally artificial
mixtures" and the statistical (risk
assessment) prediction of the mixture
from the artificial mixtures for the
environmental mixture;
Determine if the frequency of "rare
spontaneous mutations" for compounds
that elicit these rare events provides a
means for examining the dose-response
curve at extremely low doses;
Examine the hypothesis that multiple
chemicals give an additive response
when each chemical is applied at below
detectable (response) doses but the
modeled additive response gives a
minimally detectable response; and
Address the observation that genotoxic
mixtures also have other toxic effects
(e.g., immunotoxicity).
Research in this project will develop and
use experimental designs and statistical
analysis methods for mixtures of multiple
chemicals having a common MOA. Chemical
mixtures to be evaluated are a chemical
mixture comprised of 4 trihalomethanes
(chloroform, bromoform,
bromodichloromethane and
chlorodibromomethane) that are regulated by
the Agency; a chemical mixture comprised of
5 HA As regulated by the Agency
(monochloro-, dichloro-, trichloro-,
monobromo-, and dibromoacetic acid); and a
9 chemical mixture that contains the sub-
mixtures. This research will:
Extend existing methodology for simple
mixtures (Gennings et al., 1997) to
mixtures of up to 10-20 chemicals.
Additional research in project 1 will test the
interaction of persistent chemicals that are
similar in structure and assess their effects on
following developmental exposure. This
research will explore in in vitro and in vivo
models the effects of chemical mixtures such
as the PCBs and polybrominated
diphenylethers on key biological events
known to be critical for the development of
the nervous system, such as PKC. These
experiments will test the hypothesis that the
interactions of chemicals with a similar MOA
follow dose-additivity with regard to PKC and
will:
Develop neuronal cultures (hippocampal
and cerebellar granule neurons) to
examine the effects of chemical
mixtures in vitro,
Construct individual dose-response
curves in vitro for selected chemicals,
Determine presence of dose-additivity of
binary mixtures, and
Evaluate dose-additivity of complex
mixtures at the low end of the dose-
response curve.
Project 2 - Examine the Potential for
Response-Additivity of Mixtures of
Chemicals with Different MO As. With few
exceptions, the research that has been
conducted examining the validity of the
assumption of additivity has used mixtures of
5-5
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chemicals that have either the same MOA or
the same target organ. Far less work has
examined whether additivity holds for
mixtures of chemicals that contain chemicals
with different MO As (diverse mixtures), yet it
is to these mixtures that humans are exposed.
Current risk assessments assume response-
additivity as the default for mixtures of
chemicals of dissimilar action. Thus,
predictive models that operate in the
observable effects region would need to
incorporate response-addition for dissimilar
chemicals. It is possible, however, that dose-
additive and response-additive models may
yield the same answer in the very low dose
region. Research in project 2 will evaluate
models for assessment of mixtures of diverse
chemicals, again with emphasis on the lower
regions of the dose-response curves. Research
in project 2 will:
Test the null hypothesis that additivity of
effects on membrane excitability will be
observed when mixtures of pyrethroids
and organochlorines are administered to
individual cells and cells in intact brain
slices;
Test the null hypothesis that OPs and
carbamates will interact in a dose-
additive manner;
Use genotoxicity assays to examine low
dose additivity assumptions for mixtures
of compounds that act via a different
MOAs;
Analyze and interpret the results of a
full-factorial study (5x5x5) of the organ
toxicity of three diverse chemicals,
including trichloroethylene, diethylhexyl
phthalate, and heptachlor, as well as all
possible toxicant combinations;
Develop and use experimental designs
and statistical analysis methods for
mixtures of multiple chemicals having
different MOAs; and
Test the interaction of persistent
chemicals (e.g., PCBs, pesticides,
polybrominated diphenyl ethers) and
metals (methylmercury, organotins) that
are not similar in structure and assess
their effects on neuronal PKC.
