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)-O-.) by the zinc form of metallo thionein (ZnMT) was studied in HL-60 cells and in nuclei from such cel ls previously treated with ZnCl2 (ZnMT cells). Cells were frown 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-str and 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 concentratio n of 5 x 10(5)/ml to a concentration at harvest of 16 x 10(5)/ml. Cell s 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 i n 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 distanc e of 6.1 nm in control cells. SSB generation by hydroxyl radicals form ed by Cs-137-gamma rays in Zn-treated cells decreased by 12%, accompan ied by a decrease in d from 4.8 nm to 2.9 nm. Thus, ZnMT preferentiall y reacts with (OH)-O-. formed at some distance from DNA. In nuclei iso lated 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 dis tance 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. (C) 1 998 Elsevier Science Ireland Ltd. All rights reserved.