Aminoanthracene is a mechanism-based inactivator of CYP1A in channel catfish hepatic tissue.
In beta NF-induced channel catfish, hepatic ethoxyresorufin-O-deethylase (EROD) activity decreased 66.5% 24 hr after injection of 2-aminoanthracene (AA, 10 mg/kg) compared with non-AA-injected animals (p < 0.05). This difference in hepatic EROD activity was also significant 48 hr after treatment (p < 0.05), but no significant difference was observed after 4 or 7 days. Immunoblot analysis of hepatic microsomal protein from fish 24 hr after treatment with AA revealed two bands cross-reacting with CYP1A-specific monoclonal antibody 1-12-3: an apparently native CYP1A protein (52 kDa) and a 30-kDa protein. Furthermore, these two proteins were preferentially bound by [3H]AA compared with other microsomal proteins. Interestingly, the 30-kDa protein was observed only in fish exposed to AA and was immunoprecipitable with 1-12-3. In a separate in vivo experiment, hepatic EROD activity decreased and the 30-kDa protein increased with increased dose of AA. The 30-kDa protein is thought to be a CYP1A degradation product. In vitro experiments helped elucidate the mechanisms of interaction between AA and CYP1A. Incubation of microsomes with AA, prior to analysis of these microsomes for EROD activity, resulted in a NADPH- and time-dependent inhibition of EROD activity. Additionally, the P450 inhibitors 1-phenylimidazole and 3,3',4,4'-tetrachlorobiphenyl were used to decrease the binding of AA to CYP1A, suggesting that the binding of AA to CYP1A requires the enzymatic activity of CYP1A. It is proposed that mechanism-based inactivation of CYP1A by AA accounts for the observed AA-dependent decrease in hepatic EROD activity in vitro and in vivo in channel catfish.
Watson, DE; Ménard, L; Stegeman, JJ; Di Giulio, RT
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