Inhibition of cytochrome P450 2E1 expression by 2-(allylthio)Pyrazine, a Potential chemoprotective agent: hepatoprotective Effects

Abstract

Cytochrome P450 2E1 (P450 2E1) is active in both the detoxification and activation of small organic molecules. The effects of 2-(allylthio)pyrazine (2-AP) on P450 2El-catalytic activity and the expression of rat hepatic P450 2E1 were examined. 2-AP competitively inhibited 4-nitrophenol hydroxylase activity in vitro ( K i , 12 μM). 2-AP treatment of rats (200 mg/kg/day, po, 1–3 days old) resulted in 20–30% decreases in the rates of P450 2E1-specific metabolic activities. Immunoblot analysis also revealed that hepatic microsomes isolated from 2-AP-treated rats showed substantial decreases in P450 2E1 level. 2-AP-suppressed isoniazid (INH)-inducible hepatic P450 2E1 levels, as shown by both metabolic activities and immunoblot analyses. Thus, 2-AP was effective in suppressing both constitutive and inducible P450 2E1 expression. Northern blot analysis showed that 2-AP transiently suppressed the hepatic P450 2E1 mRNA level, suggesting that suppression in P450 2E1 expression by 2-AP may be mediated in part by transcriptional inactivation. Hepatoprotective effects of 2-AP against toxicants were monitored in mice. 2-AP pretreatment prior to the administration of lethal doses of acetaminophen (AAP) or INH substantially reduced toxicant-induced mortality. Whereas serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were markedly elevated after AAP administration (i.e. 9–20-fold), 2-AP pretreatment of animals before AAP administration resulted in >95% decreases in elevated serum aminotransferase activities. 2-AP was also effective against CCl 4-induced hepatotoxicity. Whereas CC1 4 treatment caused 35–70-fold increases in aminotransferase activities, treatment of mice with 2-AP (>10 mg/kg) resulted in the blocking of CCl 4-induced liver toxicity. The hepatoprotective effect of 2-AP was in part due to 2-AP-induced elevation of hepatic GSH levels. Whereas AAP or CCl 4 treatment resulted in 70–80% reduction in hepatic GSH levels, pretreatment of mice with 2-AP caused a 40–210% elevation in hepatic GSH levels, as compared with either AAP or CCl 4 alone. 2-AP pretreatment also reduced AAP- or CCl 4-induced increases in lipid peroxidation in a dose-dependent manner. The results of these metabolic activities and of immunoblot and RNA blot analyses demonstrate that 2-AP is efficacious in suppressing constitutive and inducible P450 2E1 expression and effective in protecting against toxicant-induced liver toxicity.