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.