Evaluation of the Comet Assay to assess oxidative DNA damage and repair capacity in the peripheral mononuclear cells of systemic sclerosis and systemic lupus erythematosus and silicotic patients

Evaluation Of The Comet Assay To Assess Oxidative Dna Damage And Repair Capacity In The Peripheral Mononuclear Cells Of Systemic Sclerosis And Systemic Lupus Erythematosus And Silicotic Patients

X Masoka
NIOH, University of the Witwatersrand, Chris Hani Baragwanath Hospital
Project Summary:

The single cell gel (SCG) electrophoresis or 'Comet assay' is a rapid and sensitive method to examine oxidative DNA damage and repair at individual cell level. This assay is widely used in toxicology ranging from clinical investigations to genetic toxicology, molecular epidemiology, and fundamental research in DNA damage and repair. Since the introduction of the alkaline version of the Comet assay in 1988, a number of advancements have greatly increased the flexibility and utility of this technique for detecting various forms of DNA damage (e.g., single- and double-strand breaks, oxidative DNA base damage, and DNA-DNA/DNA-protein/DNA-Drug crosslinking) and DNA repair in virtually any eukaryotic cell.

The assay derives its wide use and advantages from its simplicity, sensitivity, versatility, speed, and economy. Comet assay not only provides an estimate of how much damage is present in cells, but what form it takes. Although it is essentially a method for measuring DNA breaks, the introduction of lesion-specific endonucleases allows detection of, for example, ultraviolet (UV)-induced pyrimidines dimers, oxidized bases, and alkylation damage.

Oxidative stress, including oxidative damage to DNA is a result of interaction of DNA with reactive oxygen species (ROS), has been thought to contribute to the general decline in cellular functions that are associated with many human diseases including Alzheimer disease, Parkinson disease, atherosclerosis, multiple sclerosis, human cancers as well as the aging. Systemic sclerosis or scleroderma (SSc) has been the subject of many studies; however, its causes are largely unknown. Significant evidence is emerging that there is oxidative stress (caused by the overproduction of free radicals) in patients with scleroderma and to a lesser extent in systemic lupus erythematosus or lupus (SLE), as measured by various biomarkers, including lipid peroxidation products, antioxidant capacity, and urinary levels of F2 –isoprostanes. Also, polymorphisms in the antioxidant enzymes, glutathione S-transferase (GST) and manganese superoxide dismutase (MnSOD) have been shown as further evidence that genetic factors may have a contributory role to play in predisposing to oxidative stress in SSc. Lymphocytes from patients with various diseases associated with autoimmunity have shown impaired capacity to repair o-6-methylguanine, a powerful promutagenic directly miscoding base lesion. Also, aberrant processing of 8-oxodG, a product of oxidative DNA damage, has been reported in SLE.

In this study we intend to demonstrate that the Comet assay can be used to detect free radical induced DNA strand breaks or damage in silicotic patients and SSc and SLE patients with or without silicosis. Secondly, we intend to use the Comet assay to assess the ability of mononuclear cells from these patients to effectively repair DNA damage induced by free radicals produced by in vitro exposure to H< sub>2O2, a DNA damaging agent, compared to healthy controls.

  Contact Person: Mr. X. Masoka (xolani.masoka@nioh.nhls.ac.za)