EPA/625/3-917019A
SAB/SAP Review Draft
February 1991
ALPHA-2U-GLOBULIN: ASSOCIATION WITH CHEMICALLY-INDUCED
RENAL TOXICITY AND NEOPLASIA IN THE MALE RAT
Prepared for the
Risk Assessment Forum
U.S. Environmental Protection Agency
Washington, DC
Authors
Karl Baetcke
Imogene Sevin Rodgers
Letitia Tahan
Gordon Hard
Robert McGaughy
Karl P. Baetcke, Ph.D.*
Letitia Tahan, M.S.*
Marion Copley, D.V.M.
Technical Panel
Julie Du, Ph.D.
Robert McGaughy, Ph.D.
William PePelko, Ph.D.
*Technical Panel Co-Chairman
Project Officer: 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.
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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DRAFT — DO NOT QUOTE OR CITE
This document is a draft for review purposes only and does not
constitute Agency policy. Mention of trade names or commercial
products does not constitute endorsement or recommendation for use.
11
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TABLE OF CONTENTS
PREFACE
GLOSSARY
EXTERNAL PEER REVIEWERS
EXECUTIVE SUMMARY
vil
viii
ix
1
I. Introduction
PART 1 - NEPHROTOXICITY
II. Hyaline droplets and alpha-2u-globulin; physiology
and biochemistry
A. Filtration, reabsorption, and catabolism of
low-molecular-weight proteins by the kidney
B. Hyaline droplets in renal tubules
C. Factors affecting kidney accumulation of
low-molecular-weight proteins
D. The alpha-2u-globulin superfamily of proteins
E. Characteristics of alpha-2u-globulin
F. Sex and species comparison of urinary protein
content of the lipocalin superfamily
G. Noncovalent binding to alpha-2u-globulin and
its homologues
H. Catabolism of alpha-2u-globulin complexed
with CIGA
I. Structure-activity relationships for CIGA
17
17
17
20
23
25
29
31
33
38
41
III. Alpha-2u-globulin nephropathy
A. Pathologic features of alpha-2u-globulin
nephropathy
B. Rat urine chemistry and CIGA
43
44
48
ill
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C. Species variation in the renal response
to GIGA
D. Factors affecting the expression of
alpha-2u-globulin nephropathy
E. Chronic progressive nephropathy
F. Renal toxicity observed in chronic
bioassays of chemicals that induced
kidney tumors in rats
50
53
56
59
PART 2 - CARCINOGENICITY
IV. Pathologic features of renal carcinogenesis
induced by classical carcinogens
A. Early nephrotoxicity
B. Karyomegaly
C. Tubule cell hyperplasia
D. Adenoma
E. Adenocarcinomas and carcinomas
F. Tumor progression
G. Site of origin of renal tubule tumors
V. Neoplastic and pre-neoplastic lesions observed
in the 2-year bioassays
A. Generic considerations
B. Renal tumor incidence
C. Histogenesis of renal tumors
D. Renal tumor latency and progression
E. Induction of other tumor types
VI. Additional evidence concerning the renal
carcinogenicity of GIGA
A. Genetic toxicology studies
B. Initiation-promotion data
iv
66
66
67
68
68
69
70
70
71
71
80
82
83
:84
84
86
86
91
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VII. Comparison of CIGA with classical renal carcinogens 94
VIII. Evidence concerning human kidney cancer 97
A. Morphology and histogenesis 97
B. Incidence and mortality 98
C. Environmental and lifestyle factors 101
D. Occupational factors 102
E. Renal cancer and hydrocarbon, solvent 103
or petroleum product exposure
IX. Evidence for dose- and time-dependent
progression from early to late lesions 107
A. Association between CIGA, hyaline droplet 107
formation and alpha-2u-globulin accumulation
B. Association between hyaline droplet formation, 109
cell necrosis, and tubule cell regeneration
C. Progression to cast formation, tubule 115
dilation and mineralization
D. Association between CIGA and chronic 119
progressive nephropathy
E. Evidence concerning progression from 120
nephrotoxicity to renal neoplasia
PART 3 - EVALUATION OF THE HYPOTHESIS 123
X. Summary of the evidence on the renal effects of CIGA 123
A. Association between alpha-2u-globulin 123
and nephrotoxicity
B. Association between nephrotoxicity and 125
renal cancer
C. Information reducing confidence in the 126
conclusion that the alpha-2u-globulin
response is specific to the male rat
XI. Conclusions 128
XII. Research needs 134
v
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PART 4 - POLICY
REFERENCES
APPENDICES
137
138
161
VI
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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 guidance. 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 established 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 have reported renal tubule tumor formation
in male rats as the sequela of renal toxicity commencing with an
excessive accumulation of the protein, alpha-2u-globulin (a2u-9)/
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 Reiter rat, which
does not produce a2u-g, also fails to show a proliterative 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
two 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 a2u-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 recommendations for Agency-
wide use. This document is the product of that effort.
The literature review supporting this document is current as
of February 25, 1991.
NOTE: Except for SAB/SAP review, the scientific analysis in this
report is complete; editorial review is incomplete. Accordingly,
this draft is being submitted simultaneously to the SAB for
scientific peer review, and to technical editors for final editing,
formatting, and reference review.
VII
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GLOSSARY
AAT aspartate aminotransferase
ABS chromosome aberrations in CHO cells
a -g alpha-2u-globulin
CHO Chinese hamster ovary
Ci confidence interval
GIGA Chemical(s)Inducing alpha-2u-Globulin Accumulation
CPN chronic progressive nephropathy
1,2-DCB 1,2-dichlorobenzene
1,4-DCB 1,4-dichlorobenzene
DEN diethylnitrosamine
DMN dimethylnitrosamine
EHEN N-ethyl-N-hydroxyethylnitrosamine
FBPA N-4'-(fluoro-4-biphenylyl)acetamide
IRDC International Research and Development Corporation
MLA TK-gene mutation assay in L5178Y cells
MTD maximum tolerated dose
MUP mouse major urinary protein
NAG N-acetyl-/3-glucosaminidase
NBR NCI Black-Reiter rat
NTP National Toxicology Program
NCI National Cancer Institute
OR odds ratio
RR relative risk
P1 first convoluted segment of proximal tubule
P2 second convoluted segment of proximal tubule
P3 pars recta of proximal tubule
SAL salmonella
SCE sister chromatid exchange
SEER Surveillance, Epidemiology and End Results
Program of NCI
SLRL sex-linked recessive lethal
TFT trifluorothymidine
TK thymidine-kinase
TMP 2,2,4-trimethylpentane
TMPOH 2,4,4-trimethylpentanol
UDS unscheduled DNA synthesis
Vlll
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EXTERNAL PEER REVIEWERS
This draft report was evaluated at a two-day Peer Review
Workshop sponsored by the U.S. EPA Risk Assessment Forum. The
meeting^ held in Gaithersburg, Maryland, on November 13 and 14
1990, was chaired by Richard Griesemer, director of the Division of
Toxicology Research and Testing, National Toxicology Program (NTP).
A separate report of this meeting will be available as EPA
publication no.[ ]. In addition to plenary sessions on each
day, workgroups were asked to address specific issues on four
topics, nephropathy and biochemistry, cancer, criteria for
evaluating renal carcinogens, and risk characterization.
The Nephropathy and Biochemistry Workgroup was chaired by
Michael Olson of General Motors Research Laboratories. Other
participants in that group included: Carl Potter (Risk Reduction
Engineering Laboratory, Cincinnati) and James McKinney (Health
Effects Research Laboratory, Research Triangle Park) of EPA,
Benjamin Trump of the University of Maryland, and Dennis Lynch of
the Division of Biological and Behavioral Sciences, National
Institute for Occupational Safety and Health.
The Cancer Workgroup was chaired by John Ashby of the Central
Toxicology Laboratory at International Chemical Industries, Ltd.
Other members included: R. Daniel Benz of the Center for Food
Safety and Applied Nutrition at the Food and Drug Administration,
James Popp, head of the Department of Experimental Pathology and
Toxicology at Chemical Industries Institute of Toxicology, Michael
Elwell of NTP, and Joseph McLaughlin and Jerrold Ward of the
National Cancer Institute.
The Criteria Workgroup was composed of pathologists who had
specific research experience either in examining the lesions
hypothesized to be associated with alpha-2u-globulin accumulation
or in renal carcinogenesis. This group was chaired by Gordon Hard,
of the Medical Research Council. Other members included: William
Busey of Experimental Pathologies Laboratories, Scot Eustis of the
NTP, Lois Lehman-McKeeman of Procter and Gamble Company, and James
Swenberg of the University of North Carolina.
Participation in the Risk Characterization Workgroup was
limited to government officials except for the chair, Norbert Page
of Page Associates. Others were: William Farland and Penny Fenner-
Crisp of EPA, Deborah Barsotti of the Division od Toxicology at the
Agency for Toxic Substances Disease Registry, Murray Cohn of the
Directorate for Health Sciences at Consumer Product Safety
Commission, Elizabeth Grossman of the Office of Risk Assessment at
the Occupational Safety and Health Administration, and Lauren Zeiss
of the State of California Health Department.
Drafts prepared in advance of the Peer Review Workshop
were reviewed and commented on by the following external reviewers.
ix
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Carl Alden (first and second drafts)
The Procter and Gamble Company
Miami Valley Laboratories
Cincinnati, Ohio
William Busey (first draft)
Experimental Pathology Laboratories
Herndon, VA
Gordon Hard (first draft)
Medical Research Council Toxicology Unit
Great Britain
Michael Lipsky (first draft)
University of Maryland
School of Medicine
Michael Olson (first and second drafts)
General Motors Research Laboratories
Warren, Michigan
James Popp (second draft)
Department of Experimental Pathology and Toxicology
Chemical Industries Institute for Toxicology
Research Triangle Park, North Carolina
James Swenberg (first draft)
University of North Carolina
Department of Pathology
Chapel Hill, North Carolina
The Technical Panel and the Risk Assessment Forum also
acknowledge with appreciation the special contributions of
Lawrence Valcovic, who prepared the section on mutagenicity,
Joseph McLaughlin and Cheryl Siegel Scott, who greatly assisted in
the preparation of the epidemiology section, and Ila Cote, Margaret
M.L. Chu, and Richard N. Hill for their thoughtful comments.
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EXECUTIVE SUMMARY
A Technical Panel of the U.S. Environmental Protection Agency's
(EPA) Risk Assessment Forum advises EPA risk assessors against
using information on renal tubule tumors or nephrotoxicity that is
associated with alpha-2u-globulin (a2u-g) accumulation in hyaline
droplets in male rats to assess human risk. The scientific
information reviewed by the Technical Panel provides reasonable
evidence to suggest that the acute and chronic renal effects
observed in male rats from chemically-induced a2u-g accumulation
are unlikely to occur in the absence of a2u-g, or a protein with a
structurally similar binding domain, in the large quantities
typically seen in the male rat. Thus, if a chemical induces a2u-g
accumulation in hyaline droplets, the associated nephropathy
observed in male rats may not be an appropriate endpoint for
assessing noncancer risk in humans. Likewise, a carcinogenic
response in the male rat kidney attributable to a process involving
a2u-g accumulation in the renal proximal tubule may not be an
appropriate endpoint for assessing carcinogenic risk to humans.
The policy set out in this report provides guidance on
determining when it is reasonable to presume that renal tumors in
male rats result from a process involving a2u-g accumulation and on
selecting appropriate procedures for estimating risks to humans
under such circumstances. It also defines situations that suggest
different approaches and calls for research to clarify questions
raised because of the existence of human proteins that may be
structurally similar to o;2u-g.
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In the male rat, the production of renal tumors by chemicals
inducing a-2u-globulin accumulation (CIGA) is preceded by the renal
lesions ascribed to a2u-g-associated nephropathy. The involvement
of hyaline droplet 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 nephrotoxicity, but no apparent hyaline droplet
accumulation.
Investigations performed in multiple laboratories over the
last decade have demonstrated a consistent association between
hyaline droplets containing a2u-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:
(1) an excessive accumulation of hyaline droplets
containing a2u-g in renal proximal tubules;
(2) subsequent cytotoxicity and single cell
necrosis of the tubule epithelium;
(3) sustained regenerative tubule cell prolif-
eration, providing exposure continues;
(4) development of intralumenal granular casts
from sloughed cell debris associated with
tubule dilation and papillary mineralization;
(5) foci of tubule hyperplasia in the convoluted
proximal tubules; and finally,
(6) renal tubule tumors.
Biochemical studies with model compounds show that CIGA or
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their metabolites bind specifically, but reversibly, to male rat
a2u-g. The resulting a2u-g-CIGA complex appears to be more
resistant to hydrolytic degradation by lysosomal enzymes than
native, unbound a2u-g. Inhibition of the catabolism of a2u-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.
It is instructive to compare GIGA renal carcinogens with other
renal carcinogens. Several genotoxic chemicals 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 DNA adducts in the kidney. In contrast, GIGA renal
carcinogens are not known to react with DNA and are generally
negative in short-term tests for genotoxicity. GIGA renal
carcinogens also interact with a2u-g in a reversible and
noncovalent manner.
GIGA produced minimal changes in urine chemistry and very
little or no glomerular dysfunction in male rats. The mild tubule
toxicity of GIGA, in contrast to the obvious urinary changes
induced by renal toxins such as mercuric chloride or
hexachlorobutadiene, is characteristic of GIGA and is consistent
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with the notion that CIGA do not bind covalently to a2u-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 may be 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%. 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 rats showed that
these CIGA promoted atypical tubule cell hyperplasia and/or renal
tubule tumors in males but not in females. In contrast, d-limonene
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
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of chemically induced steps from atypical hyperplasia through
microscopic adenomas to macroscopic adehocarcinomas or carcinomas.
Renal tubule tumors induced by the eight model carcinogens are
morphologically indistinguishable from those induced 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.
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
renal tubule 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 a2u-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 cells excreted in the
urine has been observed for CIGA. Dose-response relationships
between hyaline droplet accumulation and proximal tubule cell
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proliferation 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 relationship between severity of
nephropathy and renal tubule cancer in male rats.
The Technical Panel is not aware of any epidemiological study
that has been designed or conducted specifically to examine the
applicability of the GIGA hypothesis 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 GIGA 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, it is difficult to identify specifically the agent
responsible for the increased risk. These studies, therefore, are
considered inadequate for purposes of hazard identification.
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
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:. . . . .
hydrolytic degradation, a feature apparently required for hyaline
droplet formation.
Extensive studies in mice, whose urine contains large amounts
of mouse major urinary proteins (MOP), have found no evidence of
renal lesions similar to those associated with the a2u-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 a2u-g which 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 a2u-g-like nephropathy when exposed to
GIGA. In cases where nephrotoxicity was observed in mice or female
rats, it was less severe and qualitatively different from that in
male rats and did not involve the spectrum of discrete lesions
associated with a2u-<3 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 a2u-g in the liver),
and injection of male rats with estrogen and female rats with a2u-
g, show that development of the early features of the specific
nephropathy syndrome is dependent on the presence of a2u-g- Very
limited information in dogs, hamsters, guinea pigs, and monkeys
also supports this statement. These studies further support the
hypothesis that this a2u-g-related nephropathy occurs specifically
in the male rat.
In summation, the reversible binding of the compound to a2u-
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g, which results in a shift in balance between reabsorption and
hydrolysis and the accumulation of a2u-g in hyaline droplets in the
P2 segment of the renal tubule provides a plausible explanation for
the initial steps in a seg^ience 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. This increased
proliferative response caused by chemically-induced cytotoxicity
may be a plausible reason for the development of renal tubule
tumors in male rats, and renal tubule tumors produced in male rats
where there was GIGA-induced a2u-g nephropathy should be
distinguished from other renal tumors wherever possible for
purposes of their use in human risk assessment.
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X. INTRODUCTION
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
accompanied by accumulation of the protein, alpha-2u-globulin (a2u-
g) (reviewed in 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 major regulatory and commercial interest. For example,
isophorone 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.