Project 3- Examine the Usefulness of the
Assumption of Additivity Under Repeated
Exposure Conditions. The existing literature
on the effects of chemical mixtures focuses
almost exclusively on acute exposure
scenarios. The results from these acute
experiments (additivity, synergy, antagonism)
must be used, for lack of better information, to
estimate the risk of chronic exposure to these
mixtures. However, it is well established in
biology that receptor systems will attempt to
maintain homeostasis; and, when subjected to
over-activation or chronic block, receptor
based systems will modulate accordingly to
return the system to its previous balance. This
has been observed as increases or decreases in
receptor or enzyme number; up- or down-
regulation of second messenger response
systems coupled to receptors; and alterations
in the expression of specific subunits
comprising the enzyme, receptor, or ion
channel. Beyond the level of the biomolecular
receptor, changes in metabolism may affect
the amount of active compounds in the
mixture. Thus, responses which were additive
following single, acute exposures, may
deviate from additivity following repeated
exposure to the same stimulus (mixture).
Research in this project will:
Develop models for mixtures of ChE-
inhibiting pesticides in adult animals
that determine the degree to which
repeated exposure to ChE-inhibiting
pesticides will differ from acute
exposure responses and
Determine models for mixtures of
pesticides in developing animals.
Impact
The results from this research effort are
expected to lead to improvements in the risk
assessment of chemical mixtures by (1)
testing assumptions of dose- versus response-
additivity, especially in the low dose region;
(2) examining changes in "additivity" from
acute to chronic exposure scenarios; and (3)
development of appropriate and efficient
statistical models for testing the additivity
5-6
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hypothesis in mixtures comprised of larger
numbers of chemicals. Results from this
program project will include chemical specific
data, as well as new statistical methods and
experimental designs for the conduct,
analysis, and interpretation of mixtures
experiments. In addition, the information
from this research may be used to support
guidance for the risk assessment based on the
type of mixtures (e.g., common MOA
mixtures or dissimilar MOA chemicals,
simple mixtures or complex mixtures),
conditions (e.g., higher dose versus low dose,
acute versus chronic), and endpoints (e.g.,
genotoxicity, neurotoxicity, cytotoxicity) for
which the default assumptions of dose-
addition or response-addition have been
validated.
Cross-Agency Interactions
This program project involves cross-ORD
collaborations between NHEERL and NCEA
explicitly in projects 1 and 2. The NCEA
collaborators are Ms. L. Teuschler and Dr. R.
Hertzberg. In addition to their specific work
on these projects, they will participate in the
program project research team meetings. The
planned workshops (use of dose addition to
predict interactive effects of mixtures with a
common MOA; use of response addition to
predict interactive effects of mixtures with
diverse MO As; effect of dose on interactive
toxicity) will include experts and participants
from across ORD, the Program Offices, and
Regions.
5.5 Gap Analysis and Links to Other Multi-
Year Plans
In the past several years, cumulative risk
has taken on increased importance as
evidenced by several recent publications such
as Pesticides in the Diets of Infants and
Children (NRC, 1993) and Science and
Judgment in Risk Assessment (NRC, 1994).
NHEERL research is consistent with research
needs identified in those documents and with
issues raised in the Supplementary Guidance
for Conducting Health Risk Assessment of
Chemical Mixtures (US EPA, 2001), as well
as the legislative requirement of the FQPA for
the Agency to focus on the cumulative effects
of pesticides that have a common mechanism
of toxicity. NHEERL research also focuses
on more recent concerns about predicting risk
from mixtures of chemicals with dissimilar
mechanisms, as well as issues related to
changes in mechanism as a function of
repeated exposure. NHEERL research will
examine mixtures from several chemical
classes, including the OPs, carbamates,
pyrethroids, halomethanes, persistent
bioaccumulative toxicants, conazoles, and
chloroatrazines. That NHEERL has little or
no research planned for aggregate risk is a
major gap although NHEERL will examine
the validity of route-to-route extrapolation for
selected air toxics within the Air Toxics MYP.
NHEERL research on cumulative risk is
linked to several other MYPs. In the case of
EDCs, NHEERL will examine the effects of
mixtures of dioxin-like chemicals on the
development in the rat and will examine the
mechanism(s) by which PCB mixtures affect
the development of the nervous system. In the
case of Air Toxics and PM, NHEERL
research on cumulative risk is linked to
research to characterize effects of volatile
organic compound mixtures using in vitro
assays and studies on interaction between PM
and gaseous co-pollutants in mediating
COPD. NHEERL research also is linked to
research to investigate the assumption of
additivity in assessing pesticides with
common MO As (e.g., mixtures of OPs,
pyrethroids).
