Sentjurc M, Mason R P
National Institute of Environmental Health Sciences, National Institutes of Health, Laboratory of Molecular Biophysics, Research Triangle Park, NC 27709.
Free Radic Biol Med. 1992;13(2):151-60. doi: 10.1016/0891-5849(92)90077-t.
In vivo spin trapping of radical metabolites has become a promising tool in understanding and predicting toxicities caused by different xenobiotics. However, in biological systems radical adducts can be reduced to electron paramagnetic resonance (EPR)-silent hydroxylamines. To overcome this difficulty, different procedures for reoxidation of the reduced radical adducts were systematically investigated and some metabolic inhibitors of nitroxide reduction were tested. As a test system, carbon tetrachloride (CCl4), a known hepatotoxic substance, was used. CCl4 is metabolized by liver to .CCl3 and, in the presence of the spin trap phenyl N-t-butylnitrone (PBN), forms the PBN/.CCl3 and PBN/.CO2- radical adducts. These radical adducts were measured in the bile using electron paramagnetic resonance after administration of CCl4 and PBN to the rat. We have shown that these radical adducts were reduced to the corresponding hydroxylamines in vivo, since immediately after the collection of bile only traces of the radical adducts could be detected, but after oxidation by different procedures such as bubbling with oxygen, addition of mild oxidant potassium ferricyanide or autoxidation the EPR spectra intensity increases, indicating that the hydroxylamines had been re-oxidized back to nitroxides. The collection of bile into plastic Eppendorf tubes containing the sulfhydryl reagent N-ethylmaleimide (NEM) or the enzyme ascorbate oxidase did not increase the intensity of the spectra significantly, demonstrating that neither reduction by reduced glutathione (GSH) nor ascorbic acid occurred ex vivo. However in the presence of NEM faster re-oxidation was observed. A new radical adduct that was not observed previously in any in vivo experiment and which exhibited 13C hyperfine coupling was detected when the rats were injected with 13CCl4. We have proven that this is the same adduct detected previously in vitro in microsomal incubations of CCl4, PBN, GSH, and reduced nicotinamide adenine dinucleotide phosphate (NADPH). As a general rule, we have shown that a variety of oxidation procedures should be tried to detect the different radical adducts which are otherwise not observable due to the in vivo reduction of radical adducts.
体内自由基代谢物的自旋捕获已成为理解和预测不同外源性物质所致毒性的一种有前景的工具。然而，在生物系统中，自由基加合物可被还原为电子顺磁共振（EPR）沉默的羟胺。为克服这一困难，系统研究了还原自由基加合物的不同再氧化程序，并测试了一些氮氧化物还原的代谢抑制剂。作为测试系统，使用了已知的肝毒性物质四氯化碳（CCl4）。CCl4在肝脏中代谢为·CCl3，在自旋捕获剂苯基N-叔丁基硝酮（PBN）存在下，形成PBN/·CCl3和PBN/·CO2-自由基加合物。在给大鼠注射CCl4和PBN后，使用电子顺磁共振在胆汁中测量这些自由基加合物。我们已表明，这些自由基加合物在体内被还原为相应的羟胺，因为在收集胆汁后立即只能检测到痕量的自由基加合物，但在用不同程序氧化后，如用氧气鼓泡、添加温和氧化剂铁氰化钾或自氧化，EPR光谱强度增加，表明羟胺已被重新氧化为氮氧化物。将胆汁收集到含有巯基试剂N-乙基马来酰亚胺（NEM）或酶抗坏血酸氧化酶的塑料Eppendorf管中，并未显著增加光谱强度，这表明在体外既未发生由还原型谷胱甘肽（GSH）介导的还原，也未发生由抗坏血酸介导的还原。然而，在NEM存在下，观察到更快的再氧化。当给大鼠注射13CCl4时，检测到一种以前在任何体内实验中均未观察到且表现出13C超精细偶合的新自由基加合物。我们已证明，这与先前在CCl4、PBN、GSH和还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的微粒体孵育体外实验中检测到的加合物相同。一般来说，我们已表明，应尝试多种氧化程序以检测不同的自由基加合物，否则由于自由基加合物在体内的还原，这些加合物将无法观察到。
