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Measuring the effects of antioxidants

About the Linus Pauling Institute

Operating from the core philosophy that optimum diet is the key to optimum health, researchers at The Linus Pauling Institute (LPI) at Oregon State University are actively involved in research on dietary components and their beneficial effects for human health.

The LPI is an NCCAM (National Center for Complementary and Alternative Medicine) Center of Excellence, and the institute’s mission is: to determine the function and role of micronutrients (vitamins and minerals) and phytochemicals (chemicals from plants) in promoting health and preventing and treating disease.

Researchers are particularly interested in the role of oxidative and nitrative stress and antioxidants in human health and debilitating diseases.

Exercise induced DNA damage and the effect of antioxidant vitamins

The comet assay was used to determine whether 6 weeks of supplementation with vitamins C and E can alleviate exercise-induced DNA damage [1]. The 21 runners involved in the study were randomly assigned to one of two groups and given either placebos (PL) or antioxidants (AO) (1000 mg vitamin C and 400 IU natural vitamin E) for 6 weeks prior to completing a 50 km ultra marathon. DNA damage in circulating leukocytes was assessed at selected time points: pre-, mid-, and 2 hours post race and daily for 6 days post race.

The comet assay is used to detect DNA strand breaks as well as alkaline labile lesions. For many laboratories it has become the tool of choice for detecting DNA damage in eukaryotic cells due to its versatility, and the name is derived from the shape of DNA distribution seen which bears resemblance to a celestial comet.

Perceptive Instruments comet assay system at Linus Pauling

This study used the comet assay protocol based on that of Singh et al [2] with some modification. Exposed and control cells were embedded in an agarose gel ‘sandwich’ on a microscope slide. Following overnight immersion in cold lysing solution, the slides were rinsed and the cells subjected to electrophoresis. When the electrophoresis is performed in a neutral pH condition, DNA double strand breaks are revealed. In this case, a strong alkaline solution (pH > 13) was used to reveal all DNA strand breaks and alkaline-labile lesions. The slides were rinsed and neutralized before staining with 60 µl of 20µg/ml ethidium bromide to visualize DNA migration under a fluorescence microscope. 25 randomly chosen nuclei per each duplicate slide were analyzed using the comet assay image analysis system from Perceptive Instruments. Using a highly sensitive video camera attached to a microscope, the system immediately transfers a live video image to the computer screen to provide real-time cell scoring.

Each comet is brought into sharp focus and the operator initiates the measurement, which includes correction for background with a click of the mouse cursor. Measurements are saved to an Excel spreadsheet for statistical analysis.

‘Percentage DNA in tail’ was used as the indicator of DNA damage in the published study [1]. The results showed that DNA damage in leukocytes, induced by the extreme exercise of an ultra-marathon, increased transiently at mid race but returned to baseline by 2 hours post race, indicating that the exercise bout induced non-persistent DNA damage. One day post-race, women taking antioxidant vitamin supplements had 62% less DNA damage than women taking the placebo. In contrast, there were no statistically significant differences between the two treatment groups of men at any time point. Thus, endurance exercise resulted in DNA damage as shown by the comet assay and antioxidants seemed to enhance recovery in women but not in men.

References
1. Mastaloudis A, Yu T-W, O'Donnell RP, Frei B, and Dashwood R and Traber M, Endurance exercise results in DNA damage as detected by the comet assay. Free Radical Biol Med, Free Radical Biology and Medicine, 36(8): 966-975, 2004.
2. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175: 184-191, 1988.

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