INHIBITION OF IN-VITRO AFLATOXIN B-1-DNA BINDING IN RAINBOW-TROUT BY CYP1A INHIBITORS - ALPHA-NAPHTHOFLAVONE, BETA-NAPHTHOFLAVONE AND TROUTCYP1A1 PEPTIDE ANTIBODY

Rainbow trout cytochrome P450 (CYP)1A detoxifies aflatoxin B-1 (AFB(1) ) to aflatoxin M(1) (AFM(1)), whereas CYP2K1 activates AFB(1) to AFB(1 )-8,9-epoxide. We report that alpha-naphthoflavone (ANF) and beta-naph thoflavone (BNF) both strongly inhibit CYP1A-mediated ethoxyresorufin O-deethylase (EROD) activity (K-i = 9.1 +/- 0.8 and 7.6 +/- 1.1 nM, re spectively). These inhibitors (selective for mammalian CYP1A at low co ncentrations), as well as rabbit polyclonal antibody to a trout CYP1A1 peptide (residues 277-294), also strongly inhibited trout microsome-c atalyzed AFB(1)-DNA binding and lauric acid (omega-1) hydroxylation in vitro, reactions previously established to be CYP2K1-dependent. ANF a t 0.5, 5, 50 and 500 mu M inhibited liver microsome-catalyzed AFB(1)-D NA binding by 22, 58, 84 and 91%, respectively, whereas BNF at the sam e concentrations inhibited 22, 74, 78 and 81%, respectively. The CYP1A 1 peptide and CYP2K1 polyclonal antibodies (10 mg IgG/mg microsomal pr otein) inhibited AFB(1)-DNA binding by 84 and 66%, respectively, compa red with pre-immune IgG. Lauric acid (omega-1) hydroxylation was inhib ited 61% by 5 mu M ANF, 69% by 5 mu M BNF and 100% by either antibody at 12 mg IgG/mg microsomal protein. These results demonstrate that mam malian CYP1A inhibitors also inhibit trout microsomal AFB(1)-DNA bindi ng and lauric acid (omega-1) hydroxylation, catalyzed primarily by CYP 2K1. In the absence of evidence that trout CYP1A can catalyze AFB(1)-D NA binding, the results suggest configuration similarities at, or near , the active sites for these two fish enzymes that result in antibody crossreaction and loss of the inhibitor specificity observed with mamm alian CYP1A.