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

Abstract

An ESR spin-trapping technique was used to determine whether free radical metabolites are formed as a result of the reduction of 1, 3-diphenyl-1-triazene (DPT) in vivo and in vitro by components of the cytochrome P450 (P450) mixed-function oxidase system in microsomes or by gut microflora in anaerobic cecal incubations. The ESR spectrum of the DMPO-phenyl radical adduct was detected in a microsomal incubation containing DPT, DMPO, and NADPH with the following hyperfine coupling constants: a(N) = 15.95 G and = 24.37 G. The amplitude of the spectrum from the phenyl radical adduct generated in microsomal incubations of DPT with DMPO and NADPH was not attenuated by the P450 inhibitor 1-aminobenzotriazole (ABT) or by carbon monoxide, indicating that P450 is not significantly involved in phenyl radical formation. The formation of a DMPO-phenyl radical adduct was also catalyzed by recombinant human cytochrome P450 reductase. Addition of anti-rat P450 reductase antibody led to an attenuation of the signal in incubations containing either microsomes or reductase. Low concentrations of DMPO-phenyl radical adducts were detected by ESR in the toluene extract of cecal contents containing DPT and the spin trap. In the in vivo experiments with rats treated 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 were 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 potential for this chemical.

Procainamide, but notN-Acetylprocainamide, Induces Protein Free Radical Formation on Myeloperoxidase: A Potential Mechanism of Agranulocytosis

“…The coupling constants for species 1 (Fig. 3C, a N = 15.84 G and a H = 24.80 G) with its rather high β-hydrogen coupling indicated that this species was derived from a DMPO-phenyl radical adduct (36). The smaller spectrum from species 2 (Fig.…”

A review of evidence from short-term studies leading to the prediction that diazoaminobenzene (1,3-diphenyltriazine) is a carcinogen

“…injection. Bile was collected and the phenyl radical adduct was detected by ESR spectroscopy (Kadiiska et al, 2000). These results suggest that cytochrome P450 reductase as well as gut microflora can catalyse the Mathews and DeCosta, 1999) cleavage of the triazeno linkage resulting in the formation of aniline and a phenyl diazenyl radical that decomposes to form benzene and nitrogen gas (Fig.…”

“…2; Kadiiska et al, 2000). In vitro, ESR studies demonstrated the formation of a phenyl radical from the reduction of DAAB by components of the cytochrome P450 system in microsomes or by gut microflora in anaerobic cecal incubations.…”

“…benzene and aniline. A series of metabolism studies (Mathews and DeCosta, 1999), ESR experiments (Kadiiska et al, 2000) and toxicity studies (NTP, 2002;Ress et al, 2002) were performed to investigate this prediction. The collected data show that DAAB is metabolized to benzene and aniline and exhibits some toxic effects associated with benzene and aniline, supporting the prediction that DAAB is a carcinogen.…”

Deleterious Effects of Reactive Metabolites

“…Other spin trapping agents such as 2-methyl-2-nitroso-propane (MNP) to trap carbon-centered free radicals,104 5,5-dimethyl-pyrroline-N-oxide (DMPO) to trap hydroxy, carbon-centered, and phenyl radicals,105,106 and 5-diethoxyphosphoryl- 5-methyl-1-pyrroline-N-oxide (DEPMPO) to trap S-centered radicals such as glutathionyl (GS · ) and the sulfite radical anion (SO3 · ),107 have also been used. The trapped radicals can then be studied using electron spin resonance (ESR).…”