EPA/600/R-05/024
February 2005
EpA Headquarters Libra/y
1?nnpMa"code3404T *
^Pennsylvania Avenue NW
Washington, DC 20460
202-566-0556
Trichloroethylene Issue Paper 3:
Role of Peroxisome Proliferator-Activated Receptor Agonism and
Cell Signaling in Trichloroethylene Toxicity
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC 20460
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DISCLAIMER
This issue paper does not represent and should not be construed to represent any agency
determination or policy. This issue paper has not been externally reviewed. The information is
being provided to assist the National Academy of Sciences in their review of the scientific issues
surrounding trichloroethylene health risks.
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CONTENTS
LIST OF ABBREVIATIONS AND ACRONYMS iv
PREFACE vi
AUTHORS AND CONTRIBUTORS vii
THE TCE ISSUE PAPERS viii
1. INTRODUCTION 1
1.1. TCE AND PPARa AGONISM 1
1.2. PERSPECTIVES ON PPARa AGONISM AND TUMOR INDUCTION 2
1.3. ISSUES FOR NAS TO CONSIDER REGARDING PPARa AGONISM
AND TCE 7
2. CONSIDERATIONS FOR PEROXISOME PROLIFERATION AND OTHER
EFFECTS OF TCE AND METABOLITES 9
2.1. CONTRIBUTION OF TCE METABOLITES TO TUMOR INDUCTION
AND EXPOSURE-LEVEL CONSIDERATIONS FOR THEIR EFFECTS 9
2.2. IMPLICATIONS OF PHENOTYPE FOR TCE-INDUCED TUMORS AND
HUMAN RISK 11
2.3. TRANSCRIPTIONAL REGULATION IN RESPONSE TO TCE
EXPOSURE: ADEQUACY FOR MOA :... 12
3. ISSUES RELATED TO PPAR AGONISM AND MOAs 13
3.1. PPAR RECEPTOR ACTIVITIES, INTERACTIONS, AND PLEIOTROPIC
NATURE 14
3.2. PPARa EFFECTS: RELATIONSHIP TO NONPARENCHYMAL LIVER
CELLS 17
3.3. PPAR AGONISM AND EPIGENETICS 19
3.4. PPAR ACTIVITY AND SPECIES DIFFERENCES 21
3.4.1. Recent Data on Species Differences in PPAR Expression and
Response 22
3.4.2. SAP Perspectives on Species Differences and Human Sensitivity 23
3.5. HETEROGENEITY OF PPARa EFFECTS IN PPARa KNOCKOUT MICE .. 26
3.6. INTRINSIC FACTORS AFFECTING PPAR-RELATED RISK 29
4. SUMMARY 31
REFERENCES 33
APPENDK: RECENT LITERATURE ON EFFECTS ASSOCIATED WITH PPAR
AGONISM OR RELATED TO ITS MECHANISM OF ACTION 42
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LIST OF ABBREVIATIONS AND ACRONYMS
AGO Acyl-coenzyme A oxidase
CAD Coronary artery disease
CH Chloral hydrate
DBF Dibutyl phthalate
DCA Dichloroacetic acid
2,4-D 2,4-Dichlorophenoxyacetic acid
DEHP Di(2-ethylhexyl) phthalate
DHEA Dehydroepiandrosterone
EOF Epidermal growth factor
ES Esterase
FAH Foci of altered hepatocytes
FCHL Familial combined hyperlipidemia
GJIC Gap junction intercellular communication
GEM Gemfibrozil
GH Growth hormone
GW Gestation week
IGF Insulin-like growth factor
IGFBP IGF-binding proteins
IL1 Interleukin 1
LCT Leydig cell tumor
LPL Lipoprotein lipase
Ly-6D Lymphocyte antigen 6 complex locus
MOA Mode of action
NAS National Academy of Sciences
NPC Nonparenchymal cell
PACT Pancreatic acinar cell tumor
PPAR Peroxisome proliferator-activated receptor
PPARet PPARalpha
PPAR6 PPARdelta
PPARA PPARgamma
PPRE Peroxisome proliferator response element
ROS Reactive oxygen species
SAB Science Advisory Board
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LIST OF ABBREVIATIONS AND ACRONYMS (continued)
SAP Science Advisory Panel
SOD Superoxide disrnutase
TCA Trichloroacetic acid
TCE Trichloroethylene
TNFa Tumor necrosis factor alpha
WY WY-14,643
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PREFACE
Publication of these issue papers is a part of EPA's effort to develop a trichloroethylene
(TCE) human health risk assessment. These issue papers were developed to provide scientific
and technical information to the National Academy of Sciences (NAS) for use in developing their
advice on how to best address the important scientific issues surrounding TCE health risks. As
such, these papers discuss a wide range of perspectives and scientific information (current
through Fall 2004) on some of these important issues, highlighting areas of continuing
uncertainty and data that may be relevant. They are intended to be useful characterizations of the
issues, not a presentation of EPA conclusions on these issues. The papers have undergone
internal review within EPA, but they have not been externally reviewed. The concepts presented
in these papers will eventually be addressed in EPA's revised risk assessment of TCE, after the
advice from the NAS, along with comments from the EPA Science Advisory Board and the
public, as well as recently published scientific literature, have been incorporated.
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AUTHORS AND CONTRIBUTORS
Many individuals contributed to the completion of this set of tichloroethylene (TCE)
issue papers. The TCE Risk Assessment Team identified the topics covered by the papers and
prepared them for submission to the National Academy of Sciences. The authors wish to thank
Dr. Peter Preuss, Dr. John Vandenberg, Mr. David Bussard, Mr. Paul White, Dr. Bob Sonawane,
Dr. Hugh Barton, Dr. Aparna Koppikar, Mr. David Bayliss, Dr. William Wood, and Dr. Ila Cote
for their input and comments.
TCE Risk Assessment Team
Jerry Blancato, ORD/NERL
Jane Caldwell*, ORD/NCEA
Chao Chen, ORD/NCEA
Weihsueh Chiu*, ORD/NCEA (TCE Chemical Manager)
Marina Evans, ORD/NHEERL
Jennifer Jinot, ORD/NCEA
Nagu Keshava*, ORD/NCEA
John Lipscomb*, ORD/NCEA
Miles Okino*, ORD/NERL
Fred Power, ORD/NERL
John Schaum, ORD/NCEA
Cheryl Siegel Scott*, ORD/NCEA
* Primary authors of issue papers. Entire team contributed via review and comment.
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THE TCE ISSUE PAPERS
BACKGROUND
In August 2001, a draft, Trichloroethylene (TCE) Health Risk Assessment: Synthesis and
Characterization, was released for external review. This draft assessment drew on 16 "state-of-
the-science" papers published as a supplemental issue of Environmental Health Perspectives
(Volume 108, Supplement 2, May 2000). Subsequent to its release, EPA's 2001 draft assessment
underwent a peer review by a panel of independent scientists through EPA's Science Advisory
Board (SAB), which provided a peer review report in December 2002. In addition, the public
submitted more than 800 pages of comments to EPA during a 120-day public comment period.
There are a number of important issues that EPA will need to examine as it moves
forward in revising the draft TCE assessment These include issues raised not only in the SAB
peer review and public comments, but also by new scientific literature published since the release
of the state-of-the-science papers and EPA's 2001 draft assessment Some of this research is
specific to the study of TCE or its metabolites while some of it describes advances in scientific
fields more generally but which have potential relevance to characterizing the human health risks
from TCE.
In February 2004, EPA held a symposium so that authors of some of the TCE-specific
research that had been published since the release of the draft assessment could present their
findings in more detail. This symposium represented only a limited cross section of recently
published research, but was reflective of the breadth of new relevant science that EPA will
consider in revising the assessment (the presentation slides and a transcript of the meeting are
available separately on EPA's website and have already been sent to the NAS).
In 2004, EPA, in cooperation with a number of other federal agencies, initiated a
consultation with the National Academy of Sciences (NAS) to provide advice on scientific issues
related to the health risk assessment of TCE. It was recognized that a review by an NAS panel of
the important scientific issues would be beneficial and informative to clarify the state-of-the-
science as EPA moves forward in completing its health risk assessment. A charge was
developed for the NAS through an Interagency Workgroup led by the White House Office of
Science and Technology Policy.
PURPOSE OF THE TCE ISSUE PAPERS
Although EPA will need to address all of the issues identified in the charge to the NAS
panel in updating its assessment, EPA would like to focus the NAS panel's attention on a subset
of issues that EPA believes to be most critical in developing a revised risk assessment, as
summarized in four issue papers developed by EPA staff:
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1. Issues in trichloroethylene pharmacokinetics;
2. Interactions of trichloroethylene, its metabolites, and other chemical exposures;'
3. Role of peroxisome proliferator-activated receptor agonism and cell signaling in
trichloroethylene toxicity; and
4. Issues in trichloroethylene cancer epidemiology.
Each paper provides an overview of the science issues, a discussion of perspectives on
those issues (including the SAB and public comments), and an outline of some of the recently
published scientific literature. The pharmacokinetics issue paper also summarizes results from a
recent collaboration with the U.S. Air Force on TCE pharmacokinetics, as well as EPA's planned
approach for further refinement of the pharmacokinetic modeling of TCE and its metabolites.
These scientific areas were selected because they are (a), critical to the hazard and/or dose-
response characterization of TCE; (b) scientifically complex and/or controversial; and (c) areas in
which substantial important scientific literature has been recently published. The input from the
NAS on the topics described in the issue papers, as well as other topics put forth in the charge to
the NAS, should help to strengthen EPA's revised TCE assessment.
NEXT STEPS
The advice from the NAS, along with comments already received from the EPA SAB and
the public, as well as recently published scientific literature, will be incorporated into a revised
EPA risk assessment of TCE, strengthening its scientific basis. Because of the substantial
amount of new information and analysis that is expected, the revised draft of the assessment will
undergo further peer review and public comment prior to completion.
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1. INTRODUCTION
Understanding the mode of action (MOA) by which a chemical or its metabolite induces
tumors is key to judgment about the relevance of such tumor data for human risk assessment and
is a key feature of the U.S. Environmental Protection Agency's (EPA's) new cancer guidelines.
The charge to the National Academy of Sciences (NAS) panel states that advice is being sought
regarding the strength and weight of evidence for various MOAs for trichloroethylene (TCE)
toxicity and their relevance to humans. Additional issues regarding MOAs delineated in the
charge include the identification of key events, adverse effects, potential contributions from
multiple MOAs, and MOA differences due to dose-response.
This paper discusses the issues surrounding peroxisome prollferator-activated receptor
alpha (PPARa) agonism and TCE toxicity, both in terms of the variety of perspectives that have
been put forth on these issues and in light of recently published scientific literature. In
particular, an effort is made to highlight recently published research both on PPARa and TCE as
well as on PPARa more generally, with an emphasis on areas of scientific uncertainty that have
been identified by recent reviews (e.g., SAP 2004, Klaunig et al., 2003; Melnick et al, 2001).
Although a large body of information is presented on these topics, this paper is not intended to be
a comprehensive, complete review or characterization of PPARa agonism and TCE toxicity, nor
is it intended to advocate or critique particular points of view.
1.1. TCE AND PPARa AGONISM
One of the important endpoints associated with TCE exposure is liver cancer. The
epidemiological review of Wartenberg et al. (2000) reported that the data suggested an overall
excess incidence of liver cancer in humans exposed to TCE. Liver cancer has been reported in
mice exposed to TCE but not in rats. Furthermore, TCE metabolites (trichloroacetic acid [TCA],
dichloroacetic acid [DCA], and chloral hydrate [CH]) have been shown to cause liver tumors in
mice, with DCA also causing liver tumors in rats. DCA and TCA have been the primary focus as
the potentially active agents causing hepatocarcinogenicity.
