illliiiilpiiliiiiil
-V -.-"'": ;•='-""•::: /^>/::^'';•'•>.-•,.'-;'-?'i -j;:-"--"- =-Y'->v:;""''."irf. 'V/i-,- ";- ;-=.\ /• ;;. ^j-: >." - "; /:-;•?". :- \ ,-;:'"•-'" :-":;-
-------
-------
*"54
('*' 0 EPA/625/3-91/019F
\ f fO September 1991
ALPHA2U-GLOBULIM: ASSOCIATION WITH
CHEMICALLY INDUCED RENAL
TOX8CITY AND NEOPLASIA IN THE MALE RAT
Prepared for the
Risk Assessment Forum
U.S. Environmental Protection Agency
Washington, D.C.
Principal Authors
Karl P; Baetcke, Ph.D. imogene Sevin Rodgers, Ph.D.
Gordon C. Hard, Ph.D, D.Sc. Robert E. McGaughy, Ph.D.
Letitia M. Tahan, M.S.
Technical Pane!
Karl P. Baetcke, Ph.D., Co-Chair Robert E. McGaughy, Ph.D.
Letitia M. Tahan, M.S., Co-Chair William E. Pepelko, Ph.D.
Marion P. Copley, D.V.M.. Cheryl Siegel Scott, M.S.
Julie Du, Ph.D. Lawrence R. Valcovic, Ph.D.
Risk Assessment Forum Staff
Executive Director: Dorothy E. Patton, Ph.D, J.D.
Science Coordinator: Imogene Sevin Rodgers, Ph.D.
Consultant: Gordon C. Hard,
B.V.Sc., Ph.D., D.Sc., F.R.C. Path.,
F.R.C.V.S., F.A. Tox. Sci.
Risk Assessment Forum
U.S. Environmental Protection Agency
Washington, DC 20460
Printed on Recycled Paper
-------
This documen
Protection Agency faolicy
commercial prbduc :s does
Disclaimer
has been reviewed in accordance with U.S. Environmental
and approved for publication. Mention of trade names or
not constitute endorsement or recommendation for use.
-------
List of Tables Vj
List of Figures. 1.1""""['"^" vti
List of Abbreviations ..:.......!......!....!.!....!i!....!.!....!.y. viii
External Peer Reviewers ...:3ZZZZZIZZ. jx
Preface "'.'.'.'.'.'.'.'.'.'.'.'.'.'.'."".'."."xii
I. Executive Summary 1
II. Introduction 5
Part 1-Nephrotoxicity
III. Hyaline Droplets and Alpha2u-globulin; Physiology and
Biochemistry 11
A. Filtration, Reabsorption, and Catabolism of
Low-Molecular-Weight Proteins by the Kidney 11
B. Hyaline Droplets in Renal Tubules "13
C. Factors Affecting Kidney Accumulation of
Low-Molecular-Weight Proteins.... 14
D. The Alphas-globulin Superfamily of Proteins 15
E. Characteristics of Alpha2u-globulin 19
F. Sex and Species Comparison of Urinary Protein
Content of the Lippcalin Superfamily 20
G. Noncovalent Binding to Alpha2u-globulin and its
Homologues ; ; 21
1. Chemical entities bound to alpha2u-globulin 21
2. Nature of the association 22
3. Binding of CIGAto other macromolecules 23
4. Specificity of the interaction of CIGA with
alpha2u-globulin 23
H. Catabolism of Alpha2u-globulin Complexed with CIGA ."""....".24
I. Structure-Activity Relationships for CIGA 26
IV. Alpha2u-globulin Nephropathy 27
A. Pathologic Features of Alpha2u-globulin Nephropathy .27
B. Rat Urine Chemistry and CIGA 30
C. Species Variation in the Renal Response to CIGA .30
D. Factors Affecting the Expression of Alpha2u-globulin
Nephropathy.. 32
1. Age-related effects ^32
2. Effect of hormone manipulation !.."."..33
3. Genetic variants 33.
4. Alpha2u-globulin infusion in female rats 33
iii
-------
Contents (cont.)
E. Chronic Progressive Nephropathy ...................... • ........................... 34
F Renal Toxictty Observed in Chronic Bioassays of
Chemicals that Induced Kidney Tumors in Rats.... ......................... &
Part 2-CarcinogenicIty
V. Pathologic Features of Renal Carcinogenesis Induced by
Classical Carcinogens • .........
A. Early Nephrotoxic'rty
B. Karyomegaly
C. Tubule Cell Hyperplasia ........... .
D. Adenoma
E. Adenocarcinomas and Carcinomas
F. Tumor Progression
G. Site of Origin of Renal Tubule Tumors
VI. Neoplastic and Preneoplastic Lesions Observed in the
2-Year Bioassays .............................................. ............ ....... • .............. '""AA
A. Generic Considerations [[[ ZT
B. Renal Tumor Incidence ........................................ • ......... ....... : ....... ri
C Histogenesis of Renal Tumors [[[ ~~
D. Renal Tumor Latency and Progression .......................................... »i
E. Induction of Other Tumor Types ............. . ......................... • ............. bl
VII. Additional Evidence Concerning the Renal
Carcinogenicity of GIGA .......... .
A. Genetic Toxicology Studies
B. Initiation-Promotion Sudies
VIII. Comparison of GIGA with Classical Renal Carcinogens ......................... 57
IX. Evidence Concerning Human Kidney Cancer .................. ............... , ........ 58
A. Morphology and Histogenesis .................................. • ............ • ........ £°
B. Incidence and Mortality [[[ • .......... £*
C. Environmental and Lifestyle Factors .............................................. °u
D. Occupational Factors ........................................ -•• ....................... 61
E. Renal Cancer and Hydrocarbon, Solvent, or Petroleum
Product Exposure ......................................... v ................................ 61
X. Evidence for Dose- and Time-Dependent Progression
from Early to Late Lesions [[[ 63
A Association Between GIGA, Hyaline Droplet
-------
Contents (cont.)
C. Progression to Cast Formation, Tubule Dilation,
and Mineralization 68
D. Association Between CIGA and Chronic Progressive
Nephropathy 70
E. Evidence Concerning Progression from Nephrotoxicity to
Renal Neoplasia 71
Part 3-Evaluation of the Hypothesis
XI. Summary of the Evidence on the Renal Effects of CIGA 73
A. Association Between AIpha2u-globulin Accumulation
and Nephrotoxicity . 73
B. Association Between Nephrotoxicity and Renal Cancer 74
C. Information Reducing Confidence in the Conclusion
that the Alpha2u-globulin Response is Specific to the
Male Rat 75
XII. Conclusions. 76
XIII. Research Needs 78
Part 4-Science Policy
XIV. Background and Introduction .....81
XV. Basis for Science Policy on Male Rat Kidney Tumors 81
A. Low-Molecular-Weight Proteins in the Rat 81
B. Progression from Chemically Induced Alpha2u-giobulin
Accumulation to Nephropathy and Neoplasia 82
1. Overview 82
2. Specificity of the sequence to the male rat 83
XVI. Science Policy Statement 85
XVII.' Guidance for Evaluating Chemically Induced Male Rat
Renal Tubule Tumors 85
A. Renal Tubule Tumors in Male Rats and Alpha2u-globulin
Accumulation 86
B. Additional Information Useful for the Analysis 87
C. Use of the Data for Risk Assessment 88
XVIII. Nephropathy as a Toxic Endpoint '. 89
Appendix - Non-neoplastic Effects of Hyaline Droplet Inducers 90
References 99
-------
List of Tables
Number Page
1 Examples of organic chemicals that have produced renal jnjury in
male rats characterized by hyaline droplet accumulation but ..
not in female rats or other species ....................................... • ............. '
2. Superfamily of lipophilic ligand-binding carrier proteins .:......,,...., ..... 17
3. Summary of the histppathology and lesion progression reported in
alphaau-globulin-associatednephrotoxicity ...... ...... .... ..... • ..... ••••••• ..... *<»
4. Summary of the histopathology of spontaneous "
chronic progressive nephropathy of aging rats ....... . .......... ...» .......... ^P
5 Summary of data on non-neoplastic and Pre-Neoplastic kidney .
lesions in male rats associated with eight model compounds
that induced renal tumors in 2-year bioassays ................ . ..... ?.v ........ *'
6 Summary of data from 2-year bioassays on rioh-neoplastic and
pre-neoplastic kidney lesions in mipe and female rats exposed to
eight model compounds that induced renal tumors in male.rats ....... 38
7. Incidences of renal tubule preneoplasia and
neoplasia in rats taken from 2-year bioassays
on eight model substances ........................................ • ...................... 4t>
8 Incidences of renal tubule preneoplasia and
neoplasia in rats taken from 2-year bioassays
on chlorothalonil and trichloroethylene .............................................. 4B
9. Summary of genotoxicity data for eight selected
male rat kidney carcinogens [[[ 53
1 0. Summary of genotoxicity data for two selected
non-CIGA male rat kidney carcinogens ............................................. 54
11. Incidence of medullary mineralization in male
-------
List of Figures
Number
Page
1. Diagram of zonation and tubule segmentation
in rat kidney , 10
2. Schematic representation of endocytic uptake
of filtered proteins 12
3. Schematic representation of the uptake and fate
of alpha2u-globulin complexed with a GIGA in
hydrocarbon nephrotoxicfty ...25
4. Dose-response relationship between renal hyaline
droplet accumulation (D) and [3H]-thymidine
labeling index .66
5. Effect of 0-2,000 ppm unleaded gasoline on
continuous uptake of [3H]-TdR , 67
6. Time-sequence for the development of hyaline
droplets, regenerating tubule epithelium, and
tubule dilation in male F344 rats 69
vii
-------
List of Abbreviations
AAT
ABS
aspartate aminotransf erase
chromosome aberrations in CHO cells
CHO
CI
GIGA
CRN
1,2-DCB
1,4-DCB
DEN
DMN
EHEN
FBPA
H & E
IRDC
MLA
MTD
MUP
NAG
NBR
NTP
NCI
OR
RR
P1
P2
P3
SAL
SCE
SDS
SEER
SIR
SLRL
SMR
TK
TMP
TMPOH
UDS
Chinese hamster ovary
confidence interval . ..
£hemical(s) Inducing alpha2u-£'ob"l'n Accumulation
chronic progressive nephropathy
1,2-dichlorobenzene
1,4-dichIorpbenzene
diethylnitrosamine
dimethylnitrosamine
N-ethyl-N-hydroxyethylnitrosamme
N-(4'-fluoro-4-biphenylyl)acetamide ../.'
hematoxylin and eosin . • v • '
international Research and Development Corporation
TK-gene mutation assay in L5178Y cells
maximum tolerated dose
mouse major urinary protein
N-acetyl-p-glucosaminidase
NCI Black-Reiter rat
National Toxicology Program ..:.,.
National Cancer Institute
odds ratio
relative risk
first convoluted segment of proximaj tubule
second convoluted segment of proximal tubule
pars recta of proximal tubule ,
Salmonella
sister chromatid exchange
sodium dodecyl sulfate . : .
Surveillance, Epidemiology and End Results
Program of NCI
standardized incidence ratio
sex-linked recessive lethal
standardized mortality ratio
thymidine-kinase
2,2,4-trimethylpantane -.-••: .
2,4,4-trimethylpentanol •.••,>.
unscheduled DNA synthesis
viii
-------
External Peer Reviewers
An interim draft, prepared in September 1990, was evaluated at a two-day Peer
Review Workshp sponsored by the EPA Risk Assessment Forum. The meeting,
held in Ga'rthersburg, Maryland, on November 13 and 14,1990, was chaired by Dr.
Richard Griesemer, director of the Division of Toxicology Research and Testing,
National Toxicology Program (NTP). Invited participants are listed below. The
Workshop proceedings will be published separately. Announcement of their
availability will be through the Federal Register.
Peer Review Workshop - invited Participants
John Ashby (Cancer Workgroup
Chair)
Imperial Chemical Industries
Central Toxicology Laboratory
Cheshire, England
Deborah Barsotti
Division of Toxicology
Agency for Toxic Substances
Disease Registry (ATSDR)
Atlanta, Georgia
R. Daniel Benz
Center for Food Safety
and Applied Nutrition
Food and Drug Administration
Washington, D.C.
William Busey3
Experimental Pathology
Laboratories
Herndon, Virginia
Murray Cohn
Health Effects Division
Directorate for Health Sciences
Consumer Product Safety Commission
Bethesda, Maryland
Michael Elwell
Division of Toxicology
Research and Testing
NIEHS
Research Triangle Park,
North Carolina
Scot L. Eustis
Division of Toxicology
Research and Testing
NIEHS
Research Triangle Park,
North Carolina
William Farland
USEPA
Office of Health and
Environmental Assessment
Washington, D.C.
Penny Fenner Crisp
USEPA
Health Effects Division
Office of Pesticides Products
Washington, D.C.
Richard Griesemer (Workshop Chair)
Division of Toxicological
Research and Testing
NIEHS
Research Triangle Park,
North Carolina
Elizabeth Grossman
Office of Risk Assessment
Health Standards Programs
US Department of Labor/OSHA
Washington, D.C.
IX
-------
External Peer Reviewers (cont.)
Gordon Hard (Criteria
Workgroup Chair)3
Medical Research Council
Toxicology Unit
Carshattpn, Surrey
Great Britain
Lois Lehman-McKeeman3
The Procter and Gamble Company
Miami Valley Laboratories
Cincinnati, Ohio
Dennis Lynch
National Institute for
Occupational Safety and Health
Division of Biological
and Behavioral Sciences
Cincinnati, Ohio
James McKinney
USEPA
Health Effects Research Laboratory
Research Triangle Park,
North Carolina
Joseph McLaughlin
National Cancer Institute
Bethesda, Maryland
Michael Olson (Nephropathy and
Biochemistry Workgroup Chair)3
General Motors Research
Laboratories
Warren, Michigan
Norbert P. Page (Risk
Characterization Workgroup
Chair)
Page Associates
Gaithersburg, Maryland
James Poppa
Department of Experimental .
Pathology and Toxicology .
Chemical Industries Institute for
Toxicology (CUT) ,
Research triangle Park,
North Carolina • ;
Carl Potter
USEPA
Risk Reduction Engineering
Laboratory
Cincinnati, Ohio
James Swenberg3
University of North Carolina
Department of Pathology
Chapel Hill, North Carolina
Benjamin Trump
University of Maryland
School of Medicine
Baltimore, Maryland
Jerrold Ward
National Cancer Institute
Fredrick Cancer Research
Center
Fredrick, Maryland
Lauren Zeiss
California Department
of Health Services
Reproductive and Cancer
Hazard Assessment Section
Berkeley, California
-------
External P@©r Reviewers (corat.)
On March 27,1991, EPA's Environmental Health Committee of the Science
Advisory Board reviewed and unanimously approved a draft report (EPA/625/3-91/
019A). The FIFRA Science Advisory Panel was represented by Dr. Joe H. Grisham.
The Executive Committee approved the report of the Environmental Health Commit-
tee attheir July 23,1991 meeting, although one member of the Executive Committee
encouraged greater attention to the uncertainties concerning the hypothesis. In
summary, the Science Advisory Board concurred with the position of the Risk
Assessment Forum report.
The Technical Panel and the Risk Assessment Forum also acknowledge with
appreciation the special contributions of Joseph Mclaughlin, who greatly assisted
in the preparation of the epidemiology section, and Jerry Blancato, Margaret M.L.
Chu, Ha Cote, Richard N, Hill, and the members of the Risk Assessment Forum's
Cancer Oversight Group (William Farland, Edward Ohanian, Vanessa Vu, and
Jeanette Wiftse) for their thoughtful comments.
• Also reviewed a preliminary draft. Other reviewers of this draft were Carl Alden of Procter
and Gamble's Miami Valley Laboratories, and Michael Lipsky of the University of
Maryland Medical School.
XI
-------
Preface
The U.S. Environmental Protection Agency (EPA) Risk Assessment Forum
was established to promote scientific consensus on risk assessment issues and to
ensure that this consensus is incorporated into appropriate risk assessment guid-
ance. To accomplish this, the Risk Assessment Forum assembles experts from
throughout the EPA in a formal process to study and report on these issues from an
Agency-wide perspective.
For major risk assessment activities, the Risk Assessment Forum has estab-
lished Technical Panels to conduct scientific review and analysis. Members,are
chosen to assure that necessary technical expertise is available. Outside experts
may be invited to participate as consultants or, if appropriate, as Technical Panel
members. . ."•'.
The use of male rat kidney tumors in risk assessment has been the subject of
much recent discussion. For a certain group of chemicals, investigators hay?
reported renal tubule tumor formation in male rats as the sequela of renal toxteity
commencing with an excessive accumulation of the protein, alphas-globulin (a^-g),
in renal tubules. Renal tubule tumor formation with protein accumulation has not
been observed in female rats or other tested species, most notably the mouse. The
NCI Black Re'rter rat, which does not produce a^-g, also fails to show a proliferate
response in the kidney or evidence of a promotional effect when exposed to
chemicals that induce protein droplet accumulation in male rats of other strains; its
response has not been tested in a conventional 2-year animal bioassay. Some
scientists apply the observations seen in animals to conclude that any renal tubUle
tumor in male rats observed in connection with a^-g accumulation is a species-
specific effect inapplicable to human risk assessment Other scientists argue that
more information on humans is needed and that all male rat kidney tumors should
continue to be considered as relevant to human risk as other tumors.
Because the question is relevant in assessing risk for a number of chemicals of
interest to EPA, the Risk Assessment Forum established a Technical Panel to
assemble and evaluate the current evidence and to develop science policy recom-
mendations for Agency-wide use. This document is the product of that effort- •
The literature review supporting this document is current as of July 1,1991.
XII
-------
B. Executive Summary
This report of a Technical Panel of the U.S. Environmental Protection
Agency (EPA) Risk Assessment Forum describes conditions under which
the Forum advises EPA risk assessors against using information on certain
renal tubule tumors or nephrotoxicity to assess human risk. Risk assess-
ment approaches generally assume that chemicals producing tumors in
laboratory animals are a potential cancer hazard to humans. For most
chemicals, including many rodent kidney carcinogens, this extrapolation
remains appropriate. The scientific studies reviewed by the Technical Panel
indicate, however, that some other chemicals induce accumulation of
alpha2u-globulin (o^-g), a low-molecular-weight protein, in the male rat
kidney. The «2 -g accumulation initiates a sequence of events that appears
to lead to renal tubule tumor formation. Female rats and other laboratory
mammals administered the same chemicals do not accumulate low-mo-
lecular-weight protein in the kidney and they do not develop renal tubule
tumors! Since humans appear to be more like other laboratory animals than
like the male rat, in this special situation, the male rat is not a good model
for assessing human risk.
The analysis of the scientific studies and related science policy set out
in this report each stress the need for full scrutiny of a substantial set of data
to determine when it is reasonable to presume that renal tumors in male rats
result from a process involving a2u-g accumulation and to select appropriate
procedures for estimating risks to humans under such circumstances. The
report also defines situations that suggest different approaches and it calls
for research to clarify unanswered questions regarding the mechanisms
accounting for the response in male rats.
A!pha2u-globu!in and Renal Lesions
In the male rat, the production of renal tumors by chemicals inducing
alpha^rfllobulin accumulation (CIGA) is preceded by the renal lesions
ascribed to oc2u-g-associated nephropathy. The involvement of hyaline
dropjet accumulation in the early nephrotoxicity associated with CIGA is a
major difference from the sequence seen for classical carcinogens. The
pathologic changes that precede the proliferative sequence for classical
renal carcinogens also include a form of early nephroxicity, but no apparent
hyaline droplet accumulation.
Investigations performed in multiple laboratories over the last decade
have demonstrated a consistent association between hyaline droplets
containing oc2u-g and production of certain lesions in the male rat kidney.
These renal lesions are not found in mice, female rats, or other laboratory
-------
species tested. The histopathological sequence in the male rat consists of
the following: '
• an excessive accumulation of hyaline droplets containing •>•
OL -g in renal proximal tubules;
• subsequent cytotoxicity and single-cell necrosis of the tubule
epithelium; . . /, •>•••
• sustained regenerative tubule cell proliferation, providing,-. •.
. exposure continues; .
• development of intralumenal granular casts from sloughed cell
debris associated with tubule dilation, and papillary
mineralization;
• foci of tubule hyperplasia in the convoluted proximal tubules;
and finally,
renal tubule tumors.
. Biochemical studies with model compounds show that CIGA or.their
metabolites bind specifically, but reversibly, to male rat a2u-g. The resulting;
a, -g-CIGA complex appears to be more resistant to hydrolytic degradation
by" lysosomal enzymes than native, unbound a2u-g. inhibition of the
catabolism of a2 -g, a protein only slowly hydrolyzed by renal lysosomal
enzymes under normal physiological conditions, provides a plausible basis
for the initial stage of protein overload in the nephropathy sequence.
Comparison with Classical Carcinogens
It is instructive to compare CIGA renal carcinogens with other renal
carcinogens. Several genotoxicchemicals recognized as classical inducers
of rodent kidney tumors have been used to study the pathogenesis of renal
tubule cancer in laboratory animals. In general* these prototypic renal
carcinogens produce tumors in both males and females. Although the wide
range of chemicals represented suggests multiple mechanisms of action,
many of the classical renal carcinogens or their active metabolites are
electrophilic species able to bind covalently to macromolecules and likely to
form DNAadducts in the kidney. In contrast, CIGA renal carcinogens are
not known to react with DNA and are generally negative in short-terfn tests
for genotoxicity. CIGA renal carcinogens also interact with o,u-g in a
reversible and noncovalent manner.
CIGA produced minimal changes in urine chemistry and very little or no
glomerular dysfunction in male rats. The mild tubule toxicity of C(GA, in
contrast to the obvious urinary changes induced by renal toxins such as
mercuric chloride or hexachlorobutadiene, is characteristic of CIGA and is
consistent with the notion that CIGA do not bind covalently to oc2u-g.
Classical renal carcinogens, such as certain nitrosamines, induce renal
tubule cancer in rats or mice with high incidence, minimal duration of
exposure, and clear dose-response relationships". There is usually no
absolute sex-specificity, although males and females maybe susceptible to
different degrees. In contrast, the renal tumors produced by the eight model
carcinogens examined in this report tended not to be life-threatening,:
-------
occurred late in life, usually being found at terminal sacrifice, and were
frequently microscopic. Even though the maximum tolerated dose was
exceeded for some of the eight model carcinogens, the renal tumor
incidence rate, adjusted for intercurrent mortality, was never greater than 28
percent. An increase in renal tubule tumors was not found in mice or female
rats exposed to these chemicals. Initiation/promotion studies with gasoline,
trimethylpentane (TMP) and d-limonene in Fischer 344 rats showed that
these CIGA promoted atypical tubule cell hyperplasia and/or renal tubule
tumors in males but not in females. In contrast, d-limohene did not promote
these lesions in males of the NCI Black-Reiter (NBR) strain in the same
initiation/promotion model. Such differences in potency and species-,
strain- and sex-susceptibility suggest that CIGA renal carcinogens act via
different mechanisms than classical renal carcinogens.
Renal tubule tumors produced by CIGA carcinogens also have features
in common with other renal tubule tumors observed in the male rat. For renal
carcinogens, in general, there is a continuum of chemically induced steps
from atypical hyperplasia through microscopic adenomas to macroscopic
adenbcarcinorhas or carcinomas. Renal tubule tumors induced by the eight
model carcinogens are morphologically indistinguishable from those in-
duced by classical carcinogens. Likewise, the sequence of development of
CIGA carcinogen-induced renal tumors from tubule cell hyperplasia to
carcinoma appears identical. Furthermore, none of these chemically
induced tumors can be differentiated from spontaneous tumors.
Other Considerations
All eight of the model carcinogens examined in this report were* also
capable of producing renal tubule hyperplasia in male rats. In general, this
hyperplasia became more severe with increasing dose. The occurrence of
these preneoplastic lesions together with the neoplastic lesions provides
indirect evidence of progression that is in accord with generally accepted
views on renaltubule tumor formation.
.Dose- and time-related associations between the administration of
CIGA to male rats and the various histological stages have been observed.
these relationships were demonstrated between CIGA administration for
both hyaline droplet formation and cc2u-g accumulation. Although the
relationships between increased hyaline droplets and cell necrosis or
between cell necrosis and cell regeneration have not been quantified, a
correlation between hyaline droplet response and the number of ceils
excreted in the urine has been observed for CIGA. Dose-response relation-
ships between hyaline droplet accumulation and proximal tubule cell prolif-
eration have been shown for TMP and unleaded gasoline. Clear dose-
response relationships were demonstrated between linear mineralization in
the renal medulla and incidence of renal tubule neoplasia in male rats in
several bioassays. A recent study of d-limonene demonstrated a relation-
ship betvyeeri severity of nephropathy and renal tubule cancer in male rats.
The Technical Panel is not aware of any epidemiologic study that has
been designed or conducted specifically to examine the applicability of the
-------
CIGA hypothesis to renal cell cancer in humans. Several epidemiologic
studies were reviewed for this report, but they are of limited value for this
analysis because they involved exposure to complex blends, such as
gasoline, or otherwise involved multiple exposures to both CIGA and non-
CIGA. In addition, these studies were of limited statistical power and were
not able to account for possibly confounding factors, such as smoking or
obesity which are known to influence renal cell cancer rates. In a few
studies, slight increases in risk of renal cell cancer have been observed;
however the other factors described above could have easily accounted for
the increased risk. These studies, therefore, are considered inadequate for
purposes of exploring the relevance of the a2u-g hypothesis in humans.
Low-molecular-weight proteins that probably have a three-dimensional
structure similar to a2u-g have been identified in mice and other species,
including humans. In vitro studies have shown that the active metabolite of
TMP forms complexes with some of these proteins. Other in vitro studies
indicate however, that reversible binding does not necessarily increase
resistance to hydrolytic degradation, a feature apparently required for
hyaline droplet formation.
Extensive studies of mice, whose urine contains large amounts of
mouse major urinary proteins (MUP), have found no evidence of renal
lesions similar to those associated with the ct2u-g syndrome. Thus, the
presence of a structurally related protein, even in large quantities in the
urine, does not imply that another species will respond in a manner similar
to the male rat.
The form of oc2u-gthat originates in the liver of the male rat is not detected
in the female rat. Like the mouse, the female rat shows no evidence of an
oc -g-like nephropathy when exposedto CIGA. In cases where nephrotoxicity
was observed in mice or female rats, it was less severe or qualitatively
different from that in male rats and did not involve the spectrum of discrete
lesions associated with cc2u-g accumulation in the male rat.
Specialized studies of rats, such as those involving immature, aged and
castrated male rats, males of the NCI Black Reiter(NBR) strain (which does
not synthesize o,u-g in the liver), and injection of male rats with estrogen and
female rats with a -g, show that development of the early features of the
specific nephropathy syndrome occurs only in the presence of o^ - g. Very
limited information from dogs, hamsters, guinea pigs, and monkeys also
supports this statement. These studies further support the hypothesis that
this oc2u-g-related nephropathy occurs specifically in the male rat.
Summary
In summation, the reversible binding of the compound to o,,u-g, which
results in a shift in balance between reabsorption and hydrolysis and the
accumulation of a -g in hyaline droplets in the P2 segment of the renal
tubule, provides a plausible explanation for the initial steps in a sequence of
events leading to the formation of renal tubule tumors in the male rat. A
sustained protein overload would result in single-cell necrosis in the tubule
-------
epithelium and increased cell regeneration, with granular cast formation and
papillary mineralization as indirect consequences. The increased prolifera-
tive response caused by chemically induced cytotoxicity may be a plausible
reason for the development of renal tubule tumor in male rats. Thus, renal
tubule tumors produced in male rats in association with CIGA-induced
,o,u-g nephropathy should be distinguished from other renal tubule tumors
in terms of use in human risk assessment.
-------
II. Introduction j
For most hazardous chemicals, adequate human data are not available,
and risk analyses must rely on information from laboratory studies of rats or
mice. The inference that the results of animal experiments can be applied
to humans is a fundamental principle of all toxicologic research. This paper
deals with a specific case, however, where the male rat seems to respond;
in a different manner than other laboratory species. The possibility of a
unique response in the rat among laboratory animals raises questions about
the applicability of certain rat data to other species, including humans. This
document provides guidance for the assessment of such information.
A variety of organic chemicals have produced specific renal lesions in
male rats in the form of a hyaline droplet nephropathy accwmpaniea by
accumulation of the protein, alpha2u-globulin (a2u-g) (Health Effects Institute
[HEI] 1985,1988). Among the chemicals tested are paraffins (Haider et al.,
1984; Phillips and Cockrell, 1984), decalin (decahydronaphthalene) (Alden
et al. 1984; Kanerva et al., 1987a), petroleum-based and synthetic fuels
(MacNaughton and Uddin, 1984), military aviation propellants (Bruner,
1984) and 2,2,4-trimethylpentane (TMP) (Haider et al., 1985). As seen in
Table 1 .which lists a sampling of chemicals that have been tested, many are
of considerable regulatory and commercial interest. For example, isophprqne
is a chemical intermediate of major industrial importance. Aviation and
automotive fuels fit into the category, as does the natural food product,
d-limonene, found in citrus oils.
This analysis focuses on model compounds having both an adequate
animal carcinogenesis bioassay and information on a2u-g or hyaline droplet
accumulation in the male rat. These substances are seven chemicals; 1,4-
dichlorobenzene (1,4-DCB), dimethyl methyl phosphonate, hexachloroeth-
ane, isophorone, d-limonene, pentachloroethane, and tetrachloroethylene
and a mixture, unleaded gasoline. These eight substances are compared
and contrasted with two related non-a2u-g-inducers, chiprothalonil and
trichloroethylene. The analysis also relies on research studies on two other
modelcompounds, decalin and TMP, which have extensive information on
o^-g nephropathy but no chronic bioassay data. More limited information
on 24 additional substances is also discussed where appropriate.
'•' • • ••' • *'
Of the eight model substances tested in chronic animal bioassays, all
invoked a specific type of protein droplet nephropathy in male rats and also
produced renal tumors in male rats but not in other species tested. It has
been proposed that such renal tumors are the end product in the following
sequence of functional changes in the epithelial cells of proximal tubules
(Universities Associatedfor Research and Education in Pathology [UAREP],
1983; Alden et al., 1984; Haider et ai., 1984; HEI, 1988; Swenberg et al.,
1989).
• Excessive accumulation of hyaline droplets in proximal tubules,
representing lysosomal overload, leads to tubule cell
degeneration, cell loss, and regenerative cellular proliferation.
-------
Table 1. Examples of Organic Chemicals that have Produced Renal Injury in Male
Rats Characterized by Hyaline Droplet Accumulation but not in Female Rats
or other Species.
Chemical
Decalin
Dimethyl methyl-
phosphonate
Isophorone
JP-4 jet fuel
JP-5 shale-derived
jet fuel
d-Limonene
Methyl isobutyl
ketpne
Pentachlorpethane
Unleaded gasoline
m = male
f = female
+ = positive
- = negative
Species
Rats
Mice
Dogs
Guinea
pigs
Rats
Mice
Rats
Mice
Rats
Mice
Dogs
Rats
Mice
Dogs
Rats
Mice
Dogs.
Rats
Mice
Dogs
Monkeys
Rats
Mice
Rats
Mice
Tested
(m/f)
(m/f)
(m/f)
(m/f)
(m/f)
(m/f)
(m/f)
(m/f)
(m/f)
(m/f)
(m/f)
(m/f)
(m/f)
(m/f)
(m/f)
(m/f)
(m/f)
(m/f)
(m/f)
(m)
(m)
(m/f)
(m/f)
(m/f)
(m/f)
Renal
Toxicity Reference
+/- Aldenetal, (1985)
-/- USEPA(1987)
+/- NTP(1987b)
+/- NTP(1986a),
+/- MacNaughton and
-/- Uddin(1984)
+/- MacNaughton and
-/- Uddin (1984)
+/- NTP (1990)
-/- Webb etal. (1990)
+/- Aldenetal. (1984)
-/- Phillips et al.
(1987)
+/- NTP (1983) .
+/- USEPA(1987)
-------
• Cell debris in the form of granular casts accumulates at the
"cortteomedullary" junction with associated dilation of the affected
tubule segment and more distally, mineralization of tubules
within the renal medulla.
• Single-cell necrosis accompanied by compensatory cell
proliferation and exacerbation of the chronic progressive
nephropathy (CRN) characteristically found in aging rats occurs.
- Renaltubule hyperplasia and neoplasia develop subsequently.
According to this hypothesis, the increased proliferative response
caused by the chemically induced cytotoxicity results in clonal expansion of
spontaneously initiated renal tubule cells and increased incidence of renal
tumor formation (Trump et al.. 1984a; Alden, 1989; Swenberg et al., 1989).
This line of reasoning leads supporters of the hypothesis to conclude that
the acute and chronic renal effects induced in male rats by such chemicals
will be unlikely to occur in any species not producing «2u-g, or a very closely
related protein, in the large quantities typically seen in the male rat (Alden
1989;Borghoffetal.,1990;Greenetal.,1990;Olsonetal.,1990;Flammand
Lehman-McKeeman, 1991; Swenberg, 1991).
This report examines the hypothesis that the male rat is predisposed to
the nephrotoxic effects induced by certain classes of chemicals, such as
volatile light hydrocarbons and organohalides. It also examines data that
supportorcontradicttheconceptthatthe renal tumors produced in male rats
by these chemicals are causally related to the nephrotoxicity. Based on the
Risk Assessment Forum's (RAF) conclusions regarding these data, the
document describes a uniform approach for EPA to use in risk assessments
dealing with this spectrum of lesions and category of chemicals.
Information for this RAF report was obtained initially from a 1988 review
entitled, "Evaluation of Data Concerning the Relationships among Chemi-
cally-induced Renal Alpha2 Globulin or. Hyaline Droplet Accumulation,
Nephropathy, and Renal Neoplasia" prepared for the Office of Toxic
Substances by Dr. William Richards of Dynamac Corporation, Rockville,
Maryland. Additional information considered in this report includes recent
comprehensive reviews of the subject, comments from peer reviewers, and
otheroriginal work, especially publications subsequenttothe 1988 Dynamac
review.
This report has four parts. Following this brief introduction, Part 1
addresses the characteristics of hyaline droplets, the protein, a,u-g, and the
nephropathy associated with o,u-g accumulation (Sections HI and IV).
Part 2 (Sections V-VIII) presents data on the carcinogenic potential of
chemicals inducing alpha2u-globulin accumulation (GIGA) in the male rat.
Section V describes the preneoplastic and neoplastic lesions produced by
classical renal carcinogens. Section VI considers generic factors relevant to
all studies of potential renal carcinogenicity in laboratory animals and then
analyzes and discusses data on the renal lesions observed in 2-year
bioassays with chemicals causing the hyaline droplet nephropathy. Section
VII examines additional information that assists in defining renal carcino-
-------
gens as GIGA, in particular genotoxscity and initiation-promotion data. In
Section VIII, GIGA are compared with classical renal carcinogens, while
Section IX considers the human evidence for kidney cancer, its histogenesis
and epidemiology. Section X examines evidence for the hypothesized
dose- and time-dependent progression of lesions. ,
Part 3 evaluates the evidence considered in Parts 1 and 2 with regard
to the hypothesis that a -g accumulation in the kidney is an initial step in
a succession of histopatnologic events that may culminate in renal tubule
tumorformation in male rats. This part also lists priorities forfuture research.
Part 4 comprises the Agency policy statement regarding approaches to
risk assessment for this category of Chemicals.
