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.
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 > or = 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]quinol ine(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]i midazole,3-amino-1, 4-dimethyl-5H-pyrido[4,3-b]indole, and 3-amino-1-methyl-5H-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.
Aryal
P
P
Osaka Prefectural Institute of Public Health, Osaka, Japan.
Terashita
T
T
Guengerich
F P
FP
Shimada
T
T
Oda
Y
Y
D000123:Acetyltransferases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D002243:Carbolines / Q000633:toxicity; D002273:Carcinogens / Q000633:toxicity; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase; D029968:Escherichia coli Proteins; D005818:Genetic Engineering / Q000379:methods; D006801:Humans; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity; D009251:NADPH-Ferrihemoprotein Reductase / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D011804:Quinolines / Q000633:toxicity; D013014:SOS Response, Genetics / Q000187:drug effects; D012486:Salmonella typhimurium / Q000187:drug effects / Q000235:genetics*
D001426:Bacterial Proteins; D002243:Carbolines; D002273:Carcinogens; D029968:Escherichia coli Proteins; D009153:Mutagens; D011804:Quinolines; C019133:3-amino-1-methyl-5H-pyrido(4,3-b)indole; C078202:UmuC protein, E coli; D019388:Cytochrome P-450 CYP1A2; D009251:NADPH-Ferrihemoprotein Reductase; D000123:Acetyltransferases; C019736:N-hydroxyarylamine O-acetyltransferase; D004259:DNA-Directed DNA Polymerase; C036989:2-amino-3,4-dimethylimidazo(4,5-f)quinoline; C019132:3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole