Liver nuclear CYP2E1-mediated oxidative stress promotion in rats chronically drinking an alcohol-containing liquid diet

MI DÍAZ GÓMEZ; SL FANELLI; AMA DELGADO DE LAYÑO; CR DE CASTRO; F BIETTO; JA CASTRO; GD CASTRO

Washington, D.C

Conferencia; Food, Nutrition, Physical activity, and the Prevention of Cancer. A Global Perspective.Launch Conference.; 2007

World Cancer Research Foundation - American Institute for Cancer Research

In the course of previous studies we reported the presence in highly purified nuclei of an ethanol metabolizing group of enzymes (NEMS) leading to hydroxyl and 1-hydroxyethyl radicals. NEMS metabolized [14C]-ethanol using NADPH as a factor to produce reactive metabolites covalently binding to nuclear proteins and lipids. NEMS activity was significantly enhanced by repetitive alcohol drinking . In the present study we tested the possibility that, during NEMS, an oxidative stress process harming nuclear components involving CYP2E1 was sparked. Sprague Dawley male rats (125?150 g) were fed with a nutritionally adequate liquid diet containing ethanol to provide 36% of total energy (standard Lieber-De Carli rat diet) for 28 days. Controls were pair fed with an isocaloric diet without ethanol. Animals were sacrificed, livers excised and highly purified nuclear fractions prepared. Purity of nuclei was assessed on the basis of lack of activity of marker enzymes for mitochondria (ICDh), for cytosol (LDh); by phase contrast microscopy and by electron microscopy. The presence of CYP2E1 in those nuclear preparations was further suggested by the determination of chlorzoxazone hydroxylation and its induction by ethanol.We had previously reported their ability to metabolize other CYP2E1 known substrates such as CCl4; chloroform; N-nitrosodimethylamine ; p-nitrophenol; aniline and o-toluidine. Now we report that after the repetitive ethanol administration nuclear proteins are oxidized as evidenced by protein carbonyl and protein sulfhydryl determinations. However, no lipid hydroperoxides were detected by the xylenol orange method. These protein  oxidative processes might result from decreased defenses in the nuclei as suggested by the t-butylhydroperoxide-induced chemiluminiscence test. No increase in the 8-hydroxyguanine content in DNA was found, indeed, ethanol drinking decreased it. No mutagenic effects of the ethanol drinking were detectable in liver tissue using the Comet assay.