For clarity throughout the review, nomenclature is standardized, and
abbreviations are used for frequently repeated terms. Insofar as hyaline
droplet represents a morphological entity requiring only light microscopy for
identification, this term will be used in preference to the synonymous protein
droplet1. The designation, ct2u-g nephropathy is used to connote the full
sequence of pathologic lesions from hyaline droplet formation to restorative
hyperplasia and medullary mineralization. Toxic tubular nephropathy is a
nonspecific term commonly used in rodent bioassay reports to describe
various forms of nephrotoxicity induced by chemicals, including the specific
lesions of o^-g nephropathy. The spontaneous age-related syndrome of
rat Kidney disease otherwise known in the literature as old rat nephropathy,
chronic nephrosis, glomerulosclerosis, and progressive glomerulonephrosis,
is standardized according to Barthold (1979) as chronic progressive
nephropathy (CPN). The term lipocalin is used according to the terminology
of Pervaiz and Brew (1987) to describe the superfamily of low-molecular-
weight proteins which appear to transport lipophilic substances.
In rats, the proximal tubule of the nephron is divisible morphologically
into three parts (see Figure 1). The first segment is in continuity with the
parietal epithelium of Bowman's capsule surrounding the glomerular tuft.
Together, the first and second segments represent the convoluted portion
of the proximal tubule and are situated wholly in the cortex, the outermost
zone of the rat kidney. The third segment is the straight portion of the
proximal tubule (pars recta) comprising the outer stripe of the outer medulla
but also the medullary rays arising in the cortex. The abbreviations P1, P2,
and P3 are used conventionally to denote these three segments. The term
renal tubule tumor describes neoplasms of the renal cortical tubule epithe-
lium comprising collectively adenoma, adenocarcinoma, and carcinoma
according to standardized nomenclature determined by the Society of
1 Hyaline droplets refer to spherical inclusions in the cytoplasm which are homogeneous
and eosinophilic, representing overdistended phagolysosomes. they may contain
various macromolecules including a^-globulin. The morphology of droplets containing
different proteins ms.> oe identical and therefore irhmunocytochemistry is required for
precise definitiion of contents.
-------
Distal convoluted
tubule
Thick ascending
limb of Henle
Thin descending
limb of Henle
Thin ascending
limb of Henle
Figure 1 Diagram of Zonation and Tubule Segmentation in Rat Kidney. G: Glomeru-
- lus; PI: First segment of proximal convoluted tubule; P2: Second segment of
proximal convoluted tubule; P3: Pars recta of proximal tubule. •• • .
Source: Adapted from Bachmann era/. (1986).
Toxicologic Pathologists (Hard et al., 1991). Except when specified, the
terms adenocarcinoma and carcinoma are used interchangeably. The
same neoplasms are referred to as renal cell tumors in humans, in keeping
with the general literature (Bannayam and Lamm, 1980).
10
-------
IIS. Hyaline Droplets and Aipha2U - Globulin; Physiology and
Biochemistry
Information on the renal processing of low-molecular-weight proteins,
sex and species differences in urinary proteins, and the characteristics of
o,u-g provides an explanatory basis for the accumulation of ot^-g in hyaline
droplets in the male rat following exposure to GIGA. It is pertinent, therefore,
to examine the physiological and biochemical characteristics of o,u-g and
related proteins, particularly those that occur in humans, before exploring
the possible associations between oc2u-g accumulation, renal toxicity and
renal tumor formation and their relevance to human risk assessment.
A. Filtration, Reabsosptlon, and Catabollsm of Low-Moleculaf-
Weight Proteins by the Kidney
The mammalian kidney has a major role in maintaining the plasma
concentrations of circulating low-molecular-weight proteins at their normally
low, physiological levels Thus, low-molecular-weight proteins are continu-
ally removed from the plasma by glomerularf iltration followed by reabsorption
and catabolism in the proximal tubules (Maack et al., 1985} or by excretion.
Figure 2 is a schematic representation of the cellular uptake and disposition
of filtered proteins by the renal tubule.
The normal renal glomerulus freely passes proteins with a molecular
weight of less than 20,000 daltons, including peptides such as insulin,
lysozyme, rat growth hormone, myoglobin, and cytochrpme C (Maack et al.,
1985). For larger proteins like the albumins and globulins, which have afar
greater plasma concentration and much Sower filtration rate than low-
molecular-weight proteins, the kidney has no regulating role in plasma
protein concentration.
Reabsorption of filtered protein occurs predominantly in the convoluted
part of the proximal tubule and to a lesser extent in the pars recta cells.
Tubular absorption of a protein is a complex process initiated by binding of
the protein to the microvilli of the proximal tubule epithelium. This is followed
by migration to the base of the microvilli and adsorptive endocytosis
whereby invagination of the surface membrane internalizes the protein
(Kaysen et al., 1986). While reabsorpiion was once considered largely
nonselective, high capacity, low affinity transport (Maack et al., 1985), from
recent work it now appears that interaction between the protein and the
brush border membrane is the step at which a degree of selectivity in the
absorption process occurs (Kaysen et al., 1986).
11
-------
Tubule Lumen
Degradation products
Blood |
Proximal (P2) Tubule Cell
Figure 2. Schematic representation of endocytic uptake of filtered proteins. Filtered
proteins are adsorbed to endocytic sites at the lumenal membrane and
segregated in endocytic vacuoles (EV). These EV migrate to the cell interior,
where they fuse with lysosomes (L) to form secondary lysosomes(SL) or
phagolysosomes' where digestion of the protein takes place. The products of
hydrolysis (amino acids) permeate the SL membrane, cross the
contralumenal cell membrane, and return to the circulation.
SOURCE: Adapted from Kaysen et al. (1986).
12
-------
Within proximal tubule cells, endocytic vesicles fuse to form endocytic
vacuoles which in turn coalesce with lysosomes derived from the Golgi
apparatus, forming secondary lysosomes. The hydrolysis of proteins by
protease enzymes takes place within the secondary lysosomes. The
lysosomal enzymes of renal cortical tubules include two major classes of
acid proteinases, i.e., cysteine proteinases (cathepsin B, H, and L) and an
aspartic acid proteinase, cathepsin D (Lehman-McKeeman et al., 1990a).
Lehman-McKeeman et al. (1990a) have shown that both of these endopep-
tidase classes contribute to the degradation of a2u-g.
Lysosomes have a large, but not unlimited, capacity to cope with
increased amounts of hydrolyzabie proteins, but the proteins differ in
susceptibility to hydrolysis. Protein half-lives, which are indices of their
catabolism by proteases in the kidney, depend on specific molecular
determinants in the protein. The primary amino acid sequence may be one
important factor in determining protein half-lives (Dice, 1987). The plasma
half-lives of many low-molecular-weight proteins are measured typically in
minutes (Maack et al., 1985). One of the exceptions is a2u-g, with a half-life
measured in hours (Geertzen et al., 1973).
Whether or not low-molecular-weight proteins like a2u-g accumulate in
kidney tubules depends on the balance between the rate of reabsorption by
epithelium and the rate of hydrolysis in the cells. Based on the information
presented below, it is believed that exposure to GIGA results in a shift of this
balance in male rats.
B. Hyaline Droplets in Renal Tubules
The product of protein reabsorption and accumulation in renal tubule
cells is visualized by light microscopy as hyaline droplets. Small protein
reabsorption droplets of uniform size are a constitutive feature of normal
mature male rats being particularly evident in the P2 segment of proximal
tubules (Logothetopoulos and Weinbren, 1955; Maunsbach, 1966a;
Goldsworthy et al., 1988a). Ultrastructurally, hyaline droplets are abnor-
mally large, dense, secondary lysosomes (also termed phagolysosomes),
representing fusion of endocytic vacuoles with primary lysosomes. Some
hyaline droplets show crystalloid changes by electron microscopy that are
not observed in the lysosomes of female rats (Maunsbach, 1966b). Crys-
talline formation in the normal male rat is believed to indicate the presence
of a poorly catabolized protein in pure solution (Pesce et al., 1980),
presumably a2u-g in the kidney iysosomes.
Hyaline droplets in the proximal tubules of normal male rats contain
o^-g (Alden et al., 1984; Garg et al., 1987; Goldsworthy et al., 1988a), and
their occurrence appears to parallel the variable synthesis of this protein.
Thus, hyaline droplets become apparent in male rats at the time of puberty,
but they decline progressively with increasing age after 18 months
(Logothetopoulos and Weinbren, 1955; Murty et al., .1988). In female rats,
protein droplets in proximal tubules are either absent or considerably less
frequent than in males, and they do not contain o^g (Logothetopoulos and
Weinbren, 1955; Maunsbach, 1966a; Goldsworthy et al., 1988a; Burnett et
13
-------
al.; 1989). Hyaline droplets are substantially reduced in castrated male rats
(Logothetopoulos and Weinbren, 1955).
Because an abnormal increase in hyaline droplets has more than one
etiology and can be associated with the accumulation of different proteins,
it is necessary to apply special diagnostic methods such as '"imunohista-
chemical staining to make the association between chem.cal exposure and
pathologic accumulation of o,u-g.
Abnormal accumulation of hyaline droplets in rodent *Wney te seenln
certain disease processes. Both male and female rats with histiocytac
sarcoma show hyaline droplet accumulation in the proximal tubules indis-
tinguishable from the CIGA-induced lesion. The accumulating protein in
hesetumor-bearing animals has been identified as ysozyme (Had and
Snowden,1991). Similarly ,in male and female mice w.thh.siocytic tumors
aWrmal accumulation of lysozyme-containing hyal.ne droplets sometimes
occurs in proximal tubules (Hard and Snowden, 1991).
In humans, the Bence-Jones proteins, a class of light chain immuno-
globulins, are produced in large amounts in multiple myeloma patients
(Pirani e al 1983). In human cases of mononuclear cell leukemia
ysozyme is produced (Muggia et al., 1969). The kidney injury seen with
these neoplastic diseases has been described as similar to ha produced
bv administration of decalin to male rats (Alden, 1986 , including protein
&3SSS^
and Piatts 1970;. Pirani et al., 1983). Patients with epidemic hemorrhagic
fever, infused with large amounts of concentrated human serum albumin as
atherapeutic procedure for shock have also developed a comparable form
of hyaline droplet accumulation (Oliver and MacDowell, 1958).
C. Factors Affecting Kidney Accumulation of LowrMolecular-
Weight Proteins
Protein accumulation in the proximal tubule can reach pathological
levels resulting in excessive hyaline droplet formation for several reasons:
m the rate of protein delivery to the tubule cells is abnormally high (2) the
proteins delivered are difficult to hydrolyze, or (3) the lysosomal hydrolyze
capacity is sufficiently reduced.
The rate of protein delivery to the tubule can be abnormally high under
conditions when the capillary wall of the glomerulus fails to provide the
normal filtration barrier. This happens, for example when there is immuno-
logical, inflammatory, or toxic disease in the glojnerulus or when the
permselectivity barrier is overloaded by filterable proteins (Kaysen et al.,
1986).
The increased urinary excretion of low-molecular-weight proteins seen
in diseases such as multiple myeloma in humans or histiocytic sarcoma in
rats is primarily the result of an increase in plasma concentration caused by
overproduction of specific small proteins (Maack et al., 1985). Lysozyme
(histiocytic sarcoma) and light chain immunoglobulins (multiple myeloma)
14
-------
are proteins that are relatively resistant to hydrolysis (Maack et al., 1985).
This suggests a combination of rate of delivery and difficulty of hydrolysis as
etiologic factors in the accumulation of lysozyme in rats with histiocytic
sarcoma and light chain immunoglobulins in humans with multiple myeloma.
The combination of difficult hydrolysis of the protein, as suggested by its long
half-life, coupled with high rate of protein delivery to tubule cells in the
sexually mature male rat also appears to be a factor in the accumulation of
o,u-g in the renal tubules of male rats.
The process of protein hydrolysis can be reduced or inhibited when
lysosomes are unable to maintain the low pH required for hydrolytic enzym6
function. Inhibition of the metabolically driven hydrogen ion pump, by
metabolic poisons or the presence of a weak base in tubule lysosomes,
alters the pH and results in the accumulation of proteins (Maack et al., 1985).
In the presence of a reduced lysosomal hydrolysis capacity, the most
hydrolytically resistant proteins, like o,u-g, tend to accumulate first. Testos-
terone is known to have a suppressive effect on the activity of some major
proteolytic enzymes in the male rat kidney (Kugler and Vornberger, 1986).
Consequently, the lysosomal protease activity in male proximal tubules is
lower than that of females (Jedrzejewski and Kugler, 1982; Kugler and
Vornberger, 1986) implying that the male rat could be intrinsically more
prone to protein overload in the renal tubules than the female rat.
Reduction of the hydrolytic capacity of renal lysosomes and increased
resistance of protein to hydrolysis can both be affected by exogenous
chemicals. Although CIGA may not compromise kidney lysosomal enzyme
activity per se (Murty et al., 1988; Lehman-McKeeman et al., 1990a), any
chemically induced impediment to a2u-g digestibility caused by CIGA would
be further superimposed on the causes considered previously that alone
can result in excessive protein accumulation in renal tubules.
D. The Alpha2u-globulin Superfamily of Proteins
Alphas-globulin2 is a member of a large superfamily of low-molecular-
weight proteins. The complete amino acid sequence of oc2u-g was first
deduced by Unterman et al. (1981). With the exception of a2u-g and mouse
major urinary protein(s) (MUP), the sequence homology between any pair
of proteins in this superfamily is small, about 20 percent (Akerstrom and
Logdberg, 1990). Statistical analysis shows, however, that the proteins are
related evolutionarily (AkerstrQm and Logdberg, 1990).
Of the approximately 20 proteins now considered to be potential
members of the superfamily (Akerstrom and Logdberg, 1990), the three-
dimensional structure is known fo'r only three, retinol-binding protein, p-
lactoglobulin, and insecticyanin (Sawyer, 1987). The central core of these
1 The male rat urine protein, alpha -globulin is named in accordance with immunoelectro-
. phoretic nomenclature (Roy and Neuhaus, 1967). Because of its size, some authors
refer to this protein as alpha.,- microglobulin or alpha 2\i- globulin, a term more appropri-
ately reserved for another low-molecular-weight endometrial protein associated
with pregnancy.
15
-------
three proteins is composed of eight strands with a p-barrel itructure forming
a hydrophobic pocketthat appears to enclose the l.gand (Papa at al., 1986
Sawver 1987) This structure has been described as resembling a coffee
mTp^per(AtorLSem and LSgdberg, 1990). In addition to theb-stmctural
moHf one helical rod and several other structural elements appear tobe
Served among the proteins. Protein folding patterns tend to be highly
Served in homologous proteins even though they may diverge cons.d-
-SS?h smcTure and function, suggesting that other members of he
superfamily, including 0,-g, possess a similar three-dimensional structure.
The onlv member of the protein superfamily with a clearly defined
physical function is retinol-binding protein. More circumstanUal ev,-
dpncesuaaests that the superfamily members serve as carriers of lipoph,lc
'StolpSKand Brew, 1987). The mode of binding in which the l.pid
EganS is enclosed within the ^-barrel impressed Perva.2^and Brew as not
unlike the protective role of the calyx to a flower. On this basis, they
suggeJted tPhe Srative name, lipocalins, for the superfamily of prote.ns.
Table 2 illustrates the information available on several imembers of the
lipocalin superfamily, which includes a?u-g, retmol-bindinjprotem,
Kooprotein D a -acid glycoprotein, cc.-mfcroglobulm, ruminant fi-lacto-
gtobS andpyrazine-binding protein (i.e., odorant-binding protem) rat
odVrarS-bTndingprotein, and MUP. Some of the membenj; oTthe lipocahn
superfamily, such as retinol-binding protein, arac.d glycoprotein and a
microglobuln have been identified in many spec.es and their properties
appear to be species-independent, suggesting that they share a common
vS function (Akerstrom and Logdberg, 1990). Others, such as a2u-g and
MUP seem to be species-dependent.
Several functions have been suggested for oyg. Cava99f ru_et al
(1987) speculatedthat azu-g may serve totransferocforants sucf^setheeal
ioid Dheromones from male rat urine to the air for attracting females.
G^andularTssue production of ex2u-g helps support these speculator*
^urty et al., 1987; Mancini et al., 1989). In addition, a2u-g has been
Sfied as a fatty acid-binding protein of the kidney (Kimura et al 1989)
and may serve to transport fatty acid, an important energy source in k dney
within renal epithelial cells. Brooks (1987) found a protein structurally
related to OL -g that is synthesized and secreted by the rat ep.d.dymis unde
ttInfTuen^e of androgenic hormones. He ^^.^^fSate
these proteins may be to carry retinoids within the lumen of the male
reproductive tract.
Other members of the lipocalin superfamily, such as retinol-binding
orotein apolipoprotein D, p-lactoglobulin, andafacid glycoprotein,function
fn thTiransport of lipids between cells and across hydroph.Hc barners
IPevsner et al. 1988). The lipids bound by the proteins differ considerably
n structure and range from odorants in rat nasal epithelium to human
cholesterol and retinol (vitamin A). It is not yet clear how selective these
proteins are for specific ligands or whether a given protein might bind a wide
spectrum of small hydrophobic molecules. Both cases might occur since
16
-------
«0
i
1
g>
"•6
1
JO
13
-a
t
§.
^
^
;^
I
1
1—
§
£
"S
EC
TO
o"o
•£.£.
E
«
flj jiiir
O .O)
© (j)
o £
B- *O
«J!
3 >,
F=l
w
8
(0
1
a.
o~
R i
oo •—'
a> c
•^- o
~— w
S ®
m ^
~o
i c
.N «
nj — i-
1 1
s
^
^ ° "o?
gilt
O TOO E
CO" 3 CO" §
CO
i
a.
V)
E
E
2
_
.E
i^ ff
1-Acidglycoproteii
(acute-phase pro
(Orosomucoicl)
a
v— *.
o>
-r—
13
cS
c
2
0
o
CO
T~
8
oT
CO
i
a.
u
To
i
2
f
CO
'o
polipoprotein D
(cholesterol-asso
•<
o"
&
m
CD
-d
TJ
3"
T3
C
CO
1
u>
1
CM
CO
T~
o>
CO
o"
CM
co
CO
a.
_£0
E
E
• 2
] Microglobulin
(Protein HC)
8
Hi "
"S
E
2
c
"ffl
I
0>
1
lo
1
o
•a
CD
cc
CD O) T—
o> O) co
CO
"co
N
'5.
£
CM
CD
co
'iz "c
.2.9
o> o>
co co
00
CD
co
o
Q
m
o>
13
I
••2.
CM
CO
to co
CM i-
§
co"
S
a
I
CO
(O
'
—1 Q.
.
CD
I
oo"
T3
_co
CD" TO
•§18
3 =
SSL a.
« £
.E ex
8
m
oo"
8
to
2
TJ
1
-.2
-------
(>
o
::
i
0
)
•3
>S
"1
(0
5 „
1
i
i. T
OS
51
«
a
i
a.
V)
1
I
1
H^
73
aJ
3
c\i cr> oj
(O Cn 00
S^i- o
g. 8.
0(f i— O
T- CM CM
"ST
O £
it i Ii
i ^ g*
i S *
Major urinary protein
Purpurin
Bowman's gland protein
c
%
1 s
N 0>
* *
^ W
O 5**
.3 a
m *
CM
§8 ff
CM z
^
u*
E
"o
0)
Tobacco
homworm,
butterfly
Insecticyanin
(Bilin-binding protein)
1
us
a
c
"3 o «
_r o 2^
Adapted from Pevsner et
NR = Not reported, chars
a Also occurs in other seer
b Described as a2U-globuli
18
-------
retinol-binding protein is quite specific for retinol, whereas odorani-binding
proteins may have a broad range of ligands (Godovac-Zimmermann, 1988).
The ability of a chemical to serve as a ligand for one member of the
superfamily appears to be a poor predictor of binding affinity for other
members of the superfamily. Cavaggioni et.al. (1990) measured the binding
affinities of 0,,,-g, M UP, and pyrazine-binding protein isolated from calf nasal
mucosa for a series of odorants. MUP bound only one of these chemicals,
pyrazine-binding protein bound six, arid a2u-g bound twelve. The best ligand
for a?u-g was chemically unrelated to the best ligands for the other two
proteins, which were also chemically unrelated.
E. Characteristics of Alphas-globulin •
Alpha2u-giobulin was first characterized in male rat urine (Roy and
Neuhaus, 1967). All isoforms of a -g are anionic at neutral pH although they
have varying isoelectric points. The molecular weight of a, -g has been
reported to be 18,000 to 20,000 daltons. In all rat strains tested to date
except for the NCI Black-Reiter (NBR) rat, a strain that appears to have a
tissue- and gene-specific regulatory defect involving a2U-g (Chatterjee et al.,
1989), the major urinary source of OL-g is the liver where CL -g mRNA
constitutes approximately 1 percent of the hepatic mRNA population (Sippel
et al., 1976; Kurtz and Feigelson, 1977). The hepatic isoforms of a -g may
vary throughout the lifetime (Roy et al., 1983). Synthesis of the protein in rat
liver is under multihormonal control, particularly andrbgen, but also glu-
cocorticoids, thyroid hormones, insulin and growth hormone (Feigelson and
Kurtz, 1977; Roy and Chatterjee, 1983). These hormones appear to act by
regulating the steady-state level of oc2u-g mRNA (Kurtz and Feigelson, 1977).
Neither o^-g nor its corresponding mRNA are detectable in the livers of
sexually intact female rats (Sippel et al., 1975,1976; Maclnnes et al., 1986).
However, a very low background level of the mRNA has been indicated in
the ovariectomized female rat (Chatterjee et al., 1979), and ovariectomy in
concert with androgen treatment induces a parallel increase in a, -g and its
mRNA in female rat liver (Roy and Neuhaus, 1967; Sippel et al.,u1975).
Although plasma and urinary a2u-g derive predominantly from the liver
in male rats, high levels of o^-g and its mRNA are also present in the
preputial gland of both male and female rats, and neither castration nor
ovariectomy significantly alter the preputial concentration of this protein and
its mRNA (Murty et al, 1988). Alpha2u-globulin mRNA has also been
detected in the female mammary gland during pregnancy, and in the
submaxillary, lachrymal, Meibomian, and perianal glands of rats of both
sexes (Maclnnes et al., 1986; Mancini et al., 1989). The female forms of
o,u-g show distinct differences from male rat a -g suggesting that they are
encoded by different genes (Vandoren et al., 1983).
Low levels of a2u-g first become detectable in the male rat liverunderthe
stimulus of testosterone at 35 to 40 days, reaching maximum adult levels by
60 to 80 days (Roy eftal., 1983;Motwanietal., 1984; Maclnnes etal., 1986).
Due to the development of hepatic insensitivity to androgen during aging,
hepatic synthesis of a2u-g begins to fall gradually in male rats sometime after
19
-------
* mnnthc of aoe Bv 22 months of age, there has been a drop of over 90
synthesis in the liver.
fs^B^^^^y^^^
zme Charbonneau et al., W"™*
bn nanaogoun were - to 1 00,000-fold more easily
sZerfS « sS in the balance between reabsorption and hydrolys,s
occur.
F Sex and Specfes Comparison of Urinary Protein Content of
the Llpocalin Superfamlly
Relative to the female rat/and other species including humans the
1978) Tn?he B6C3F1 strain, males have been shown to excrete 14.9 mg
S&cert SKS2, -S rsyreabsoTbed by the kidney, MUP is not reabsorbed
S 'IS mSSa . aS appears to be totally excreted (Caudill et al;, 1991).
In contrast, normal human urine contains relatively little protein, only 1
percent of the total concentration present in mature male rtf t unnc 5 pson et
al 1990) Human urinary proteins are predominantly high-molecular-
20
-------
weight species with only minor components weighing less than 66,0i
daltons. Within the low-molecular-weight fraction, trace amounts of proteii
represent the Sippcalin superfamily, but none appear to share molecul
weight identity with o,0-g. The urinary excretion of retinol-binding protei
a,-acid glycoprotein and armicroglobulin has been measured at 0.0001
0.0007, 0.0006 to 0.002, and 0.02 to 0.05 mg/day/g kidney, respective
(Berggard, 1970; Peterson and Berggard, 1971; Ekstrom and Berggai
1977). Thus, the urinary excretion of ct2u-g in the male rat is approximate
two orders of magnitude greaterthan the human urinary content of the thn
superfamily proteins combined.
Recently, a sex-dependent protein of unknown origin and functio
termed urine protein 1, was identified in normal human urine (Bernard et a
1989). The molecular features of protein 1 are similar to a,u-g as it has
molecular weight of approximately 21,000 daltons and an isoelectric poi
around 4.8 although its amino acids have not been fully sequence
(personal communication, R. Lauwerys, Catholic University of Lowai
Belgium, to I. Rodgers, March 25,1991). Protein! occurs in both sexes fro
an early age, but increases substantially in males after puberty, reaching i
to a 50-fold difference over females during late adolescence. A 5-fold ma
to female differential persists through adulthood. Average urinary conce
trations of protein 1 have been determined as 108 and 3.2 jig/L respective
for males and females aged 15 to 20 years, and 24.7 and 5.8 u.g/L for mal<
and females in the 20 to 60 year age range (Bernard et al., 1989). Su<
levels of proteinl in human male urine, however, are calculated as four
five orders of magnitude less than o,u-g concentrations in the urine of ma
rats.
G. Noncovalent Binding to Alphas-globulin and Its Homologues
It has been suggested that GIGA bind reversibly and noncovalently
a,0-g, forming a resultant complex that is even more poorly digested in tl
male rat kidney than a,u-g (Swenberg, 1989).
1. Chemical entities bound to alpha2u-globulin
In a few instances, the specific chemical entity complexed with a2u-g hi
been identified. TMP, a branched chain aliphatic hydrocarbon present
gasoline was the first model CIGA to be studied in this manner. When [14C
TMP was administered in a single oral dose to rats, radioactivity w?
retained in the kidneys of males, but not of females (Kloss et al., 198
Charbonneau et al., 1987). The major metabolite of TMP in the male r
kidneys was identified as 2,4,4-trimethyI-2-pentanol (TMPOH) (Charbonne*
et al., 1987). In a separate report, TMPOH was shown to be the only ligar
for a,u-g whenever TMP was administered to the male rat (Lock et a
1987a). TMPOH was not detected in the kidney tissue of the female rat
which excreted more conjugated TMPOH (glucuronides and sulfates) the
the males (Charbonneau et al., 1987). Later studies confirmed, as su
pected, that the TMPOH-o,u-g complex is cleared slowly from male r
kidney (Swenberg, 1989).
21
-------
Fnr d-limonene d-limonene-1,2-oxide has been shown to be the
Xp»e^^^
ma,e Fauo 1,4-DCB.|Uthe• P"**^OT«T&i&
.. *S* i u^^—^nn «»iifsMiot a aHministered
^'^^s^Xxw^^M^exlmMXervi
ISlJ^Sta the Vmma-lactone of 3,5,5-trimethyl hexanoic acid.
component being the
2. Nature of the association
The nature of the
kidneys. Cytosol.obta
' riMiw •
issociation of CIGA with o^-g was explored initially
i/ho dosed sexually mature male Fischer 344 (F344)
ed them 8 to 72 hours later, and homogenized the
ned by centrifugation of the homogenate at 116,000
- - -— ^-75 column. About 26 percent of the cytosol
• • in the kidney) eluted in the fraction
\ percent of the radiolabel in the cytosol
•-" equilibrium dialysis against phos-
aKHjiinjiv - u- dialyzed cytosol showed that the
Deled material coeluted with the peak containing
F "--~v a detergent which
c^Sedw^^
etal 1989), d-limdnene (Lehman-McKeeman et al., 1989 , 355-
trimethylhexanoloxyb^nzene sulfonate (Lehman-McKeeman et al., 1991),
and lindane (Dietrich and Swenberg, 1991 a).
Inthe d-limonene study (Lehman-McKeeman et al., 1989), the amount
of ra^actlvSTobserYed in the kidneys of Spragu^Dawley rats 24 hours
after oral administration of P4C]-d-limonene was about 2.5 times higher in
males han iSalei Equilibrium dialysis, in the presence or absence p
SDSinSed Sit Approximately 40 percent of the radioact.ve material
fetained in^he mate ^kidney was associated with proteins in a reversible
mannlr Gel fHtratioh high-performance liquid chromatography HPLC),
mveree-phase HPLC and amino acid sequencing demonstrated that this
SX materiai was associated with « -g. No d-«.monene i or
d-nmonene metabol4 coeluted with female rat kidney prote.ns. Inthe 355-
trimethylhexanoloxyb^nzene sulfonate study (Lehman-McKeeman , rt ta L,
1991 distribution of he chemical was examined in mice and rats of both
sexes The male rat kidney contained roughly 10 times the concentration
^chemical ™ ^ the f Late rat kidney, and the concentrations ,n mouse
kidney were even lower than those in the female rat.
22
-------
1990). Twenty-four
mg dose by gavage, liver
binding. Neither
[j)NA. In addition,
into the
3. Binding of CIGA to other macromolecules
Reversible binding generally implies a dissociable Chemical-protein
interaction in which the free chemical can be liberated from the protein
without having produced molecular damage. In contrast, in covalent binding
a reactive chemical species, usually an electrophile, reacts with nucleophilic
centers in target molecules comprising enzymes, other proteins, nucleic
acids, or lipids. CIGA appear to differ from many known bhemical toxins,
nephrotoxins included, which bind covalently and irreversibly to proteins
and/or DMA and through this process cause cellular injury.
A DNA binding study with F344 rats and B6C3F1 mice o1! both sexes was
performed using [1,3,5,-14C]-is6phorone (Thier et al.,
hours after the animals were administered a 500 mg c. _
and kidneys were processed for determination of DNA
isophorone nor its metabolites showed covalent binding to
metabolically formed degradation products were not incoriporated ...„,»,«
DNA bydanoVQ synthesis of DNA from labeled fragments of the xenobiotic.
In contrast to 1,4-DCB, which is a CIGA, 1,2-DCB, a closely related
isomer, does not induce hyaline droplets and appears to b nd covalently to
proteins in the male rat liver, plasma, and kidney (Charbonneau et al., 1989).
When administered orally to male rats, 1,4-DCB (and its metabolite 2,5-
dichlorophenol) in the kidney cytosol eluted as a single peak in the low-
molecular-weight fraction containing a,-g. Dialysis of the kidney cytosol
with SDS led to a substantial loss of 1,4-DCB, demonstrating the reversible
nature of the CIGA-protein binding. 1,2-DCB bound to low-molecular-
weight proteins in the kidney cytosol of male rats, but it also bound to proteins
in the 64,000 to 70,000 dalton range. Dialysis of the kidney 4ytosol with SDS
failed to remove approximately half the 1,2-DCB, suggesting substantial
covalent binding of this chemical in the male rat kidney.
4. Specificity of the interaction of CIGA with alpha2u-globvlin
The capacity of CIGA to serve as ligands for other lipocalins, some of
which are found in humans, has been investigated. Prejiminary studies
designed to determine the accumulating protein in the kidhey of male rats
exposed to decalin employed two-dimensional gel electrophoresis of rat
kidney homogenate (Alden et al., 1984). Although other proteins in the
lipocalin superfamiiy are present in the male rat, decalin (was associated
solely with a.y-g. Other preliminary studies involving the in vitro binding of
TMPOH to lipocalins suggest that TMPOH, which binds reversibly to OL -g
in vitro, may also bind reversibly to three other members of he superfamlily,
i.e., retinol-binding protein, a, -acidglycoprotein and p-lactog obulin (Borghoff
et al., 1988). TMPOH did not bind to p2-microglobulin or lysozyme, low-
molecular-weight proteins that are not members of the superfamiiy.
D-limonene-1,2-oxide also does not appear to bind to or^-apid glycoprotein
or urine protein 1 in in vitro studies (personal communication, L. Lehman-
McKeeman, Procter and Gamble, to I. Rodgers, February 27,1991).
Gas chromatographic analysis in experiments with livter microsomes
have shown that mice are able to oxidize d-limonene to cis-a-limonene-1,2-
23
-------
ovide as in the rat although some qualitative and quantitative species
specificity of the interaction between C1GA and a^-g.
When PHl-retino! was administered to male rats, retinol- derived radfo-
flrti c^eluted ! w ith the protein traction in cytosol containing
-------
hyaline
droplets
Tubule Lumen
Alphas-globulin + GIGA
Blood
Proximal (P2) Tubule Cell
Figure 3: Schematic representation of the uptake and fate of alphas-globulin
complexed with a CIGA in hydrocarbon nephrotoxicity.
Source: Modification of Figure 2, above.
25
-------
producing hyaline droplet accumulation.
delated SSSwcKSS. but rather an increase, in rat kidney lysosomal
proteolytic enzyme activity.
/ structure-Activity Relationships for CIGA
^
Thowever, restricts the present utility of
and92^
function for isophorone.
Another recent study employed a quantitative approach to determine
Sln^SbSto frSude cycloaliphatics by substituting the requ.rement
or an isopentyl structure with a requirement for the presence of at leas one
tertte JSSton atom. Usihgthis binding site model, Bomhard et ai. pred^ed
the hyaline-droplet inducing activity of 18 prev.ousy untested ' hydrocar-
bon ?rS^ Tchemicals were then testec Tfor ^toi^^g'wclrgrt
accumulation in adult male Wistar rats. ^nthWh*«^^rjB^ model
was based on the structure of the parent compound and did not alow for
active metabolites, the results in the rats were described as being in good
agreement with the predictions.
26
-------
Borghoff et al. (1991) determined the apparent binding affinity to OL -g
for a number of chemicals associated with a,u-g-nephropathy and measured
their ability to compete with TMPOH. Using molecular modeling and
information on the most active compounds, these investigators concluded
that the presence of an electronegative atom for hydrogen bonding is a
critical factor in determining binding affinity. Lipophilicity also seemed
crucial for hydrophobic interactions, but the presence of an electronegative
atom was necessary for greater activity. Steric volume was also considered
to play an essential role in binding activity.
The conclusions of Borghoff et al. (1991) are consistent with the notion
that 0,,,-g is capable of transporting lipophilic compounds within a binding
site pocket of specific dimensions. Since binding affinity does not correlate
well with hyaline droplet formation, however, the ability of this structural
feature to serve as a predictive tool would appear limited.
IV. A!pna2U-globu!in Nephropathy
Substances reported to induce increased formation of hyaline droplets
in proximal tubule cells of male rats are listed in Table A-1, along with
available information on whether the accumulating protein is a -g. The
nephrotoxicity that can ensue from hyaline droplet accumulation is novel
because it is associated with excessive o^-g accumulation. This a -g
accumulation is believed to initiate a sequence of events resulting in chronic
proliferation of tubule epithelium, as well as an exacerbation of CRN.
:• Because a2u-g is a male rat-specific protein, nephropathy induced by
.accumulation of a2u-g would not be expected to occur in female rats, mice
of either sex, or other species.
; The proposed sequence of histopathological changes is based mainly
on research studies with four model substances, unleaded gasoline and
TMP (Short et al., 1986; 1987; 1989a), decalin (Aiden et al., 1984; Kanerva
etal., 1987a,b,c; Stone etal., 1987),andd-iimonene(Kanervaetal., 1987b;
Webbet al., 1989). For even these four substances, not all of the individual
lesions in the proposed progression have been shown to belong to a
sequence of interrelated events. Specific information pertaining to lesion
nature and sequence is lacking for many of the hyaline-droplet inducers
listed in Table A-1.
Much of the information useful for defining the pathologic sequelae to
a^-g accumulation does not require chronic exposure. Accumulation of
oc2u-g is visible within a matter of days and the response to chronic
, administration of GIGA might even diminish since oc2u-g levels decline in
aging male rats (Murty et al., 1988). The nephrotoxicity associated with
(X,u-g accumulation might also be influenced by age. Certainly, the age-
related progression of CPN obscures the lesions directly related to CIGA
administration, making evaluation of the chronic sequence of lesions
especially difficult.
A. Pathologic Features of Alpha2u-globulin Nephropathy
Renal lesions described in scientific studies as being associated with
0,,,-g nephropathy are listed in Table 3. The first morphological manifesta-
27
-------
Tables.
Summ,
Alpha^
ayofthe Histopathology and Lesion Progression Reported in
Globulin-Associated Nephrotoxicity
1. Excessive
region of ki
exposure cease
accumulation of hyaline droplets in the P2 segment of the proximal tubule
" -"" or 2 days. This is reversible within 3 days to 2 weeks after
C.Avv7Oul VO C»WN*MI 11**I**M**» • f • •,
region of kidney accurs after 1
exposure cease 5.
