Abstract
Liver microsomal cytochrome P4502E1-dependent p-nitrophenol (PNP) hydroxylation and expression of cytochrome P4502E1 were studied in rats subjected to gamma-hexachlorocyclohexane (HCCH) or L-3,3,5-triiodothyronine (T3) administration as a possible mechanism contributing to superoxide radical (O2.-) generation. HCCH treatment (a single dose of 40 mg/kg body wt) produced a 43% increase in the content of total cytochrome P450, whereas T3 (daily doses of 0.1 mg/kg body wt for two consecutive days) led to a 37% decrease. NADPH-dependent O2.- generation was elevated by HCCH and T3, expressed as either per mg of protein or per nmol of cytochrome P450, with a 135% enhancement in the O2.- production/superoxide dismutase (SOD) activity ratios being observed in both conditions. This was partly due to depression of SOD activity. Concomitantly, the molecular activity of NADPH-cytochrome p450 reductase was enhanced by 90 and 69% by HCCH and T3, respectively. In these conditions, microsomal PNP hydroxylation showed increases of 58 and 45% in HCCH- and T3-treated rats over control values, respectively, with a parallel 31% (HCCH) and 41% (T3) enhancement in the content of cytochrome P4502E1 assessed by western immunoblotting. We conclude that HCCH and T3 enhance the expression and activity of cytochrome P4502E1 and that of NADPH-cytochrome P450 reductase in rat liver, regardless of the changes in total cytochrome P450 content, representing major contributory mechanisms to microsomal NADPH-dependent O2.- generation.
Highlights
Reactive species derived from chemicals, oxygen, or nitrogen have been implicated as putative noxious intermediates responsible for cellular damage (Comporti, 1989; Radi et al, 1991)
Because electrophilic metabolites or radicals and excited species can readily interact with essential biomolecules, covalent binding to cellular components and/or their oxidative modification can occur, leading to structural and functional alterations when protective mechanisms are overcome or exhausted (Comporti, 1989; Kappus, 1987)
From the toxicological point of view, cytochrome P4502E1, the ethanol-inducible form (Lieber, 1997), is considered of particular interest due to (a) its capacity to oxidize several chemicals to reactive intermediates that are hepatotoxic (Guengerich et al, 1991; Koop, 1992; Lieber, 1997), and (b) its poor coupling with NADPH-cytochrome P450 reductase (Gorsky et al, 1984; Ekström and Ingelman-Sundberg, 1989). The latter feature of cytochrome P4502E1 involves a high NADPH oxidase activity, with enhanced production of O2.- and hydrogen peroxide (H2O2) during microsomal NADPH oxidation, and represents an effective catalyst for lipid peroxidation (Ekström and Ingelman-Sundberg, 1989; Dai et al, 1993). In view of these considerations, the aim of this study was to evaluate the influence of HCCH treatment on liver microsomal O2.production in relation to cytochrome P4502E1-dependent activity and content of this specific isoenzyme
Summary
Reactive species derived from chemicals, oxygen, or nitrogen have been implicated as putative noxious intermediates responsible for cellular damage (Comporti, 1989; Radi et al, 1991). Previous work by our group has shown that acute γ-hexachlorocyclohexane (HCCH or lindane) treatment enhances the oxidative stress status of the liver, an effect that is both dose- (Junqueira et al, 1986) and time-dependent (Junqueira et al, 1988; Barros et al, 1988) and that seems to be primarily related to its biotransformation by the microsomal P450 system. Induction of cytochrome P450 at later times after HCCH intoxication (24 h) further enhances the oxidative stress status of the liver due to acceleration of xenobiotic biotransformation (Videla et al, 1990) and higher microsomal NADPH-dependent superoxide radical (O .-)
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