PPARand Effects of TCE

Referencing: Key Issues in the Role of Peroxisome Proliferator–Activated Receptor Agonism and Cell Signaling in Trichloroethylene Toxicity

We would like to offer a different opinion on the ideas presented in the article by Keshava and Caldwell (2006).The authors indicated that their article summarized scientific literature published since an earlier U.S. Environmental Protection Agency (EPA) risk assessment of trichloroethylene (TCE), with an emphasis on the possible role of proliferator-activated receptor (PPAR) agonism relevant to TCE risk assessment. Interestingly, in the section on recent data on PPAR agonism, Keshava and Caldwell failed to establish any gene expression signature relating TCE and PPAR.

Keshava and Caldwell (2006) contended that it is difficult to identify a clear pattern of common gene expression changes for TCE and PPAR agonists in general. However, they did not consider numerous reports and reviews (e.g., Klaunig et al. 2003; Peters et al. 2005) illustrating that there are common and reproducible changes in gene expression associated with PPAR agonists. Further, extensive characterization has definitively demonstrated specific, direct targets of PPAR-retinoid X receptor heterodimers (reviewed by Klaunig et al. 2003). Keshava and Caldwell (2006) also did not discuss the possibility that the effect of TCE on gene expression could be mediated by mechanisms independent of PPAR, which likely explains the disparity described in their article. Keshava and Caldwell did not critically discuss the data summarized in their Table 2 (Keshava and Caldwell 2006), failing to note that many of these gene targets have no clear linkage with the PPAR agonist mode of action (MOA) and may be mediated either via different ligand–receptor–coactivator complexes that form on the promoter regions of the regulated genes by secondary events downstream of the initial events associated with PPAR activation, or by mechanisms that are independent of PPAR. In addition, the authors failed to describe the limitations of the various gene array platforms and to correctly interpret the findings in the context of gene targets by other PPAR agonists, especially when more comprehensive data sets exist but were not cited (Anderson et al. 2004a, 2004b).

Keshava and Caldwell (2006) further raised concerns regarding the use of PPAR-null mice to evaluate the MOA of PPAR by indicating that the physiologic differences observed in PPAR-null mice relative to wild-type mice suggest that the null mouse is an inadequate model to study the PPAR MOA. The data they cited, however, appears selective because they failed to mention that liver regeneration in PPAR-null mice is reportedly unchanged compared with wild-type mice (Rao et al. 2002), and age-related, sexually dimorphic obesity has not been observed in congenic PPAR-null mice (Akiyama et al. 2001). Thus, although the null mouse exhibits changes consistent with the critical role of PPAR in modulating fatty acid catabolism, this phenotype does not preclude its application for determining the critical role of this receptor in the MOA of PPAR agonists. Importantly, Keshava and Caldwell (2006) did not comprehensively discuss significant findings a) that PPAR-null mice are refractory to liver tumors induced by two different PPAR agonists (Hays et al. 2005; Peters et al. 1997); b) that they are refractory to increased markers of replicative DNA synthesis and suppression of apoptosis after exposure to numerous PPAR ligands (summarized by Peters et al. 2005); or c) that PPAR-null mice expressing the human PPAR in the liver respond to PPAR agonists by increasing expression of genes encoding proteins that catabolize lipids, but they fail to show increases in markers of cell proliferation and are resistant to liver cancer (Cheung et al. 2004; Morimura et al. 2006). To dismiss these findings through lack of discussion or citation does little to advance our understanding and suggests that Keshava and Caldwell’s article is unbalanced.

Keshava and Caldwell (2006) also misrepresented an earlier review by Klaunig et al. (2003) regarding the MOA of PPAR agonists. Keshava and Caldwell (2006) incorrectly suggested that Klaunig et al. (2003) placed substantial weight on the associative event of peroxisome proliferation with this MOA, when, in fact, peroxisome proliferation was strongly—but not causally—associated, as noted for sustained increased cell proliferation. Keshava and Caldwell (2006) also misconstrued this review (Klaunig et al. 2003), focusing on DNA damage as a possible contributor to the MOA. Citing one manuscript that examined the effect of one, nonspecific PPAR ligand (DHEA) is not sufficient to refute the comprehensive review by Klaunig et al. (2003). Finally, Keshava and Caldwell (2006) also suggested that the effects of PPAR ligands on mitochondrial function are part of the MOA, but they provided no direct evidence to support their contention that PPAR agonists or TCE causes mitochondrial dysfunction.