Evidence of single-cell necrosis in the P2 segment epithelium and exfoliation afterSdays
of continuous e> posure
3. Accumulation _. 0
dilation, at the ju notion of the
days of continuous exposure
commencing ex >osure
ofi qranular casts formed from the cellular debris and subsequent tubule
" ' 5 P3 segment and the thin loop of Henle, following 20 to 40
„„ ». Granular casts have been observed at 3 to 1:3 weeks after
and sometimes beyond, up to 2 years.
increase .n ce.i proliferation within the P2 segment following 3 weeks of continuous
exposure, rema ning elevated above normal at 48 weeks of exposure.
Linear mineralization of tubules within the renal papilla, appearing between 3 and 12
months after a 28-day exposure, and sometimes observed at the end of a 2 year stuay.
Hyperplasia of he renal pelvic epithelial lining observed around 1 year. •
Exacerbation of the spontaneous chronic progressive nephropathy syndrome common
in aging rats. *
Formation of occasional hyperplastic foci within cortical epithelium atchronictime^points.
Indirect conseque ice of progression of lesions.
tion of o .
proximal tubule
compounds (Webb
nephropathy is the rapid accumulation of hyaline droplets in
cells, developing within 24 hours of dosing with some
etal., 1989).
The droplets
negative for periodic
et al., 1984). "
stain positively with Mallory's Heidenhain stain but are
die acid Schiff, indicating their protein composition (Alden
allory's Heidenhain stain is therefore more useful than
conventional hematoxylin and eosin (H & E) for visualizing and quantitating
hey represent lysosome-derived entities, the droplets are
•escent (yellow) in paraffin sections under ultraviolet illumi-
3d observations, G.C. Hard). In plastic-embedded tissue,
:an be visualized easily with Lee's methyiene blue basic
al., 1986).
the droplets. As
strongly autofluo _.
nation (unpublished observations
hyaline droplets — — -•'-—«—
fuschin (Short et
Excessive
segment,
seen in the P1
hyal
line droplet formation occurs primarily in cells of the P2
but smkll increases in the number of hyaline droplets may also be
and P3 cells (Short et al., 1987). By light microscopic
immunohistoche -nistry, oc2u-g has been clearly and'specif ically localized to
the hyalinedroplotswrthinproximaltubules (Burnettetal., 1989). Ultraslruo
•..—n.. 4u~ h.,«itT,e droplets are enlarged secondary lysosomes partially
g (Garg et al., 1989a). Many are polyangular or irregular
ig a condensed crystalline core suggestive of aggregated
i. Although the a2u-g-associated hyaline droplet accumu-
turally, the hyali
composed of
in shape, containin
protein in pure form
28
-------
lation persists during chronic exposure, the severity apjpears to
increasing duration of exposure (Short et al., 1989a).
of the response with continued exposure could be
«2U-g production by the male rat beginning at some
age (Roy et al., 1983; Motwani et al., 1984; Richardson
lessen with
Tjiis apparent waning
elated to declining
months of
etal.,1987).
stage after 5
With continued exposure, the initial accumulation
hyaline droplets may be followed by a sequence of inte
events, (f) Scattered single-cell necrosis occurs pre
segment cells (Short et al., 1987) with subsequent
of o,u-g-containing
related pathological
ominantly in the P2
exfoliation of these
w^,.. .v... wi.w \viissil \*l Ul., ItJUlf Kllll OUUO^V^UOI II CAIUIICUIUil Wi U IC?Ot?
degenerate cells and cell fragments laden with crystalloid phagolysosomes
InT^s "frr*!^ ti ir\i il A In ttnnf\s^ lA/!^(*» ^Is^^^liv^ *^ ••*•*! V^IB>W « S «J.n «M •».**. >•* - *.£ .•*._. IB ^s «..>____._ A? »
into the tubule lumen. With decalin, a minimal degree
necrosis was reported to be present in the proximal con
5 days of exposure, becoming maximal at 19 days,
minimal level after 31 days of exposure (Kanerva et al.
exfoliation of droplet-affected cells was observed witt
exposure to unleaded gasoline or TMP (Short et al.
sustained single-cell loss while exposure to CIGA con
(2) Epithelial cell proliferation primarily involving
curs as a regenerative response to cell damage and los
as increased numbers of mitotic figures or demon
>f cell degeneration/
'oluted tubules after
but reverting to the
1987a). Scattered
up to 48 weeks of
1989a), indicating
inues.
he P2 segment oc-
s. This can be seen
itrated by labeling
techniques for DNA-synthetic activity. Increased prolrferative activity has
been recorded after only three weeks of petroleum hydrocarbon exposure
(Short et al., 1987) but it persisted during 48 weeks of chronic exposure
(Short etal.,1989a).
. (3) GranularcastscomposedofsloughedceHdebfisaccumulateatthe
junction between the P3 segment of the proximal tubule and the descending
thin loop of Henle, that is, at the junction between the inraerand outer stripes
of outer medulla, with consequent tubule dilation at this
(Alden et al., 1984). This can occur as early as two to thr 3e weeks after initial
exposure (Alden et al., 1984; Kanerva et al., 1987a). A
recognizable cell debris, the granular casts stain pos
> well as comprising
ively for o^-g (per-
sonal communication, J.R. Foster, Central Toxicology Laboratory, ICI,
Macclesfield, to G. Hard, November1990) indicating p
the debris from cells which had accumulated this pro
formation appears to be associated with higher doses o
than with the lowest doses that can induce increa
accumulation. An absence of casts after treatment m
a dose-related decrease in the severity of cell necr
(Short etal., 1986,1987).
(4) At chronic timepoints, linear mineralization dT
papilla, outlining affected medullary tubules, along with
pelvic epithelial lining (urotheHum) (Alden, 1989). With
this lesion was first observed at 6 months of exposure (
mineralization appears to form within the loops of H
identified as calcium hydroxyapatite (Trump et al., I984|a)
between papillary mineralization and the proximal tubule
undetermined but the medullary lesion is presumed to
obable derivation of
ein. Granular cast
a compound rather
ed hyaline droplet
ght therefore reflect
sis and exfoliation
29
part of the nephron
velops in the renal
hyperplasia of the
unleaded gasoline,
JSEPA.1987). The
nle and has been
The relationship
lesion remains
represent mineral-
-------
ized remnants of debris from disintegrating.granular casts; thailodge in the
prebend segments of the loops of Henle (Bruner, 1984; Alden 1989)v£
turn urothelial hyperplasia, which mainly affects the surface of the renal
papilla may be a response of the renal pelvis lining to pap.llary mineraliza-
tion (Bruner, 1984; Alden and Frith, 1991).
e. Rat Urine Chemistry and C!GA ;
Several studies have examined renal function in rats treated with GIGA
and subsequently developing «2u-g nephropathy. Two^days of reatment
with TMP resulted in mild urinary increase in the lysosomarenzyme
N^acetyl-B-g^cosaminidase (NAG) and alkaline phosphatase, a decrease
£ creatininl and mild increase in urinary cell debris. Other parameters
aspartale aminotransferase (AAT), urine osmolality and volume werenot
affected (Fowlie et al., 1987). A single, oral dose of TMP had ™ .effect on
renal function (Stonard et al,, 1986). In a 14-day study with decal.r^of s x
urinary enzymes tested, only AAT, lactate dehydrogenase, and NAG were
altered (increases) at days 21 and/or 28 (Evans and Morgan, 1986). Similar
Ss werobtaned for levamisole except that AAT remained normal
Hans et 1,1988). During prolonged treatment with eye,, isoparafte
solvent up o 8 weeks, the only urinary changes observed were mild
l?Sns of glucose and albumin, slightly ^creased concentrating power
and osmolality, and epithelial cell debris in the urine. There was no alteration
in urinary p2-microglobulin content (Phillips and Egan, 1984).
Taken together, these studies suggest that CIGA produce minimal
changes in urinary chemistry and very little or no glomerular dysfunction or
damage. The minor alterations seen in urine composition in the days
following administration of CIGA suggest also that hyaline droplet accumu-
lation is not related to increased passage of serum proteins ^ the gbmeru-
lus The mild tubule toxicity identified by clinical chemistry is a characteristic
of CIGA which contrasts with the obvious urinary changes associated witn
the nephrotoxicity induced by such classical renal toxins as -mercuric
chloride, hexachlorobutadiene, aminoglycosides, and papillotoxic agents
(Stonard, 1987).
C. Species Variation in the Renal Response to CIGA
The male-specific effects of hyaline-droplet inducers have been demon-
strated over a range of rat strains including F344, Sprague-Dawley, Buffalo,
and Brown Norway rats (Ridder et al., 1990). Hyaline droplet accumulation
or the spectrum of lesions comprising a2u-g nephropathy have not been
observed in female rats or mice of either sex, following treatment with hese
chemicals (Alden et al., 1984; Swenberg et al., 1989). In addition to these
studies, other hyaline-droplet inducers have been tested or toxicity in
hamsters (jet fuels), guinea pigs (decalin), dogs (decalm, jet, fuels
d-limonene, and methyl isobutyl ketone), and monkeys (gasoline and
methyl isobutyl ketone). No renal pathology was demonstrated in these
species at doses known to cause nephropathy in male rats (Alden et al.,
1984- Kuna and Ulrich, 1984; MacFarland, 1984; MacNaughton and Uddm,
1984: Phillips et al., 1987) except for one report of minor changes in.dogs
30
-------
treated for 6 months with d-Iimonene (Tsuji et al., 1975). In this chronic
study, an increased incidence of proteinaceous casts was observed in male
and female beagles, but no tubule epithelium changes, tubule lumen
dilation, or mineralization was observed. However, Webb et al. (1990) were
unable to demonstrate any renal pathology in dogs after 6 months of
d-limonene treatment at comparable dose levels. The highest dosage
tested in the dogs, 1.2 mg/kg (Webb et al., 1990; Tsuji et al., 1975), is more
than ten times the doses that have caused frank nephropathy in male rats.
f '
Knowledge concerning renal effects of GIGA in humans is hampered by
the lack of data on specific chemicals in this category, and the limitations
imposed by a multiplicity of types of occupational and non-occupational
exposures. Case studies have reported a link between chronic renal
disease with gasoline, solvents, and jet and diesel fuels including rare cases
of acute tubular necrosis (proximal and distal tubule epithelium) following
severe exposure to petroleum distillates (e.g., Barrientos et al., 1977; Crisp
et al., 1979). Case reports cannot be used to establish a causal relationship
but may serve to initiate formal epidemiologic investigation (Churchill et al.,
1983).
Epidemiologic studies concerning non-neoplastic kidney disease and
occupational exposure to hydrocarbons and solvents have been conducted
only since 1975 (reviewed by Askergren, 1986; Daniell etal., 1988; Phillips
et al., 1988). A majority of these studies have indicated an association
between glomerulonephritis and exposure to hydrocarbons, especially
organic solvents or gasoline. Some have suggested a positive association
between the presence of glomeruiar disease and duration and severity of
occupational exposure to hydrocarbon solvents, including tetrachloroethylene
which is a CIGA in male rats (Kluwe et al., 1984). However, many of the
earlier studies are considered to be methodologically limited (Churchill et al.,
1983; Askergren, 1986; Phillips et al., 1988). Major shortcomings have been
heterogeneous case definition, use of inappropriate control groups or
nonblinded interviewers, and failure to consider recall bias or to adequately
define hydrocarbon exposure (Phillips et al., 1988).
More recently, Steenland et al. (1990), investigating specific occupa-
tional exposures associated with end-stage renal disease in male workers,
found elevated risks for solvents used as cleaning agents or degreasers
(odds ratio (OR) 2.5; 95% confidence interval (Cl) 1.56-3.95) but not for
exposure to gasoline and diesel fuel (OR 0.98; 95% Cl 0.49-1.06) or motor
and fuel oi! (OR 1.13; 95% Cl 0.69-1.84). Harrington et al. (1989) found no
association (OR 1.0; 95% Ci 0.16-6.3) between occupational exposure to
inorganic solvents and glomerulonephritis, but the authors also concluded
that the statistical power of this case-referent study was not sufficient to
detect other than large risk estimates.
The glomerulonephritis reported in the positive epidemiologic studies
has involved thickening of glomeruiar basement membranes or deposition
of antibodies against glomeruiar basement membrane, a mild degree of
albuminuria, and sometimes tubule atrophy and tubular basement mem-
brane thickening (Kluwe et al., 1984; Phillips et al., 1988).
31
-------
Other indicators of renal function have also been assessed in
epidemiologic studies. Levamisole, a drug used as an antihelminthic, in
cancer chemotherapy, and in the treatment of rheumatoid arthritis in
humans, falls intojthe CIGAcategory because it induces both hyaline droplet
and a -g accumulation in male rats (Read et al., 1988). Based on an
absence of elevated levels of urinary NAG in patients receiving 150 mg
levamisole per dcky for 26 weeks, there is little evidence to indicate that this
compound is nephrotoxic in humans (Dieppe et al., 1978). In addition, no
positive association between urinary NAG and acute or chronic exposure
was noted in a prevalence study of 180 dry-cleaning workers exposed to
tetrachloroethylene (Solet and Robins, 1991). Since urinary NAG is only
slightly elevated iin male rats exposed to CIGA, however, urine chemistry
may not be a goo^J biological monitor of the type of nephrotoxicity associated
with CIGA. ! ;
In a study ojf 16 females exposed to tetrachloroethylene from their
employment in dry-cleaning shops for an average of 11 years (range 1 to 25
years) Vyskocil et al. (1990) found no evidence of renal damage except for
an increase in ly$ozyme in the urine. No statistically significant increase in
urinary excretiod of p2-microglobulin, lactate dehydrogenase, or glucose,
which are other markers of tubular dysfunction, were noted. The authors
believe these latter findings, in addition to the lack of correlation.between
intensity of exposure and change in biochemical parameters, support the
conclusion that j-enal damage is not associated with tetrachloroethylene
exposure. i
The evidence regarding renal injury in humans from chronic organic
chemical exposure is inadequate to demonstrate whether or not CIGA
exposure can affect the human renal tubule cell. Existing reports imply that,
if the associatiori is real, it is the glomerulus that is pathologically involved.
However, this may simply reflect study designs which concentrated on
clinical detection^ glomerular effects. Since the injury to the rat tubule cells
is relatively mild, insensitive tests, such as urine chemistry, which are
generally used Ifor evaluating humans might be inadequate to detect
changes.
D. Factors Affecting the Expression ofAlpha2u-gIobulln Nephmpathy
Various conditions, including age, hormone manipulation and genetics,
have the potential for altering the expression of CIGA-induced a2u-g
nephropathy. Experimental studies have investigated the influence of these
factors on CIGAJnephrotoxicity as well as determining the effects of a2u-g in
female rats. ;
1. Age-related ipffects
As discussed earlier, the hepatic synthesis and urinary excretion of
a, -g in the male rat are highly age-dependent, with prepubertal and aged
animals showing negligible amounts of this protein (Neuhaus .and Flory,
1978; Royetal.,>1983; Richardson etal., 1987). Accordingly, administration
of either decalirji to immature male rats (Alden et al., 1984) or unleaded
32
-------
gasoline to aged, 26-month old, male rats (Murty et al., 1988) failed to
produce renal cortical a2u-g accumulation or an increas^ in hyaline droplets.
2. Effect of hormone manipulation ' • • • •
As a2u-g synthesis is primarily under androgenic control, the effects of
castration, which depresses hepatic synthesis of a2 -g:(Roy and Neuhaus,
1967), were explored by Hobson et al. (1986) using IMP. Although a
significant increase in hyaline droplet formation wais observed in both
castrated and uncastrated male F344 rats exposed to ja single oral dose of
TMP, the severity of the lesion was less in the former. Thus, castration
diminished but did not abolish the TMP-induced nephj-otoxicity.
'. Estrogen is known to inhibit the hepatic synthesis of a,,u-g in the rat (Roy
et al., 1975). Garg and coworkers (1988) used estradiol administration to
study the influence of inhibition of new synthesis of a2j-g on recovery from
CIGA-induced renal tubule changes. Commencing treatment on the ninth
and final day of unleaded gasoline exposure, estradiol reduced renal cortical
aj,u-g content by 25,41, and 52 percent on post-exposure days 3,6, and 9,
respectively, compared to rats receiving no hormone treatment. At the same
time, hyaline droplet removal appeared to be accelerated in rats treated
conjointly with hormones. Hyaline droplet number a|nd'size (qualitative
observations) in hormone-treated rats approached corjitrol levels at 3 days
.post-exposure, compared with up to 9 days for corbplete resolution in
unleaded gasoline-exposed rats not receiving estradiol.
i
In a subsequent study, Garg et al. (1989b) demonstrated that pretreat-
ment of mature male rats with subcutaneous injections of estradiol for 10
days before gasoline exposure completely inhibited thej renal accumulation
of a2u-g and hyaline droplets normally induced by gasoline.
3. Genetic variants
The NBR rat has no detectable levels of hepatic o^ -g mRNA in either
sex and, therefore, is unable to synthesize a,u-g in thdi liver although high
constitutive levels of the mRNA are present in the preputjat gland (Chatterjee
et al., 1989). The NBR rat is capable of developing (chemically induced
nephropathies, but under exposureconditionsthat produce o^ -gnephropathy
in F344 rats, d-limonene, TMP, isophorone, and 1,4-DC^ did not induce any
detectable cc2u-g accumulation, hyaline droplets or other lesions in the male
NBR rat (Dietrich and Swenberg, 1991 a). Identical results were obtained for
decalin (Ridder et ai., 1990) and lindane (Dietrich an|d Swenberg, 1990,
1991a). | .
4. Alphas-globulin infusion in female rats
Ridder et al. (1990) intraperitoneally administered) o,u-g (purified from
mature male rat urine) at hourly intervals to decalin-treated female Sprague-
Dawley rats for a total of 8 injections and examined jkidney samples for
hyaline droplets and oc2 -g one hour after the last protein injection (9 hours
after decalin treatment). Although droplet formation'was not evident in
kidney sections from the oc2u-g-infused female rats stained with Mallory's
33
-------
Heidenhain, hyalirJe droplet and a, -g accumulation were clearly demon-
strated in females exposed to both hydrocarbon and male urinary protein.
By means of two-dimensional gel electrophoresis, the investigators showed
slight, but apparent, renal cortical accumulation of 0,,,-g in the infused
females. Accumulation of the protein greatly increased in females that were
both infused with cL-9 and decalin-treated.
These various
renal lesion exprei
studies indicate a direct dependence of CIGA-induced
sion on the presence of cc2u-g.
E. Chronic Progressive Nephropaffiy
Rats are particularly predisposed to an age-related spontaneous
nephropathy, CRN, that is more severe in males than in females and that
affects certain stra ns more than others. CPN is more common in Sprague-
Dawley and F344 fats than in the Wistar strain (Gray, 1986), and it is also
common in the Osborne-Mendel rat (Goodman et al., 1980). The etiology
of CPN is not known but the severity of the syndrome is influenced by a
number of factors, particularly dietary manipulation affecting protein content
or caloric intake (tylasoro and Yu, 1989).
Exacerbated 4pN, involving enhanced severity and earlier onset of the
disease, is generally observed after chronic administration of GIGA to male
rats (Trump et al., I984a). It has been stated that exacerbated CPN is one
component (together with hyaline droplet accumulation and granular cast
formation) of a triad of lesions that specifies the nephropathic response to
GIGA (Kanerva et al., 1987a; Webb et al., 1989). Exacerbated CPN is
usually recognizeq after months of continuous treatment (Trump et al.,
1984a; Short et al.11989a) although Alden et al. (1984) reported early signs
after 2 to 3 weeks with decalin. These authors (Alden et al., 1984) consider
that exacerbated OPN develops as a tertiary response to nephron obstruc-
tion caused by the] GIGA-induced granular casts.
The pathologic features of CPN (listed in Table 4) include certain lesions
that are also found in o,u-g nephropathy, as well as lesions that are
distinctive. Single-pell necrosis, regenerating tubules, andfocal hyperplasia
of proximal tubule epithelium are common to spontaneous CPN and to
a, -g nephropathy i(UAREP, 1983). CPN is characterized by certain lesions
that are not components of cc2u-g nephropathy, including conspicuous
thickening of tubule and glomerular basement membranes, hyaline casts
consisting of home geneous, proteinaceous material (distinct from granular
casts containing cellular debris), interstitial mononuclear cell infiltration,
fibrosis, tubule atrophy and sclerotic glomeruli. Conversely, early and late
stages of a -g nephropathy exhibit a number of characteristics unlike CPN,
such as hyaline droplet accumulation associated with a2u-g in the P2
segment, granular casts at the "corticomedullary" junction, and linear
mineralization in the papilla (Trump et al., 1984a). In very advanced cases
of spontaneous CPN, sporadic tubules may contain excessive numbers of
hyaline droplets similar in appearance to those induced by GIGA. However,
these do not show immunochemical evidence of o,,u-g (unpublished obser-
vations, G.C. Hard). The urine and serum chemistry of advanced CPN also
34
-------
Table 4. Summary of ffre Histopafootogy of Spontaneous Chror,io Progressive
;; Naphropathy of Aging Rats
7.
8.
9.
indicative of tubule cell
1: Thickening of tubular and glomerular basement membranes.
''•'•}• •
2. Basophilic segments of proximal tubules with sporadic mitoses
proliferation.
3. Tubular hyaline casts of proteinaceous material originating in the hnore distal portion of the
hephron, mainly in the medulla, and later plugging a considerable length of the tubule.
4. Focal interstitial aggregations of mononuclear inflammatory cells within areas of affected
tubules. !
5, Glomerular hyalinization and sclerosis. j
. - , • '
6. Interstitial fibrosis and scarring.
Tubular atrophy involving segments of proximal tubules.
Chronically in advanced cases, occasional hyperplastic foci in Affected tubules.
In some advanced cases, accumulation of protein droplets in sporadic proximal tubules.
f Chemicals That
differs from o,u-g nephropathy. Albuminuria, hypojalbuminemia, and
hypochoiesterolemia typify CPN, with increases in serum creatinine and
urea nitrogen levels in end-stage disease (Barthold, 1979).
F. . Renal Toxicity Observed In Chronic Bi
' , Ihdufced Kklhey Tianois In Rats '
For the purpose of the current review, bioassays were identified and
data were examined on seven chemicals tested for chronic toxicity and
carclnogenicity by the National Toxicology Program (NFP) or the National
Cancer Institute (NCI). All seven produced accumulation of hyaline drop-
lets, nephropathy, and kidney tumors in male rats. These model compounds
are d-limonene, dimethyl methylphosphonate, hexachlo oethane, 1 ,4-DCB,
tetrachloroethylene, pentachloroethane, and isophofon 3. Information was
also examined on unleaded gasoline (tested by inhalation as a totally
vaporized form at the I niernational Research and Development Corporation
[IRDG] for the American Petroleum Institute). Gasoline is a complex blend
with CIGA properties (MacFarland et at., 1984). Data on the two non-CIGA,
trichloroethylene and chlorothalonil, are included f or corr parative purposes.
Although extensive acute and subchronic studies have l>een performed on
twootherchemicals (decalin andTMP), both of which cause a2u-g nephropathy
in the male rat, carcinogenicity bioassay data are not available for these
compounds.
i
3 'Several of these seven chemicals cannot be described as true "Cl GA carcinogens" since
'tine accumulating protein in the hyaline droplets has not been conf rmed to be a^-g.
Their use as model compounds for purposes of developing the discussion on cancer
should not be construed to mean that all seven chemicals fit the Policy Statement
. developed in Part IV of this document. T
35
-------
Trichtoroethylene, which was tested by the NTP, induces kidney tumors
in male rats only (NTP, 1988) but does not cause an accumulation of hyaline
droplets or an increase in a, -g levels (Goldsworthy et al., T988a). There is
also some evidence that tnchloroethylene metabolites bind covalently to
renal macromolecules (Bruckner et al., 1989). Consequently, this com-
pound would not be considered a CIGA.
Chlorothalonil, af ungicide tested on separate occasions by industry and
a Government agency, induced renal tubule tumors in male and female rats
arid in male mice (NCI, 1978a). It also induced hyaline droplet accumulation
fn proximal convoluted tubules of male rats (USEPA, 1988), but hese may
not become apparent during the first few weeks of treatment (Killeen et al.,
1990) Electron microscopic studies of male rat kidney following subchronic
Chlorothalonil exposure revealed angular membrane-bound lysosomes
containing crystalline structuressimilar to those observed in a, -gnephropathy
(written communication, William M. Busey and James C. Killeen, Experi-
mental Pathology Laboratories, Inc., to Office of-Pesticides Programs
1988) However, a, -g has not been detected in the renal tubules of
chlorothalonil-exposed rats (Swenberg, 1989). The progression o
Chlorothalonil nephrotoxicity involves initially, vacuolar degeneration of
Droximal tubule epitheliumfollowed4weekslaterbytubuleceli hypertrophy,
hvperplasia, and tubule dilation (Killeen et al., 1990). Therefore, this
compound appears not to produce the same spectrum or sequence of
lesions induced by CIGA. Furthermore, Chlorothalonil has been shown to
interact with cellular macromolecules including histones and thiol proteins,
possibly through covalent binding of a metabolite with sulfhydryl groups
(Rosanoff and Siegel, 1981). Chlorothalonil also induces overt renal
dysfunction in both sexes of rats. At doses from 40 mg/kg/day,. blood urea
nitroaen and creatinine were increased while circulating levels of glucose
and albumin were decreased (USEPA, 1988). For these various reasons,
Chlorothalonil is not considered a member of the CIGA class.
A summary of the non-neoplastic and preneoplastic kidney effects
observed in male rats after administration of the eight substances selected
as possible CIGA is presented in Table 5.
Non-neoplastic and preneoplastic lesions reported in female rats sind
mice of both sexes are summarized in Table 6. The data in these two Tables
were extracted from the NTP Technical Reports (see Table A-2 in the
Appendix) and other relevant literature.
In male rats, at least one case of renal tubule cell hyperplasia was
reported in the 2-year bioassays for the seven renal carcinogens tested by
the NTP The incidence was generally much higher and dose responsive.
Although not reported in the IRDC bioassay, this lesion was observed in later
research studies of unleaded gasoline (Short et al., 1989b). None of the
eight bioassayed substances produced tubule cell hyperplasia in female
rats although this lesion was reported in male mice exposed to
tetrachloroethylene. In male rats, renal changes described as "toxic tubular
nephropathy" (encompassing degeneration of tubule epithelium, necrosis,
36
-------
3 « Q
"i S 1
to J2 ^*
•S »
in —
•0 £.
1 8. «=
Q. >. £
1 x I
O 0.
"*"* O "••
m"~* *• 5^
CO w
£ * £
"i
TJ m
•2 JL **
c
•J3 fg W O
u o O w
SI
ir "S
. S> 7:
03 p^ ij
5 S «
2 £
.2
(O
. J3
1- 1
1 ||
'S u.
"a.
§
1 it
C ^* t" S
ca •£ —
•S 8. „
w S. O -S)
— a £ o> c
s-s I- s a
| § =• Q S
2! *^ O
O i fli W
CM *•• *^
(rt C Q}
Q "e >, o"
*g o i -a
E
CG fr^
ll
I
® E
S $
19 -C
j2 o
+ + + + -.-i* c
+ +-•«•+• + + + -^.
cc cc £cc CCDC+<
z z zz zz ^f
«!• -,.^
4- 4- 4-5 + + 2 "*"
+ + + i + + i +
+ + ++ + + .+ +
f*
+• + ++.SP + 55 +
0) '^ *-
+ + + • + + + +
+ + + + + + + +
.2
eg i
_^ CO 5
S J Q Q3 i 2 g
yjiif iiifp
m
= ""5
« ™
co *-r
+ Positive
Negative
NR Not reported
a Localized to rei
D Data from rese
37
-------
U]
s
CO
O
f
I
-a
I
•g
CO
CO
CO
"5.
Q.
. X
'co
0)
o
CO
JO
ra
MH
2
01
c
o
11
o
u.
j;
0 §_
o 2
r"" "5?
a>
Z
CD S
e o
"5 S-
>•«
Chemical
1
CE
Z
CO-
S"
CO
1 -~
1tO
J)
1
c
CO
"to '
CD
co
E
CD
1 ,4-Dichlorobenzen
8
•••'•-, , , , E
' . . : . &
' " ' .- ~
tr cc , cc a: 5 +.2
2: z z z . z T «
: ig.
•I
8'§
•— Q. II'1
. ' £ 0>
CO
9.
+sl , , , +S
1 + '~ —3 1 1 * f •— ;
^ CO £
-C
«
S" 2?
= • +•!'*= • • • z
) 2 ^3
j m c
r vffi ™
, ' g S i
1 ~S TO 1^
>• 2 jc 5-
££> ^E ^5 4g
CD CO g _ tD O S
. E g 8 g c b 2
I! 1 111!
.£ Q. CD 91®.®
Q I J2 T3 O. H-
a
0
2
CD
"CO
CD
g>
1 ®
II
$
a
+ Posi
Neg
NP Not reported
Ca Calcium
en
Inciden
Highes
a
b
38
-------
following admin-
ie aspect of toxic
ice administered
6). For example,
administration of
st formation was
loroethane and
epithelial cell regeneration, and cast formation) were see
istration of all eight of the renal carcinogens (Table 6). So
tubular nephropathy was also observed in female rats or
hexachloroethane, 1,4-DCB, ortetrachloroethylene (Tabl
calcium deposition or mineralization was seen after
hexachloroethane to mice or 1,4-DCB to female rats. C
reported in mice following administration of hexac
tetrachloroethylene.
Several difficulties arise in the interpretation and utilization of the
bioassay-derived data when mouse and female rat lesioras are considered.
The nature of casts (granular vs. hyaline) is not always described, and for
mineral deposits, the site (papillary vs. corticomedullary) and form (linear
vs. globular) may not be specified. The range of lesions encompassed by
the term "toxic nephropathy" is not always defined, and there is sometimes
no clear distinction from CPN. Nevertheless, it appears prom the data that
female rats and mice do not develop as broad a spectrum of nephrotoxic
lesions as those proposed to be associated with a2u-g nephropathy and
renal tumor formation in the male rat. Furthermore, whore nephrotoxicity
was reported in both male and female rats, the males had more lesions and
the female response never demonstrated the characteristics seen in the
male response to CIGA. Therefore, the lesions caused bjr CIGA seemto be
both qualitatively and quantitatively different for male rats
and female rats.
compared to mice.
39
-------
-------
Part 2. Carol nogenlcSty
The second major part of this document compares and contrasts
information on kidney tumors induced by classical renal carcinogens with
information from the NTP (or NCI) assays for renal neoplasia induced by
chemicals that produced hyaline droplets and/or accumulation of a,u-g. In
addition, other information, such as mutagenic activity and tumor-promoting
ability, which help to define a GIGA carcinogen or point to possible mecha-
nism of action, are evaluated.
Epidemiological studies of human renal cell cancer are reviewed for
consistency with the hypothesis that GIGA-induced renal cancer in male rats
is an inappropriate endpoint for assessing human risk. Implicit in this
evaluation is a presumption of male rat-to-human tumor site concordance,
a supposition EPA generally does not make. In this special case, however,
the hypothesized mechanism being examined depends on the accumula-
tion of low-molecular-weight protein in the renal tubule, regardless of
species. Hence, the predicted target site for cancer in hu mans, as in the rats,
would be the renal tubule.
V. Pathologic Features of Renal Carcinogenesis induced by Classical
Carcinogens
Among the many chemicals recognized as inducers of rodent cancer,
several have been used as model kidney carcinogens for studying the
pathogenesis of renal tubule tumors in rats. These are dimethylnitrosamine
(DMN), diethylnitrosamine (DEN), N-nitrosomorpholine, N-ethyl-N-
hydroxyethylnitrosamine (EHEN), lead acetate, N-(4-fluoro-4-
biphenylyl)acetamide (FBPA), and aflatoxin B, (Hard, 1990). In the mouse,
certain nitrosamines, streptozotocin, and ochratoxin A are strong inducers
of renal tubule tumors, while the classical renal carcinogen in hamsters is
diethylstilbestrol (Hard, 1987). In general, these prototypic renal carcino-
gens are active in both males and females.
Studies on the pathogenesis of renal tubule tumor formation using
model carcinogens in rats demonstrate that a continuum of chemically
induced steps leads from atypical hyperplasia in tubules (also termed
hyperplastic tubules, tubule dysplasia, and atypical cell foci) through micro-
scopic adenomas, to macroscopic adenocarcinomas or carcinomas (Hard,
1987; Lipsky and Trump, 1988).
In addition, there are invariably pathologic changes which precede the
proliferative sequence of preneoplastic and neoplastic lesions including a
41
-------
period of early nephrotoxicity and, often, karyomegaly. These various
lesions are described below in chronological sequence.
A. Early Nephrotoxicity
Acute toxic changes occur in the proximal tubules shortly after the
administration of classical renal carcinogens. They include rriifcl lipid droplet
accumulation and scattered single-cell necrosis (Hard, 1987). Depending
on the carcinogen used, this early damage can be observed in different
segments of the renal tubule. For instance, with DMN it is localized to the
P2 segment (Hard et al., 1984) and with FBPA, to the P3 segment (Dees et
al., 1980a,b).
Detailed histological and/or ultrastructural observation shows that hya-
line droplet accumulation is not induced by DMN (Hard and Butler, 1971;
Hard etal., 1984) or DEN (G.C. Hard, unpublished observations); nor has
it been described in studies using other carcinogens, such as FBPA (Dees
et al., 1980a,b), as models for renal carcinogenesis.
More is known about DMN than other classical renal carcinogens
concerning molecular interactions during the time that acute toxic changes
are seen in the proximal tubules. DNA adduct formation in rat renal tissue
occurs rapidly following a single administration of DMN. 06-Methylguanine
formed in the renal cortex (Fan et al., 1989) persists at least 4 days post-
injection (Nicoll et al., 1975), which is consistent with the notion that
methyiation of the O6 position of guanine in DNA is the most likely initiating.
event (Pegg, 1984).
B. Karyomegaly
Conspicuous nuclear enlargement, indicative of increased chromo-
some number without completion of mitosis (Jackson, 1974), may occur in
scattered proximal tubule cells during the weeks preceding development of
carcinogen-induced prolif erative foci. Although karyomegaly is produced by
many, but perhaps not all renal carcinogens, there is no evidence that these
cells participate in the initial formation of prolif erative foci. Hence karyomegaly
is not regarded as a preneoplastic lesion (Dees et al., 1980a; Hard, 1987;
Lipsky and Trump, 1988).
C. Tubule Cell Hyperplasia
Tubule cell hyperplasia leads to the appearance of tubules with prolif-
erating epithelium, usually multilayered, that partially or completely fills the
tubular lumen. Although lumenal dilation may be pronounced (sometimes
to cystic proportions), the structure of the individual tubule remains intact
with a confluent basal lamina. Affected cells may be eosinophilic, baso-
philic, or pale-staining and often with vesicular nuclei and prominent
nueleoli. Mitotic figures are variable. As a preneoplastic lesion, the
hyperplastic tubule is usually associated with some degree of cellular atypia
(dysplasia) in the form of cell pleomorphism and increased nuclear to
cytoplasmic area ratio (Hard, 1987; Lipsky and Trump, 1988). Preneoplastic
tubule hyperplasia is generally considered to be distinguishable from the
42
-------
background tubular regeneration that is a component of spontaneous CRN
(Lipsky and Trump, 1988; NTP, 1988).
D. Adenoma
Adenomas are small neoplastic foci representing epithelial cell prolif-
eration beyond the well-defined structure of individual tubules. These
lesions are solid or cystic in form and the cellular morphology and architec-
tural appearance is similarto that of adenocarcinomas, which are described
belpw, particularly the well-differentiated variants. Whereas adenomas and
hyperplastic tubules can be differentiated on the basis of finite structure, the
distinction between adenomas and adenocarcinomas/carcinomas is an
arbitrary one based primarily on size. Neoplasms in the rat kidney paren-
chyma less than approximately 0.5 cm tend to lack significant vasculariza-
tion, hemorrhage, and degeneration, although there may be single-cell
necrosis, mitosis, and cell pleomorphism (Hard, 1990).