EPA's draft TCE assessment concluded that the MOA for liver cancer for TCE or its
metabolites was unknown and considered peroxisome proliferation as one of several possible
MOAs for TCE-induced liver tumors. TCE, DCA, and TCA induce peroxisome proliferation in
rodents via PPARa activation, although they are considered to be weak peroxisome proliferators
(e.g., as compared with the model pharmaceutical drug WY-14,463 [WY]). EPA's draft
assessment stated that the role of PPARa activation as a possible MOA was more plausible for
TCA than for DCA because TCA induces a more sustained proliferative response than DCA, and
, 1
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DCA appears to induce effects on cell-signaling processes and on carbohydrate handing at lower
concentrations than those associated with peroxisome proliferation or PPARa activation. It was
noted that the role of PPARa activation and the sequence of events following activation of this
receptor that are important to tumorigenesis are not well defined and, furthermore, that the key
events critical to tumor induction and the cross-species relevance of these key events had yet to
be identified.
The question of what human health risks may be posed by peroxisome proliferators, both
in general and in the case of TCE, remains controversial. The EPA Science Advisory Board
(SAB) review of EPA's draft TCE risk assessment recommended that EPA consider giving less
weight to peroxisome proliferation as a possible MOA for TCE-induced liver cancer than to
some other hypotheses. By contrast, among public comments, some commenters stated that
more emphasis should be placed on TCA and a mechanism involving interaction with PPARa
and that humans are more generally unresponsive to peroxisome proliferators with regard to the
factors that may contribute to liver tumors.
1.2. PERSPECTIVES ON PPARa AGONISM AND TUMOR INDUCTION
Substantial scientific interest exists regarding the role of peroxisome proliferation in
rodent hepatocarcinogenesis and its relevance for human carcinogenesis in both the liver and
other potential sites. Because of the uncertainty of human cancer risk associated with
peroxisome proliferators, delineating the mechanisms of carcinogenesis by these agents is of
great interest (Kiss et al., 2001). Despite scientific advances, the mechanism by which PPARa
agonists induce liver tumors in rodents is unknown (Melnick et al., 2003; Melnick, 2002, 2001;
Miller et al., 2001; Kiss et al., 2001; Bull, 2000; Peters et al., 2000; Zhou and Waxman, 1998;
Bojesetal., 1997).
It was initially proposed that the MOA for liver tumors caused by peroxisome
proliferators is due to oxidative damage caused by marked increases in free radical-generating
enzymes and peroxisomal p-oxidation might initiate carcinogenesis. Under this hypothesis, it
was generally believed that because peroxisome proliferation has not been observed in humans,
agents that produced this result in rodents would not present a carcinogenic hazard to humans.
For instance, Cattley et al. (1998) state:
A core set of biochemical and cellular events has been identified in the rodent
strains that are susceptible to the hepatocarcinogenic effects of peroxisome
proliferators, including peroxisome proliferation, increases in fatty acyl-CoA
oxidase levels, microsomal fatty acid oxidation, excess production of hydrogen
peroxide, increases in rates of cell proliferation, and expression and activation of
the alpha subtype of the peroxisome proliferator-activated receptor (PPAR-a).
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Such effects have not been identified clinically in liver biopsies from humans
exposed to peroxisome proliferators or in in vitro studies with human hepatocytes,
although PPAR-a is expressed at a very low level in human liver. Consensus was
reached regarding the significant intermediary roles of cell proliferation and
PPAR-a receptor expression and activation in tumor formation. Information
considered necessary for characterizing a compound as a peroxisome proliferating
hepatocarcinogen include hepatomegaly, enhanced cell proliferation, and an
increase in hepatic acyl-CoA oxidase and/or palmitoyl-CoA oxidation levels.
Given the lack of genotoxic potential of most peroxisome proliferating agents, and
since humans appear likely to be refractive or insensitive to the tumorigenic
response, risk assessments based on tumor data may not be appropriate. However,
nonrumor data on intermediate endpoints would provide appropriate toxicological ,
endpoints to determine a point of departure such as the LED 10 or NOAEL which
would be the basis for a margin-of-exposure (MOE) risk assessment approach.
Pertinent factors to be considered in the MOE evaluation would include the slope
of the dose-response curve at the point of departure, the background exposure
levels, and variability in the human response.
A number of public comments on the draft TCE assessment expressed support for this
point of view.
However, more recent scientific developments have led some to reevaluate the state of
the science concerning the MOA and human relevance of rodent tumors induced by certain
peroxisome- proliferating agents. One recent debate on the risk posed to humans by peroxisome
proliferators has been focused by the risk assessment of di-(2-ethylhexyl) phthalate (DEHP), a
mild peroxisome proliferator in rodent liver (Melnick, 2002, 2001). Melnick et al. (2003) have
argued that human cancer risk from PPAR agonists cannot be dismissed. In particular, Melnick
(2001) states;
The literature review presented in this commentary reveals that, although our
knowledge of the mechanism of peroxisome proliferation has advanced greatly
over the past 10 years, our understanding of the mechanism(s) of carcinogenicity
of peroxisome proliferators remains incomplete. Most important is that published
studies have not established peroxisome proliferation per se as an obligatory
pathway in the carcinogenicity of DEHP. No epidemiologic studies have been
reported on the potential carcinogenicity of DEHP, and cancer epidemiologic
studies of hypolipidemic fibrate drugs (peroxisome proliferators) are inconclusive.
Most of the pleiotropic effects of peroxisome proliferators are mediated by the
peroxisome proliferator activated receptor (PPAR), a ligand-activated
transcription factor that is expressed at lower levels in humans than in rats and
mice. In spite of this species difference in PPAR expression, hypolipidemic
fibrates have been shown to induce hypolipidemia in humans and to modulate
gene expression (e.g., genes regulating lipid homeostasis) in human hepatocytes
by PPAR activation. Thus, humans are responsive to agents that induce
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peroxisome proliferation in rats and mice. Because peroxisome proliferators can
affect multiple signaling pathways by transcriptional activation of PPAR-
regulated genes, it is likely that alterations in specific regulated pathways (e.g.,
suppression of apoptosis, protooncogene expression) are involved in tumor
induction by peroxisome proliferators. In addition, because DEHP also induces
biological effects that occur independently of peroxisome proliferation (e.g.,
morphologic cell transformation and decreased levels of gap junction intercellular
communication), it is possible that some of these responses also contribute to the
carcinogenicity of this chemical. Last, species differences in tissue expression of
PPARs indicate that it may not be appropriate to expect exact site correspondence
for potential PPAR-mediated effects induced by peroxisome proliferators in
animals and humans. Because peroxisome proliferation has not been established
as an obligatory step in the carcinogenicity of DEHP, the contention that DEHP
poses no carcinogenic risk to humans because of species differences in
peroxisome proliferation should be viewed as an unvalidated hypothesis.
In addition, the International Life Sciences Institute's Risk Science Institute
independently, convened a workgroup to update the state of the science regarding PPARa
agonist-induced rodent liver tumors and to evaluate the MOA for Leydig cell tumors (LCTs) and
pancreatic acinar cell tumors (PACTs), which also are observed frequently in rats with PPARa
agonists (Klaunig et al., 2003). They described a proposed mode of action for rodent liver
carcinogenesis, including a discussion of key events that were either causal (PPARa activation, '
perturbation of cell proliferation and/or apoptosis, and selective clonal expansion) or associative
(expression of peroxisomal genes; PPARa mediated gene expression of cell cycle, growth, and
apoptosis; nonperoxisome lipid gene expression; peroxisome proliferation; inhibition of gap
junction intercellular communication [GJIC]; hepatocyte oxidative stress; and Kuppfer cell-
mediated events). The workgroup concluded that although the PPARa receptor can be activated
in humans, substantial species differences in toxicodynamics make it very unlikely for the
downstream key events, and therefore hepatocarcinogenesis, to occur in humans. For instance, in
contrast to Melnick et al. (2003), the ILSI workgroup concluded the following with respect to
DEHP (Klaunig et al., 2003):
Using the human relevance framework, the data are sufficient to support the
conclusion that a mode of action for the DEHP-induced liver tumor has been
established in animals. Comparing the key events of this MOA to humans (and
nonhuman primates), the key event of perturbation of cell proliferation and/or
apoptosis does not occur in nonhuman primates. In addition, some associative
events such as increased peroxisomal enzyme activity and inhibition of GJIC do
not occur. This leads to a conclusion that key events of the MOA are not
plausible in nonhuman primates (and humans). The strength of this conclusion
rests on only one study of a causal key event and several studies of associative
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events. However, dynamic and kinetic factors contribute to this decision. The
data lead to a conclusion that a carcinogenic response induced via the MOAs for
liver tumorogenesis in the rodent is not likely to occur in humans following
exposure to DEHP.
In addition, the workgroup concluded that, at present, there is insufficient information to firmly
establish an MOA for LCTs and PACTs in rats (Klaunig et al., 2003).
Most recently, on December 9,2003, the Federal Insecticide, Fungicide, and Rodenticide
Act Scientific Advisory Panel (SAP) met to discuss its review of the set of scientific issues being
considered by EPA pertaining to Proposed Science Policy: Peroxisome Prolifemtor Activated
Receptor-alpha (PPAR-a) Agonist-Mediated Hepatocarcinogenesis in Rodents and Relevance to
Human Health Risk Assessment. The SAP was asked to comment on several issues for which
science policy conclusions had been proposed. These issues (and the proposed conclusions)
included the following:
1. An MOA for rodent hepatocarcinogenesis (i.e., PPARa agonists activate PPARa
leading to an increase in cell proliferation, a decrease in apoptosis, and eventual
clonal expansion of preneoplastic cells leading to liver cancer);
. 2. The relative sensitivity of the fetal, neonatal, and adult rodent (i.e., conclusions about
MOA in adults would apply to the young in rodents and humans);
3. The human relevance of PPARa agonist-induced hepatocarcinogenesis (i.e., that
humans and nonhuman primates are refractory to the hepatic effects of PPARa
agonists);
4. The data requirements sufficient to demonstrate the proposed MOA for rodent liver
tumors (i.e., evidence of PPARa agonism, in vivo evidence of increase in size and
number of peroxisomes, increases in the activity of acyl-coenzyme A oxidase [AGO]
and hepatic cell proliferation, and adequacy of dose-response and temporal sequence
between precursor events and liver tumor formation); and
5. The MOAs for other tumors induced by PPARa agonists (i.e., the'data are insufficient
to support MOAs for other tumors induced by PPARa agonists, such as LCTs and
PACTs in rats).
The summary minutes of the meeting became publicly available March 5,2004 (SAP
2004). In brief, the panel conclusions were as follows:
1. Regarding the MOA for rodent hepatocarcinogenesis, the SAP wrote:
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Overall, the majority of the Panel felt the evidence in support of the
proposed MOA for PPAR-a agonist induced rodent hepatocarcinogenesis
was adequate, though the opinions of individual Panel members ranged
from full agreement to complete disagreement. The key event in the MOA
is PPAR-a activation. PPAR-a activation triggers multiple events leading
to tumorigenesis but the PPAR-a-altered genes in the causal pathway for
tumor induction have not been identified. While some of the key events
that occur after PPARa activation, such as increased cell proliferation,
inhibition of apoptosis, and the clonal expansion of preneoplastic lesions
are known, the PPAR-a dependent mechanism for the perturbation of
these key events is less well established. Specifically, mechanisms and
steps linking key events downstream of PPAR-a activation are not known.
