EVIDENCE OF DIRECT GENERATION OF OXYGEN FREE-RADICALS FROM HETEROCYCLIC AMINES BY NADPH CYTOCHROME-P-450 REDUCTASE INVITRO

Highly purified cytochrome P-450 reductase (also called cytochrome c reductase; EC 1.6.2.4.) and NADPH were used to generate superoxide radical (O2.-) from 11 different heterocyclic amines (HCAs) as identified by electron spin resonance spectroscopy using the spin trapping method with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The signal intensity of DMPO-OOH(-O2-) (i.e., the DMPO spin adduct of O2.) was strongest for 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ). The O2.- generation with HCAs decreased in the following order: 2-amino-3,8-demethylimidazo-4,5-f]quinoxaline (MeIQx) = 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) > 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (diMeIQx) greater-than-or-equal-to other HCAs; O2.-generation was lowest with 2-amino-3-methyl-9H-pyrido[2,3-b]indole . CH3COOH (MeAaC). By using Lineweaver-Burk plots, K(m) values of cytochrome P-450 reductase for mitomycin C, IQ. and MeIQ were determined to be 1.60 X 10(-6) M, 1.97 x 10(-5) M, and 2.83 x 10(-6) M, respectively. The present findings have important implications for carcinogenesis because of the known effect of oxygen radicals on cell proliferation.