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

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Donald H. Atha ◽  
Erdem Coskun ◽  
Onur Erdem ◽  
Alessandro Tona ◽  
Vytas Reipa ◽  
...  
Keyword(s):  
Comet Assay ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Ethyl Methanesulfonate ◽  
Genotoxic Effects ◽  
Strand Breaks ◽  
Mammalian Cell Lines ◽  
Chromatography Tandem Mass Spectrometry ◽  
Wide Range ◽  
Dna Strand

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.

scholarly journals Alkaline Comet Assay using the monocytic cell line THP-1

2019 ◽  
Author(s):  
Ana Neves-Costa ◽  
Dora Pedroso ◽  
Luis F Moita
Keyword(s):  
Comet Assay ◽  
Cell Line ◽  
Adherent Cell ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Strand Breaks ◽  
Human Monocytic Cell Line ◽  
Human Monocytic Cell ◽  
Dna Strand

Abstract This protocol details the experimental procedure for performing the comet assay, a very sensitive DNA break assay based on single cell gel electrophoresis.The analysis of DNA strand breaks, both single- and double-strand breaks (SSBs and DSBs, respectively), was performed in immune responsive cells. The cell line used was the human monocytic cell line THP-1, an adherent cell type with many known applications in in vitro studies of innate immunity. The comet assay is a robust procedure that allows the accurate and reproducible quantification of DNA damage. Here we describe not only the comet assay step-by-step protocol, but also some important aspects related to troubleshooting.

scholarly journals Protective Activity of C-Geranylflavonoid Analogs from Paulownia tomentosa against DNA Damage in 137Cs Irradiated AHH-1Cells

2014 ◽  
Vol 9 (9) ◽  
pp. 1934578X1400900
Author(s):  
Hyung-In Moon ◽  
Min Ho Jeong ◽  
Wol Soon Jo
Keyword(s):  
Dna Damage ◽  
Protective Effects ◽  
Strand Breaks ◽  
Human Lymphoblastoid Cell ◽  
Wide Range ◽  
Radiation Induced ◽  
Anti Oxidant ◽  
Dna Strand ◽  
Cellular Dna

Radiotherapy is an important form of treatment for a wide range of cancers, but it can damage DNA and cause adverse effects. We investigated if the diplacone analogs of P. tomentosa were radio-protective in a human lymphoblastoid cell line (AHH-1). Four geranylated flavonoids, diplacone, 3′- O-methyl-5′-hydroxydiplacone, 3′- O-methyl-5′- O-methyldiplacone and 3′- O-methyldiplacol, were tested for their antioxidant and radio-protective effects. Diplacone analogs effectively scavenged free radicals and inhibited radiation-induced DNA strand breaks in vitro. They significantly decreased levels of reactive oxygen species and cellular DNA damage in 2 Gy-irradiated AHH-1 cells. Glutathione levels and superoxide dismutase activity in irradiated AHH-1 cells increased significantly after treatment with these analogs. The enhanced biological anti-oxidant activity and radioprotective activity of diplacone analogs maintained the survival of irradiated AHH-1 cells in a clonogenic assay. These data suggest that diplacone analogs may protect healthy tissue surrounding tumor cells during radiotherapy to ensure better control of radiotherapy and allow higher doses of radiotherapy to be employed.

scholarly journals DNA Repair Functions That Control Sensitivity to Topoisomerase-Targeting Drugs

2004 ◽  
Vol 3 (1) ◽  
pp. 82-90 ◽  
Author(s):  
Mobeen Malik ◽  
John L. Nitiss
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Topoisomerase I ◽  
Topoisomerase Ii ◽  
Excision Repair ◽  
Dna Strand Breaks ◽  
Dna Topology ◽  
Strand Breaks ◽  
Wide Range ◽  
Dna Strand

ABSTRACT DNA topoisomerases play critical roles in a wide range of cellular processes by altering DNA topology to facilitate replication, transcription, and chromosome segregation. Topoisomerases alter DNA topology by introducing transient DNA strand breaks that involve a covalent protein DNA intermediate. Many agents have been found to prevent the religation of DNA strand breaks induced by the enzymes, thereby converting the enzymes into DNA-damaging agents. Repair of the DNA damage induced by topoisomerases is significant in understanding drug resistance arising following treatment with topoisomerase-targeting drugs. We have used the fission yeast Schizosaccharomyces pombe to identify DNA repair pathways that are important for cell survival following drug treatment. S. pombe strains carrying mutations in genes required for homologous recombination such as rad22A or rad32 (homologues of RAD52 and MRE11) are hypersensitive to drugs targeting either topoisomerase I or topoisomerase II. In contrast to results observed with Saccharomyces cerevisiae, S. pombe strains defective in nucleotide excision repair are also hypersensitive to topoisomerase-targeting agents. The loss of DNA replication or DNA damage checkpoints also sensitizes cells to both topoisomerase I and topoisomerase II inhibitors. Finally, repair genes (such as the S. pombe rad8+ gene) with no obvious homologs in other systems also play important roles in causing sensitivity to topoisomerase drugs. Since the pattern of sensitivity is distinct from that seen with other systems (such as the S. cerevisiae system), our results highlight the usefulness of S. pombe in understanding how cells deal with the unique DNA damage induced by topoisomerases.

Detection of DNA strand breaks by comet assay in sputum leucocytes of bitumen-exposed workers: A pilot study

2010 ◽  
Vol 29 (9) ◽  
pp. 721-729 ◽  
Author(s):  
B. Marczynski ◽  
M. Raulf-Heimsoth ◽  
B. Pesch ◽  
B. Kendzia ◽  
HU Käfferlein ◽  
...  
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Rank Correlation ◽  
Dna Strand Breaks ◽  
Strand Breaks ◽  
Blood Lymphocytes ◽  
Exposed Workers ◽  
Before And After ◽  
Dna Strand ◽  
Post Shift

DNA strand breaks were determined in leucocytes of induced sputum (IS) and compared with DNA strand breaks in blood lymphocytes from 42 bitumen-exposed workers pre and post shift. Comet assay results were expressed in arbitrary units based on visual scoring (sputum leucocytes) and Olive tail moment (OTM, blood lymphocytes). DNA damage in IS leucocytes was overall high but did not change during shift. Level of DNA strand breaks in IS samples correlated with total cell count and neutrophil content (Spearman rank correlation coefficient rs = 0.47, p = 0.001, rs= 0.48, p = 0.001, respectively) and with IL-8 concentration before and after shift (rs = 0.31, P = 0.048, and rs = 0.43, P = 0.005). DNA damage in IS was not associated with DNA strand breaks in blood lymphocytes (rs = —0.04, p = 0.802 before shift, rs = 0.27, p = 0.088 after shift). A higher level of DNA strand breaks was measured in blood lymphocytes before shift (median OTM 1.7 before and 1.3 after shift, p = 0.023). A strong correlation was found between the number of neutrophils and IL-8 concentration in IS before and after shift (rs = 0.77 and rs= 0.75, p < 0.001). This study showed an association between genotoxic and inflammatory effects in the lower airways and compared simultaneously DNA strand breaks in IS and blood of bitumen-exposed workers.

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