Characterization of DNA lesions induced by CaCrO4 in synchronous and asynchronous cultured mammalian cells

Abstract

Alkaline elution studies demonstrated CaCrO4-induced DNA single strand breaks and DNA-protein crosslinks. DNA single strand breaks increased following treatment with 10-400 microM CaCrO4 in Chinese hamster ovary cells maintained with a minimal salts/glucose medium. DNA single strand breaks were rapidly repaired when extracellular CaCrO4 was removed even following exposure levels of CaCrO4 (200 microM for 2 hr) which reduced survival to 0.6%. Under these exposure conditions the trypan blue exclusion was greater than 80%, whereas cell growth was inhibited by 46% within 24 hr. The DNA-protein crosslinks induced by 10 microM CaCrO4 were repaired in the absence of metal within 24 hr. In contrast, the amount of DNA-protein crosslinks measured 24 hr after a 2-hr treatment with 50, 100, and 200 microM CaCrO4 remained unchanged at the 50 microM level or increased at the two higher concentrations. Thus, at concentrations of 50 microM or greater there was no repair of the DNA protein crosslinks, and this may have been due to cytotoxicity of the metal. CaCrO4 at 10 or 25 microM exposure for 6 hr also induced DNA-protein crosslinking in Chinese hamster ovary cells maintained in normal tissue culture growth media. The lack of repair of DNA-protein crosslinks at the 25 microM level, which did not substantially reduce cell survival, indicated the persistence of these lesions in a noncytotoxic form. Uptake of CaCrO4 was linear with all of the concentrations tested. Analysis of the cell cycle sensitivity to CaCrO4 revealed that cells in early S phase were the most sensitive to the cytotoxic and strand breaking activity of CaCrO4. Compared with other phases of the cell cycle, there was also an elevated level of DNA-protein crosslinks when cells were treated in early S phase and incubated 24 hr without CaCrO4. These results implicate the DNA-protein crosslink as an important lesion that may be responsible for the cytotoxic and carcinogenic properties of chromate.