| pubmed-article:7083159 | pubmed:abstractText | The metabolic N-oxidation of the carcinogen 2-aminofluorene was examined in vitro using fortified hepatic microsomes from a variety of species. Rat, dog, human, and pig liver microsomes catalyzed the formation of N-hydroxy-2-aminofluorene (N-OH-AF) from AF at rates of 1.6, 1.0, 1.2, and 3.5 nmol/min/mg protein, respectively. The involvement of both cytochrome P-450 and the flavin-containing monooxygenase was demonstrated with hepatic microsomes and with purified enzymes by using specific enzyme inhibitors. 2-[(2,4-Dichloro-6-phenyl)phenoxy]ethylamine, a potent cytochrome P-450 inhibitor, decreased microsomal N-OH-AF formation by 96, 83, 70, and 46% in the rat, dog, human, and pig, respectively; and further addition of methimazole, a high-affinity flavin-containing monooxygenase substrate, abolished the residual N-hydroxylating activity. Using the purified porcine flavin-containing monooxygenase, metabolic formation of N-OH-AF occurred at a rate of 4.9 nmol/min/nmol flavin adenine nucleotide and was insensitive to 2-[(2,4-dichloro-6-phenyl)phenoxy]ethylamine inhibitor. In addition, purified rat liver cytochrome P-450 (isolated from 5,6-naphthoflavone-induced animals) N-hydroxylated AF (1.1 nmol/min/nmol P-450) and was completely inhibited by 2-[(2,4-dichloro-6-phenyl)-phenoxy]ethylamine, but the reaction was insensitive for methimazole. To determine whether or not the metabolic formation of N-OH-AF could lead directly to covalently bound adduct(s) with DNA under these incubation conditions (30 min, pH 7.5), the binding of synthetic and metabolically formed [3H]-N-OH-AF to added calf thymus DNA and to DNA in isolated rat liver nuclei was investigated. In all cases, the amount of DNA-bound carcinogen accounted for 0.08 to 0.15% of the N-OH-AF present in the incubation mixtures. These data, when compared to the levels of AF bound to hepatic nuclear DNA reported in vivo, suggest that the nonenzymatic reaction of N-OH-AF with nuclear DNA may be sufficient to account for a substantial portion of the observed in vivo binding of this carcinogen. | lld:pubmed |