E. Adenocarcinomas and Carcinomas
Renal tubule tumors comprise histological variants based on staining
characteristics and architectural organization. In the rat, renal tubuletumors
consist mainly of lightly basophilic, granular, and/or clear cells organized in
tubujar, lobular, solid, or papillary patterns. Glandular differentiation as
opposed.to solid sheets of cells distinguishes adenocarcinomas from
carcinomas but any clear distinction between adenocarcinomas and carci-
nom^as is often meaningless because of admixture of both well-differenti-
atedand poorly* differentiated areas within the same tumor. Increased
cellularpleomorphism tends to correlate with adecreasing degreeof tubular
differentiation and anaplastic variants occur occasionally. ,
Cells within adenocarcinomas maintain many of the light and electron
microscopic characteristics of proximal tubule epithelium, in particular,
microvilli resembling brush border, basement membrane, and cytoplasmic
vesicles, Brush border may occur inappropriately between adjacent cells,
along any cell border, or as intracellular profiles. Adenocarcinomas/
carcinomas are well vascularized and usually display areas of hemorrhage
and degeneration (UAREP, 1983; Lipsky and Trump, 1988; Hard, 1990).
F. Tumor Progression
Renal tubule tumors of the rat are slowly growing neoplasms usually
requiring about 40 weeks to become clinically palpable in most experimental
systems (Hard, 1987). They can grow to large dimensions, several
centimeters in diameter.
Unlike their spontaneously occurring human counterparts, renal tubule
tumors induced in rats by chemical carcinogens metastasize infrequently
(Lipsky and Trump, 1988). However, lifespan of the animal in chronic-
exposure regimens may be a limiting factor. Single-dose studies with DMN,
whlqti maximize the life-span following tumor initiation, have demonstrated
a link between, survival period, tumor size, and incidence of metastasis in
renal, carcinogenesis (Hard, 1984). For example, rats that survived at least
1.5 years after dosing with DMN showed a high rate of metastasis,
43
-------
approximately 50 percent, whenever epithelial tumor dimensions exceeded
2.4 cm. These data confirm the malignant potential of renal tubule tumors
induced in the rat by a classical carcinogen.
G. Site of Origin of Renal Tubule Tumors
The precise location within the nephron from which experimental renal
tubule tumors arise varies with the carcinogen, and correlates with the site
of the induced early nephrotoxicity. Thus, the P3 segment is the site of origin
for FBPA-induced tumors (Dees et al., 1980a,b), while DMN tumors arise
from the convoluted segments of proximal tubules, probably P2 (Hard,
1990). Lead-acetate and DEN-induced tumors appear to originate in both
P2 and P3 segments (Nogueira, 1987).
VI. Neoplastic and Preneoplastic Lesions Observed in the 2-Year
Bioassays
Data for all reported renal tubule tumors and tubule hyperplasia in male
rats from the 2-year bioassays on the eight model substances are summa^
rized in Table 7. For tumors occurring at sites other than the renal tubule,
only statistically significant incidences are mentioned. Table 8 provides
similar information for trichloroethylene and chlorothalonil. For eight of the
ten substances, exposure was by the oral route; for two, it was by inhalation.
The experiments were conducted over approximately a decade, which may
account for the lack of standardized terminology in describing lesions and
differences in attention paid to the possible role of chemically induced
accumulation in the male rat kidney.
In a separate set of animal bioassays conducted for the military, male
rats were exposed 6 hours/day, 5 days/week for 1 year to the synthetic
hydrocarbon missile fuels, RJ-5 and JP-10. At terminal sacrifice after 2
years, these animals had evidence of nephropathy characteristic of a2u-g
accumulation and significant increases in renal tubule tumors (9 in 65
animals exposed to RJ-5 at 150 mg/m3; 9 in 50 animals exposed to JP-10
at 562 mg/m3) (Bruner, 1 984; MacNaughton and Uddin, 1 984). In contrast,
the kidneys of female rats andfemale C57/BL6 mice similarly exposedto RJ-
5 or JP-1 0 were unaffected. Likewise, none of the animals, including male
rats, exposed to RJ-5 continuously for 90 days and held 19 additional
months before sacrifice developed renal tubule tumors.
In addition to the specific results obtained from individual bioassays,
there are considerations generic to all bioassays conducted by the NTP. For
example, the NTP positions with regard to evaluation of rare tumors and the
use of historical controls influence NTP interpretation of the evidence for
carcinogenicity of GIGA (Haseman et al., 1984). Likewise, survival rates
influence the ability to analyze information from animal bioassays. These
generic issues are explored before describing the results of individual
studies.
A. Generic Considerations
Renal tubule tumors are neoplasms with a low background incidence
in laboratory animals including the rat strains used in the chronic bioassays
44
-------
Table 7.
Chemical
Incidences of Renal Tubule Preneoplasia andNeoplasia in Rats taken from 2-
year Bioassays on Eight Model Substances
Strain
Sex
Changes
Doses (mg/kg/day)
0 150 300
1 ,4-Dichloro-
benzene
(NTP-TR-319)
(NTP, 1987a)
Gavage
Other Tumors:
Chemical
Dimethyl
rnethyl-
phdsphonate
(NtP-TR-323)
(NTP, 1987b)
., .-.. .
Gavage
Other Tumors:
Chemical
Hexachloro-
ethane
(NTP-TR-68)
(NCI, 1978b)
Gavage
F344 M Survival (%)
Hyperplasia (%)
Adenomas
Incidence
Adj. Rate (%)
Incidence
Adj. Rate (%)
c_pjnbjned
Incidence
Adj. Rate (%)
Hepatocellular tumors in mice.
Strain Sex Changes
F344 M Survival (%)
Hyperplasia (%)
Adenomas
Adepocarcinomas
Incidence
Adj. Rate (%)
77 69 43
0 2 18
0/50 0/50 1/50
00 4
1/50 3/50 7/50
3 9 26
1/50 3/50 8/50
3 9 28
Doses (mg/kg/day)
0 500 1000
56 34 19
0 16 18
None
0/50 2/50 3/49
0 .9 19
Mononuclear cell leukemia; transitional cell papillomas of the renal pelvis.
Strain Sex Changes
0
Osborne- M Survival (%) 56
Mendel Hyperplasia (%)
Adenomas
Incidence 0/20
: Adj. Rate 0
Doses (mg/kg/day)
0 212 423
65 20 1.8
Not Reported
0/18 4/37 0/29
0 11 0
Carcinoma None
Other Tumors: Hepatocellular tumors in mice.
(cont)
45
-------
Table 7. (cont)
Chemical Strain
Sex
Change®
Doses (mg/kg/day)
0 10 20
Hexachloro-
ethane
(NTP-TR-361)
(NTP, 1989)
Gavage
Other Tumors:
Chemical
Isophorone
(NTP-TR-291)
(NTP, 1986a)
Gavage
Other Tumors:
Chemical
d-Limonene
(NTP-TR-347)
(NTP, 1990)
Gavage
F344 M Survival (%)
Hyperpiasia (%)
Adenomas
. Incidence
Adj. Rate (%)
AdenocarstDomas.
Incidence
. Adj. Rate (%)
Combined
Incidence
Adj. Rate (%)
Marginal increase in pheochromocytomas in male
Strain Sex Changes
F344 M Survival (%)
Hyperpiasia (%)
Adenomas
Incidence
Adj. Rate (%)
Adenocarcinomas.
' Incidence
Adj. Rate (%)
Combined
Incidence
Adj. Rate (%)
62
4
1/50
3
0/50
0
1/50
3
rats.
.Doses
0
66
0
0/50
0
0/50
0
0/50
0
58
8
2/50
6
0/50
0
2/50
6
52
22
4/50
15
3/50
9
7/50
24
(mg/kg/day)
250
66
2
0/50
0
3/50
9
3/50
9
500
28
8
2/50
8
1/50
4
3/50
12
Preputial gland tumors in male rats; hepatocellular tumors, mesenchymal
tumors, and malignant lymphomas in male mice
Strain Sex Changes
F344 M Survival (%)
Hyperpiasia (%)
Adenomas
Incidence
Adj. Rate (%)
Adenocarcinomas
Incidence
Adj. Rate (%)
Combined
Incidence
Adj. Rate (%)
Doses
0
60
0
0/50
0
0/50
0
0/50
0
(mg/kg/day)
75
68
4
4/50
12
4/50
12
8/50
23
150
69
7
8/50
19
3/50
7
11/50
25
Other Tumors: None in mice or rats
46
-------
TmbfsT. (cont.)
Chemical Strain
Sex
Changes
Doses (mg/kg/day)
0 75
Pentachloro-
ethane
(NTP-TR-232)
. (NTP, 1983)
Gavage
Other Tumors:
Chemical
Tetrachloro-
ethylene
(NTP-TR-311)
(NTP, 1986b)
Inhalation
Other Tumors:
Mixture
Unleaded
gasoline
(USEPA, 1987)
Inhalation
F344 M Survival (%)
Hyperplasia (%)
Adenomas
Incidence
Adj. Rate {%)
Adenocara'nomas
Incidence
Adj. Rate (%)
Combined
Incidence
Adj. Rate (%) .
Hepatocellular tumors in mice.
Strain Sex Changes
F344 M Survival (%)
Hyperplasia (%)
Adenomas
Incidence
Adj. Rate (%)
Adenocarcinomas
Incidence
Adj. Rate (%)
Combined
Incidence
Adj. Rate (%)
Leukemia in rats; hepatocellular tumors in mice.
Strain Sex Changes
0
F344 M Survival {%)
Hyperplasia (%)
Adenomas
Incidence 0/49
Adj. Rate (%) 0
Carcinomas
Incidence 0/49
Adj. Rate (%) 0
Combined
Incidence 0/49
Adj. Rate (%) 0
82 68 52
0 0 2
0/50 1/49 4/50
0 3 14
1/50 1/49 0/50
2 30
1/50 2/49 4/50
2 6 14
•
Doses (ppm)
0 200 400
48 40 24
0 6 10
1/49 3/49 2/50
4 11 11
0/49 0/49 2/50
0 0 11
1/49 3/49 4/50
4 11 22
Doses (ppm)
67 292 2050
Not affected
Not reported in the
bioassay
1/59 2/56 1/45
24 2
1/59 2/56 6/45
2 4 14
1/59 5/56 7/45
29 16
Other Tumors: Hepatocellular tumors in female mice.
47
-------
TableB. Incidan
year Bit
Chemical Stn
Chlorotha- Osbo
lonil Men
(NTP-TR-41)
(NCI, 1978a)
Diet
Other Tumors: noi
Chemical . S
Trichloro- Os
ethylene N
(NTP-TR-273)
(NTP.1988)
Gavage
Tumors in Other Str
Other Tumors: Ma
fer
:es of Renal Tubule PisnoopSassa andNeoplasia in Rats taken from 2-
tassays on Chlorothalonil and TrichloroathylBne
In Sex Changes Doses (ppm)
0 5063 10126
me- M Survival (%) 82 40 40
del Hyperplasia (%) none
Adenomas
Incidence 0/10 2/46 1/49
Rate(%) 0 4 2
Carcinomas
Incidence 0/10 1/46 3/49
Rate(%) 02 6
Qombined "•
Incidence 0/10 3/46 4/49.
Rate (%) 0 68
F Survival (%) 50 62 72
Hyperplasia (%) none
Adenomas
Incidence 0/10 0/48 3/50
Rate(%) 0 06
Carcinomas
Incidence 0/10 1/48 2/50
Rate(%) 0 24
Combined
Incidence 0/10 1/48 5/50
Rate(%) 0 2 10
ie, _ . • . . . •
rain Sex Changes Doses (mg/kg/day)
00 500 1000
borne- M Survival (%) 42 44 34 30
endel Hyperplasia (%) 0 0 10 6
Adenomas
Incidence 0/50 0/50 6/50 1/50
Adj. Rate (%) 0 0 32 6
Carcinomas
Incidence 0/50 0/50 0/50 1/50
Adj. Rate (%) 0006
Combined
Incidence 0/50 0/50 6/50 2/50
Adj. Rate (%) 0 0 32 11
ins: 2-4% renal tubule tumors in three other strains.
ignant mesothelioma in male rats; hepatocellular tumors in male and
lale mice, and lymphoma in female mice.
48
-------
on GIGA, namely F344 and Osbome-Mendel. The overall historical inci-
dence of these tumors in mal© F344 rats is considered by t le NTP to be 0.5
percent based on data reported on 1,943 animals which served as vehicle
controls in studies involving administration of chemicals via corn oil gavage
(NTP, 1990). In a larger historical control database, invo
and 2,370 female F344 rats used as untreated controls
bioassays, the incidence was 0.35 percent for males and
ving 2,320 male
n NTP two-year
0.17 percent for
females suggesting a male predilection for renal tubule tun tors (Solleveid et
al., 1984). This is supported by spontaneous renal tubule
tumor incidence
rates recorded for Osborne-Mendel rats used as controls in the NCI
Carcinogenesis Testing Program (Goodman etal., 1980). n975 males and
970 females, the incidence was 0.3 percent and 0 percent, respectively.
Because of the in!requency of renal tubule tumors, even marginal increases
in their incidence in treated animals (statistically significant when compared
to historical rather than concurrent controls) is regarded! by the NTP as
biologically significant and attributable to compound administration (Haseman
etal., 1984; NTP, 1989).
In the 2-year studies with the eight selected renal carcinogens, the
observed incidences of renal tumors for individual chemically dosed groups
were less than 25 percent, and no higher than 16 percent for most. Because
of the low background rate in both concurrent and historical controls,
however, development of renal tubule tumors at these
ascribed to an effect of the chemical.
The NTP bioassays provide little insight into the hi
renal tumors as they were designed and performed with th]
of determining the presence or absence of carcinogenic
chemical. Although an industry-sponsored study of u
included interim sacrifices, even this bioassay did not i
sacrifices designed to provide information on the site of o
esis of tumors.
incidences was
genesis of the
prime objective
ictivity of the test
leaded gasoline
rporate serial
igin or histogen-
Survival rates in high-dose male rats were poor ins
bioassays, which complicates interpretation of the data. ~
rate observed in some of these studies cannot be attributed
tumors (Hoel et al., 1988). In fact, poor survival rates
excessive toxicity. For the 1,4-DCB bioassay, survival
males, 40 percent at termination, became significantly
vehicle controls after week 97 (NTP, 1987a). Nearly
nonaccidental. A similar situation pertains to isophoronje
percent of high dose males survived to termination (NTP
veral of the NTP
he high mortality
to the renal
jsuaily indicated
of the high-dose
lower than that of
all deaths were
where only 28
1986a).
The decreased survival rates suggest that a maximu T» tolerated dose
(MTD) was exceeded since the early deaths could not be attributed to
tumors. Administration of a chemical at doses exceeding an MTD may alter
responses that would be seen at lower dose levels (Office of Science and
Technology Policy [OSTP], 1985). However, exceeding an MTD, by itself,
is not compelling evidence that tumors are produced only when detoxifica-
tion mechanisms are overwhelmed. Intact, survival of mal3 rats in low-dose
groups administered isophorone, 1,4-DCB, hexach oroethane and
tetrachloroethane was equivalent to that of the concurrent control groups
49
-------
and renal tumor incidence was elevated in these animals. Survival was
excellent for all dose groups of male rats administered d-limonene or
unleaded gasoline. However, it is difficult to compare tumor incidences
among studies with marked differences in survival rates, especially when
there is the potential for development of slow-growing tumors, such as renal
neoplasms.
B. Renal Tumor Incidence
Among the eight model carcinogens, the overall unadjusted incidence
rates for renal tubule tumors (adenomas and adenocarcinomas/carcinomas
combined) in male rats ranged from 3 percent to 11 percent at low-dose
levels and 0 percent to 22 percent at the high dose. The highest unadjusted
incidence (22%) was associated with d-limonene. For the remainder of the
chemicals, incidences of renal tumors were 16 percent or less. When
adjusted for intercurrent mortality, the incidence rates for combined renal
tumors ranged frdm 0 percent to 28 percent with 1,4-DCB being the highest
(Table?).
Forall of the eight model carcinogens, and also fortrichloroethylene and
chlorothalonil, the increase in the incidences of renal tubule tumors, where
adjusted for intercurrent mortality, was dose related. Because the incidence
of renal tubule tumors was low and there were confounding factors such as
toxicity occurring at all dose levels in most studies, it is not possible, from
the NTP bioassay data, to determine if there was a relationship between
increasing dose and percentage of tumors classified as adenocarcinomas
rather than, adenomas. In the 1986 Guidelines for Carcinogen Risk
Assessment, EPA discussed its strategy for analyzing combinations of
benign and malignant tumors (USEPA, 1986). In general, the Agency stated
that it would consider the combination of benign and malignant tumors to be
scientifically defensible if the benign tumors have the potential to progress
to the associated malignancies of the same histogenic origin. The weight-
of-evidence that a chemical is potentially carcinogenic for humans would
increase when there is a dose-related increase in the proportion of tumors
that are malignant. Conversely, if only benign tumors were observed, this
would constitute less evidence of human cancer potential. Since the
distinction between adenomas and adenocarcinomas for renal tubule
tumors fn rats is rather arbitrary, based mainly on size, these general
principles cannot be rigidly applied.
C. Hlstogenesis of Renal Tumors
As previously indicated, NTP bioassays are designed to determine
whether or not a chemical is a carcinogen. They are not designed with the
intent of providing information to evaluate the developmental stages of renal
neoplasia. Although renal tubule hyperplasia was reported in the male rat
for seven of the eight bioassays and incidences of this lesion generally
increased with increasing dose, further insight with respect to histogenesis
into possible interrelationships between hyperplasia, adenomas, and carci-
noma^ is not possible because of the low overall frequency of these lesions.
The occurrence together of preneoplastic and neoplastic lesions in most
50
-------
studies with the eight chemicals does provide indirect evidence of progres-
sion from tubule cell hyperplasia via adenomas to adenocarcinomas. In
studies with d-limonene (NTP, 1990) and hexachloroethane (NTP, 1989),
these lesions were stated to be part of a continuous morphologic spectrum.
This accords with the generally accepted view on renal tubule tumor
formation and progression (Lipsky and Trump, 1988; Hard, 1990).
D. Renal Tumor Latency and Progression
Renal tubule tumors produced by administration of CIGA appear to be
late developing neoplasms. Times at which such tumors were first observed
in bioassays of the eight model carcinogens usually exceeded 18 months.
In general, the first renal tumor observed in each of the bioassays occurred
about 5 to 10 weeks earlier in the high-dose than in low-dose animals.
Because renal tubule tumors are not immediately life-threatening, they were
usually detected in bioassays at terminal sacrifice or at death of the animal
from other causes. Out of the eight bioassays, there was only one case of
renal tumor metastasis, occurring in the high-dose group of hexachloroeth-
ane (NTP, 1989).
E. Induction of Other Tuinor Types
Six of the eight model substances produoed liver tumors in male and/or
female mice but not in male or female rats. These chemicals were
hexachloroethane, unleaded gasoline, isophorone, 1,4-DCB,
pentachloroethane, andtetrachloroethylene. A different mechanism, inde-
pendent of hyaline droplet accumulation, may be involved.in the production
of livertumors by these six chemicals. Some authors suggest a mechanism
involving peroxisome proliferation to account for the production of such liver
tumors (Elcombe et al., 1985; Goldsworthy and Popp, 1987).
An alternative explanation forthe livertumors is that both CIGA-induced
liver and kidney tumors are produced by a common mechanism (direct or
indirect) not involving a2u-g. Available data do not tend to support this
hypothesis, although a recent inhalation toxicity study of 1,4-DCB illustrates
other types of data needed before these questions can be resolved. In this
study, significantly higher levels of 1,4-DCB were found in the kidneys of
male rats and in the livers of female rats following exposure at 500 ppm for
24 hours (Umemura et al., 1990). Although the Umemura study may simply
demonstrate reaction of 1,4-DCB with a2u-g, it may also indicate metabolic
differences among species and sexes that influence the effective doses
delivered to the tumor sites.
Primary tumors in addition to those in the male rat renal tubule were not
consistently produced in rats or mice at organ sites other than the liver
following administration of the eight chemicals. The production of tumors at
othersites, however, raisesthe possibility that other mechanisms could also
be contributing to the overall kidney tumor incidence in male rats. This
possibility has been suggested for tetrachloroethyiene (Green et al., 1990;
Dekant et al., 1989). Dekant and colleagues have proposed a mechanism
involving hepatic glutathione S-conjugate formation and, ultimately,
51
-------
bioactivation of renal cysteine conjugate by p-lyase in the nephrotoxic and
carcinogenic response to halogenated alkenes, including tetrachloroethy-
lene although th£y alsodo not rule out a role for o,u-g-induced nephrotoxicity.
Within this content, it is noteworthy that in the tetrachloroethylene bioassay
a renal tubule adenocarcinoma was observed in a single low-dose male
mouse, clearly alstatistically nonsignificant event, but less readily regarded
as biologically irrelevant.
VII. Additional' Evidence Concerning the Renal Carcinogenicity of
GIGA 1
Key evidence relevant to providing information on carcinogenic mecha-
nisms can be derived from short term tests, such as assays for gene
mutations and DfsJA damage, and from studies testing the tumor-promoting
effects of GIGA.!
A. Genetic Toxicology Studies
The availablb genotoxicity data for the eight model carcinogens and for
trichloroethylene and chlorothalonil are summarized in Tables 9 and 10.
The four assays listed in the tables (Salmonella [SAL], chromosome
aberrations in [ABS] Chinese hamster ovary cells, sister chromatid ex-
change [SCE] in! Chinese hamster ovary cells, and thymidine-kinase [TK]-
gene mutations Jin L5178Y cells [MLA]) are the only ones with enough
common data fdr comparative purposes. It is not coincidental that these
assays are empjoyed by NTP. Consequently, this analysis of genotoxicity
data was limitedjforthe main part, to the 10 model substances with bioassay
data Data from prosophila tests conducted by the NTP (Yoon et al., 1985)
and in human lymphoblasts (Richardson et al., 1986) are also cited in Tables
9 and 10 where .available.
The eight 4nal carcinogens selected as possible CIGA have been
tested for chromosome aberrations in Chinese hamster ovary (CHO) cells
(Galloway et al., i 987a) and in Salmonella (Haworth et al., 1983; Mortelmans
et al., 1986; Ashby and Tennant, 1988; NTP, 1987b). All results were
negative both ip the absence and presence of exogenous activation
provided by S9 elxtracts from rat liver. Two presumed intermediate metabo-
lites of d-limonene, (the 1,2- and 8,9-epoxides) were also tested in Salmo-
nella with and without induced S9, and no increase in revertants was
observed (Watabe et al., 1981). Several chemicals have tested positive, at
least under somfe conditions, for SCE in CHO ceils (Galloway et al., 1987a)
and in the mouse lymphoma TK-gene mutation assay (McGregor et al.,
1988). Four of the eight possible CIGA and both non-CIGAs were positive.
Richardson et al j (1986) reported negative results for unleaded gasoline and
its known CIGAj component, TMP, in assays for TK-gene mutations and
SCE in the TK6 human lymphoblast cell line. A cursory appraisal of only
positive and negative responses leads to the conclusions that there is
significant heterogeneity and the CIGA groups are not distinguishable from
non-CIGA by tneir genotoxic activity. Upon more detailed analysis, it
becomes apparent that the majority of the positive responses of the eight
hyaline-droplet inducing carcinogens were observed in the absence, but not
52
-------
Comments
ens
Kidney Ca
§
x»
£
Ul
•S (0
a 5-
c ^
0) CO
CD
I
8
i i
TD
CO
"55
-Q
ive
S9 - there was a marginal positi result in one o
ments. Negative in vivo chromosome aberrations, rr)
ant lethals.
MLA wi
experi
domin
LU
0)
— -
I|3|
III!
§
UJ
g
CJ
C
o
5
f CD
||
£2
Is-
E o
O CL
o,£
5
IE
1~
.2
I
•s
o
8
•5
'5
•>^
c
o
'55
*-%
1>
9 -a ^-i.
o. ta o>
S® <-.
c ^
8tJ 0
•— QJ Ju
HI
SS:I
•Sg'i
ill
& 2 c
W 03
S'o..--
•aJH-^e
•o z o
3
0) c c
Fsl
"s^f
0 5 S
.^-5 2
o
1
^
.
o
Q-a
53
-------
Comments
^
s
Ul
O
OT
OT
CD
<
OT
1
1
4^
S
LA positive without S9; MLA n
Drosophila SLRL.
2.E
||
+
+ .
'•;'."
!
y=s
§
ro
t"
^
£1
O
c
•*~
'S c .
= .£ .. o . '
^ <• o oj •=: .a
•^ «- — I Q- t o ' — '
O3 C ^5 S*^^ ' 03
iJUl^li
mint
~fr*
'
.-,-'•••" •••'
• c • •'
•sr
f
.2
o
o ' '
' -c
h-
TJ
X
8 . • .
ii
_i
_of
8 .
>-••'.
to to
\n T-
— J
.E ' "<6
>» 'S
C/3 .«•
01 o
o O
1 ^
03 00
S 2
5! _•
vj* co
I |
c 5
jt =
| 0
!§ ^
I '*
u To
.3 . o.
ID O
8~ w
. X
c . c
•—(/)«=
in ~ aj .
|||
•!">-Gr
'ffl^-g
|S.I.
Ill
II 3 "^1
W II X
-.3a §
Ja u t3
ITB S1
o« c
E^'5.
^ ~ _+_
CO .2 of
.« !2-S
— J 5? "w
^* Ql ^
C/) i_ Q-
54
-------
in the presence of, exogenous S9 activation and at concentrations greater
than I00u.g/ml_.
Dimethyl methylphosphonate appears to present a unique genotoxicity
profile among the eight model carcinogens. Because this chemical has high
water solubility and low toxicity, in vitro assays have employed very high
concentrations, as high as 30 mg/mL. Galloway et al. (1987b) reported that
at least some of the observed in vitro mutagenic activity seen for dimethyl
methylphosphonate occurred at levels that decreased cell growth and
greatly increased the osmotic strength. Similar levels of osmolality and
chromosome aberrations were observed, for example, with 160 mM of
potassium chloride and 30 mg/mL of dimethyl methylphosphonate. The
SCE increases observed for dimethyl methylphosphonate, however, oc-
curred at concentrations causing only slight increases in osmolality.
Of particular relevance are those studies in which rodent kidney or
kidney extracts are combined with a genotoxic endpoint. Loury et al. (1987)
reported that unleaded gasoline was negative in an in vivo/in vitro kidney
unscheduled DNA synthesis assay indicative of DNA damage and repair.
Similar resu Its were reported for pentachloroethane and tetrachloroethy lene
by Goldsworthy et al. (1988b). However, both studies reported significant
elevation of replicative DNA synthesis in kidneys of male rats treated with
these compounds.
Recently, Vamvakas et al. (1989) reported clear dose-related positive
results in Salmonella TA100 with tetrachloroethyiene in the presence of
glutathione and rat kidney microsomes. The glutathione conjugate S-(1,2,2-
trichlorovinyl)glutathione was also mutagenic in the presence of kidney
microsomes and the activity was reduced in the presence of a p-lyase
inhibitor. The importance of these findings in the formation of the kidney
tumors of male rats exposed to tetrachloroethyiene is yet unclear, but similar
studies with other apparently nongenotoxic kidney carcinogens seem to be
in order before direct interaction with DNA can be excluded.
In summary, the preponderance of available data suggest that the CIGA
group possess little, if any, genotoxic activity. However, the shortage of data
in the kidney or with glutathione conjugates for these chemicals precludes
closure on the question.
B. Initiation-Promotion Studies
The multistage concept of carcinogenesis, involving in its simplest form,
an irreversible initiation phase followed by a stage of tumor promotion (Pitot,
1982), implies that chemicals may play a role in assisting, as well as directly
causing, cancer formation. There have been two research studies testing
the potential of CIGA for promotion or cocarcinogenic activity in an estab-
lished initiation-promotion model of renal carcinogenesis.
Using 2 weeks exposure to 170 pprn of EHEN in the drinking water as
the initiating agent, the first initiation-promotion experiment of Short et al.
(1989b) included both sexes of F344 rats, multiple dose levels of the test
55
-------
substances, short-term versus long-term promotion exposures, and a
sequence-reversal study to discriminate any cocarcinogenic from promo-
tional effects. The;test substances were unleaded gasoline (3 inhalation
concentration-levels of 10,70, and 300 ppm), and IMP (one oraldose-level
of 50 ppm). Treatment groups, comprised of approximately 30 animals,
included a control, 2i promotion controls, an EHEN initiation control, reverse-
sequence initiation: control, initiation-promotion group with a pro™>{'on
phase of 24 weeks, initiation-promotion group with a promotion phase ot 59
weeks, and a reverse-sequence test group where 24 weeks; of exposure to
unleaded gasoline 6r IMP preceded the 2-week period of EHEN admmis-
tration. All animals |were killed at 65 to 67 weeks after the^ommencemen
of the experiment. The results were assessed in terms of the incidence o
foci of tubule hyperplasia (called atypical cell foci by the authors) and renal
tubule tumors. Dosle-related increases in hyperplastic. foci were observed
in male rats promoted with unleaded gasoline or TMP for both the short- and
long-term promotion periods. A significant linear trend in the mcidence.of
renal tubule tumors with increasing gasoline dose was also observed jn
male rats promoted with unleaded gasoline for. 24 weeks but not for 59
weeks. The latter discrepancy reflects an experimental design weakness in
the study, namely under-estimation of an optimal initiating dose of EHEN,
which resulted in a very low basal incidence of renal tumors. Nevertheless,
the results with th4 single dose level of TMP, and the absence of renal
tumors in any negative control group, supported the observed trends with
unleaded gasoline;
In the sequence-reversal study, there was no increase in renal tumors
although the incidence of hyperplastic foci was significantly elevated for both
compounds Foci df CPN were also scored in these various groups with an
increase upon GIGA exposure apparent in male rats. However, no corre-
lation of incidence'of CPN lesions with numbers of hyperplastic foci or
incidence of renal tubule tumors was found.
On the basis of the results, the authors' conclusions that unleaded
gasoline and TMP have promoting activity for renal tubule tumors in the male
rat, rather than acting as cocarcinogens, appear reasonable. Furthermore,
there was no elevation of either hyperplastic foci or renal tumors in female
rats in the study, emphasizing once again, the male specificity of the renal
response to GIGA.1
A second initiation-promotion assay using the same EHEN model was
conducted with d-llmonene (Dietrich and Swenberg, 1991 b). This study
specificallyaddressedthecomparisonofresponsesbetweenthemaleF344
rat and the OL -g-deficient NBR strain. The initiating dose of EHEN was 500
Dpm administered in the drinking water for two weeks, followed by
d-limonene by daily gavage (5 days a week) at 150 mg/kg/day for 30 weeks.
An initiation control (EHEN), promotion control (d-limonene), and a vehicle
control were included for both strains. In the F344 rats administered EHEN
and d-limonene, atypical tubule cell hyperplasia arid renal tubule adenomas
were increased 10-fold as compared to the EHEN control group. In contrast,
no tumors were observed in any of the NBR groups. Such negative results
56
-------
in the NBR rat strongly suggest a clear dependence' on a, -g for the
promoting activity of d-Iimonene.
The promotional effect of unleaded gasoline, TMP, and d-limonene may
be occurring through the influence of sustained tubulq cell proliferation
which has been demonstrated with these same compounds (Short et al,
1989a; Dietrich and Swenberg, 1991b). The extent of pell proliferation Ss
regarded as an important factor in chemical carcinogene^is (Grisham et al.,
1983; Cohen and Ellwein, 1990; Cunningham et al., 1991) and stimulation
of cell turnover is one of the key mechanisms believed to operate in tumor
promotion (Farber, 1988).
VIII. Comparison of GIGA with Classical Renal Carcinogens
In general, classical renal carcinogens or their activjs metabolites are
electrophilic species binding covalently to macromolecules and forming, in
particular, DNA adducts (Hard, 1987; Lipsky and Trump] 1988; Alden and
Frith, 1991). Such DNA reactivity is putatively the mechanistic basis of renal
carcinogenesis induced by these chemicals. For example, carcinogenic
nitrosamines can form various alkylation products in DjNA, including O8-
alkylguanine which is a promutagenic lesion (Pegg, 1984). Accordingly,
classical renal carcinogens are usually positive in short-tierm mutagenicity
assays. In contrast, CIGA are not known to react with DNA;and are generally
negative in short-term tests for genotoxicity. As described previously
(Section IIIG), CIGA binding to a,u-g is reversible and not covalent in nature.
Classical renal carcinogens can induce renal tubule! cancer in rats or
mice in high incidences, with minimal duration of exposure, clear dose-
response relationships, and with decreased latent period of development
(Hard, 1987; Alden and Frith, 1991). Tumor frequencies Jare often over 50
percent and up to 100 percent, much higher than the low incidences (2% to
28% adjusted) recordedforCIGA. Unlike CIGA-induced ren&l carcinogenesis,
there is usually no absolute sex specificity, with males $nd females both
susceptible, but sometimes to varying degree. These differences in potency
and species- and sex-susceptibility, suggest that classical renal carcino-
gens and CIGA act via different mechanisms in kidney cprcinogenesis.
In addition, some classical carcinogens are effective renal tumor
inducers following abbreviated dosing regimens. For example, DMN, DEN,
and streptozotocin require only a single injection to produce tumors, while
the EHEN regimen utilizes a 2-week period of oral exposure. In contrast,
certain military fuels induced renal tubule tumors in male rats following
lifetime observation after 1 year of intermittent exposure:, but not after 90
days of continuous exposure (Bruner, 1984). ,
The lack of involvement of hyaline droplet accumulation in the early
nephrotoxicity associated with classical carcinogens (definite with DMN and
DEN and apparent with the others) is a major difference from the sequence
of early pathological events induced by CIGA in the male rat.
Pathology reports indicate that renal tubule tumors induced by CIGA are
morphologically indistinguishable from spontaneous tumors or those in-
57
-------
duced by classical carcinogens, With both granular and clear cell types
occurring Likewise, despite differences in toxicity observed, the sequence
ofdevetopment of Conduced renal tumors from tubule**)£«£»•»
carcinoma appears identical. However, some evidence from the ^assays
suggests that the GIGA tumors may, in general, have a smaller size
probably becauseof the difference in potency between these chemicals and
classical carcinogens, affecting the latent period of tumor development.
As withclassical carcinogens, metastases have been rarely'^ported for
renal tubule tumors related to treatment by chemicals inducing hyaline
drop ets and/or a, -g. The one case of metastasis noted with hexachloro-
ethane suggests," however, that a malignant potential exists for such
neoplasms.
Although the specific site of origin for the renal tubule tumors produced
by GIGA is not known, the P2 region of the proximal tubule as the primary
site would be consistent with existing information. Based on stud.es with
classical carcinogens this does not represent an unusual location.
IX. Evidence Concerning Human Kidney Cancer
Although not one of the most common neoplasms in the United States
renal cell adenocarcinoma/carcinoma is regarded as an importsint human
cancer This is because the disease is unpredictable and a significant
portion of patients, approximately one ^^^««$»T£
the time of diagnosis (Bennington and Beckwith, 1975; NCI, 1987). The
moS^
with renal cell cancer is 48 percent (Devesa et al., 1990). In addition, the
etiology of kidney cancer in humans is poorly understood.
A. Morphology and Histogenesis
Human renal cell tumors, which are morphologically similar to those of
rodents, are classified according to cell type and cellular arrangement
Thus, two main cell forms are recognized, granular and clear and the usual
oattems of organization are tubular, solid, papillary, and cystic Individual
tPurw ^may show an admixture of patterns and of cell types. Infrequent,
renal cell carcinoma presents as a sarcomatoid form composed of sp.ndle
cells (Bennington and Beckwith, 1975; Bannayam and Lamm, 1980,
Tannenbaum, 1983).