The data are sufficient to demonstrate a PPAR-a activation dependence to
the MOA, but are inadequate to provide the quantitative linkages
associated with a more defined mechanism of action. The Panel members
agreed that additional evidence of specific alterations associated with
PPAR-a activation would greatly strengthen the proposed MOA.
2. Regarding the relative sensitivity of fetal, neonatal, and adult rodents, the SAP wrote:
The Panel does not support the OPPTS conclusions. Although fetal and
embryonic rats and mice respond to PPAR-a agonists as demonstrated by
changes in peroxisomal enzyme activities, strong evidence demonstrating
that fetal and neonatal rats do not exhibit an increased sensitivity to
PPAR-a agonist-induced hepatocarcinogenesis is lacking. Moreover,
conclusions regarding this MOA for human hepatocarcinogenesis should
not be applied to developing humans.
3. Regarding the human relevance, the SAP wrote:
Overall, the majority of the Panel agreed that there are relevant data
indicating that humans are less sensitive than rodents to the hepatic effects
of PPAR-a agonists. However, the opinions of individual Panel members
ranged from full agreement with the proposed OPPTS policy statement, as
currently written, to complete disagreement. The majority of the Panel
recognized weaknesses in the data that supported the policy noting in
particular that the case for lack of human relevance was deficient in the
human data.
4. With regard to data requirements, the SAP wrote:
There was general consensus among the Panel that the proposed data set
was adequate and provided a straight forward approach to classifying a
chemical as a PPAR-a agonist. The Panel also concurred that the use of
PPAR-a knockout mice would be definitive evidence to ascribe a chemical
as a PPAR-a agonist, but that the proposed data set would be sufficient in
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lieu of the use of this rather costly tool. While the Panel agreed with these
data needs, they suggested some clarifications and additional supportive
approaches.
In response to a clarification by EPA that data demonstrating PPAR-a agonist activity
could be submitted in the absence of testing in long-term carcinogenesis studies, the
SAP wrote that:
[A] Panel member observed that in the absence of testing in standard
long-term rodent carcinogenicity studies, it is not possible to determine
whether the chemical would operate through a PPAR-a agonist MOA
producing rodent liver tumors. A chemical with PPAR-a agonist activity
may either: 1) not cause cancer in rodents, 2) cause liver cancer in rodents
by the proposed PPAR-a agonist MOA, 3) cause liver cancer by a MOA
other than the proposed PPAR-a agonist MOA (e.g., cytotoxicity), or 4)
cause cancer at sites other than the liver (with or without liver cancer). The
Panel concurred that an overriding requirement is that other MOAs have
been excluded. For example, rigorous tests must be performed to exclude
mutagenicity, other forms of DNA damage (clastogenicity), or overt
cytotoxicity directly produced by the test compound, or its metabolic
products.
5. Finally, with regard to other tumors, the SAP wrote:
In addition to the hepatic tumors that appear to be a general occurrence in
rats and mice, nine PPAR-a agonists have been reported to induce Leydig
cell tumors (LCTs) and pancreatic acinar cell tumors (PACTs) in rats.
Together with the hepatic tumors, this is referred to as the tumor triad. The
Panel was in agreement with the OPPTS conclusion that chemicals that
induce pancreatic or Leydig cell tumors may pose a carcinogenic hazard
for humans.
1.3. ISSUES FOR NAS TO CONSIDER REGARDING PPARa AGONISM AND TCE
NAS input regarding the state of the science on PPARa agonism as it relates to the
toxicity of TCE and its metabolites would be particularly important for several reasons as EPA
revises its risk assessment of TCE. First, as noted above, a variety of divergent perspectives exist
regarding the role of PPARa in TCE toxicity and, more generally, the actions of and human
health risks from PPARa agonists. Second, scientific interest in PPARa has greatly increased in
recent years owing to possible pharmaceutical applications, and research on the effects resulting
from activation of this receptor is expanding at an extremely rapid pace.
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Because of the quantity and quality of recent scientific research on PPARa, EPA needs to
assess both the possible role(s) of PPARa in TCE toxicity and the potential human health hazard
from TCE exposure in the light of this new literature. In particular, some of this new information
may be important in addressing areas of scientific uncertainty that have been identified in the
recent reviews cited above. Therefore, EPA would like to seek advice from NAS on the
following issues/questions:
1. What effects from PPARa agonism, either peroxisomal or extra-peroxisomal, may be
plausibly involved in TCE-induced rodent liver tumors?
2: Can changes in gene expression or methylation status be adequately characterized to
determine whether the changes induced by peroxisome proliferators and the changes
induced by TCE are consistent with each other?
3. Have PPARa agonism and related downstream effects been demonstrated to be both
necessary and sufficient to account for TCE-induced liver tumors in rodents?
a. How adequate are studies in PPARa knockout mice for demonstrating the
dependency of TCE-induced hepatocarcinogenesis on PPARa agonism?
b. Is it possible the TCE-induced biological effects related to PPARa agonism that
occur independently of peroxisome proliferation contribute to carcinogenicity?
c. What cell-signaling or other mechanisms independent of those resulting from
PPARa agonism may be plausibly involved in TCE-induced liver carcinogenesis?
4. What does the available evidence tell us about the relative sensitivity of humans to
possible MO As of TCE or its metabolites to induce liver tumors, particularly as
related to PPARa agonism?
a. What can be concluded about the actions of PPARa agonism in humans and its
relationship to MOAs of cancer?
b. What can be concluded about human sensitivity to the adverse effects from
PPARa agonism by TCE and its metabolites, given species differences in
response to PPARa agonism and the state of knowledge of effects in various
species?
c. What other factors (such as baseline actions and responses related to PPARa,
genetic polymorphisms, gender, and life stage) modulate human sensitivity to
PPARa agonism by TCE or its metabolites?
5, Are there other cancer or noncancer effects of TCE for which PPARa agonism may
be plausibly involved?
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This issue paper summarizes and outlines some of the recently published scientific
literature identified by EPA that may be informative as to TCE toxiciry. Section 2 discusses the
limited recent information on TCE or its metabolites and PPAR, and Section 3 focuses on recent
information on PPARot agonists in general that may be relevant to TCE toxicity. In addition,
throughout Section 3, we have drawn on the SAP discussion of areas of scientific uncertainty
potentially relevant to the addressing the TCE-related questions above, focusing in particular on
their discussion of the data they reviewed and what additional data they thought could help to
clarify the science. Although there may not be definitive answers to the above questions given
the current state of the science, NAS input regarding the interpretation and characterization of
existing evidence would help to strengthen EPA's revised assessment.
2. CONSIDERATIONS FOR PEROXISOME PROLIFERATION AND
OTHER EFFECTS OF TCE AND METABOLITES
EPA will need to integrate information specific to TCE with the broad database of
information on PPARot agonists (presented in Section 3) to characterize the potential role of
PPARce agonism in TCE-induced effects. Several TCE-specific issues related to this integration
are summarized in this section. Section 2.1 discusses recent evidence regarding which TCE
metabolite(s) may be involved in TCE-induced liver tumors and to what extent PPARa agonism
may be involved for different TCE metabolites. Section 2.2 discusses independent information
on liver tumor phenotypes that may inform cross-species extrapolation and thus bear on the
plausibility of various mechanisms related to PPARa agonism. Finally, Section 2.3 discusses
recent data on TCE's effects on transcriptional regulation and their potential relationship to gene
expression effects observed from PPARa agonists more generally.
2.1. CONTRIBUTION OF TCE METABOLITES TO TUMOR INDUCTION AND
EXPOSURE-LEVEL CONSIDERATIONS FOR THEIR EFFECTS
TCE and its metabolites TCA and DCA have all been shown to activate PPARs to
varying degrees. Maloney and Waxman (1999) reported TCA and DCA at high concentrations in
vitro activated PPARa in humans. There did not appear to be a difference between species in
activation of PPARa, but there was a difference in activation of PPAR gamma (PPARy). TCA
and DCA showed no PPARy activity in humans, but TCA activated PPARy in mice (Maloney
and Waxman 1999). Recent data have helped to elucidate the potential roles of each of these
compounds in TCE liver tumor induction and the extent to which PPARa agonism may play a
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role, although definitive conclusions do not appear to have been reached. As described in a
separate issue paper, the work of Bull et al. (2004) and Bull et al. (2002) suggests that neither
TCA nor DCA can be assigned individually as the agent causing TCE-induced liver tumors in
mice, that both TCA and DCA may be involved, and that TCA and DCA may affect each other's
toxicity.
With regard to PPARa, both TCA and DCA can elicit peroxisome proliferation in rodents
at high enough exposure levels. However, is peroxisome proliferation a key step in TCE-induced
carcinogenesis? Consideration needs to be given to the other effects that have been reported in
conjunction with liver tumor induction by TCE and its metabolites, and the exposure levels at
which they occur, to examine the plausibility of these effects as key events.
A study by Pereira et al. (2004a) investigated a number of effects of DCA related to liver
tumor induction. They reported DCA-induced hypomethylation in mouse liver at exposure levels
that also induced glycogen accumulation and peroxisome proliferation. To test the involvement
of DNA hypomethylation in the carcinogenic activity of DCA, the effect of methionine on both
activities was examined. Following 8 weeks of exposure, methionine altered DCA-induced
DNA hypomethylation and marginally reduced glycogen accumulation; however, it did not alter
the increased liver/body weight ratio or the proliferation of peroxisomes. After 44 weeks of
exposure, DCA induced foci of altered hepatocytes and hepatocellular adenomas with
methionine having varying effects on the multiplicity of foci that were dependent on dose. Other
studies concerning the hypomethylation of DCA and TCA (Tao et al., 2004; Tao et al., 2000) are
discussed in a separate issue paper. Tao et al. (2004) reported no difference in hypomethylation
between DCA and TCA.
A recent study by Laughter et al. (2004) has used the PPARa knockout mouse to try to
establish the role of PPARa in responses to TCE and its metabolites. Overall, the study authors
concluded that "[t]hese data support the hypothesis that PPARa plays a dominant role in
mediating the effects associated with hepatocarcinogenesis upon TCE exposure." Male wild-
type and knockout mice received either TCE for either 3 days (1,500 mg/kg) or 3 weeks (0 -
1,500 mg/kg). TCE treatment increased liver weight in wild-type mice. Knockout mice did not
show a statistically significant effect from TCE treatment but had greater liver-to-body weight
ratios than wild-type mice at all levels of exposure, including controls. This study also examined
liver to body weight ratios after 1 week of either TCA or DCA exposure in wild-type or PPARa
knockout male mice. There was no difference in liver weight ratios between the two types of
mice after TCA exposure and only a small difference at the highest dose of DCA. Liver weight
changes were suggested as a surrogate for peroxisomal proliferative activity, although neither
peroxisome proliferation nor changes in glycogen content of the liver was directly measured.
Differences in experimental protocol and relatively high baseline measures of liver-to-body
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weight ratios in control PPARa knockout mice made comparisons between TCE effects and
those of its metabolites difficult in this study. Additional discussion of this study is in Sections
2.3 and 3.5, with Section 3.5 also discussing general issues related to interpreting results using
PPARa knockout mice.
Based on relevancy of exposure levels to reported effects, Bull (2004) and Bull et al.