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5.6 References
Gennings, C., P. Schwartz, H. Carter, and J.E.
Simmons. An efficient approach for detecting
departure from additivity in mixtures of many
chemicals with a threshold additivity model. J
Agr Bio Env Stat 2:198-211 (1997). Erratum
5:275-259(2000).
National Research Council. Pesticides in the Diets
of Infants and Children. Washington, DC,
National Academy of Sciences. (1993).
National Resarch Council. Science and Judgment
in Risk Assessment. Washington, DC, National
Academy of Sciences. (1994).
Simmons, J.E. Chemical mixtures: challenge for
toxicology and risk assessment. Toxicology
105:111-119(1995).
Teuschler, L., J. Klaunig, E. Carney, J. Chambers,
R. Connolly, C. Gennings, J. Giesy, R. Hertzberg,
C. Klaassen, R. Kodell, D. Paustenbach, and R.
Yang. Support of science-based decisions
concerning the evaluation of the toxicology of
mixtures: a new beginning. Reg Toxicol
Pharmacol 36:34-39. (2002).
U.S. Environmental Protection Agency.
Guidelines for the Health Risk Assessment of
Chemical Mixtures. Federal Register
51(185):34014-34025. (1986).
U.S. Environmental Protection Agency. Technical
Support Document on Health Risk Assessment of
Chemical Mixtures. Washington, DC.
EPA/600/8-90/064. 1990.
U.S. Environmental Protection Agency.
Supplementary Guidance for Conducting Health
Risk Assessment of Chemical Mixtures.
Washington, DC. EPA/630/R-00/002. 2001.
5-S
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APPENDIX A
Resource Allocation for Program Projects
HARMONIZATION
TITLE
Harmonization of Cancer and Non-cancer
Risk Assessment: Disruption of Mitogen-
Activated (MARK) Signaling as a Common
Mode of Action for Environmental
Toxicants
Disruption of Luteinizing Hormone (LH)
Secretion as a Common Mode of Action for
Altered Fertility, Reproductive Disease, and
Cancer of the Reproductive System
Modulation of Cytochrome P-450s and
Other Xenobiotic Metabolizing Enzymes
(XMEs) Leading to Common Mode of
Action for Multiple Toxicities
TEAM LEADERS
William Mundy (NTD)
Barbara Abbott (RTD)
Ralph Cooper (RTD)
Stephen Nesnow (ECD)
FY03 FTE (ESTIMATES )
5.7
3.1
10.0
SUSCEPTIBLE SUBPOPULATIONS
Identifying and Validating Biologic
Indicators of Susceptibility and Sensitivity
among Children to Assess Potential Risk of
Adverse Outcomes Associated with
Environmental Exposure
Extrapolating Across Windows of
Vulnerability to Assess Children's Health
Risks Using Rodent Toxicity Data
Long-Term Effects of the Developmental
Environment
Susceptible Subpopulations: Susceptibility
Associated with the Aged
Environmental Risk Factors for Asthma
Environmental and Genetic Interactions in
Hypertensive Rats: Oxidative Stress as a
Common Susceptibility Attribute for Non-
cancer Risks
Genotype and Phenotype in the Metabolism,
Toxicity and Carcinogenicity of Arsenic
Stanley Barone (NTD)
Pauline Mendola
(BSD)
Hugh Barton (ETD)
John Rogers (RTD)
Chris Lau (RTD)
Andrew Geller (NTD)
Ian Gilmour (ETD)
Urmilla Kodavanti
(ETD)
Kirk Kitchin (ECD)
David Thomas (ETD)
14.4
2.7
8.5
4.2
4.8
5.9
3.3
CUMULATIVE RISK
Exploring the Limits of Additivity
Jane E Simmons (ETD)
Stephanie Padilla
(NTD)
7.5
A-l
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APPENDIX B
Annual Performance Goals (APGs) and Measures (APMs)
Harmonization Research
ANNUAL PERFORMANCE GOALS AND MEASURES
APG - Identify potential common modes of actions that underlie
different toxic effects
APM
APM
Report on immunohistochemical methods for the detection of
signal transduction activation in vivo in animals and humans
exposed to pollutants
Report summarizing biological basis for common precursors for
cancer and non-cancer effects by prototypic agents
APG - Determine utility of emerging technologies in harmonizing risk
assessment
APM
APM
APM
Report on the development and application of emerging
technologies to detect changes in signal transduction in vitro and
in vivo
Report on the use of toxicogenomic and related technologies to
define common modes of action of P-450 modulating chemicals
Summary report on the use of emerging technologies in risk
assessment
APG - Provide scientific basis for use of mechanistic data in