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TABLE 1. SOME EXAMPLES OP ORGANIC CHEMICALS THAT PRODUCE RENAL
INJURY IN MALE RATS CHARACTERIZED BY HYALINE DROPLET
ACCUMULATION BUT NOT IN FEMALE RATS OR OTHER SPECIES
CHEMICAL
SPECIES TESTED
RENAL
TOXICITY
REFERENCE
Decalin
Dimethyl methyl
phosphonate
Isophorone
JP-5 shale-derived
jet fuel
JP-4 jet fuel
d-Limonene
Methyl isobutyl
ketone
Pentachloroethane
Unleaded gasoline
Rats (m/f)
Mice (m/f)
Dogs (m/f)
Guinea
pigs (m/f)
Rats (m/f)
Mice (m/f)
Rats (m/f)
Mice (m/f)
Rats
Mice
Dogs
Rats
Mice
Dogs
Rats
Mice
Dogs
(m/f)
(m/f)
(m/f)
(m/f)
(m/f)
(m/f)
(m/f)
(m/f)
(m)
Rats (m/f)
Mice (m/f)
Dogs (m)
Monkeys (m)
Rats
Mice
(m/f)
(m/f)
— /—
Rats (m/f)
Mice (m/f)
Alden et al. (1985)
USEPA (1987)
NTP-TR-323 (1987b)
NTP-TR-291 (1986a)
MacNaughton &
Uddin (1984)
MacNaughton &
Uddin (1984)
NTP-TR-347 (1990)
Webb et al. (1991)
Alden et al. (1984)
Phillips et al.
(1987)
NTP-TR-232 (1983)
USEPA (1987)
m = male
f = female
-f = positive
- = negative
10
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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, hexachloroethane, isophorone, d-limonene,
pentachloroethane, tetrachloroethylene, and a mixture, unleaded
gasoline. These eight substances are compared and contrasted with
two related non-a2u-g-inducers, chlorothalonil and
trichloroethylene. The analysis also relies on research studies on
two other model compounds, decalin and 2,2,4-trimethylpentane
(TMP), which have extensive information on a2u-g nephropathy but no
chronic bioassay data. More limited data on 22 additional
substances is also discussed where appropriate.
Among the eight model chemicals tested in chronic animal
bioassays, all invoked a specific type of protein droplet
nephropathy in male rats and all 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
(UAREP, 1983; Alden et al., 1984; Haider et al., 1984; HEI, 1988;
Swenberg et al., 1989a).
• Excessive accumulation of hyaline droplets in proximal
tubules, representing lysosomal overload, leads to tubule
cell degeneration, cell loss, and regenerative cellular
proliferation.
• Cell debris in the form of granular casts accumulates at
the "corticomedullary" junction with associated dilation
of the affected tubule segment and more distally,
mineralization of tubules within the renal medulla.
11
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Single cell necrosis accompanied by compensatory cell
proliferation and exacerbation of the chronic progressive
nephropathy (CPN) characteristically found in aging rats '
occurs.
Renal tubule hyperplasia and neoplasia develop subse-
quently .
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., 1984b;
Alden, 1989; Swenberg et al., 1989a). This line of reasoning leads
supporters of the hypothesis to conclude that the acute and chronic
renal effects induced in male rats by these chemicals will be
unlikely to occur in any species not producing a2u-g or a very
closely related protein in the large quantities typically seen in
the male rat (Alden 1989; Borghoff et al., 1990; Green et al.,
1990; Olson et al., 1990; Flamm and Lehman-McKeeman, 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 support or contradict
the concept that the renal tumors produced in male rats by these
chemicals are causally related to the nephrotoxicity. Based on the
Risk Assessment Forum's conclusions regarding these data, the
document proposes a uniform approach for EPA to use in risk
assessments dealing with this spectrum of lesions and category of
chemicals.
Information for this Risk Assessment Forum report was obtained
12
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initially from a 1988 review entitled "Evaluation of Data
Concerning the Relationships among Chemically-induced Renal
Alpha2uGlobulin 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 other original work, especially publications subsequent to the
1988 Dynamac review.
The document has four parts. Following this brief
introduction, Part 1 addresses the characteristics of hyaline
droplets and the protein, a2u-g, and the nephropathy associated
with a2u-g accumulation (Sections II and III) .
Part 2 (Sections IV-IX) presents data on the carcinogenic
potential of GIGA in the male rat. Section IV describes the
preneoplastic and neoplastic lesions produced by classical renal
carcinogens. Section V 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 VI examines additional information that
assists in defining renal carcinogens as GIGA, in particular
genotoxicity and initiation-promotion data. In Section VII, CIGA
are compared with classical renal carcinogens, while Section VIII
considers the human evidence for kidney cancer, its histogenesis
and epidemiology. Section IX examines evidence for dose- and time-
13
-------
dependent progression of the lesions hypothesized to lead to this
nephropathy.
Part 3 evaluates the evidence considered in Parts 1 and 2 with
regard to the hypothesis that a2u-g accumulation in the kidney is
an initial step in a succession of histopathologic events that may
culminate in renal tubule tumor formation in male rats. This part
also lists priorities for future 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, alPha-2u-alobulin (a,.,-g) nephropathy is used to
connote the full sequence of pathologic lesions from hyaline
droplet formation to restorative hyperplasia and medullary
mineralization. Toxic tub"i*T- nenhropathv is a non-specific term
commonly used in rodent bioassay reports to describe various forms
of nephrotoxicity induced by chemicals, including the specific
lesions of a2u-g nephropathy. The spontaneous age-related syndrome
of rat kidney disease otherwise known in the literature as old rat
1Hvaline droplets refer to spherical inclusions in the
cytoplasm which are homogeneous and eosinophilic, representing
overdistended phagolysosomes. They may contain various
macromolecules including a-2u-globulin. The morphology of droplets
containing different proteins may be identical and therefore
iSmunoSy??chemistry is required for precise definition of contents.
14
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nephropathy, chronic nephrosis, glomerulosclerosis, and progressive
glomerulonephrosis, is standardized according to Barthold (1979)
as chronic progressive nephropathv (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 PI. P2. P3 are used conventionally to denote
these three segments. The term renal tubule tumor describes
neoplasms of the renal cortical tubule epithelium comprising
collectively adenoma, adenocarcinoma and carcinoma according to
standardized nomenclature determined by the Society of Toxicologic
Pathologists (Hard et al., 1991). The same neoplasms are referred
to as renal cell tumors in humans, in keeping with the general
literature (Bannayan and Lamm, 1980).
15
-------
Distal convoluted
x
UJ
I-
cc
O
o
DC -J
UJ 3
t- Q
=3 UJ
O s
< Outer strjpe_
a Inner stripe
a
UJ
5
DC
UJ
z
z
Pa pi I la
.Thjc.k
limb of Henle
Collecting duct
Thin descending
I jmb of Henle
Thi n ascending
limb of Henle
FIGURE 1. DIAGRAM OF ZONATION AND TUBULE SEGMENTATION IN RAT
KIDNEY.
G: Glomerulus; P,,: First segment of proximal convoluted
tubule; P2: Second segment of proximal convoluted
tubule; P3: Pars recta of proximal tubule.
(Adapted from Bachmann et al., 1986)
16
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PART 1. NEPHROTOXICITY
II. HYALINE DROPLETS AND ALPHA-2U-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 a2u-g provides an explanatory basis for the
accumulation of a2u-g in hyaline droplets in the male rat following
exposure to GIGA. It is pertinent, therefore, to examine the
physiological and biochemical characteristics of a2u-g and related
proteins, particularly those that occur in humans, before exploring
the possible associations between a2u-g accumulation, renal toxicity
and renal tumor formation and their relevance to human risk
assessment.
A. Filtration, reabsorption, and catabolism of low-molecular-
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 continually removed from the plasma by
glomerular filtration followed by reabsorption and catabolism in
the proximal tubules (Maack et al., 1985), or excretion. Figure
2 is a schematic representation of the cellular uptake and
disposition of filtered proteins by the renal tubule.
17
-------
Proximal_[P2) Tubule Cell
EV
Tubule
Lumen
QEGBAD.AI
FLBLQLQUCTS
GOLGI
APPARATUS
FIGURE 2: schematic representation of eadocytic 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 phagolysosome 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. (Adapted from Kaysen et
al., 1986).
18
-------
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
cytochrome C (Maack et al., 1985). For larger proteins like the
albumins and globulins, which have a far greater plasma
concentration and much lower 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 reabsorption was once considered
largely non-selective, 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).
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
19
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tubules include two major classes of acid proteinases - 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 endopeptidase classes
contribute to the degradation of a2u-g.
Lysosomes have a large, but not unlimited, capacity to cope
with increased amounts of hydrolyzable 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). Alpha-2u-globulin, with a half-life
measured in hours (Geertzen et al., 1973), is one of the
exceptions.
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
20
-------
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 abnormally large, dense,
secondary lysosomes (also termed phagolysosomes), representing
fusion of endocytic vacuoles with primary lysosomes. Some hyaline
droplets show crystalloid changes by electron microscopy which are
not observed in the lysosomes of female rats (Maunsbach, 1966b).
Crystalline 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
lysosomes.
Hyaline droplets in the proximal tubules of normal male rats
contain a2u-g (Alden et al., 1984; Garg et al., 1987; Goldsworthy
et al., 1988), 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 ot2u-g
(Logothetopoulos and Weinbren, 1955; Maunsbach, 1966a; Goldsworthy
et al., 1988a; Burnett et 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
21
-------
one etiology and can be associated with the accumulation of
different proteins, it is necessary to apply special diagnostic
methods such as immunohistochemical staining to make the
association between chemical exposure and pathologic accumulation
of a2u-g.
Abnormal accumulation of hyaline droplets in rodent kidney is
seen in certain disease processes. Both male and female rats with
histiocytic sarcoma show hyaline droplet accumulation in the
proximal tubules, indistinguishable from the CIGA-induced lesion.
The accumulating protein in these tumor-bearing animals has been
identified as lysozyme (Hard and Snowdon, 1991). Similarly, in
male and female mice with histiocytic tumors, abnormal accumulation
of lysozyme-containing hyaline droplets sometimes occurs in
proximal tubules (Hard and Snowdon, 1991).
In humans, the Bence-Jones proteins, a class of light chain
immunoglobulins, are produced in large amounts in multiple myeloma
patients (Pirani et al., 1983). In cases of mononuclear cell
leukemia, lysozyme is produced (Muggia et al., 1969). The kidney
injury seen with these neoplastic diseases has been described as
similar to that produced by administration of decalin to male rats
(Alden, 1986), including protein droplet accumulation in renal
tubules (Oliver and MacDowell, 1958; Pirani et al, 1983; Pruzansky
and Platts, 1970). Patients with epidemic hemorrhagic fever,
infused with large amounts of concentrated human serum albumin as
a therapeutic procedure for shock have also developed a comparable
form of hyaline droplet accumulation (Oliver and MacDowell, 1958).
22
-------
C. Factors affecting kidney accumulation of low-molecular-
weight proteins
Protein accumulation in the proximal tubule can reach
pathological levels resulting in excessive hyaline droplet
formation for several reasons: (1) the rate of protein delivery
to the tubule cells is abnormally high, (2) the proteins delivered
are difficult to hydrolyze, or (3) the lysosomal hydrolysis
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 where there is immunological, inflammatory, or toxic
disease in the glomerulus 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) are proteins also
relatively resistant to hydrolysis (Maack et al., 1985). This
suggests a combination of features (1) and (2) as an etiologic
factor in the accumulation of protein observed in rats with
histiocytic sarcoma (lysozyme) and in human patients with multiple
myeloma (light chain immunoglobulins). 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
23 •
-------
in the sexually mature male rat also appears to be a factor in the
accumulation of o:2u-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 enzyme 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 a2u-g, tend to accumulate first.
Testosterone 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 those 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 GIGA may not compromise kidney
lysosomal enzyme activity per se (Murty et al., 1988; Lehman-
McKeeman et al., 1990), any chemically-induced impediment to a2u-
g digestibility caused by CIGA would be further superimposed on the
factors considered above that alone can cause excessive protein
accumulation in renal tubules.
24
-------
D. The alpha-2u-globulin superfamily of proteins
Alpha 2u-globulin is a member of a large superfamily of low
molecular weight proteins. The complete amino acid sequence of
a2u-g was first deduced by Unterman et al. (1981). Even though,
with the exception of Q!2U-g and mouse major urinary protein(s)
(MUP), the sequence homology between any pair of proteins in this
superfamily is small, about 20 percent, statistical analysis shows
that the proteins are related evolutionarily (Akerstrom 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 for only three,
retinol-binding protein, J3-lactoglobulin, and insecticyanin
(Sawyer, 1987). The central core of these three proteins is
composed of eight strands with a 6-barrel structure forming a
hydrophobic pocket that appears to enclose the ligand (Papiz et
al., 1986; Sawyer, 1987). This structure has been described as
resembling a coffee filter paper (Akerstrom and Logdberg, 1990).
In addition to the B-structural motif, one helical rod and several
other structural elements appear to be conserved among the
proteins. Protein folding patterns tend to be highly conserved in
homologous proteins even though they may diverge considerably in
structure and function, suggesting that other members of the
superfamily, including a2u-g, possess a similar three dimensional
structure.
The only member of the protein superfamily with a clearly
25
-------
defined physiological function is retinol-binding protein. More
circumstantial evidence suggests that the superf amily members serve
as carriers of lipophilic molecules (Pervaiz and Brew, 1987). The
mode of binding in which the lipid ligand is enclosed within the R-
barrel impressed Pervaiz and Brew as not unlike the role of the
calyx to a flower. On this basis, they suggested the illustrative
name, lipocalins. for the superfamily of proteins.
Table 2 illustrates the information available on several
members of the lipocalin superfamily, which includes a2u~9'
retinol-binding protein, apolipoprotein D, a1-acid glycoprotein and
c^-microglobulin of humans, bovine 6-lactoglobulin and pyrazine-
binding protein (i.e., odorant-binding protein), rat odorant-
binding protein and major urinary protein(s) (MUP) of mice. Some
of the members of the lipocalin superf amily, such as retinol-
binding protein, 6-lactoglobulin, and o^-microglobulin have been
identified in many species, and their properties appear to be
species independent, suggesting that they share a common vital
function (Akerstrom and Logdberg, 1990) . Others, such as a2u-g and
MUP seem to be species-specific.
26
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TABLE 2. SUPERFANILY OF LIPOPHILIC LIGAHD-BIKDING CARRIER PROTEINS
SPECIES
Human
Cow
Rat
Mouse
Chick
Frog
Insect5
PROTEIN
a^-Acid glycoprotein
Apolipoprotein D
Pregnancy-associated
endometrial ag-globulin
Protein HC; a. microglobulin
Retinal-binding protein
B-lactoglobulin
Pyrazine-binding protein
(^-globulin
Androgen - dependent
secretory protein
Odorant-binding protein
Fatty-acid-binding protein
Major urinary protein
Purpurin
Bowman's gland protein
Insecticyanin
TISSUE OR
BODY FLUID
Plasma
Plasma
Placenta
Plasma, urine,
spinal fluid
Liver
Milk
Nasal epithelium
Primarily male
liver, urine
Epididymis
Nasal epithelium
Liver
Liver (both
(sexes), urine
Retina
Olfactory
epithelium
Memo lymph
MOLECULAR
WEIGHT
18,944 2
19,300
25,000
20,619
22,868
18,281
19,000
18,709
18,500
18,091
14,000
18,730
21,924
20,300
21,382
NO. OF
AMINO ACIDS
167
169
Not known
182
199
162
Not known
162
184
172
Not known
162
196
182
189
' Adapted from Pevsner et at, 1988, except as noted.
