The high-affinity binding of [H-3]Norharman ([H-3]beta-carboline) to the ethanol-inducible cytochrome P450 2E1 in rat liver

High-affinity binding sites of [H-3]norharman (synonymous: [H-3]beta-carbol ine) were characterized in microsomal membranes from rat liver utilizing va rious beta-carboline (BC) derivatives and substances binding to enzymes of the cytochrome P450 (CYP) superfamily (EC 1.14.14.1). Saturation experiment s demonstrated that [H-3]norharman binds with high-affinity (dissociation c onstant 20.86 nM; maximum binding 21.40 pmol/mg protein). Displacement expe riments with the beta-carboline derivatives 6-methyl-BC and 6-hydroxy-BC re vealed a better adaptation to the two-site model, indicating that [H-3]norh arman binds to at least two sites, with an affinity of the high-affinity si te in the low nM range. Substances binding with relative preference to isoz ymes of the CYP superfamily displaced [H-3]norharman with a lesser potency than unlabeled norharman. Imidazole, pyrazole, and 4-methylpyrazole, known as inducers of the ethanol-inducible CYP2E1, displaced [H-3]norharman with relative high potency. Furthermore, binding experiments with microsomes fro m human lymphoblast expressed rat CYP2E1 revealed a high-affinity binding s ite [inhibition constant (K-i) 13.21 nM] comparable to that of microsomal m embranes for norharman. It was displaceable by ethanol (K-i 14.25 mu M), in dicating that norharman and ethanol bind to the same binding site on CYP2E1 . In vivo experiments with rats which had ingested ethanol fur two weeks re vealed that norharman blood plasma levels were significantly elevated at th e end of this period, supporting the notion of an interaction of norharman and ethanol metabolism. Since it has been demonstrated in the Ames test tha t norharman's comutagenic action is connected with microsomal membranes (co ntaining CYP isozymes), the present findings suggest that the observed incr ease in the levels of norharman in alcoholics leads to further CYP enzyme i nduction and thereby contributes to the increased risk of carcinomas in the se patients. BIOCHEM PHARMACOL 57;5:511-520, 1999. (C) 1999 Elsevier Scienc e Inc.