It is generally accepted that the origin of renal cell carcinoma is the
proximal tubule, based on both immunological study (WaHace and Nairn
1972) and ultrastructural features (Tannenbaum, 1971; Benn.ngton and
Beckwith, 1975). Electron microscopy reveals many similarities between
the tumor cells and normal proximal.tubule epithelium, including brush
border elements, membrane-associated vesicles, and basilar mfoldings of
the plasma membrane (Tannenbaum, 1971). Ultrastructurally, the amount
of intracellular lipid, paniculate glycogen, and organelles distinguishes clear
from granular cells.
It is widely considered that human renal adenomas represent small
adenocarcinomas or carcinomas as there are no microscopic, histocnemi-
58
-------
cal, or immunologic features which discriminate them, other than size, which
is not an absolute biologic parameter (Bennington and Beckwith, 1975;
Ritchie and Chisholm, 1983; Tannenbaum, 1983). Adenomas are, there-
fore, considered part of an evolutionary continuum from hyperplasia through
adenoma to adenocarcinoma/carcinoma, as in rodents. As a general
observation, there is a direct relationship between tumor size and frequency
of metastasis (Bell, 1950; Hellsten et al., 1981; Ritchie and Chisholm, 1983).
Kidney cancer statistics are usually reported in a form which encom-
passes all types of malignant cancer affecting kidney* renal pelvis, and
sometimes ureter and urethra. Renal cell cancer rarely occurs under the age
of 40 years (Mclaughlin and Schuman, 1983; Asal et al., 1988a) and
represents about 70 percent of all kidney tumors in adults (Devesa ei al.,
1990). Kidney cancer statistics, therefore, provide an approximation only of
renal cell tumor prevalence.
The number of new cases of kidney and urinary tract cancer (excluding
bladder) estimated for 1991 in the US is 25,300 with a mortality estimate of
10,600 deaths (Boring et al.., 1991). These figures represent approximately
2 percent of both new cancer cases at all sites and total cancer deaths. The
age-adjusted incidence rates in the US for the period between 1975 and
1985 obtained from the NCi Surveillance, Epidemiology and End Results
Program (SEER) data for renal cell cancer are 8.4 per 100,000 for males and
3.7 per 100,000 for females, with no difference among racial groups
(Devesa et al., 1990). Most studies indicate a consistent male to female ratio
of 2:1 forthe incidence of renal cell tumors (Asal etal., I988a; Devesa etal.,
1990).
In considering renal cell tumors specifically, the highest rates interna-
tionally have been reported from Iceland and other Scandinavian countries.
Renal cell carcinoma is the fifth most common malignant tumor of males in
Iceland although it ranks only tenth in females (Thorhallson and Tulinius,
1981). The lowest rates for renal cancer are recorded in Africa, Asia, and
South America (Mclaughlin and Schuman, 1983). Within the US, mortality
surveys indicate that the North Central region and some areas in the
Northeast have the highest incidence rate for renal cell carcinoma (Pickle et
al., 1987). It has been suggested that the clustering in the North Central
region may be partially explained by the predominantly German and
Scandinavian origin of the area's population (Mclaughlin et al., 1984).
Several studies reported that the urban rates for renal cell tumor incidence
are higher than for rural areas, but the correlation is considered to be weak
(Newsom and Vugrin, 1987).
In contrast to the relatively low incidence and mortality figures for
malignant kidney and related tumors provided by cancer statistics data, the
occurrence of renal cell adenomas at autopsy is common. The reported
incidence has ranged from 15 percent (Bannayam and Lamm, 1980) to 25
percent, the latter for males over the age of 50 (Reese and Winstanley,
1958). These findings have led to speculation that a proportion of adenomas
59
-------
may reach a limit of growth and/or remain quiescent (Bannayam and Lamm,
1980; Warter, 1983).
During the period 1950 to 1985, the US Cancer Statistics data indicated
an increase of 82 percent in the incidence of kidney and renal pelvis cancer
combined (NCI, 1987). For renal cell cancer alone, the increase among
whrteTbLedoncx^
to 1985 was about 30 percent; this would be an average annual percent
change'in incidence of 2.0 for males and 1.8 for females (Devesa et al
1990) Data from Cancer Registries in Scotland also indicated an increased
incidence of renal cell carcinoma of approximately 37 percent for males
between 1967 and 1979, although there was no corresponding increase in
females (Ritchie and Chisholm, 1983). Despite an|improvement .mmortality
rates since 1950 compared to incidence rates (NCI, 1987) the relative 5-
vear survival rates, which are close to 50 percent, have not altered since the
early 1970's (Boring et al., 1991), suggesting little improvement in treatment
over the past two decades. On the other hand, diagnostic detection
measures have improved dramatically during this time which may explain,
at least in part, the observed increase in renal cancer incidence (Higginson
etal., 1984; NCI, 1987).
Renal cell carcinoma has been diagnosed with increasing frequency in
patients with chronic renal failure (Hughson et al., 1986; Newsom_ and
Vuarin 1987). In particular, this appears to reflect an association with the
development of acquired renal cystic disease which frequently occurs in
patients on long-term hemodialysis. The incidence of renal cell carcinoma
in patients with acquired cystic disease has been estimated as approxi-
mately 6 percent (Hughson et al., 1986). Thus, current data suggest that a
growing population of humans receiving maintenance dialysis may be at nsk
for developing renal cell tumors.
C. Environmental and Lifestyle Factors
Potential etiological associations between renal cell cancer and exog-
enous and endogenous environmental factors, lifestyle, and occupation,
have been sought in cohort and case-control studies. Of all the environmen-
tal and lifestyle factors investigated, tobacco use in the form of cigarette,
cigar or pipe smoking has been the one most consistently associated with
renal'cell carcinoma (Dayal and Kinman, 1983; McLaughlin and Schuman,
1983- Yu et ah, 1986; Asal et al., 1988a; Brownson, 1988; La Vecchia et al.,
1990) Although af ew studies have failed to identify a statistical association
between smoking and renal cell cancer, it has been estimated that 30
percent of renal cell carcinomas in males and 24 percent in females may be
attributable to cigarette smoking (McLaughlin et al., 1984) and that there is
evidence for a moderate dose response (McLaughlin and Schuman, 1983).
One study has also linked use of chewing tobacco with renal cell carcinoma
in males (Goodman et al., 1986), and another has associated smoking with
renal adenoma (Bennington et al., 1968).
Other possible risk factors that have been reported include coffee and
tea consumption, artificial sweeteners, high body mass index (maintained
60
-------
from 20 years of age), high dietary animal protein and fat, lower educational
levels, long-term analgesic use, and diuretics (reviewed in DayaS and
Kinman, 1983; Mclaughlin and Schuman, 1983; McLaughlin, 1984;
Mclaughlin et al., 1984; Goodman et al., 1986; Yu et al., 1986; Asal et al.,
1988a; McCredie et al., 1988). Of these, the evidence is least consistent for
beverage consumption, artificial sweeteners, other dietary factors, and
socioeconomic status, and is strongest for high body mass index and drug
use (phenacetin and diuretics).
D. Occupational Factors
Although a number of epidemiological studies have reported some
association between occupation and renal cancer, clear occupational
determinants have yet to be demonstrated and it is considered that much
epidemiological research is needed to further define and quantify potential
risks (Mclaughlin and Schuman, 1983). Occupational exposures in North
"America, where at least one study has reported an association with
increased kidney cancer rates, include asbestos (Selikoff et al., 1979; Smith
ef al., 1989), coke-oven emissions in the steel industry (Redmond et al.,
1972), printing press chemicals (Paganini-Hilletal., 1980), laundry- and dry-
cleaning agents (Blair et al., 1979; Katz and Jowett, 1981; Duh and Asal,
1984; Asal et al., 1988b), exhaust fumes in truck drivers (Brownson, 1988),
petroleum, tar, and pitch products (Thomas et al., 1980; Hanis et al., 1982;
Wen et al., 1983; McLaughlin et al., 1984; Savitz and Moure, 1984;
Kadamani et al., 1989), and aviation and jet fuels (Siemiatycki et al., 1987).
Inthese studies, information on smoking history was rarely available, so that
its possible influence could not be determined.
A study that examined the relationship between renal cancer and
occupation as defined in the 1960 Census in Sweden, where the incidence
rates are higher than in the US, did not detect increased risk for hearth and
furnace workers in the steel industry, printing workers, laundry and dry-
cleaning workers, or workers in petroleum refineries and gasoline stations
(Mclaughlin et al., 1987). Instead, the Swedish study reported an increase
in incidence of renal cell cancer among health care professionals.
E. Renal Cancer and Hydrocarbon, Solvent or Petroleum Product
Exposure
Several of the occupations listed above involve exposure to certain
Classes of chemicals that may fall into the CIGA category. Besides GIGA,
however, non-CIGA compounds are also present, making it difficult to
attribute elevations in risk with a unique exposure (e.g., CIGA). In a recent
population-based case-control study, Kadamani et al. (1989) did not ob-
serve statistically significant associations between renal cell carcinoma and
high occupational exposure to hydrocarbons in males (OR 1.6; 95% Cl 0.7-
3.6) or in females (OR 0.8; 95% Cl 0.3-2.3). The authors, however, noted
a positive exposure-response relationship for those with older ages and for
workers with the greatest duration of exposure.
In a case-referent study of occupational risk indicators for renal cell
adenocarcindmas, Partanen et al. (1991) examined all cases reported in
61
-------
Finland in 1977 to 1978. These investigators found an elevated risk and an
exposure-response relationship for gasoline exposure. Since thejjostu-
lated average latency period was about SOyears, arolefor lead compounds
could not be ruled out. V
Synthetic solvents widely used in dry-cleaning include the GIGA
tetrachloroethylene, as well as Stoddard and 140F solvents.* Severa,
studies analyzing proportional mortality data on laundry- and dry-cleaning
workers in various parts of the US reported elevated risks for kidney cancer
(Blair et al., 1979; Katz and Jowett, 1981; Duh and Asal 1984; Asal et al.,
1988b) More recent studies that were better designed, however, have not
substantiated the earlier findings. No statistically significant elevations in
kidney cancer risks have been detected in the studies °^ry-clean,ng
workers by Blair et al. (1990) (Standardized Mortality Ratio [SMR] 50,95 k
Cl 10-180) Lynge and Thygesen (1990) (Standardized Incidence Ratio
^IR]males 1 5;95%CI0.6-3.3;fernales0.6;95%CI0.2-1.4)IorBrdwnand
Kaplan (1987) (SMR 200; 95% Cl 55-517). In considering occupational
exposure to solvents as a general chemical category Harrington et al
(1989) found no relationship with renal cancer (OR 1.0; 95% Cl 0.2-4.9)
although the statistical power of this study, as with most others was
acknowledged by the authors as sufficient to identify only large risk esti-
mates. * . •
Siemiatycki et al. (1987) conducted a population-based case-referent
study in Montreal on cancer associations with exposure to 12 petroleum-
derived liquids. These various mixtures included automotive and aviation
gasolines and distillate jet fuel. Aviation gasoline differs in composition from
the automotive counterpart by its high content of alkylate naphthas consti-
tuted mainly of branched alkanes (Siemiatycki et al., 1987). No statistically
siqnificant risk of renal cancer was found with exposure to automotive
qasoline (OR 1.2; 90% Cl 0.8-1.6). Statistically significant elevations,
however, were noted at the 90 percent confidence level with exposure to
aviation gasoline (OR 2.6; 90% Cl 1.2-5.8) and to jet fuel (OR 2.5; 90% Cl
1 1-54) Six of the seven cases with exposure to aviation gasoline also naa
exposure to jet fuel, making it difficult to distinguish a unique exposure. Jn
depth analyses of the two associations using logistic regression methods
indicated, however, a greater role for aviation gasoline than for jet 'fuel.
Wong and Raabe (1989) conducted a quantitative meta-analysis by
cancer site of petroleum industry employees from the US, Canada, United
Kingdom, Europe, Australia, and Japan, critically reviewing almost 100
published and unpublished epidemiological reports. Standardized mortality
ratios observed for kidney cancer in the industry as a whole were similar to
those for the general population. Results from refinery studies ranged from
nonsignificant deficits to nonsignificant excesses. However, the possibility
of an elevated kidney cancerrisk was raisedforonespecificgroupwithmthe
Stoddard and 140F solvents are mixtures of hydrocarbons including straight and
branched chain paraffins suggesting that they may also be CIGA.
62
-------
industry. Drivers among British distribution workers (Rushton and Alderson,
as reported by Wong and Raabe) showed borderline significance for excess
kidney cancer mortality. Wong and Raabe (1989) concluded that additional
data, particularly involving exposure to downstream gasoline, are needed to
resolve the issue.
In a large population-based case-control study adjusted for the con-
founding factors of age and cigarette smoking, no overall association (OR
1.0;,95% Gl 0.7-1.4) was observed between risk for renal cell cancer and
employment in a range of occupations with potential for exposure to
petroleum products (Mclaughlin etal., 1985). There was, however, a small
excess risk among gasoline station attendants (OR 1.2; 95% Cl 0.6-2.3)
which increased with duration of employment, although individual point
estimates and tests for trends were not statistically significant. A case-
control study on a combined cohort of approximately 100,000 male refinery
workers from five petroleum companies, sponsored by the American Petro-
leum Institute (Poole et al., 1990), suggested increases in kidney cancer risk
for laborers (Relative Risk [RR] 1.9; 95% Cl 1.0-3.9), workers in receipt,
storage, and movements (RR 2.5; 95% Cl 0.9-6.6), and refinery unit
cleaners (RR 2.3; 95% Cl 0.5-9.9) when compared with a reference group
of office workers, professionals, and technicians. In the cohort there were
102 kidney cancer cases among 18,323 deaths.
In evaluating unleaded gasoline, 55 relevant studies were reviewed by
the USEpA (1987) to determine whether there was any epidemiologic
evidence for an association between gasoline exposure and cancer risk.
The evidence for drawing causal inferences between unleaded gasoline
and .cancer was considered inadequate under EPA's guidelines for cancer
risk assessment (USEPA, 1986). As Enterline and Viren (1985) have
emphasized in their review on the epidemiology of renal cancer and gasoline
exposure, most of the studies have not been designed or analyzed with an
hypothesis specifically associating gasoline exposure and renal cancer.
The cohort studies of petroleum workers do not lend themselves for a
comparison since they shed no light on gasoline exposure, per se. Expo-
sures in these studies have been varied, and the only common element is
the place of work. Thus, the individuals in the cohort who had the exposure
of interest, i.e., gasoline or a specific fraction, cannot be identified.
As a general conclusion from the foregoing, small risks cannot be
excluded for specific job categories; but the association between human
kidney cancer and exposure to petroleum distillates, if there is one, does not
suggest high risks for the types of exposures that have occurred in the past.
X. Evidence lor Dose- and Time-Dependent Progression from
Early to Late Lesions
An important aspect for examining the hypothesis that renal tumor
formation is directly associated with accumulation of a^-g in the male rat
kidney is a demonstration of the progression of lesions proposed to
culminate in neoplasia. For some of the steps, clear dose-response
relationships have been shown. In other cases, histopathologically observ-
es
-------
able events are of a secondary nature and not in the direct Pr°9ress!°.n-n^ -
Slowing information shows, however, data demonstrating the existence
of other steps in the proposed progression are limited, restricting confidence
in judgments on the nature of the association.
Evaluation of the events leading to neoplasia is further complicated by
the low incidence of renal tumors induced by the GIGA studied. Such
information makes it difficult to identify possible relationsh.ps between the
induced nephropathy and renal carcinogenesis.
A. Association Between CIGA, Hyaline Droplet Formation, and
AIpha2 -globulin Accumulation
Dose-dependent relationships have been demonstrated between the
administratiohofd-lirronen
foominick et al., 1990) and excessive formation of hyaline droptoand
between unleaded gasoline or IMP and o,u-g accumulation (Olson.et a.,
^ChiSSmeau et al., 1987). Inthe d-ifmonene study, W™W$*
were araded on a scale of 0 to 12 according to size, eosmophilic intensity,
aS tffSSSr oftubules loaded with droplets The droplett scores> for
d-Hmonene doses of 0,0.1,0.3,1.0, and 3.0 mmol/kg were, control to high
dose 3 45 ca7 8,and10(Lehman-McKeemanetal.,1989). Thedose-
?es£ns^relaSip with ^-g accumulation is exemplified by measure-
ments following administration of IMP, which, fl™" at single •doses of
0.044,0.440, and 4.000 mmol/kg, induced a2u-g concentrations; irrat tadney
tissue at 24 hours of 10.3,17.3, and 28.1 mg/g wet weight, respectively,
against a control value of 9.5 mg/g wet weight (Charbonneau et al., 1987).
With orally administered gasoline, the a -g concenrations were^dose-
responsive only in the range of 0.04 to 1.(fu mL/kg (Olson et al., 1987).
In a special NTP study/male and female F344 rats were exposed to
d-limonenebygavagefor14daysovera21-daypenod(NTP, 1990). The
1-^content! quantitated withan ELISA test in kidney homogenaes
Screased significantly in dosed male rats relative to vehicle controls. At 75
mo/ka the low dose employed for male rats in the 2-year bioassay, c^-g
levels were approximately double those in controls. In females, increasing
the dose as high as 1,200 mg/kg had no measurable effect on o^-g levels
in the kidney. Although microscopic examination of kidney sections stained
wtth hematoxylln and eosin (H & E) showed no vteWed«erer^sbe^«en
dose and vehicle control male rats, in plastic embedded sections sta ned
with Lee's methylene blue basic fuchsin, differences in the distribution,
amount, and shape of intracytoplasmic granules in the proximal tubules
were detected.
In contrast to the 21-day follow-up study, the 13-week range.-finding
study conducted before the d-limonene bioassay failed to detect an accu-
mulation of hyaline droplets. TheNTPreport(1990)acknowledged[that this
failure might have been related to the fact that several days passed between
the time the chemical was last administered and the time the animals were
killed for histological examination. Other studies have shown that renal
az -g concentrations decline rapidly, reaching pre-exposure levels by the
64
-------
third day aftertreatmeni, although hyaline droplets, being structural entities,
require up to 9 days for complete resolution (Garg et al., 1988). This
suggests that the interval between the time the chemical was last adminis-
tered and the time the animals were killed for histological examination is
critical to finding hyaline droplets and probably accounts for discrepancies
found among some studies.
These various observations, along with the results of o,u-g localization
studies and binding studies considered earlier, support a causa! association
between the administration of GIGA and o^-g accumulation in hyaline
droplets.
B. Association Between Hyaline Droplet Formatlop, Cell Necrosis and
tubule Cell Regeneration
Hyaline droplet accumulation, single-cell necrosis, and cell proliferation
occur predominantly in the P2 segment of the proximal tubule following
CIGA administration (Short et al., 1987, 1989a,b). Although single-cell
necrosis has been clearly demonstrated in association with cellular hyaline
droplet accumulation (Kanerva et al., 1987a; Short et al., 1987), there are
no dose-response studies quantitating the relationship between increased
hyaline droplets and cell necrosis in histological sections, or between cell
necrosis and cell regeneration. However, Alden has shown a correlation
between the hyaline droplet response, increased mitotic index in proximal
convoluted tubules, and elevation of the number of cells excreted hourly in
the urine (an index of exfoliated necrof ic tubule cells), using two dose levels
of d-limonene given orally for 3 weeks (Alden and Frith, 1991).
Dose-response relationships between hyaline droplet accumulation
and proximal tubule cell proliferation have been observed. Short and
coworkers (1987) exposed male rats for 3 weeks to TM P (oral) or unleaded
gasoline (inhalation) and then measured [3H]-thymidine labeling indices.
The extent and severity of hyaline droplet accumulation paralleled the extent
and localization of cell proliferation in proximal tubule cells, and both
parameters were increased in dose-dependent fashion (Figures 4 and 5). In
an extended study of the same compounds, Short et al. (1989a) observed
6-to 11-fold increases in labeling indices in the P2 segment of the rat kidney
after the rats received 3,10, and 22 weeks of exposure to 300 ppm unleaded
gasoline or 50 ppmTMP. These labeling indices remained 4-to 6-fold higher
than control values during the 48th week of exposure.
In contrast, Viau et al. (1986) did not observe a sustained regenerative
response in the kidneys of male rats exposed to an isoparaffinic solvent
consisting of saturated C10-C12 aliphatic hydrocarbons beyond 5.5 weeks.
Labeling indices in the cortex of treated rats at 46 and 68 weeks were no
different from the controls. This apparent discrepancy between the gasoline
and TMP results undoubtedly reflects differences in the technique of
radioactive labeling. Viau et al. (1986) used a single injection of [3Hj-
thymidine 1 hour prior to sacrifice, whereas, Short et al. (1989a) labeled
continuously by subcutaneous osmotic minipump infusion over a 7-day
period, the preferred method for cell populations with a low cell turnover,
thereby increasing the amount of radioactivity incorporated into renal tissue.
65
-------
_co
"flj
.0
TJ
jO>
0>
_Q
O
O.
60
40
20
0
Labeled Cells
Droplets
10
Dose IMP (mg/kg)
o
o
•o^
CD
2 in
o
—i
CD
o
0
100
Figure 4: Dose-response relationship between renal hyaline droplet accumulation (D)
and pH]-lhymidine labeling index of proximal tubule P2 cells in male F344 rats
gavaged with TMP for 5 consecutive days per week for 3 weeks. Seven-day
osmotic minipump implanted on twelfth day after start of dosing. Rats killed
and fixed on 22nd day.
Source: Adapted from Short et al., 1987.
66
-------
0.1 1 10 100 1000
Dose Gasoline (ppm)
Figure 5: Effect ofO to 2,000 ppm unleaded gasoline on continuous uptake offHJ-TdR
by P1,P2, and P3 segments of the proximal tubule epithelium. Each test
point is the mean value determined from 3 rats. Dosage is presented on a log
scale.
Source: Adapted from Short et a/., 1987.
67
-------
In recovery studies with unleaded gasoline and IMP, Short and cowork-
ers (1989a) showed that neither increased hyaline droplets norcell prolifera-
tion were observable 7 days after discontinuing the 3-week exposures,
indicating complete recovery. However, after 10 and 22 week periods of
exposure, recovery was only partial, labeling indices remaining nearly three
times above controls following 10 days in a gasoline- or TMP-free environ-
ment. Thus, proximal tubule cell proliferation is a persistent phenomenon
in chronic exposure to CIGA, becoming less amenable to recovery with
increasing duration of exposure.
Furthermore, in promotion studies with d-limonene, cell proliferation,
assessed by bromodeoxyuridine labeling via subcutaneous osmotic
minipump implants, was not induced beyond background by d-limonene
after 5 or 30 weeks of exposure in the a -g-def icient NBR rat, compared to
a 5-fold increase in the tubule cell labeling of d-limonene-promoted F344
rats initiated with EHEN (Dietrich and Swenberg, 1991b). This result
suggests that the sustained proliferative response induced by a CIGA is
dependent on the a,u-g syndrome.
Thus, the sequence of events following CIGA administration involves
lysosomal overload, cell necrosis, and cell replication. All three of these
occur in the same segment of the nephron in conventional strains of rats, but
none occur in the NBR rat. Whereas these events are temporally correlated,
it is not yet clear whether the lysosomal overload causes necrosis or whether
necrosis can be linked with replication. These questions need further
investigation and hypothesis development in orderto establish mechanisms
of action.
C. Progression to Cast Formation, Tubule Dilation, and Mineralization
Since few chronic studies incorporated serial sacrifices, it is difficult to
assess the time-dependence of the development and progression of the
sequential lesions proposed to be associated with a2u-g nephropathy.
Granular cast formation was recorded exclusively in male rats for most
of the selected chemicals evaluated in 13-week toxicity studies by the NTP
and sometimes in the 2-year bioassays. In another study, Viau et al. (1986)
exposed male ratsto C10-C12 aliphatic hydrocarbons by inhalationfor 5.5,46,
or68 weeks and found granularcasts atthe earliest time-point, butthey were
absent at the later time-points. One explanation for these results is that
certain lesions in the sequence are transitory in nature. Granular casts, for
example, are assumed to be linked to the active hyaline droplet overload.
Once OL -g levels become low because of age, after approximately 18
months, the number of new hyaline droplets being formed should become
minimal. A second explanation is that subtle changes such as granular cast
formation and the associated tubule dilation can be obscured by the
development of CPN in later stages.
Tubule dilation is presumed to follow obstruction of the nephron by the
accumulation of granular casts composed of sloughed epithelial cell debris
in the tubule lumen. Figure 6 shows one example of the interrelationships
68
-------
Hyaline Droplets
Regenerating
Epithelium
Tubule Dilation
1 23
Weeks of treatment
Figure 6: Time-sequence for the development of hyaline droplets, regenerating tubule
epithelium, and tubule dilation, in male F344 rats administered 2 g/kg
unleaded gasoline daily by gavage for a 28-day period.
Source: Adapted from Thomas etal., 1985.
69
-------
observed between hyaline dropletformation, epithelial ceil proliferation, and
tubule dilation. In this study, male rats were administered unleaded gasoline
(2 o/ko/day) for a period of 28 days and examined at 5 interim time-posnts
Thomas et al., 1985). An initial accumulation of hyaline droplets, commenc-
ing on the first day of exposure and persisting throughout, was followed at
14,21, and 28 days, by increases in epithelial cell proliferation and tubule
dilation associated with lumenal accumulation of granular debris.
Linear mineralization in the renal papilla of male rats has been consis-
tently observed in a number of NTP and other 2-year bioassays with
potential CIGA carcinogens but not in the 13-week toxicity'Studies. Clear
dose-response relationships were demonstrated for 1,4-DCB (NTP, i98/a|,
JP-4 mixed distillate (MacNaughton and Uddin, 1984), and unleaded
aasoline (USEPA, 1987). In the 2-year unleaded gasoline study there were,
interim sacrifices at 3, 6,12, and 18 months permitting quantitative obser-
vation on the incidence of mineralization (USEPA, 1987). Although this
lesion was termed pelvic rather than medullary mineralization in the original
report from IRDC, it was qualified as referring to material located within
tubules of the renal pelvis, thus conforming to the medullary site seen with
otherCIGA. Table 11 presents a summary of these data which shows a clear
dose-related progression in the incidence of mineralization from 6 months
up to, and including, the 2-year sacrifice. Parallel dose-response increases
have been demonstrated for medullary mineralization and urothelial
hvperplasia with JP-5 jet fuels, Diesel Fuel Marine, and decalin (Bruner
1986), supporting the notion that the pelvic hyperplasia is a urothelial
response to mineralization in the papilla.
D. Association Between CIGA and Chronic Progressive Mephropathy
Although exacerbation of spontaneous CPN by CIGA has been noted
in many studies, quantitation of this response has been attempted on few
Table 11. Incidence of Medullary Mineralization in Male Rats During Inhalation
Exposure to Unleaded Gasoline
Exposure levels of unleaded
Observation
time-points
(months)
3
6
12
18
24
0
0
0
0
0
0
gasoline
67
0
0
0
0
5
vapor (ppm)
292
0
0
20
20
63
'
2056
0
20* ..,..
80
80
91
1 The incidence of medullary mineralization is reported as percent of animals affected:
Source: Adapted from USE PA, 1987.
70
-------
occasions. Short etal. (1989a) compared the number of CRN foci per kidney
section in male rats at three dose levels of unleaded gasoline exposure a^d
two chronic time-points, with control specimens. ^»**<*"*™£2
0 10 70 and 300 ppm unleaded gasoline, the numbers of foci observed at
22 weeks of exposure were 0.4, 0,1.0, and 6.3, respectively, and at 48
weeks of exposure, 5.0,4.0,10.0 and 9.0. This study, therefore, supports
the conclusion that there is an earlier onset of CPN, demonstrable by 5
months, and a higher incidence of disease in the middle- and high-dose
groups.
In the SMTP bioassay of d-limonene (NTP, 1990), treated male rats
showed a spectrum of compound-related kidney lesions, including exacer-
bation of CPN, mineralization in the renal medulla, hyperplasia of the
epithelium liningthe renal papilla, and proliferative lesions of the renal tubule
epithelium. The severity of CPN on a scale of 0 to 4 was graded as not
present, minimal, mild, moderate, or marked." The mean value increased
with increasing d-limonene dose from 1.5 in vehicle controls to 1.8 and 2.2
in animals dosed at 75 and 150 mg/kg, respectively.
As CPN is exacerbated by CIGA administration, and CPN-affected
tubules have a high cell turnover rate, it has been suggested that CPN may
play a role in renal tumor production following OLU-Q nephropathy because
enhanced regeneration is considered a risk factor for carcinogenesis
(Trumpetal., I984b; Short etal.. 1989b). There is no firm evidence available
to date that substantiates or disproves a link between CPN and renal tubule
tumor induction. Nevertheless, in a specialized initiation-promotion study
with unleaded gasoline and TMP, where the authors quantified foci of CPN,
some adenomas were described as arising within foci of CPN (Short et al.,
1989b).
E. Evidence Concerning Progression from Nephrotoxlclty to Renal
Neoplasla
For the eight selected CIGA carcinogens examined in this report, there
was an overall pattern indicative of dose-related increases in the incidences
of toxic nephropathy, hyperplasia, and renal tubule tumors in male rats. For
two CIGA unleaded gasoline and TMP, dose-related increases in renal
tubule proliferation were sustained throughout chronic administration. It is
believed that the likelihood of producing a cancerous cell is increased, not
only if there is a probability of a genetic transition, but also if the rate of cell
replication is increased (Cohen and Ellwein, 1990; Deal et al., 1989). Thus,
a sustained state of cell turnover in the target cell population as a mecha-
nistic link between a, -g nephropathy and renal neoplasia should be
considered a plausible, but unproven, explanation of the observed results.
The hyperplastic tubules and adenomas produced by CIGA carcino-
gens appear to arise from the cortex, which includes the P2 segment of the
proximal tubule, the main site of cellular injury in cc2u-g nephropathy,
providing further support for their linkage. Furthermore, studies that
examined cell regeneration in the different segments of the male rat kidney
have shown an increase in cell replication rates specifically in the histologi-
71
-------
cally damaged P2
pentachloroethane
line (Short et al.,
replication in female
nor in rats of both
(Goldsworthy et al.
Recent studies
egments after administration of tetrachioroethylene or
(Goldsworthy et al., 1988a) or TMP or unleaded gaso-
11987, 1989a). Under the same conditions, tubule cell
rats did not differ from controls in any of these studies,
sexes treated with a non-CIGA, trichloroethylene
1988a).
,-g nephropathy
(1991 b) demonstrs
male F344 rats, an
fold increase of P2-
of the promotion potential of d-limonene, TMP, and
gasoline also provijde convincing evidence to support a linkage between
and renal tubule neoplasia. Dietrich and Swenberg
ted that d-limonene promoted renal tubule tumors in
animal that produces cc2u-g. In addition, there was a 5-
abeling index in the F344 rats treated with d-limonene.
In contrast, no response was recorded for proliferation, hyperplasia, or renal
the NBR rat, an o,u-g-deficient animal which does not
develop the charao eristic nephropathy. These results substantiate those
of an earlier study where dose-related increases in atypical cell foci were
observed in male re is promoted with unleaded gasoline or TMP for 24 or 60
weeks (Short etal.,
in incidence of ren
1989b). In that study, there was a significant linear trend
I tubule tumors in the male rat promoted with unleaded
gasoline for 24 weeks. In contrast, none of these changes was observed in
similarly treated female rats.
Finally, the
bioassays of renal
to result from cell
whenever nephrotdx
i.e., for hexachloro
were not character
an independent me
nephrotoxicity seen in male rats in the selected 2-year
ubule carcinogens was characteristic of that proposed
damage caused by oc2u-g accumulation. In contrast,
icity was observed in female rats, or mice of either sex,
ithane, tetrachloroethylene, and 1,4-DCB, the lesions
stic of GIGA and probably were a response caused by
chanism.
72
-------
Part 3 - Evaluation ©I the Hypothesis
XI. Summary of the Evidence on the Renal Effects
Several lines of evidence establish an association between exposure of
of GIGA
accumulation (GIGA) and
association between this
the male rat to chemicals that induce a2u-g
nephrotoxicity, and strongly support an
nephrotoxicity and renal tubule tumors.
A. Association Between Alpha^-globUlln Accumulation and
Nephrotoxicity
The information that supports an association between ex -g accumula-
tion and male rat-specific renal toxicity following GIGA administration is
summarized below.
Although hyaline droplet accumulation per se is not necessarily
diagnostic of a GIGA until proven to represent a2ui 3 accumulation,
34 organic compounds including fuels, solvents, and other
chemicals (listed in Table A-1), examined in this report, have
been shown to induce an excessive accumulation of hyaline
droplets in the renal proximal tubule epithelium of male rats. In
contrast, where tested, mice and female rats showed no
evidence of hyaline droplet accumulation from chemical
treatment.
There is convincing evidence that the excessivfe accumulation
of hyaline droplets is followed sequentially by t Jbule epithelial
cell necrosis, granular cast formation, and other aspects of
a2u-g nephropathy in the male rat. Five of the 34 hyaline-droplet
inducers were tested in species otherthan the mouse orthe rat,
although possibly not as rigorously. Characteristic lesions
were observed in the male rat kidney for these five substances,
but there was no apparent nephrotoxic response in the female
rat or any other species tested, which included mice (all 5
substances), hamsters (jet fuels), guinea pigs (decalin), dogs
(jet fuels, decalin, d-limonene, and methyl isobutyl ketone), and
monkeys (methyl isobutyl ketone and gasoline;).
The increase in hyaline droplets, tubule dilation caused by
granular cast formation, tubule cell proliferation, and medullary
mineralization is dose-dependent as shown by research studies
conducted to date with four model GIGA, (decajin, d-limonene,
unleaded gasoline, and TMP). j
In general, the chronic administration of GIGA tp male rats and
the ensuing nephrotoxicity enhanced the agio-related renal
degenerative process by exacerbating spontaneous CRN.
73
-------
Specialized studies involving rats of varying age, castrated or
estrogen-treated rats, the NBR strain, and o^-g-treated female
rats have shown that development of the early features of
o^-g nephropathy is dependent on the presence of o^-g
formed in the liver.
• For three of the eight model carcinogens (hexachloroethane,
tetrachloroethylene, and 1,4-DCB), renal toxicity was observed
in chronic studies of female rats or mice, but the renal toxicity
appeared to be less severe orqualitatively different, not involving
the same spectrum of discrete lesions associated with o,u-g
nephropathy.
• GIGA bind reversibly to o,u-g as atarget molecule, andthe renal
accumulation of a,u-g and hyaline droplet formation may be
explained by chemically induced impairment of cc^-g catabolism
after reabsorption of the complex by the proximal tubule.
• TMPOH, the active metabolite of IMP may be able to form in
vitro complexes with retinol-binding protein and a,-acid
glycoprotein, members of the lipocalin superfamily found in
humans. Invivodataonretinolandoc^-g, however,demonstrate
that such an association does not necessarily lead to a,u-g
accumulation or hyaline droplet formation.
EL Association Between Nephrotoxicity and Renal Cancer
Based on information from the rodent bioassays examined in this report
and additional key data, features of renal tumors occurring subsequent to
the development of nephropathy in the male rat can be identified.
• The eight model carcinogens produced hyperplasia, adenomas,
and adenocarcinomas in the renal tubule of the male rat.
All eight that produced renal tumors in male rats also produced
nephrotoxicity in male rats.
• Specifically, the nephrotoxicity that preceded renal tumor
formation in male rats was characteristic of the form associated
with a2u-g and distinguishable from other forms of toxicity
associated with non-CIGA renal toxicants.
• The incidence of renal tumors produced in the male rat by the
eight model carcinogens was relatively low. These tumors
were morphologically indistinguishable from other chemically
induced renal tubule neoplasia and renal tubule neoplasia that
occurs rarely, but spontaneously, in male and female rats.