£ In rat (Pervaiz and Brew, 1987)
f Cavaggioni, et at., 1987
* Kimura, et at., 1989
Tobacco hornworm
27
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Several functions have been suggested for a2u-g. Cavaggioni
et al. (1987) speculated that a2u-g may serve to transfer odorants
such as ethereal lipid pheromones from male rat urine to the air
for attracting females. Glandular tissue production of a2u-g helps
support these speculations (Murthy et al., 1987; Mancini et al.,
1989) . In addition, c*2u-g has been identified 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 kidney,
within renal epithelial cells. Brooks (1987) found a protein
structurally related to a2u-g which is synthesized and secreted by
the rat epididymis under the influence of androgenic hormones. He
speculated that the function of 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 protein, apolipoprotein D, /3-lactoglobulin, and o^-acid
glycoprotein, function in the transport of lipids between cells and
across hydrophilic barriers (Pevsner et al., 1988). The lipids
bound by the proteins differ considerably in 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 hydrophobia molecules. Both cases
might occur since retinol-binding protein is quite specific for
retinol, whereas odorant-binding proteins may have a broad
specificity (Godovac-Zimmermann, 1988).
Cavaggioni et al. (1990) reported substantial differences in
28
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the binding affinities of a2u-g, MUP 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; and a2u-g bound twelve. The best ligands for each of the
three proteins were chemically unrelated; close structural analogs
of the best ligands were also only weak ligands, much weaker than
structurally unrelated chemicals. This study suggests that
structure-activity and binding affinities for one lipocalin are
poor predictors for other members of the superfamily.
E. Characteristics of alpha-2u-globulin
Alpha-2u-globulin was first characterized in male rat urine
(Roy and Neuhaus, 1967) . All isoforms of a2u-g are anionic at
neutral pH although they have varying isoelectric points. The
molecular weight of a2u-g has been reported to be 18,000-20,000
daltons. In all known rat strains, except for the NCI Black-Reiter
(NBR) rat (Chatterjee et al., 1989), the major urinary source of
a2u-g is the liver where a2u-g mRNA constitutes approximately 1% of
the hepatic mRNA population (Sippel et al., 1976; Kurtz and
Feigelson, 1978). The hepatic isoforms of a2u-g may vary
throughout the lifetime (Roy et al., 1983). Synthesis of the
protein in rat liver is under multihormonal control, particularly
androgen, but also glucocorticoids, 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 a2u-g mRNA (Kurtz and Feigelson, 1977) . Neither a2u-
g nor its corresponding mRNA are detectable in the livers of
29
-------
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 a2u-g and its mRNA in female rat
liver (Roy and Neuhaus, 1967; Sippel et al., 1975).
Although plasma and urinary a2u-g derives predominantly from
the liver in male rats, high levels of a2u-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) . Alpha-2u-globulin mRNA has also been detected in the female
mammary gland during pregnancy, and in the submaxillary, lacrymal,
Meibomian, and perianal glands of rats of both sexes (Maclnnes et
al., 1986; Mancini et al., 1989). The female forms of a2u-g show
distinct differences from male rat a2u»g suggesting that they are
encoded by different genes (Vandoren et al., 1983).
Low levels of <*2u-g first become detectable in the male rat
liver under the stimulus of testosterone at 35-40 days, reaching
maximum adult levels by 60-80 days (Roy et al., 1983; Maclnnes et
al., 1986; Motwani et al., 1984). At some stage after 100-150 days
of age, due to the development of hepatic insensitivity to androgen
during senescence, hepatic synthesis of a2u-g falls gradually to 50
percent of peak levels in 600 day old male rats, beyond 750 days
of age, becoming undetectable (Roy et al., 1983; Motwani et al.,
1984). Renal cortical tissue content (Murty et al., 1988) and
30
-------
urinary excretion (Neuhaus and Flory, 1978; Motwani et al., 1984)
of aZu-g reflect the same age related trends as synthesis in the
liver.
In the mature male rat, approximately 50 mg of a2u-g is
filtered per day, 40% of the filtered protein being excreted in the
urine and 60% undergoing reabsorption and catabolism (Neuhaus et
al., 1981; Caudill et al., 1991). It is catabolized slowly
relative to most other proteins in the glomerular filtrate, the
half-life in plasma or kidney cytosol or lysosomal preparations
being 5-8 hours (Geertzen et al., 1973; Ekstrom, 1983; Lehman-
McKeeman et al., 1990a). In vitro studies indicate that a2u-g is
more resistant to lysosomal enzyme digestion than bovine 0-
lactoglobulin and lysozyme (Charbonneau et al., 1988). In another
study comparing members of the protein superfamily, a2u-g and a.,-
acid glycoprotein were the most resistant to proteinase K digestion
while retinol-binding protein and 0-lactoglobulin were 1000- to
100,000-fold more easily hydrolysed (Borghoff et al., 1990) . These
data indicate that a2u-g may be more likely to accumulate in the
kidney than most other members of the superfamily if shifts in the
balance between reabsorption and hydrolysis occur.
F. Sex and species comparison of urinary protein content of
lipocalin superfamily
Relative to the female rat, and other species including
humans, the normal mature male rat is physiologically proteinuric.
This is due to the amount of a2u-g in male rat urine, 1.36-8.64
mg/day/gm kidney (Neuhaus and Lerseth, 1979), which is 100 to 300
times more than observed in female rat urine (Shapiro and
31
-------
Sachchidananda, 1982; Vandoren et al., 1983). The mouse can also
be described as physiologically proteinuric because of a high
urinary content of MUP (Thung, 1962). MUP shows the greatest
similarity to a2u-g in the lipocalin superfamily, sharing 90% amino
acid sequence homology (Dolan et al., 1982). Representing a group
of proteins encoded by a multigene family, MUP is synthesized in
the liver of mice of both sexes but at rates four to five times
greater in males than females (Hastie et al., 1979; Roy and
Chatterjee, 1983). Daily urinary excretion of MUP varies
considerably among strains (Szoka and Paigen, 1978). In the B6C3F1
strain, males have been shown to excrete 14.9 mg of MUP/day in the
urine, and females, 2.1 mg/day (Lehman-McKeeman et al., 1990b).
Adjusted for body weight, a male B6C3F1 mouse therefore excretes
approximately 600 mg/kg/day of MUP, some 12 fold higher than a2u-g
urinary excretion by the male rat. Unlike the rat, however, where
60% of filtered a2u-g is reabsorbed by the kidney, MUP is not
reabsorbed in the mouse and appears to be totally excreted (Caudill
et al., 1991).
In contrast, normal human urine contains relatively little
protein, only 1% of the total concentration present in mature male
rat urine (Olson et al., 1990). Human urinary proteins are
predominantly high-molecular-weight species with only minor
components weighing less than 66,000 daltons. Within the Iqw-
molecular-weight fraction, trace amounts of proteins represent the
lipocalin superfamily, but none appear to share molecular weight
identity with a2u-g. The urinary excretion of retinol-binding
32
-------
protein, a,-acid glycoprotein and c^-microglobulin has been measured
at 0.0001-0.0007, 0.0006-0.002, and 0.02-0.05 mg/day/gm kidney,
respectively (Berggard, 1970; Peterson and Berggard, 1971; Ekstrom
and Berggard, 1977). Thus, the urinary excretion of a2u-g in the
male rat is approximately two orders of magnitude greater than the
human urinary content of the three superfamily proteins combined.
Recently, a sex-dependent protein of unknown origin and
function, termed urine protein 1, was identified in normal human
urine (Bernard et al., 1989, 1990). The molecular features of
protein 1 are similar to a2u-g as it has a molecular weight of
approximately 21,000 daltons and an isoelectric point around 4.8
(Bernard et al., 1990). As its amino acids have not been
sequenced, it cannot be placed in the lipocalin superfamily.
Protein 1 occurs in both sexes from an early age, but increases
substantially in males after puberty, reaching up to a fifty-fold
difference over females during late adolescence. A five-fold male
to female differential persists through adulthood. Average urinary
concentrations of protein 1 have been determined as 108 and 3.2
jug/liter respectively for males and females aged 15 to 20 years,
and 24.7 and 5.8 /zg/liter for males and females in the 20 to 60
year age-range (Bernard et al., 1989). Such levels of protein 1
in human male urine, however, are calculated as four to five orders
of magnitude less than a2u~g concentrations in the urine of male
rats (Bernard et al., 1990).
G. Noncovalent binding to alpha-2u-globulin and its homologues
It has been suggested that GIGA bind reversibly and
33
-------
noncovalently to <*2u-g in the male rat, forming a resultant complex
that is even more poorly digested than a2u-g (Swenberg et al.,
1989a). ;
In a few instances, the specific chemical entity complexed
with a2u-g has been identified. TMP, a branched chain aliphatic
hydrocarbon present in gasoline (Haider et al., 1985) was the first
model GIGA to be studied in this manner. When [14-C]TMP was
administered in a single oral dose to male or female rats,
radioactivity was retained in the kidneys of males, but not of
females (Kloss et al., 1985; Charbonneau et al., 1987). The major
metabolite of TMP detected in the kidneys of male rats was
identified as 2,4,4-trimethyl-2-pentanol (TMPOH) (Charbonneau et
al., 1987). In a separate report, it was demonstrated that TMPOH
is the only compound that binds to a2u-g whenever TMP is
administered to the male rat (Lock et al., 1987a). TMPOH was not
detected in the kidney tissue of the female rats, which excreted
more conjugated TMPOH (glucuronides and sulfates) than the males
(Charbonneau et al., 1987). Later studies confirmed, as suspected,
that the TMPOH-a2u-g complex is cleared slowly from male rat kidney
(Swenberg, 1989b).
It has been shown since for d-limonene, that the metabolite
interacting predominantly with a2u-g is d-limonene-l,2-oxide
although there is also some binding to the parent material (Lehman-
McKeeman et al., 1989). For isophorone, the bound material is the
parent compound (Strasser et al., 1988). Following exposure of the
male rat to 1,4-DCB, both the parent chemical and the metabolite,
34
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2,5-dichlorophenol, bind reversibly to a2u-g (Charbonneau et al.,
1989).
The nature of the association of CIGA with a2u-g was explored
initially by Lock et al. (1987a) who dosed sexually mature Fischer
344 rats with [3-H]TMP, killed them 8-72 hours later, and
homogenized the kidneys. Cytosol, obtained by centrifugation of
the homogenate at 116,000 g, was applied to a Sephadex G-75 column.
For the males, 26% of the cytosol radiolabel (15% of all radiolabel
in the kidney) eluted in the fraction containing a2u~9-
Approximately 19% of the radiolabel present in male rat kidney
cytosol was nondialyzable following overnight equilibrium dialysis
against phosphate buffer. Chromatography of the dialyzed cytosol
showed that the nondialyzable radiolabeled material coeluted with
the peak containing a2u-g. When 0.1% sodium dodecyl sulfate, a
detergent which affects the secondary and tertiary structure of
proteins, was added to the dialysis buffer, there was a significant
loss of binding. These results suggest a reversible binding
between TMP metabolite and the protein fraction containing a2u-g
(Lock et al., 1987a) . The reversibility of the chemical binding
with <*2u~9' whether parent compound or metabolite, has been
confirmed with isophorone (Strasser et al., 1988), 1,4-DCB
(Charbonneau et al., 1989) , and d-limonene (Lehman-McKeeman et al. ,
1989) .
In the d-limonene study (Lehman-McKeeman et al., 1989), the
amount of radioactivity observed in the kidneys of Sprague Dawley
rats 24 hours after oral administration of [14-C]d-limonene was
35
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about 2.5 times higher in the males than in the females.
Equilibrium dialysis in the presence or absence of sodium dodecyl
sulfate indicated that approximately 40% of the radioactive
material retained in the male rat kidney was associated with
proteins in a reversible manner. Gel filtration high performance
liquid chromatography (HPLC), reverse phase HPLC, and amino acid
sequencing demonstrated that this radioactive material was
associated with a2u-g. No d-limonene or d-limonene metabolite was
seen to coelute with female rat kidney proteins.
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. GIGA appear to differ from many known chemical toxins,
nephrotoxins included, which bind covalently and irreversibly to
proteins and/or DNA and through this process cause cellular injury.
A DNA binding study with F344 rats and B6C3F1 mice of both
sexes was performed using [l,3,5,-14C]-isophorone (Thier, et al.,
1990) . Twenty-four hours after the animals were administered a 500
mg dose by gavage, liver and kidneys were processed for
determination of DNA binding. Neither isophorone nor its
metabolites showed covalent binding to DNA. In addition,
metabolically formed degradation products were not incorporated
into the DNA by de novo synthesis of DNA from labelled fragments
36
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of the xenobiotic.
The non-CIGA, 1,2-dichlorobenzene (1,2-DCB), unlike its
closely related isomer, 1,4-DCB, binds to a2u-g and other proteins
in the kidney cytosol without inducing an increase in hyaline
droplets. The binding of 1,2-DCB to a2u-g was less reversible than
it was for the hyaline droplet inducer, 1,4-DCB. This finding is
consistent with the more severe and different nephrotoxicity seen
for 1,2-DCB compared with 1,4-DCB these two compounds (NTP, 1987a;
Charbonneau et al., 1989).
Gas chromatographic analysis in experiments with liver
microsomes have shown that mice are able to oxidize d-limonene to
cis-d-limonene-1,2 oxide, as in the rat, although some quantitative
and qualitative species differences were noted (Lehman-McKeeman,
1990b). However, equilibrium saturation binding studies then
demonstrated a lack of any interaction between d-limonene or its
metabolites and MUP in male or female mice (Lehman-McKeeman, 199Ob;
Caudill et al., 1990). These results add further support to the
specificity of the interaction between GIGA and a2u-g.
The capacity of CIGA for association with other low-molecular-
weight proteins, some of which are found in humans, that share some
homology with <*2u-g has been investigated in in vitro assays. For
example, the alcohol metabolite of TMP, TMPOH, which binds
reversibly to a2u-g in vitro, also binds reversibly to three other
members of the superfamily, ie., retinol-binding protein, o^-acid
glycoprotein, and 6-lactoglobulin (Borghoff et al., 1988). It did
not bind to the 62-microglobulin or lysozyme, low-molecular-weight
37
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proteins that are not members of the superfamily.
When [H3]-retinol was administered to male rats, retinol-
derived radioactivity coeluted with the protein fraction in cytosbl
containing a2u-g. However, retinol did not produce hyaline droplet
or a2u-g accumulation (Borghoff et al., 1989). In vitro studies on
the binding affinities of retinol and several GIGA for a2u-g show
that retinol can compete with GIGA for binding to a2u-g (Borghoff
et al., 1991). These studies suggest that hyaline droplet
accumulation may not depend on how strongly a chemical binds to
CL- -g, but on whether the chemical causes a conformational change
in the protein (Borghoff et al., 1990) which inhibits protein
catabolism.
Binding affinities measured in in vitro studies generally have
not correlated well with the efficacy of chemicals for causing
hyaline droplet accumulation. Other factors affecting the
development of hyaline droplet accumulation are the protein
concentration in the tubule lumen, the rate of breakdown of the
protein-hydrocarbon complexes in the tubule cells, the death of
cells resulting from abnormal accumulation of hyaline droplets, and
the subsequent appearance of cell debris in the lumen of tubule
cells. These factors are discussed in the following sections.
H. Catabolism of alpha-2u-globulin complexed with GIGA
Reduced renal lysosomal catabolism of the CIGA-a2u-g complex
leads to its accumulation in the cells of the proximal renal
tubule, causing lysosomal protein overload and individual cell
death (Swenberg et al., 1989a). Figure 3 illustrates this proposed
38
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Proximal (P2) Tubule Cell
Tubule
Lumen
2u **
CIGA
endocytic
yesicle
6
s ecor>.daty.
ly so some
hy_anne.
d_ro_p.lets
Blood
FIGURE 3: Schematic representation of the uptake and fate of
alpha-2u-globulin complexed with a GIGA in hydrocarbon
nephrotoxicity.
39
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sequence of events.
