REGULATION OF ETHANOL INDUCIBLE CYTOCHROME P450 2E1 (CYP2E1)

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We have demonstrated multiple regulatory mechanisms for CYP2E1: induction via transcription, mRNA stabilization, activation of mRNA translation and protein stabilization, suppression via transcription, mRNA degradations and protein degradation. We have recently reported transcriptional suppression of CYP2E1 gene by an exogenous compound, YH439. The potential beneficial effect of this synthetic inhibitor of CYP2E1 were studied in an in vivo model of acute hepatitis by treatment with carbon tetrachloride. In vivo hepatobiliary imaging analyses revealed that YH439 efficiently protects liver injury from carbon tetrachloride. These results were confirmed by the corresponding changes in the levels of serum transaminases and by histological evaluations. The protective effect of YH439 appears to be the result of effective suppression of CYP2E1, which catalyzes the metabolism of carbon tetrachloride, leading to the initiation of free-radical mediated tissue damage. The level of acetaldehyde adduct was also studied. Our immunoblot data, using polyclonal antibody against acetaldehyde-protein adduct, showed that an immunoreactive band (apparent Mr 37 kDa on SDS-polyacrylamide gel) was not detected in animals treated with control diet while this band was clearly detected in rats pair-fed with alcohol liquid diet. However, the immunoreactive band was virtually absent upon treatment with YH439. Immunocytological analyses revealed that immunoreactive antigen is primarily found in the pericentral region where CYP2E1 is mainly localized. These data suggest that the acetaldehyde-protein adduct (Mr 37 kDa) was produced in a CYP2E1-dependent manner. Since CYP2E1 -mediated metabolism is known to cause oxidative stress resulting in DNA and protein damage, the levels of DNA-adducts in rat tissues from different treatments, including ethanol in the absence and presence of YH439, are being measured by HPLC. We have also searched for potential mutations in the human CYP2E1 gene by analyzing DNA samples obtained from individuals with low and high levels of CYP2E1 activity. Our data suggest that the different levels of CYP2E1 activity do not correspond with CYP2E1 gene polymorphism. In addition, over-production of CYP2E1 in baculovirus expression system is being performed to study the biological role of each of the four exposed lysine residues, as viewed in a modeled CYP2E1 protein structure. These lysine residues are good candidates for ubiquitin conjugation, leading to the proteasomal degradation of CYP2E1. Each of these lysine residues is, therefore, being mutated with alanine to study the rates of ubiquitin conjugation and subsequent CYP2E1 degradation as well as to elucidate the exact mechanism of CYP2E1 stabilization by ethanol.