Direct reaction of H2O2 with sulfhydryl groups in HL-60 cells: zinc-metallothionein and other sites.

The reaction of the sulfhydryl groups in metallothionein with hydrogen peroxide was examined in HL-60 cells. Partial purification of cell cytosol using Sephadex G-75 chromatography showed that zinc-metallothionein (Zn-MT) was induced by 24-h treatment with 100 microM ZnCl2, but the cellular glutathione content and glutathione peroxidase and catalase activities were unaffected. The ratio of H202 concentrations needed to reduce cell survival 50% in Zn-induced cells compared to normal cells was 1.65 to 1. According to alkaline elution experiments, the average ratio of single-strand breaks caused by H202 at 37 degrees C in Zn-induced vs normal cells was 0.5 to 1. A similar reduction in strand breakage was seen in nuclei from Zn-treated cells exposed to H202; however, at 4 degrees C protection against DNA strand breakage by Zn pretreatment was not seen. Incubation of Zn-pretreated cells with H202 at 37 degrees C but not 4 degrees C was accompanied by loss of Zn bound to MT and a reduction in the number of MT sulfhydryl groups. In the absence or presence of Zn-MT, sulfhydryl groups from glutathione and protein fractions were also reduced by exposure of cells to H202. However, thiolate groups in the MT fraction were preferentially lost compared to the other pools of sulfhydryl residues. Zn-MT also spared glutathione sulfhydryl groups in vitro from oxidation by H202. Protection against strand breakage correlated with the ability of Zn-MT to react in vitro with H202 at 37 degrees C, but not at 4 degrees C. The reaction was slow and was not inhibited by the presence of an hydroxyl radical scavenger, dimethyl sulfoxide. Similarly, in cells dimethyl sulfoxide did not prevent the loss of sulfhydryl groups from glutathione or protein. Incubation of MT or higher molecular weight fractions from cells exposed to H202 with either 2-mercaptoethanol or dithiothreitol in the presence of Cd failed to regenerate any detectable, reduced MT, suggesting that MT sulfhydryl groups were oxidized by H202 beyond the disulfide oxidation state.