Molecular mechanism of trichloroethylene-induced hepatotoxicity mediated by CYP2E1

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Abstract

Cytochrome P450 (CYP) 2E1 was suggested to be the major enzyme involved in trichloroethylene (TRI) metabolism and TRI-induced hepatotoxicity, although the latter molecular mechanism is not fully understood. The involvement of CYP2E1 in TRI-induced hepatotoxicity and its underlying molecular mechanism were studied by comparing hepatotoxicity in cyp2e1+/+ and cyp2e1−/− mice. The mice were exposed by inhalation to 0 (control), 1000, or 2000 ppm of TRI for 8 h a day, for 7 days, and TRI-hepatotoxicity was assessed by measuring plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and histopathology. Urinary metabolites of trichloroethanol and trichloroacetic acid (TCA) were considerably greater in cyp2e1+/+ compared to cyp2e1−/− mice, suggesting that CYP2E1 is the major P450 involved in the formation of these metabolites. Consistent with elevated plasma ALT and AST activities, cyp2e1+/+ mice in the 2000 ppm group showed histopathological inflammation. TRI significantly upregulated PPARα, which might function to inhibit NFκB p50 and p65 signalling. In addition, TRI-induced NFκB p52 mRNA, and significantly positive correlation between NFκB p52 mRNA expression and plasma ALT activity levels were observed, suggesting the involvement of p52 in liver inflammation. Taken together, the current study directly demonstrates that CYP2E1 was the major P450 involved in the first step of the TRI metabolism, and the metabolites produced may have two opposing roles: one inducing hepatotoxicity and the other protecting against the toxicity. Intermediate metabolite(s) from TRI to chloral hydrate produced by CYP2E1-mediated oxidation may be involved in the former, and TCA in the latter.

Introduction

Trichloroethylene (TRI) is a chlorinated solvent that has been used to remove grease from metal parts or lenses and as a constituent in other chemicals. Because it is extensively used in consumer products and industrial applications, more than 3.5 million individuals are estimated to be exposed to TRI annually (NTP, 1990). The occupational exposure to TRI is declining in fully industrialized countries due to technological innovation and legislation (Grote et al., 2003), but it is increasing in the emerging industrialized countries, especially in Asia (Huang et al., 2002).

Studies conducted in animals reported that exposure to TRI causes renal damage (Mensing et al., 2002) and impaired reproductive function in male mice (Forkert et al., 2002). Moreover, TRI exposure at high concentrations was found to affect the immune system (Kaneko et al., 2000) and to induce autoimmune hepatitis in autoimmune-prone MRL mouse model (Griffin et al., 2000). Human exposure to TRI leads to damaging health effects such as disturbances in the central nervous system, cardiac arrhythmias, and renal and hepatic damage (ASTDR, 1997). A retrospective study of aircraft workers has suggested a correlation between TRI and renal disease (Radican et al., 2006). Low-level exposure to TRI negatively impacted hepatic function among Japanese workers (Nagaya et al., 1993). In addition to these adverse effects, a recent clinical study revealed that TRI causes severe hypersensitive skin damage associated with impaired hepatic function (Huang et al., 2006, Kamijima et al., 2007).

TRI is metabolized through two pathways: either oxidation by cytochrome P450 (CYP) as a major one or conjugation with glutathione (GSH) as a minor one. The liver is the primary site where TRI is oxidatively metabolized to the initial metabolite TRI-epoxide, and this intermediate metabolite is then converted to chloral hydrate (CH), which is rapidly metabolized to trichloroacetic acid (TCA) and trichloroethanol (TCE) via aldehyde dehydrogenase and alcohol dehydrogenase, respectively (Lash et al., 2000). Some CYPs such as CYP1A1/2, CYP2B1/2, CYP2C11 and CYP2E1 were reported to be involved in the hepatic metabolism of TRI (Nakajima et al., 1990, Guengerich et al., 1991). Among these P450s, CYP2E1 is the most important due to its role in the conversion of TRI to CH in both humans and rodents (Nakajima et al., 1992), suggesting that CYP2E1 may also be responsible for the hepatotoxicity induced by TRI exposure.

Nakajima et al. (1988), Nakajima (1997) suggested an important role for CYP2E1 in TRI-induced hepatotoxicity by determining the relationship between induction of CYP2E1 by ethanol treatment and the elevation of TRI-induced hepatotoxicity. However, concerns remained with regard to the potential effects of the presence or absence of residual alcohol in the body. To avoid these potential effects, the role of CYP2E1 in chemical-induced hepatotoxicity can be investigated directly using cyp2e1−/− mice lacking CYP2E1 expression (Wong et al., 1998, Nakajima et al., 2005). Recently, it was reported that the hepatotoxicity following chemical exposure was contributed by the induction of nuclear factor kappa B (NFκB) (Jaeschke et al., 2002, Brasil et al., 2006). Activation of NFκB produced pro-inflammatory cytokines that are associated with liver injury (Laskin and Laskin, 2001). Nevertheless, it is not known whether NFκB activation contributes to TRI-induced hepatotoxicity.

In determining the role of CYP2E1 in TRI-induced liver damage and its molecular mechanism, TRI-induced hepatotoxicity was investigated by comparing toxicity in cyp2e1+/+ and cyp2e1−/− mice. NFκB-related signalling pathways were also determined in both mouse lines. The involvement of CYP2E1 in TRI-induced hepatotoxicity and the possible involvement of p52 could be demonstrated by resistance to TRI-induced hepatotoxicity in mice lacking CYP2E1.

Section snippets

Chemicals

TRI was a kind gift from the Japan Association for Hygiene of Chlorinated Solvent. Its purity was at least 99% as judged by gas chromatography-mass spectrometry (GC-MS). All other chemicals were purchased from commercial sources and were of the highest purity available.

Animals

This study was conducted according to the Animal Experimental Guidelines of the Nagoya University Graduate School of Medicine. A pair of parental stocks of cyp2e1−/ and cyp2e1+/+ mice was shipped from the Laboratory of

Liver and body weight

Table 1 shows the changes in the body weight, liver weight and liver/body weight ratios in cyp2e1+/+ and cyp2e1−/− mice after TRI treatment. Significant differences in liver weight and liver/body ratios were seen between the non-exposed control group of cyp2e1−/ and cyp2e1+/+ mice. Body weight was not significantly increased in either mouse line following TRI exposure. In contrast, liver weight and liver/body ratios were significantly increased in cyp2e1+/+ mice but not in cyp2e1−/− mice,

Discussion

In the present study, the mechanism of TRI-induced hepatotoxicity was investigated and the following findings were revealed: 1) CYP2E1 is a major P450 involved in the in vivo metabolism of TRI and is associated with TRI-induced hepatotoxicity; 2) p52 may be involved in the signalling pathways of TRI-induced inflammation, i.e., hepatotoxicity; and 3) the TRI metabolite, TCA, may be involved in PPARα-related anti-inflammation pathway. The induction of PPARα and its target genes was only seen in

Acknowledgments

The authors would like to express their gratitude to the Japan Association for Hygiene of Chlorinated Solvents for their kind gift of trichloroethylene. This study was supported in part by Grants-in-Aid for Scientific Research (18659169, B 15406026, 17659175) from the Japan Society for the Promotion of Science (JSPS).

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