Human cytochrome P450 2E1 and sulfotransferase 1A1 coexpressed in Chinese hamster V79 cells enhance spontaneous mutagenesis.
Genetic engineering of target cells for investigating the genotoxicity associated with specific xenobiotic-metabolizing enzymes is useful for elucidating metabolic activation and inactivation processes. We constructed a V79-derived cell line expressing both human cytochrome P450 (CYP) 2E1 and human sulfotransferase (SULT) 1A1. We previously reported that this cell line (V79-hCYP2E1-hSULT1A1) efficiently activates various important pro-genotoxicants. Here we present data on the expression level and stability of the heterologous enzymes, measured by immunoblotting, enzyme activities, and mutagenic responses to CYP2E1- and SULT1A1-dependent promutagens. Unexpectedly, these cells demonstrated greatly elevated spontaneous gene mutation frequencies (determined at the Hprt locus), and elevated frequencies of sister chromatid exchange, as compared with control V79 cells and V79-derived lines engineered for other enzymes. Therefore, V79-hCYP2E1-hSULT1A1 cells require regular cleansing in aminopterin-containing medium when used for Hprt gene mutation assays. In a 4-week time course without such selection, V79-hCYP2E1-hSULT1A1 demonstrated a progressive increase in the spontaneous mutant frequency from 2.9 to 155 x 10(-6). This phenomenon was moderately, strongly, and completely prohibited in the presence of CYP2E1 inhibitor 1-aminobenzotriazole, SULT1A1 inhibitor pentachlorophenol and both in combination, respectively. This protection indicates that the enhanced spontaneous mutagenicity involves the activity of the expressed enzymes rather than being caused by an accidental genetic alteration that might have occurred during transfection. We postulate that human CYP2E1 and SULT1A1 activate an endogenous cellular molecule or a medium component to become mutagenic. It will be challenging to identify this compound and to see whether it is involved in spontaneous mutagenesis and carcinogenesis in vivo.
Liu
Yungang
Y
Department of Nutritional Toxicology, German Institute of Human Nutrition (DIfE) Potsdam Rehbruecke, 14558 Nuthetal, Germany. yungang1@hotmail.com
Glatt
Hansruedi
H
D000818:Animals; D015239:Arylsulfotransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics / Q000378:metabolism*; D065691:Cytochrome P-450 CYP2E1 Inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005818:Genetic Engineering; D005822:Genetic Vectors; D006801:Humans; D016296:Mutagenesis / Q000187:drug effects / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014230:Triazoles / Q000494:pharmacology
D065691:Cytochrome P-450 CYP2E1 Inhibitors; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D014230:Triazoles; C033020:1-aminobenzotriazole; D019392:Cytochrome P-450 CYP2E1; D015239:Arylsulfotransferase; C117544:SULT1A1 protein, human