Development of a new genotoxicity test system with Salmonella typhimurium OY1001/1A2 expressing human CYP1A2 and NADPH-P450 reductase.
In order to develop a new tester strain detecting environmental promutagens and procarcinogens, we introduced two plasmids into Salmonella typhimurium TA1535; one contains the cDNAs of human cytochrome P450 (P450 or CYP) 1A2 and NADPH-P450 reductase and the other (pOA101) a umuC"lacZ fusion gene. The newly developed tester strain, S. typhimurium OY1001/1A2, was found to express P450 at a level of 0.15 nmol/ml in whole cell culture. Membrane fractions, when isolated from this tester strain, contained 0.04 P450 nmol/mg protein and a reductase activity of 170 nmol cytochrome c reduced/min/mg protein and were active in catalyzing CYP1A2-dependent 7-ethoxyresorufin O-deethylation and metabolic activation of heterocyclic aromatic amines to DNA-damaging products in a conventional tester S. typhimurium NM2009 strain, only when NADPH was added as a reducing equivalent. In the OA1002/1A2 strain, heterocyclic aromatic amines (e.g., IQ, MeIQ, and MeIQx) were found to be activated to reactive metabolites that cause induction of umuC gene expression in a dose-dependent manner, without addition of external NADPH. These results indicate that the newly established strain can be of use to detect mutagenic and carcinogenic potencies of environmental chemicals without addition of metabolic activation system.
Aryal
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P
Osaka Prefectural Institute of Public Health, Osaka, 537-0025, Japan.
Yoshikawa
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K
Terashita
T
T
Guengerich
F P
FP
Shimada
T
T
Oda
Y
Y
D000588:Amines / Q000633:toxicity*; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006841:Hydrocarbons, Aromatic / Q000633:toxicity*; D007546:Isoquinolines / Q000633:toxicity; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011804:Quinolines / Q000633:toxicity; D011810:Quinoxalines / Q000633:toxicity; D012486:Salmonella typhimurium / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics
D000588:Amines; D006841:Hydrocarbons, Aromatic; D007546:Isoquinolines; D009153:Mutagens; D011804:Quinolines; D011810:Quinoxalines; C036990:2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline; D019388:Cytochrome P-450 CYP1A2; D009251:NADPH-Ferrihemoprotein Reductase; C036989:2-amino-3,4-dimethylimidazo(4,5-f)quinoline; C039109:isoquinoline