Toxicology 203 (2004) 69–76
H
2
S cytotoxicity mechanism involves reactive oxygen species
formation and mitochondrial depolarisation
Mohammad A. Eghbal, Peter S. Pennefather, Peter J. O’Brien
∗
Department of Pharmaceutical Sciences, Faculty of Pharmacy,
University of Toronto, Toronto, Ont., Canada M5S 2S2
Received 21 October 2003; received in revised form 25 May 2004; accepted 26 May 2004
Available online 8 July 2004
Abstract
A number of scavengers of reactive oxygen species (ROS) were found to be protective against cell death induced by hydrogen
sulfide (H
2
S) in isolated hepatocytes. The H
2
O
2
scavengers -ketoglutarate and pyruvate, which also act as energy substrate
metabolites, were more protective against H
2
S toxicity than lactate which is only an energy substrate metabolite. All of these
results suggest that H
2
S toxicity is dependent on ROS production. We measured ROS formation directly in hepatocytes using
the fluorogenic dichlorofluorescin method. H
2
S-induced ROS formation was dose dependent and pyruvate inhibited this ROS
production. Non-toxic concentrations of H
2
S enhanced the cytotoxicity of H
2
O
2
generated by glucose/glucose oxidase, which
was inhibited by CYP450 inibitors. Furthermore, hepatocyte ROS formation induced by H
2
S was decreased by CYP450 inhibitors
cimetidine and benzylimidazole. These results suggest that CYP450-dependant metabolism of H
2
S is responsible for inducing
ROS production. H
2
S-induced cytotoxicity was preceded by mitochondrial depolarization as measured by rhodamine 123
fluorescence. Mitochondrial depolarization induced by H
2
S was prevented by zinc, methionine and pyruvate all of which
decreased H
2
S-induced cell death. Treatment of H
2
S poisoning may benefit from interventions aimed at minimizing ROS-induced
damage and reducing mitochondrial damage.
© 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cell death; CYP450; H
2
S; Hepatocytes; Mitochondria; ROS; ROS scavengers
1. Introduction
Exposure to high concentrations of H
2
S, even for a
brief period, is extremely toxic to humans. The delete-
rious effects of H
2
S have been recognized in a myriad
of natural and industrial settings, including the oil
∗
Corresponding author. Tel.: +1 426 978 2716;
fax: +1 416 978 8511.
E-mail address: peter.obrien@utoronto.ca (P.J. O’Brien).
and gas industry, where the majority of H
2
S-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 en-
zyme of oxidative phosphorylation and this is gener-
ally considered to be its primary mechanism of toxic-
ity (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 H
2
S is similar to that of hydrogen
0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.tox.2004.05.020