| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1994: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1993: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
Cytochrome P4502E1 (2E1) is a major ethanol-oxidizing enzyme of the non-alcohol dehydrogenase metabolic pathway in liver. Recently, a restriction fragment length polymorphism (RFLP) termed PstI/PsaI was found within the 5'-franking region of the human CYP2E1 structural gene. One of the CYP2E1 allelic variants characterized, namely c2, exhibited enhanced transcriptional activity upon transfection into Hep G2 cells when compared to the wild-type c1 allele. However, it is not known whether these allelic variations of the CYP2E1 gene influence the expression of 2E1 protein in human liver. Thus, we sought to describe the relationship between CYP2E1 genotype and hepatic expression of both 2E1 mRNA and 2E1 protein in man. Liver biopsy samples were obtained from non-alcoholic subjects without overt liver disease. CYP2E1 genotypes, including type A (homozygous for c1) , type B (heterozygous for c1 and c2) , and type C (homozygous for c2) , were determined by PCR amplification of the CYP2E1 5'-upstream region, followed by restriction digestion of the amplified DNA fragment with PstI and RsaI.2E1 mRNA was quantified by first subjecting total RNA extracted from the liver specimens to RT-PCR,and then hybridizing the amplified 2E1 cDNAs on slot blots with a digoxigenin-labeled 2E1 cDNA probe. 2E1 protein content in microsomes prepared from the biopsy samples was measured by Western blots using human 2E1 antibody while 2E1 enzyme activity was assesed using p-nitrophenol as a metabolic probe. Subjects with the CYP2E1 B genotype express significantly higher levels of both hepatic 2E1 mRNA and 2E1 protein. Our results indicate that the CYP2E1 PstI/RsaI polymorphism markedly influences not only the transcription of this human P450 gene but also the levels at which the corresponding enzyme is expressed in liver.
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