RT Journal Article
SR Electronic
T1 Mechanism of <em>N</em>-Hydroxy-2-Acetylaminofluorene Mutagenicity in the <em>Salmonella</em> Test System
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 337
OP 344
VO 19
IS 2
A1 PETER J. WIRTH
A1 SNORRI S. THORGEIRSSON
YR 1981
UL http://molpharm.aspetjournals.org/content/19/2/337.abstract
AB N-Hydroxy-2-acetylaminofluorene N-O acyltransferase and sulfotransferase were purified from male Sprague-Dawley rat liver, and the partially purified enzyme fractions were used to evaluate the role of each enzyme in the metabolic and mutagenic activation of N-hydroxy-2-acetylaminofluorene in the Salmonella mutagenesis system. Partially purified N-O acyltransferase was approximately 5 times as active as rat liver 105,000 x g supernatant in its ability to activate N-hydroxy-2-acetylaminofluorene to a mutagen in the Salmonella test system. The addition of either ascorbate (2 mM) or NADPH (1 mM) markedly increased the mutagenicity of N-hydroxy-2-acetylaminofluorene, whereas the addition of either paraoxon (10-6 M) or 3'-phosphoadenosine-5'-phosphosulfate had no effect on the mutagenicity of N-hydroxy-2-acetylaminofluorene mediated by N-O acyltransferase. N-O acyltransferase also activated N-hydroxy-2-acetylaminofluorene in vitro to a species which formed covalent nucleic acid-aminofluorene adducts with yeast tRNA. Adduct formation was markedly inhibited by either ascorbate or NADPH but unaffected by the addition of either paraoxon or 3'-phosphoadenosine-5'-phosphosulfate. Similarly, partially purified sulfotransferase catalyzed the covalent binding in vitro of N-hydroxy-2-acetylaminofluorene to tRNA. The addition of 3'-phosphoadenosine-5'-phosphosulfate markedly increased (60-fold) the extent of adduct formation, whereas ascorbate (2 mM) and NADPH (1 mM) markedly inhibited the covalent binding of N-hydroxy-2 acetylaminofluorene to tRNA. The addition of 3'-phosphoadenosine-5'-phosphosulfate in combination with either ascorbate or NADPH resulted in a marked increase (50- to 70-fold) in the extent of covalent binding of N-hydroxy-2-acetylaminofluorene, although the total amount bound was only 10-20% of that observed with 3'-phosphoadenosine-5'-phosphosulfate alone. However, in contrast to N-O acyltransferase, purified rat liver sulfotransferase was completely inactive in its capacity to activate N-hydroxy-2-acetylaminofluorene to a mutagen in the Salmonella test system. The addition of either ascorbate or NADPH with or without 3'-phosphoadenosine-5'-phosphosulfate had no effect (no revertants observed) on the mutagenicity of N-hydroxy-2-acetylaminofluorene mediated by sulfotransferase. These data indicate that the initial step in the mutagenic activation of N-hydroxy-2-acetylaminofluorene in the Salmonella test system by rat liver 105,000 x g supernatant is deacetylation via N-O acyltransferase rather than sulfate formation via sulfotransferase.