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Genotoxic Effects of Etoposide, Bleomycin, and Ethyl Methanesulfonate on Cultured CHO Cells: Analysis by GC-MS/MS and
Donald H Atha1, Erdem Coskun2,3, Onur Erdem2,4
1National Institute of Standards and Technology, Biosystems and Biomaterials Division, Materials Measurement Laboratory, Gaithersburg, MD 20899, USA.
Abstract:
To evaluate methods for analysis of genotoxic effects on mammalian cell lines, we tested the effect of three common genotoxic agents on Chinese hamster ovary (CHO) cells by single-cell gel electrophoresis (comet assay) and gas chromatography-tandem mass spectrometry (GC-MS/MS). Suspension-grown CHO cells were separately incubated with etoposide, bleomycin, and ethyl methanesulfonate and analyzed by an alkaline comet assay and GC-MS/MS. Although DNA strand breaks were detected by the comet assay after treatment with all three agents, GC-MS/MS could only detect DNA nucleobase lesions oxidatively induced by bleomycin. This demonstrates that although GC-MS/MS has limitations in detection of genotoxic effects, it can be used for selected chemical genotoxins that contribute to oxidizing processes. The comet assay, used in combination with GC-MS/MS, can be a more useful approach to screen a wide range of chemical genotoxins as well as to monitor other DNA-damaging factors.
Insights
The comet assay effectively detects DNA damage from various genotoxins in mammalian cells. Gas chromatography-tandem mass spectrometry (GC-MS/MS) shows limitations but can identify specific oxidative DNA lesions.
Area of Science:
- Toxicology
- Molecular Biology
- Genetics
Background:
- Assessing genotoxic effects is crucial for understanding chemical toxicity.
- Mammalian cell lines provide a model for evaluating DNA damage.
- The comet assay and GC-MS/MS are analytical techniques used in genotoxicity studies.
Purpose of the Study:
- To compare the efficacy of the comet assay and GC-MS/MS in detecting genotoxic effects.
- To evaluate the performance of these methods using common genotoxic agents.
- To determine the utility of combining these techniques for comprehensive genotoxicity screening.
Main Methods:
- Chinese hamster ovary (CHO) cells were exposed to etoposide, bleomycin, and ethyl methanesulfonate.
- DNA strand breaks were analyzed using the alkaline comet assay.
- DNA nucleobase lesions were analyzed using gas chromatography-tandem mass spectrometry (GC-MS/MS).
Main Results:
- The comet assay detected DNA strand breaks induced by all three genotoxic agents.
- GC-MS/MS successfully identified oxidative DNA nucleobase lesions induced by bleomycin.
- GC-MS/MS demonstrated limitations in detecting genotoxic effects from etoposide and ethyl methanesulfonate.
Conclusions:
- The comet assay is a sensitive method for detecting DNA strand breaks from diverse genotoxins.
- GC-MS/MS is valuable for identifying specific oxidative DNA lesions caused by certain chemicals.
- Combining the comet assay with GC-MS/MS offers a more comprehensive approach for genotoxicity assessment and monitoring DNA-damaging factors.
