Modulation of aromatic amine mutagenicity in Salmonella typhimurium with rat-liver 9000 g supernatant or monolayers of rat hepatocytes as an activation system

Abstract

2-Aminofluorene (AF), 2-acetylaminofluorene (AAF) and N-hydroxy-2-acetylaminofluorene (N-OH-AAF) were studied for mutagenic activity in S. typhimurium and either liver 9000 g supernatant fractions (S9) or monolayer cultures of hepatocytes isolated from Wistar rats were used as an activation system. All 3 compounds were converted into mutagens excreted into the incubation medium by the cell-culture system, with N-OH-AAF greater than AF greater than AAF. Cultures used 24 h after plating were less efficient in promutagen conversion than were cultures used after 2 h. Phenobarbital, but not 3-methylcholanthrene, pretreatment of the rats caused similar effects on AF, AAF and N-OH-AAF mutagenicity with both S9 and hepatocyte cultures. The mutagenicities of AF and AAF were reduced by the cytochrome-P-450 inhibitors metyrapone and alpha-naphthoflavone, whereas the mutagenicity of N-OH-AAF was increased by using both inhibitors. Further, the microsomal deacetylase inhibitor paraoxon caused only a moderate reduction in N-OH-AAF mutagenicity, but a total inhibition of AAF mutagenicity. No significant effect of paraoxon on AF mutagenicity was seen. With the S9 system, no effect of ascorbate on the mutagenicity of AF, AAF or N-OH-AAF was observed. In contrast, the mutagenicity of all 3 compounds was increased by ascorbate when hepatocyte cultures were used as activation system. Incubation of hepatocyte monolayers in a sulfate-free medium did not change the mutagenicity of AF, AAF or N-OH-AAF. Galactosamine, an inhibitor of glucuronidation in cells, increased the mutagenicity of AF, AAF and N-OH-AAF with hepatocyte cultures. The addition of cofactor for glucuronidation in the S9 system, however, had no effect. A reduction in mutagenicity of AF and AAF, but not that of N-OH-AAF, was observed with the addition of glutathione (GSH) in both the S9 and the hepatocyte systems. On the other hand, no effect of cellular GSH depletion was seen on aromatic-amine mutagenicity in the hepatocyte system. The data indicate that the hepatocyte culture system offers advantages over the conventional liver-sub-fraction activation system as a model, in vivo, for the metabolism of the aromatic amine mutagens/carcinogens.