(2004) have recently suggested that peroxisome proliferation is not a key step in liver tumor
induction. They report that a direct comparison in the no-effect level or low-effect level for
induction of liver tumors in the mouse and several other endpoints shows that, for TCA, liver
tumors occur at lower concentrations than peroxisome proliferation in vivo but that PPAR«
activation occurs at a lower dose than either tumor formation or peroxisome proliferation. A
similar comparison for DCA shows that liver tumor formation occurs at a much lower exposure
level than peroxisome proliferation, PPARa activation, or hypomethylation. However, apoptosis
is suppressed at levels that also induce liver tumors as well as decreases in insulin and increases
in glycogen. In addition, they report that these chemicals are effective as carcinogens at doses
that do not produce cytotoxicity. Thus, the authors suggest that TCA and DCA encourage clonal
expansion'of initiated cells through subtle, selective mechanisms.
2.2. IMPLICATIONS OF PHENOTYPE FOR TCE-INDUCED TUMORS AND
HUMAN RISK
Tumor phenotype has been used to investigate the MOA of TCE through inferences of
the assignment of metabolite(s) as the active agent in rodent liver tumor production. It has also
been used to examine the differences in tumors produced by peroxisome proliferators and other
agents. Tumor phenotype is discussed in Section 2.2 of Interactions ofTrichloroethylene, Its
Metabolites, and Other Chemical Exposures with regard to the potential active agents of TCE
toxicity and to the effects of potential co-exposures on the toxicity pattern of TCE. Consistent
with SAP advice to investigate other MOAs besides PPARa agonism, phenotype may help in
determining the relevancy of TCE-induced rodent tumors to human risk and may be informative
regarding the plausibility of contributions from PPARa agonism. Among the different types of
liver tumor, hepatocellular neoplasms predominate by far in both animals and humans. Foci of
altered hepatocytes (FAH) precede both hepatocellular adenomas and carcinomas in rodents and,
in humans with chronic liver diseases that predispose them, hepatocellular carcinomas. Striking
similarities in specific changes of the cellular phenotype of preneoplastic FAH are emerging in
experimental and human hepatocarcinogenesis, irrespective of whether this was elicited by
chemicals, hormones, radiation, viruses, or, in animal models, by transgenic oncogenes or
Helicobacter hepaticus. Several authors have noted that the detection of phenotypically similar
FAH in various animal models and in humans prone to developing or bearing hepatocellular
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carcinomas favors the extrapolation from data obtained in animals to humans (Bannasch et al,,
2003; Su and Bannasch, 2003; Bannasch et al., 2001).
2.3. TRANSCRIPTIONAL REGULATION IN RESPONSE TO TCE EXPOSURE:
ADEQUACY FOR MOA
Information has recently emerged on the effects both of TCE and its metabolites and of
PPARce agonists more generally on gene expression. Gene expression studies have been
expected to hold great promise for the elucidation of MO As in general and, in particular, with
relationship to the potential contributions from PPARa agonism. As mentioned in Section 1.3,
one of the key issues for these studies is whether gene expression changes can be adequately
characterized for TCE and its metabolites specifically as well as for peroxisome proliferators in
general, so as to provide profiles which can be compared. Discussion of these issues in relation
to peroxisome proliferators in general follows in Sections 3.3 and 3.5.
With regard to TCE specifically, although there is some information on changes in DNA
methylation from TCE metabolites, as discussed above, TCE-specific data using DNA arrays
remain limited. Studies attempt to examine changes in transcriptional regulation following TCE
exposure to establish a pattern of response that can be related to an MOA. However, such
changes must be considered within the context of the experimental paradigm used for the test
condition and may be difficult to interpret. Furthermore, these data may be not only limited for
examination of changes induced by TCE exposure but also inadequate for comparison to a
standard set of changes expected by PPARa agonism—if such a profile exists (see Section 3,3).
In a screening analysis of 148 genes for xenobiotic-metabolizing enzymes, DNA repair
enzymes, heat shock proteins, cytokines, and housekeeping genes, Bartosiewicz et al. (2001)
report gene expression patterns in the liver in response TCE. TCE-induced gene induction was
highly selective; only Hsp 25 and 86 and Cyp2a were up-regulated at the highest dose tested.
Collier et al. (2003) report differentially expressed mRNA transcripts in embryonic hearts from
Sprague-Dawley rats exposed to TCE and show that sequences down-regulated with TCE
exposure appear to be those associated with cellular housekeeping, cell adhesion, and
developmental processes, while TCE exposure up-regulated expression of numerous stress-
response and homeostatic genes.
Laughter et al, (2004) recently examined transcription profile using macroarrays
containing approximately 1,200 genes in response to TCE exposure. Forty three genes were
significantly altered in the TCE-treated wild-type mice and 67 genes significantly altered in the
TCE-treated PPARa knockout mice. Out of the 43 genes expressed in wild-type mice upon TCE
exposure, 40 genes were dependent on PPARa. These genes included CYP4al2, epidermal
growth factor receptor and additional genes involved in cell growth. The interpretation of this
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information is difficult because PPARcc knockout mice are more sensitive to a number of
hepatotoxins partly because of defects in the ability to effectively repair tissue damage in the
liver (Shankar et al., 2003a; Mehendale, 2000). Further, a comparison of gene expression
profiles between controls (wild-type and PPARcc knockout) were not reported. A more general
discussion of the heterogeneity of PPARa effects in knockout mice follows in Section 3.5.
3. ISSUES RELATED TO PPAR AGONISM AND MOAs
Chronic diseases (such as diabetes, obesity, atherosclerosis, and cancer) are responsible
for most deaths in developed societies. The evidence that PPAR activity may be involved in
these ailments and can be modulated by such drugs as thiazolidinediones and fibrates has
generated a huge research effort into PPARs (Kersten et al., 2000). The generation of data
relevant to characterization of the human response to PPARa has been extensive since release of
the draft EPA assessment of TCE.
A wide range of chemicals induce tumors when tested in laboratory strains of rats and
mice of both genders. The classes of chemicals with such activity include DEHP and other
phthalates; chlorinated paraffins; chlorinated solvents (such as TCE and perchloroethylene); and
certain pesticides, hypolipidemic Pharmaceuticals, and endogenous hormones and fatty acids
(Klaunig et al., 2003; Gonzalez, 2002). Several different MOAs for tumor induction have been
postulated, some beginning with PPARcc activation as a causal first step (Klaunig et al., 2003).
An examination of the full spectrum of PPARa activity is necessary to make a
comprehensive comparison with TCE-induced effects and thereby assess the potential role of
PPARa agonism in TCE toxicity and its relevance to human risk. For instance, although humans
are responsive to PPARa agonism, as evidenced by the efficacy of hypolipidemic fibrate drugs,
studies of TCA and DCA in human hepatocyte cultures seem to indicate that the human liver is
refractory to markers of peroxisome proliferation (Walgren et al., 2000a, b). However, as
discussed previously, other studies have noted that extra-peroxisomal actions consistent with
PPARa agonism by TCE metabolites occur at concentrations much lower than those producing
peroxisome proliferation and tumor induction (Bull, 2004).
Some of the issues and perspectives surrounding this spectrum of activity are outlined in
the following sections, including the variety of observed responses and potential species, strain,
and inter-individual differences in those responses. Section 3.1 discusses the pleiotropic nature
of responses to PPARa agonism beyond peroxisome proliferation alone and presents data on how
these varied effects may be related to disease endpoints. Section 3.2 discusses data related to
effects of PPARa agonists on nonparenchymal cells that may be PPARa-independent and their
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potential role in carcinogenesis. Section 3.3 discusses information on PPARcc agonism and
changes in gene expression, with particular emphasis on changes in DNA methylation and their
potential role in carcinogenic transformation. Section 3.4 presents recent information and
perspectives on species differences in response to PPARa agonists, including a detailed
discussion of the SAP's perspectives on the question of human sensitivity. Section 3.5 discusses
the adequacy of data from PPARa knockout mice on the MOA for PPARa agonists. Finally,
Section 3.6 describes recent data on intrinsic factors, including genetic polymorphisms, gender,
and life stages, that may modulate responses to PPARa agonism. NAS input regarding
interpretation of these data and perspectives will be valuable as EPA revises its draft TCE risk
assessment.
3.1. PPAR RECEPTOR ACTIVITIES, INTERACTIONS, AND PLEIOTROPIC
NATURE
This section discusses the pleitropic nature of responses to PPARa agonism beyond
peroxisome proliferation alone and presents data on how these varied effects may be related to
disease endpoints. Although early hypotheses regarding the MOA for PPARa agonists had
focused on induction of peroxisomes, more recent data suggest a broader spectrum of effects that
may be related to adverse outcomes. A large body of scientific evidence has been recently
published to describe the functions and actions of PPARa and its relationship to other receptors.
Although chemicals that are agonists for the PPARa receptor have been traditionally
identified in rodents by their induction of peroxisomes, many actions other than peroxisome
proliferation are associated with PPARa activation. As noted by the ILSI workgroup (Klaunig et
al., 2003), "While the term 'peroxisome proliferator' has been very useful in providing a
shorthand term for a group of chemically diverse agents that induce a common pleiotropic
response, the term is also somewhat misleading since it acknowledges only a very limited
component of the responses caused by this group of agents." Scatena et al. (2003) further .
suggest that PPARa agonists produce myriad extraperoxisomal effects that are not necessarily
dependent on their interaction with PPARa and that, therefore, the biochemical profile and a
therapeutic role of this class of PPAR ligands is more complex than those previously proposed.
Peroxisome proliferators are heterogeneous in action and the receptors extremely pleiotrophic.
This pleotropic effect has been shown by the phenotypes of PPARa knockout mice.
Some agonists have been shown to display activity toward more than one receptor, which
complicates data interpretation across chemicals. Shimizu et al. (2004) reported that expression
of these genes (PPARa or PPARy2) is induced through the same peroxisome proliferator
response element in the liver and adipose tissue, where the two PPAR subtypes are specifically
expressed. Blanquart et al. (2003) report that transcriptional activity of the PPARs is regulated
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by post-translational modifications, such as phosphorylation and ubiquitination. Phosphorylation
of PPARs is controlled by environmental factors activating different kinase pathways leading to
the modulation of their activities. PPAR degradation by the ubiquitin-proteasome system
modulates the intensity of the ligand response by controlling the level of PPAR proteins in the
cells.
Thus, although the SAP concluded that the concordance of the hypothesized MOA of
PPARa activation, increased cell proliferation, decreased apoptosis, and clonal expansion of
preneoplastic cells is supported with data for several PPARa agonists, they also wrote:
One Panel member noted several inconsistencies in the supporting data however.
These include observations from long-term carcinogenicity studies of the PPAR-cc
agonist gemfibrozil, where a dose-related increase in liver tumors was observed in
male rats, while in females, a dose-dependent decrease in liver tumors was seen
(IARC, 1996). In another example, studies in rats with two PPAR-ce agonists,
WY-14,463 and DEHP, demonstrated that doses that produced equivalent levels
of hepatic peroxisome proliferation, measured as peroxisome number and
peroxisomal enzyme activity, produced markedly different liver tumor incidences
(Marsman et al., 1988). Another Panel member noted that these differences may
be due to sex, species, and strain differences in pharmacokinetics.
The heterogeneity effects from PPARa agonism have been supported by recent studies,
which suggest chemical, gender, and dose-specific effects on gene regulation. For example, Fan
et al. (2003) investigated peroxisome proliferator effects on other components of the P450-
metabolizing system that are often a rate-limiting component in P450-dependent reactions. The
down-regulation of the P450R protein was gender- and tissue-specific, in that exposure to
peroxisome proliferators led to increases in P450R protein in female rat livers (di-n-butyl
phthalate [DBF] only) and male rat kidneys (WY, gemfibrozil [GEM], and DBF). Poole et al.
