Optimized noninvasive procedures to measure DNA damage in comet assay

The comet assay is a well-established, simple and sensitive method to measure DNA damage in single cell and is commonly used in human trials to investigate the effects of pollution, occupational hazards and potential genoprotective agents. Peripheral blood lymphocytes are most commonly used in human...

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Bibliographic Details
Published in:Human & experimental toxicology 2012-11, Vol.31 (11), p.1144-1150
Main Authors: Szeto, YT, Lee, AKH, Benzie, IFF, Obied, HK
Format: Article
Language:English
Subjects:
Adult
Antioxidants
Antioxidants - pharmacology
Assays
Bioassays
Bioindicators
biomonitoring
Buffers
Cells, Cultured
Comet assay
Comet Assay - methods
DNA
DNA Damage
Electrophoresis
Female
Humans
Hydrogen peroxide
Hydrogen Peroxide - pharmacology
Hydrogen-Ion Concentration
Lymphocytes
Lymphocytes - drug effects
Lymphocytes - metabolism
Male
Mouth
Mouth Mucosa - cytology
Mouthwashes
Occupational hazards
Oxidants - pharmacology
Oxidative stress
Peripheral blood
pH effects
Pollution
Pollution effects
Quercetin
Quercetin - pharmacology
Sampling
Toxicity
Unwinding
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Summary:The comet assay is a well-established, simple and sensitive method to measure DNA damage in single cell and is commonly used in human trials to investigate the effects of pollution, occupational hazards and potential genoprotective agents. Peripheral blood lymphocytes are most commonly used in human biomonitoring studies, but lymphocytes collected from the mouth offer a potentially attractive, noninvasive alternative. The aim of the current study was to develop a buccal cell lymphocyte comet assay procedure. Cells were collected from mouthwash of three healthy volunteers and tested individually. The comet assay was performed under different pH and times of alkaline treatment, electrophoresis run times and hydrogen peroxide concentrations. Optimal conditions for buccal lymphocytes in comet assay were found to be pH >13 for unwinding and electrophoresis buffers, 10-min alkaline unwinding treatment and 20-min electrophoresis run time. We successfully utilized our optimized assay conditions to demonstrate the genoprotective activity of quercetin. This newly established procedure offers an alternative noninvasive sampling method for the investigation of DNA protection and/or damaging effect.
ISSN:0960-3271
1477-0903
DOI:10.1177/0960327112446816