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
Mb. Kadiiska et al., 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, CHEM RES T, 13(11), 2000, pp. 1082-1086
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.