(2001) report that chronic exposure to WY and GEM, but not to DBF, led to decreases in
carboxyesterase ES-4 in male rat livers, but only GEM increased it in females. WY exposure led
to decreased ES-10 in male and female rat livers, while DBF increased ES-10 in females. In the
kidney, chronic exposure to WY or DEHP in wild-type mice had down-regulation of ES-4 and
ES-10. These decreases in kidney esterase (ES) expression were not observed in PPARa
knockout mice. The authors concluded that ES protein expression is under complex sex- and
compound-dependent control by peroxisome proliferators. Recent information has been reported
for gender differences in TCE-induced peroxisome proliferation in mice (Nakajima et al., 2000).
No remarkable sex difference was observed in induction of peroxisome proliferation, as
measured morphologically, but a markedly higher induction of several enzymes and PPARa
protein and mRNA was found in males.
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Other extraperoxisomal effects of PPARa agonists have been reported relating to changes
in cell bioenergetics. Zhou and Wallace (1999) report that GEM and WY induced the
mitochondrial permeability transition as characterized by calcium-induced swelling and
depolarization of membrane potential, both of which were inhibited by cyclosporine A.
Fenofibrate, clofibrate, ciprofibrate, and DEHP, on the other hand, caused a direct dose-
dependent depolarization of mitochondrial membrane potential. However, the mechanism of
membrane depolarization varied among the test chemicals. Bezafibrate and TCE elicited no
effect on succinate-supported mitochondrial bioenergetics. The authors concluded that most, but
not all, peroxisome proliferators they studied interfered with mitochondria! bioenergetics, and the
specific biomolecular mechanism differed among the individual compounds.
' A related effect of peroxisome proliferators that has been reported is pronounced
mitochondrial proliferation and increased activity of mitochondrial enzymes in liver tumors
(Bannasche et al., 2001; Zhou and Wallace, 1999). Bannasch (1996) reports that the
hypothesized DNA damage caused by marked increases in free radical-generating enzymes of the
peroxisomal p-oxidation through H202 (Reddy and Rao, 1989) is not supported by the findings in
rats treated with the peroxisome proliferator dehydroepiandrosterone (DHEA). The
adrenocortica! hormone DHEA is a potential natural regulator of the peroxisomal compartment
(Depreter et al., 2002). Amphiphillic cell foci preceding the appearance of hepatocellular
neoplasms do not develop from the perivenular zones, in which the most pronounced peroxisome
proliferation occurs, but from the periportal areas in which the prevailing cellular alteration is
proliferation of mitochondria. Polyak et al, (1998) have examined mitochondria with regard to
neoplasia, largely because of their role in apoptosis and other aspects of tumor biology. The
mitochondrial genome is particularly susceptible to mutations because of the high level of
reactive oxygen species (ROS) generation in this organelle, coupled with a low level of DNA
repair. They report mutations in the mitochondrial genome in the majority of human colorectal
cancer examined. Possible effects of peroxisome proliferators on mitochondrial genomics have
not been investigated.
PPARa agonism is also thought to be involved in several different diseases, effects, and
receptor pathways. The table presented as an appendix to this document cites some of the recent
literature investigating such relationships and demonstrates the pleiotropic nature of the receptor.
Along with the liver, other target organs and systems affected include the muscle, cardiovascular
system, small intestine, testes, ovary, thyroid, adrenal axis,.and immune system. A large variety
of cells have also been noted to be involved with PPARa responses that not only include the
parenchyma! cell of the liver (hepatocytes) but also macrophages. Processes affected include
lipid and glucose metabolism; inflammatory cytokine production and recruitment to
inflammatory sites; and control of glucocorticoids, growth hormones (GHs), P450 genes
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(including CYP2B, CYP2C, and CYP4a family members), fasting, bile acid synthesis,
macrophage cholesterol homeostasis, key proteins involved in all stages of atherosclerosis, liver
fatty-acid binding protein, thyroid hormone and estrogen action, male rat-specific alpha 2u
globulin, a mouse homologue of alpha 2u, glutathione S-transferase, and glutathione reductase.
Effects on the vulnerability of the liver to other insults such as acetominophen have also been
reported.
3.2. PPARa EFFECTS: RELATIONSHIP TO NONPARENCHYMAL LIVER CELLS
Another issue raised recently with respect to the role of PPARa agonism in liver
carcinogenesis has been whether such agonists also independently (of PPARs) activate
nonparenchymal liver cells (such as Kupffer cells), and whether such activation may be necessary
for tumor induction. For instance, the SAP wrote that "[one] Panel member observed that there
is a large body of data demonstrating that PPAR-a agonists activate Kupffer cells through a
PPAR-a independent mechanism, resulting in the release of cytokines capable of stimulating
parenchymal cell mitosis and suppressing apoptosis (Rolfe et al., 1997; Rusyn et al., 2001;
Parzefall et al., 2001; Hasmall et al., 2001)." The ILSI workgroup (Klaunig et al., 2003) also
noted some of the issues raised by these same studies, stating that "[i]n light of these in vitro
results, one should be mindful of the potential meaning of results with respect to the
responsiveness (or lack thereof) in human hepatocyte assay systems that would have the Kupffer
cells removed during preparation."
The role of oxidants in the mechanism of tumor promotion by peroxisome proliferators
remains controversial, but recent data suggest a possible relationship between oxidants and
Kupffer cell activities. Although the early idea that induction of ACO leads to increased
production of H2O2, which damages DNA, seems unlikely now, free radicals might be important
in signaling in specialized cell types such as Kupffer cells, which produce mitogens (Rusyn et al.,
2001). Current data support a role for cytokines such as the mitogenic cytokine tumor necrosis
factor alpha (TNFa) and interleukin 1 (IL1) in hepatocarcinogenesis. Specifically, Rusyn et al.
(2000) state the following:
Oxidative DNA damage caused by leakage of H2O2 from peroxisomes was
hypothesized initially as the mechanism by which these compounds cause liver
tumors. It seems unlikely that oxidants of peroxisomal origin explain the
mechanism of action of peroxisome proliferators because treatment with these
compounds in vivo does not lead to increased H2O2 production. On the other
hand, Kupffer cell-derived oxidants, such as superoxide, may play a role in
initiating TNFa production that leads to hepatocyte proliferation.
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The authors also report that peroxisome proliferators activate the transcription factor NF-
kappaB, one of the major regulators of TNFa expression, in Kupffer cells. Rusyn et al. (2001,
2000) report a body of work that suggests that peroxisome proliferator-induced cell proliferation
and tumors require the PPARa, but that PPARa is not involved in TNFa production by Kupffer
cells because it is not expressed in this cell type. They suggest that both Kupffer cell TNFa and
parenchymal cell PPARa are required and suggest that phthalate peroxisome proliferators
increase free radicals in the liver before peroxisomal oxidases are induced.
Hasmall et al. (2001) report results consistent with nonparenchymal cells (NPCs) being
required for peroxisome proliferator-induced growth but not for peroxisome proliferation. These
data support a role for NPCs in facilitating a response of hepatocytes to peroxisome proliferators
that is ultimately dependent on the presence of PPARa in the hepatocyte. Holden et al. (2000)
report that TNFa acts downstream or independently of PPARa to mediate the suppression of
apoptosis and induction of DNA synthesis by peroxisome proliferators. In their in vitro model,
the peroxisome proliferator nafenopin does not appear to mediate de novo TNFa gene
expression, suggesting that the response to nafenopin may be mediated by bioactivation or
release of pre-existing TNFa protein from Kupffer cells. Bojes et al. (1997) reported that
neutralizing antibodies to TNFa blocks increases in protein kinase C and cell proliferation due to
WY, and Peters et al. (2000) have suggested that Kupffer cells play a central role in peroxisome
proliferator-induced carcinogenesis, most likely via mechanisms involving increases in
superoxide, activation of nuclear factor kappaB, and production of TNFa. Recent evidence
suggests that responses of hepatocytes to peroxisome proliferators is not only dependent on
PPARa but also on the trophic environment provided by nonparenchymal cells and by cytokines
such as TNFa (Roberts et al., 2002; Parzefall et al., 2001). Additionally, the ability of
peroxisome proliferators to suppress apoptosis and induce proliferation depends on survival
signaling mediated by p38 mitogen-activated protein kinase.
The gene expression of TNFa has been compared with that of nafenopin and epidermal
growth factor (EOF) to study the potential relationship between both. Chevalier et al. (2000)
report that proteins showing an altered expression pattern in response to nafenopin differed from
those showing altered expression in response to EOF. However, many proteins showing altered
expression following stimulation with TNFa were common to both the EOF and nafenopin
responses. They report 32 proteins with altered expression following stimulation with nafenopin,
including muscarinic acetylcholine receptor 3, intermediate filament vimentin, and the beta
subunit of the ATP synthase, and suggest that these nonperoxisomal protein targets offer insights
into the mechanisms of peroxisome proliferator-induced carcinogenesis in rodents.
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The SAP noted that "the mechanism for the induction of cell proliferation and apoptosis
suppression induced by PPAR-c agonists is not known. One significant factor to consider is the
role of nonparenchymal hepatic cells in these process." They also wrote more generally:
The Panel agreed that there were considerable uncertainties as to the significance
of associated key events, such as hepatic acyl CoA oxidase induction, with regard
to the tumor forming potential of PPAR-a agonists in rats and mice. PPAR-ct
agonists can bind directly to PPAR-a, but may also perturb interactions with the
RXR [retinoid-X receptor] binding partner, the binding of co-activators and
co-repressors to the receptor, or the availability and action of endogenous ligands
or inhibitors.
3.3. PPAR AGONISM AND EPIGENETICS
Recent data have also suggested that PPARa agonism affects gene expression,
particularly with respect to DNA methylation. These effects are potentially important because of
the role played in carcinogenesis by gene expression changes. As discussed in Section 2.3,
attempts have also been made to explore the effects of TCE on transcriptional regulation. Pereira
et al. (2004a) have reported that DCA induces DNA demethylation at concentrations that also
induce peroxisomes. Questions naturally arise, then, as to whether changes in gene expression or
methylation status induced by peroxisome proliferators can be adequately characterized and
whether they are consistent with effects from TCE. This section discusses recent literature both
on the potential role that DNA methylation plays in carcinogenesis and on reported effects by
PPARa agonists on DNA methylation and gene expression.
The role of chromatin in mediating the transformation of a normal cell into a malignant
state is particularly interesting. On one hand, there is the discovery that aberrant methylation
patterns occur in an increasing number of tumor suppressor and DNA repair genes that determine
carcinogenic transformation; on the other hand, there is the existence of a series of methyl-DNA
binding activities that recruit co-repressor complexes and modify the structure of the chromatin
to produce a transcriptionally silenced state. Many nuclear receptor genes can be silenced
through aberrant methylation in tumors; epigenetic silencing, therefore, represents an additional
mechanism that modifies expression of key genes during carcinogenesis (Berger and
Daxenbichler, 2002; Ballestar and Esteller, 2002). Inactivation of tumor suppressor genes is
central to the development of all common forms of human cancer. This inactivation often results
from epigenetic silencing associated with hypermethylation rather than intragenic mutations. In
human cells, the mechanisms underlying locus-specific or global methylation patterns remain
unclear (Rhee et al., 2002).
