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Use of genetically engineered Salmonella typhimurium OY1002/1A2 strain coexpressing human cytochrome P450 1A2 and NADPH‐cytochrome P450 reductase and bacterial O ‐acetyltransferase in SOS/ umu assay
Author(s) -
Aryal Pramod,
Terashita Takao,
Guengerich F. Peter,
Shimada Tsutomu,
Oda Yoshimitsu
Publication year - 2000
Publication title -
environmental and molecular mutagenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1
H-Index - 87
eISSN - 1098-2280
pISSN - 0893-6692
DOI - 10.1002/1098-2280(2000)36:2<121::aid-em6>3.0.co;2-p
Subject(s) - cyp1a2 , reductase , acetyltransferase , microbiology and biotechnology , biochemistry , chemistry , subcloning , genotoxicity , strain (injury) , 2 acetylaminofluorene , mutagen , cytochrome p450 , biology , enzyme , carcinogen , gene , acetylation , recombinant dna , microsome , toxicity , organic chemistry , anatomy
The major pathway of bioactivation of procarcinogenic heterocyclic aromatic amines (HCAs) is cytochrome P450 1A2 (CYP1A2)–catalyzed N ‐hydroxylation and subsequent esterification by O ‐acetyltransferase ( O ‐AT). We have previously reported that an umu tester strain, Salmonella typhimurium OY1001/1A2, endogenously coexpressing human CYP1A2 and NADPH‐P450 reductase (reductase), is able to detect the genotoxicity of some aromatic amines [Aryal et al., 1999, Mutat Res 442:113–120]. To further enhance the sensitivity of the strain toward HCAs, we developed S. typhimurium OY1002/1A2 by introducing pCW″/1A2:hNPR (a bicistronic construct coexpressing human P450 1A2 and the reductase) and pOA102 (constructed by subcloning the Salmonella O ‐AT gene in the pOA101‐expressing umuC ″ lacZ gene) in S. typhimurium TA1535. In addition, as an O ‐AT–deficient strain, we developed the OY1003/1A2 strain by introducing pCW″/1A2:hNPR and pOA101 into O ‐AT–deficient S. typhimurium TA1535/1,8‐DNP. Strains OY1001/1A2, OY1002/1A2, and OY1003/1A2 expressed, respectively, about 150, 120, and 140 nmol CYP1A2/l culture (in whole cells), and respective cytosolic preparations acetylated 15, 125, and ≧0 nmol isoniazid/min/mg protein as the O ‐AT activities of cytosolic preparations, respectively. We compared the induction of umuC gene expression as a measure of genotoxicity and observed that the OY1002/1A2 strain was more sensitive than OY1001/1A2 strain toward the genotoxicity of 2‐amino‐1,4‐dimethylimidazo[4,5‐ f ]quinoline (MeIQ), 2‐amino‐3‐methylimidazo[4,5‐ f ]quinoline (IQ), 2‐amino‐3,8‐dimethylimidazo[4,5‐ f ]quinoxaline (MeIQx), 2‐aminoanthracene, 2‐amino‐6‐methyldipyrido[1,2‐ a ::3,2′‐ d ]imidazole, 3‐amino‐1,4‐dimethyl‐5 H ‐pyrido[4,3‐ b ]indole, and 3‐amino‐1‐methyl‐5 H ‐pyrido[4,3‐ a ]indole. However, the genotoxicity of MeIQ, IQ, and MeIQx was not detected with the OY1003/1A2 strain. These results indicate that the newly developed strain OY1002/1A2 can be employed in detecting potential genotoxic aromatic amines requiring bioactivation by CYP1A2 and O‐ acetyltransferase. Environ. Mol. Mutagen. 36:121–126, 2000. © 2000 Wiley‐Liss, Inc.