Lysosomal degradation of ct2u-g bound to GIGA has been studied
by measuring the digestion rate of the protein recovered from
treated male rat kidney (Charbonneau et al., 1988) or of purified
urine-derived protein conjugated with GIGA in vitro (Lehmah-
McKeeman et al, 1990a). Charbonneau et al. (1988) found that both
a mixture of standard protease enzymes of non-rat origin or
proteinase K digested <*2u-g from rats treated with TMP at a much
slower rate than a2u-g from untreated rats.
Using an in vitro incubation system with renal cortex
lysosomes prepared from male rats, Lehman-McKeeman et al. (1990b)
demonstrated that the reversible binding of three other GIGA or
their metabolites impaired the degradation of a2u-g by one-third.
Under the experimental conditions employed, this was equivalent to
an extension of the apparent half-life of a2u-g from 6.67 to 10
hours. The study is particularly interesting in that it shows
reversible binding of a GIGA to a2u-g does not necessarily alter the
rate of protein degradation but that this may be a function of a
metabolite. Thus, d-limonene and 1,4-DCB did not impair hydrolysis
of a- -g but their respective bound metabolites, d-limonene-1,2-
oxide and 2,5-dichlorophenol, did. With isophorone, however, it
was the parent compound alone which produced the effect. This
apparent need to reduce protein degradation might offer an
explanation to describe why chemicals, such as retinol, have been
shown to bind to a2u~9' without producing hyaline droplet
accumulation.
40
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Other evidence (Olson et al, 1988) shows that administration
to male rats of leupeptin (an inhibitor of the lysosomal peptidase
cathepsin B) , causes a rapid a2u-g accumulation in the kidney,
indistinguishable from that induced by TMP and gasoline. These
various observations provide evidence that CIGA-induced hyaline
droplet accumulation may result from a reduced protein degradation
rate either by 1) making the protein harder to digest or 2)
inhibiting enzymatic components of the proteolytic process.
Studies by Charbonneau et al. (1988) and Lehman-McKeeman et al.
(1990a) support the former by indicating that the TMP metabolite-
protein complex is more resistant to hydrolysis than free a2u-g.
Furthermore, Murty et al. (1988) found that unleaded gasoline was
not associated with a reduction, but rather an increase, in rat
kidney lysosomal proteolytic enzyme activity.
I. Structure-Activity Relationships for GIGA.
An ability to predict those chemicals that will induce
accumulation of a2u~9 in the male rat through structural
relationships would be clearly advantageous. The fact that
relatively minor metabolites such as d-limonene 1,2-epoxide can
account for the majority of the association with a2u-g, however,
restricts the present utility of structure activity calculations
as a predictive tool. Nevertheless, some associations have been
observed. Lehman-McKeeman et al. (1990a) noted that retarded
degradation of a2u-g correlates with the presence on the active
CIGA or metabolite of an oxygen function of one type or another,
i.e., a hydroxyl group for TMPOH and 2,5-dichlorophenol, an epoxide
41
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for d-limonene-l,2-oxide, and a ketone function for isophorone.
Another recent study employed a quantitative approach to
determine the structural features necessary to induce excessive
hyaline droplet activity in male rats (Bomhard et al.f 1990).
Based on data for a number of light hydrocarbons, Bomhard et al.
surmised that an n-octanol water partition coefficient above 3.5
and the presence of an isopentyl structural moiety are associated
with increased hyaline droplet formation in male rats. A binding
site model for aliphatics was derived from this information. The
model was then generalized to include cycloaliphatics by
substituting the requirement for an isopentyl structure with a
requirement for the presence of at least one tertiary carbon atom.
Using this binding site model, Bomhard et al. predicted the
hyaline-droplet inducing activity of 18 previously untested
hydrocarbons. These chemicals were then tested for ability to
induce hyaline droplet accumulation in adult male Wistar rats.
Although the binding site model was based on the structure of the
parent compound and did not allow for active metabolites, the
results in the rats were described as being in good agreement with
the predictions.
Borghoff et al. (1991) determined the apparent binding
affinity to ct2u-g for a number of chemicals associated with a2u-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
42
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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 <*2u-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.
III. ALPHA-2U-GLOBULIN NEPHROPATHY
Substances reported to induce increased formation of hyaline
droplets in proximal tubule cells of male rats are listed in
Appendix 1, along with available information oh whether the
accumulating protein is a2u-g. The nephrotoxicity that can ensue
from hyaline droplet accumulation is novel because it is associated
with excessive a2u-g accumulation. This ct2u-g accumulation is
believed to initiate a sequence of events resulting in chronic
proliferation of tubule epithelium, as well as an exacerbation of
CPN. 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 (Alden
43
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et al., 1984; Kanerva et al., 1987a,b,c; Stone et al., 1987) and
d-limonene (Kanerva et al., 1987b; Webb et 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 Appendix 1.
Much of the information useful for defining the pathologic
sequelae to <*2u-g accumulation does not require chronic exposure.
Accumulation of a2u-g is visible within a matter of days and the
response to chronic administration of GIGA might even diminish
since ct2u-g levels decline in aging male rats (Murty et al., 1988) .
The nephrotoxicity associated with a2u-g accumulation might also be
influenced by age. Certainly, the age-related progression of CPN
obscures the lesions directly related to GIGA administration,
making evaluation of the chronic sequence of lesions especially
difficult.
A. Pathologic features of alpha-2u-globulin nephropathy
Renal lesions that have been associated with a2u-g nephropathy
are listed in Table 3. The first morphological manifestation of
a2u-g nephropathy is the rapid accumulation of hyaline droplets in
proximal tubule cells, developing within the first 24 hours after
dosing (Webb et al., 1989).
The droplets stain positively with Mallory's Heidenhain stain
but are negative for periodic acid Schiff, indicating their protein
composition (Alden et al., 1984). Mallory's Heidenhain stain is
44
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TABLE 3. SUMMARY OF THE HISTOPATHOLOGY AKD LESION PROGRESSION
REPORTED IN ALPHA-2U-GLOBULIN-ASSOCIATED NEPHROTOXICITY
1. Excessive accumulation of hyaline droplets in the P2 segment
of the proximal tubule region of kidney occurs after 1 or 2
days. This is reversible within 3 days to 2 weeks after
exposure ceases.
2. Evidence of single cell necrosis in P2 segment epithelium and
exfoliation after 5 days of continuous exposure.
3. Accumulation of granular casts formed from the cellular debris
and subsequent tubule dilation, at the junction of the P3
segment and the thin loop of Henle, following 20 to 40 days
of continuous exposure. Granular casts have been observed at
3 to 13 weeks after commencing exposure and sometimes beyond,
up to two years. *
4. Increase in cell proliferation within the P2 segment following
3 weeks of continuous exposure, remaining elevated above
normal at 48 weeks of exposure.
5. 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 two year study. *
6. Hyperplasia of the renal pelvic urothelium observed around
1 year. *
7. Exacerbation of the spontaneous chronic progressive
nephropathy syndrome common in aging rats. *
8. Formation of occasional hyperplastic foci within cortical
epithelium at chronic time-points.
*Indirect consequence of progression of lesions.
45
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therefore more useful than conventional hematoxylin and eosin for
visualizing and quantitating the droplets. As they represent
lysosome-derived entities, the droplets are strongly
autofluorescent (yellow) in paraffin sections under ultraviolet
illumination (unpublished observations, G.C. Hard). In plastic-
embedded tissue, hyaline droplets can be visualized easily with
Lee's methylene blue basic fuschin (Short et al., 1986).
Excessive hyaline droplet formation occurs primarily in cells
of the P2 segment, but small increases in the number of hyaline
droplets may also be seen in PI and P3 (Short et al., 1987). By
light microscopic immunohistochemistry, a2u-g has been clearly and
specifically localized to the hyaline droplets within proximal
tubules (Burnett et al., 1989). Ultreistructurally, the hyaline
droplets are enlarged secondary lysosomes partially composed of
a2u-g (Garg et al., 1989a). Many are polyangular or irregular in
shape, containing a condensed crystalline core suggestive of
aggregated protein in pure form. Although the a2u-g-associated
hyaline droplet accumulation persists during chronic exposure, the
severity becomes less with increasing duration of exposure beyond
about three weeks (Short et al., 1989a). This apparent waning of
the response with continued exposure could be related to declining
a2u-g production by the male rat beginning at some stage after 100-
150 days of age (Roy et al., 1983; Motwani et al.,1984).
With continued exposure, the initial accumulation of a2u-g-
containing hyaline droplets may be followed by a sequence of
interrelated pathological events. (1) Scattered single cell
46
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necrosis occurs predominantly in the P2 segment cells (Short et
al., 1987) with subsequent exfoliation of these degenerate cells
and cell fragments laden with crystalloid phagolysosomes into the
tubule lumen. With decalin, a minimal degree of cell
degeneration/necrosis was reported to be present in the proximal
convoluted tubules after 5 days of exposure, becoming maximal at 19
days, but reverting to the minimal level after 31 days of exposure
(Kanerva et al., 1987a). Occasional exfoliation of droplet-
affected cells was observed after 48 weeks of exposure to unleaded
gasoline or TMP (Short et al., 1989a), indicating sustained single
cell loss while exposure to GIGA continues.
(2) Epithelial cell proliferation primarily involving the P2
segment occurs as a regenerative response to cell damage and loss.
This can be seen as increased numbers of mitotic figures or
demonstrated by labeling techniques for DNA-synthetic activity.
Increased proliferative 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 et al.,
1989a).
(3) Granular casts composed of sloughed cell debris
accumulate at the junction between the P3 segment of the proximal
tubule and the descending thin loop of Henle, that is, at the
junction between the inner and outer stripes of outer medulla, with
consequent tubule dilation at this part of the nephron (Alden et
al., 1984). This can occur as early as two to three weeks after
initial exposure (Alden et al., 1984; Kanerva et al., 1987a). As
47
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well as comprising recognizable cell debris, the granular casts
stain positively for a2u-g (unpublished observations, R.J. Foster,
Central Toxicology Laboratory, ICI, Macclesfield) indicating
probable derivation of the debris from cells which had accumulated
this protein. Granular cast formation appears to be associated
with higher doses of compound rather than with the lowest doses
that can induce increased hyaline droplet accumulation. An absence
of casts after treatment might therefore reflect a dose-related
decrease in the severity of cell necrosis and exfoliation (Short et
al., 1986, 1987). '
(4) At chronic timepoints, linear mineralization develops in
the renal papilla, outlining affected medullary tubules, along with
hyperplasia of the pelvic urothelium (Alden, 1989). The
mineralization appears to form within the loops of Henle and has
been identified as calcium hydroxyapatite (Trump et al., 1984b).
The relationship between papillary mineralization and the proximal
tubule lesion remains undetermined but the medullary lesion is
presumed to represent mineralized remnants of debris from
disintegrating granular casts that lodge in the prebend segments of
Henle's loop (Bruner, 1984; Alden, 1989). In turn, urothelial
hyperplasia, which mainly affects the surface of the renal papilla,
may be a response of the renal pelvis lining to papillary
mineralization (Bruner, 1984; Alden, 1991).
B. Rat urine chemistry and GIGA
Several studies have examined renal function in rats treated
with GIGA and subsequently developing a2u-g nephropathy. Two days
48
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of treatment with TMP resulted in mild urinary increase in the
lysosomal enzyme N-acetyl-/3-glucosaminidase (NAG) and alkaline
phosphatase, a decrease in creatinine, and mild increase in urinary
cell debris. Other parameters, aspartate aminotransferase (AAT),
urine osmolality and volume, were not affected (Fowlie et al.,
1987). A single, oral dose of TMP had no effect on renal function
(Stonard et al., 1986). In a 14-day study with decalin, of six
urinary enzymes tested, only AAT, lactate dehydrogenase and NAG
were altered (increases) at days 21 and/or 28 (Evans et al., 1986).
Similar results were obtained for levamisole except that AAT
remained normal (Evans et al., 1988). During prolonged treatment
with C10-C11 isoparaffinic solvent, up to 8 weeks, the only urinary
changes observed were mild elevation of glucose and albumin,
slightly decreased concentrating power and osmolality, and
epithelial cell debris in the urine. There was no alteration in
urinary /32-microglobulin content (Phillips and Egan, 1984) .
Taken together, these studies suggest that GIGA produce
minimal changes in urinary chemistry and very little or no
glomerular dysfunction or damage in the days following their
administration. The minor alterations seen in urine composition
following administration of GIGA suggest also that hyaline droplet
accumulation is not related to increased passage of serum proteins
by the glomerulus. The mild tubule toxicity identified by clinical
chemistry is a characteristic of GIGA, which contrasts with the
obvious urinary changes associated with the nephrotoxicity induced
by such classical renal toxins as mercuric chloride,
49
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hexachlorobutadiene, aminoglycosides and papillotoxic agents
(Stonard, 1987).
C. Species variation in the renal response to GIGA
The male-specific effects of hyaline droplet inducers have
been demonstrated over a range of rat strains including Fischer
344, 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 these chemicals
(Alden et al., 1984; Swenberg et al., 1989a) . In addition to these
studies, other hyaline-droplet inducers have been tested for
toxicity in hamsters (jet fuels), guinea pigs (decalin), dogs
(decalin, 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 Uddin, 1984; Phillips et
al., 1987) except for one report of minor changes in dogs treated
for 6 months with d-limonene (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. 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.
Knowledge concerning the renal effects of CIGA in humans is
hampered by the lack of data on specific chemicals in this
50
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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, 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).
Epidemiological studies concerning non-neoplastic kidney
disease and occupational exposure to hydrocarbons and solvents have
been conducted only since 1975 (Reviewed by Askergren, 1986;
Daniell et al., 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 glomerular disease and duration and severity of
occupational exposure to hydrocarbon solvents, including
tetrachloroethylene which is a GIGA 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). Their major shortcomings have been
heterogeneous case definition, use of inappropriate control groups
or non-blinded 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
51
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occupational 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
(CI) 1.56-3.95) but not for exposure to gasoline and diesel fuel
(OR 0.98; 95% CI 0.49-1.06) or motor and fuel oil (OR 1.13; 95% CI
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 glomerular basement membranes or
deposition of antibodies against glomerular basement membrane,; a
mild degree of albuminuria, and sometimes tubule atrophy and
tubular basement membrane thickening (Kluwe et al., 1984; Phillips
et al., 1988).
Levamisole, a drug used as an anthelmintic, in cancer
chemotherapy, and in the treatment of rheumatoid arthritis in
humans, falls into the GIGA category because it induces both
hyaline droplet and a2u~9 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 day for 26 weeks, there is
little evidence to indicate that this compound is nephrotoxic in
humans (Dieppe et al., 1978). Since urinary NAG is only slightly
elevated in male rats exposed to GIGA, however, urine chemistry may
not be a good biological monitor of the type of nephrotoxicity
52
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associated with CIGA.
In a study of 16 females exposed to tetrachloroethylene from
their employment in dry-cleaning shops an average of 11 years
(range 1-25 years), Vyskocil et al. (1990) found no evidence of
renal damage except for an increase in lysozyme in the urine.
Although a high concentration of lysozyme in the urine can be a
measure of decreased tubular reabsorption, the authors discounted
this explanation because there was no statistically significant
increase in urinary excretion of /?2-microglobulin, lactate
dehydrogenase, or glucose, other markers of tubular dysfunction.
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 association is real, it is the
glomerulus that is pathologically involved. However, this may
simply reflect study designs which concentrated on detection of
glomerular effects. Since the injury to the rat tubule cells is
relatively mild, insensitive tests, such as urine chemistry, which
are generally used for evaluating humans might be inadequate to
detect changes.
D. Factors affecting the expression of alpha-2u-globulin
nephropathy
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 CIGA nephrotoxicity as well as
determining the effects of a2u-q in female rats.