In summary, Keshava and Caldwell (2006) missed an excellent opportunity to critically and objectively examine the data that support or refute the role of PPAR in TCE-induced effects. In our opinion, their article did not advance our understanding of the MOA of PPAR agonists or TCE.

The views expressed in this article are those of the authors and do not necessarily reflect the views or policies of the U.S. Consumer Product Safety Commission.

J.C.C. is employed by Pfizer, which is developing PPAR agonists for treatment of disease indications. R.M.D. is a member of the American Chemistry Council, Phthalate Ester Panel. J.G.D. is employed by Merck Research Laboratories, which has an interest in the development of PPAR agonists as therapeutic agents, and he owns stock and stock options in Merck. R.H.M. is employed by ExxonMobil, a manufacturer of PPAR agonists (but not TCE). R.A.R. is employed by AstraZeneca, which has an active research program in PPAR/ agonists for potential treatment of lipid and glucose abnormalities associated with diabetes. The remaining authors declare they have no competing financial interests.

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PPAR and TCE: Keshava et al. Respond

We appreciate the opportunity to discuss the issues raised by Klaunig et al. in their letter. First, we reiterate that, given the mini-monograph’s scope (Chiu et al. 2006), our article (Keshava and Caldwell 2006) was intended not to comprehensively review the role of peroxisome proliferator-activated receptor (PPAR) agonism in trichloroethylene (TCE) toxicity but rather to “highlight some of the recently published literature on PPAR ... to help inform and illustrate the key scientific issues relevant to TCE risk assessment.” In addition, we considered not just hepatocarcinogenesis, but a broader range of modes of action (MOAs) and toxicity effects, necessitating a brief discussion of the article by Klaunig et al. (2003). Furthermore, because of the pending National Academy of Sciences report and revision of the TCE assessment, Klaunig et al.’s suggestion to examine whether the data “support or refute the role of PPAR in TCE-induced effects” would have been premature in the mini-monograph.

In their letter, Klaunig et al. state that there are “common and reproducible changes in gene expression associated with PPAR agonists.” However, as described by Klaunig et al. (2003), the well-characterized changes are largely peroxisomal or related to lipid metabolism, and thus not causally related to hepatocarcinogenesis. Hays et al. (2005) and the Federal Insecticide, Fungicide, and Rodenticide Act Science Advisory Panel [FIFRA SAP (2004)] suggested that the MOA underlying PPAR agonist-induced hepatocarcinogenesis has not been fully elucidated in that the specific target genes modulated by PPAR leading ultimately to liver cancer have not been identified. We share the concerns of Klaunig et al. about critically interpreting gene array data and the concerns of Voss et al. (2006) about also considering dose-, time course–, species-, and strain-related differences. Given reports that PPAR agonists have zonal differences in hepatocyte, peroxisomal, and mitochondrial proliferation, and in foci development (Anderson et al. 2004a; Bannasch 1996), zone-dependent and nonparenchymal cell responses (e.g., Kupffer cells) should also be taken into account. Finally, Table 2 of our article (Keshava and Caldwell 2006) illustrated the pleiotropic and varying liver responses of the PPAR receptor to various agonists, but we did not imply that these responses were responsible for carcinogenesis.

We agree with Klaunig et al. that PPAR-null mice have been useful in investigating the MOA for hepatocarcinogenesis, particularly for the strong agonist WY-14,643 {[4-chloro-6-(2,3-xylidino)-2-pyrimidinylthiol]acetic acid}. However, possible limitations of genetically modified mice, such as lack of complete tumor development or manipulation of the carcinogenic process, should be adequately characterized [U.S. Environmental Protection Agency) (EPA) 2005]. Maronpot et al. (2004) noted the need for lifetime studies to characterize background or spontaneous tumor patterns and life spans (including those of the background strain) for these models.

