The objective of this study is to determine the role played by free radicals in the reductive metabolism of xenobiotics. The anaerobic incubation of almost all nitroaromatic xenobiotics, e.g., nitrobenzene, by the microsomal, mitochondrial, or cytosolic fractions of rat liver in the presence of either NADH or NADPH, leads to a multiple-line electron spin resonance spectrum characteristic of the nitro anion free radical. We have now demonstrated nitro anion radical formation by mitochondria using endogenous cofactors. Nitro drugs do not affect mitochondrial respiration, in particular the coupling to ADP. The sites of nitro reduction, as determined by inhibitors of the mitochondrial transport chain, appear to be NADH dehydrogenase and outer-membrane NAD(P)H cytochrome c reductase. Halogen-substituted nitro compounds are radiosensitizers and are among the most toxic nitro compounds. Loss of halide by the nitroaromatic anion forms a very reactive carbon-centered free radical, detected by spin trapping, which reacts with cellular macromolecules. The irreversible binding of these nitro compounds to DNA, protein, etc. may be inhibited by spin traps. Free radical formation by hepatic microsomal cytochrome P-450 reduction of gentian violet, SO2, CCl4 and O2 has also been investigated.
