Characterization of DNA lesions produced by HgCl 2 in cell culture systems

Abstract

HgCl 2 is extremely cytotoxic to Chinese hamster ovary (CHO) cells in culture since a 1-h exposure to a 75-μM concentration of this compound reduced cell plating efficiency to 0 and cell growth was completely inhibited at 7.5 μM. The level of HgCl 2 toxicity depended upon the culture incubation medium and has previously been shown to be inversely proportional to the extracellular concentration of metal chelating amino acids such as cysteine. Thus, HgCl 2 toxicity in a minimal salts/glucose maintenance medium was about 10-fold greater than the toxicity in McCoy's culture medium. The HgCl 2 toxicity in the latter medium was 3-fold greater than that in α-MEM which contains more of the metal chelating amino acids. When cells were exposed to HgCl 2 there was a rapid and pronounced induction of single strand breaks in the DNA at time intervals and concentrations that paralleled the cellular toxicity. The DNA damage was shown to be true single strand breaks and not alkaline sensitive sites or double strand breaks by a variety of techniques. Consistent with the toxicity of HgCl 2, the DNA damage under an equivalent exposure situation was more pronounced in the salts/glucose than in the McCoy's medium and more striking in the latter medium than in α-MEM. Most of the single strand breaks occurred within 1 h of exposure to the metal. We believe that the DNA damage caused by HgCl 2 leads to cell death because the DNA single strand breaks are not readily repaired. DNA repair activity measured by CsCl density gradient techniques was elevated above the untreated levels at HgCl 2 concentrations that produced little measurable binding of the metal to DNA or few single strand breaks assessed by the alkaline elution procedure. DNA repair activity decreased at HgCl 2 concentrations that produced measurable DNA binding and single strand breaks. These irreversible interactions of HgCl 2 with DNA may be responsible for its cytotoxic action in cells.