Kadiiska M B, De Costa K S, Mason R P, Mathews J M
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Chem Res Toxicol. 2000 Nov;13(11):1082-6. doi: 10.1021/tx000098v.
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.
采用电子自旋共振（ESR）自旋捕获技术，以确定在体内和体外，微粒体中细胞色素P450（P450）混合功能氧化酶系统的成分或厌氧盲肠培养中的肠道微生物群将1,3 - 二苯基 - 1 - 三氮烯（DPT）还原时，是否会形成自由基代谢物。在含有DPT、DMPO和NADPH的微粒体孵育体系中检测到了DMPO - 苯基自由基加合物的ESR谱，其超精细偶合常数如下：a(N) = 15.95 G和 = 24.37 G。DPT与DMPO和NADPH在微粒体孵育中产生的苯基自由基加合物的谱线幅度，不会被P450抑制剂1 - 氨基苯并三唑（ABT）或一氧化碳减弱，这表明P450在苯基自由基形成过程中没有显著作用。重组人细胞色素P450还原酶也能催化形成DMPO - 苯基自由基加合物。添加抗大鼠P450还原酶抗体后，在含有微粒体或还原酶的孵育体系中，信号减弱。在含有DPT和自旋捕获剂的盲肠内容物甲苯提取物中，通过ESR检测到了低浓度的DMPO - 苯基自由基加合物。在用DPT和自旋捕获剂DMPO处理大鼠的体内实验中，在大鼠经DPT和DMPO处理后40 - 60分钟，胆汁中很容易检测到DMPO - 苯基自由基加合物的六线ESR信号。结果首次表明，DPT的还原会形成苯基自由基，这可能表明该化学物质具有潜在毒性。
