Dataset Information

Genotoxic Effects of Etoposide, Bleomycin, and Ethyl Methanesulfonate on Cultured CHO Cells: Analysis by GC-MS/MS and Comet Assay.

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.

SUBMITTER: Atha DH

PROVIDER: S-EPMC7414336 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Genotoxic Effects of Etoposide, Bleomycin, and Ethyl Methanesulfonate on Cultured CHO Cells: Analysis by GC-MS/MS and Comet Assay.

Atha Donald H DH Coskun Erdem E Erdem Onur O Tona Alessandro A Reipa Vytas V Nelson Bryant C BC

Journal of nucleic acids 20200730

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 ...[more]

PMID: 32802493

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Project description:Highlights • Malondialdehyde (MDA) is an important biomarker of oxidative stress (lipid peroxidation)• MDA and [1,3-dideutero]MDA (d2-MDA) for use as internal standard react with pentafluorobenzyl bromide (PFB-Br) in aqueous acetone to form di-PFB derivatives.• GC–MS/MS allows for highly accurate measurement of MDA in human plasma, serum and urine.• Many pre-analytical factors including hemolysis may compromise measurement of biological MDA.• Sample storage and study design of long-term clinical studies are important factors to be considered. Malondialdehyde (MDA; 1,3-propanedial, OHC-CH2-CHO) is one of the most frequently measured biomarkers of oxidative stress in plasma and serum. L-Arginine (Arg) is the substrate of nitric oxide synthases (NOS), which convert L-arginine to nitric oxide (NO) and L-citrulline. The Arg/NO pathway comprises several members, including the endogenous NOS-activity inhibitor asymmetric dimethylarginine (ADMA) and its major metabolite dimethyl amine (DMA), and nitrite and nitrate, the major NO metabolites. Reliable measurement of MDA and members of the Arg/NO pathway in plasma, serum, urine and in other biological samples, such as saliva and cerebrospinal fluid, is highly challenging both for analytical and pre-analytical reasons. In our group, we use validated gas chromatography-mass spectrometry (GC–MS) and gas chromatography-tandem mass spectrometry (GC–MS/MS) methods for the quantitative determination in clinical studies of MDA as a biomarker of oxidative stress, and various Arg/NO metabolites that describe the status of this pathway. Here, the importance of pre-analytical issues, which has emerged from the use of GC–MS and GC–MS/MS in clinico-pharmacological studies, is discussed. Paradigmatically, two studies on the long-term oral administration of L-arginine dihydrochloride to patients suffering from peripheral arterial occlusive disease (PAOD) or coronary artery disease (CAD) were considered. Pre-analytical issues that were addressed include blood sampling, plasma or serum storage, study design (notably in long-term studies), and the alternative of measuring MDA in human urine.

Dataset Information

Genotoxic Effects of Etoposide, Bleomycin, and Ethyl Methanesulfonate on Cultured CHO Cells: Analysis by GC-MS/MS and Comet Assay.

Publications

Genotoxic Effects of Etoposide, Bleomycin, and Ethyl Methanesulfonate on Cultured CHO Cells: Analysis by GC-MS/MS and Comet Assay.

Similar Datasets

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