1,3-Dinitrobenzene Metabolism and GSH Depletion

Abstract

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.

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“…3). Electron-deficient nitroaromatic compounds are known to be reduced in biological systems (Spain 1995) and to readily react with thiols (Ellis et al 1992) and also to covalently modify cysteine residues in proteins (Reeve et al 2002;Callan et al 2009). As the mutation of Cys387 in DprE1 leads to BTZ resistance, Trefzer et al (2010) speculated that the nitroso derivatives of BTZs form a semimercaptal with Cys387.…”

Nitroaromatic compounds: Environmental toxicity, carcinogenicity, mutagenicity, therapy and mechanism

“…In the final reduction step, the NPHA is converted to nitroaniline, by two electrons (Scheme ). Subsequent work by the same authors revealed that, nitrosonitrobenzene and NPHA nonenzymatically reacted with nonprotein sulfhydryls and mitochondrial glutathione (GSH), leading to GSH depletion and adduct formation with proteins (Reeve and Miller, ; Reeve et al ., ).…”

Proteomic identification of carbonylated proteins in 1,3-dinitrobenzene neurotoxicity

“…The primary cellular targets of 1,3-DNB are Type 1 brainstem astrocytes, with secondary involvement of oligodendrocytes and neurons (Philbert et al, 1987). 1,3-DNB induced significant perturbations in metabolic function and increased ROS production within susceptible brainstem astrocytes (Romero et al, 1995, Tjalkens et al, 2000, Reeve et al, 2002, Romero et al, 1996). In in vitro models of neurotoxicity, 1,3-DNB exposure led to significantly increased levels of ROS, loss of the mitochondrial membrane potential (ΔΨ m ), ATP depletion, and onset of the MPT which preceded cell death.…”

“…The resemblance between the lesions that result from exposure to 1,3-DNB and the regional distribution of lesions that develop in other acute energy deprivation syndromes suggests a common cellular pathogenic mechanism, at least in the early stages, that may result from oxidative stress-induced protein modification. Like other nitroaromatic chemicals, 1,3-DNB undergoes a nitroreductive metabolism that, in the presence of molecular oxygen, produces superoxide anion (Reeve et al, 2002, Tay et al, 2005). Additional intracellular sources of ROS, such as mitochondria, could conceivably increase the oxidative burden of a cell thus increasing the likelihood that protein damage may result from an oxidative mechanism.…”

“…Increased protein carbonylation is known to occur in conjunction with increased mitochondrial ROS production (Nyström 2005). The involvement of oxidative stress and mitochondrial dysfunction in mitochondrial encephalopathy is also well-established (Tjalkens et al, 2000, Reeve et al, 2002, Romero et al, 1996). …”