The renal tumors produced by the eight model carcinogens
occurred late, usually being found at the time of sacrifice,
metastasized rarely, and were not life-threatening.
For d-limonene, the one CIGA examined in an initiation-
promotion study comparing male rats of the NBR strain with a
conventional strain, «2u-g accumulation was necessary for
promotion of male rat renal tubule tumors initiated by EHEN.
74
-------
• CIGA appear to be nongenotoxic or only marginally so and, ,
therefore, may not depend on direct genetic injury as the
mechanism for tumor induction.
« Trichloroethylene, a compound structurally similar to
hexachloroethane and tetrachloroethyiene produced renal
.tumors apparently by mechanisms, such as cpvalent binding to
DNA, which do not appear applicable to the.CIGA hypothesis.
C. Intpmiattori Reducing Confkferico In the Conclusion that the
Alpha2a-glQ$3utiF$ Is 'Specific to the. Mai@ Rat
Although the evidence available to date supports the hypothesized
association between a2u-g accumulation and renal tubule tumors in the male
rat, confidence in this assertion would be improved if the same results were
found in an expanded database. In addition, the paucity of data on the
lipocalin superfamily, in general, leaves several questions unanswered
regarding the specificity of the response to the male rat.
Pathological accumulation of hyaline droplets is a reaction to
excessive protein load not exclusively related to cc2}J-g
accumulation. Although there are 34 hyaline droplet-inducing
compounds identified inTable A-1 of this report, the accumulating
protein responsible for hyaline droplet formation has been
identified for only 17 of these compounds.
. •.:. Data sufficient to demonstrate interdependence of the lesions
, , in the proposed pathological sequence from hyaline droplet
accumulationto chronic toxicity exist foronly a few substances.
Data to define dose-response relationships for tubule cell
necrosis and its association with cell proliferation are even
more limited, as is dose-related information on increased cell
proliferation rates over chronic exposure periods.
• The mechanism whereby a2u-g accumulation leads to cell
death has not been established.
• Hexachloroethane.tetrachloroethylene, and 1,,4-DGB produced
renal toxicity in female rats or mice indicating that some CIGA
may have additional effects on rodent kidney not limited to the
. a2 -g-induced sequence of lesions.
•'- :- Information on a possible association between renal cell tumors
and CIGA exposure in humans is inconclusive since exposures
in the reviewed epidemiologic studies have been to both CIGA
and non-ClGA compounds.
• Information on the in vivo binding of CIGA with other lipocalins
in the ot2u-g superfamiiy of proteins suggests, but does not
conclusively demonstrate, that toxicity in humans could not
occur via this mechanism.
« Althoughthere are major quantitative and qualitative differences
between male rats and humans in the amounts of protein
excreted in urine, little is known concerning the relative quantities
of low-molecular-weight proteins that are normally filtered by
the human glomerulus and reabsorbed by the renal tubules for
catabolism.
75
-------
The scientific data summarized above were used to draw conclusions
concerning the role of a, -g accumulation and hyaline droplet formation in
producing male rat-specific nephropathy and renal tubule neoplasia, and to
determine the relevance of this information to assessing human risk.
XII. Conclusions
The available information on CIGA-associated renal tubule
carcinogenesis in the male rat can be described by a suggested sequence
of critical cellular and molecular events. According to this description, the
reaction of a lipophilic compound with the low-molecular-weight protein,
a, -g, appears to lead to the formation of a complex that is more resistant to
lysosomal degradation than the unreacted protein. This results in a shift in
balance between reabsorption and hydrolysis leading to an abnormal
accumulation of the protein in the P2 segment of the renal tubule of male
rats. If exposure ceases after a short time period, recovery is. complete.
Continued exposure, however, results in a nephrotoxic response that is less
readily reversible and a sustained increase in cell turnover, enhancing the
chance that molecular alterations in DNA occurring in the kidney may be
replicated rather than repaired.
Because there are substantial data gaps, especially with regard to the
expected response in humans and the critical linkages between single-cell
necrosis and increased cell turnover, and tubule hyperplasia and renal
tubule cancer, the a2u-g syndrome should be considered a satisfactory
working hypothesis but not a proven mechanism of action to describe renal
tubule cancer in male rats exposed to GIGA. As such, it provides an
empirical description of a series of observed events in laboratory animals
which could be modified or expanded upon as additional information
becomes available.
Despite these limitations and the fact that
-------
have been administered a, -g isolated from male rat urine. NBR rats which
do not possess the mRNA for liver o,u-g, and castrated male rats, also
respond differently from conventional male rats. Of the other species, the
mouse is the most thoroughly tested. Although the mouse produces large
amounts of a structurally similar lipocalin, MUP, this protein is not known to
bind with CIGA; it is not reabsorbed from the urine; and the mouse does not
develop kidney tumors or the characteristic nephropathy seen in male rats.
Limited testing in dogs, hamsters, guinea pigs and monkeys has not shown
hyaline droplet accumulation or nephropathy in these species, further
suggesting that the o^-g syndrome occurs specifically in the male rat.
With regard to the potential for a chemical to produce renal tubule
neoplasia in the male rat, there are common characteristics among the
substances evaluated in this report. First, these compounds (and their
CIGA-binding metabolites) possess little or no mutagenic activity in stan-
dard batteries of tests, they are lipophiles and not electrophilic substances,
and they do not appear to bind covalently to DNA. Second, the nephrotoxic
response characteristic of CIGA always preceded renal tumor formation in
the male rat, a finding not characteristic of classical renal carcinogens.
Third, for all eight model compounds examined in this report, additional
sexes/species/strains were tested, and the increased incidence of renal
tumors was found only in the male rat.
the manner in which the human male responds to CIGA has not been
tested directly although there are human proteins that, like cc^-g, are
members of the lipocalin superfamily. Human urine also contains small
amounts of a sex-linked urinary protein. Epidemiologic studies have
focused on glomerulonephritis or renal cancer and organic chemical expo-
sure, in general, and not on renal tubule damage and CIGA exposure, and
they do not yield results useful for testing the hypothesized mechanism in
humans. Protein overload can result in formation of hyaline droplets in
human kidneys, although there is no evidence that this response has
occurred from lipocalin accumulation. While it is not possible to resolve the
issue of how the human renal tubule responds to CIGA exposure from the
available data, the uniqueness of the male rat response among the tested
laboratory species and the high doses needed to produce an effect, even in
the male rat, suggest that this reaction would not occur in humans, especially
under typical conditions of exposure.
Several factors complicate the analysis of data on the renal effects of
CIGA. For the compounds examined to date, not all of the administered
substance has been found to bind to oL-g. Thus additional CIGA/CIGA
metabolites potentially exist in the kidney along with the a -g-bound
material. The possibility that these other moieties are toxic to the kidney
needs to be taken into account. For example, tetrachloroethylene, in
addition to showing a2u-g nephropathy, displays evidence of renal toxicity
typical of chlorinated hydrocarbons. This example demonstrates how other
mechanisms may play some role in the observed results.
At present, there is insufficient information on CIGA and their metabo-
lites to confident!/ predict activity on the basis of structural analogy. Recent
77
-------
research on structural correlations suggests that the presence of an electro-
negative atom for hydrogen bonding, lipophilicity, and steric volume are
important considerations. Conformational changes or other structural
alterations to the protein may also be necessary since binding of the
compound in the protein pocket, alone, appears to be an insufficient
condition to cause reduced digestibility of the protein.
Evidence of dose-responsiveness between CIGA administration and
the degree of hyaline droplet or aau-g formation has been demonstrated in
several studies. However, these findings are frequently based on subjective
histopathological criteria, limiting their usefulness for making quantitative
judgments about the relative hazard potential of different chemicals.
It is also important to recognize that for various reasons (e.g., doses
administered too low, animals killed before the latency period of these slow-
growing tumors is attained, number of specimens and histological sections
insufficient, competing toxicity in kidney or other organs), the entire patho-
logical sequence culminating in renal tubule neoplasia may not be demon-
strated in all cases of CIGA administration. Thus, not all CIGA would be
expected to demonstrate renal tubule neoplasia in the male rat in a 2-year
animal bioassay. Such a finding would not negate the applicability of the
hypothesized CIGA syndrome to the evaluation of nephropathy data.
Based on the cancer bioassays and other research studies of CIGA, an
increased prolif erative response caused by chemically induced cytotoxicity
appears to play a role in the development of renal tubule tumors seen
exclusively in male rats. The male rat specificity of the response to CIGA
administration is emphasized by negative findings in mice and female rats.
These conclusions can probably be extended to analysis of human hazard
potential, especially whenever human exposure to CIGA is not excessively
high for sustained periods of time, when short-term tests for genotoxicity of
the compound are negative, when the nephrotoxic response and increased
cell turnover characteristic of CIGA have been demonstrated in the male rat,
and other species/sex combinations were tested but renal tubule tumors
were observed only in male rats.
XIII. Research Needs
Certain studies, suggested to fill key data gaps, are listed below. There
has been no attemptto outline all the possible avenues for research on CIGA
and on lipocalins, since a vast array of useful experiments could be
envisioned. Instead, recommended studies would greatly improve the
database on these chemicals, provide needed information to answer
questions of human relevance, and set up a framework for improving the
testing of chemicals that are potentially male rat renal tubule tumorigens.
These research needs are listed as follows:
'.• Extend studies in humans, wherever possible, to determine
directly the effects of hydrocarbon and solvent exposure,
focusing on specific jobs with relatively pure CIGA exposure.
Any human renal pathology found should be compared with
78
-------
Oau-Q nephropathy in the male rat, and urine should be examined
for the presence of cells and casts since this noninvasive
technique is readily applied to humans.
Examine human subpopulaiions that excrete abnormal amounts
of low-molecular-weighf protein in the urine to determine if they
are at risk of renal disease or renal cell cancer.
Examine additional active GIGA metabolites for binding to
lipocalins, such as retinol-binding protein, a,-acid glycoprotein,
and urine protein 1. If there is binding, determine if the protein
complex has a slower degradation rate.
Thoroughly characterize any protein droplet nephrotoxicity
observed as the result of administering known CIGA (e.g.,
d-Iimonene, TMP) to additional species (e.g., dog, hamster,
rabbit, guinea pig, and especially nonhuman primate).
Develop a standardized short-term protocol (e.g., in the 2-week
subacute study) that will detect any abnormal accumulation of
hyaline droplets in the male rat kidney before suspected CIGA
are placed on chronic study. (If hyaline droplet accumulation is
found, this information should betaken into account in designing
the bioassay.)
Further characterize the response of the NBR rat, which does
not appear to synthesize azu-g, to CIGA and to classical renal
carcinogens. These studies should verify, in a two-year chronic
bioassay, that the NBR rat kidney is responsive to classical
renal carcinogens already tested in other strains, and they
should evaluate the suitability of this strain of rat as a test
species. If the NBR rat meets these two criteria, the possibility
of employing a separate test group, consisting of male NBR
rats, should be considered for conventional bioassays whenever
excessive hyaline droplet formation has occurred in shorter-
term tests.
Conduct serial-sacrifice studies of CIGA and non-CIGA renal
carcinogens to determine if a distinctly different progression
from a^g nephropathy to tumor formation can be seen for
CIGA. Studies should involve chronic exposures, examine the
histogenesis of the renal tubule tumors, and include "stop"
experiments and time-dependent appearance of tumor markers.
Perform dose-response studies designed to quantitate the
relationship between increased hyaline droplets and ceil necrosis
and between cell necrosis and cell regeneration. In addition,
explore the possibility of additional steps in the progression that
might further define the expression of cancer in the male rat and
the cause of cell death.
Conduct metabolism and disposition studies of CIGA in other
species, compared with male rats, to determine the causative
chemical for the nephropathy, and to clarify sites of
biotransformation and deposition and fate of these compounds.
79
-------
Additional work, not as critical asthe above, butwhteh would also assist
in understanding this disease process, includes the following:
• Identify the accumulating material contained in hyaline droplets
ofproximaltubulesforchernicalsthatareapparent.butunverrfied
CIGA and conduct 2-year bioassays for decalin and TMP.
- Perform additional in vitro assays using rodent kidney extracts
to more specifically determine mutagenic potential of GIGA (or
• Conduct studies on the genesis of CPN and its relationship to
OL, -g nephropathy and examine the possible role of CPN as a
cocarcinogenic factor for renal tumor induction. H
• Obtain more information on the renal catabolism of cyg ana
the rate and efficiency of protease-mediated hydrolysis in
control and CIGA-treated rats.
• Study the binding relationships between CIGA and a,u-g (e.g.
affinity, concentration ranges, binding effectors) and determine
the site at which binding of CIGA to o^-g occurs (e.g., liver,
plasma, or urine) to investigate the hypothesis that the protein-
CIGA complex is only formed at high concentrations of the
ch6ffliCcii
• Determine the reasons why the amount of low-molecular-
weight protein in human urine is much less than it is in male rats.
80
-------
Part 4. Selenee Policy
XIV. Background and introduction
An increased incidence of neoplasms in laboratory animals adminis-
tered test chemicals is customarily viewed by scientists as an indication of
carcinogenicity in animals and as some signal that .humans may be similarly
affected. From this line of reasoning, EPA generally presumes that animal
tumor findings indicate there may be a cancer hazard to humans, although
a final judgment as to human carcinogenic potential can be made only in
relation to all other relevant information. Recent studies suggest, however,
that tumors produced in the tubule of the male rat kidney following the
accumulation of alpha2u-globulin (a,-g) might involve a process that occurs
only in the male rat. Because of the implications to cancer risk assessment,
the Risk Assessment Forum (RAF) established a Technical Panel to
examine the available information on o:2u-g accumulation in the kidney,
associated renal disease, and kidney cancer. The scientific data supporting
the Technical Panel's conclusions regarding the cL0-g sequence of lesions5
are covered in depth in the preceding sections (Parts 1 through 3) of this
document.
Part 4 provides guidance to EPA risk assessors regarding evaluation of
male rat kidney tumors and presents RAF conclusions regarding potential
human hazard and risk for a special subset of these tumors, that is, renal
tubule tumors in the male rat resulting from chemically induced o,u-g
accumulation. Criteria for demonstrating this relationship are set forth below
for use and discussion in all EPA assessments in which data on renal tubule
tumors in the male rat are used to assess human risk.
XV. Basis for Science PoSIcy on Male Rat Kidney Tumors
The information that follows highlights critical data and outlines inferen-
tial bridges used to select the most plausible explanation forthe information
available on male rat kidney tumors.
A. Low-Molecular-W@ighf Proteins in the Rat
In rat kidneys, as in those of other mammals, naturally occurring low-
molecular-weight proteins are transferred from the plasma into the urine by
giomerular filtration. The proteins are then partially reabsorbed from the
urine into the renal tubule of the kidney where they are eventually broken
down by catabolism (see section III-A). One of these low-molecular-weight
proteins, «2u-g produced by the liver under the stimulus of testosterone,
In this report, lesion is a morphological alteration, due to disease.
81
-------
reaches very high levels in the plasma and urine of young adult male rats,
gradually declining with age.
Alpha, -globulin is regarded as a member of a large superfamily of
proteins thought to be carriers of lipophilic molecules (see section IH-D).
Some of these proteins, e.g., retinol-binding protein and a,-acid glycopro-
tein, are found in many species, including humans. Others, like o,0-g, are
found in specific species. The only member of the superfamily with a clearly
defined physiological role is retinol-binding protein, the carrier protein for
vitamin A. Although these low-molecular-weight proteins are believed to
have similar three-dimensional structures, the alignment of amino acid
residues between any pair of proteins in the superfamily is small, roughly 20
percent. The exception is a2u-g and mouse major urinary protein(s) (MUP)
which are approximately 90 percent homologous.
AIpha2u-globulin derived from hepatic synthesis is not known to occur in
the female"rat or any other species, including humans. Although similar
forms of a, -g are synthesized at nonhepatic sites in female rats and in the
male NCI "Black Reiter (NBR) rat, a strain whose males lack hepatic
synthesis of o,u-g, none of these other forms of cc2u-g nor M UP accumulates
in the renal tubule following administration of the compounds discussed in
Parts 1 through 3.
B. Progression from Chemically Induced Alphasu-gl0bulln
Accumulation to Nephropathy and Neoplasla
1. Overview
The information available provides a plausible, although probably
incomplete, picture of a sequence of events occurring in the male rat kidney
following chemical administration. This sequence can be portrayed on a
cellular and molecular level. Initially, the test chemical appears to bind
reversibly to o,u-g, seemingly forming a complex more resistant to lyspsomal
degradation than the unreacted protein itself (see section III-H). This shifts
the balance between reabsorption and catabolism and appears to result in
accumulation of the protein complex in a specific area of the renal tubule, the
P2 segment. Continued compound administration results in a cytotoxic
response from the sustained protein overload to the renal tubule, causing
single-cell necrosis of cells lining the surface of the tubule and other kidney
pathology. The dead cells are replaced by cell division. As the cycle of cell
death and cell replacement continues, with time tubule hyperplasia (in-
crease in number of cells) and neoplasia may occur. It is presumed, but
certainly not proven, that continued cell proliferation plays a role in the
neoplastic process.
Morphologically, the sequence of events begins with an increase in
the number and size of hyaline droplets6 containing a2u-g. The next
6 Spherical inclusions in the cytoplasm that may contain various proteins (see section
B).
82
-------
characteristic lesion, single-cell necrosis in the renal tubule, may not be
seen but can be confirmed by observation of exfoliated degenerate cells in
the tubule lumen7 and granular casts.8 Enhanced cell replication in response
to cell death can be seen as increased cell division or demonstrated by
labeling techniques that measure increased DNA synthesis. In chronic
laboratory animal bioassays, tubule hyperplasia, linear mineralization in the
renal papilla (possibly representing remnants of debris from disintegrating
granular casts), and renal tubule tumors are observed. •
2. Specificity of the sequence to the male rat
Consistent results from hypothesis-testing experiments conducted over
the last decade in various laboratories establish the association between the
accumulation of abnormal amounts of oc2u-g in the P2 segment of the renal
tubules and a specific form of kidney disease, and they support an associa-
tion between this nephropathic response and renal tubule tumors. Specifi-
cally, the male rat responds to administration of oc2u-g inducers with a
characteristic nephropathy. The severity of this kidney disease is dose-
dependent, not only with respect to the amount of compound administered,
but also with respect to the concentration ofa,u-g in the kidney. This alpha2u-
gtobulin nephropathy differs sufficiently from chronic progressive nephropathy
(see section IV-E) commonlyfound spontaneously in aging male rats so that
the two effects can be distinguished. In contrast, mice and female rats
administered
-------
The specificity of the male rat response has been tested to a limited
extent in a number of other species, with no evidence of hyaline droplet
nephropathy in dogs, guinea pigs, hamsters, or monkeys. Smcethese
species (and the mouse and female rat) have proteins similar in structure to
oL-g the lack of damage to their kidney cells is consistent with the
presumption that the specific CLu-g produced by the liver of male rats is
necessary for the expression of the renal effects.
Male rats of the NBR strain provide a unique opportunity for testing the
OL -a hypothesis since this animal has no detectable levels of hepatic
messenger RNA for a2u-g. Under conditions of exposure that produced
a, -a nephropathy in male rats of other strains, several chemicals adminis-
tered to the NBR rat did not induce detectable accumulation of «2u-g in the
renal tubules.
Mice and female rats exposed to a2u-g-inducers in chronic bioassays did
not develop an increased incidence of renal tubule tumors. In contrast, male
rats developed a dose-dependent neopiastic response in the kidney.
Additional experimentation using a nitrosamine as the initiator of cancer and
an OL -g-inducer as the promoter also support the observation that a,u-g is
involved in the process leading to renal tubule tumors in the male rat. Irfone
of these studies, the promotion potential of an c^-g-inducer in NBR rats was
contrasted with the response in a conventional strain, the F344 rat. Consis-
tent with the hypothesis that o^-g is necessary to induce a response, the
promoter did not enhance renal tubule tumor formation in the a,u-g-deficient
NBR rat, but it did promote renal tubule tumor formation in the F344 rat.
It is clear that not all renal tubule cancer in laboratory animals occurs
through the hypothesized a2u-g sequence. Other inducers of rodent renal
tubule cancer are well known. These include, for example, certain
nitrosamines in the rat and mouse and diethylstilbestrol in hamsters. In
general these prototypic renal carcinogens are active in both males and
females. The acute nephrotoxic changes in the renal tubules include mild
lipid droplet accumulation and scattered single-cell necrosis, but hyaline
droplet accumulation and its specific associated nephropathy are not
characteristic.
Based on available information, a2u-g-inducers appear to have addi-
tional features that distinguish them from other rodent kidney carcinogens,
such as the nitrosamines. Alpha2u-globulin inducers appear to be
nongenotoxic, or only marginally so, suggesting that the mechanism for
tumor induction does not depend on direct genetic injury. So far, the
incidence of renal tumors produced in the male rat by o^-g inducers has
been relatively low, occurring late in life, and metastasizing rarely. In
contrast, certain rodent carcinogens induce a high incidence of kidney
tumors after as little as a single dose.
Distribution studies of compounds and information on chemical binding
to CL -g indicate that, of the total chemical administered to the animal, only
a small portion of the metabolites (possibly the parent compound) can
account for all of the a2u-g accumulation. Considerable amounts of the
84
-------
chemical and other metabolites may also be present in the male rat kidney,
not bound to o,u-g. These other moieties may, at times, cause toxic effects
in the kidney, possibly even cancer, that are unrelated to the accumulation
of o,u-g. Such information does not preclude a determination that the
a,u-g sequence is involved in some manner with the renal tumor response.
XVI. Science Policy Statement
Based on the analysis of the scientific literature in Parts I through III, the
RAF Technical Panel reached three major conclusions. First, the sequence
of events proposed to link oLu-g accumulation to nephropathy and renal
tubule tumors in the male rat is plausible, although not totally proven.
Second, the o,u-g response following chemical administration appears
to be unique to the male rat. Even though closely related proteins are
present in other species, there is no evidence that these species respond to
-g inducers in a manner similar to the male rat.
Third, the male rat kidney response to chemicals that induce ct^-g
accumulation is probably not relevant to humans for purposes of nsk
assessment.
The RAF Technical Panel's findings provide the basis for a two part EPA
science policy statement regarding use of data on male rat renal tubule
tumors for human risk assessment. This science policy applies to individual
chemicals or chemical mixtures.
(1) Male rat renal tubule tumors arising as a result of a process
involving a2u-g accumulation do not contribute to the
qualitative weight-of-evidence that a chemical poses a
human carcinogenic hazard. Such tumors are not included
in dose-response extrapolations for the estimation of
human carcinogenic risk.
(2) If a chemical induces o,u-g accumulation in male rats, the
associated nephropathy is not used as an endpoint for
determining non-carcinogenic hazard. Estimates of non-
carcinogenic risk are based on other endpoints.
Even when chemically induced a2u-g-related kidney tumors are
present, other tumors in the male rat and any tumor in other exposed
laboratory animals may be important in evaluating the carcinogenic poten-
tial of the chemical. Likewise, the role of chemically induced a2)J-g accumu-
lation in the induction of renal tubule tumors in the male rat is assessed
independently of evaluations made regarding tumors at other sites.
XVSi. Guidance for Evaluating Chemically Induced Male Rat Renal
Tubule Tumors
To determine the appropriate use of the data for EPA risk assessments,
chemicals inducing renal tubule tumors in the male ratare examined interms
of three categories.
(1) The oc2u-g sequence of events accounts for the renal
tumors.
85
-------
(2) Other potential carcinogenic processes account for the renal
Y3V The a, -g-associated events occur in the presence of other
potentfal carcinogenic processes, both of which result in renal :
tumors.
Two questions need to be answered. The first and simplest question is
whether or not the a2u-g process is involved in the tumor deye opment The
• second moredifficullquestion, given an affirmative answer to the firsj .s;the
extent to which ^-g-associated events, rather than other processes,
account for the tumor increase.
A determination of the extent to which the a^u-g process is involved in
tumor development requires a substantial database, and not just a limited
set of information confined to the male rat. For example cancer bioassay
data are needed from the mouse and the female rat to be able to demon-
strate that the renal tumors are male-rat specific. Even to answer the first
question affirmatively, information from toxicity stud.es mus ^monstrato
whether or not the OL -g process is operative (see section XVII-A below). In
the absence of this minimum information, there is no basis for judging the
aDDlicabilitv of the a -g process, and it would be assumed that the male rat
XfS Additional data are
necessary (see section XVII-B below) to.answer the second question and
to assign a chemical to categories 1, 2, or 3. .
A. -Renal nibule Tumors In Male Rats and Alphas-globulin
Accumulation
The following information from adequately conducted studies of male
rats shows that the a -g process could be a factor in the observed renal
effects; a*affirmative response in each of the three categories is required
If data do not meet the criteria in any one category, the available renal tumor
data should be analyzed in accordance with standard risk assessment
principles.. . .....'..... : :
- (1) Increased number and size of hyaline droplets in renal proximal
tubule cells of treated male rats •
..'•" the abnormal accumulation of hyaline droplets in the P2 segment of the
renal tubule is necessary to attribute the renal tubule tumors to the
-------
Typical lesions include: single-cell necrosis, exfoliationof epithelial cells
into the proximal tubular lumen, formation of granular casts, linear mineral-
ization of papillary tubules, and tubule hyperpiasia. if the response is mild,
all of these lesions may not be observed; however, some elements! consis-
tent with the pathological sequence must be demonstrated to be pi resent.
S. Additional Information Useful for the Analysis
If the preceding analysis (section XVII-A) indicates that the o,u-g
process is operative, then other information is reviewed to determine if the
renal effects are solely a,u-g-associated, a combination of the a, -g process
and other potential carcinogenic processes, or due primarily to other
processes. Many kinds of information can assist in confirming that chemi-
cally induced cc2u-g accumulation is involved in the renal tumor response or
that other processes cannot be ruled out. Some of these findings a|re listed
below; the information may not always be available, nor should this list be
considered exhaustive. :
Hypothesis-testing data: Data from specialized tests can; greatly
increase confidence that the o2u-g sequence is involved in the rendil tubule
tumor response. Such information might include: modification of the
nephrotoxic response through use of the NBR rat, or manipulation of sex
hormones (e.g., androgens) or a,u-g levels (e.g., a2u-g administration to
female rats). Other information might include initiation-pro motion; studies
comparing males of the NBR strain with males of other rat strains:
Additional biochemical information: Certain in vivo and in vitro data help
characterize a chemical as one that induces accumulation of cc2u-g. Such
information might include: reversible binding of the chemical (or ijnetabo-
lites) to o^-g, reduction in the lysosomal degradation of the a.u-g-cpmplex,
and disposition studies demonstrating sex- and species-specific retention of
the test compound in the male rat kidney. !
Sustained celldivision in the proximal tubule of the male rat: A sustained
increase in cell replication in the P2 segment of the renal tubule at doses
used in the cancer bioassay and a dose-related increase in Atypical
hyperpiasia of the renal tubule is consistent with the oc2u-g process, espe-
cially if other laboratory animals were tested and did not show; similar
responses. These endpoints are nonspecific for a_u-g-inducers, however,
since other renal carcinogens may also affect the P2 segment of the renal
tubule.
Structure-activity relationships: Structure-activity relationshipsfo;r chemi-
cals that induce a?u-g accumulation in the male rat kidney are not well
defined, although there appear to be dimensional requirements to fit the
protein pocket, a requirement for a degree of lipophilicity, and a need for an
electronegative atom in the molecule or its active metabolite.; Other
structural features might suggest that a chemical belongs to a different class
of suspected carcinogens. •
Covalent binding to macromolecules: Some inducers of renal tubule
cancer in rodents (e.g., nitrosamines) are known to bind covalently'to DNA
87
-------
or other macromolecules. Others do not appear to bind to DNA (e.g.,
isophprone) suggesting that such information may assist in distinguishing
different processes leading to renal cancer.
Genotoxicity: Although renal tubule neoplasia associated with clearly
qenotoxic chemicals is a well known response, information to date supports
a conclusion that a, -g inducers are essentially nongenptoxic and do not
depend on direct genetic injury for the production of tumors. Thus,
information on potential genotoxicity in a standard battery of short-term tests
relevant to the evaluation of potential carcinogenicity provides a possible
device for helping to distinguish between these processes.
Nephrotoxicity: Chronic progressive nephropathy (CRN) in the aging
male rat can complicate the analysis of other renal lesions. However,
nephrbtoxicity in the male rat not attributable to either CPN or ct^-g
accumulation, or a nephrotoxic response in the female rat or the mouse,
suggests that the possibility of other processes leading to renal cancer
should be considered.
Animal bioassay data in other species-, sex-combinations: The a,u-g-
syndrbme is specific to the male rat. Positive cancer responses in the renal
tubule' in female rats, mice of either sex, or any other laboratory animal imply
that ttjie oLu-g syndrome alone does not account for the renal tubule tumor
response in.the male rats.
Confidence in determining which of the three categories applies de-
pends on the comprehensiveness and consistency of available data. If all
the data (two species, two sex combination bioassay, all elements in xvn-
A and additional information such as that described in XVII-B) are consis-
tent with a role for chemically induced a^-Q. there is a high degree of
confidence that the a,-g syndrome, alone, accounts for the renal tubule
tumors. In contrast, if information from adequate testing is inconsistent with
the a, -g syndrome (e.g., renal tubule tumors are present in female rats or
miceTother carcinogenic processes probably account for all or most of the.
renal Itumors. Sometimes, the information will indicate that more than one
carcinogenic process is occurring; in these cases, as a minimum, the criteria
in subport of a, -g involvement (section XVII-A) are present, but there is also
evidence consistent with other mechanisms. Decisions on the applicability
of the' three categories can only be made on a case-by-case basis, taking
ail of the information into account. Whatever the finding, the risk assessor
should clearly delineate and thoroughly document the basis for any deci-
sions! made.
C. Use of the Data for Risk Assessment
Once a decision on the applicability of the three categories is made, it
becomes possible to determine how the response in the male rat renal
tubule would apply to evaluating human hazard and to estimating human
cancer risk. In general, the following guidance applies, recognizing that
tumors occurring at other sites in laboratory animals administered com-
pounjdsthat induce cc^-g accumulation in the male rat will be judged on their
own merits.
88
-------
Compounds producing renal tubuie tumors in maie rats attribut-
able solely to chemically induced o^-g accumulation: these renal tubule
tumors will not be used for human cancer hazard identification or for dose-
response extrapolations.
Compounds producing renal tubule tumors that are not linked to
o^-g accumulation: these renal tubule tumors are an appropriate endpoint
for human hazard identification and are considered, along with other
appropriate endpoints, for quantitative risk estimation.
Compounds producing some renal tubuie tumors In male rats
attributable to the a,-g process and some attributable to other carci-
nogenic processes: In general, the information needed to make a quanti-
tative determination of the relative contribution of each process to tumor
development will not be available. Thus, even though the information on the
renal tubule tumors remains relevant for purposes of hazard identification,
a meaningful dose-response estimate based on renal tubule tumors in the
male rat is generally not possible and should not be performed. If there is
enough information to determine the relative contribution of each process to
the overall renal tubule cancer response in male rats, the non-o,u-g-induced
component may be used, as appropriate, for dose-response evaluation as
well as hazard identification.
XVIIE. Nephropathy as a Toxic Endpoint
If a compound induces oc2 -g accumulation in hyaline droplets, the
associated nephropathy in male rats is not an appropriate endpoint to
determine noncancer (systemic) effects potentially occurring in humans.
Likewise, quantitative estimates of noncancer risk (e.g., reference doses
and margin-of-exposure determinations) are based on other endpoints.
It should not be anticipated that a compound that produces nephropathy
in the male rat through the sequence of events beginning with the accumu-
lation of oL^-g will always be found to induce renal tubule tumors in the male
rat. The aoility to detect renal tumors depends on many features that may
not be present in any individual experiment, e.g., sufficient dose to induce
effect without early deaths of the animals, competing toxicity from other
moieties not bound to (X,u-g, insufficient length of exposure or followup, and
incomplete histopathology. Even in the absence of renal tubule tumors in
the male rat, if the sequence of lesions characteristic of the ct^-g syndrome
are present, the associated nephropathy in the male rat does not contribute
to determinations of noncarcinogenic hazard or risk.
89
-------
•s
o
if
5*
-g.
S
|cl
•§£•£
f!
o-oT»
*J!
8|,8
g£*
Sog
uj S
S
I
|
OT
§
0
s
i
cc
z
cc
tr
^J T-
O> "^ Co
« 5 ,
D. DO C5
S =
I? I I
°1 f «
ii zf.s
S 5 *> a e
? n ro eft ra
mO 2C.S
oc
o
.S
-------
«9
1 §
i e
. « S3
fc 3
a jo
— ^
5^
O (8
1|
§
.
•'• : .
.
03 "^
§ Jl
»-6«
•2=1
§1.2
l°i
Substance/Chemical
8
M
g
0
cc
§
1
£
1
CO
S
&
i
e
8
«
i
u.
§
CO .
DC
CC
Z
ig
13
I
1
If-
It
.
4-
RJ-5 synthetic jet fuel
(hydrogenated dimers
of norbomadiene)
DC
cc
z
•S
JZ
15 :
i.
c
t— O>
&_ *^»*
III
1
+
JP-7 distillate jet fuel
/
or
z
cc
z
"8
-s
•s
Q.
C
I 1
i_ ~^
111
•.
+
JP-TS distillate jet fuel
-
S
CD
rt"
"«
ia
^TO
i -
DC .
Z *
Q) (D
o '*o S"
Q Q CO
CO C3 "eg
wj *n n -
f i f i §
O.-C- Q. Oi»
i i
+
Stoddard solvent
C10-CnandC10-C12
isoparaffinic solvents
(saturated aliphatic
hydrocarbons)
«•- |
•— • "c5
i~ 4-«
co CD
® . £:
•S TO ' C
<*^
i
. cc
z
S s-_
2. {§5>R _
i ^^s §
_: 52 co *- — : —i
tO 2-2 CD CO CO
Qj »_ ^ ^J ® ©
§8§|g |
2 1 i g S J
• i
+ •" . • +
_c _c
Jo
(_
91
-------
i
. 1
il &
«.£
11 $
•2 3 E
on u.
i-i.
11 S
UJ « To
S S
,.il
2 "~ d> ^
80 3
•Q.-O
S-5^ j
£ c— \
^1.2 *
e> —
2-Sg
u £ |
i
Substance/Chemical
J
0?
J;
15
E!
§
+
Ridder etal. (1987)
>
+
d-Limonene
_•
>
! '^
j ^
: tn
iSTJ3
"S
Kanervaet si. (198/3)
NTP (1990)
Lehman-McKeeman eti
(1989)
Webb etal. (1989)
^
-ft
I
c
O
cc cc cc
z z z
cc cc +
•z. -z.
1
C.2
""" S" "c?^ «
O> Q> °5 "D ^
g) O) m o
i— • i— 1— o -C
Z Z ZOQO
i i i
+ + +
Pentachlorobenzene
1 ,2,4,5-Tetrachloro-
benzene
1,4-Dichlorobenzene
92
of
i.
"3 "S
CD ®
t t
o o
•o "^95
3 3*
i i
+ +
0?
Goldsworthy etal. (198
Green etal. (1990)
Goldsworthy et al.
(1988a)
1 '
+ +
Tetrachtoroethylene
(Perchloroethylene)
Pentachloroethane
.8 |
« §
"& -o^
* Is
8 £o-
1 l|
CO Q ^>
DC DC ,
Z Z
K + +'
.&.
NTP (1989)
Strasser etal. (1988)
Dietrich and Swenberg
(1990, 1991 b)
, '<£
. . z
+ + +
Hexachloroethane
Isophorone
Lihdane
-------
a>
n M
B a
•2 c — E
8ti 4
13
ffl
73
ffl
i^ .
— 2. §
o "?* "3
"e S §
If ""
•"! «
| s
^ i
49 *5»
*™ gy-
SI fit ^^
DC
Z
cc
Z
^^
§
a.
t—
cc
Z
cc
Z
f^"
§
^
in
,9.
