REGULATION OF ETHANOL INDUCIBLE CYTOCHROME P450 GENE

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We have previously isolated both the cDNA and genomic clones encoding the ethanol-inducible cytochrome P450IIE1 and have demonstrated six distinct types of regulation of its expression. During the past year, we have continued to investigate the effect of an experimental anti-cirrhotic agent YH439 and have further characterized a novel mechanism of P450IIE1 regulation by an exogenous compound, YH439. P450IIE1 activity and protein level were effectively suppressed by YH439 in a time- and dose-dependent manner. Nuclear run-on transcription assays confirmed that YH439 inhibited transcription of P450IIE1 gene as early as two hours post-YH439 administration. In contrast, P450IA1/2 genes were transcriptionally induced by YH439. To elucidate the mechanisms of the transcriptional regulations by YH439, the promoter regions of P4502E1 and P4501A1/2 genes are being actively studied for negative and positive elements, respectively. We have shown that YH439 activates the P450IA1/2 genes in the human hepatocarcinoma cell line, HepG2. In this cell line, YH439 activates the aromatic hydrocarbon receptor (Ah receptor), previously shown to mediate the toxic effects of dioxine as well as other environmental contaminants. We have previously published that P450IIE1 was pretranslationally suppressed during pregnancy and that it returned to normal level (within 1 day) upon parturition. We have also investigated the expression of other P450s during pregnancy and their mechanism of gene expression. Nuclear run-on transcription analyses revealed that P450IIE1 and other P450 genes are mainly regulated at the post-transcriptional level. In collaboration with Drs. Roberts and Shoaf, the levels of P450IIE1 and other P450 isoenzymes in liver, brain, and other tissues were studied during alcohol consumption and after alcohol withdrawal period. Alcohol elevated the levels of P450IIE1 5- fold in all tissues examined. After withdrawal of alcohol, P450IIE1 level rapidly (within 12 hours) returned to the normal level, supporting our earlier report on the relatively short half-lives of P450IIE1 and its protein stabilization by ethanol and acetone. The P450IIE1 stabilization by ethanol was mediated by interference of the conjugation of P450IIE1 with ubiquitin, which renders rapid degradation of P450IIE1.