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Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay
Published on: December 19, 2012
Optimized noninvasive procedures to measure DNA damage in comet assay
Y T Szeto1, A K H Lee, I F F Benzie
1Department of Applied Science, Hong Kong Institute of Vocational Education (Shatin), Sha Tin, New Territories, Hong Kong. savio.yim.tong.szeto@inet.polyu.edu.hk
Human & Experimental Toxicology
|June 2, 2012
Summary
A new buccal cell lymphocyte comet assay offers a noninvasive method to measure DNA damage. This optimized technique successfully demonstrated the protective effects of quercetin against genotoxicity.
Area of Science:
- Biomonitoring
- Genetics
- Cell Biology
Background:
- The comet assay is a standard method for detecting DNA damage in single cells.
- Peripheral blood lymphocytes are commonly used, but a noninvasive alternative is desirable.
- Buccal cells offer a promising, noninvasive source of lymphocytes for biomonitoring.
Purpose of the Study:
- To develop and optimize a buccal cell lymphocyte comet assay.
- To establish reliable conditions for DNA damage assessment using mouthwash samples.
- To validate the assay's utility in detecting genoprotective effects.
Main Methods:
- Cells were collected from mouthwash of healthy volunteers.
- The comet assay was optimized for pH, alkaline treatment time, and electrophoresis parameters.
- Genoprotective activity was assessed using quercetin.
Main Results:
- Optimal conditions included pH >13 for buffers, 10-min unwinding, and 20-min electrophoresis.
- The optimized assay successfully detected the genoprotective effects of quercetin.
- The developed method provides a viable alternative for DNA damage assessment.
Conclusions:
- A noninvasive buccal cell lymphocyte comet assay procedure has been successfully established.
- This assay provides an alternative to blood sampling for DNA damage and protection studies.
- The optimized method is sensitive and effective for evaluating genoprotective agents.