;=
a.
cc
z
cc
z
§F
2
^u^»
"«
ffi
1
cc
z
cc
z
(i"
2
13
S
(§
EC
Z
+
g"
o>
1
(D
CC
cc
z
+
§"""
^
13
1
EC
Z
+
§"~"
_
"S
o
1
•B +
+ + +
I
8
£
2
|
to
I,
Eg
Is-
: O
ci-i.
co
CD
I
f f
S S
I
t
-8
.S2
5
a,
.i
f A
0)
"o
u>
o
CO
AI aj *•*
W r^ ^-^
°t
CNf
Ills
-3 :§.§.§
93
-------
1
',*:
I
•5>2
5 o>
§ c
§Ji
.— 2.
£. Xj
S
^ v ^
0) O ^
OcL-g
x 2 ~
*" » p
•2 c •—
•S X
81.2
C 5*0.
. e J~ —
log
> a>
UJ £
Substance/Chemical
References
1
E
«
u.
1
a
S
References
S
7
i
<
UL
i
4
"
;
o"
8"
"*"**
"Dominick eta
l '
;+.'
r*?
JaJ
^
•5
Dominick et ,
1
+
Gabapentin
CO
aS
1
E
to
1 '• .
11
DC
• z
"eS
m
Q>
C
.
m
1
. CO ^
li
Si
cc
z
+
o
'5
3,5,5-Trimethyl-hexani
add derivatives
§F
O)
"*^*_
75 '
Daugherty et
cc
z .
^^
§,
.
^r
co
Daugherty e
cc
z
4-
s
.'a
is
CO
00
CD
^
"irt
4..
Henningsen e
cc
z
i?
o>
"to
m
Henningsen
DC
z
+
Isopropylcydohexane
cc
z
cc
z • •.
*^^ .
oo
^oo
Barhett et al
Johnson (19
+
1 ,3,6-Tricyanohexane
'-.
+ Positive
Negative
NR = Not reiported
94
-------
3
•s
1
I
1
§
o
•«
j>
o
•s:
(9
_«
O.
8
1
i
A
I
i
•s
>,
€
g
lili
c\i O.TO .E
3 ±
i TJ
!
0
! <^ _
.&»»•§
-?M2£'5^
«J 2 ™ r^C. .5 5
.2 «» a, E o 3 g-
**lf|iS
\u _ {fl •
CO £ —"~ ^c o
~^
O
X o.
jj g S .g QJ
S ^ ® *3 O
-------
es
n-Neoplas
I
^
I
-p i
X (A (0
» « -S-TJ^
p tO T3 g eg
£ 2= w <2 E
II 12
I Ill-ill
§|§iJ|i
simii
r= £ JO ~ Q.
-------
in"
!<§!
.2 .E J2~ S T= "m" P "C1
1 Q>£-S c o>E«
mill"!
Jo co J= ^ >• to to
C _ — «•• « . - Q.
.:=••- oj re Js co -
•D"g ""o-D^f S
fllflfll
ii^lls.si
Ip&lHI
CSc
c . 35 to to -o o —
ip.»!l?
£ o £ g-Q* ^
9 -2- o ^ ^ a jf J
S
01
§
lo
1
o
p
CI
s
d
i
I
I
£
I
§
o
6
i
-
vu i - v: co *w
il Q. Q. «2 LL J= !
.S 11- 2. c '
to w B^_-
O Q* O ffl
•^ W C ST
I
lit
2= co
O
P P
C •-
2S«4
97
-------
-------
References
Akerstrom, B., and LCgdberg, L. (1990) An intriguing memberof the iipocalin
protein family: a^microglobulin. Trends Biochem. Sci. 15:240-243.
Alden, C.L. (1986) A review of unique male rat hydrocarbon nephropathy.
Toxicologic Pathol. 14:109-111.
Alden, C.L. (1989) Male rat specific alpha globulin nephropathy and renal
tumorigenesis. In: Bach, P.H., and Lock, E.A. (eds.), Nephrotoxicity. In
Vitro to In Vivo. Animals to Man. Plenum Press, New York, pp. 535-541.
Alden, C.L, and Frith, C.H. (1991) Toxicologic pathology of the kidney. In:
Haschek, W.M., and Rousseaux, C.G. (eds.), Handbook of Toxicologic
Pathology. Academic Press, New York. In press.
Alden, C.L., Kanerva, R.L., Ridder, G., and Stone, LC. (1984) The
pathogenesis of the nephrotoxicity of volatile hydrocarbons in the male
rat. In: Mehlman, M.A., Hemstreet, G.P., Thorpe, J.J., and Weaver, N.K.
(eds.), Advances in Modern Environmental Toxicology. Vol. VII. Renal
Effects of Petroleum Hydrocarbons. Princeton Scientific Publishers,
Inc., Princeton, New Jersey, pp. 107-120.
Alden, C.L, Ridder, G., Stone, L., and Kanerva, R.L. (1985) Pathology of
petrochemical fuels in male rats. Acute toxicity. In: Bach, P.H., and
Lock, E.A. (eds.), Renal Heterogeneity and Target Cell Toxicity. John
Wiley and Sons, New York, pp. 461-472.
Arnold, F.J., and Meyerson, L.R. (1990) Radial immunodiffusion assay for
rat Oj-acid glycoprotein. Pharmacol. Biochem. Behavior 37:485-491.
Asal, N.R., Risser, D.R., Kadamani, S., Geyer, R., Lee, E.T., and Chemg,
N. (1988a) Risk factors in renal cell carcinoma: I. Methodology,
demographics, tobacco, beverage use, and obesity. Cancer Detection
Prevention 11:359-377.
Asal, N.R., Geyer, J.R., Risser, D.R., Lee, E.T., Kadamani, S., and Chemg,
N. (1988b) Risk factors in renal cell carcinoma II. Medical history,
occupation, multivariate analysis, and conclusions. Cancer Detection
and Prevention 13: 263-279.
Ashby, J., and Tennant, R.W. (1988) Chemical structure. Salmonella
mutagenicity and extent of carcinogenicity as indicators of genotoxic
carcinogenesis among 222 chemicals tested in rodents by the U.S. NCI/
NTP. Mutat. Res. 204:17-115.
Askergren, A. (1986) Solvents and the kidney. Progr. Clin. Biol. Res. 220:
155-167.
Bachmann, S., Sakai, T., and Kriz, W. (1986) Nephron and collecting duct
structure in the kidney, rat. In: Jones, T.C., Mohr, U., and Hunt, R.D.
(eds.), Urinary System. ILSI Monographs on Pathology of Laboratory
Animals. Springer-Veriag, New York, pp. 3-24.
99
-------
Bannayam, G.A., and Lamm, D.L. (1980) Renal cell tumors. Pathol. Ann.
15: part 2, 271-308.
Barnett, J.W., Johnson, D.E., Boothe, A.D., and Jphannsen, F.R. (1987)
Protein accumulation as the initial event in tricyanohexane induced
nephropathy in male rats. Toxicologist 7:27 (abstract).
Barrientos, A., Ortuno, M.T., Morales, J.M., Martinez Tello, F., and Rodicio,
J.L.(1977) Acute renal failure after use of diesel fuel as shampoo. Arch.
Intern. Med. 137:1217.
Barthold, S.W. (1979) Chronic progressive nephropathy in aging rats.
Toxicol. Pathol. 7:1-6.
Bennington, J.L., Ferguson, B.R., and Campbell, P.B. (1968) Epidemiologic
studies of carcinoma of the kidney. II. Association of renal adenoma
with smoking. Cancer 22:821-823.
Bennington, J.L., and Beckwith, J.B. (1975) Tumors of the Kidney, Renal
Pelvis, and Ureter. Atlas of Tumor Pathology. Second Series, Fascicle
12. Armed Forces Institute of Pathology, Washington, ti.C., pp. 93r199.
Bell, E.T. (1950) Renal Diseases. Lea and Febiger, Philadelphia, p. 428.
Berggard, I. (1970) Plasma proteins in normal human urine. In: Manuel, Y.,
Revillard, J.P., and Betuel, H. (eds.), Proteins in normal and pathological
urine. S. Karger, Basel, pp. 7-19.
Bernard, A.M., Lauwerys, R.R., Noel, A., Vandeleene, B., and Lambert A.
(1989) Urine protein 1:asex dependent marker of tubular or glomerular
dysfunction. Clin. Chem. 35: 2141-2142.
Blair, A., Decoufle, P., and Grauman, D. (1979) Causes of death among
laundry and dry cleaning workers. Am. J. Public Health 69:508-511.
Blair, A., Stewart, P.A.,ToIbert, P.E., Grauman, D., Moran, F.X.,Vaught, J.,
and Rayner, J. (1990) Cancer and other causes of death among a
cohort of dry cleaners. Brit. J. Industr. Med. 47:162-168.
Bomhard, E., Luckhaus, G., Voight, W-H., and Loeser, E. (1988) Induction
of light hydrocarbon nephropathy by p-dichlorobehzene. Arch. Toxicol.
61:433-439.
Bomhard, E., Marsmann, M., Ruhl-Fehlert, Ch., and Zywietz, A. (1990)
Relationships between structure and induction of hyaline droplet
accumulation in the renal cortex of male rats by aliphatic and alicyclic
hydrocarbons. Arch. Toxicol. 64:530-538. ....,-
Borghoff, S.J., Strasser, J., Charbonneau, M., and Swenberg, J.A. (1988)
Analysis of 2,4,4-trimethyl-2-pentanol (TMP-OH) binding to male rat
kidneys-globulin (oj and other proteins. Toxicologist 8:135 (abstract).
Borghoff, S.j", Upton, P.B., and Swenberg, J.A. (1989) Characteristics of
2,4,4-trimethyl-2-pentanol (TMPOH) binding to o20-globulin and other
compounds that cause protein droplet nephropathy. Toxicologist 9:79
(abstract).
Borghoff, S.J., Short, B.G., and Swenberg, J.A. (1990) Biochemical
mechanisms and pathobiology of a,u-globulin nephropathy. Ann. Rev.
Pharmacol. Toxicol. 30:349-367.
Borghoff, S.J., Miller, A.B., Bowen, J.P., and Swenberg, J.A. (1991)
Characteristics of chemical binding to oc2u-globulin in vitro - evaluating
structure-activity relationships. Toxicol. Appl. Pharmacol. 107: 228-
238.
100
-------
Boring, C.C., Squires, T.S., andTong.T. (1991) Cancer Statistics. CA.41:
19-36.
Brooks, D.E. (1987) The major androgen-regulated secretory proteins of
the rat epididymis bear sequence homoiogy with members of the o,u-
globulin superfamily. Biochem. Internat. 14:235-240.
Brown, DP., and Kaplan, S.D. (1987) Retrospective cohort mortality study
of dry cleaner workers using perchloroethylene. J. Occup. Med. 29:
535-541. (1987).
Brownson, R.C. (1988) A case-control study of renal cell carcinoma in
relation to occupation, smoking, and alcohol consumption. Arch.
Environ. Health. 43: 238-241.
Bruckner, J.V., Davis, B.D., and Blancato, J.N. (1989) Metabolism, toxicity,
and carcinogenicity of trichloroethylene. CRC Critical Rev. Toxicol. 20:
31-50:
Bruner, R.H. (1984) Pathologic findings in laboratory animals exposed to
hydrocarbon fuels of military interest. In: Mehlman, M.A.. Hemstreet,
G.P., Thorpe, J.J., and Weaver, N.K. (eds.), Advances in Modem
Environmental Toxicology. Vol. VII. Renal Effects of Petroleum
Hydrocarbons. Princeton Scientific Publishers, Inc., Princeton, New
Jersey, pp. 133-140.
Bruner, R.H. (1986) Chronic consequence of alpha2u-globulin nephropathy:
review. Proceedings, Toxicology Forum. Aspen, Colorado, July 15, pp.
161-166.
Burnett, VI.., Short, B.G., and Swenberg, J.A. (1989) Localization of o,,,-
glbbulin within protein droplets of male rat kidney: immunohistochemistry
using perfusion-fixed, GMA-embedded tissue sections. J. Histochem.
Cytochem. 37:813-818.
Caudill, D., Asquith.T.N., andLehman-McKeeman, L.D. (1991) Biochemical
differences between o2u-globulin (cc2u) and mouse urinary protein
(MUP): explanation of murine resistance to develop hyaline droplet
nephropathy (HDN). Toxicologist 11:137 (Abstract).
Cavaggioni, A., Sorbi, R.T., Keen, J.N., Pappin, D.J.C., and Findlay, J.B.C.
(1987) Homoiogy between the pyrazine-binding protein from nasal
mucosa and major urinary proteins. FEBS Lett. 212: 225-228.
Cavaggioni, A., Findlay, J.B.C., and Tirindelli, R. (1990) Ligand binding
/ characteristics of homologous rat and mouse urinary proteins and
pyrazine-binding protein of calf. Comp. Biochem. Physiol. 96B: 513-
520.
Charbonneau, M., Lock, E.A., Strasser, J., Cox, M.G., Turner, M.J., and
Bus, J.S. (1987) 2,2,4-Trimethylpentane-induced nephrotoxicity. I.
Metabolic disposition of TMP in male and female Fischer 344 rats.
Toxicol. Appl. Pharmacol. 91:171 -181.
Charbonneau, M., Strasser, J., Borghoff, S.J., and Swenberg, J.A. (1988)
In vitro hydrolysis of [14C]-a,u-globulin (a2u) isolated from male rat
kidney. Toxicologist 8:135 (abstract).
Charbonneau, M., Strasser, J., Lock, E.A., Turner, M.J., and Swenberg, J.A.
(1989) Involvement of reversible binding to a2u-globulin in 1,4-
dichlorobenzene-induced nephrotoxicity. Toxicol. Appl. Pharmacol.
99:122-132.
101
-------
Chatterjee, B., Hopkins. J., Dutchak, D., and Roy, A.K.,(1979) Superinductionlv
of OL -globulin by actinqmycin D: evidence fpr drug-mediated increase
in 2" mRNA. Proc. Natl. Acad.Sci. 76:1833-1837. .. . - .-
Chatte?eue, B., Demyan, W.F., Song, C.S., Garg, B;D., and Roy, A.K. (1989)
Loss of androgenic induction of (vQtobulin gene family in the Itver of
NIH Black rats. Endocrinology 125:1385-1388.
Churchill, D.N., Fine, A., and Gault, M.H. (1983) Association between
hydrocarbon exposure and glqmerulonephritis. An appraisal of the
evidence. Nephrbn 33:169-172. ;
Cohen, S.M., and Ellwein, L.B. (1990) Cell proliferation in carcinogenesis.
Science 249:1007-1011. v ,
Crisp* A.J., Bhalla, A.K., and Hoffbrand, B.I. (1979) Acute tubular necrosis
after exposure to diesel oil. Brit. Med. J. 2: .177. ,n , Maronpot, R.R., and Matthews, H.B. (1991);
Correlation of hepatocellular proliferation with hepatocarcinogenicity
induced by the mutagenic noncarcinogen:carcinogen pair - 2,6- and
2 4-diaminotoluene. Toxicol. Appl. Pharmacol. 107:562-567.
Daniell, W.E., Couser, W.G., and Rosenstock, L (1988) Occupational;
solvent exposure and glomerulonephritis.. A case report and review of
the literature. J. Am. Med. Assoc. 259:2280-2283.
Daugherty, W.C., Smith, J.H., Hinz.J.P., and Biles, R.W. (1990) Subchromc
toxicHy evaluation of tridecyl acetate in rats. Fundam. Appl. Toxicol, 14:
104-112. , ••-' . ^
Dayal, H., and Kinman, J. (1983) Epidemiology of kidney cancer. Semm.
• OncoLI 0:366-377. '
Deal, F.H., Richardson, F.C., and Swenberg, J.A. (1989) Dose response of
hepatocyte replication in rats following continuous exposure to
diethylnitrosamine. Cancer Res. 49:6985-6988. ---.^
Dees, J.H., Heatfield, B.M., Reuber, M.D., and Trump, B.F. (1980a)
Adenocarcinoma of the kidney. III. Histogenesis of renal
adenocarcinomas in rats by N-(4'-fluoro-4-bipheny!yl)acetamide. J.
Natl. Cancer Inst. 64:1537-1545. : :;
Dees, J.H., Heatfield, B.M., and Trump, B.F. (1980b) Adenocarcinoma of
the kidney. IV. Electronmicroscopic study of the development of renal
adenocarcinomas induced in rats by N-(4'-f luoro-4-biphenylyl)acetamide.
J. Natl. Cancer Inst. 64:1547-1562. •• .- :
Dekant, W., Vamvakas, S., and Anders, M.W. (1989) Bioactivation of
nephrotoxic haloalkanes by glutathione conjugation — formation of
toxic and mutagenic intermediates by cysteine conjugate (5-lyase. Drug
Metab. Rev. 20:43-83. ^ .
Devesa, S.S., Silverman, D.T., Mclaughlin, J.K., Brown, C.C., Connelly,
R.R., and Fraumeni, J.F. (1990) Comparison of the descriptive
epidemiology of urinary tract cancers. Cancer Causes and Control 1.:
133-141.
Dice J F. (1987) Molecular determinants of protein half-lives in eukaryotic
cells. FASEB J. 1:349-357. ^ .
Dieppe, P.A., Doyle, D.V., and Burry, H.C. (1978) Renal damage during
treatment with antirheumatic drugs. Brit. Med. J. 2:664.
102
-------
Dietrich, D.R., and Swenberg, J.A. (1990) Lindane induces nephropathy
and renal accumulation of o2u-globulin in male but not infemale Fischer
344 rats or male NBR rats. Toxicol. Lett. 53:179-181.
Dietrich, D.R., and Swenberg, J.A. (1991a) NCI-Black-Reiter (NBR) male
rats fail to develop renal disease following exposure to agents that
induce a^ -globulin (oj nephropathy. Fundam. Appl.Toxicol. 16:749-
762. " '
Dietrich, D.R., and Swenberg, J.A. (1991 b) Alphas-globulin is necessary for
d-limonene promotion of male rat kidney tumors. Cancer Res. 51:3512-
3521
Dodd, D.E., Losco, P.E., Troup, C.M., Pritts, I.M., and Tyler, T.R. (1987)
Hyaline droplet nephrosis in male Fischer 344 rats following inhalation
of diisobutyl ketone. Toxicol. Indust. Health 3:443-457.
Dolan, K.P., Unterman, R., McLauglin, M,, Nakhasi, H.L., Lynch, K.R., and
Feigelson, P. (1982) The structure and expression of very closely
related members of the o^-globulin gene family. J. Biol. Chem. 257:
13527-13534. ^ ,.
Dominick, NLA., Robertson, D.G., Sifler, M.R., Susick, R.L., and Bobrowski,
W.F. (1990) Absence of nephrocarcinogenesis in male rats administered
gabapentin, a compound that causes alpha-2u-globulin nephropathy.
Proceedings of the American College of Veterinary Pathology, December
9-14,1990, p. 45 (abstract). . .
Drayna, D.T., McLean, J.W., Wion, K.L., Trent, J.M., Drabkin, H.A., and
Lawn, R.M. (1987) Human apolipoprotein D gene: gene sequence,
chromosome localization, and homology to the o2u-globulinsuperfamily.
DNA 6:199-228. _,_, ,
Duh R-W., and Asal, N.R. (1984) Mortality among laundry and dry cleaning
workers in Oklahoma. Am. J. Public Health 74:1278-1280.
Ekstrom R.C. (1983) Characterization and metabolism of oc^-globulin - a
male sex-dependent protein of the rat. Ph.D. thesis, University of
Wisconsin-Madison. . .
Ekstrom, B., and Berggard, I. (1977) Human armicroglobulin: purification
procedure, chemical and physicochemical properties. J. Biol. Chem.
252:8048-8057. • , u- * i '• i
Elcombe, C.R., Rose, M.S., and Pratt, I.S. (1985) Biochemical, histological,
and ultrastructural changes in rat and mouse liver following the
administration of trichloroethylene: possible relevance to species
differences in hepatocarcinogenicity. Toxicol. Appl. Pharmacol. 79:
365-376. ^ • '-.
Enterline, P.E., and Viren, J. (1985) Epidemiologic evidence for an
association between gasoline and kidney cancer. Environ. Health
Persp. 62:303-312.
Evans, G.O., and Morgan, R.J.I. (1986) Urinary enzyme measurements in
male rats after oral administration of decalin. Human Toxicol. 5:120
Evans, aO., Goodwin, D.A., Parsons, C.E., and Read, N.G. (1988) The
effects of levamisole on urinary enzyme measurements and proximal
tubule cell inclusions in male rats. Brit. J. Exp. Pathol. 69:301-308.
103
-------
Fan, C.Y., Butler, W.H., and O'Connor, PJ. (1989) Cell and tissue specific
localization of 06-methylguanine in the DNA of rats given N-
nitrosodimethylamine: effects of protein deficient and normal diets.
Carcinogenesis 10:1967-1970.
Farber, E. (1988) Cancer development and its natural history. A cancer
prevention perspective. Cancer 62:1676-1679.
Feigelson, P., and Kurtz, D.T. (1977) Hormonal modulation of specific
messenger RNA species in normal and neoplastic rat liver. Adv.
Enzymol.47:275-312.
Flamm, W.G., and Lehman-McKeeman, L.D. (1991) The human relevance
of the renal tumor-inducing potential of d-limonene in male rats:
implications for risk assessment. Regulatory Toxicol. Pharmacol. 13:
70-86.
Fowlie, A.J., Grasso, P., and Bridges, J.W. (1987) Renal and hepatic lesions
induced by 2,2,4-trimethylpentane. J.Appl. Toxicol. 7:335-341.
Galloway, S.M., Armstrong, M.J., Reuben, C., Colman, S., Brown, B.,
Cannon, C., Bloom, A.D., Nakamura, F., Ahmed, M., Duk, S., Rimpo, J.,
Margolin, B.H., Resnick, M.A., Anderson, B., and Zeiger, E. (1987a)
Chromosome aberrations and sister chromatid exchanges in Chinese
hamster ovary cells: evaluations of 108 chemicals. Environ. Mol.
Mutagen. 10(Suppl. 10): 1-175.
Galloway, S.M., Deasy, D.A., Bean, C.L., Kraynak, A.R., Armstrong, M J.,
.and Bradley, M.O. (I987b) Effects of high osmotic strength on
chromosome aberrations, sister-chromatid exchanges and DNA strand
breaks, and the relation to toxicity. Mutat. Res. 189:15-25.
Garg, B.D., Olson, M.J., Li, L.C., Mancini, M.A-, and Roy, A.K. (1987)
Immunoelectron microscopic localization of oc -globulin in the male rat
kidney after gasoline exposure. In: Bailey, G.w. (ed.), Proceedings of
the45th Annual Meeting of the Electron Microscopic Society of America.
San Francisco, California, pp. 872-873.
Garg, B.D., Olson, M.J., Demyan, W.F., and Roy, A.K. (1988) Rapid post-
exposure decay of o^-globulin and hyaline droplets in the kidneys of
gasoline-treated male rats. J. Toxicol. Env. Health 24:145-160.
Garg, B.D., Olson, M.J., and Li, L.C. (1989a) Phagolysosomal alterations
induced by unleaded gasoline in epithelial cells of the proximal convoluted
tubules of male rats: effect of dose and treatment duration. J. Toxicol.
Env. Health 26:101-118.
Garg, B.D., Olson, M.J., Li, L.C., Mancini, M.A., and Roy, A.K. (1989b)
Estradiol pretreatment of male rats inhibits gasoline-induced renal
hyaline droplet and alpha-2u-globulin accumulation. Research
Publication, General Motors Research Laboratories. GMR-6557. March
1989.
Gaworski, C.L., MacEwen, J.D., Vernot, E.H., Bruner, R.H., and Cowan,
M.J. (1984) Comparison of the subchronic inhalation toxicity of petroleum
and oil shale JP-5 jet fuels. In: Mehlman, M.A., Hemstreet, G.P.,
Thorpe, J.J., and Weaver, N.K. (eds.), Advances in Modern
Environmental Toxicology. Vol. VII. Renal Effects of Petroleum
Hydrocarbons. Princeton Scientific Publishers, Inc., Princeton, pp. 3-
47.
104
-------
Gaworski, C.L., MacEwsn, J.D., Vernot, E.H., Haun, C.C., Leahy, H.F.,
Bruner, R.H., Hail, A., Lateradresse, J.R., Mattie, D.R., and Pitts, LL.
(1985a) Evaluation of 90-day inhalation toxtcity of petroleum and oil
shale diesel fuel marine (DFM). Harry G. Armstrong Aerospace Medical
ResearchLaboratory,AMRL-TR-85-074,NMRI85-57,Decernber1985.
Gaworski, C.L, Haun, C.C., MacEwen, J.D., Vernot, E.H., Bruner, R.H.,
Amster, R.L., and Cowan, MJ. (1985b) A 90-day vapor inhalation
toxicity study of decalin. Fundam. Appl. Toxicol. 5:785-793.
Geertzen, H.G.M., van den Ouderaa, F.J.G., and Kassenaar, A.A.H. (1973)
isolation and metabolism of male sex-dependent urinary protein from
rats. ActaEndocrinotogica 72:197-208.
Godovac-Zimmermann, J. (1988) The structural motif of p-iactoglobu|in and
retinol-binding protein: a basic framework for binding and transport of
i small hydrophobic molecules? 1988. Trends Biochem. Sci. 13:64-66.
Goldsworthy, T.L., and Popp, J.A. (1987) Chlorinated hydrocarbon-induced
peroxisomal enzyme activity in relation to species and organ
carcinogenic'rty. Toxicol. Appl. Pharmacol. 88:225-233.
Goldsworthy, T.L., Lyght, O., Burnett, V.L., and Popp, J.A. (1988a)
Potential role of o2u-globulin, protein droplet accumulation, and cell
replication in the renal carcinogenicity of rats exposed to trichloroethylene,
perchloroethylene, and pentachloroethane. Toxicol. Appl. Pharmacol.
96:367-379.
Goldsworthy, T.L., Smith-Oliver, T., Loury, D.J., Popp, J.A., and Butterworth,
B.E. (1988b) Assessment of chlorinated hydrocarbon-induced
genotoxicity and cell replication in rat kidney cells. Environ. Mol.
Mutagen. 11(Suppl. 11): 39.
Goodman, D.G., Ward, J.M., Squire, R.A., Paxton, M.B., Reichardt, W.D.,
Chu, K.C., and Linhart, M.S. (1980) Neoplastic and non-neoplastic
lesions in aging Osborne-Mendel rats. Toxicol. Appl. Pharmacol. 55:
433-447.
Goodman, M.T., Morgenstern, H., and Wynder, E.L. (1986) A case-control
study of factors affecting the development of renal cell cancer. Am. J.
Epidemic!. 124:926-941.
Gray, J.E. (1986) Chronic progressive nephrosis, rat. In: Jones, T.C., Mohr,
U-.andHunt, R.p.(eds.), Urinary System. ILSI Monographs on Pathology
of Laboratory Animals. Springer-Verlag, New York, pp. 174-179.
Green, T., Odum, J., Nash, J.A., and Foster, J.R. (1990) Perchloroethylene-
induced rat kidney tumors: an investigation of the mechanisms involved
and their relevance to humans. Toxicol. Appl. Pharmacol. 102:77-89.
Grisham, J.W., Kauf mann, W.K., and Kaufmann, D.G. (1983) The cell cycle
and chemical carcinogenesis. Survey Synth. Pathol. Res. 1:49-66.
Haider, C.A., Warne, T.M., Hatoum, N.S. (1984) Renal toxicity of gasoline
and related petroleum naphthas in male rats. In: Mehlman, M.A.,
Hemstreet, G.P., Thorpe, J.J., and Weaver, N.K. (eds.), Advances in
Modern Environmental Toxicology. Vol. VII. Renal Effects of Petroleum
Hydrocarbons. Princeton Scientific Publishers, Inc., Princeton, New
Jersey, pp. 73-88.
Haider, C.A., Holdsworth, C.E., Cockrell, B.Y., and Piccirillo, V.J. (1985)
Hydrocarbon nephropathy in male rats: identification of the nephrotoxic
components of unleaded gasoline. Toxicol. Indust. Health 1:67-87.
105
-------
Hanis, N.M., Holmes, T.M., Shallenberger, L.G., and Jones, K.E. (1982)
Epidemiologic study of refinery and chemical plant workers. J. Occup.
Med. 24:203-212.
Hard, G.C. (1984) High frequency, single-dose model of renal adenoma/
carcinoma induction using dimethylnitrosamine in CrI:(W)BR rats.
Carcinogenesis 5:1047-1050.
Hard, G.C. (1987) Chemically induced epithelial tumors and carcinogenesis
of the renal parenchyma. In: Bach, P.H., and Lock, E.A. (eds.)
Nephrotoxicity in the Experimental and the Clinical Situation, Part I.
Martinus Nijhoff Publishers, Lancaster,;pp. 211-250.
Hard, G.C. (1990) Tumours of the kidney, renal pelvis and ureter. In:
Turusov, V.S., and Mohr, U. (eds.), Pathology of Tumours in Laboratory
Animals. Vol. 1 - Tumours of the Rat. Second Edition. International
Agency for Research on Cancer, Lyon. IARC Scientific Publications No.
99, pp. 301-344.
Hard, G.C., and Butler, W.H. (1971) Morphogenesis of epithelial neoplasms
induced in the rat kidney by dimethylnitrosamine. Cancer Res. 31:
1496-1505.
Hard, G.C., and Snowden, R.T. (1991) Hyaline droplet accumulation in
rodent kidney proximal tubules: an association with histiocytic sarcoma.
Toxicol. Pathol. (in press).
Hard, G.C., Mackay, R.L., and Kochhar, O.S. (1984) Electron microscopic
determination of the sequence of acute tubular and vascular injury
induced in the rat kidney by a carcinogenic dose of dimethylnitrosamine.
Lab. Invest. 50:659-672.
Hard, G.C., Alden, C.L., Stula, E.F., and Trump, B.F. (1991) Proliferate
lesions of the kidney in rats. Society of Toxicologic Pathologists.
Standard System of Nomenclature and Diagnostic Pathology. In press.
Harrington, J.M., Whitby, H., Gray, C.N., Reid, F. J., Aw, T.C., and Waterhouse,
J.A. (1989) Renal disease and occupational exposure to organic
solvents: a case-referent approach. Brit. J. Industr. Med. 46:643-650.
Haseman, J.K., Huff, J., and Boorman, G.A. (1984) Use of historical control
data in carcinogenicity studies in rodents. Toxicol. Pathol. 12:126-135.
Hastie, N.D., Held, W.A., and Toole, J.J. (1979) Multiple genes coding for
the androgen-regulated major urinary proteins of the mouse. Cell 17:
449-457.
Haworth, S., Lawlor, T., Mortelmans, K., Speck, W., and Zeiger, E. (1983)
Salmonella mutagenicity test results for 250 chemicals. Environ.
Mutagen. 5 (Suppl. 1): 1-142.
Health Effects Institute (HEI). (1985) Gasoline Vapor Exposure and Human
Cancer: Evaluation of Existing Scientific Information and
Recommendations for Further Research. Health Effects Institute,
Report of the Institute's Health Review Committee, September 1985.
Health Effects Institute (HEI). (1988) An Update on Gasoline Vapor
Exposure and Human Cancer: An Evaluation of Scientific Information
Published Between 1985 and 1987. Health Effects Institute, Report of
the Institute's Health Review Committee, January 6,1988.
Hellsten, S., Berge, T., and Wehlin, L. (1981) Unrecognised renal cell
carcinoma. Clinical and pathological aspects. Scand. J. Urol. Nephrol.
8:273-278.
106
-------
Higginsoh, J., Muir, C.S., and Buff ler, PA (1 984) The epidemiology of renal :
Carcinoma in humans with a note on the effect of exposure to gasoline.
In: Mehlman, MA, Hemstreet, G.P., Thorpe, J.J., and Weaver, :N;K.
(eds.), Advances in Modern Environmental Toxicology. Vol. VII. Renah
Effects of Petroleum Hydrocarbons. Princeton Scientific Publishers,
Inc., Princeton, New Jersey, pp. 203-226. : ; >
Hobson, D.W;, Mattie, D.R., Bruner, R.R, Uddini D.E., Eurell,T., and Olson,
:,C.T. (1986) Effect of castration on the development of 2,2,4-
trimethylpentane-induced hydrocarbon nephrOpathy in the male Fr344
rat. Toxicologist 6: 1 73 (abstract),
Hoel, D.G., Haseman, J.K., Hogan, M.D., Huff, J., and McConnell, E.E.
= (1988) The impactof toxicity on carcinogenicity studies: implications for
risk assessment. Garcinogenesis 9: 2045-2052.,
Hughson, M:D:, Buchwold, D., and Fox, M. (1986) Renal rieoplasia and
acquired cystic kidney disease in patients receiving long term dialysis.
Arch. Pathol. Lab. Med. 110:592-600.
Jackson; M.R. (1974) The nature of dimethylnitrosarnine induced
enlargement of rat hepatocyte nuclei. J. Pathol. 113: 197-208.
Jedrzejewski, K., and Kug!erv P. (1982) Peptidases in the kidney and urine
rof^rats after castration. Histochemistry 74: 63-84.
Johnson, D.E. (1987) Protein induced nephropathy in male rats. In:
Holcenberg, J.S., and Winkeihake, J:L.; (eds.), The. Pharmacology and
Toxicology of Proteins. Alan R. Liss lncM New York, pp.1 65-1 71.
Kadamani, S., Asal, N.R.,andNelson, R.Y. (1989) Occupational hydrocarbon
exposure and risk of renal cell carcinoma. Am* J. Industr. Health 15:
' " ' ' " "
. . •
Kanerva, R.L., McOrackeri, M;S:i Alden, C.L., and Stone, L.C. (1987a)
Morphogenesis of decalin-indueed renal alterations in the male rat; Fd.
^Chem. Toxicol. 25: 53-61. •'•'•'••
Kaherva, R.L., Ridder, G.M:, Lefever, F.R., and Alden, C.L. (1987b)
Comparison of short-term renal effects due to oral administration of
:decalinord-limonene in young adult male Fischer-344rats. Fd. Ghem.
Toxicol. 25: 345-353; ;• v :•:
Kanerya, R.L., Ridder, G.M., Stone, L.C., and Alden, ^C.L. (1987c)
Characterization of spontaneous and deealinrinduced hyaline droplets
in kidneys of adult male rats. Fd. Chem. Toxicol. 25: 63-82. :?••
Katz, R.M., and,Jowett, D. (1 981 ) Female laundry and dry cleaning workers
.; in Wisconsin: a mortality analysis. Am. J. Public Health 71 : 305-307.
Katz, G.V., Renner, E.R., and Terharr, C.J. (1986) Subchronic inhalation
toxicity of methyl isoamyl ketone in rats. Fundam; Appl. Toxicol. 76:
'• 498-505. -; •• ' • "' '.• •••"' • •" ':"' ' >'• •'•-'• - • '•'- ' -'
Kaysen, G.A., Myers, B.D., Couser, W.G., Rabkiri, R., and Felts.U.M. (1986)
Biology of Disease. Mechanisms and consequences of proteinuria.
= Lab. Invest. 54: 479-498.
Killeen, J.C., Wilson, N.H., Ford, W.H., Siou, G., Busey, W.M., and Eilnch,
: G.L. (1990) Progression of renal and forestomach effects following
administration of chlorothalonil to rats. Toxicologist 10:269 (abstract).
Kimura, H., Odani, S., Suzuki, J-l., Arakawa, M., and Ono, T. (1989) Kidney
fatty acid-binding protein: identification as a2il-globulin. FEBS Letters
246: 101-104.
107
-------
Kloss, M.W., Cox, M.G., Norton, R.M., Swenberg, J.A., and Bus, J.A.
(1985). Sex-dependent differences in the disposition of [14C-5]2,2,4-
trimethylpentane in Fischer 344 rats. In: Bach, P.H., and Lock, E.A.
