Metabolism of 2-acetylaminofluorene by hepatocytes isolated from rainbow trout

The metabolism of 2-acetyl-[⁹-¹⁴C]aminofluorene (AAF) by hepatocytes isolated from rainbow trout (Oncorhynchus mykiss), Shasta strain, was investigated in order to assess the competing activation and detoxification pathways which may explain the resistance of this species and strain to the initiation of carcinogenesis by this model carcinogenic aromatic amide. Freshly isolated hepatocytes (per milliliter: 1.0 mg dry wt; 1.5 (10⁶) hepatocytes) incubated with 65 μM AAF for 4 hr converted 15.4 nmol AAF to metabolites, including 7.8 nmol of water-soluble compounds. AAF-derived radioactivity extracted from the incubation mixtures, before and after hydrolysis by β-glucuronidase and arylsulfatase, was analyzed by reversed-phase HPLC. The metabolite profile following incubation of hepatocytes with 6.5 μM AAF for 4 hr included (as percentage of total metabolites): 7-OH-AAF, 5-/8-/9-OH-AAF and 2-aminofluorene (AF) (17, 2.4, and 2.7%, respectively); conjugates of these respective primary metabolites (39,9, and 4%, respectively). Glucuronides amounted to 49% of the total metabolites. N-OH-AAF and its conjugates always amounted to < 1% of total metabolites. The relative amount of (unconjugated) AF increased considerably (to 26%) following incubation of hepatocytes with 65 μM AAF, with a corresponding decrease in the total amount of glucuronides formed. Following incubation with 65 μM AAF, 1.6% of AAF metabolites was covalently bound to macromolecules, giving a ratio of covalently bound derivatives to detoxification products of 0.028. These data are consistent with the hypothesis that rainbow trout are resistant to AAF-induced hepatocarcinogenesis, in part, because trout liver efficiently detoxifies AAF and forms only relatively small amounts of active intermediates capable of binding to macromolecules, including DNA.