Application of the comet assay for investigation of oxidative DNA damage in equine peripheral blood mononuclear cells.
Abstract: Oxidative stress occurs when antioxidant defense mechanisms are overwhelmed by free radicals and may lead to DNA damage, which has been implicated in processes such as aging and diseases such as cancer. The two main techniques presently used to quantify DNA damage are measurement of 8-hydroxydeoxyguanosine and the Comet assay (also known as single-cell gel electrophoresis). The aim of this study was to apply the comet assay to equine peripheral blood mononuclear cells (PBMCs) and identify two conditions in which we hypothesized that oxidative DNA damage would be increased in PBMCs: aging and equine recurrent airway obstruction (RAO, a condition similar to human asthma). The images obtained were similar to those previously published for humans, cats, and dogs. The optimum concentration of H(2)O(2) to estimate susceptibility to exogenous damage was 50 microM. Mean intraassay coefficients of variation were 4.7 and 9.7% for endogenous and exogenous tail-DNA quantities, respectively, and 7.3 and 8.3%, respectively, for interassay coefficients. There was no significant difference in either endogenous or exogenous percentages of tail DNA for samples collected from six ponies on three consecutive days. There was no significant difference in endogenous, exogenous, or exogenous (corrected for endogenous) oxidative DNA damage between mature and aged ponies. However, young pony foals had significantly less endogenous DNA damage than mature or aged ponies (P < 0.05). RAO-affected horses without airway inflammation (i.e., in clinical remission) had significantly greater endogenous damage compared with non-RAO-affected control animals (P = 0.009). There was a significant correlation between endogenous percentage of tail DNA in PBMCs and red blood cell hemolysate glutathione concentration (r = 0.720; P < 0.001). In conclusion, the comet assay appears to be suitable for investigating DNA damage in equine PBMCs.
Publication Date: 2004-07-31 PubMed ID: 15284420DOI: 10.1093/jn/134.8.2133SGoogle Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research study evaluated the use of a method known as the comet assay to measure oxidative DNA damage in horses. The researchers investigated whether this method could detect increased DNA damage in horses as they age and in horses with a disease called recurrent airway obstruction.
Methodology of Comet Assay
- The Comet assay, also known as single-cell gel electrophoresis, is one of the major techniques used to measure and quantify DNA damage. This tool is used along with another method, measurement of 8-hydroxydeoxyguanosine, for gauging DNA damage.
- This study aimed at using the Comet assay to study DNA damage in equine peripheral blood mononuclear cells (PBMCs). PBMCs are blood cells that possess a round nucleus and include cells such as lymphocytes, monocytes, and macrophages.
Investigating Cases of Aging and Equine Recurrent Airway Obstruction
- The research evaluated two potential conditions where they hypothesized an increase in oxidative DNA damage: aging and the disease equine recurrent airway obstruction (RAO). RAO, a disease similar to human asthma, often affects horses, creating respiratory difficulties.
- For comparison, the study included a control group of non-RAO affected animals and another group of RAO-affected horses without any signs of airway inflammation (i.e., in clinical remission).
Observations and Findings
- The comet assay results showed images similar to those previously reported in humans, cats, and dogs, indicating the applicability of the method in horses.
- The optimal concentration of hydrogen peroxide (H2O2) for estimating susceptibility to external DNA damage was determined to be 50 microM.
- Younger horses (pony foals) showed significantly less DNA damage compared to mature and older horses.
- RAO-affected horses exhibited significantly greater DNA damage compared to non-RAO control animals.
- Interestingly, there was no significant difference in either intrinsic (endogenous) or extrinsic (exogenous) percentages of DNA tail for samples taken from six ponies on three consecutive days.
- Furthermore, a significant correlation was found between intrinsic DNA damage (represented as percentage of DNA tail in PBMCs) and the concentration of a molecule called glutathione in red blood cell hemolysate. Glutathione is an antioxidant that protects cells against oxidative stress.
Conclusion
- The study concludes that the comet assay can be effectively used as a tool to investigate DNA damage in equine PBMCs. This could potentially aid in understanding diseases and conditions where DNA damage plays a significant role.
Cite This Article
APA
Marlin DJ, Johnson L, Kingston DA, Smith NC, Deaton CM, Mann S, Heaton P, Van Vugt F, Saunders K, Kydd J, Harris PA.
(2004).
Application of the comet assay for investigation of oxidative DNA damage in equine peripheral blood mononuclear cells.
J Nutr, 134(8 Suppl), 2133S-2140S.
https://doi.org/10.1093/jn/134.8.2133S Publication
Researcher Affiliations
- Centers for Equine Studies, Animal Health Trust, Kentford, Newmarket, Suffolk, CB8 7UU, UK. david.marlin@aht.org.uk
MeSH Terms
- Aging / metabolism
- Airway Obstruction / blood
- Airway Obstruction / etiology
- Animals
- Antioxidants / administration & dosage
- Antioxidants / metabolism
- Antioxidants / therapeutic use
- Comet Assay
- DNA Damage
- Diet
- Horses
- Hydrogen Peroxide / blood
- Leukocytes, Mononuclear / metabolism
- Oxidative Stress
Citations
This article has been cited 6 times.- Sani A, Abdullahi IL, Khan MI, Cao C. Analyses of oxidative DNA damage among coal vendors via single cell gel electrophoresis and quantification of 8-hydroxy-2'-deoxyguanosine. Mol Cell Biochem 2023 Aug 18;.
- Bullone M, Lavoie JP. The Contribution of Oxidative Stress and Inflamm-Aging in Human and Equine Asthma. Int J Mol Sci 2017 Dec 5;18(12).
- Steinberg ML, Hubbard K, Utti C, Clas B, Hwang BJ, Hill HZ, Orlow I. Patterns of persistent DNA damage associated with sun exposure and the glutathione S-transferase M1 genotype in melanoma patients. Photochem Photobiol 2009 Jan-Feb;85(1):379-86.
- Bissett W Jr, Smith L, Thompson JA. Geostatistical analysis of DNA damage in oysters, Crassostrea virginica, in Lavaca Bay, Texas. Ecotoxicology 2009 Jan;18(1):69-74.
- Bissett W Jr, Smith R, Adams LG, Field R, Moyer W, Phillips T, Scott HM, Thompson JA. Geostatistical analysis of biomarkers of genotoxicity in cattle, Bos taurus and Bos taurus x Bos indicus, sentinels near industrial facilities. Ecotoxicology 2009 Jan;18(1):87-93.
- Paraš S, Paspalj J, Baghdad K, Janković O, Škrbić R, Gajanin R, Massiani P, Launay F, Gotovac Atlagić S. Biocompatibility of nano/micro-sized pyrophyllite particles by pulmo, liver, kidney and gastric mucosis cells. J Mater Sci Mater Med 2024 Jun 17;35(1):30.
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