harmonized risk assessment
APM
APM
APM
APM
APM
APM
APM
APM
Report on the use of mechanistic data to define common modes
of action for risk assessment of P450 modulating
chemicals
Report on the range of chemicals that modify regulation of
luteinizing hormone
Report on the mechanisms involved in altering luteinizing
hormone
Report on determining common modes of action for
developmental reproductive and neural toxicities induced by P-
450 modulating chemicals
Report summarizing use of cell signaling data as common mode
of action for harmonization
Report on use of cell signaling data for extrapolation of mode of
action information from in vitro to the whole animal
Report on the integration of toxic effects, P-450 modulation,
structure-activity analysis, and gene expression profiling for
application to risk assessment
Report on characterization of cancer and non-cancer
reproductive effects following modulation of luteinizing
hormone secretion
YEAR
2003
2003
2003
2008
2005
2006
2008
2012
2004
2005
2006
2006
2007
2008
2008
2008
Program Project
PP#1 MAP-Kinase
All Program Projects
PP#1 MAP-Kinase
PP#3 P-450 and XME
All Program Projects
PP#3 P-450 and XME
PP#2-LH
PP#2-LH
PP#3 P-450 and XME
PP#1 MAP-Kinase
PP#1 MAP-Kinase
PP#3 P-450 and XME
PP#2-LH
A-2
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ANNUAL PERFORMANCE GOALS AND MEASURES
APM
APM
APM
APM
APM
APM
Report summarizing data from prototypic compounds
acting through p-450/XME modulation
Report on evaluation of risk associated with multiple exposures
to chemicals having differential effects on secretion of luteinizing
hormone
Report on the use of cell signaling data for extrapolation of mode
of action information for interspecies extrapolation
Report summarizing harmonized risk assessment for chemicals
modifying luteinizing hormone secretion
Report summarizing risk assessment for chemicals acting by
oxidative stress
Summary report on use of cell signaling data for use in
harmonization of risk assessment
APG - Provide a framework for harmonization of extrapolation factors
and mechanistic data in risk assessment
YEAR
2008
2010
2010
2012
2012
2012
2012
Program Project
PP#3 P-450 and XME
PP#2-LH
PP#1 MAP-Kinase
PP#2-LH
PP#9 Oxidative Stress
PP#1 MAP-Kinase
All Program Projects
A-3
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APPENDIX C
Annual Performance Goals (APGs) and Measures (APMs)
Susceptible Subpopulations
ANNUAL PERFORMANCE GOALS AND MEASURES
APG
2005
APM
APM
APM
APM
APM
APM
APM
APM
APG
100
APM
APM
APM
By 2005, provide risk assessors and managers with methods
and tools for measuring exposure and effects in children,
characterizing risk to children, and reducing risks to children
in schools from harmful environmental agents to support EPA
risk assessment and risk management
Provide proof of concept for use of a biologically based, dose
response model of a specific cellular, developmental event to
predict the risk of adverse outcome (i.e., toxicant induced limb
defects)
Report on the biological basis of childhood asthma
Report on the health effects associated with exposure of
animals and humans with asthma to diesel exhaust particles
External review draft report on conducting risk assessments
for children as a sensitive subpopulation and summary of
supporting ORD research
Report on the health effects associated with exposure of
exhaust particles in asthma animal model
Report on at least 1 method to test for effects in the National
Children's Study, including the assessment of respiratory
health outcomes such as asthma
Report on assessing health effects in children under five years
of age exposed to pesticides
Report on methods to collect, store, and transport biologic
specimens from surrogate tissues in humans and rodent
models for protein and gene expression analysis
By 2009, complete two or more targeted epidemiological or
exposure studies on children to test hypotheses, collect data,
and validate methods and tools for measuring, characterizing,
and reducing real world risks from exposures to harmful
environmental agents to support EPA risk assessment and risk
management
Report on determinants of susceptibility to the acute
