Formation of mutagenic urinary metabolites from 1-nitropyrene in germ-free and conventional rats: Role of the gut flora

1-Nitropyrene (NP), an environmental pollutant, a potent mutagen and an animal carcinogen, undergoes reduction, acetylation, ring-hydroxylation and conjugation in the rat in vivo to form mutagenic metabolites which are excreted in the urine. In order to investigate the role of the gut flora in the generation of these metabolites, germ-free rats of the AGUS strain, and conventional AGUS rats matched for sex and age, were injected i.p. with NP labelled with ¹⁴C. The germ-free rats excreted significantly less of the dose in urine than did the conventional rats. When urines were examined for mutagenicity with the Ames plate incorporation assay, the highest mutagenic activity was seen in the presence of S9 in 8-24 h urine from conventional rats. The conventional urines exceeded the germ-free urines by 10-fold in their content of 6-hydroxy-1-acetamidopyrene (NAAP-6-OH), previously identified as the predominant contributor to the mutagenicity of the urines of rats dosed with NP and excreted mainly as its β-glucuronide conjugate. Conventional Charles River CD rats treated orally with D-glucaro-1,4-lactone, an inhibitor of β-glucuronidase activity, excreted somewhat less NP-derived ¹⁴C in their urines over 48 h than did matched untreated rats, and their 8-24 h urines contained less than half as much of the mutagenic NAAP-6-OH as was found in the urines of the control rats. These results indicate that the gut flora are necessarily involved in the formation of NAAP-6-OH, and that both nitroreduction and the hydrolysis of glucuronides released for enterohepatic recirculation are essential in generating mutagenic metabolites from NP.