MECHANISM OF HGCL2 CYTO-TOXICITY IN CULTURED MAMMALIAN-CELLS

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  • 1 January 1984
    • journal article
    • research article
    • Vol. 26  (2) , 360-368
Abstract
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 significanlty reduced the extent of DNA damage caused by HgCl2 without a similar attenuation of its uptake into cells, as did the autoclaved enzymes. Addition of radical scavengers such as glycerol or ascorbate inhibited the DNA damage but also reduced the 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. DNA damage in cells rendered sensitive to radicals was greater when these cultures were subsequently treated wth 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 .apprx. 7-35 Hg atoms per 104 base pairs (bp) at concentrations of HgCl2 which produce between 1 SSB/107 bp and 1 SSB/106 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. 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. In addition to the ability of HgCl2 to enhance oxygen radical formation in cells nad to bind with DNA, the HgCL2 uptake, binding to DNA, and strand breaks were reduced by a temperature shift from 37 to 4.degree., as compared with 37.degree., a 50% reduction in both uptake and DNA binding of Hg occurred, but the lower temperature resulted in almost complete suppression of DNA strand breaks. Evidently DNA lesions caused by HgCl2 in intact cells were temperature-dependent, possibly involving free radicals and enzymatic systems.