respiratory health effects of combustion-related pollutants
among asthmatic children in 7 US communities
Report on field ready test system based on classical
conditioning for use in testing developmental neurological
disorders in human infants
Peer-reviewed scientific publication on intra-urban variations
in the prevalence of childhood asthma associated with intra-
urban gradients of combustion-related pollutants and air
toxics in El Paso, TX
YEAR
2005
2002
2004
2004
2005
2005
2004
2004
2004
2009
2003
2004
2004
Program Project
PP#4 Indicators
PP#8 Asthma
PP#8 Asthma
PP#4 Indicators
PP#8 Asthma
PP#4 Indicators
PP#4 Indicators
PP#4 Indicators
PP#8 Asthma
PP#4 Indicators
PP#8 Asthma
A-4
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ANNUAL PERFORMANCE GOALS AND MEASURES
APM
APM
APM
APM
APG
2012
APM
APM
APM
APM
APM
APM
APM
APM
APM
Report on the characterization of physicochemical properties
of the allergenic protein from selected indoor fungi and
compare to other well-characterized proteins for hazard
identification
Report on prevalence of asthma and low pulmonary function
levels among 4th and 5th grade schoolchildren in a second
major urban area
Report on analysis of National Children's Study data on
relationship between exposure to environmental agents and
adverse health outcomes
Report on a validation of field-collected diesel exposure
biomarkers
By 2012, provide risk assessors and managers with methods
and tools for measuring exposure and effects in children,
including adolescents, characterizing cancer and non-cancer
hazards and risk to children, and reducing risks to children in
schools from harmful environmental agents to support EPA
risk assessment and risk management
Report on the use of genomics for monitoring expression of
health status through analysis of accessible tissues and cells
Report on the long-term persistent effects of developmental
exposure to environmental chemicals on cancer and non-
cancer endpoints
Report on proteins from several indoor fungi that pose a
hazard with respect to allergenicity
Report on long-term effects of in utero insult on adult health
and reproduction to assess adequacy of current testing
guidelines for reproductive toxicity
Report on the validation of a human blood ex vivo model to
evaluate dose, genetic and age specific factors impacting inter-
and intra-individual phenotypic response measurements
Report on the assessment of the relative potency of several
indoor fungal allergens to obtain quantitative information for
risk assessments
Report on age-related pharmacokinetic and
pharmacodynamic changes in developing animals
Report on the reliability of assays to measure endogenous and
exogenous constituents of breast milk that are implicated in
altering health status of women or their breast-fed children
Report on feasibility of measuring early postnatal serum
measures of neurotrophic factors (NTF), neurotransmitters,
and cytokines in human infants
YEAR
2006
2007
2008
2008
2012
2004
2004
2004
2005
2006
2005
2006
2006
2006
Program Project
PP#8 Asthma
PP#8 Asthma
PP#4 Indicators
PP#8 Asthma
PP#4 Indicators
PP#6 Developmental
Environment
PP#8 Asthma
PP#6 Developmental
Environment
PP#$ Indicators
PP#8 Asthma
PP#5 Windows
PP#4 Indicators
PP#4 Indicators
A-5
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ANNUAL PERFORMANCE GOALS AND MEASURES
APM
APM
APM
APM
APM
APG
135
APM
APM
APM
APM
APM
APM
APG
APM
APM
APM
APG
126
Report on development of principles to be used to assess
cancer risks in children
Report on the application of mechanistic information in
assessing cancer risk in children
Report on the mechanism of chemical-induced childhood
asthma
Summary report on state of science and application to risk
assessment of pharmacokinetics and pharmacodynamics as it
relates to children's health
Report on the predictive validity of early postnatal serum
measures of neurotrophic factors (NTF), neurotransmitters,
and cytokines in human infants for developmental
neurological disorders
By 2008, provide risk assessors and managers with methods
and tools for assessing differences in exposures and responses
to harmful environmental agents between the elderly and
younger adults.