53
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1. Age-related effects
As discussed earlier, the hepatic synthesis and urinary
excretion of a2u-g in the male rat are highly age-dependent, with
prepubertal and aged animals showing negligible amounts of this
protein (Neuhaus and Flory, 1978; Roy et al., 1983). Accordingly,
administration of either decalin to immature male rats (Alden et
al.r 1984) or unleaded gasoline to aged, 26 month old, male rats
(Murty et al., 1988) failed to produce renal cortical a2u-g
accumulation or an increase 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 a2u-g
(Roy and Neuhaus, 1967), were explored by Hobson et al. (1986)
using TMP. Although a significant increase in hyaline droplet
formation was observed in both castrated and uncastrated male F344
rats exposed to a 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 nephrotoxicity.
Estrogen is known to inhibit the hepatic synthesis of a2u-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 a2u-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 a2u-g
content by 25%, 41% and 52% on post-exposure days 3, 6, and 9
respectively, compared to rats receiving no hormone treatment. At
54
-------
the same time, hyaline droplet removal appeared to be accelerated
in rats treated conjointly with hormone. Hyaline droplet number
and size (qualitative observations) in hormone-treated rats
approached control levels at 3 days post-exposure, compared with up
to 9 days,for complete resolution in unleaded gasoline-exposed rats
not receiving estradiol.
In a subsequent study, Garg et al. (1989b) demonstrated that
pretreatment of mature male rats with subcutaneous injections of
estradiol for 10 days before gasoline exposure completely inhibited
the renal accumulation of a2u-g and hyaline droplets normally
induced by gasoline.
3. Genetic variants
The NBR rat is a strain that appears to have a tissue- and
gene-specific regulatory defect involving a2u-g. This rat has no
detectable levels of hepatic a2u-g mRNA in either sex and,
therefore, is unable to synthesize a2u-g in the liver although high
constitutive levels of the mRNA are present in the preputial gland
(Chatterjee et al., 1989). Under exposure conditions that produce
a2u-g nephropathy in Fischer 344 rats, d-limonene, TMP, isophorone,
and 1,4-DCB did not induce any detectable a2u-g accumulation,
hyaline droplets or other lesions in the male NBR rat (Dietrich and
Swenberg, 1990a). Identical results were obtained for decalin
(Ridder et al., 1990) and lindane (Dietrich and Swenberg, 1990b).
4. Alpha-2u-globulin infusion in female rats
Ridder et al. (1990) intraperitoneally administered a2u-g
(purified from mature male rat urine) at hourly intervals to
55
-------
decalin-treated female Sprague-Dawley rats for a total of 8
injections and examined kidney samples for hyaline droplets and a2u-
g one hour after the last protein injection (9 hours after decalin
treatment). Although droplet formation was not evident in kidney
sections from the a2u-g-infused female rats stained with Mallory's
Heidenhain, hyaline droplet and a2u-g accumulation were clearly
demonstrated 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 a2u~9 in the infused females. Accumulation of the
protein greatly increased in females that were both infused with
a2u-g and decalin-treated.
These various studies indicate a direct dependence of CIGA-
induced renal lesion expression on the presence of a2u-g.
E. Chronic progressive nephropathy
Rats are particularly predisposed to an age-related
spontaneous nephropathy, CPN, that is more severe in males than in
females and that affects certain strains more than others. CPN is
more common in Sprague-Dawley and Fischer 344 rats than 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 (Masoro and Yu, 1989).
Exacerbated CPN, involving enhanced severity and earlier onset
of the disease, is generally observed after chronic administration
56
-------
of CIGA to male rats (Trump et al., 1984b) . 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 CIGA (Kanerva
et al., 1987a; Webb et al., 1989). Exacerbated CPN is usually
recognized after months of continuous treatment (Trump et al.,
1984b; Short et al., 1989a) although Alden et al. (1984) reported
early signs after 2-3 weeks with decalin. These authors (Alden et
al., 1984) consider that exacerbated CPN develops as a tertiary
response to nephron obstruction caused by the CIGA-induced granular
casts.
The pathologic features of CPN (listed in Table 4) include
certain lesions that are also found in a2u-g nephropathy, as well
as lesions that are distinctive. Single cell necrosis,
regenerating basophilic tubules and focal hyperplasia of proximal
tubule epithelium are common to spontaneous CPN and to a2u-g
nephropathy (UAREP, 1983). CPN is characterized by certain lesions
which are not components of a2u-g nephropathy, including
conspicuous thickening of tubule and glomerular basement membranes,
hyaline casts consisting of homogeneous, 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 a2u-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
57
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TABLE 4. SUMMMARY OF THE HISTOPATHOLOGY OF SPONTANEOUS
CHRONIC PROGRESSIVE NEPHROPATHY OF AGING RATS
1. Thickening of tubular and glomerular basement membranes.
2. Basophilic segments of. proximal tubules with sporadic mitoses
indicative of tubule cell proliferation.
3. Tubular hyaline casts of proteinaceous material originating in
the more distal portion of the nephron, 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.
i
6. Interstitial fibrosis and scarring.
7. Tubular atrophy involving segments of proximal tubule.
8. Chronically in advanced cases, occasional hyperplastic foci in
affected tubules.
9. In some advanced cases, accumulation of protein droplets in
sporadic proximal tubules.
58
-------
junction, and linear mineralization in the papilla (Trump et al.,
1984b). 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 c*2u-g (unpublished observations, G.C.
Hard). The urine and serum chemistry of advanced CPN also differs
from <*2u-g nephropathy. Albuminuria, hypoalbuminemia, and
hypocholesterolemia typify CPN, with increases in serum creatinine
and urea nitrogen levels in end-stage disease (Barthold, 1979).
F. Renal toxicity observed in chronic bioassays of chemicals that
induced kidney tumors in rats
For the purpose of the current review, bioassays were
identified and the data examined on seven chemicals tested for
chronic toxicity and carcinogenicity by the National Toxicology
Program (NTP) or the National Cancer Institute (NCI). All seven
produced accumulation of hyaline droplets, nephropathy, and kidney
tumors in male rats. These model compounds are d-limonene,
dimethyl methylphosphonate, hexachloroethane, 1,4-DCB,
tetrachloroethylene, pentachloroethane, and isophorone1.
Information on unleaded gasoline (tested at International Research
and Development Corporation (IRDC) for the American Petroleum
Institute), which is a mixture regarded as a GIGA, was also
1Several of these seven chemicals cannot be described as true
"GIGA carcinogens" since the accumulating protein in the hyaline
droplets has not been confirmed to be a2u-g. They are occasionally
described as "potential CIGA" or "potential GIGA carcinogens" for
purposes of developing the discussion on cancer. This should not
be construed to mean that all seven chemicals fit the Policy
Statement developed in Part IV of this document.
59
-------
examined. The two non-CIGA, trichloroethylene and chlorothalonil
are included for comparative purposes. Although extensive acute
and subchronic studies have been performed on two other chemicals
(decalin and TMP), both of which cause the sequence of nephropathy
in male rat kidney beginning with <*2u-g accumulation,
carcinogenicity bioassay data are not available for these
compounds.
Trichloroethylene, which was tested by NTP, induces kidney
tumors in male rats only (NTP, 1988a) but does not cause an
accumulation of hyaline droplets or an increase in a2u-g levels
(Goldsworthy et al, 1988a). There is also some evidence that
trichloroethylene metabolites bind covalently to renal
macromolecules (Bruckner et al, 1989). Consequently, this compound
is not considered to be a GIGA.
Chlorothalonil, a fungicide tested on separate occasions by
industry and a government agency, induced renal tubule tumors ,in
male and female rats and in male mice (NCI, 1978a) . It also
induced hyaline droplet accumulation in proximal convoluted tubules
of male rats (USEPA, 1988) , but these 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 structures similar to those
observed in a2u-g nephropathy (personal communication, William M.
Busey and James C. Killeen). However, a2u-g has not been detected
in the renal tubules of chlorothalonil-exposed rats (Swenberg,
60
-------
1989a). The progression of chlorothalonil nephrotoxicity involves
initially, vacuolar degeneration of proximal tubule epithelium
followed 4 weeks later by tubule cell hypertrophy, hyperplasia, 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 nitrogen and creatinine were
increased while circulating levels of glucose and albumin were
decreased (U.S. EPA, 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 ten
selected chemicals is presented in Table 5. Non-neoplastic and
preneoplastic lesions reported in female rats and mice of both
sexes are summarized in Table 6. The data in these two Tables were
extracted from the NTP Technical Reports (see Appendix 2) and other
relevant literature.
61
-------
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BIOASSAY!
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In male rats, renal tubule cell hyperplasia was reported in
the 2-year bioassays for the 7 renal carcinogens tested by NTP.
Although not reported in the bioassay for unleaded gasoline, this
lesion was observed in later research studies with the mixture
(Short et al., 1989b). None of the eight bioassayed chemicals
produced tubule cell hyperplasia in female rats, although this
lesion was reported in male mice exposed to tetrachloroethylehe.
In male rats, renal changes described as "toxic tubular
nephropathy" (encompassing degeneration of tubule epithelium,
necrosis, epithelial cell regeneration, and cast formation) were
seen following administration of all 8 of the renal carcinogens
(Table 6) . Some aspect of toxic tubular nephropathy was also
observed in female rats or mice administered hexachloroethane, 1,4-
DCB, or tetrachloroethylene (Table 6) . For example, calcium
deposition or mineralization was seen after administration of
hexachloroethane to mice or 1,4-DCB to female rats. Cast formation
was reported in mice following administration of hexachloroethane
and tetrachloroethylene.
Several difficulties arise in the interpretation and
utilization of the bioassay-derived data when mouse and female rat
lesions 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 from the
64
-------
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, where 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
GIGA. Therefore, the lesions caused by CIGA seem to be both
qualitatively and quantitatively different for male rats compared
to mice and female rats.
65
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PART 2.
CARCIKOGENICITY
The second major part of this document describes information
from NTP (or NCI) assays for renal neoplasia induced by chemicals
that produced hyaline droplets and/or accumulation of a2u-g and
compares and contrasts this information with the kidney tumors
induced by classical renal carcinogens. In addition, other
information, such as mutagenic activity and tumor-promoting
ability, which help to define a GIGA carcinogen or point to
possible mechanism of action, are evaluated.
Epidemiological studies of human renal cell cancer are
reviewed for consistency with the hypothesis that CIGA-induced
renal cancer in male rats is an inappropriate endpoint for
asssessing 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,
I
however, the hypothesized mechanism being examined depends on the
accumulation of low-molecular-weight protein in the renal tubule,
regardless of species. Hence, the predicted target site in humans,
as in the rats, would be the renal tubule.
IV. PATHOLOGIC FEATURES OP 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
66
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acetate, N-(4'-fluoro-4-biphenylyl)acetamide (FBPA), and aflatoxin
B1 (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 carcinogens 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), to tubule dysplasia and
atypical cell foci through microscopic 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 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 mild 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).
67
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Detailed histological and/or ultrastructural observation shows
that hyaline droplet accumulation is not induced by DMN (Hard and
Butler, 1971; Hard et al, 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 methylation
of the O6 position of guanine in DNA is the most likely initiating
event (Pegg, 1984).
B. Karyomegaly
Conspicuous nuclear enlargement, indicative of increased
chromosome number without completion of mitosis (Jackson, 1974),
may occur in scattered proximal tubule cells during the weeks
preceding development of carcinogen-induced proliferative 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 proliferative 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
68
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with proliferating 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, basophilic or pale-
staining and often with vesicular nuclei and prominent nucleoli.
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 background tubular
regeneration that is a component of spontaneous CPN (Lipsky and
Trump, 1988; NTP, 1988a).
D. Adenoma
Adenomas are small neoplastic foci representing epithelial
cell proliferation beyond the well-defined structure of individual
tubules. These lesions are solid or cystic in form and the
cellular morphology and architectural appearance is similar to that
of adenocarcinomas, which are described below, 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 on size. Neoplasms in the rat kidney
parenchyma less than approximately 0.5 cm tend to lack significant
vascularization, hemorrhage and degeneration, although there may be
69
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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 tubule tumors consist mainly of lightly basophilic,
granular and/or clear cells organized in tubular, lobular, solid or
papillary patterns. Glandular differentiation as opposed to solid
sheets of cells distinguishes adenocarcinomas from carcinomas.
Increased cellular pleomorphism tends to correlate with a
decreasing degree of 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);.
p. 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
70
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infrequently (Lipsky and Trump, 1988). However, effective life-
span in chronic-exposure regimens may be a limiting factor.
Single-dose studies with DMN, which 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, approximately 50%, 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).
V. NEOPLASTIC AND PRE-NEOPLASTIC 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 chemicals are summarized in Table 7. Information on tumors
at non-renal sites with a statistically significant increase are
also mentioned. Table 7-a provides similar information for
trichloroethylene and cholorothalonil.
71
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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 position with regard to
evaluation of rare tumors and the use of historical controls
influences 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.
