Studies of species differences in liver effects of TCE are important for assessing the risk of human exposure (Lash et al., 2002
; Lumpkin et al., 2003
; Sano et al., 2009
). It is widely accepted that oxidative metabolism of TCE is a key event for liver toxicity, and there are important quantitative differences among species with regard to formation of two major metabolites, TCA and DCA (NRC, 2006
). These molecules are hepatocarcinogenic themselves in the mouse, yet are thought to operate through different molecular pathways (Corton, 2008
). TCA-induced mouse liver tumors have molecular features similar to those induced by typical peroxisome proliferators (Bull et al., 2002
; Latendresse and Pereira, 1997
). Even though TCE induces peroxisome proliferation, TCE-induced tumors exhibit a number of important dissimilarities as compared with TCA-induced tumors, which led to a suggestion that the mode of action for TCE hepatocarcinogenesis is different from that of TCA (Corton, 2008
). Thus, the issue of quantitative assessment of TCE metabolism is an important challenge in relating adverse health effects in rodents to humans (NRC, 2006
). Importantly, we show that in addition to differences in TCE metabolism between species, there are important differences between individuals within the same species.
The relative amounts of TCA and DCA being formed as a result of TCE administration has been determined in several studies. Although some still debate whether DCA is formed in vivo
in quantifiable levels (Merdink et al., 2008
), several recent studies showed that DCA can be detected; however, serum concentrations are more than 1000-fold lower than those for TCA (Delinsky et al., 2005
; Kim et al., 2009a
; Sano et al., 2009
). Our study confirms this and shows that DCA and TCA serum concentrations do not correlate with each other, supporting the pharmacokinetic model suggesting that the metabolism of TCA to DCA is not a major pathway for the formation of DCA in the mouse (Kim et al., 2009b
). At the same time, we show that the interstrain differences in serum TCA correlate well with liver Cyp2e1, an enzyme which is known to be a major oxidase for metabolizing TCE to TCA (Lash et al., 2000a
Furthermore, this study provides additional evidence that, in the mouse, the glutathione pathway of TCE metabolism plays a minor role. We show that very low levels (~10,000-fold less than TCA) of DCVG and DCVC are formed, that this phenomenon is reproducible across multiple inbred mouse strains, and that these metabolites are rapidly eliminated after a single large dose of TCE when administered in corn oil vehicle. It has been suggested that the extent of formation of glutathione conjugates from TCE in humans is much higher than in rodents based on high blood concentrations of DCVG reported in humans after inhalation of TCE (Lash et al., 1999
). The same group studied formation of glutathione conjugates in rats after single large oral dose of TCE and reported that DCVG concentrations in blood and urine were high, yet not dose or time dependent (Lash et al., 2006
). Other groups have reported a very low rate of glutathione conjugates’ formation in vivo
in rats (Dekant et al., 1990
) and mice (Kim et al., 2009a
). Thus, the findings reported herein provide additional important information for rodent-to-human extrapolations with regard to TCE metabolism, which is critical for dose-response analysis and derivation of the reference values in risk assessment.
To better understand the differences in the molecular events elicited by TCE in the mouse liver, we evaluated both strain-dependent and independent gene expression changes. Genetic polymorphisms play a major role in influencing gene expression in liver (Gatti et al., 2007
) and other tissues (Bystrykh et al., 2005
; Chesler et al., 2005
), an effect that is often stronger than that of a toxicant (Harrill et al., 2009
). When genetic background–dependent gene expression differences between strains are not taken into the account, the population-wide TCE-induced gene expression changes were found to be related to the peroxisomal proliferation mode of action. Indeed, TCE is well known to cause many liver effects in mice in a PPARα-dependent manner (Laughter et al., 2004
; Nakajima et al., 2000
). Even though we did not find PPAR genes themselves to be induced 24 h after treatment with TCE, similar to the observation of Sano et al. (2009)
, the targets of these transcription factors were strongly upregulated in the overwhelming majority of the strains. This is indicative of the fact that the peroxisomal proliferation effect of TCE is occurring consistently in all strains.
