1,3-Dinitrobenzene metabolism and protein binding.

1,3-Dinitrobenzene is a testicular toxicant, which produces a lesion in the seminiferous tubules of the rat. In the present study, we investigated which subcellular fractions of the seminiferous tubules are capable of 1,3-dinitrobenzene metabolism and protein adduct formation. Subcellular fractions of the liver were used as positive controls and to further investigate potentially important binding proteins. Microsomes, cytosol, and mitochondria prepared from each tissue were incubated with 200 microM [(14)C]1,3-dinitrobenzene and 2 mM NADH or NADPH. Since nitroreduction is an oxygen sensitive metabolic pathway, incubations were carried out in the presence and absence of oxygen. Under anaerobic conditions, 1,3-dinitrobenzene was metabolized to nitroaniline and/or nitrophenylhydroxylamine. Metabolite formation was inhibited under aerobic conditions, suggesting the presence of an oxygen-dependent redox-cycle. For the seminiferous tubules, no metabolites were generated under aerobic conditions. In the absence of oxygen, only the mitochondria produced 1,3-dinitrobenzene metabolites. For the liver, under anaerobic conditions, all three subcellular fractions produced 1,3-dinitrobenzene metabolites with the microsomes containing the greatest activity. However, under aerobic conditions, only the microsomes generated metabolites. One-dimensional gel electrophoresis demonstrated that protein adduct formation within the liver and seminiferous tubule subcellular fractions correlated with metabolite formation. Addition of GSH to seminiferous tubule mitochondrial incubations decreased the amount of (14)C-labeled protein. Moreover, when seminiferous tubule mitochondria were incubated with 1,3-dinitrobenzene at an increased protein concentration, radioactive labeling of a 54 kDa protein became more prominent. Two-dimensional gel electrophoresis of liver mitochondrial protein incubated with [(14)C]1,3-dinitrobenzene and NADPH yielded three predominantly radiolabeled proteins of the same approximate size (54 kDa). Amino acid sequencing identified each of these proteins as rat mitochondrial aldehyde dehydrogenase.

1,3-Dinitrobenzene metabolism and GSH depletion.

Previous work demonstrated that the mitochondrial fraction of rat seminiferous tubules is capable of metabolizing 1,3-dinitrobenzene, using NADPH as a cofactor. Moreover, 1,3-dinitrobenzene treatment of rat tubules caused a decrease in mitochondrial GSH levels. In situ mitochondrial metabolism of 1,3-dinitrobenzene may have caused this depletion through the production of reactive oxygen intermediates, generating oxidative stress and/or one or more metabolites of 1,3-dinitrobenzene which reacted nonenzymatically with GSH. The goal of this study is to investigate which of these two potential mechanisms may have caused the observed GSH depletion. Liver microsomes, known to rapidly metabolize 1,3-dinitrobenzene, generated the superoxide anion radical when incubated with 1,3-dinitrobenzene and NADPH. However, with the seminiferous tubule mitochondria, no oxygen radicals were detected. Hence, the aforementioned GSH depletion is unlikely due to the production of reactive oxygen intermediates from in situ mitochondrial metabolism of 1,3-dinitrobenzene. To investigate the ability of 1,3-dinitrobenzene metabolites to deplete seminiferous tubule mitochondrial GSH, mitochondria were incubated with 1,3-dinitrobenzene and NADPH. Loss of GSH correlated with the appearance of the 1,3-dinitrobenzene metabolites, nitrophenylhydroxylamine and nitroaniline. Subsequent investigation demonstrated that the metabolites, nitrosonitrobenzene, known to react nonenzymatically with nonprotein sulfhydryls, and nitrophenylhydroxylamine both oxidized seminiferous tubule mitochondrial GSH. Further studies suggested that nitrophenylhydroxylamine could deplete GSH via a free radical mechanism. In aqueous solution, this metabolite was shown to exist in equilibrium with a radical form, thought to be the hydronitroxide radical. The addition of GSH eliminated the signal, implying that the radical reacted nonenzymatically with GSH. In conclusion, the data in this study suggest that the decrease in mitochondrial GSH observed in DNB-treated seminiferous tubules is due to the formation of NPHA and NNB and not reactive oxygen intermediates.