Cytotoxic mechanisms of hydrosulfide anion and cyanide anion in primary rat hepatocyte cultures.

Hydrogen sulfide and hydrogen cyanide are known to compromise mitochondrial respiration through inhibition of cytochrome c oxidase and this is generally considered to be their primary mechanism of toxicity. Experimental studies and the efficiency of current treatment protocols suggest that H(2)S may exert adverse physiological effects through additional mechanisms. To evaluate the role of alternative mechanisms in H(2)S toxicity, the relative contributions of electron transport inhibition, uncoupling of mitochondrial respiration, and opening of the mitochondrial permeability transition pore (MPTP) to hydrosulfide and cyanide anion cytotoxicity in primary hepatocyte cultures were examined. Supplementation of hepatocytes with the glycolytic substrate, fructose, rescued hepatocytes from cyanide anion induced toxicity, whereas fructose supplementation increased hydrosulfide anion toxicity suggesting that hydrosulfide anion may compromise glycolysis in hepatocytes. Although inhibitors of the MPTP opening were protective for hydrosulfide anion, they had no effect on cyanide anion toxicity, consistent with an involvement of the permeability transition pore in hydrosulfide anion toxicity but not cyanide anion toxicity. Exposure of isolated rat liver mitochondria to hydrosulfide did not result in large amplitude swelling suggesting that if H(2)S induces the permeability transition it does so indirectly through a mechanism requiring other cellular components. Hydrosulfide anion did not appear to be an uncoupler of mitochondrial respiration in hepatocytes based upon the inability of oligomycin and fructose to protect hepatocytes from hydrosulfide anion toxicity. These findings support mechanisms additional to inhibition of cytochrome c oxidase in hydrogen sulfide toxicity. Further investigations are required to assess the role of the permeability transition in H(2)S toxicity, determine whether similar affects occur in other cell types or in vivo and evaluate whether this may provide a basis for the design of more effective therapeutic measures for hydrogen sulfide intoxication.

In vivo and in vitro hepatotoxicity and glutathione interactions of N-methyldithiocarbamate and N,N-dimethyldithiocarbamate in the rat.

The ability of N-methyldithiocarbamate (NMDC) to generate methylisothiocyanate and HS(-) together with its greater acid stability suggest that NMDC may exert greater acute toxicity following oral exposure than its dialkyl analog,N,N-dimethyldithiocarbamate (DMDC). To assess this possibility, cell culture, perfused liver, and in vivo studies were performed to delineate differences in the hepatotoxicity and thiol interactions of NMDC and DMDC in the rat. The role of methylisothiocyanate and HS(-) in NMDC-induced hepatotoxicity was evaluated and glutathione interactions characterized through analysis of reduced glutathione (GSH), glutathione disulfide (GSSG), and S-methylthiocarbamoylglutathione (GSMITC) using HPLC and liquid chromatography tandem mass spectrometry (LC/MS/MS). Following oral administration, centrilobular hepatocyte necrosis and enzyme leakage was observed for NMDC but not for DMDC. Dose dependent decreases of intracellular GSH were produced by both dithiocarbamates in primary hepatocytes but DMDC appeared to deplete GSH through the generation of GSSG whereas NMDC produced GSMITC consistent with the generation of a methylisothiocyanate intermediate. In primary hepatocytes, both NMDC and DMDC cytotoxicity was increased by prior depletion of intracellular GSH and diminished by prior supplementation of GSH. The results obtained using perfused livers were similar for NMDC in that elevated levels of GSMITC were detected in the bile; however, DMDC produced only a modest increase of GSSG over controls that was not significantly different to that produced by NMDC. Results obtained from isolated liver mitochondria and primary hepatocytes were not consistent with NMDC producing HS(-)-mediated inhibition of mitochondrial respiration. These data support a greater potential for hepatotoxicity to result following oral exposure to NMDC relative to DMDC and that glutathione may play a role in cytoprotection for NMDC, presumably through detoxification of a methylisothiocyanate metabolite.