INHIBITION OF IN-VITRO AFLATOXIN B-1-DNA BINDING IN RAINBOW-TROUT BY CYP1A INHIBITORS - ALPHA-NAPHTHOFLAVONE, BETA-NAPHTHOFLAVONE AND TROUT CYP1A1 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-naphthoflavone (BNF) both strongly inhibit CYP1A-mediated ethoxyresorufin O-deethylase (EROD) activity (K-i = 9.1 +/- 0.8 and 7.6 +/- 1.1 nM, respectively). These inhibitors (selective for mammalian CYP1A at low concentrations), as well as rabbit polyclonal antibody to a trout CYP1A1 peptide (residues 277-294), also strongly inhibited trout microsome-catalyzed AFB(1)-DNA binding and lauric acid (omega-1) hydroxylation in vitro, reactions previously established to be CYP2K1-dependent. ANF at 0.5, 5, 50 and 500 mu M inhibited liver microsome-catalyzed AFB(1)-DNA binding by 22, 58, 84 and 91%, respectively, whereas BNF at the same concentrations inhibited 22, 74, 78 and 81%, respectively. The CYP1A1 peptide and CYP2K1 polyclonal antibodies (10 mg IgG/mg microsomal protein) inhibited AFB(1)-DNA binding by 84 and 66%, respectively, compared with pre-immune IgG. Lauric acid (omega-1) hydroxylation was inhibited 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 mammalian CYP1A inhibitors also inhibit trout microsomal AFB(1)-DNA binding and lauric acid (omega-1) hydroxylation, catalyzed primarily by CYP2K1. In the absence of evidence that trout CYP1A can catalyze AFB(1)-DNA 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 mammalian CYP1A.