The Xanthine Oxidase Inhibitor Allopurinol Results In A Loss Of Cytoprotection Conferred by Pharmacologic Pre-conditioning With Hydrogen Sulfide

Abstract

Introduction: A growing body of literature has shown that pharmacologic pre-conditioning with hydrogen sulfide (HS) mitigates damage caused by ischemia-reperfusion injury (IRI). However, its protective mechanism remains unclear. As a reversible inhibitor of cytochrome c oxidase, HS reduces ATP synthesis by diminishing oxidative phosphorylation, resulting in increased xanthine oxidase (XO) activity and the generation of reactive oxygen species (ROS). It is hypothesized that this non-lethal level of free radicals is sufficient to upregulate cellular antioxidant enzymes and reduce IRI. Our laboratory has shown that a 10μM dose of the XO inhibitor allopurinol abrogates the protective effects of HS on skeletal muscle in the setting of IRI. However, other studies suggest that allopurinol protects from IRI by directly scavenging free radicals and decreasing ROS production. We hypothesize that higher concentrations of allopurinol are required to restore and complement the protective effects of pharmacologic preconditioning with HS in an in vitro model of IRI. Methods: Murine myoblasts were differentiated into myotubes and then treated with either 10μM HS, 100μM allopurinol, both 10μM HS and 100μM allopurinol, or vehicle alone (non-treated control). The experiment was replicated with a higher concentration of allopurinol (1mM). Cells were exposed to 3h of anoxia followed by 3h of normoxia (21% O2). Parallel control groups were exposed to normoxia for 6 hours. A TUNEL assay was performed to determine the apoptotic index (AI; number of TUNEL-positive cells divided by the total number of cells) for each group. Results: The AI of myotubes treated with 100μM allopurinol alone was unchanged from that of non-treated controls (9.1%±1.1% v. 10.2%±1.4%, p=0.194). For cells treated with 1mM allopurinol alone, the AI was similarly unchanged (9.5%±1.2%, p=0.317). Ten μM HS alone resulted in a significant decrease in AI when compared to non-treated controls (4.0%±0.4%, p<0.001). However, treatment with both 100μM allopurinol and HS resulted in an AI of 11.3%±0.8%, significantly greater than that of myotubes treated with HS alone (p<0.001) and unchanged from non-treated controls (p=0.974). Likewise, treatment with both 1mM allopurinol and HS resulted in an AI of 10.5%±0.9%, significantly greater than that of HS alone (p<0.001) and not significantly different from that of the untreated controls (p=0.448). Conclusions: Even in the presence of increased concentrations of allopurinol, the protective effects of HS in the setting of IRI are lost. Furthermore, in contrast to others' reports, allopurinol alone was found not to have a cytoprotective effect. These data suggest that the protective mechanism of HS is due at least in part to a xanthine oxidase-mediated pathway and the generation of low levels of ROS. These findings importantly expand our understanding of the pathophysiology of IRI and may help to delineate the clinical utility of HS.