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Regarding epigenetic mechanisms, the SAP wrote:
One member of the Panel expressed a concern, which was shared by some other
Panel members, that the MOA and evaluation of human relevance was lacking in
its assessment of altered gene expression that could be associated with altered
methylation of DNA. There is evidence that DNA methylation is modified in
rodents following exposure to PPAR-a agonists (Ge et al., 2001, Ge et al., 2002,
and Pereira, et al., 2004[a]).
Given the accepted role for DNA methylation in gene imprinting and the loss
of imprinting in cancer etiology (see for example McClachlan et al., 2001), such a
role for PPAR-a agonists in causing similar alterations in humans should be
explored before human relevance can be appropriately evaluated, particularly for
exposure during early life stages and for questions regarding site concordance.
In addition, the SAP also noted that
PPAR-a agonists not only modulate the expression of genes with PPREs
[peroxisome proliferator-response elements] but they may also regulate gene
expression by altering levels of gene methylation (Ge, et al., 2001). Such DNA
methylation is known to be involved in imprinting and alterations or loss of
imprinting can directly or indirectly impact disease risk at later life stages (Cui, H.
et al., 2003).
Gene imprinting is an epigenetic mechanism for accomplishing persistent change in gene
expression (McLachlan et al., 2001). Pereira et at (2004b) report that the ability of nongenotoxic
colon carcinogens to cause DNA hypomethylation is correlated with their carcinogenic activity in
the colon of the mouse and rat. In humans, a striking correlation between genetic instability and
methylation capacity suggested that methylation abnormalities may play a role in chromosome
segregation processes in cancer cells (Lengauer et al., 1997). Herman et al. (1998) suggest that
genes involved in DNA mismatch repair are associated with microsatellite instability in
colorectal cancer and with instability resulting from epigenetic inactivation in association with
DNA methylation.
Effects of genetic disruption of PPARdelta (PPAR6) have also been implicated as an
effector of the tumorigenicity of human colon cancer cell with suppression of PPAR5
contributing to growth-inhibitory effects of the adenomatous polypsis coli/beta-catenin pathway
(Park et al., 2001). PPAR6 expression has been reported to be elevated in human colorectal
cancers (He et al., 1999).
Evidence suggests that the peroxisome proliferates 2,4-dichlorophenoxyacetic acid (2,4-
D), dibutyl phthalate (DBF), GEM, and WY have the ability to alter the methylation and
expression of the c-myc protooncogene. All four peroxisome proliferators caused
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hypomethylation of the c-myc gene in the liver, which supports the hypothesis that the
peroxisome proliferators prevent methylation of hemimethylated sites (Ge et al., 2002, 2001),
Genes that may be altered in response to PPARa agonism leading to a tumorigenic
response have yet to be identified. The same limitations stated for this approach in Section 2.3
are applicable here. Hasmall et al. (2002) report gene expression profiling of mRNA from wild-
type versus PPARa-null mice to show a three- to sevenfold down-regulation of hepatic
lactoferrin in response to DEHP. The authors suggest that the regulation of iron-binding proteins
by PPARa ligands plays a role in peroxisome proliferator-mediated liver growth but not in
peroxisome proliferation.
Depreter et al. (2002) have reported modulation of the peroxisomal gene expression
pattern by DHEA and vitamin D. After 1 and 6 days of treatment with DHEA and 25-
hydroxycholecalciferol, relative transcription levels of 39 selected genes were evaluated in
female rats. Expression levels of 16 (of which 11 were peroxisomal) genes were altered. Pex 11,
AGO, multifunctional enzyme type 1, thiolase 1, phytanoyl-CoA hydroxylase, 70 kDa
peroxisomal membrane protein, and very long chain AGO synthetase were up-regulated; three
others were down-regulated. Vitamin D caused down-regulation of six genes.
Meyer et al. (2003) used cDNA microarrays to study the expression profiles of 26
hepatocellular carcinomas developing spontaneously in peroxisomal fatty ACO-null mice. The
development of liver tumors in these null mice is due to sustained activation of PPARa by the
unmetabolized substrates of AGO, which serve as natural PPARa ligands. Comparisons of the
null mouse liver tumor expression profiles with those induced by ciprofibrate or
diethylnitrosamine showed that these mice share a number of deregulated (up- or down-
regulated) genes with ciprofibrate-induced liver tumors. Some genes identified previously as
PPARa regulated were CD36, lymphocyte antigen 6 complex locus (Ly-6D), and C3f, For all 3
classes of liver tumors, 12 genes were up-regulated and included an uncharacterized RIKEN
cDNA, lipocalin 2, insulin-like growth factor-binding protein 1, Ly-6D, and CD63 among others.
3.4. PPAR ACTIVITY AND SPECIES DIFFERENCES
This section presents recent information and perspectives on species differences in
response to PPARa agonists, including a detailed discussion of the SAP's perspectives on the
question of human sensitivity. Marked species and tissue differences in the expression of PPARs
and responses to PPAR agonists complicate the extrapolation of preclinical data to humans. For
example, despite the observation that these compounds are rodent carcinogens, PPARa ligands
such as the hypolipidemic fibrates have been used extensively in the clinic over the past 20 years
to treat cardiovascular disease and side effects of clinical fibrate use have not been widely
reported. Graham et al. (2004), however, recently reported significantly increased incidence of
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hospitalized rhabdomyolysis in patients treated with fibrates both alone and in combination with
statins. Adverse clinical responses have also been seen with PPARy ligands that were not
predicted by preclinical models (Boitier et al, 2003).
Concerns have been raised about the adequacy of human data to determine the
carcinogenic potential in humans for drugs that cause peroxisome proliferation in rodents
(Melnick, 2001; Newman and Hulley, 1996). Newman and Hulley (1996) provided a review of
methods and interpretation of carcinogenicity studies in rodents and results of clinical trials in
humans and concluded that all members of the two most popular classes of lipid-lowering drugs
(the fibrates and the statins) cause cancer in rodents, in some cases at levels of animal exposure
close to those prescribed to humans. They also concluded that evidence of carcinogenicity of
lipid-lowering drugs from clinical trials in humans is inconclusive because of inconsistent results
and insufficient duration of followup. While the ISLI Workgroup (Klaunig et al., 2003) also
stated that "[t]he available epidemiological and clinical studies are inconclusive," the group
concluded that these studies "nonetheless, do not provide evidence that peroxisome proliferators
cause liver cancer in humans."
3.4.1. Recent Data on Species Differences in PPAR Expression and Response
Several different hypotheses regarding the mechanisms behind species differences have
been investigated. Roberts et al. (2000) suggest that these species differences between humans
and rats and mice can be attributed to a reduced quantity of full-length functional PPARa in
human liver. Hasmall et al. (2000) have suggested that the human AGO gene promoter differs
from the rat AGO promoter at three bases within the PPRE and appears to be refractory to
peroxisome proliferators from studies of DEHP.
Pugh et al. (2000) report that short-term studies of peroxisome proliferators di-isononyl
phthalate and DEHP at high exposure levels in young adult male cynomolgus monkeys after 14
days of treatment showed no distinctive treatment-related effects in the liver, kidney, or testes
upon histological examination. No changes were noted in any of the hepatic markers for
peroxisomal proliferation.
Both the hamster and the guinea pig have PPARa, and the guinea pig receptor has been
characterized to be fully functional, as demonstrated in reporter gene expression assays.
However, the guinea pig PPARa is expressed at low levels in the liver, and the currently favored
hypothesis to explain species differences in hepatic peroxisome proliferation invokes the low
level of PPARa as the principal determinant of species responsiveness. On the other hand, the
demonstration that guinea pigs and humans undergo hypolipidemia induced by PPARa agonists
calls into question the MOA of PPARa agonists in "nonresponsive" species (Choudhury et al.,
2000).
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O'Brien et al. (2001) treated rats and hamsters with WY and observed decreases in alpha-
tocopherol content and total superoxide dismutase (SOD). DT-diaphorase was decreased in
activity following WY treatment in rats but only sporadically affected in hamsters. Rats and
hamsters treated with DBF demonstrated increased SOD activity at 6 days; however, in the rat,
DBF decreased SOD activity at 90 days and alpha-tocopherol content was decreased throughout.
In GEM-treated rats and hamsters, a decrease in alpha-tocopherol content and an increase in DT-
diaphorase activity were observed.
3.4.2. SAP Perspectives on Species Differences and Human Sensitivity
This section outlines SAP perspectives on the issues of species differences and human
sensitivity, specifically noting the discussion of the data they reviewed, and of what data they
believed may help to clarify these issues. As previously noted in Section 1.2, overall, the
majority of the SAP members agreed that relevant data indicate humans being less sensitive than
rodents to the hepatic effects of PPARct agonists, but opinions of the individual panel members
on the proposed OPPTS policy statement as written ranged from full agreement to complete
disagreement. The SAP further elaborated:
In addition, the Panel members agreed that the MOA and its application to
addressing human relevance would be greatly strengthened by additional evidence
of the specific alterations associated with PPAR-a activation that lead to the more
general steps of hepatocellular proliferation, clonal expansion of initiated
hepatocytes and tumor development.
Considering the proposed MOA, there was agreement that PPAR-a is present in
humans and that the receptor is activated in human liver following exposure to
known agonists. Accordingly, the proposed MOA for PPAR-a agonist-induced
hepatocellular carcinogenesis in rodents is plausible for humans. There was also
agreement that the nature of gene expression associated with hepatocellular
PPAR-a activation is qualitatively different between humans and rodents. This
difference may result from species differences in PPREs, but there are few data
available that identify these potentially important differences, particularly in
humans. Humans are at least as sensitive to activation end-points that lead to
hypolipidemia but are much less sensitive to other end-points normally associated
with peroxisome proliferation.
Whereas PPAR-a activation is a very specific component of the MOA, the other
steps deemed to be causally-related, namely increased hepatocellular proliferation
and clonal expansion of initiated hepatocytes leading to tumor development were
very general and non-specific. Overall, the Panel members agreed that additional
evidence of specific alterations associated with PPAR-a activation in primates and
especially humans would greatly strengthen the proposed MOA.
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On this last subject, the linkage between PPARa activation and cell proliferation, the
SAP further noted:
There was a general consensus that the data linking PPAR-cc activation to
increased cell proliferation in all species was relatively weak. The strongest
evidence in support of the importance of this step in subsequent tumor
development is derived from the PPAR-cc knockout mouse studies in which no
increase in hepatic cell proliferation and no tumors are observed after 11 months
of treatment (Peters et al., 1997).
Discussion of the limitations of the knockout model, including SAP perspectives and discussion
of some new studies, follows in Section 3.5.
With regard to data from other animals besides rats, mice, and humans, the SAP wrote:
The available data from other animals includes guinea pigs, hamsters, dogs and
non-human primates. In all cases, these animals demonstrate reduced liver
sensitivities to PPAR-a agonists....Collectively, the Panel was split on the
applicability of data from other animals to contribute to a weight of evidence
regarding the hepatocarcinogenic effects of PPAR-a agonists in humans.
The SAP discussed human data in some detail, writing:
Although much of the data cumulatively support the hypothesis that
agonist-induced human PPAR-a (hPPAR-a) activation fails to follow the MOA
seen in rodent livers, namely, increased liver cell proliferation, decreased
apoptosis, formation of preneoplastic foci and clonal expansion of these foci into
liver tumors, the weight of evidence for this MOA and consequences of
agonist-induced PPAR-a activation events in humans is less well defined than in
rodents. Human liver biopsy data, while limited, indicate that clinical
administration of PPAR-a agonists results in increases in the number and volume
density of hepatic peroxisomes. The Panel agreed that the available cancer
epidemiological data on pharmacologic PPAR-a agonists are too limited in study
size and duration to provide any relevant information to evaluate human
relevance. As such, data from other animals, including non-human primates, along
with in vitro studies in human hepatocytes, or cell lines, provide the basis for
evaluating the relevance of the proposed MOA in humans.
