Protection of DNA in HL-60 cells from damage generated by hydroxyl radicals produced by reaction of H2O2 with cell iron by zinc-metallothionein.

Scavenging of hydroxyl radicals (.OH) by the zinc form of metallothionein (ZnMT) was studied in HL-60 cells and in nuclei from such cells previously treated with ZnCl2 (ZnMT cells). Cells were grown for 48 h to label DNA for alkaline elusion experiments. During the last 24 h 0.1 mM ZnMT was included to induce ZnMT. Generation of DNA single-strand breaks (SSBs) by H2O2 in cells (5 x 10(5)/ml) treated at 4 degrees was increased by approximately 70% in Zn-treated cells by comparison with control cells. These cells had grown from an initial concentration of 5 x 10(5)/ml to a concentration at harvest of 16 x 10(5)/ml. Cells started at 6 x 10(5)/ml and growing to a final concentration of 20 x 10(5)/ml did not exhibit a similar increase in SSBs. This elevation in SSBs was traced to an increase in cell Fe content which exhibited a sharp dependence upon concentrations of cells and of ZnCl2 at the time of addition. The diffusion distance (d) from Fe to DNA of ZnMT cells treated with H2O2 was found to be 3.4 nm. This compares with a distance of 6.1 nm in control cells. SSB generation by hydroxyl radicals formed by 137Cs-gamma rays in Zn-treated cells decreased by 12%, accompanied by a decrease in d from 4.8 nm to 2.9 nm. Thus, ZnMT preferentially reacts with OH formed at some distance from DNA. In nuclei isolated from ZnMT cells started at 5 x 10(5)/ml, SSB generation by H2O2 increased by 60%. The d in these nuclei was 4.9 nm, similar to the distance in control nuclei reported previously. These data suggest that, in addition to altering the scavenging environment, treatment of cells with Zn leads to an increase in reactive Fe in cells and in isolated nuclei which can generate DNA damage through reaction with H2O2.

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.

Induction of cytochrome P-450 in a selective subpopulation of hepatocytes.

The objective of this study was to determine whether the inductive effect of phenobarbital (PB) on liver cytochrome P-450 was the result of the action of this drug on all or some hepatocytes. For this purpose, a light (cell band I) and a heavy (cell band II) subpopulation of hepatocytes were separated from rat liver in a continuous density gradient. To determine the location of these hepatocytes in tissue, [14C]bromobenzene, which binds covalently to centrilobular hepatocytes, was administered. The specific activity (14C dpm/mg protein) was greater in cells of band I than in cells of band II, suggesting a predominant contribution of centrilobular hepatocytes to the lighter cell band. Microsomes were separated from each cell subpopulation after 3 days of PB administration and cytochrome P-450 was measured. Although a fivefold increment in cytochrome P-450 content of light hepatocytes was noted, the content of heavy hepatocytes was similar to that of the respective subpopulation in controls. Concomitantly, PB administered for 3 days induced the smooth endoplasmic reticulum of centrilobular hepatocytes only, as revealed by electron microscopy of whole tissue. These results indicated that PB induces cytochrome P-450 in a selective subpopulation of hepatocytes, most likely located near the terminal hepatic venule.