CYP2E1 catalyzes the metabolic activation of numerous protoxicants and procarcinogens and its expression is elevated rapidly in response to xenobiotics and altered pathophysiology. Both transcriptional and post- transcriptional mechanism(s) govern CYP2E1 expression. Mechanistic studies on post-transcriptional regulation of CYP2E1 expression have been limited owing to a lack of a cell culture system which expresses CYP2E1 mechanistically comparable to hepatic tissue. We have shown that primary hepatocytes plated on Vitrogen and cultured with Chee's media express CYP2E1 and that expression is elevated in response to xenobiotics or metabolic alterations comparable to hepatic tissue in vivo. Treatment of cultured rat hepatocytes with CIPRO elevates CYP2E1, 2B1/2B2 mRNA and protein, while PYR, acetone or thiazole (5-50 mM) treatment enhance expression of CYP2E1 protein and 2B1/2B2 mRNA and protein. Modification of either the 5' or 3' UTR of CYP2E1 mRNA results in dramatic alterations in CYP2E1 protein synthesis (e.g. from 150% to 10% of control), while inhibition of protein synthesis decreases mRNA degradation and turnover in a cell free translation system. The hypothesis of this research is that CIPRO-mediated increases in CYP2E1 mRNA and protein occur via mRNA stabilization and translation, whereas xenobiotic-mediated increases in CYP2E1 protein occur through enhanced translation of CYP2E1 mRNA because of unique CYP2E1 mRNA structural characteristics. Thus, the specific aims of this research are; 1. To identify the mechanisms which regulate CYP2E1, 2B1/2B2 gene expression in response to CIPRO, fatty acids and ketone bodies; 2. To examine the mechanisms of PYR-enhanced expression of CYP2E1 in primary cultured hepatocytes and in CIPRP-trated primary cultured hepatocytes; 3. To examine whether co-culture of hepatocytes with Kupffer cells alone and with nonparenchymal cells (endothelial cells, kupffer cells, and lipocytes in combination) affects responsiveness to the inducers CIPRO an PYR; 4. To examine the role of the CYP2E1 5' and 3' untranslated regions (UTRs) and poly(A) tail length in regulating CYP2E1 mRNA translation and turnover in cell free translation systems and in transiently transfected primary cultured hepatocytes. Nuclear run on assays, 3H-uridine mRNA stabilization experiments, as well as 35S- methionine labelling, immunoaffinity isolation, immunoblot and autoradiographic analysis of labelled protein, in conjunction with inhibitors of transcription or translation, will be used for mechanistic studies. Immunocytochemistry and metabolic competency will be used in studies on CYP2E1 expression in hepatocytes and in hepatocyte co-culture. CYP2E1 mRNAs with modified 5' UTRs, CYP2E1 mRNAs of defined poly(A) tail length (e.g. 25, 70, 90 bases) and CYP2E1 mRNAs with modified 3'UTRs (i.e. CYP2E1 3""""""""UTRs with specific deletions and chimeric 3' UTRs containing part CYP2B1 and part CYP2E1 sequence) will be prepared, translated in cell-free translation systems and examined for rates of translation and mRNA turnover. Western and Northern blot analysis will be used to monitor protein and mRNA levels, respectively. The results of this research will provide novel data on CYP2E1, 2B1/2B2 expression in primary cultured hepatocytes and in hepatocyte co-culture, metabolic competency of this system and seminal information on the role of translation in CYP2E1, 2B1/2B2 expression in response to xenobiotics.
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