Although the human data are limited, the existing data do provide some important
information for consideration. Human liver contains functional PPAR-a receptors
and the fibrate class of drugs is able to activate this receptor to alter the expression
of genes involved in lipid metabolism that induce hypolipidemia. Chronic
exposure data reported in humans for two different PPAR-a agonists suggest that
humans do not respond to PPAR-a agonists by an increase of the associated key
events (such as cell proliferation, suppressed apoptosis, and clonal expansion of
^
! 24
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preneoplastic hepatic lesions) observed during PPAR-a activation in rats and mice
exposed to these agonists. In addition to the short duration of exposure and the use
of therapeutic doses (lower than the doses used in studies with rats and mice), the
limitations of these studies include the use of weak agonists. The human
epidemiology data from short duration follow up (5 year time period) indicated an
early increase in GI tract tumors, although liver cancer was not reported
independently. However, no differences were noted after 8 years of follow up.
Evidence for peroxisome proliferation and increased cell proliferation was lacking
in human liver biopsies. Problems with these observations include the high
variability in assessing peroxisome increases in biopsy material that are not
representative of all zones of the liver, and whether the timing of biopsy sample
acquisition was appropriate for detecting an increase in cell proliferation.
Finally, regarding in vitro data with human hepatocytes, the SAP wrote:
The strength of the hypothesis that humans are less sensitive to agonist-induced
PPAR-a-mediated hepatocarcinogenesis lies in the human primary hepatocyte
data. The Panel was again divided on the interpretation and utility of these data.
First, there was a difference of opinion on the applicability of the in vitro studies
used to assess the ability of human hepatocytes to proliferate in response to
treatment with a PPAR-a agonist. Although limited in total sample size, these
studies have shown that in vitro cultured human hepatocytes respond differently to
PPAR-a agonists when compared to in vitro cultured rodent hepatocytes. As
discussed in more detail below, whether these differences are attributable to true
interspecies differences or reflect differences in human and rodent hepatocyte
culture preparations remains an open question, hi parallel experiments with in
vitro cultured rodent hepatocytes, in vitro cultured human hepatocytes fail to
display several of the key responses deemed essential for the MOA in
agonist-induced PPAR-a-mediated rodent hepatocarcinogenesis, those being
increased cell proliferation and decreased apoptosis. Furthermore, in vitro cultured
human hepatocytes appear to be less responsive to upregulation of peroxisomal
genes and proliferation of peroxisomes, two key associative events of
agonist-induced PPAR-a-mediated rodent hepatocarcinogenesis. Several Panel
members suggested that further experiments in human primary hepatocytes
(co-cultured with and without Kupffer cells; see comments below) would be
useful if they provide additional biochemical data that demonstrate reduced levels
of PPAR-a expression in human liver and an inability for agonist-induced
PPAR-a to modulate the gene expression for several key peroxisomal enzymes.
Evidence for Kupffer cell involvement in PPARa agonist liver effects was discussed previously
in Section 3.2, and touched on again in Section 3.5. The importance of this issue with regard to
human relevance was noted by the SAP:
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For example, the growth permissive factors released from activated Kupffer cells
following PPAR-a agonist exposure are absent and may explain the lack of
induction of DNA synthesis seen in cultured human hepatocytes. Support for this
possibility has been demonstrated in rodent cultures in vitro (Rose, et al., 1999).
In these studies, PPAR-a agonists were unable to induce DNA synthesis in
purified preparations of rodent hepatocytes (devoid of nonparenchymal cells),
while PPAR-a agonist-induced DNA synthesis was restored upon the addition of
nonparenchymal cells, or medium derived from activated Kupffer cells, to the
purified hepatocyte cultures.
3.5. HETEROGENEITY OF PPARa EFFECTS IN PPARa KNOCKOUT MICE
Although studies using PPARa knockout mice have been used to support the dependency
of PPARa agonism on liver tumor induction, several concerns have been raised regarding the
adequacy of this model. These are related to both existing study designs and to whether the
intrinsic characteristics of these knockout mice mean that they exhibit differing responses from
those of wild-type mice independent of effects related to PPARa agonism. PPARa knockout
mice can be useful in describing effects that are associated only with activation of the receptor
but not necessarily the effects associated only with peroxisome proliferation. Animal studies in
PPARa-null mice also may be inadequate to fully describe effects of lack of the receptor on
cancers because they show an absence of nonspecific effects only at the high doses associated
with peroxisome proliferation.
Some SAP members expressed concerns over the adequacy of the "knockout" or "null"
mouse model to demonstrate the dependency on PPARa agonism to induce hepatocarcinogenesis
in mice. The SAP wrote:
There was agreement among most, but not all, of the Panel that data from the
PPAR-a -/- mouse indicate the requirement for the activation of PPAR-a in the
MOA of the hepatocarcinogenic effect of these agents. A few Panel members
expressed concern over the short duration of the studies in the PPAR-a -/- mouse .
(i.e., 11 months vs. 24 months in standard cancer bioassays), which rendered the
studies incapable of assessing the lifetime liver cancer risk of PPAR-a agonists in
this knockout mouse model, and thus, inadequate to conclusively demonstrate that
PPAR-a activation is required for hepatocarcinogenesis.
Regarding the interplay between Kupffer cells and knockout mice, the SAP wrote:
It was noted that arguments against the involvement of the Kupffer cells comes
from studies in the PPAR-a null mice. In these mice, agonists failed to elicit a
DNA synthetic response. Since this model is replete with Kupffer cells, the lack of
DNA synthesis has been interpreted as indicating that the Kupffer cell is not
required. On the other hand, some members of the Panel felt that the
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communication and/or interplay between PPAR-cc agonism and Kupffer cells has
not been fully characterized and as such, the null mouse, lacking PPAR-a, is not
directly applicable to the human situation in which PPAR-a is present and can be
activated.
The recent study by Laughter et al. (2004), which as discussed previously used PPARa
knockout mice to try to investigate the role of PPARa in response to TCE and its metabolites,
also illustrates the potential difficulties in interpreting studies which use knockout mice. For
instance, as mentioned previously, knockout mice did not show a statistically significant effect
from TCE treatment but had greater liver-to-body weight ratios than wild-type mice at all levels
of exposure, including controls. Moreover, measures of induction of hepatocyte proliferation
(BrdU incorporation) showed that baseline levels were also elevated in PPARa knockout mice
compared with wild-type mice. At the second highest exposure level (1,000 mg/kg), both wild-
type and PPARa knockout mice had elevated levels of hepatocyte proliferation with high
variability in response. These increases did not appear to be statistically different from each
other, but such an analysis was not made by the authors. In the 3 week study, TCE toxicity was
observed at the highest dose in the knockout mice that was not observed in the wild-type mice —
all knockout mice were moribund and had to be removed from the study. Inspections of livers
and kidneys from the group did not reveal overt signs of toxicity that would lead to morbidity.
At the same dose, wild-type mice exhibited mild granuloma formation with calcification or mild
hepatocyte degeneration. Kidney-to-body weight ratios were increased by TCE in wild-type but
not in knockout mice, with WY having no effect on the kidney in either strain. There results
suggest that the knockout mouse may exhibit differences in response with the wild-type mouse
that may be independent of the peroxisomal effects of PPARa agonism.
Many phthalates are considered to be relatively weak peroxisome proliferators and have
been studied in knockout mice. These studies help illustrate that elimination of PPARa activity
has effects on expression of other genes. Valles et al. (2003) report that exposure of di-isononyl
phthalate in SV129 wild-type, SV129 PPARa-null, and B6C3F1 mice shows a varied pattern of
gene expression that was dependent on gender and age, with some changes in gene expression
dependent on PPARa activity and others not. An additional gene was shown to be down-
regulated in wild-type mice but up-regulated in PPARa-null mice, indicating more complex
regulation by PPARa and additional factors. Macdonald et al. (2001) carried out.quantitative
proteomic analyses of DEHP-treated wild-type or PPARa-null mouse livers. Fifty-nine proteins
were identified where altered expression was both PPARa- and peroxisome proliferation-
dependent. In addition, six proteins regulated by the deletion of PPARa were identified, possibly
indicating an adaptive change in response to the loss of this receptor. Proteins identified as being
regulated by PPARa are known to be involved not only in lipid metabolism pathways but also in
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amino acid and carbohydrate metabolism, mitochondria! bioenergetics, and stress responses,
including several genes not previously reported to be regulated by PPARa.
The report of Anderson et al. (2002) on liver regeneration and hepatic gene expression
following partial hepatectomy in wild-type and PPARa-null mice showed that PPARa-null mice
had a 12- to 24-hour delay in liver regeneration associated with a delayed onset and lower peak
magnitude of hepatocellular DNA synthesis. Furthermore, these mice had a 24-hour lag in the
hepatic expression of the G(1)/S checkpoint regulator genes Ccndl and cMyc and increased
expression of the IL-lbeta cytokine gene. Hepatic expression of Ccndl, cMyc, IL-lrl, and IL-6r
was induced in wild-type mice, but not in PPARa-null mice, following acute exposure to the
potent PPARa agonist WY, indicating a role for PPARa in regulating the expression of these
genes. The authors suggest that liver regeneration in PPARa-null mice is transiently impaired
and is associated with altered expression of genes involved in cell cycle control, cytokine
signaling, and fat metabolism.
Jia et al. (2003) report disruption of the inducible beta-oxidation pathway in mice at the
level of fatty AGO, the first and rate-limiting enzyme, resulting in spontaneous peroxisome
proliferation and sustained activation of PPARa. Mice with complete inactivation of
peroxisomal beta-oxidation at the level of the second enzyme, enoyl-CoA hydratase/L-3-
hydroxyacyl-CoA dehydrogenase (L-PBE) of the inducible pathway and D-3-hydroxyacyl-CoA
dehydratase/D-3-hydroxyacyl-CoA dehydrogenase (D-PBE) of the noninducible pathway (L-
PBE-/-D-PBE-/-), exhibit severe growth retardation and postnatal mortality with none surviving
beyond weaning. L-PBE-/-D-PBE-/- mice that survived exceptionally beyond the age of 3 weeks
exhibited overexpression of PPARa-regulated genes in the liver, despite the absence of
morphological evidence of hepatic peroxisome proliferation.
There is a gender difference in expression of PPARa in rodents that has been explored
using knockout mice. Lewitt et al. (2001) report mice lacking PPARa knockout have a sexually
dimorphic phenotype with PPARa influencing the insulin-like growth factor (IGF)/IGF-binding
proteins (IGFBP) response to feeding, particularly in males. Following fasting and refeeding,
IGFBP-1 and insulin concentrations were higher in males than in females and were further
increased in PPARa knockout, suggesting significant hepatic insulin resistance. The authors
suggest that gender differences in the IGF system contribute to the PPARa knockout phenotype.
It has been suggested that elevated serum levels of 1GF-1 and leptin are associated with increased
risk of developing cancer (Bursting et al., 2003; Sandhu et al., 2002; Liu et al., 2001; Thompson
etal.,1999).