72
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TABLE 7. INCIDENCES OF RENAL TUBULE PRENEOPLASIA AND NEOPLASIA
IN RATS TAKEN FROM 2-YEAR BIOASSAYS ON EIGHT MODEL COMPOUNDS
Chemical
Strain
Sex Changes
Doses (mg/kg/day)
0 150 300
1,4-Dichloro-
benzene
(NTP-TR-319)
1987a
Gavage
F344 M Survival (%)
Hyperplasia (%)
Adenomas
Incidence
Adj. Rate (%)
Adenocarcinomas
Incidence
Adj. Rate (%)
Combined
Incidence
Adj. Rate (%)
Other Tumors; Hepatocellular tumors in mice
77
0
69
2
43
18
0/50 0/50 1/50
004
1/50 3/50 7/50
3 9 26
1/50 3/50 8/50
3 9 28
Chemical
Strain
Sex Changes
Doses (mg/kg/day)
O 500 1000
Dimethyl
methyl
phosphonate
(NTP-TR-323)
1987b
Gavage
Other Tumors:
F344
M Survival (%)
Hyperplasia (%)
Adenomas
Adenocarcinomas
Incidence
Adj. Rate (%)
56
34
16
None
19
18
0/50 2/50 3/49
0 9 19
Mononuclear cell leukemia; transitional cell
papillomas
continued
73
-------
TABLE 7. INCIDENCES OF RENAL TUBULE PRENEOPLASIA AND NEOPLASIA
IN RATS TAKEN FROM 2-YEAR BIOASSAYS ON EIGHT MODEL COMPOUNDS
(continued)
Chemical
Strain
Sex Changes
Doses (mg/kg/day)
0 0 212 423
Hexachloro-
ethane
(NTP-TR-68)
NCI 1978b
Gavage
Osborne-
Mendel
M Survival (%) 56 65 20 18
Hyperplasia (%) Not Reported
Adenomas
Incidence
Adj. Rate
Carcinoma
0/20 0/18 4/37 0/29
0 0 11 0
None
Other Tumors: Hepatocellular tumors in mice
Chemical Strain
Hexachloro- F344
ethane
(NTP-TR-361)
1989
Gavage
Sex Changes Doses (mg/kg/day)
0 10 20
M Survival (%) 62
Hyperplasia (%) 4
Adenomas
Incidence 1/50
Adj. Rate (%) 3
Adenocar c inoma s
Incidence 0/50
Adj. Rate (%) 0
Combined
Incidence 1/50
Adj. Rate (%) 3
58
8
2/50
6
0/50
0
2/50
6
52
22
4/5,0
15
3/50
9
7/50
24
Other Tumors: Marginal increase in pheochromocytomas in M rats
continued
74
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TABLE 7. INCIDENCES OF RENAL TUBULE PRENEOPLASIA AND NEOPLASIA
IN RATS TAKEN FROM 2-YEAR BIOASSAYS ON EIGHT MODEL COMPOUNDS
(continued)
Chemical Strain
Isophorone F344
(NTP-TR-291)
1986a
Gavage
Sex Changes
M Survival (%)
Hyperplasia (%)
Adenomas
Incidence
Adj. Rate (%)
Adenocarcinomas
Incidence
Adj. Rate (%)
Combined
Incidence
Adj. Rate (%)
Doses (mg/kg/day)
O 250 500
66 66 28
028
0/50 0/50 2/50
00 8
0/50 3/50 1/50
094
0/50 3/50 3/50
0 9 12
Other Tumors: Preputial gland tumors in male rats; hepatocellular
tumors, mesenchymal tumors & malignant lymphomas in male mice
Chemical Strain
d-Limonene F344
(NTP-TR-347)
Sex Changes
M Survival (%)
Hyperplasia (%)
Doses (mg/kg/day)
0 75 150
60 68 69
0 4 7
1990
Gavage
Adenomas
Incidence
Adj. Rate (%)
Adenocarcinomas
Incidence
Adj. Rate (%)
Combined
Incidence
Adj. Rate (%)
Other Tumors; None in mice or rats
0/50 4/50 8/50
0 12 19
0/50 4/50 3/50
0 12 7
0/50 8/50 11/50
0 23 25
continued
75
-------
TABLE 7. INCIDENCES OF RENAL TUBULE PRENEOPLASIA AND NEOPLASIA
IN RATS TAKEN FROM 2-YEAR BIOASSAYS ON EIGHT MODEL COMPOUNDS
(continued)
Chemical
Strain Sex Changes
Doses (mg/kg/day)
.0 75 150
Pentachloro-
ethane
F344
M
Survival (%)
Hyperplasia (%)
82
0
68
0
52
2
(NTP-TR-232) Adenomas
1983
Incidence
Adj. Rate (%)
Gavage
Adenocarcinomas
Incidence
Adj. Rate (%)
Combined
Incidence
Adj. Rate (%)
Other Tumors; Hepatocellular tumors in mice
0/50 1/49 4/50
0 3 14
1/50 1/49 0/50
230
1/50 2/49 4/50
2 6 14
Chemical
Tetrachloro-
ethylene
(NTP-TR-311)
1986b
Strain Sex Changes
F344 M Survival (%)
Hyperplasia (%)
Adenomas
Doses (ppm)
0 200 400
48 40 24
0 6 10
Inhalation
OtherTumors;
Incidence
Adj. Rate (%)
Adenocarcinomas
Incidence
Adj. Rate (%)
Combined
Incidence
Adj. Rate (%)
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
Leukemia in rats; hepatocellular tumors in mice
continued
76
-------
TABLE 7. INCIDENCES OF RENAL TUBULE PRENEOPLASIA AND NEOPLASIA
IN RATS TAKEN FROM 2-YEAR BIOASSAYS ON EIGHT MODEL COMPOUNDS
(continued)
Mixture
Strain Sex Changes
Doses (ppm)
67 292 2056
Unleaded F344
gasoline
(US EPA)
1987
M Survival (%)
Hyperplasia (%)
Adenomas
Incidence
Adj. Rate (%)
Not affected
0/49 1/59 2/56
0.2 4
1/45
2
Carcinomas
Incidence 0/49
Adj. Rate (%) 0
Combined
Incidence 0/49
Adj. Rate (%) 0
Other Tumors; Hepatocellular tumors in F mice
1/59
2
2/56 6/45
4 14
1/59 5/56 7/45
2 9 16
77
-------
TABLE 7a. INCIDENCES OF RENAL TUBULE PRENEOPLASIA AND NEOPLASIA
IN RATS TAKEN FROM 2-YEAR BIOASSAYS ON CHLOROTHALONIL AND
TRICHLOROETHYLENE
Chemical Strain Sex Changes
Chlorotha-
lonil
(NTP-TR-41)
NCI 1978a
Osborne- M Survival (%)
Mendel Hyperplasia (%)
Adenomas
Incidence
Rate (%)
Carcinomas
Incidence
Rate (%)
Combined
Incidence
Rate (%)
F Survival (%)
Hyperplasia (%)
Adenomas
Incidence
Rate (%)
Carcinomas
Incidence
Rate (%)
Combined
Incidence
Rate (%)
Doses (ppm)
0 5063 10126
82
0/10
0
..
0/10
0
0/10
0
50
0/10
0
0/10
0
0/10
0
40
none •
2/46
4
1/46
2
3/46
6
62
none
0/48
0
1/48
2
1/48
2
40
1/49
2
3/49
6
4/49
8
72
3/50
6
2/50
4
5/50
10
Other Tumors; none
(continued)
78
-------
TABLE 7a. INCIDENCES OP RENAL CELL PRENEOPLASIA AND NEOPLASIA
IN RATS TAKEN PROM 2-YEAR BIOASSAYS ON CHLOROTHALONIL AND
TRICHLOROETHYLENE
(continued)
Chemical
Strain Sex Changes
Doses (mg/kg/day)
0 0 500 1000
Trichloro-
ethylene
Osborne
Mendel
M Survival (%) 42
Hyperplasia (%) 0
44
0
34
10
30
6
(NTP-TR-273)
1988a
Gavage
Incidence 0/50 0/50 6/50 1/50
Adj. Rate (%) 0 0 32 6
Care inomas
Incidence 0/50 0/50 0/50 1/50
Adj. Rate (%) 0 0 06
Combined
Incidence 0/50 0/50 6/50 2/50
Adj. Rate (%) 00 32 11
Tumors in Other Strains; 2-4% Renal tubule tumors in 3 other
strains .
Other Tumors ; Malignant mesothelioma in M rats; hepatocellular
tumors in male and female mice and lymphoma in F mice
79
-------
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 on GIGA, namely Fischer 344 and Osborne-
Mendel. The overall historical incidence of these tumors in male
Fischer 344 rats is considered by the NTP to be 0.5% 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 data-base, involving
2,320 male and 2,370 female Fischer 344 rats used as untreated
controls in NTP two-year bioassays, the incidence was 0.35% for
males and 0.17% for females suggesting a male predilection for
renal tubule tumors (Solleveld 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 et al., 1980). In 975 males and 970
females the incidence was 0.3% and 0% respectively. Because of the
infrequency 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 et al., 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%, and no higher than 16%
for most. Because of the low background rate in both concurrent
80
-------
and historical controls, however, development of renal tubule
tumors at these incidences was ascribed to an effect of the
chemical.
The NTP bioassays provide little insight into the histogenesis
of the renal tumors as they were designed and performed with the
prime objective of determining the presence or absence of
carcinogenic activity of the test chemical. Although an industry-
sponsored study of unleaded gasoline included interim sacrifices,
even this bioassay did not incorporate serial sacrifices designed
to provide information on the site of origin or histogenesis of
tumors.
Survival rates in high dose male rats were poor in several of
the NTP bioassays, which complicates interpretation of the data.
The high mortality rate observed in some of these studies cannot be
attributed to the renal tumors (Hoel et al., 1988). In fact poor
survival rates usually indicated excessive toxicity. For the 1,4-
DCB bioassay, survival of the high-dose males, 40% at termination,
became significantly lower than that of vehicle controls after week
97 (NTP, 1987a). Nearly all deaths were non-accidental. A similar
situation pertains to isophorone where only 28% of high dose males
survived to termination (NTP, 1986a).
The decreased survival rates suggest that a maximum tolerated
dose (MTD) was exceeded since the early deaths could not be
attributed to tumors. Administration of a chemical at dose-levels
exceeding an MTD may alter responses that would be seen at lower
dose-levels (OSTP, 1985) . However, exceeding an MTD, by itself, is
81
-------
not compelling evidence that tumors are produced only when
detoxification mechanisms are overwhelmed. In fact, survival of
male rats in low-dose groups administered isophorone, 1,4-DCB,
hexachloroethane and tetrachloroethane was equivalent to that of
the concurrent control groups 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%
to 11% at low-dose levels and 0% to 22% 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% or less. When adjusted for intercurrent mortality, the
incidence rates for combined renal tumors ranged from 0% to 28%
with 1,4-DCB highest (Table 7).
For all of the eight model carcinogens, and also for
trichloroethylene 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
82
-------
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 its 1986 Cancer
Guidelines, EPA discussed its strategy for analyzing combinations
of benign and malignant tumors (U.S. EPA, 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 in rats is rather
arbitrary, based mainly on size, these general principles cannot be
rigidly applied.
C. Histogenesis 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
83
-------
possible interrelationships between hyperplasia, adenomas, and
adenocarcinomas is not possible because of the low overall
frequency of these lesions. The occurrence together of pre-
neoplastic and neoplastic lesions in most studies with the eight
chemicals does provide indirect evidence of progression 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 GIGA 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 5-10 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
hexachloroethane (NTP, 1989).
E. Induction of other tumor types
Six of the eight model compounds produced liver tumors in male
and/or female mice but not in male or female rats. These chemicals
84
-------
were hexachloroethane, unleaded gasoline, isophorone, 1,4-DCB,
pentachloroethane, and tetrachloroethylene. A different mechanism,
independent of hyaline droplet accumulation, may be involved in the
production of liver tumors 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 for the liver tumors 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 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 other sites, however,
raises the possibility that other mechanisms could also be
contributing to the overall kidney tumor incidence in male rats.
This possibility has been suggested for perchloroethylene (Green et
al., 1990; Dekant et al., 1989). Dekant and colleagues have
85
-------
proposed a mechanism involving hepatic glutathione S-conjugate
formation and, ultimately, bioactivation by renal cysteine
conjugate B-lyase in the nephrotoxic and carcinogenic response to
halogenated alkenes, including perchloroethylene, although they
also do not rule out a role for a-2u-g-induced nephrotoxicity.
Within this context, it is noteworthy that in the
tetrachloroethylene bioassay a renal tubule adenocarcinoma was
observed in a single low dose male mouse, clearly a statistically
nonsignificant event, but less readily regarded as biologically
irrelevant.
VI. ADDITIONAL EVIDENCE CONCERNING THE RENAL CARCINOGENICITY
OP GIGA
Key evidence relevant to providing information on carcinogenic
mechanisms can also be derived from short term tests, such as
assays for gene mutations and DMA damage, and from studies testing
the tumor-promoting effects of GIGA.
A. Genetic toxicology studies
The available genotoxicity data for the eight model
carcinogens and for trichloroethylene and chlorothalonil are
summarized in Table 8. The four assays listed in the table
(Salmonella (SAL), chromosome aberrations in Chinese hamster ovary
cells (ABS), sister chromatid exchange in Chinese hamster ovary
cells (SCE), and thymidine-kinase (TK)-gene mutations in L5178Y
cells (MLA)) are the only ones with enough common data for
comparative purposes. It is not coincidental that these are the
assays employed by the NTP. Consequently, this analysis of
86
-------
genotoxicity data was limited, for the main part, to the 10
chemicals with bioassay data. Data from Drosophila tests conducted
by the NTP (Yoon et al., 1985) and in human lymphoblasts
(Richardson et al., 1986) are also cited in Table 8 when available.
All eight renal carcinogens selected as potential GIGA have
been tested for chromosome aberrations in Chinese hamster ovary
(CHO) cells (Galloway et al., 1987a) and in Salmonella (Haworth et
al., 1983; Mortelmans et al., 1986; Ashby and Tennant, 1988; NTP,
1987). All results were negative both in the absence and presence
of exogenous activation provided by S9 extracts from rat liver.
Two presumed intermediate metabolites of the GIGA, d-limonene, (the
1,2- and 8,9-epoxides) were also tested in Salmonella with and
without induced S9, and no increase in revertants was observed
(Watabe et al., 1981). Several chemicals have tested positive, at
least under some conditions, for sister chromatid exchange in CHO
cells (Galloway et al., 1987a) and in the mouse lymphoma TK gene
mutation assay (McGregor et al., 1988). Four of the eight
potential CIGA and both the non-CIGAs were positive. Richardson et
al. (1986) reported negative results for unleaded gasoline and its
known CIGA 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 their genotoxic activity. Upon
more detailed analysis, it becomes apparent that the majority of,
the positive responses of the eight model carcinogens selected as
87
-------
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hyaline droplet inducers were observed in the absence, but not in
the presence, of exogenous S9 activation and at concentrations
greater than 100 /ig/ml.
Dimethyl methylphosphonate appears to present a unique
genotoxicity profile among the eight model carcinogens. Because
dimethyl methylphosphonate has high water solubility and low
toxicity, in vitro assays have employed very high concentrations of
dimethyl methylphosphonate, 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, occurred at concentrations
causing only slight increases in osmolality.
Of particular relevance to this report 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 UDS assay
indicative of DNA damage and repair. Similar results were reported
for pentachloroethane and tetrachloroethylene 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
90
-------
positive results in Salmonella TA100 with tetrachloroethylene 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 6-lyase inhibitor. The importance of
these findings in the formation of the kidney tumors of male rats
exposed to tetrachloroethylene is yet unclear, but similar studies
with other 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 GIGA group possess little, if any, genotoxic activity.
However, the dearth 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 promoting or cocarcinogenic activity in an established
initiation-promotion model of renal carcinogenesis.
Using 2 weeks exposure to 170 ppm of EHEN in the drinking
water as the initiating agent, the first initiation-promotion
experiment of Short et al. (1989b) included both sexes of Fischer
344 rats, multiple dose-levels of the two test compounds (unleaded
gasoline and TMP) , short-term versus long-term promotion exposures,
91
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and a sequence-reversal study to discriminate any cocarcinogenic
from promotional effects. The test compounds were unleaded
gasoline (3 inhalation concentration-levels of 10, 70 and 300 ppm),
and TMP (one oral dose-level of 50 ppm). Treatment groups,
comprised of approximately 30 animals, included a control, 2
promotion controls, an EHEN initiation control, reverse-sequence
initiation control, initiation-promotion group with a promotion
phase of 24 weeks, initiation-promotion group with a promotion
phase of 59 weeks, and a reverse-sequence test group where 24 weeks
of exposure to unleaded gasoline or TMP preceded the 2-week period
of EHEN administration. All animals were killed at 65 to 67 weeks
after the commencement of the experiment. The results were
assessed in terms of the incidence of foci of tubule hyperplasia
(called atypical cell foci by the authors) and renal tubule tumors.
Dose-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 incidence of renal tubule tumors with increasing gasoline
dose was also observed in 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 the single dose-level of TMP, and the absence of renal
tumors in any negative control group, supported the observed trends
with unleaded gasoline.
92
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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 of CPN were also
scored in these various groups with an increase upon CIGA exposure
apparent in male rats. However, no correlation 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 CIGA.
A second initiation-promotion assay using the same EHEN model
was conducted with d-limonene (Deitrich and Swenberg, 1991). This
study specifically addressed the comparison of responses between
the male Fischer 344 rat and the a2u-g-deficient NBR strain. The
initiating dose of EHEN was 500 ppm administered in the drinking
water for two weeks, followed by d-limonene by daily gavage at 150
mg/kg for 30 weeks. An initiation control (EHEN), promotion
control (d-limonene), and a vehicle control was included for both
strains. In the Fischer rats administered EHEN and d-limonene,
atypical tubule cell hyperplasia and renal tubule adenomas were
increased ten-fold as compared to the EHEN control group. In
contrast, no tumors were observed in any of the NBR groups. Such
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negative results in the NBR rat strongly suggest a clear dependence
on ct2u-<3 for the promoting activity of d-limonene.
The promotional effect of unleaded gasoline, TMP, and d-
limonene may be occurring through the influence of sustained tubule
cell proliferation which has been demonstrated with these same
compounds (Short et al., 1989a; Dietrich and Swenberg, 1991). The
extent of cell proliferation is regarded as an important factor in
chemical carcinogenesis (Grisham et al., 1983; Cohen and Ellwein,
1990) and stimulation of cell turnover is one of the key mechanisms
believed to operate in tumor promotion (Farber, 1988).
VI. COMPARISON OP GIGA WITH CLASSICAL RENAL CARCINOGENS
In general, classical renal carcinogens or their active
metabolites are electrophilic species binding covalently to
macromolecules and forming, in particular, DMA adducts (Hard, 1987;
Lipsky and Trump, 1988; Alden, 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 DNA, including 06-alkylguanine which
is a promutagenic lesion (Pegg, 1984). Accordingly, classical
renal carcinogens are usually positive in short-term mutagenicity
assays.
