CO-METABOLISM OF ETHANOL, ETHANOL-DERIVED ACETALDEHYDE, AND 4-HYDROXYNONENAL IN ISOLATED RAT HEPATOCYTES

Our laboratory has previously reported on the ability of 4-hydroxynone nal (4-HNE), a primary product of lipid peroxidation, to inhibit aceta ldehyde metabolism in isolated mouse liver mitochondria. The purpose o f the present study is to determine whether the cc-metabolism of ethan ol and 4-HNE compromises the elimination of either substrate in isolat ed rat hepatocytes, Hepatocytes were isolated and incubated with ethan ol and 4-HNE. Ethanol elimination and acetaldehyde accumulation were m onitored by gas chromatography, whereas 4-HNE elimination and metaboli te accumulation were measured by UV detection and reversed-phase HPLC at 202 nm, In the absence of 4-HNE, hepatocytes metabolized ethanol at an initial rate of 9.4 nmol/min/million cells, Ethanol elimination wa s moderately inhibited by the presence of 4-HNE, Accumulation of ethan ol-derived acetaldehyde was not apparent in incubations with only etha nol, In contrast, in incubations containing both substrates, ethanol-d erived acetaldehyde accumulation exceeded that observed in hepatocytes exposed only to ethanol and was proportional to the 4-HNE concentrati on in the incubations. In all instances, the rate of 4-HNE elimination was not compromised by the presence of ethanol, Accordingly, ethanol metabolism did not alter the oxidative or conjugative metabolism of 4- HNE, However, the reductive metabolism of 4-HNE was affected by the pr esence of ethanol, wherein accumulation of 1,4-dihydroxy-2-nonene incr eased >2-fold of that observed in incubations with only 4-HNE, To dete rmine further if 4-HNE and ethanol are metabolized through the same me tabolic pathways, cells were preincubated with either 4-methylpyrazole or cyanamide to inhibit alcohol dehydrogenase (E.C, 1.1.1.1.) and ald ehyde dehydrogenase (E,C, 1.2.1.2.), respectively, Expectantly, 4-meth ylpyrazole blocked the formation of 1,4-dihydroxy-2-nonene, but had no effect on the rate of 4-HNE elimination. In contrast, cyanamide subst antially inhibited the formation of 4-hydroxy-2-nonenoic acid, decreas ed the rate of 1,4-dihydroxy-2-nonene formation, but did not decrease the elimination rate of 4-HNE. Overall, these results support our prev ious observation that 4-HNE inhibits acetaldehyde metabolism and estab lish that ethanol and 4-HNE are metabolized through the same alcohol d ehydrogenase- and aldehyde dehydrogenase-mediated pathways, These data continue to suggest that, as a consequence of enhanced lipid peroxida tion resulting from chronic ethanol consumption, increased 4-HNE level s could compromise cellular elimination of ethanol-derived acetaldehyd e and thus function in the potentiation of alcoholic liver fibrosis.