Reduction of 1,3-diphenyl-1-triazene by rat hepatic microsomes, by cecal microflora, and in rats generates the phenyl radical metabolite: An ESR spin-trapping investigation

An ESR spin-trapping technique was used to determine whether free radical m etabolites are formed as a result of the reduction of 1,3-diphenyl-1-triaze ne (DPT) in vivo and in vitro by components of the cytochrome P450 (P450) m ixed-function oxidase system in microsomes or by gut microflora in anaerobi c cecal incubations. The ESR spectrum of the DMPO-phenyl radical adduct was detected in a microsomal incubation containing DPT, DMPO, and NADPH with t he following hyperfine coupling constants: alpha (N) = 15.95 G and alpha (H )(beta) = 24.37 G. The amplitude of the spectrum from the phenyl radical ad duct generated in microsomal incubations of DPT with DMPO and NADPH was not attenuated by the P450 inhibitor 1-aminobenzotriazole (ABT) or by carbon m onoxide, indicating that P450 is not significantly involved in phenyl radic al formation. The formation of a DMPO-phenyl radical adduct was also cataly zed by recombinant human cytochrome P450 reductase. Addition of anti-rat P4 50 reductase antibody led to an attenuation of the signal in incubations co ntaining either microsomes or reductase. Low concentrations of DMPO-phenyl radical adducts were detected by ESR in the toluene extract of cecal conten ts containing DPT and the spin trap. In the in vivo experiments with rats t reated with DPT and the spin trap DMPO, the six-line ESR signal of the DMPO -phenyl radical adduct was readily detected in bile 40-60 min after rats we re treated with DPT and DMPO. The results show for the first time that the phenyl radical is formed by the reduction of DPT and may indicate a toxic p otential for this chemical.