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Role of Arylhydroxamic Acid Acyltransferase in the Mutagenicity of N-Hydroxy-N-2-fluorenylacetamide in Salmonella typhimurium¹

Divisions of Molecular Biology [C. E. W., W. T. A., C. M. K.] and Microbiology and Immunology [S. C. L., E. J. L.], National Center for Toxicological Research, Jefferson, Arkansas 72079

Partially purified fractions of rat liver arylhydroxamic acid acyltransferase were used as the external metabolic activation system for N-hydroxy-N-2-fluorenylacetamide in a Salmonella typhimurium mutagenicity assay system. Salmonella strain TA 1538, which is sensitive to arylamine derivatives, was utilized as the indicator strain. Acyltransferase, partially purified by fractional precipitation with ammonium sulfate, greatly enhanced the mutagenicity of N-hydroxy-N-2-fluorenylacetamide. Further purification by ion-exchange chromatography on diethylaminoethyl cellulose followed by gel permeation chromatography on Sephadex G-100 yielded acyltransferase preparations that retained mutagenic activation potential.

Experiments were performed for the purpose of elucidating the relationship between acyltransferase-induced nucleic acid adduct formation and mutagenic potential with the carcinogen N-hydroxy-N-2-fluorenylacetamide. Activation of the carcinogen to a reactive electrophilic species by acyltransferase-mediated N—O acyl transfer was determined by quantitation of tRNA:fluorenylamine adduct formation. N-2-Fluorenylamine, which competes for the acyl group during intramolecular N—O acyl transfer, or guanosine monophosphate, which competes with nucleic acid for the reactive electrophile generated from N-hydroxy-N-2-fluorenylacetamide by acyltransferase, decreased the acyltransferase-catalyzed nucleic acid: fluorenylamine adduct formation by 90 and 70%, respectively. Addition of N-2-fluorenylamine or guanosine monophosphate to the external media in the Salmonella system had no effect on the acyltransferase-catalyzed mutagenicity of N-hydroxy-N-2-fluorenylacetamide.

For further investigation of the relationship(s) between nucleic acid adduct formation and mutagenicity, S. typhimurium strain TA 1538 was incubated with N-2-fluorenylacetamide, mouse liver supernatant preparations obtained on centrifugation of a homogenate at 9000 x g, and varied amounts of transfer RNA in the external media. The exogenous transfer RNA, up to 10 mg/plate, had no effect on the mutagenicity of N-2-fluorenylacetamide.

These data support the conclusions that (a) S. typhimurium strain TA 1538 does not detect the reactive electrophile, N-acetoxy-N-2-fluorenylamine, formed extracellularly from the carcinogen N-hydroxy-N-2-fluorenylacetamide by N—O acyl transfer; (b) a metabolite of N-hydroxy-N-2-fluorenylacetamide that is not capable of spontaneous covalent reaction with nucleic acids is generated by acyltransferase and is responsible for induction of mutations; and (c) bacterial activation pathways may play a role in mutations produced by arylamine derivatives.

¹ Presented in part at the American Association for Cancer Research, Denver, Colo., May 1977 (35).

² Veterans Administration Hospital, Little Rock, Ark. 72206. Supported in part by Interagency Agreement 224-750002 between the Veterans Administration Hospital and the Food and Drug Administration.

³ Present address: Oak Ridge National Laboratory, Biology Division, Oak Ridge, Tenn. 37830.

⁴ Present address: National Center for Toxicological Research, Division of Carcinogenesis, Jefferson, Ark. 72079.

⁵ To whom requests for reprints should be addressed.

⁶ Appointee of the National Cancer Institute. Current address: Michigan Cancer Foundation, Detroit, Mich. 48201.

Received 7/15/77. Accepted 11/23/77.

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