论文部分内容阅读
AIM:Human cytochrome P-450 2E1(CYP2E1)takes part inthe biotransformation of ethanol,acetone,many small-molecule substrates and volatile anesthetics.CYP2E1 isinvolved in chemical activation of many carcinogens,procarcinogens,and toxicants.To assess the metabolic andtoxicological characteristics of CYP2E1,we cloned CYP2E1cDNA and established a HepG2 cell line stably expressingrecombinant CYP 2E1.METHODS:Human CYP2E1 cDNA was amplified withreverse transcription-polymerase chain reaction(RT-PCR)from total RNAs extracted from human liver and cloned intopGEM-T vector.The cDNA segment was identified by DNAsequencing and subcloned into a mammalian expressionvector pREPg.A transgenic cell line was established bytransfecting the recombinant plasmid of pREP9-CYP2E1to HepG2 cells.The expression of CYP2E1 mRNA wasvalidated by RT-PCR.The enzyme activity of CYP2E1catalyzing oxidation of 4-nitrophenol in postmitochondrialsupernate(S9)fraction of the cells was determined byspectrophotometry.The metabolic activation of HepG2-CYP2E1 cells was assayed by/V-nitrosodiethylamine(NDEA)cytotoxicity and micronucleus test.RESULTS:The cloned CYP2E1 cDNA segment was identicalto that reported by Umeno et al(GenBank access No.J02843).HepG2-CYP2E1 cells expressed CYP2E1 mRNA andhad 4-nitrophenol hydroxylase activity(0.162±0.025nmol·min~(-1)·mg~(-1)S9 protein),which were undetectable inparent HepG2 cells.HepG2-CYP2E1 cells increased thecytotoxicity and micronucleus rate of NDEA in comparisonwith those of HepG2 cells.CONCLUSION: The cDNA of human CYP2E1 can be successfully cloned, and a cell line, HepG2-CYP2E1, which can efficiently express mRNA and has CYP2E1 activity, is established. The cell line is useful for testing the cytotoxicity, mutagenicity and metabolism of xenobiotics, which may possibly be activated or metabolized by CYP2E1.
AIM: Human cytochrome P-450 2E1 (CYP2E1) takes part in the biotransformation of ethanol, acetone, many small-molecule substrates and volatile anesthetics. CYP2E1 is involved in chemical activation of many carcinogens, procarcinogens, and toxicants. To evaluate the metabolic andtoxicological characteristics of CYP2E1, we cloned CYP2E1 cDNA and established a HepG2 cell line stably expressing recombinant RNA2 CYP2E1.METHODS: Human CYP2E1 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR) from total RNAs extracted from human liver and cloned intopGEM-T vector.The cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREPg.A transgenic cell line was established by transfer of the recombinant plasmid of pREP9-CYP2E1to HepG2 cells. The expression of CYP2E1 mRNA was verified by RT-PCR. The enzyme activity of CYP2E1catalyzing oxidation of 4- nitrophenol in postmitochondrialsupernate (S9) fraction of the cells was determined by specific photometry. metabolites. ic activation of HepG2-CYP2E1 cells was assayed by / V-nitrosodiethylamine (NDEA) cytotoxicity and micronucleus test. RESULTS: The cloned CYP2E1 cDNA segment was identical to that reported by Umeno et al (GenBank access No. JOZ2843) .HepG2-CYP2E1 cells expressed CYP2E1 mRNA andhad 4-nitrophenol hydroxylase activity (0.162 ± 0.025nmol · min -1 (-1) S9 protein), which were undetectable inparent HepG2cells.HepG2-CYP2E1cells increased thecytotoxicity and micronucleus rate of NDEAin comparisonwith those of HepG2 cells. CONCLUSION: The cDNA of human CYP2E1 can be successfully cloned, and a cell line, HepG2-CYP2E1, which can express express mRNA and has CYP2E1 activity, is established. The cell line is useful for testing the cytotoxicity, mutagenicity and metabolism of xenobiotics, which may possibly be activated or metabolized by CYP2E1.