Establishment of a Salmonella tester strain highly sensitive to mutagenic heterocyclic amines.
Heterocyclic amines (HCAs) that are present in cooked foods require metabolic activation to exert their genotoxicity. They undergo activation via N-hydroxylation by cytochrome P450 1A2 (CYP1A2), followed by O-esterification by O-acetyltransferase (OAT). To develop a Salmonella tester strain that is highly sensitive to mutagenic HCAs, we introduced a coexpression plasmid (p1A2OR) carrying human CYP1A2 and NADPH-CYP reductase cDNAs and an expression plasmid (pOAT) carrying Salmonella OAT to Salmonella typhimurium TA1538 to yield a TA1538/ARO strain. The TA1538/ARO strain was proven to express the enzymes, as indicated by high activities of 7-ethoxyresorufin O-deethylase and isoniazid N-acetylase. The TA1538/ARO strain exhibited very high sensitivity to mutagenic HCAs 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and a somewhat higher sensitivity to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine compared with the parent Ames tester strain TA1538. The minimum concentrations of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, IQ, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine giving positive results were defined by evidence that the number of colonies increased in a dose-dependent manner and reached a number two times higher than that obtained by vehicle alone as a control in the TA1538/ARO strain at concentrations of 0.3, 3, 30, and 1000 pM, respectively. When the membrane and cytosol fractions prepared from TA1538/ARO were added to a mixture containing the parental TA1538, the sensitivity of TA1538 to IQ was much lower than that seen with TA1538/ARO. These results indicate that the intracellular expression of drug-metabolizing enzymes makes the established strain of Salmonella highly sensitive to mutagenic HCAs.
Suzuki
A
A
Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo-shi, Japan.
Kushida
H
H
Iwata
H
H
Watanabe
M
M
Nohmi
T
T
Fujita
K
K
Gonzalez
F J
FJ
Kamataki
T
T
D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism*; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D006571:Heterocyclic Compounds / Q000633:toxicity*; D006801:Humans; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D012486:Salmonella typhimurium / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D012680:Sensitivity and Specificity; D014162:Transfection
D017931:DNA Primers; D006571:Heterocyclic Compounds; D009153:Mutagens; D019388:Cytochrome P-450 CYP1A2; D009251:NADPH-Ferrihemoprotein Reductase; D000123:Acetyltransferases