Genetic polymorphism and regulation of cytochrome P450 2E1

Abstract: Cytochrome P450 2E1 (CYP2E1) participates in the metabolism of several xenobiotics. A physiological role of CYP2E1 is its ability to convert acetone into gluconeogenetic precursors. Due to a pronounced oxidase activity CYP2E1 can generate substantial amount of oxygen free radicals and may contribute to oxidative stress. The regulation of CYP2E1 expression is relatively complex and includes control at transcriptional, mRNA and protein levels. The interindividual variability of CYP2E1 expression might be caused by genetic as well as by environmental and physiological factors. In the present investigation the regulation and polymorphism of the rat and human CYP2E1 genes has been studied. Oxidative stress caused by CYP2E1 might contribute to cell injury under various conditions including alcohol liver disease. We found that treatment of rats with chlomethiazole (CMZ), an antiischemic drug, potently inhibited of the increased expression of CYP2E1 during starvation seen both at the enzymatic, protein, mRNA and transcriptional levels. This finding suggests the possibility to use CMZ as an efficient, non-toxic inhibitor of CYP2E1 in vivo. Next, we investigated the mechanisms of induction of CYP2E1 in rat liver during starvation and/or exposure to ethanol in the Lieber DeCarli model. The results showed that starvation caused an elevation of CYP2E1 mRNA, whereas alcohol primarily caused stabilisation of the enzyme at the posttranslational level. No evidence was obtained in the rat for any increased pretranslational CYP2E1 gene expression in the presence of ethanol even at intoxication levels. Due to the interindividual variation of CYP2E1 expression, we considered of interest to investigate the presence of any functional mutations in the human CYP2E1 gene. Using genomic DNA from 200 individuals in three populations, two different very rare mutations in the CYP2E1 gene causing amino acid substitutions were identified: Arg76His (CYP2E1 '2) and Val389Ile (CYP2E1 '3). The functional significance of the two alleles was evaluated by expressing the CYP2E1 cDNAs generated by site-directed mutagenesis in COS 1 cells. The Arg76His, caused the formation of lower levels of enzyme probably due to decreased stability of the protein whereas the other mutation was of no functional significance. It was concluded that the human CYP2E1 gene is highly conserved which suggests an important physiological role for the CYP2E1 enzyme. We sequenced and characterised the 5'-flanking region of the human and rat CYP2E1 genes. The identity between the human and rat sequences (-3.8 kb to +1 kb) was generally between 35 and 60%, and the most similar regions were found in the proximal part of the sequence (90% identity). A polymorphic repeat sequence in the human gene was found between -2178 to -1945 bp. The common allele (CYP2E1 '1C) contained 6 repeats (each 42-60 bp long) and the rare allele (CYP2E1 '1D) had 8 repeats with an allele frequency of I % among Caucasian and 23 % in Chinese. The functional properties of the two alleles were examined in reporter gene systems with plasmids transfected into human B16A2 hepatoma cells, but no difference was found in the constitutive expression of the alleles. In human hepatoma cell line B16A2 IL-4 caused a 2.5 fold increase of CYP2E1 mRNA level within 4 hrs whereas no effect was seen in the rat hepatoma Fao cells. To identify the genetic response element(s) involved in IL-4 induction, several nested deletions of the 5'-flanking region of the human CYP2E1 gene were inserted in the front of a luciferase reporter gene and the plasmids transfected into B 16A2 cells. The results revealed the presence of an IL-4 responsive element at a position between -669 and -519 bp in the human CYP2E1 gene. This was further confirmed by electrophoretic mobility shift assay. It is concluded that the CYP2E1 gene is well conserved at both the regulatory and functional levels and that environmental factors are of major importance for regulation and form basis for the interindividual differences in expression.