Comet assay in cataract research

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• Created: Wednesday, 20 March 2013 09:54

Cataract development is the leading cause of blindness and visual impairment in the world and the incidence of cataracts continues to increase with the growing global elderly population. Cataract surgery is still the only effective treatment for this disease. Strategies to reduce the risk and/or to delay the development of senile cataracts could improve visual impairments and reduce the cost associated with this disease. Improvement of dietary intake of micronutrients might be one of the strategies for reducing the risk for senile cataract. Lutein and zeaxanthin are among the micronutrients that have been reported to be associated with reduced risk for senile cataract development.

During the investigation, researchers evaluated their modulation of oxidative damage in human lens epithelial cells (HLEC). To comprehensively investigate the protective effects of lutein and zeaxanthin against oxidative DNA damage, the researchers measured DNA damage in HLEC using the comet assay. Subconfluent HLEC were incubated with or without 5 µM of lutein, zeaxanthin or a-tocopherol (the positive control) for 48 hours and then exposed to 100 µM H2O2 for 1 hour. The comet assay was performed and the slides were coded and analysed blind to remove any experimental bias. DNA damage was measured using Comet Assay IV from Perceptive Instruments. At least 100 cells from each sample were counted and the amounts of DNA in the tail and main body were measured by fluorescence intensity. The percentage of DNA in the tail was used to express the amount of DNA damage.

It was reported that supplementation with lutein or zeaxanthin suppressed H2O2-induced DNA damage in the HLEC. Several other experimental methods were used in the study and the researchers suggested that lutein or zeaxanthin supplementation protects lens protein, lipid, and DNA from oxidative damage and improves intracellular redox status upon oxidative stress.

Case study based upon:

photooxidative damage and modulates the expression of inflammation-related genes in retinal pigment epithelial cells Gao S, Qin T, Liu Z, Caceres MA, Ronchi CF, Chen CY, Yeum KJ, Taylor A, Blumberg JB, Liu Y, Shang F.
Free Rad Bio Med. 2012;53.6:1298-1307. 15 Sept 2012.