| The inhibition of mitochondrial respiration by chemicals, drug metabolites or hypoxia results in cytotoxicity, but the molecular cytotoxic mechanisms that cause membrane disruption, the endpoint of cytoxicity are not well understood. The cytotoxic mechanisms of mitochondrial respiratory inhibitors, mainly cyanide (CN) and nitric oxide (NO) have, therefore, been studied using in vitro experiments with isolated rat hepatocytes and in vivo experiments with mice with a goal of developing more effective antidotes. (1) The sequence of events found, when hepatocytes are treated with mitochondrial respiratory inhibitors or hypoxia:reoxygenation involves ATP depletion, an increased lactate/pyruvate ratio, iron release, and oxygen activation. Furthermore, NADH producing nutrients, which further increased lactate/pyruvate ratio, markedly enhanced the loss of cell viability induced by mitochondrial respiratory inhibitors, even though ATP levels could be restored. On the other hand, NADH utilizing substrates, which normalized the hepatocyte lactate/pyruvate ratio prevented cytotoxicity and iron release. (2) Hepatocytes were found to be more resistant to the toxic effects of mitochondrial respiratory inhibitors, particularly cyanide, at high oxygen levels. CN also inhibited the respiration of hepatocytes more effectively at lower oxygen levels. It was concluded that oxygen competes with CN for binding to cytochrome oxidase. The mitochondrial enzyme rhodanese, which is involved in CN detoxification, was less active at high oxygen levels. (3) The glycolytic nutrients dihydroxyacetone (DHA) and glyceraldehyde prevented CN-induced cytotoxicity, restored mitochondrial respiration and restored ATP levels immediately upon addition, even when added 60 min after CN. The cytoprotective mechanisms against CN principally involved trapping CN to form cyanohydrins, and supply ATP via glycolysis. These nutrients also prevented antimycin A cytotoxicity by normalizing redox state of the cell and supplying ATP through glycolysis. (4) DHA was found to effectively antagonize the lethal effects of CN in vivo in mice and to restore brain, heart and liver cytochrome oxidase activity after CN poisoning in vivo. Sodium thiosulfate greatly potentiated the antidotal effect of DHA against CN. This new antidote could prove useful against CN toxicity particularly in smoke inhalation. The antidotal mechanism of DHA is mainly by trapping CN to form a cyanohydrin, however, DHA can also supply ATP through glycolysis which may be involved in the antidotal effect of DHA against CN. (5) Sodium nitroprusside (SNP) toxicity was accompanied by peroxynitrite formation, lipid peroxidation, iron release, CN release, and partial inhibition of mitochondrial respiration. Antioxidants, the iron chelator desferoxamine, and nontoxic concentrations of CN could act as antidotes to the cytotoxicity. CN-trapping agents and thiosulfate increased iron release, lipid peroxidation, and cytotoxicity. It was concluded that NO rather than CN is the cytotoxic metabolite of SNP in hepatocytes. (6) The sequence of events when NO donors N-methyl-N{dollar}spprime{dollar}-nitro-N-nitrosoguanidine or butyl nitrite was incubated with hepatocytes were found be rapid GSH depletion, S-nitrosyl glutathione (GSNO) formation, mitochondrial respiration inhibition, ATP depletion, lipid peroxidation and finally cell death. Desferoxamine or various antioxidants acted as antidotes to the cytotoxicity. GSNO seemed to be the responsible cytotoxic metabolite as prior GSH depletion prevented the cytotoxic effects. |