Report on modeling for age-dependent pharmacokinetics in
risk assessment
Report on age-dependence of protective repair and plasticity
mechanisms in aged animal models
Report on latent adverse health effects of developmental
exposures
Report on differential response of older animals and cells to
environmental agents
Report on gene expression changes correlated with latent
adverse health effects of developmental exposures
Report on mechanisms of susceptibility to environmental
insult in models of aging
By 2007, analyze and demonstrate the role of genetic factors in
causing cancer and non-cancer endpoints
Report on genetic polymorphisms that might alter human risk
of arsenic carcinogenesis
Report on the role of genetic factors in cancer endpoints
Report on gene array data from animal and human
polymorphisms and relation to disease susceptibility and
underlying oxidative stress
By 2006, evaluate the variation in vulnerability to
environmental agents as a result of health status as reflected
by nutritional status and pre-existing disease.
YEAR
2006
2007
2007
2008
2010
2008
2004
2005
2005
2008
2006
2010
2007
2002
2007
2008
2006
Program Project
PP#4 Indicators
PP#4 Indicators
PP#8 Asthma
PP#5 Windows
PP#4 Indicators
PP#5 Windows
PP#7 Aging
PP#7 Aging
PP#6 Developmental
Environment
PP#7 Aging
PP#7 Aging
PP#6 Developmental
Environment
PP#7 Aging
PP#10 Genetics
PP#10 Genetics
PP#9 Oxidative Stress
A-6
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ANNUAL PERFORMANCE GOALS AND MEASURES
APM
APM
Report on the effects of pre-existing respiratory disease on
response to air pollutants
Report on comparative dose-response toxicity data for
susceptible hypertensive rats and role of oxidative stress
YEAR
2005
2006
Program Project
PP#8 Asthma
PP#9 Oxidative Stress
A-7
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APPENDIX D
Annual Performance Goals (APGs) and Measures (APMs)
Additivity
ANNUAL PERFORMANCE GOALS AND MEASURES YEAR
APG 134 - Develop methods and measurement data to support models
of cumulative exposures, dose, and effects
APM
APM
APM
APM
APM
APM
APM
APM
APM
APM
APM
APM
APM
APM
APM
Report on studies examining interactions of carbamate
pesticides in mixtures
Complete analysis and report on full factorial 5x5x5 study of
effects of three diverse chemicals on several non-cancer
endpoints
Report on cumulative risk of exposure to two high use
pesticides on female reproductive system
Report of studies on modeling of dose-response curves of
prototypic chemicals having similar or different modes or
mechanisms of action
Develop in vitro preparations to study interaction of
pyrethrin pesticides in vitro
Report on effects of disinfectant by-product mixtures in vivo
and in vitro
Develop parameters to model dose-additivity for mixtures of
organophosphate and carbamate pesticides
Develop in vitro methods to predict interactions of persistent
environmental toxicants
Demonstrate effects of pesticide mixtures after repeated
dosing scenarios in vivo are different than those following
short-term exposure
Report on studies to predict interactions between persistent
environmental toxicants
Report on studies to predict interactions between
environmentally relevant mixtures of pyrethrins
Report on prediction of interactions of chemicals in vivo
based on principles developed in vitro
Proceedings from workshop on principles of dose additivity
and response-additivity to predict interactions between
chemicals with similar and dissimlar modes of action,
respectively
Summary report on methods and models to predict
interactions of environmental chemicals
Chemical mixtures report on the use of dose-additivity to
predict interactions of chemicals with a common mode of
action, and use of response additivity to predict interaction of
chemicals having different mechanisms of action
2009
2004
2004
2004
2004
2005
2005
2005
2005
2006
2006
2007
2008
2008
2009
2011
Program Project
PP#11 Additivity
PP#11 Additivity
PP#11 Additivity
PP#11 Additivity
PP#11 Additivity
PP#11 Additivity
PP#11 Additivity
PP#11 Additivity
PP#11 Additivity
PP#11 Additivity
PP#11 Additivity
PP#11 Additivity
PP#11 Additivity
PP#11 Additivity
PP#11 Additivity
A-8
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Office of Research and Development
National Health and Environmental
Effects Research Laboratory
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