Mechanism of HgCl2 cytotoxicity in cultured mammalian cells

Treatment of intact Chinese hamster ovary cells with HgCl2 produced a rapid, concentration-dependent induction of DNA single-strand breaks (SSB) as revealed by alkaline elution analysis. Direct addition of HgCl2 to cell lysates did not result in DNA strand breaks. HgCl2 treatment of cells also caused a rapid leakage of superoxide radicals that were detected in their media by measurement of the reduction of exogenously added cytochrome c. There was a linear relationship between the production of radicals and the induction of DNA strand breaks, and there were also excellent temporal correlations in these parameters. Addition of oxygen radical scavengers, such as the enzymes superoxide dismutase and catalase, to the extracellular media significantly reduced the extent of DNA damage caused by HgCl2 without a similar attenuation of its uptake into cells, as did the autoclaved enzymes. Similarly, addition of radical scavengers such as glycerol or ascorbate inhibited the DNA damage but also reduced the uptake of the metal by almost the same degree. Thus, because of secondary effects on uptake of the metal, the radical scavenger experiments could not address the importance of oxygen radicals in the DNA damage caused by HgCl2. SSB were enhanced when cells were treated with HgCl2 and diethylmaleate or diethyldithiocarbamate, agents that deplete cellular reduced glutathione or inhibit the intracellular activity of superoxide dismutase, respectively. Thus, DNA damage in cells rendered sensitive to radicals was greater when these cultures were subsequently treated with HgCl2. The binding of 203HgCl2 to the DNA of intact Chinese hamster ovary cells was also studied. These studies were made possible by the relatively high stability of Hg(II) interaction with DNA and by utilizing a gentle method of DNA isolation that minimized redistribution of intracellular Hg(II) complexes after cells were lysed. The amount of Hg(II) bound to DNA varied from approximately 7 to 35 Hg atoms per 10(4) base pairs (bp) at concentrations of HgCl2 that have been previously shown to produce between 1 SSB/10(7) bp and 1 SSB/10(6) bp. The Hg(II)-DNA adducts were relatively stable complexes, since they resisted treatment with 0.1 M EDTA and 1 M NaCl and were stable to precipitation of the DNA with ethanol and trichloroacetic acid. However, the Hg(II) was released from the DNA when it was degraded enzymatically to mononucleosides, suggesting that the Hg(II)-DNA bonds formed in the cell were not truly covalent and that the strength of Hg(II) binding to DNA depended upon polynucleotide structure.(ABSTRACT TRUNCATED AT 400 WORDS)