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Nitroreductase-mediated Metabolic Activation of 2-Amino-4-(5-nitro-2-furyl)thiazole and Binding to Nucleic Acids and Proteins¹

Division of Clinical Oncology, Department of Human Oncology, Wisconsin Clinical Cancer Center, Madison, Wisconsin 53792

The reductive metabolism of 2-amino-4-(5-nitro-2-furyl)-thiazole (ANFT), a rat urinary bladder and forestomach carcinogen, was examined in vitro using rat liver tissues. ANFT was reduced by rat liver microsomes or cytosol on anaerobic incubation with reduced nicotinamide adenine dinucleotide phosphate. The intermediate(s) produced during the microsomal reduction bound to microsomes and to exogenously added proteins or yeast transfer RNA (tRNA). The binding to tRNA was a function of the concentration of tRNA, microsomes, reduced nicotinamide adenine dinucleotide phosphate, and ANFT. At optimal conditions, up to 10 nmol [2-¹⁴C]ANFT metabolite(s) were bound per mg tRNA. Polyacrylamide gel electrophoresis, chromatography, and sensitivity to RNase treatment of the adduct revealed covalent binding between [2-¹⁴C]ANFT metabolite(s) and tRNA. Similar covalent binding was observed with proteins. On microsomal reduction of [2-¹⁴C]-ANFT, about 7% of the reduced metabolite was bound to a trichloroacetic acid-insoluble fraction, most of which was associated with proteins. Addition of mercaptoethanol, dithiothreitol, reduced glutathione, iodoacetamide, N-ethylmaleimide, or p-chloromercuribenzoate to the incubation mixture significantly reduced the amount of protein binding, suggesting an involvement of sulfhydryl groups in binding. These data demonstrate that reactive intermediate(s) are generated by anaerobic incubation of ANFT with rat liver microsomes. It is suggested that the binding might involve interaction between the nitroso or N-hydroxylamino intermediate(s) and susceptible target sites in yeast tRNA or proteins.

¹ Supported in part by Grants CA 11946, CA 14520, and CA 14524 through the National Bladder Cancer Project. Preliminary reports of this work have been made (18, 20).

² To whom requests for reprints should be addressed, at Department of Human Oncology, Wisconsin Clinical Cancer Center, University of Wisconsin Center for Health Sciences, K4/548 CSC, 600 Highland Avenue, Madison, Wis. 53792.

³ Present address: Center for Human Systems, Institute for Environmental Studies, WARF Building, 610 Walnut Street, Madison, Wis. 53706.

Received 4/12/82. Accepted 8/10/82.