(eds.), Renal Heterogeneity and Target Cell Toxicity. Wiley, Chichester,
pp. 489-492. '
Kluwe, W.M., Abdo, K.M., and Huff, J. (1984) Chronic kidney disease and
organic chemical exposures: evaluations of causal relationships in
humans and experimental animals. Fundam. Appl. Toxicol. 4:889-901.
Kugler, P., and Vornberger, G. (1986) Renal cathepsin-B activities in
ratsafter castration and treatment with sex hormones. Histochemistry
85:157-161.
Kuna, R.A., and Ulrich, C.E. (1984) Subchronic inhalation toxicity of two
motor fuels. J. Am. Coll. Toxicol. 3:217-229.
Kurtz, D.T., and Feigelson, P. (1977) Multihormonal induction of hepatic
a -globulin mRNA as measured by hybridization to complementary
DWA. Proc. Natl. Acad. Sci. 74:4791-4795.
LaVecchia, C., Negri, E., D'Avanzo, B,, and Franceschi, S. (1990) Smoking
and renal cell carcinoma. Cancer Res. 50:5231-5233.
Lee K-H., Wells, R.G., and Reed, R. R. (1987) Isolation of an olfactory cDNA:
similarity to retinol-binding protein suggests a role in olf action. Science
235:1053-1056.
Lehman-McKeeman, L.D., Rodriguez, P.A., Takigiku, R., Caudill, D., and
Fey, M.L (1989) d-Limonene-induced male rat-specific nephrotoxicity:
evaluation of the association between d-limonene and o,u-globulin.
Toxicol. Appl. Pharmacol. 99:250-259.
Lehman-McKeeman, L.D., Rivera-Torres, M.I., and Caudill, D. (1990a)
Lysosomal degradation of a2u-globulin and a -globulin-xenobiotic
conjugates. Toxicol. Appl. Pharmacol. 103:539-548.
Lehman-McKeeman, L.D., Caudill, D., Takigiku, R., Schneider, R.E., and
Young, J.A. (1990b) Comparative disposition of d-limonene in rats and
mice: relevance to male-rat-specific nephrotoxicity. Toxicol. Lett. 53:
193-195.
Lehman-McKeeman, L.D., Rodriguez, P.A., Caudill, D., Fey, M.L, Eddy,
C.L., and Asquith, T.N. (1991) Hyaline droplet nephropathy resulting
from exposure to 3,5,5-trimethylhexanoyloxybenzene sulfonate. Toxicol.
Appl. Pharmacol. 107:429-438.
Lipsky, M.M., and Trump, B.F. (1988) Chemically induced renal epithelial
neoplasia in experimental animals. Intl. Rev. Exptl. Pathol. 30:357-383.
Lock, E.A., Charbonneau, M., Strasser, J., Swenberg, J.A., and Bus, J.S.
(1987a) 2,2,4-Trimethylpentane-induced nephrotoxicity. II. The
reversible binding of a TMP metabolite to a renal protein fraction
containing oc2u-globulin. Toxicol. Appl. Pharmacol. 91:182-192.
Lock, E.A., Stonard, M.D., and Elcombe, C.R. (1987b) The induction of
[omega] and p-oxidation of fatty acids and effect on oc2t,globulin content
in the liver and kidney of rats administered 2,2,4-trimethylpentane.
Xenobiotica17:513-522.
Logothetopoulos, J., and Weinbren, K. (1955) Naturally occurring protein
droplets in the proximal tubule of the rat's kidney. Brit. J. Exptl. Pathol.
36:402-406.
108
-------
Loury, D.J., Smith-Oliver, T-, and Butterworth, B.E. (1987) Assessment of
unscheduled and replicative DNA synthesis in rat kidney cells exposed
in vitro or in vivo to unleaded gasoline. Toxicol. Appl. Pharmacol. 87:
127-140.
Lynge, E., and Thygesen, L. (1990) Primary liver cancer among women in
laundry and dry-cleaning work in Denmark. Scand. J. Work Environ.
Health 16:108-112.
Maack, T., Park, C.H., and Camargo, M.J.F. (1985) Renal filtration,
transport, and metabolism of proteins. In: Seldin, D.W., and Giebisch,
G. (eds.), The Kidney: Physiology and Pathophysiology. Raven Press,
New York, pp. 1773-1803.
MacFarland, H.N. (1984) Xenobiotic induced kidney lesions: hydrocarbons.
The 90-day and 2-year gasoline studies. ln:Mehlman,M.A.,Hemstreet,
GP Thorpe, J.J., and Weaver, N.K. (eds.), Advances in Modern
Environmental Toxicology. Vol. VII. Renal Effects of Petroleum
Hydrocarbons. Princeton Scientific Publishers, inc., Princeton, New
Jersey, pp. 51-56.
MacFarland, H.N., Ulrich, C.E., Holdsworth, C.E., Kitchen, D.N., HaHiwell,
W.H., and Blum. S.C. (1984) A chronic inhalation study with unleaded
gasoline vapor. J. Am. Coll. Toxicol. 3:231 -248.
MacInnes,J.I.,Nozik,E.S.,andKurtz,D.T.(1986)Tissue-specificexpression
of the rat alpha globulin gene family. Molec. Cell. Biol. 6:3563-3567.
MacNaughton, M.G.", and Uddin, D.E. (1984) Toxicology of mixed distillate
and high-energy synthetic fuels. In: Mehlman, M.A., Hemstreet, G.P.,
Thorpe J.J., and Weaver, N.K. (eds.), Advances in Modern
Environmental Toxicology. Vol. VII. Renal Effects of Petroleum
Hydrocarbons. Princeton Scientific Publishers, Inc., Princeton, New
Jersey, pp. 121-132.
Mancini, M.A., Majumdar, D., Chatterjee, B., and Roy, A.K. (1989) Alpha2u
globulin in modified sebaceous glands with pheromonal functions:
localization of the protein and its mRNA in preputial, Meibomian, and
perianal glands. J. Histochem. Cytochem. 37:149-157.
Masoro, E.J., and Yu, B.P. (1989) Diet and nephropathy. Lab. Invest. 60:
165-167.
Mattie D.R., Bruner, R.H., Hoeflich, T.J., and Kendrick, J.M. (1988) Effects
of'decalin and JP-10 on the function and morphology of male rat
kidneys. Toxicologist 8:187 (abstract).
Maunsbach, A.B. (1966a) Observations on the segmentation of the
proximal tubule in the rat kidney. Comparison of results from phase
contrast, fluorescence and electron microscopy. J. Ultrastruct. Res. 16:
239-258. •-.,.'
Maunsbach, A.B. (1966b) Electron microscopic observations of cytoplasmic
bodies with crystalline patterns in rat kidney proximal tubule cells. J.
Ultrastruct. Res. 14:167-189.
McCredie, M., Ford, J.M., and Stewart, J.H. (1988) Risk factors for cancer
of the renal parenchyma. Int. J. Cancer 42:13-16.
McGregor D.B., Brown, A., Cattanach, P., Edwards, I.A., McBride, D.,
Riach,C.,Caspary,W.J.(1988) Responses of the L5178Ytk/tk mouse
lymphoma cell forward mutation assay. III. 72 coded chemicals.
Environ. Mol. Mutagen. 12; 85-154.
109
-------
Mclaughlin, U.K. (1984) Risk factors from a population-based case-control
study of renal cancer. In: Mehlman, MA, Hemstreet, G.P., Thorpe, J.J.,
and Weaver, N.K. (eds.), Advances in Modern Environmental Toxicology.
Vol. VII. Renal Effects of Petroleum Hydrocarbons. Princeton Scientific
Publishers, Inc., Princeton, New Jersey, pp. 227-244.
McLaughlin, J.K., and Schuman, L.M. (1983) Epidemiology of renal cell
carcinoma. Rev. Cancer Epidemiol. 2:170-210.
Mclaughlin, U.K., Mandel, J.S., Blot, W.J., Schuman, L.M., Mehl, E.S., and
Fraumeni, J.F. (1984) A population-based case-control study of renal
cell carcinoma. J. Natl. Cancer Inst. 72:275-284.
McLaughlin, J.K., Blot, W.J., Mehl, E.S., Stewart, P.A., Venable, F.S., and
: Fraumeni, J.F. (1985) Petroleum-related employment and renal cell
cancer. J. Occup. Med. 27:672-674.
McLaughlin, J.K., Malker, H.S.R., Stone, B.J., Weiner, J.A., Malker, B.K.,
Ericsson, J.L.E., Blot, W.J., and Fraumeni, J.F. (1987) Occupational
risks for renal cancer in Sweden. Brit. J. Industr. Med. 44:119-123.
Mortelrnans, K., Haworth, S., Lawlor, T., Speck, W., Tainer, B., and Zeiger,
E. (1986) Salmonella mutagenicity tests. II. Results from the testing of
270 chemicals. Environ. Mutagen. 8 (Suppl. 7): 1-119.
Motwani, N.M., Caron, D., Demuan, W.F., Chatterjee, B., Hunter, S., Poulik,
M.D., and Roy, A!K. (1984) Monoclonal antibodies to a,u-globulin and
immunocytofluorometric analysis of cc?u-globulin-synthesizing
hepatocytes during androgenic induction and aging. J. Biol. Chem. 259:
3653-3657.
Muggia, F.M., Heinemann, H.O., Farhangi, M., psserman, E.F> (1969)
Lysozymuria and renal tubular dysfunction in monocytic and
myelomonocytic leukemia. Am. J. Med. 47:351-366.
Murty, C.V.R., Sarkar, F.H., Mancini, M.A., and Roy, A.K. (1987) Sex-
independent synthesis of o^-globulin and its messenger ribonucleic
acid in the rat preputial gland: biochemical and immunocytochemical
analysis. Endocrinology 121:1000-1005.
Murty, C.V.R., Olson, M.J., Garg, B.D., and Roy, A.K. (1988) Hydrocarbon-
induced hyaline droplet nephropathy in male rats during senescence.
Toxicol. Appl. Pharmacol. 96:380-392.
National Cancer Institute (NCI) (1978a) Bioassay of chlorothalonil for
possible carcinogenicity. Carcinogenesis Testing Program, National
Cancer Institute. DHEW Publication No. (NIH) 78-841 (NTP-TR-41).
National Cancer Institute (NCI) (1978b) Bioassay of hexachloroethane for
possible carcinogenicity. Carcinogenesis Testing Program, National
Cancer Institute. CAS No. 67-72-1. DHEW Publication No. (NIH) 78-
1318. (NTP-TR-68).
National Cancer Institute (NCI) (1987) Annual Cancer Statistics Review:
National Cancer Institute, US DHHS. NIH Publication No. 88-2789.
National Toxicology Program (NTP) (1983) Carcinogenesis bioassay of
pentachloroethane (CAS No. 76-01-7) in F344/N rats and B6C3F1 mice
(gavage study). NTP-81-34. NIH Publication No. 83-1788. NTP-TR-
232, April 1983.
National Toxicology Program (NTP) (1986a) Toxicology and Carcinogenesis
studies of isophorone (CAS No. 78-59-1) in F344/N rats and B6C3F1
110
-------
••;.• -mice (gavage studies). NIH Publication No. 86-2547. NTP-TR-291,
January 1986.
NationalToxicology Program (NTP) (1986b) Toxicology and carcinogenesis
: oftetrachloroethylene (perchloroethylene) (CAS No. 127-18-4) in F344/
N rats and B6C3F1 mice (inhalation studies). NIH Publication No. 86-
, 2567. NTP-TR-311, August 1986.
NationalToxicology Program (NTP) (1987a) Toxicology and carcinogenesis
.'..-• studies of 1,4-dichlorobenzene (CAS No. 106-46-7) in F344/N rats and
-.-: B6C3F1 mice (gavage studies). NIH Publication No. 87-2575. NTP-
TR-319, January 1987.
NationalToxicology Program (NTP) (I987b) Toxicology and carcinogenesSs
; - studies of dimethyl methylphosphonate (CAS No. 756-79-6) in F344/N
rats and B6C3F1 mice (gavage studies). NIH Publication No. 87-2579.
: NTP-TR-323, November 1987.
National Toxicology Program (NTP) (1988) Toxicology and carcinogenesSs
-. studies of trichloroethylene (CAS No. 79-01-6) in four strains of rats
(AGI, August, Marshall, Osborne-Mendei) (gavage studies). NIH
Publication No. 88-2529. NTP-TR-273, April 1988.
National Toxicology Program (NTP) (1989) Toxicology and carcinogenesis
: .studies of hexachloroethane (CAS No. 67-72-1) in F344/N rats (gavage
; studies). NIH Publication No. 89-2816. NTP-TR-361, August 1989.
National Toxicology Program (NTP) (1990) Toxicology and carcinogenesis
studies of d-limonene (CAS No. 5989-27-5) in F344/N rats and B6C3F1
mice (gavage studies). NIH Publication No. 88-2902. NTP-TR-347,
; January 1990.
National Toxicology Program (NTP) (1991 a) Toxicity studies of
pentachlorobenzene in F344/N rats and B6C3F, mice (feed studies)
(NTP Tox 6). NIH Publication No. 91 -3125, January 1991...
.NationalToxicology Program (NTP) (1991b) Toxicity studies of 1,2,4,5-
tetrachlorobenzene in F344/N rats and B6C3F, mice (feed studies)
(NTP Tox 7). NIH Publication No. 91-3126, January 1991.
Neuhaus, O.W., and Flory, W. (1978) Age-dependent changes in the
excretion of urinary proteins by the rat. Nephron 22:570-576.
Neuhaus, O.W., and Lerseth, D.S. (1979) Dietary control of the renal
reabsorption and excretion of a2u-globulin. Kidney Intl. 16:409-415.
^Neuhaus, O.W., Flory, W., Biswas, N., and Hollerman, C.E. (1981) Urinary
excretion of o^f globulin and albumin by adult male rats following
treatment with nephrotoxic agents. Nephron 28:133-140.
Newsom, G.D., and Vugrin, D. (1987) Etiologic factors in renal cell
-; adenocarcinoma. Semin. Nephrol. 7:109-116.
Nkx)ll, J.W., Swann, P.F., and Pegg, A.E. (1975) Effect of dimethylnitrosamine
on,persistence of methylated guanines in rat liver and kidney DNA.
Nature 254: 261-262.
Nogueira, E, (1987) Rat renal carcinogenesis after chronic simultaneous
exposure to lead acetate and N-nitrosodiethylamine. Virchows Arch.
: (Cell Pathol.) 53: 365-374.
Office of Science and Technology Policy (OSTP) (1985). Chemical
Carcinogens; A Review of the Science and its Associated Principles.
111
-------
Office of Science and Technology Policy, February 1985. Federal
Register, March 14,1985, pp. 10371-10442.
Oliver, J., and MacDowell, M. (1958) Cellular mechanisms of protein
metabolism in the nephron. VII. The characteristics and significance of
the protein absorption droplets (hyaline droplets) in epidemic hemorrhagic
fever and other renal diseases. J. Exptl. Med. 107:731-754.
Olson, M.J., Garg, B.D., Murty, C.V.R., and Roy, A.K. (1987) Accumulation
of o^-giobulin in the renal proximal tubules of male rats exposed to
unleaded gasoline. Toxicol. Appl. Pharmacol.90:43-51.
Olson, M.J., Mancini, MA, Garg, B.D., and Roy, A.K. (1988) Leupeptin-
mediated alteration of renal phagolysosomes: similarity to hyaline
droplet nephropathy of male rats exposed to unleaded gasoline. Toxicol.
Lett. 41:245-254.
Olson, M J., Johnson, J.T., and Reidy, C.A. (1990) A comparison of male
rat and human urinary proteins: implications for human resistance to
. hyaline droplet nephropathy. Toxicol. Appl. Pharmacol. 102:524-536.
Paganini-Hill, A., Glazer, E., Henderson, B.E., and Ross, R.K. (1980)
Cause-specific mortality among newspaper web pressmen. J. Occup.
.... Med. 22:542-544.
Papiz, M.Z., Sawyer, L, Eliopoulos, E.E., North, A.C.T., Findlay, J.B.C.,
Sivaprasadarao, R., Jones, T.A., Newcomer, M.E., and Kraulis, PJ.
(1986) The structure of p-lactoglobulin and its similarity to plasma
retinol-binding protein. Nature 324:383-385.
Parker, G.A., Bogo, V., and Young, R.W. (1981) Acute toxicity of conventional
versus shale-derived JP5 jet fuel: light microscopic, hematologic, and
serum chemistry studies. Toxicol. Appl. Pharmacol. 57:302-317.
Partanen,,T., Heikkila, P., Hernberg, S., Kauppinen, T., Moneta, G., and
Ojajciarvi, A. (1991) Renal cell cancer and occupational exposure to
chemical agents. Scand. J. Work Environ. Health 17:231-239.
Pegg, A.E. (1984) Methylation of the Opposition of guanine in DNA is the
most likely initiating event in carcinogenesis by methylating agents.
Cancer Investig. 2:223-231.
Pervaiz, S., and Brew, K. 1987. Homology and structure-function correlations
between c^-acid glycoprotein and serum retinol-binding protein and its
relatives. FASEB J. 1:209-214.
Pesce, A.J., Clyne, D.H., Pollak, V.E., Kant, S.F., Foulkes, EC., and
Selenke, W.M. (1980) Renal tubular interactions of proteins. Clin.
Biochem. 13:209-215.
Peterson, P.A., and Berggard, I. (1971) Isolation and properties of a human
.'.'•'. retinol-transpbrting protein. J. Biol. Chem. 246:25-33.
Pevsner, J., Reed, R.R., Feinstein, P.G., andSnyder, S.H. (1988) Molecular
cloning of odorant-binding protein: member of a ligand carrier family.
Science 241:336-339.
Phillips, R.D., and Cockrell, B.Y. (1984) Kidney structural changes in rats
following inhalation exposure to C^-C^ isoparaff inic solvent. Toxicology
3:261-273.
Phillips, R.D., and Egan, G.F. (1984) Effect of C^-C^ isoparaffinic solvent
on kidney function in Fischer 344 rats during eight weeks of inhalation.
Toxicol. Appl. Pharmacol. 73:500-510.
112
-------
Phillips, R.D., Moran, E.J., Dodd, D.E>, Fowler, E.H., Kary, C;D., and
O'Dpnoghue, J. (1987) A 14-week vapor inhalation toxicity study of
methyl isobutyl ketone. Fundam. Appl. Toxicoi. 9:380-388.
Phillips, S.C., Petrone, R.L., Hemstreet, G.P. (1988) A review of the non-
neoplastic kidney effects of hydrocarbon exposure in humans. Occupatl.
Med: State of the Art Reviews 3:495-509.
Pickle, L.W., Mason, T.J., Howard, N., Hoover, R., and Fraumeni, J.F.
(1987) Atlas of U.S. cancer mortality among whites: 1950-1980.
Washington, D.C. U.S. Department of Health and Human Services
Publication 46 (NIH 87-2900).
Pirani, C.L., Silva, F.G., Appel, G.B. (1983) Tubulb-inierstitial disease in
multiple myeloma and other nonrenal neoplasias. In: Gotran, R.S.,
Brenner, B.M., and Stein, J.H. (eds.), Tubuio-lnterstitial Nephropathies.
Churchill Livingstone, New York. pp. 287-334.
Pitot, H.C. (1982) The natural history of neoplastic development: the
relation of experimental models to human cancer. Cancer 49:120§-
1211.
Poole, C., Satterfield, M.H., Levin, L., Rothman, K.J., and Dreyer, N.A.
(1990) A case-control study of kidney cancer among petroleum refinery
workers. Final Report to American Petroleum Institute, Washington
D.C. by Epidemiology Resources Inc. Chestnut Hill, Massachusetts.
January 2nd, 1990.
Pruzanski, W., and Plaits, M.E. (1970) Serum and urinary proteins,lysozyme
(muramidase), and renal dysfunction in mono- and myelomonocytic
leukemia. J. Clin. Invest. 49:1694-1707. ;
Read, N.G., Astbury, P J., Morgan, R.J.I., Parsons, D.N., and Port, C.J.(1988)
Induction and exacerbation of hyaline droplet formation in the proximal
tubular cells of the kidneys from male rats receiving a variety of
pharmacological agents. Toxicology 52:81-101.
Redmond, C.K., Ciocco, A., Lloyd, W., and Ruch, H.W. (1972) Long-term
mortality study of steelworkers. VI, Mortality from malignant neoplasms
among coke oven workers. J. Occup. Med. 14:621-629.
Reese, A.J.M., and Winstanley, D.P. (1958) The small tumour-like lesions
of the kidney. Brit. J. Cancer 12:507-516.
Richardson, K.A., Wilmer, J.L., Smith-Simpson, D.t and Skopek, T.R.(1986)
Assessment of the genotoxic potential of unleaded gasoline and 2,2,4-
trimethylpentane in human lymphoblasts in vitro. Toxicpl. Appl.
Pharmacol. 82:316-322.
Richardson, A., Butler, J.A., Rutherford, M.S., Semsei, I., Gu, M-Z.,
Fernandes, G., and Chaing, W-H. (1987) Effect of age and dietary
restriction on the expression of o2u-globulin. American Soc. Biochem.
Mol.Biol. 12821-12825.
Ridder, G., Von Bargen, E., Parker, R., Alden, C. (1987) A comparative
; study of the acute effects of d-limoriehe and decalin on male rat
: nephropathy. Toxicologist 7:238 (abstract).
Ridder, G.M., Von Bargen, E.G., Parker, R.D., and Alden, C.L (1988)
Spontaneous and induced accumulation of ot2u globulin in the kidney
cortex of rats and mice. Toxicologist 8:89 (abstract).
113
-------
Ridder, G.M., Von Bargeri, E.G., Alden, C.L, and Parker, R.D. (1990)
Increased hyaline droplet formation in male rats exposed to decalin is
dependent on the presence of a2u globulin. Fundam. Appl. Toxicoi. 15:
732-743.
Ritchie, A.W.S., and Chisholm, G.D. (1983) The natural history of renal
carcinoma. Semin. Oncol. 10:390^400.
Rosanoff, K.A., and Siegel, M.R. (1981) Mechanism of action and fate of the
fungicide chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile) in biological
' •' systems 3. Interaction with mammalian DNA, histones, and isolated rat
liver nuclei. Pesticide Biochem. Physiol. 16:120-128.
Roy, A.K., and Neuhaus, O.W. (1967) Androgenic control of a sex-
dependent protein in the rat. Nature 214:618-620.
Roy, A.K., and Chatterjee, B. (1983) Sexual dimorphism in the liver. Ann.
Rev. Physiol. 45:37-50.
Roy, A.K., McMinn, D.M., and Biswas, N.M. (1975) Estrogenic inhibition of
the hepatic synthesis of a2u-globulin in the rat. Endocrinology 97:1501 -
1508.
Roy, A.K., Math, T.S., Motwani, N.M., and Chatterjee, B. (1983) ; Age-
dependent regulation of the polymorphic forms of a2u-globulin. J. Biol.
Chem. 258:10123-10127.
Savitz, D.A., and Moure, R. (1984) Cancer risk among oil refinery workers:
a review of epidemiological studies. J. Occup. Med. 26: 662-670.
Sawyer, L. (1987) Protein structure - One fold among many. Nature 327,
659.
Selikoff, I.J., Hammond, E.C., and Seidman, M. (1979) Mortality experience
of insulation workers in the United States and Canada, 1943-76. Ann.
N.Y. Acad.Sci.330:91-116.
Serve, M.P., Olson, C.T., Llewellyn, B.M., Bruner, R.H., Yu, K.Q., and
Hobson, D.W. (1988) The metabolism and nephrotoxicity of tetralin in
Fischer 344 rats. Toxicologist 8:180 (abstract).
Shapiro, L.E., and Sachchidananda, J. (1982) Regulation of proteins by
thyroid hormone and glucocorticoid: the responses of hepatic (x,u-
globulin and pituitary growth hormone differ in adult male hypothyrpid
rats. Endocrinology 111:653-660.
Short, B.G., Burnett, V.L., and Swenberg, J.A. (1986) Histopathology and
cell proliferation induced by 2,2,4-trimethylpentane in the male rat
kidney. Toxicoi. Pathol. 14:194-203. ' ,
Short, B.G., Burnett, V.L., Cox, M.G., Bus,.J.S., and Swenberg, J.A. (1987)
Site-specific renal cytotoxicity and cell proliferation in male rats exposed
to petroleum hydrocarbons. Lab. Invest. 57:564-577.
Short, B.G., Burnett, V.L., and Swenberg, J.A. (1989a) Elevated proliferation
of proximal tubule cells and localization of accumulated a2u-globulin in
F344 rats during chronic exposure to unleaded gasoline or 2,2,4-
trimethylpentane. Toxicoi. Appl. Pharmacol. 101:414-431.
Short, B.G., Steinhagen, W.H., and Swenberg, J.A. (1989b) Promoting
effects of unleaded gasoline and 2,2,4-trimethylpentane on the
development of atypical cell foci and renal tubular cell tumors in rats
exposed to N-ethyl-N-hydroxyethylnitrosamine. Cancer Res. 49:6369-
6378.
114
-------
Siemiatycki, J., Dewar, R., Nadon, L, Gerin, M., Richardson, L., and
Wacholder, S. (1987) Associations between several sites of cancer and
twelve petroleum-derived liquids. Scand. J. Work Environ. Health 13:
493-504.
Sippel, A.E., Feigelson, P., and Roy, A.K. (1975) Hormonal regulation of the
hepatic messenger RNA levels for a2u globulin. Biochemistry 14:825-
829.
Sippel, A.E., Kurtz, D.T., Morris, HP., and Feigelson, P. (1976) Comparison
. of in vivo translational rates and messenger RNA levels of a^-globulin
in rat liver and Morris hepatoma 5123D. Cancer Res. 36:3588-3593.
Smith, A.H.,Sheam, V.I., and Wood, R. (1989) Asbestos and kidney cancer:
the evidence supports a causal association. Am. J. Industr. Med. 16:
159-166.
Solet, D., and Robbins, T.G. (1991) Renal function in dry cleaning workers
exposed to perchloroethylene. Am. J. Industr. Med. (In press).
Solleveld, H.A., Haseman, J.K., and McConnell, E.E. (1984) Natural history
of body weight gain, survival, and neoplasia in the F344 rat. J. Natl.
. Cancer Inst. 72:929-940.
Steenland, N.K., Thun, M.J., Ferguson, C.W., and Port, F.K. (1990)
Occupational and other exposures associated with male end-stage
renal disease: a case/control study. Am. J. Public Health 80:153-157.
Stonard, M.D., Foster, J.R., Phillips, P.G.N., Simpson, M.G., Lock, E.A.
(1985) Hyaline droplet formation in rat kidney induced by 2,2,4-
trimethylpentane. In: Bach, P.H., and Lock, E.A. (eds.), Renal
Heterogeneity and Target Cell Toxicity. John Wiley and Sons, Chichester,
pp. 485-488.
Stonard, M.D., Phillips, P.G.N., Foster, J.R., Simpson, M.G., and Lock, E.A.
(1986) o^-Globulin: measurement in rat kidney following administration
of 2,2,4-trimethylpentane. Toxicology 41:161-168. ,
Stonard, M.D. (1987) Proteins, enzymes and cells in urine as indicators of
the site of renal damage. In: Bach, P.H-, and Lock, E.A. (eds.),
Nephrotoxicity in the Experimental and Clinical Situation. Part 2.
Martinus Nijhoff, Lancaster, pp. 563-592.
Stone, L.C., Kanerva, R.L., Burns, U.L., and Alden, C.L. (1987) Decalin-
. , induced nephrotoxicity: light and electronmicroscopic examination of
the effects of oral dosing on the development of kidney lesions in the rat.
Fd. Chem. Toxicol. 25: 43-52.
Strasser, J., Charbonneau, M., Borghoff, S.J., Turner, M.J., and Swenberg,
J.A. (1988) Renal protein droplet formation in male Fischer 344 rats
after isophorone (IPH) treatment. Toxicologist 8:136 (abstract).
Swenberg, J A. (1989) Lecture sponsored by International Life Sciences
Institute, presented at Brookings Institution, Washington D.C., December
13.
Swenberg, J.A. (1991) Editorial. Risk assessment of chemicals causing a,u-
globulin nephropathy. Reg. Toxicol. Pharmacol. 13:1-2.
Swenberg, J.A., Short, B., Borghoff, S., Strasser, J., and Charbonneau, M.
(1989) The comparative pathobiology of cc2u-globulin nephropathy.
Toxicol. Appl. Pharmacol. 97: 35-46.
115
-------
Szoka, P.R., and Paigen, K. (1978) Regulation o
protein productkDn by the MUP-A gene. Genetics 90:597-612.
Tannenbaum, M. (1971) Ultrastructural pathology of human renal cell
tumors. Pathol. Ann. 6:249-277. _
Tannenbaum, M. (1983) Surgical and histopathology of renal tumors. Sem.
Thier.R^Peter, H., Wiegand, H.J., and Bolt, H.M. (1990) DNA binding study
•'•" of isophorone in rats and mice. Arch. Toxicol. 64:684-685.
Thomas, T.L., Decoufle, P., and Moure-Eraso, R. (1980) Mortality among
workers employed in petroleum refining and petrochemical plants. J.
(Dccup. Med.22:97-103.
Thomas, F.B., Haider, C.A., Holdsworth, C.E., and Cockrell, B.Y. (1985)
Hydrocarbon nephropathy in male rats. Temporal and morphologic
characterization of the renal lesions. In: Bach, P.H., and Lock, E.A.
(eds.), Renal Heterogeneity and Target Cell Toxicity. John Wiley and
Sons, New York, pp. 477-480. .,„..,.
Thorhallson, P., and Tulinius, H. (1.981) Tumors in Iceland. 3. Malignant
tumors of kidney. A histological classification. Acta Path. Microbiol.
Scand. Sect. A 89:403-410.
Thung, P.J. (1962) Physiological proteinurea in mice. Acta Physiol.
Pharmacol. Neerl. 10:248-261. .
Trump B.F., Lipsky, M.M., Jones, T.W., Heatfield, B.M., Higgmson, J.,
Endicott, K., and Hess, H.B. (1984a) An evaluation of the significance
of experimental hydrocarbon toxicity to man. In: Mehlman, M.A.,
Hemstreet, G.P., Thorpe, J.J., and Weaver, N.K. (eds.), Advances in
Modern Environmental Toxicology, Vol. VII, Renal Effects of Petroleum
Hydrocarbons. Princeton Scientific Publishers, Inc., Princeton, pp. 273-
288
Trump B.F., Jones, T.W., and Heatfield, B.M. (1984b) The biology of the
kidney. In: Mehlman, M.A., Hemstreet, G.P., Thorpe, J.J., and Weaver,
N.K. (eds.), Advances in Modern Environmental Toxicology, Vol. VII,
Renal Effects of Petroleum Hydrocarbons. Princeton Scientific
Publishers, Inc., Princeton, pp. 27-49.
Tsuji M., Fujisaki, Y., Arikawa, Y., Masuda, S., Tanaka, T., Sato, K., Noda,
K., Ide, H., and Kikuchi, M. (1975) Studies on d-limonene, as gallstone
solubilizer (IV). Chronic toxicity in dogs. Pharmacometrics 9:775-808.
(Japanese)
Umemura, T., Takada, K., Ogawa, Y., Kamata, E., Saito, M., and Kurokawa,
Y. (1990). Sex difference in inhalation toxicity of p-dichlorobenxene (p-
DCB) in rats. Tox. Lett. 52:209-214.
Universities Associated for Research and Education in Pathology (UAREP)
(1983) Hydrocarbon toxicity: acute, subchronic, and chronic effects in
relation to unleaded gasoline exposure of rodents with comments on the
significance to human health. Universities Associated for Research and
Education in Pathology, Inc. Contract No. PS-6 UAREP (504-2) with the
American Petroleum Institute. December 1983.
Unterman, R.D., Lynch, K.R., Nakhasi, H.L., Dolan, K.P., Hamilton, J.W.,
Cohn, D.V., and Feigelson, P. (1981) Cloning and sequence of several
a, -globulin cDNAs. Proc. Natl. Acad. Sci. U.S.A. 78, 3478-3482.
116
-------
US Environmental Protection Agency (USEPA) (1985) Health Assessment
DocumentforTetrachloroethylene (Perchloroethylene). Office of Health
and Environmental Assessment, U.S. Environmental Protection Agency,
July 1985. pp. 8-23, 8-24.
US Environmental Protection Agency (USEPA) (1986) Guidelines for
Carcinogen Risk Assessment, 51 FR 33992-34002, September 24,
1986.
US Environmental Protection Agency (USEPA) (1987) Evaluation of the
carcinogenicity of unleaded gasoline. Carcinogen Assessment Group,
United States Environmental Protection Agency, September 1987.
US Environmental Protection Agency (USEPA) (1988) Chlorothalonil.
Health advisory. Off ice of Drinking Water, U.S. Environmental Protection
Agency, June 1988.
Vamvakas, S., Herkenhoff, M., Dekant, W., and Henschler, D. (1989)
Mutagenicity of tetrachloroethene in the Ames test—metabolic activation
by conjugation with glutathione. J. Biochem. Toxicol. 4:21 -27.
.Vandoren, G., Mertens, B., Heyns, W., Van Baelen, H., Rombauts, W., and
Verhoeven, G. (1983) Different forms of a,u-globulin in male and female
rat urine. Eur. J. Biochem. 134:175-181.
Vyskobil, A., Emminger, S., Tejral, J., Fiala, Z., Etterova, E., andCermanova,
A. (1990) Study on kidney function in female woekers exposed to
perchloroethylene. Human Exptl. Toxicol. 9:377-380.
Viau, C., Bernard, A., Gueret, F., Maldague, P., Gengoux, P., and Lauwerys,
R. (1986) Isoparaffinic solvent-induced nephrotoxicity in the rat.
Toxicology 38:227-240.
Wallace, A.C., and Nairn, R.C. (1972) Renal tubular antigens in kidney
tumors. Cancer 29: 977-981.
Waiter, H.L. (1983) Recent progress inthe pathological anatomy of cancers
of the kidney. Progr. Surg. (Base!) 17:32-57.
Watabe.T., Hiratsuka, A., Ozawa, N., and Isobe, M. (1981) A comparative
study on the metabolism of d-limonene and 4-vinyIcyclohex-1-ene by
hepatic microsomes. Xenobiotica 11:333-344.
Webb, D.R., Ridder, G.M., and Alden, C.L. (1989) Acute and subchronic
nephrotoxicity of d-limonene in Fischer 344 rats. Fd. Chem. Toxicol. 27:
639-649.
Webb, D.R., Kanerva, R.L., Hysell, O.K., Alden, C.L., and Lehman-
McKeeman, L.D. (1990) Assessment of the subchronic oral.toxicity of
d-limonene in dogs. Fd. Chem. Toxicol. 28:669-675.
Wen, C.P., Tsai, S.P., McLellan, W.A., and Gibson, R.L. (1983) Long-term
mortality study of oil refinery workers: mortality of hourly and salaried
workers. Am. J. Epidemiol. 118:526-542.
Wong, O., and Raabe, G.K. (1989) Critical review of cancer epidemiology
in petroleum industry employees, with a quantitative meta-analysis by
cancer site. Am. J. Industr. Med. 15: 283-310.
Yoon, J.S., Mason, J.M., Valencia, R., Woodruff, E.G., and Zimmering, S.
(1985) Chemical mutagenesis testing in Drosophila. IV. results of 45
coded compounds tested for the National Toxicology Program, Environ.
Mutagen. 7:349-367.
117
-------
Yu, M.C., Mack, T.M., Hanisch, R., Cicioni, C., and Henderson, B.E. (1986)
Cigarette smoking, obesity, diuretic use, and coffee consumption as risk
factors for renal .cell carcinoma. J. Natl. Cancer Inst. 77:351-356.
' ,„_, 1591 5t8 187.
*U.S. GOVERNMENT PJUNT1NC OFFICE: -
118
-------
|