The model Ah-receptor agonist β-naphthoflavone inhibits aflatoxin B₁—DNA binding in vivo in rainbow trout at dietary levels that do not induce CYP1A enzymes

beta-Naphthoflavone (BNF), a well-known Ah-receptor agonist, has been believed to inhibit aflatoxin B-1 (AFB(1)) carcinogenesis in rats and rainbow trout primarily through induction of the cytochrome P450 1A (CYP1A) enzyme subfamily and consequent diversion of AFB(1) to the less carcinogenic phase I metabolite aflatoxin M(1) (AFM(1)). This study investigates the dose responsive effects of dietary BNF treatment on CYP1A induction, AFM(1) formation, AFB(1)-8,9-epoxide formation and AFB(1)-DNA binding in the trout model. Pre-feeding diet containing 10-200 p.p.m. BNF after AFB(1) i.p. injection provided dose-dependent induction of CYP1A-dependent ethoxyresorufin-O-deethylase (EROD) activity and inhibition of in vivo AFB(1)-DNA binding. However, most of the observable inhibition of DNA adduction (45% inhibition) had occurred at 10 p.p.m. BNF without detectable EROD induction; higher doses of BNF up to 200 p.p.m. induced EROD >6-fold but provided only another 15% inhibition of DNA adduction in vivo. When in vitro AFB(1)-DNA binding was assessed using liver microsomes from trout fed 10-100 p.p.m. BNF, induced microsomal EROD activity correlated moderately with reduction of in vitro AFB(1)-DNA binding activity. However, BNF treatment in a low dose range (0.2-10 p.p.m.) also strongly inhibited in vivo hepatic AFB(1)-DNA binding (69% inhibition at 5 p.p.m. BNF in this experiment), in a dose-dependent manner, in the complete absence of detectable EROD induction. The microsomes from 5 p.p.m. BNF-treated trout had no more EROD activity than control microsomes, and no less capacity for catalyzing AFB(1) - DNA binding in vitro than control microsomes. Thus, the potent inhibition of hepatic AFB(1)-DNA binding in vivo by 5 p.p.m. BNF was a result of neither CYP1A enzyme induction nor irreversibly reduced catalytic capacity for AFB(1)-8,9-epoxide formation. Direct analysis of AFB(1) metabolites formed in vitro by liver microsomes from trout fed 10, 100 and 500 p.p.m. BNF showed that low dietary BNF (10 p.p.m.) neither induced microsomal CYP1A-mediated AFM(1) formation nor altered AFB(1)-8,9-epoxide formation compared to the control. By comparison, 100 and 500 p.p.m. BNF pretreatment significantly elevated microsome-catalyzed AFM(1) formation in vitro (P < 0.001), and this increase was highly correlated with increased EROD activity (r(2) = 0.999, P < 0.001). Upon in vitro addition, BNF was found to be a potent inhibitor of microsome-mediated AFB(1)-8,9-exo-epoxide formation (IC50 = 2.6 +/- 0.1 mu M) and AFB(1)-DNA binding (inhibition constant K-i = 3.03 +/- 0.25 mu M). These findings indicate that CYP1A enzyme induction can contribute modestly to BNF protection against AFB(1) in this species both in vivo and in vitro at higher BNF doses, but does not do so at lower doses. Instead, enzyme inhibition by BNF against AFB(1) 8,9-epoxidation appears to be the predominant protective mechanism at higher BNF doses, and the sole protective mechanism at low doses, in the rainbow trout. These findings demonstrate that mechanisms of chemoprevention can change with anticarcinogen dose, and caution that even potent induction of phase I or phase II activities does not assure that pathway to be a predominant protective mechanism in vivo.