Interestingly, when correlations between TCE metabolism to TCA or to gene expression changes were considered in the context of strain, we found that almost all the genes were also significantly impacted by the genetic background effect. This observation is in agreement with the fact that major strain-dependent differences in TCE metabolism exist. Induction of a lipid and drug metabolism network of genes centered on PPARs was the major molecular signature of this correlation analysis, consistent with the fact that TCA was the major metabolite of TCE in all strains and that it is a ligand for PPARs (Maloney and Waxman, 1999
The interstrain variability in TCE metabolism observed in this study resulted in little observable liver toxicity (marginal increases in ALT observed in 2 out of 15 strains) when liver histopathology was considered. Acute single-dose exposures have not been associated with liver injury. The studies of Ramdhan et al.
are the shortest that showed liver toxicity in mice following 7 days of inhalation exposure to TCE, an effect that was dependent on Cyp2e1 (Ramdhan et al., 2008
), but not PPARα (Ramdhan et al., 2010
The mechanism for TCE-induced liver damage has not been well studied, but it has been suggested that induction of nuclear factor-kappa-light-chain enhancer of activated B cells-activated cytokine pathways may be responsible for hepatotoxicity (Ramdhan et al., 2008
). Thus, it is interesting that we find that cell death, liver necrosis, and inflammatory-mediated response networks are altered by TCE treatment, and it is genetic background independent. This further suggests that liver damage may not depend solely on TCE metabolism to potentially hepatotoxic intermediates, such as chloral hydrate or DCA, and that sufficient amounts of hepatotoxic intermediates are formed in all strains in spite of a four- to sixfold difference in TCE metabolites.
The transcription factor analysis of the genes that were altered by TCE independent of genetic background revealed several inflammation-related regulatory proteins, MAZR, AP2α, and SP1, which may be common regulators involved in TCE-induced inflammatory response in mouse liver. MAZR transactivates the Myc
gene in B cells and is important for the development of B cells in association with Bach2 (Kobayashi et al., 2000
). AP2 is involved in a number of inflammatory pathways including NFκB, cyclooxygenase-2, and inducible nitric oxide synthase, as well as PPARγ (Makowski et al., 2005
). SP1 is one of key regulators of acute-phase response inflammatory genes in hepatocytes (Cantwell et al., 1998
). Because these transcription factors have been shown to be associated with activation of macrophages and lymphocytes, the gene expression signature reported in this study suggests that TCE may have an effect on Kupffer cells, a hypothesis which needs to be tested.
It has been suggested that PPARα may be protective against liver injury, at least in the short-term exposure scenarios (Ramdhan et al., 2008
). Interestingly, a recent study investigating the effects of TCE in Ppar
α-null and humanized mice showed that PPARα and PPARγ may be important factors in TCE-induced lipid accumulation in the liver but that TCE-induced liver toxicity was largely independent of PPARα status and also occurred in humanized mice (Ramdhan et al., 2010
). The authors also reported that human PPARα may afford only weak protection against TCE-mediated effects as compared with mouse PPARα (Ramdhan et al., 2010
). This observation is of importance for consideration of species-specific differences in TCE toxicity as data exist from human occupational exposure studies suggesting that exposures to TCE may lead to acute and chronic liver injury (Pantucharoensri et al., 2004
; Thiele et al., 1982
). Our observation that induction of PPARα-mediated pathways by TCE is dependent on strain and thus an individual's genetic background and further supports a need for additional studies elucidating the role of PPARα beyond peroxisome proliferation effects.
We also observed that Myc was induced by TCE. This observation is in agreement with the studies of Tao et al. (2000)
who found decreased methylation in the promoter regions of the Jun
genes and increased levels of their mRNA and proteins in mice exposed to TCE, DCA, and TCA. It has been suggested that a rapid increase in proliferation caused by most peroxisome proliferators would prevent the methylation of the newly synthesized strands of DNA (Ge et al., 2001
). Indeed, the temporal relationship between increased cell proliferation and DNA hypomethylation of the Myc
gene was observed in a study after single administration of another peroxisome proliferative agent WY-14643 to mice (Ge et al., 2001
). Importantly, this short-term effect is not sustained because subchronic and chronic administration of WY-14643 has no effect on Myc
methylation; however, major changes in DNA methylation occurred (Pogribny et al., 2007
). Thus, it must be established whether upregulation of Myc
by TCE is playing a role in long-term effects and what is a temporal relationship between cell proliferation and its promoter methylation and expression levels.
In conclusion, this study shows that important interindividual differences exist in TCE metabolism and molecular signaling in the liver. The biologically relevant exposure metrics collected in this study, both assessment of TCE metabolites and toxicogenomic-based evaluation, allow for better understanding of the association between key events and an individual's genotype. Although it is widely accepted that toxicogenomics has great potential to yield a new generation of exposure metrics and mode of action information (Cui and Paules, 2010
), it is also important to understand which of the gene expression changes may be indicative of the population-wide versus an individual's response. Although further research is needed to find genetic and genomic markers that could identify individuals susceptible to TCE toxicity, this study provides important clues which point to liver inflammatory responses as being a population-wide response, although the extent of TCE metabolism and peroxisomal proliferation, at least in the mouse, may be dependent on an individual's genotype. A careful evaluation of gene expression–based biomarkers of response through multistrain experiments can assist in understanding the molecular basis of interindividual differences in metabolism and toxicity.