Evidence of Direct Generation of Oxygen Free Radicals from Heterocyclic Amines by NADPH/Cytochrome P‐450 Reductase in vitro

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 JV‐oxide (DMPO). The signal intensity of DMP0‐OOH(‐O₂⁻) (i.e., the DMPO spin adduct of ‐O₂⁻) was strongest for 2‐amino‐3,4‐dimethy]imidazo[4,5‐f]quinoline (MeIQ). The O₂⁻ generation with HCAs decreased in the following order: 2‐amino‐3,8‐dimethyI‐imidazo[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) ≥ other HCAs; O₂⁻ generation was lowest with 2‐amino‐3‐methyl‐9H‐pyrido[2,3‐b]indole CH₃COOH (MeAαC). 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 × 10⁻⁶ M, 1.97 × 10⁻⁵M, and 2.83 × 10⁻⁶ M, respectively. The present findings have important implications for carcinogenesis because of the known effect of oxygen radicals on cell proliferation.