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3.6. INTRINSIC FACTORS PPAR-RELATED AFFECTING RISK
Another key element to evaluating of risk that PPAR agonists may pose to humans is how
intrinsic factors may modulate that risk. Among the key factors for which some recent data exist
regarding differential responses are genetic polymorphisms, gender, and life stages. Intrinsic
susceptibility is also a factor for epidemiologic investigations of fibrate drugs because patients
who are being studied are generally taking the drug for a disturbance that has been already
manifested in lipid metabolism. In general with such studies, an awareness of what human
samples are being studied, and under what conditions, is needed to understand the possibility of a
false negative signal.
The possibility of genetic polymorphism and the association of PPARa polymorphism
with metabolic diseases is the subject of a number of recent studies. Familial combined
hyperlipidemia (FCHL) is a common genetic lipid disorder present in 10% of patients with
premature coronary artery disease (CAD). Eurlings et al. (2002) report that the PPARa gene is a
modifier of the FCHL phenotype. Lacquemant et al. (2000) screened the PPARa gene for
mutations to test the genetic contribution of the PPARa in diabetes and its vascular
complications. They concluded that it is unlikely that PPARa gene has a major role in diabetes
and CAD in their populations, although they cannot exclude a minor contribution of the PPARa
gene to the risk of coronary heart disease associated with Type 2 diabetes through a modulation
of atherogenic plasma lipids.
Regarding gender differences, some of the effects on gender dependencies related to gene
regulation were discussed in Section 3.1. In addition, male rats have been reported to be more
responsive to fibrates than female rats. Jalouli et al. (2003) report that male rats had higher
levels of hepatic PPARa mRNA and protein than female rats. Fasting increased hepatic PPARa
mRNA levels to a similar degree in both sexes. Hypophysectomy increased hepatic PPARa
mRNA and protein levels and was.more pronounced in females than in males but was not
mediated by GH. The authors suggest that sex hormones regulate the sex difference in hepatic
PPARa levels but not via the sexually dimorphic GH secretory pattern.
Regarding questions about life stages, Michalik et al. (2002, 2001) and Wahli (2002)
report that in rodents,.PPARa, PPAR6, and PPARy show specific time- and tissue-dependent
patterns of expression during fetal development and in adult animals, hi addition to citing
evidence that suggested differences in p-oxidation capabilities in developing rodents and
humans, the SAP wrote
It was also considered that differences in cell proliferation, xenobiotic
metabolism, and other factors in the developing rodent (or human) could affect
sensitivity to PPAR-a hepatocarcinogenesis. Therefore, information on the
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expression of the PPAR-a during ontogeny as well as responses of embryonic and
fetal human hepatocytes to PPAR-a agonists should be evaluated before
concluding that the developing human conceptus is unresponsive to PPAR-a
agonist exposures.
More specifically, the SAP summarized a number of results relating to early life stages as
follows:
Published reports have shown that both the expression of PPAR-a and the
assembly of peroxisomes occur late in the development of rats and mice.
Furthermore, it has been shown that, as in adult livers, embryonic, fetal and
neonatal livers of rats and mice respond to PPAR-a agonists by increasing
peroxisome number, peroxisome volume density, liver weight, and the expression *
of the peroxisomal enzyme palmitoyl CoA oxidase. This suggests that at least
some of the cellular macromoiecules involved in the proposed PPAR-a agonist
MOA are functional and responsive to PPAR-a agonists in rat and mouse
embryonic, fetal, and neonatal livers. However, data on the hepatocarcinogenic
response of rat and mouse embryonic, fetal, and neonatal livers to PPAR-a
agonists are lacking and, therefore, no conclusions can be made at this time as to
the relative sensitivity of these early life stages to PPAR-a agonist induced
hepatocarcinogenicity.
Although the exposure of pregnant rats and mice led to increases in
peroxisomal enzyme activities and increases in liver weight in embryonic, fetal,
and neonatal liver tissues, other parameters involved in the proposed MOA, such
as cell proliferation, inhibition of apoptosis and clonal expansion of preneoplastic
cells, were not examined in these studies, hi addition, responses to PPAR-a
agonists in the fetal and neonatal rat and mouse, as measured by the peroxisomal
enzyme expression levels, suggest that there are differences in young animals
relative to adults. It is unclear how these differences in enzyme expression levels
might translate into differences in sensitivity to hepatocarcinogenesis. Regarding
the comparison of changes in liver weights across early and later life stages, it is
inappropriate to assume that a given proliferative response seen at one stage of life
is equivalent to a similar proliferative response at another stage of life. For
example, an increase in liver weight during the neonatal period might result in a
much greater lifetime risk of cancer than an equivalent increase occurring during
adulthood, because a larger number of cells in the neonatal liver will undergo
multiple cell divisions than in the adult. Finally, none of the studies examining the
response of the rodent in utero or during early life stages were carried out with the
late onset of tumors as a specific endpoint.
Conclusions regarding the relevance of the PPAR-a agonist MOA for human
hepatocarcinogenesis applied to adults may not apply to the young. In contrast to
adult human liver, there are no data establishing PPAR-a expression levels in
embryonic, fetal and neonatal human liver.
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In contrast to embryonic and fetal rodent liver in which cytochrome P45.0
enzymes are expressed near, during and after birth (Ring et al. 1999), embryonic
and fetal human livers possess metabolic activation capabilities resulting from the
early developmental expression of cytochrome P450 enzymes. Moreover, the
expression profiles of xenobiotic metabolizing enzymes and isozymes are
different in embryonic, fetal, neonatal and adult human livers. Like the gene
expression profile of xenobiotic metabolizing enzymes, it is difficult to disregard
the possibility that there could be differences between the expression of PPAR-a
and its transcriptional co-factors in the human conceptus and adult human liver.
In addition, metabolic differences in rats and mice play an important role in
determining the degree of response to some PPAR-a agonists (Lake, 1995) and
that could also apply to the human conceptus.
Differences in peroxisome biogenesis have been reported during the ontogenic
* development of rodents and humans. While the assembly of peroxisomes in rats
and mice, including the insertion of p-oxidation enzymes into the peroxisomes,
occurs near birth, the assembly of human peroxisomes has been observed as early
as 8 weeks of gestation (Espeel, et al, 1997). The number and density of
peroxisomes plateau by 17 weeks of gestation in humans. Moreover, acyl-CoA
oxidase and 3-ketoacyl CoA thiolase are immunodetectable in the peroxisomes by
10 and 9 weeks of gestation, respectively. These observations suggest differences
in P-oxidation capabilities in developing rodents and humans and therefore
information on the expression of the PPAR-a during ontogeny, as well as
responses to PPAR-a agonists in embryonic and fetal human hepatocytes should
be evaluated before concluding that the developing human conceptus is
unresponsive to PPAR-a agonist exposures.
Finally, there is also evidence that peroxisome proliferators are much more potent in
producing tumors in older rats than in younger ones, even though effects on peroxisome
proliferation and cell proliferation were the same (Youssef et al., 2003; Chao et al., 2002;
Youssef and Badr, 2002). Promotional effects of PPAR agonists for tumors induced by other
MOAs are described in the issue paper on interactions. However, a promotion effect in older
animals with already initiated foci could be the MOA for increased sensitivity of older rats to
PPARa effects.
4. SUMMARY
As scientific information that can aid in the hazard characterization of TCE has increased
since the last assessment, so has the field of PPARa activation and its nonperoxisomal effects in
humans. The receptor appears to be pleiotropic in its actions, and those actions also seem to be
chemical, gender, age, and concentration dependent. There is additional insight as to what types
of cell signals and relationships exist with PPARa activation. Recent work on the role of the
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Kupffer cell in effects resulting from PPARa appears to be important to understanding
hepatocarcinogenesis. However, the way in which peroxisome proliferators induce tumors in
rodents is still unknown, and the relevancy of those tumors to human risk is controversial. The
task of NAS is as follows: Given the substantial amount of new information on PPARa agonism
in general and more limited information regarding its relationship to TCE, advise EPA on the
interpretation of new data and on whether conclusions can be made about the role of PPARa
agonism in TCE toxicity and its relevance to human health risks.
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APPENDIX
Recent Literature on Effects Associated With PPAR Agonism or Related to
Its Mechanisms of Action
t
Effects
Obesity and atherosclerosis
Diabetes
Cardiomyopathies
Familial combined hyperlipidemia
Increased susceptibility from aging
Atherosclerosis, inflammation, cancer, infertility,
and demyelination
Acetaminophen hepatotoxicity
Cardiac cell metabolism
Lipid and glucose metabolism
Muscle lipid homeostasis
Lipoprotein lipase (LPL)
Fatty acid oxidation
Liver fatty-acid-binding protein (liver and small
intestine)
Triglyceride and fatty acid metabolism
Macrophage cholesterol homeostasis
Hypoglycemia, role for the development of insulin
resistence in response to a Western-type high-fat
diet
Regulation during fasting
Genes implicated in the inflammatory response
(NFkappaB, AP-1, C/EBP beta, STAT-1 and
NFAT)
Tumor necrosis factor-alpha
Reference
Kersten et al. (2000)
Vohl et al. (2000); Shankar et al. (2003a, b, c);
Holden et al. (1999); Lacquemant et al. (2000)
Nohammer et al. (2003)
Eurlings et al. (2002)
Chao et al. (2002); Youssef and Badr (2002);
Youssefetal. (2003)
Berger and Moller (2002); Berger and Wagner
(2002), Barbieretal. (2002)
Chen et al. (2000)
Jiang et al. (2004)
Brisson et al. (2002)
Muoio et al. (2002)
Nohammer et al. (2003)
Kersten et al. (2001); Watanabe et al. (2000)
Poireretal. (2001)
Barbier et al. (2002)
Barbier et al. (2002)
Guerre-Millo et al. (2001)
Escher et al. (2001); Poirer et al. (2001)
Blanquart et al. (2003)
Bojes et al. (1997); Rusyn et al. (2000); Holden
et al. (2000); Chevalier et al. (2000); Peters et al.
(2000); Roberts et al. (2002)
42
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Effects
Early inflammation phase of the heating
Control by glucocorticoids (corticosterone)
Connexin32 (major gap junction forming protein
in liver)
Fetal or neonatal CYP4A mRNA expression
P450 genes, including CYP2B, CYP2C, and
CYP4A family members
Glutathione S-transferase glutathione peroxidase
and glutathione reductase
Growth hormone and STATSb
Carboxylesterases in the liver
L-pyruvate kinase (glycolytic enzyme)
Bile acid synthesis and catabolism in the liver
(UDP-glucuronosyltransferase)
Constitutive myocardial beta-oxidation of the
medium and long chain fatty acids, octanoic acid,
and palmitic acid
Proteins not involved in lipid metabolism but are
implicated in the pathogenesis of heart disease
Ovarian function
Estrogen action
Testicular degeneration
Thyroid hormone action
Regulation of CYP1A1
Rat male-specific alpha 2u-globulin
Reference
Michalik etal. (2001)
Lemberger et al. (1996); Plant et al. (1998)
Moennikes et al. (2003)
Simpson et al. (1996); Simpson et al. (1995)
Fan et al. (2003)
O'Brien etal. (200 la)
Zhou and Waxman (1999); Zhou et al. (2002)
Poole et al. (2001) v
Pan et al. (2000)
Barbier et al. (2003a, b); Sinai et al. (2001)
Watanabe et al. (2000)
Vosper et al. (2002)
Komar etal. (2001)
Klotz et al. (2000); Zhu et al. (1999); Xu et al.
(2001)
Dufour et al. (2003); Gazouli et al. (2002)
Miller etal. (2001)
Seree et al. (2004)
Cortonetal. (1997, 1998)
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