Reactive oxygen species do not cause arsine-induced hemoglobin damage.

Previous work suggested that arsine- (AsH3-) induced hemoglobin (HbO2) damage may lead to hemolysis (Hatlelid et al., 1996). The purpose of the work presented here was to determine whether reactive oxygen species are formed by AsH3 in solution, in hemoglobin solutions, or in intact red blood cells, and, if so, to determine whether these species are responsible for the observed hemoglobin damage. Hydrogen peroxide (H2O2) was detected in aqueous solutions containing AsH3 and HbO2 or AsH3 alone but not in intact red blood cells or lysates. Additionally, high-activity catalase (19,200 U/ml) or glutathione peroxidase (68 U/ml) added to solutions of HbO2 and AsH3 had only a minor protective effect against AsH3-induced damage. Further, the differences between the visible spectra of AsH3-treated HbO2 and H2O2-treated HbO2 indicate that two different degradative processes occur. The presence of superoxide anion (O2-) was measured by O2(-)-dependent reduction of nitro blue tetrazolium (NBT). The results were negative for O2-. Exogenous superoxide dismutase (100 micrograms/ml) did not affect AsH3-induced HbO2 spectral changes, nor did the hydroxyl radical scavengers, mannitol, and DMSO (20 mM each). The general antioxidants ascorbate (< or = 10 mM) and glutathione (< or = 1 mM) also had no effect. These results indicate that the superoxide anion and the hydroxyl radical (OH) are not involved in the mechanism of AsH3-induced HbO2 damage. The results also indicate that although AsH3 contributes to H2O2 production in vitro, cellular defenses are adequate to detoxify the amount formed. An alternative mechanism by which an arsenic species is the hemolytic agent is proposed.