H₂S cytotoxicity mechanism involves reactive oxygen species formation and mitochondrial depolarisation

Abstract

A number of scavengers of reactive oxygen species (ROS) were found to be protective against cell death induced by hydrogen sulfide (H₂S) in isolated hepatocytes. The H₂O₂ scavengers α-ketoglutarate and pyruvate, which also act as energy substrate metabolites, were more protective against H₂S toxicity than lactate which is only an energy substrate metabolite. All of these results suggest that H₂S toxicity is dependent on ROS production. We measured ROS formation directly in hepatocytes using the fluorogenic dichlorofluorescin method. H₂S-induced ROS formation was dose dependent and pyruvate inhibited this ROS production. Non-toxic concentrations of H₂S enhanced the cytotoxicity of H₂O₂ generated by glucose/glucose oxidase, which was inhibited by CYP450 inibitors. Furthermore, hepatocyte ROS formation induced by H₂S was decreased by CYP450 inhibitors cimetidine and benzylimidazole. These results suggest that CYP450-dependant metabolism of H₂S is responsible for inducing ROS production. H₂S-induced cytotoxicity was preceded by mitochondrial depolarization as measured by rhodamine 123 fluorescence. Mitochondrial depolarization induced by H₂S was prevented by zinc, methionine and pyruvate all of which decreased H₂S-induced cell death. Treatment of H₂S poisoning may benefit from interventions aimed at minimizing ROS-induced damage and reducing mitochondrial damage.

Introduction

Exposure to high concentrations of H₂S, even for a brief period, is extremely toxic to humans. The deleterious effects of H₂S have been recognized in a myriad of natural and industrial settings, including the oil and gas industry, where the majority of H₂S-induced poisonings occur (Arnold et al., 1985, Burnett et al., 1977). Hydrogen sulfide is recognized to be a potent inhibitor of cytochrome c oxidase, the terminal enzyme of oxidative phosphorylation and this is generally considered to be its primary mechanism of toxicity (Smith et al., 1977, Holland and Kozlowski, 1986, Dorman et al., 2002, Reiffenstein et al., 1992). The proposed mechanism for the inhibition of cytochrome c oxidase by H₂S is similar to that of hydrogen cyanide (HCN) and involves binding to the heme iron of the enzyme, with the greatest affinity being for the oxidized (Fe³⁺) state. This process results in a nearly complete inhibition of aerobic metabolism that is followed by ATP depletion and lactic acid accumulation (Beauchamp et al., 1984). However, the more limited efficacy of cyanide intoxication therapies against hydrogen sulfide intoxication suggest that cytotoxic mechanisms of hepatocytes other than inhibition of cytochrome c oxidase may contribute to hydrogen sulfide intoxication (Thompson et al., 2003, Eghbal, 1999).

In this work, we have investigated the role of increased reactive oxygen species (ROS) formation and mitochondrial toxicity in cytotoxicity induced by H₂S. The mitochondrial electron transport chain (ETC) has been recognized as one of the major cellular generators of reactive oxygen species (ROS). Some of the electrons passing through the mitochondrial ETC leak out to reduce molecular oxygen (O₂) to form superoxide (O₂⁻) which is quickly dismutated by the mitochondrial superoxide dismutase (Mn-SOD) to form hydrogen peroxide (H₂O₂) (Loschen et al., 1974, Raha and Robinson, 2000). Because hydrogen sulfide is a cytochrome oxidase inhibitor, we expected a small increase in hepatocyte ROS formation similar to what we previously found with cyanide (Siraki et al., 2002). In the following, we used the fluorogenic probe, 2′,7′-dichlorofluorescin diacetate (DCFH-DA) to assess intracellular ROS formation by freshly isolated hepatocytes (Siraki et al., 2002) when incubated with NaHS. Our results show that a metabolic activation of H₂S also contributed to ROS formation. Since ROS production is known to compromise mitochondrial function (see Gyulkhandanyan et al., 2003), we examined the effect of NaHS on mitochondrial membrane potential. We found that cell death was preceded by mitochondrial depolarization and that both were prevented by zinc (sulfide scavenger),methionine (methyl donor), pyruvate (hydrogen peroxide scavenger) or dihydroxyacetone (a glycolytic substrate). A number of ROS scavengers were effective at preventing NaHS-induced cytotoxicity, whereas interventions that increased ROS production potentiated NaHS cytotoxicity.. We conclude that a significant component of hydrogen sulfide-induced toxicity is mediated by its ability to generate ROS (see also Tapley et al., 1999).