In contrast, GIGA are not known to react with DNA and are
generally negative in short-term tests for genotoxicity. As
described previously (Section IIG) GIGA binding to a2u-g is
reversible and not covalent in nature.
Classical renal carcinogens can induce renal tubule cancer in
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rats or mice in high incidence, with minimal duration of exposure,
clear dose-response relationships, and with decreased latent period
of development (Hard, 1987; Alden, 1991). Tumor frequencies are
often over 50% and up to 100%, much higher than the low incidences
(2-28% adjusted) recorded for CIGA. Some genotoxic carcinogens,
e.g. DMN, DEN and streptozotocin, are highly effective by single
dose. Unlike CIGA-induced renal carcinogenesis, there is usually
no absolute sex-specificity, with males and females both
susceptible, but sometimes to varying degree. These differences in
potency and species- and sex-susceptibility, suggest that classical
renal carcinogens and CIGA act via different mechanisms in kidney
carcinogenesis.
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 induced by classical carcinogens, with both granular and
clear cell types occurring. Likewise, despite differences in
toxicity observed, the sequence of development of CIGA-induced
renal tumors from tubule hyperplasia to carcinoma appears
identical. However, some evidence from the bioassays suggests that
the CIGA tumors may, in general, have a smaller size, probably
because of the difference in potency between these chemicals and
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classical carcinogens, affecting the latent period of tumor
development.
As with classical carcinogens, metastases have been rarely
reported for renal tubule tumors related to treatment by chemicals
inducing hyaline droplets and/or a2u-g. The one case of
metastasis noted with hexachloroethane 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 studies with classical carcinogens this does
not represent an unusual location.
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VIII. 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
important human cancer. This is because the disease is
unpredictable and a significant proportion of patients,
approximately one third, have distant metastasis at the time of
diagnosis (Bennington and Beckwith, 1975; NCI, 1987). The
mortality rate in these cases is high, and overall, the survival
rate for patients with renal cell cancer is 48% (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 patterns of organization are
tubular, solid, papillary and cystic. Individual tumors may show
an admixture of patterns and of cell types. Infrequently, renal
cell carcinoma presents -as a sarcomatoid form composed of spindle
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
(Wallace and Nairn, 1972) and ultrastructural features (Tannenbaum,
1971; Bennington and Beckwith, 1975). Electron microscopy reveals
many similarities between the tumor cells and normal proximal
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tubule epithelium, including brush border elements, membrane-
associated vesicles, and basilar infoldings of the plasma membrane
(Tannenbaum, 1971). Ultrastructurally, the amount of intracellular
lipid, particulate 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,
histochemical or immunologic features which discriminate them,
other than size, and this is not an absolute biologic parameter
(Bennington and Beckwith, 1975; Ritchie and Chisholm, 1983;
Tannenbaum, 1983). Adenomas are therefore 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).
B. Incidence and mortality
Kidney cancer statistics are usually reported in a form which
encompasses 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., 1988) and represents about 70% of all kidney tumors in
adults (Devesa et 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 1989 in the U.S. is 23,100 with
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a mortality estimate of 10,000 deaths (Silverberg and Lubera,
1989). These figures represent approximately 2% of both new cancer
cases at all sites and total cancer deaths. The age-adjusted
incidence rates in the U.S. for the period between 1975-1985
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 for the incidence of renal
cell tumors (Asal et al., 1988; Devesa et al., 1990).
In considering renal cell tumors specifically, the highest
rates internationally 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 U.S., 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 have
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).
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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% (Bannayam
and Lamm, 1980) up to 25%, the latter for males over the age of 50
(Reese and Winstanley, 1958). These findings have led to
speculation that a proportion of adenomas may reach a limit of
growth and/or remain quiescent (Bannayam and Lamm, 1980; Warter
1983).
Over the period 1950-1985, the U.S. Cancer Statistics data
indicate an increase of 82% in the incidence of kidney and renal
pelvis cancer combined (NCI, 1987). For renal cell cancer alone,
the increase among whites was estimated at about 30% between 1969-
1971 and 1983-1985 representing 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 between 1967 and
1979 also indicate an increase of approximately 37% in the
incidence of, renal cell carcinoma for males, although no overall
increase in females (Ritchie and Chisholm, 1983). Despite an
improvement in mortality rates since 1950 compared to incidence
rates (NCI, 1987), the relative 5-year survival rates, which are
close to 50%, have not altered since the early 1970's (Silverberg
and Lubera, 1989), 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
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incidence (Higginson et al., 1984; NCI, 1989).
Renal cell carcinoma has been diagnosed with increasing
frequency in patients with chronic renal failure (Hughson et al.,
1986; Newsom and Vugrin, 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 approximately 6%
(Hughson et al., 1986). Thus, current data suggest that a growing
population of humans receiving maintenance dialysis may be at risk
for developing renal cell tumors.
C. Environmental and lifestyle factors
Potential etiological associations between renal cell cancer
and exogenous and endogenous environmental factors, lifestyle and
occupation, have been sought in cohort and case-control studies.
Of all the environmental 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 al.,
1986; Asal et al., 1988; Brownson, 1988). Although a few studies
have failed to identify a statistical association between smoking
and renal cell cancer, it has been estimated that 30% of renal cell
carcinomas in males and 24% 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
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cell carcinoma in males (Goodman et al., 1986) and another has
associated smoking with renal adenoma (Bennington et al., 1968).
Other possible risk factors which have been reported include
coffee and tea consumption, artificial sweeteners, high body mass
index (maintained from 20 years of age), high dietary animal
protein and fat, lower educational levels, long-term analgesic use,
and diuretics (reviewed in Dayal and Kinman, 1983; McLaughlin and
Schuman, 1983; McLaughlin, 1984; McLaughlin et al., 1984; Goodman
et al., 1986; Yu et al., 1986; Asal et al., 1988; McCredie et al.,
1988). Of these, the evidence is least consistent for beverage
consumption, artificial sweeteners, other dietary factors, and
socioeconomic status, and 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 et al., 1989), coke-
oven emissions in the steel industry (Redmond et al., 1972),
printing press chemicals (Paganini-Hill et al., 1980), laundry- and
dry-cleaning agents (Blair et al., 1979; Katz and Jowett, 1981; Duh
and Asal, 1984), exhaust fumes in truck drivers (Brownson, 1988),
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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). In these studies, information on
smoking history was rarely available, so that its possible
influence could not be determined.
A study of renal cancer by occupation in Sweden, where the
incidence rates are higher than in the U.S., did not detect
increased risk for hearth and furnace workers in the steel
industry, printing workers, laundry-and dry-cleaners, 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 GIGA category.
Besides CIGA, 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) reported a weak positive association (OR
1.6; 95% CI 0.7-3.6) between renal cell carcinoma and high
occupational exposure to hydrocarbons in males but not in females
(OR 0.8; 95% CI 0.3-2.3). The authors reported a dose-response
relationship for the older age groups and for workers with the
greatest duration of exposure.
The synthetic solvents that have been widely used in dry-
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cleaning include one chemical shown in rodent tests to be a CIGA,
namely tetrachloroethylene, as well as Stoddard and 14OF solvents
which are mixtures of hydrocarbons including straight and branched
chain paraffins. Three studies analyzing proportional mortality
data on laundry- and dry-cleaning workers in several U.S. states
reported elevated risks for kidney cancer (Blair et al., 1979; Duh
and Asal, 1984; Katz and Jowett, 1981). More recently, however, a
better designed cohort mortality study on a larger population of
dry cleaning workers by Blair et al. (1990) revealed no excess
kidney cancer (Standardized Mortality Ratio (SMR) 0.5; 95% CI 0.1-
1.8). 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% CI 0.2-4.9) although
the statistical power of this study was acknowledged by the authors
as sufficient to identify only large risk estimates.
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, constituted
mainly of branched alkanes (Siemiatycki et al., 1987). No
statistically significant risk of renal cancer was found with
exposure to automotive gasoline (OR 1.2; 90% CI 0.8-1.6).
Statistically significant elevations, however, were noted at the
90% confidence level with exposure to aviation gasoline (OR 2.6;
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90% CI 1.2-5.8) and to jet fuel (OR 2.5; 90% CI 1.1-5.4). Six of
the seven cases with exposure to aviation gasoline also had
exposure to jet fuel, making it difficult to distinguish a unique
exposure. In 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 U.S.,
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 non-
significant deficits to non-significant excesses. However, the
possibility of an elevated kidney cancer risk was raised for one
specific group within the industry. Drivers among British
distribution workers showed borderline significance for excess
kidney cancer mortality. These authors 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 confounding factors of age and
cigarette smoking, no overall association (OR 1.0; 95% CI 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 et al., 1985). There was, however, a small
excess risk among gasoline station attendants (OR 1.2; 95% CI 0.6-
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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 Petroleum Institute
(Poole et al., 1990), suggested increases in kidney cancer risk for
laborers (Relative Risk (RR) 1.9; 95% CI 1.0-3.9), workers in
receipt, storage and movements (RR 2.5; 95% CI 0.9-6.6), and
refinery unit cleaners (RR 2.3; 95% CI 0.5-9.9) when compared with
a reference group of office workers, professionals and technicians.
In this study there were 102 kidney cancer cases among 18,323
deaths.
In evaluating unleaded gasoline, 55 relevant studies were
reviewed by 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 the EPA guidelines for epidemiologic evidence. 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 a specific
hypothesis associating gasoline exposure and renal cancer in mind.
The cohort studies of petroleum workers do not lend themselves for
a comparison since they shed no light on gasoline exposure, per se.
Exposures in these studies have been varied, and the only common
element is the place of work. Thus, who in the cohort had the
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exposure of interest, i.e. gasoline, 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.
IX. EVIDENCE FOR 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 a2u-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. As the
information presented below shows, however, data demonstrating the
existence of other steps in the proposed progression are extremely
limited, hindering the ability to reach 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 relationships between the induced nephropathy and renal
carcinogenesis.
A. Association between GIGA, hyaline droplet formation, and
alpha-2u-globulin accumulation
Dose-dependent relationships have been demonstrated between
the administration of d-limdnene (Lehman-McKeeman et al. , 1989) and
gabapentin (Dominick, et al., 1990) and excessive formation of
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hyaline droplets, and between unleaded gasoline or TMP and a2u-g
accumulation (Olson et al., 1987; Charbonneau et al., 1987). In
the d-limonene study, hyaline droplets were graded on a scale of 0-
12 according to size, eosinophilic intensity, and the number of
tubules loaded with droplets. The droplet scores for d-limonene
doses of 0, 0.1, 0.3, 1.0 and 3.0 mmol/kg were, control to high
dose, 3, 4.5, ca.7, 8 and 10 (Lehman-McKeeman et al., 1989). The
dose-response relationship with a2u-g accumulation is exemplified
by measurements following administration of TMP, which, given at
single doses of 0.044, 0.440, and 4.000 mmol/kg, induced a2u-g
concentrations in rat kidney 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 a2u-g concentrations were dose-
responsive only in the range of 0.04 to 1.00 ml/kg (Olson et al.,
1987).
In a special NTP study, male and female F344 rats were exposed
to d-limonene by gavage for 14 days over a 21-day period (NTP,
1990) . The a2u-g content, quantitated with an ELISA test in kidney
homogenates, increased significantly in dosed male rats relative to
vehicle controls. At 75 mg/kg, the low dose employed for male rats
in the 2-year bioassay, a2u-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 a2u-g levels in the kidney.
Although microscopic examination of kidney sections stained with
hematoxylin and eosin showed no visible differences between dose
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and vehicle control male rats, in plastic embedded sections stained
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 accumulation of hyaline droplets. The NTP report (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 ct2u-g
concentrations decline rapidly, reaching pre-exposure levels by the
third day after treatment, 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 administered 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 a2u-g
localization studies and binding studies considered earlier,
support a causal association between the administration of CIGA and
a2u-g accumulation in hyaline droplets.
B. Association between hyaline droplet formation, cell
necrosis, and tubule cell regeneration.
Hyaline droplet accumulation, single cell necrosis and cell
proliferation occur predominantly in the P2 segment of the proximal
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tubule following GIGA administration (Short et al., 1987; 1989a;
1989b). 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 (1991) 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 necrotic tubule cells), using two dose-
levels of d-limonene given orally for 3 weeks.
Dose-response relationships between hyaline droplet
accumulation and proximal tubule cell proliferation have been
observed. Short and coworkers exposed male rats for 3 weeks to TMP
(oral) or unleaded gasoline (inhalation) and then measured [3H]-
thymidine labeling indices (1987). 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 ppm TMP. These
labeling indices remained 4- to 6-fold higher than control values
during the 48th week of exposure. !
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60
Q
W
UJ
CD
40 •
20 '
-l 1-
o
o
O
o
3
0.2
50
Dose TMP (mg/kg)
FIGURE 4: Dose-response relationship between renal hyaline droplet
accumulation (D) and [3H]-thymidine labeling index (L) of proximal
tubule P2 cells in male P-344 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. (Adapted from short et al., 1987).
Ill
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0.1 1 10 100 1000
Log Dose Gasoline PPm
FIGURE 5: Effect of 0-2,000 ppm unleaded gasoline on continuous
uptake of [3H]TdR by Pi, P2, amd 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. (Adapted from Short et
al., 1987)
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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 with the gasoline and TMP
results undoubtedly reflects differences in the technique of
radioactive labeling. Viau et al. (1986) used a single injection
of tritiated-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 radiolabel incorporated into renal tissue.
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In recovery studies with unleaded gasoline and TMP, Short and
coworkers (1989a) showed that neither increased hyaline droplets
nor cell proliferation 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 environment.
Thus, proximal tubule cell proliferation is a persistent phenomenon
in chronic exposure to GIGA, 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
a2U-g-deficient NBR rat, compared to a five-fold increase in the
tubule cell labeling of d-limonene-promoted Fischer rats initiated
with EHEN (Dietrich and Swenberg, 1991). This result suggests
that the sustained proliferative response induced by a GIGA is
dependent on the a2u-g syndrome.
Thus, the sequence of events following GIGA 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 dissociated from replication. These questions need further
• 114
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investigation and hypothesis development in order to 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 rats to C10-C12
aliphatic hydrocarbons by inhalation for 5.5, 46, or 68 weeks and
found granular casts at the earliest time-point, but they 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 a2u-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 observed between
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Hyaline Dxoplets
0 1 2 3
WEEKS OF TREATMENT
FIGURE 6: Time-sequence for the development of hyaline droplets
(o), regenerating tubule epithelium (+), and tubule dilation (*),
in male F-344 rats administered 2 g/kg unleaded gasoline daily by
gavage for a 28-day period. (Adapted from Thomas et al., 1985).
' 116
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hyaline droplet formation, epithelial cell proliferation, and
tubule dilation. In this study, male rats were administered
unleaded gasoline (2 g/kg/day) for a period of 28 days and examined
at 5 interim time-points (Thomas et al., 1985). An initial
accumulation of hyaline droplets, commencing 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 consistently observed in a number of NTP and other 2-year
bioassays with potential GIGA carcinogens but not in the 13-week
toxicity studies. Clear dose-response relationships were
demonstrated for 1,4-DCB (NTP, 1987a), JP-4 mixed distillate
(MacNaughton and Uddin, 1984) , and unleaded gasoline (USEPA, 1987) .
In the 2-year unleaded gasoline study there were interim sacrifices
at 3, 6, 12 and 18 months permitting quantitative observation on
the incidence of mineralization (USEPA, 1987). Although this
lesion was termed pelvic rather than medullary mineralization in
the original report from the IRDC, it was qualified as referring to
material located within tubules of the renal pelvis, thus
conforming to the medullary site seen with other GIGA. Table 9
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 hyperplasia with JP-5 jet fuels, Diesel Fuel Marine and
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decalin (Bruner 1986), supporting the notion that the pelvic
hyperplasia is a urothelial response to mineralization in the
papilla.
