Effect of rat liver cytosolic enzymes and cofactors on mutagenicity of 1-amino-8-nitropyrene

Abstract

1-Amino-8-nitropyrene (1,8-ANP), a product of 1,8-dinitropyrene metabolism by either bacterial or mammalian enzymes, is weakly mutagenic to the 'classical nitroreductase'-deficient Salmonella tester strain TA98NR. The addition to the test system of rat liver cytosol without cofactors did not produce any effect on the 1,8-ANP mutagenic response toward TA98NR strain. Conversely, when both rat hepatic cytosol and NADPH (1 mM) were added to the mutagenicity assay, a 10-fold increase in 1,8-ANP mutagenic activity was observed. This suggests the involvement of rat hepatic cytosolic NADPH-dependent nitroreductase(s) in 1,8-ANP mutagenic activation. The addition to the mutagenesis assay of pentachlorophenol, an inhibitor of O-acetyltransferase and sulfotransferase, produced a dose-dependent decrease of 1,8-ANP mutagenic activation, whereas 2,6-dichloro-4-nitrophenol, a more specific inhibitor of sulfotransferase than O-acetyltransferase, did not affect the activation of 1,8-ANP to a mutagen at concentrations that selectively inhibit only bacterial sulfotransferase. This indicates that bacterial O-acetyltransferase but not sulfotransferase plays a role in the mutagenic activation of 1,8-ANP. Addition of acetyl co-enzyme A (AcCoA) and adenosine 3'-phosphate 5'-phosphosulfate (PAPS), cofactors for O-acetyl-transferase and sulfotransferase respectively, to the test system caused a dose-dependent inhibition of 1,8-ANP mutagenic activation by rat liver cytosol and NADPH, probably due to the formation of highly reactive O-acetoxy and N-sulfate ester derivatives of 1,8-ANP, which react with nucleophilic sites before reaching bacterial DNA. This hypothesis was confirmed by DNA covalent binding in in vitro experiments showing that both the cofactors AcCoA and PAPS enhanced the NADPH/rat liver cytosol-mediated covalent binding of 1,8-ANP to DNA from calf thymus 10- and 3-fold respectively. It seems likely that rat hepatic cytosolic nitroreductases activate 1,8-ANP to an N-hydroxyarylamine derivative which can be further metabolized to mutagenic species by either bacterial or mammalian O-acetyltransferase.