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Establishment of a Salmonella Tester Strain Highly Sensitive to Mutagenic Heterocyclic Amines¹

Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo-shi, 060, Japan [A. S., H. K., H. I., K-i. F., T. K.]; Division of Biochemicals, National Cancer Center Reserch Institute, Tokyo, 104, Japan [M. W.]; Division of Genetics and Mutagenesis, National Institute of Health Sciences, Tokyo, 158, Japan [T. N.]; and Laboratory of Metabolism, NIH, Bethesda, Maryland 20892 [F. J. G.]

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 NADPHCYP 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 tomutagenic 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.

¹ Supported by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Drug ADR Relief, Research and Development Promotion and Product Review, Japan. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan.

² To whom requests for reprints should be addressed, at Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Hokkaido University, N12 W6 Kita-ku, Sapporo-shi, 060-0812, Japan. Phone/Fax: 81-11-706-4978; E-mail: kamataki@pharm.hokudai.ac.jp.

Received 10/ 6/97. Accepted 2/27/98.

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