TABLE 9. INCIDENCE OF MEDULLARY MINERALIZATION IN
MALE RATS DURING INHALATION EXPOSURE TO
UNLEADED GASOLINE
Exposure levels of U.G. Vapor (ppm)
Observation
Time-Points
(months)
3
6
12
18
24
67
292
2056
0
0
0
0
0
0
0
0
0
5
0
0
20
20
63
0
20*
80
80
91
*The incidence figures are percent of animals affected.
The data was taken from USEPA, 1987.
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D. Association between GIGA and chronic progressive nephropathy
Although exacerbation of spontaneous CPN by GIGA has been
noted in many studies, guantitation of this response has been
attempted on few occasions. Short et al. (1989a) compared the
number of CPN foci per kidney section in male rats at three dose-
levels of unleaded gasoline exposure and two chronic time-points,
with control specimens. For a daily dose range of 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 with the middle and
high doses of unleaded gasoline in male rats.
In the NTP bioassay of d-limonene, (NTP, 1990), treated male
rats showed a spectrum of compound-related kidney lesions,
including exacerbation of CPN, mineralization in the renal medulla,
hyperplasia of the epithelium lining the renal papilla, and
proliferative lesions of the renal tubule epithelium. The severity
of CPN 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 a2u-g
nephropathy because enhanced regeneration is considered a risk
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factor for carcinogenesis (Trump et al., 1984a: Short et al.,
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 nephrotoxicity to renal
neoplasia
For the eight selected 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 finding that a sustained state of cell
turnover in the target cell population is a mechanistic link
between cc2u-<3 nephropathy and renal neoplasia should be considered
a plausible, but unproven, description of the observed results.
The hyperplastic tubules and adenomas produced by CIGA
carcinogens appear to arise from the cortex, which includes the P2
segment of the proximal tubule, the main site of cellular injury in
a2u-g nephropathy, providing further support for their linkage.
Furthermore, Goldsworthy et al. (1988a) have shown an increase in
• 120
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cell replication rates specifically in the histologically damaged
P2 segments after tetrachloroethylene or pentachloroethane exposure
in male rats. Under the same conditions, cell replication did not
differ from controls in female rats given these chemicals nor in
rats of both sexes treated with a non-CIGA, trichloroethylene.
Recent studies of the promotion potential of d-limonene, TMP,
and gasoline also provide convincing evidence to support a linkage
between a2u-g nephropathy and renal tubule neoplasia. Dietrich and
Swenberg (1991) demonstrated that d-limonene promoted renal tubule
tumors in male F-344 rats, an animal that produces a2u-g. In
addition, there was a five-fold increase of P2-labeling index in
the F-344 rats treated with d-limonene. In contrast, no response
was recorded for proliferation, hyperplasia, or renal tubule
adenomas in the NBR rat, an a2u-g-deficient animal which does not
develop the characteristic nephropathy. These results substantiate
those of an earlier study where dose-related increases in atypical
cell foci were observed in male rats promoted with unleaded
gasoline or TMP for 24 or 60 weeks (Short et al., 1989b) . In that
study, there was a significant linear trend in incidence of renal
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 nephrotoxicity seen in male rats in the selected
two year bioassays of renal tubule carcinogens was characteristic
of that proposed to result from cell damage caused by a2u~^
accumulation. In contrast, whenever nephrotoxicity was observed in
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female rats or mice of either sex, ie. for hexachloroethane,
tetrachloroethylene, and 1,4-DCB, the lesions were not
characteristic of GIGA and probably were a response caused by an
independent mechanism.
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PART 3 -EVALUATION
O P
THE
HYPOTHESIS
X. SUMMARY OF THE EVIDENCE ON THE RENAL EFFECTS OF CI6A
Several lines of evidence establish an association between
exposure of the male rat to chemicals that induce <*2u-g
accumulation (CIGA) and nephrotoxicity, and strongly support an
association between this nephrotoxicity and renal tubule tumors.
A. Association between a2u-g accumulation and nephropathy
The information that supports an association between a2u-g
accumulation and male rat-specific renal toxicity following CIGA
administration is summarized below.
(1) Thirty-two organic compounds including fuels, solvents,
and other chemicals (listed in Appendix 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. The results in female rats for many of these compounds and
the results in mice for about half were also examined, with no
finding of hyaline droplet accumulation. However, hyaline droplet
accumulation per se is not necessarily diagnostic of a CIGA until
proven to represent a2u-g accumulation. Of the 32 substances, the
presence of a2u-g has been confirmed in male rats for 17.
(2) There is convincing evidence that the excessive
accumulation of hyaline droplets is followed sequentially by tubule
epithelial cell necrosis, cast formation and other aspects of a2u-g
nephropathy in the male rat. Five of the 32 hyaline-droplet
inducers were tested in species other than the mouse or the rat,
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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).
(3) 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 CIGA (decalin, d-
limonene, unleaded gasoline, and TMP).
(4) In general, the chronic administration of CIGA to male
rats and the ensuing nephrotoxicity enhanced the age-related renal
degenerative process by exacerbating spontaneous CPN.
(5) Specialized studies involving rats of varying age,
castrated or estrogen-treated rats, the NBR strain, and a2u-g-
treated female rats have shown that development of the early
features of <*2u-<3 nephropathy is dependent on the presence of a2u-g
formed in the liver.
(6) 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 and qualitatively
different, not involving the same spectrum of discrete lesions
associated with a2u-g nephropathy.
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(7) GIGA bind reversibly to a2u-g as a target molecule, and
the renal accumulation of a2u-g and hyaline droplet formation may
be explained by chemical inhibition of a2u-g catabolism after
reabsorption of the complex by the proximal tubule.
(8) TMPOH, the active metabolite of TMP can form in vitro
complexes with retinol-binding protein and o^-acid glycoprotein,
members of the lipocalin superfamily found in humans. In vivo data
on retinol and a2u-g, however, demonstrate that such an association
does not necessarily lead to a2u-g accumulation or hyaline droplet
formation.
B. Association between alpha-2u-globulin nephropathy 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.
(1) The eight model carcinogens produced hyperplasia,
adenomas, and adenocarcinomas in the renal tubule of the male rat.
(2) All eight that produced renal tumors in male rats also
produced nephrotoxicity.
(3) In general, the nephrotoxicity that preceded renal tumor
formation in male rats was characteristic of the form associated
with a2u-g as distinguished from other forms of toxicity associated
with non-CIGA renal toxicants.
(4) The incidence of renal tumors produced in the male rat by
the eight model carcinogens was relatively low. These tumors were
morphologically indistinguishable from renal tubule neoplasia that
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occurs rarely, but spontaneously, in male and female rats.
(5) The renal tumors produced by the eight model carcinogens
occurred late usually being found at sacrifice, metastasized
rarely, and were not life-threatening.
(6) For d-limonene, the one GIGA studied in an initiation-
promotion in male rats of the NBR and another strain, a2u-g
accumulation was necessary for promotion of male rat renal tubule
tumors initiated by EHEN.
(7) GIGA appear to be non-genotoxic or only marginally so and
may, therefore, not depend on direct genetic injury as the
mechanism for tumor induction.
(8) Trichloroethylene, a compound structurally similar to
hexachloroethane and tetrachloroethylene produced renal tumors.
apparently by mechanisms, such as covalent binding to DNA, which do
not appear applicable to the GIGA hypothesis.
C* Information reducing confidence in the conclusion that the
a2u-g response is specific to the male rat.
Although the evidence available to date supports the
hypothesized association between a2u-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.
(1) Pathological accumulation of hyaline droplets is a
reaction to excessive protein load not exclusively related to a2u-g
accumulation. Although there are 32 hyaline droplet-inducing
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compounds identified in Appendix 1 of this report, the accumulating
protein responsible for hyaline droplet formation has not been
identified for about half of these compounds.
(2) Data sufficient to demonstrate interdependence of the
lesions in the proposed pathological sequence from hyaline droplet
accumulation to chronic toxicity exist for only 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.
(3) Hexachloroethane, tetrachloroethylene, and 1,4-DCB
produced renal toxicity in female rats or mice indicating that some
GIGA may have additional effects on rodent kidney not limited to
the a2u-g-induced sequence of lesions.
(4) Information on a possible association between renal cell
tumors and GIGA exposure in humans is inconclusive since exposures
in the reviewed epidemiologic studies have been to both GIGA and
non-CIGA compounds.
(5) Information on the in vivo binding of GIGA with other
lipocalins in the a2u-g superfamily of proteins is too limited to
demonstrate conclusively that toxicity in humans does not occur via
this mechanism.
(6) Although there 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
127
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filtered by the human glomerulus and reabsorbed by the renal
tubules for catabolism.
(7) The mechanism whereby a2u-g accumulation leads to cell
death has not been established.
The scientific data summarized above were used to draw
conclusions with regard to the role of a2u-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.
XI. CONCLUSIONS
The available information on CIGA-associated renal tubule
carcinogenesis in the male rat can be described by a suggested
sequence of critical molecular and cellular events. According to
this description, the reaction of a lipophilic compound with the
low-molecular-weight protein, Oi2u-gt appears to lead to the
formation of a complex which 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
lesions occurring in the kidney may be replicated rather than
repaired.
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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, this 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 a2u-g accumulation
also exacerbates CPN, chemically induced a2u-g-associated
nephropathy in the male rat can be distinguished
histopathologically from other chemically-induced nephrotoxicities
and also from CPN. Excessive hyaline droplet formation is the
earliest morphologic manifestation and an important characteristic,
although a chemical can be described as a GIGA with certainty only
when there is a positive identification of a2u-g in the hyaline
droplets. Other observable characteristics indicative of possible
CIGA-induced nephrotoxicity include single cell necrosis of the
tubule epithelium, granular casts at the corticomedullary junction
caused by sloughing of necrotic cells, mitotic figures indicative
of regeneration or increased cell turnover, and often medullary
mineralization.
The hepatic synthesis of the lipocalin, a2u-g , is not known
to occur normally in any species other than the male rat. Alpha-
129
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2u-globulin-induced nephropathy is also a distinct entity specific
to the male rat among the laboratory species and genders tested to
date. The characteristic nephropathy has been found only when a2u-
g formed in the liver is present. Thus, female rats do not develop
hyaline droplets when exposed to GIGA unless they have been
administered a2u-g isolated from male rat urine. NBR rats which do
not carry the mRNA for liver a2u-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 a2u-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 standard 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
130
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carcinogens. Third, for all eight model compounds examined in this
report, additional sexes/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 GIGA has not
been tested directly although there are human proteins that, like
a2u-g, are members of the lipocalin superfamily. Human urine
contains small amounts of a sex-linked urinary protein.
Epidemiological studies have focused on glomerulonephritis or renal
cancer and organic chemical exposure, in general, and not on renal
tubule damage and GIGA 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 in the human kidney. While it
is not possible to resolve the issue of how the human renal tubule
responds to GIGA 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 GIGA. Unbound moieties, either the GIGA or its
metabolites that do not bind to a2u-g, can exist in the kidney
along with the protein-bound material. The potential toxicities of
these unbound moieties to the kidney need to be taken into account.
For example, perchloroethylene, in addition to showing a2u-g
131
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nephropathy, displays evidence of renal toxicity typical of
chlorinated hydrocarbons. This example demonstrates how other
mechanisms may play some role in the observed results. Since not
all GIGA present in the male rat kidney is protein-bound, the
possibility that the toxicity of the moieties not bound to o^u"*?
may also play some role should be kept in mind when evaluating CIGA
for purposes of human risk assessment.
At present, there is insufficient information on CIGA and
their metabolites to confidently predict activity on the basis of
structural analogy. Recent research on structural correlations
suggests the presence of an electronegative atom for hydrogen
bonding, lipophilicity, and steric volume are important con-
siderations. Conformational changes or other structural altera-
tions to the protein may also be necessary since binding of the
compound in the protein pocket, alone, appears to be an insuf-
ficient condition to cause reduced digestibility of the protein.
Evidence of dose-responsiveness between CIGA administration
and the degree of hyaline droplet or a2u-g formation has been
demonstrated in several studies. These findings are frequently
based on subjective histopathological criteria, however, 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
(eg., doses administered too low, animals killed before the latency
period of these slow growing tumors is attained, number of
specimins and histological sections insufficient, competing
132
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toxicity in kidney or other organs) , the entire pathological
sequence culminating in renal tubule neoplasia may not be
demonstrated in all cases of CIGA administration. Thus, not all
GIGA 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 laboratory data, an
increased proliferative response caused by chemically-induced
cytotoxicity appears to play a role in the development of renal
tubule tumors seen in male rats. Among the laboratory animals
tested to date, this response to CIGA administration seems to be
specific to the male rat. These conclusions can prqbably 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.
XII. RESEARCH NEEDS
Certain studies, suggested to fill key data gaps, are listed
below. There has been no attempt to 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 data base on these chemicals,
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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.
(1) Extend studies in humans, wherever possible, to determine
directly the effects of hydrocarbon and solvent exposure,
focusing on specific jobs known to have relatively pure GIGA
exposure. Any human pathology found should be compared with
ct2 -g 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.
(2) Examine human subpopulations that excrete abnormal amounts of
low-molecular weight protein in the urine to determine if they
are at risk of renal disease or renal cell cancer.
(3) Examine the binding of GIGA to lipocalins, such as retinol-
binding protein, o^-acid glycoprotein, and urine protein 1, to
be followed with a determination of those complexes that have
a slower degradation rate as a result of binding.
(4) Thoroughly characterize potential protein droplet
nephrotoxicity resulting from administration of known GIGA
(eg., d-limonene, TMP) to additional species (eg., dog,
hamster, rabbit, guinea pig, and especially non-human
primate).
(5) Further characterize the kidney response to GIGA and non-CIGA
renal carcinogens in the NBR rat which appears not to
synthesize a2u-g- 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 it is suspected that the a2u~9 syndrome
would influence the results.
(6) Develop a standard short-term protocol (eg. the 2-week study)
to look for the presence of hyaline droplets in the male rat
kidney before potential nephrotoxins are placed on chronic
study. If hyaline droplets are discovered, this information
should be taken into account in designing the chronic study to
ensure that the maximum information is attained during the
study.
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(7) Serial-sacrifice studies of GIGA and non-CIGA renal
carcinogens designed to determine if a distinctly different
progression from a2u-g nephropathy to tumor formation can be
seen for the GIGA. Studies should involve chronic exposures,
examine the histogenesis of the renal cell tumors, and include
"stop" experiments and time-dependent appearance of tumor
markers.
(8) Dose-response studies designed to quantitate the relationship
between increased hyaline droplets and cell necrosis and
between cell necrosis and cell regeneration. In addition, 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 should be explored.
(9) Metabolism and disposition studies of GIGA 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.
Additional work, not as critical as the above, but which would
also assist in understanding this disease process includes the
following.
(1) Identification of the accumulating material contained in
hyaline droplets of proximal tubules for chemicals that are
apparent, but unverified GIGA, and 2-year bioassays for
decalin and TMP.
(2) In vitro assays using rodent kidney extracts to more
specifically determine mutagenic potential of GIGA (or active
metabolites).
(3) Studies on the genesis of CPN and its relationship to a2u-g
nephropathy as well as the possible role of CPN as a co-
carcinogenic factor for renal tumor induction.
(4) More information on the renal catabolism of o^y-g and the rate
and efficiency of protease-mediated hydrolysis in control and
CIGA-treated rats.
(5) Studies on the binding relationships between GIGA and a2u-g
(e.g. affinity, concentration ranges, binding effectors) and
determination of the site at which binding of GIGA to a2u-g
occurs (eg., liver, plasma, or urine) to investigate the
hypothesis that the protein-CIGA complex is only formed at
high concentrations of the chemical.
(6) Determination of the reasons that the amount of low molecular
135
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weight protein in the human urine is much less than it is in
male rats.
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PART 4. POLICY
THIS PART WILL BE DISTRIBUTED LATER UNDER SEPARATE COVER.
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