Role of Cytochrome P–450 2E1 in Ethanol–, Carbon Tetrachloride— and Iron–Dependent Microsomal Lipid Peroxidation

ABSTRACT This study investigated the role of cytochrome P–450 2E1 in enhanced microsomal lipid peroxidation in experimental alcoholic liver disease. We also examined the contribution of this isoform to the increased microsomal injury in alcoholic liver disease caused by carbon tetrachloride—induced or iron–induced oxidant stress. Adult male Wistar rats were intragastrically infused with a high–fat diet and ethanol or glucose for 16 wk; this resulted in hepatic lipid peroxidation and fibrogenesis in the ethanol–fed animals. Microsomes were isolated by differential centrifugation in the presence of 100 μmol/L deferoxamine, washed twice in buffer without deferoxamine and incubated in the absence or presence of ethanol (50 mmol/L), carbon tetrachloride (150 μmol/L), ferric citrate (50 μmol/L) or ferric citrate plus ethanol at 37° C for 30 min in an NADPH–generating system. The basal rate of lipid peroxidation in microsomes isolated from ethanol–fed rats was increased by 52% compared with that in microsomes from controls. Carbon tetrachloride—induced and ferric citrate—induced lipid peroxidation were also accentuated in microsomes from ethanol–fed rats, by 76% and 108%, respectively. Ethanol added in vitro significantly reduced basal (-58%) and ferric citrate—induced (-48%) lipid peroxidation in microsomes from ethanol–fed rats, whereas it had an insignificant effect on that in control microsomes. In fact, this protective effect of ethanol on microsomes from ethanol–fed rats resulted in attenuation of the difference in the level of microsomal lipid peroxidation between the two groups. P–450 2E1 IgG antibody added to the incubation completely blocked the enhanced lipid peroxidation observed in microsomes from ethanol–fed rats under the basal, ferric citrate—induced and carbon tetrachloride—induced conditions. Immunoblot analysis of microsomal proteins with P–450 2E1 IgG antibody demonstrated a large increase in the level of this cytochrome in microsomes from ethanol–fed rats. The catalytic activity of P–450 2E1, measured by antibody–inhibitable p –nitrophenol hydroxylation, was increased 20–fold over control levels by the ethanol feeding. These results demonstrate (a) induction of P–450 2E1 at an advanced stage of experimental ALD, (b) a major role of this cytochrome in the enhanced basal lipid peroxidation of microsomes obtained from livers with ALD; (c) a major contribution of P–450 2E1 to the increased vulnerability of these microsomes to ferric citrate and carbon tetrachloride—induced peroxidation and (d) the possible implication of ethanol withdrawal in further peroxidative injury caused by the absence of the protective effect of ethanol. (HEPATOLOGY 1992;16:992-996.)