Effects of inhibiting antioxidant pathways on cellular hydrogen sulfide and polysulfide metabolism.

Elaborate antioxidant pathways have evolved to minimize the threat of excessive reactive oxygen species (ROS) and to regulate ROS as signaling entities. ROS are chemically and functionally similar to reactive sulfur species (RSS) and both ROS and RSS have been shown to be metabolized by the antioxidant enzymes, superoxide dismutase and catalase. Here we use fluorophores to examine the effects of a variety of inhibitors of antioxidant pathways on metabolism of two important RSS, hydrogen sulfide (HS with AzMC) and polysulfides (HS, where n = 2-7, with SSP4) in HEK293 cells. Cells were exposed to inhibitors for up to 5 days in normoxia (21% O) and hypoxia (5% O), conditions also known to affect ROS production. Decreasing intracellular glutathione (GSH) with l-buthionine-sulfoximine (BSO) or diethyl maleate (DEM) decreased HS production for 5 days but did not affect HS. The glutathione reductase inhibitor, auranofin, initially decreased HS and HS but after two days HS increased over controls. Inhibition of peroxiredoxins with conoidin A decreased HS and increased HS, whereas the glutathione peroxidase inhibitor, tiopronin, increased HS. Aminoadipic acid, an inhibitor of cystine uptake did not affect either HS or HS. In buffer, the glutathione reductase and thioredoxin reductase inhibitor, 2-AAPA, the glutathione peroxidase mimetic, ebselen, and tiopronin variously reacted directly with AzMC and SSP4, reacted with HS and HS, or optically interfered with AzMC or SSP4 fluorescence. Collectively these results show that antioxidant inhibitors, generally known for their ability to increase cellular ROS, have various effects on cellular RSS. These findings suggest that the inhibitors may affect cellular sulfur metabolism pathways that are not related to ROS production and in some instances they may directly affect RSS or the methods used to measure them. They also illustrate the importance of carefully evaluating RSS metabolism when biologically or pharmacologically attempting to manipulate ROS.