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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.
Insights
Calcium chromate (CaCrO4) causes DNA damage, including strand breaks and protein crosslinks, in cells. While strand breaks are repaired, persistent DNA-protein crosslinks, especially in early S phase cells, may drive chromate
Area of Science:
- Molecular toxicology
- Cellular and molecular biology
- Environmental health sciences
Background:
- Chromate compounds are known environmental toxicants.
- Understanding the genotoxic mechanisms of calcium chromate (CaCrO4) is crucial for assessing its health risks.
Purpose of the Study:
- To investigate the DNA damaging effects of CaCrO4 in Chinese hamster ovary cells.
- To determine the repair kinetics of CaCrO4-induced DNA lesions.
- To identify the role of DNA-protein crosslinks in CaCrO4 cytotoxicity and carcinogenicity.
Main Methods:
- Alkaline elution assays were used to detect DNA single strand breaks and DNA-protein crosslinks.
- Chinese hamster ovary cells were treated with varying concentrations of CaCrO4.
- Cell survival, growth inhibition, and cell cycle progression were monitored.
- Trypan blue exclusion was used to assess cell viability.
Main Results:
- CaCrO4 induced both DNA single strand breaks and DNA-protein crosslinks in a dose-dependent manner.
- DNA single strand breaks were rapidly repaired upon removal of CaCrO4.
- DNA-protein crosslinks showed limited repair at higher concentrations (≥50 μM) and persisted even at non-cytotoxic levels (25 μM).
- Cells in early S phase were most sensitive to CaCrO4's cytotoxic effects and exhibited increased DNA-protein crosslinking.
Conclusions:
- DNA-protein crosslinks are a persistent lesion induced by CaCrO4.
- The lack of repair and accumulation of DNA-protein crosslinks, particularly in sensitive S-phase cells, may underlie the cytotoxic and carcinogenic potential of chromate.