PPAR-null mice have baseline difference from wild-type mice that may render them more susceptible to toxic responses [e.g., reduced glycogen stores, altered responses to fasting, elevated plasma free fatty acids, fatty liver, impaired gluconeogenesis, significant hepatic insulin resistance (Lewitt et al. 2001)], or potentially shorten their life spans with chemical exposure (Anderson et al. 2004b; Hays et al. 2005) or with further genetic modification (Nohammer et al. 2003). A comparison of their life spans with those of background strains without treatment has not been reported. Moreover, in PPAR-null mice, Wheeler et al. (2003) reported alteration of cyclin-dependent kinase/cyclin complexes necessary for cell cycle progression and DNA synthesis, whereas Voss et al. (2006) found increased apoptosis and decreased mitosis with fumonisin treatment. Thus, the question remains whether PPAR-null mice may have different susceptibility to hepatocarcinogenesis not specific to the proposed PPAR MOA.

Furthermore, bioassay study designs need adequate sensitivity to detect carcinogenic responses or elucidate MOAs. Morimura et al. (2006) and Hays et al. (2005) used high concentrations (with mortality), few (and differing numbers of) animals in treated versus control groups, and differing periods of exposure (all ? 1 year) complicating study interpretation. Interestingly, in the “humanized” PPAR-null mouse after 44 weeks of treatment, Morimura et al. (2006) noted (along with decreased toxicity) a WY-14,643–induced adenoma resembling spontaneous tumors rather than those seen in PPAR agonist-treated wild-type mice; no tumors were observed in controls. This raises the question of whether, if tested for longer periods of time, the humanized mice might show significant responses with tumors more consistent with those induced by a variety of non-PPARagonists and those observed in humans (Bannasch 1996; Su and Bannasch 2003).

We acknowledge the importance of Peters et al. (1997) demonstrating in vivo effects of WY-14,643 on replicative DNA synthesis– and hepatocarcinogenesis–involved PPAR activation. Furthermore, we agree that peroxisome proliferation per se is an associative rather than causal event in the MOA for hepatocarcinogenesis (described by Rusyn et al. 2000). However, Klaunig et al. (2003) proposed a “minimal set of data elements” to support their PPAR MOA in rodents that consists of “PPAR agonism combined with light- or electron-microscopic evidence of peroxisome proliferation” or other markers of peroxisome proliferation. In addition, Klaunig et al.’s claim that we (Keshava and Caldwell 2006) misconstrued their review (Klaunig et al. 2003) as focusing on DNA damage as a possible contributor to the MOA is incorrect; that hypothesis was discussed by Reddy and Rao (1989). We believe it is important to identify changes both specific to PPAR activation and related to carcinogenesis.

Voss et al. (2006) reported fumonisin-induced apoptosis, cell proliferation, gene changes, and liver lesions to be PPAR-independent but having some common target genes with PPAR agonists. Thus, we should not only understand a particular agent’s effects on the cell cycle and proliferation but also establish dependence on PPAR. Another issue is the applicability of the proposed MOA across PPAR agonists. Hays et al. (2005) noted that much of the literature on the PPAR MOA used WY-14,643, which induces sustained cell proliferation, whereas weaker agonists produce more transient responses (Marsman et al. 1988). Kraupp-Grasl et al. (1990) noted differences among agonists in their abilities to promote tumors and suggested that they should not necessarily be considered a uniform group. Finally, the discussion of the effects of PPAR agonists on mitochondrial function in our article (Keshava and Caldwell 2006) was intended to raise the issue for further investigation.

Similar issues with respect to PPAR have been discussed by recent scientific panels (FIFRA SAP 2004; U.S. EPA Science Advisory Board 2006). We believe that our article (Keshava and Caldwell 2006), Klaunig et al.’s letter, and this response help to further elucidate these complex issues for the assessment of TCE as well as other chemicals.

The views expressed in this article are those of the authors and do not necessarily reflect the views or policies of the U.S. EPA.

The authors declare they have no competing financial interests.