Activation of the O-glucuronide of the carcinogen N-hydroxy- N-2-fluorenylacetamide by enzymatic deacetylation in vitro: Formation of fluorenylamine-tRNA adducts

Abstract

The O-glucuronide of N-hydroxy- N-2-fluorenylacetamide (N-GIO-FAA) was deacetylated by guinea pig liver. tRNA reacted with the product of this deacetylation, the O-glucuronide of N-2-fluorenylhydroxylamine (N-GIO-FA), to give fluorenylamine-substituted nucleic acid adducts. The quantity of adduct formation was used to determine deacetylase activity. Of the various guinea pig tissues assayed, only the liver contained enzyme activity, and this activity was confined to the microsomal fraction of the cell. Guinea pig liver microsomes were about four times as active as rabbit liver microsomes and about fourteen times as active as rat liver microsomes in promoting fluorenylamine-tRNA adduct formation. Adduct formation induced by guinea pig microsomes was about seven times greater at pH 8.5 than at pH 7.0. The aglycone of the O-glucuronide, N-hydroxy- N-2-fluorenylacetamide ( N-hydroxy-FAA) also yielded flourenylamine-substituted nucleic acid adducts following deacetylation at pH 8.5 by guinea pig liver microsomes in the presence of tRNA. In contrast to resuls obtained with N-GIO-FAA, adduct formation with N-hydroxy-FAA was not as efficient, and it was independent of pH over the range 7.0–8.5. Rabbit and rat liver microsomes were more active in promoting adduct formation of tRNA with N-hydroxy-FAA than with N-GIO-FAA. The differential inhibition of the microsome-induced formation of adducts of N-GIO-FAA and N-hydroxy-FAA with tRNA affirms that the first step in the binding mechanism of N-GIO-FAA with tRNA is enzymatic deacetylation and not hydrolysis to the aglycone N-hydroxy-FAA.