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Detecting DNA Damage With In-Vivo Alkaline Comet Assay

The comet assay has gained rapid importance in detecting various forms of DNA damage and repair.// The conventional methods have numerous disadvantages and are time consuming, resource intensive and require a large proliferating cell population.

The comet assay attracts adherents by it’s simplicity, speed, versatility and economy and is an ideal alternative supplementary in-vivo study. The Comet assay can be used to detect various types of DNA damage induced by different classes of genotoxic compounds.

The principle is that strand breakage of the duplex DNA leads to difference in size of the molecule and these fragments can be stretched out by electrophoresis. The image obtained consists of a head made up of intact DNA and a tail, which represents the fragmented DNA. Under alkaline conditions, unwinding of the double strand takes place, which enables the visualization of single strand breaks and alkali labile sites.

The main requirement of the assay is a single cell or nuclei suspension, which can be obtained from the tissue without adding to the DNA damage by the applied isolation technique. However, the assay only measures DNA damage and it remains unclear if mutations will result or whether the detected damage will be repaired before the next cell division.

There is a positive movement towards formal regulatory acceptance of the assay, which has resulted in increasing demand for the in vivo Comet assay. In response to this demand, RCC Cytotest Cell Research has established and validated protocols for different cell types.

In order to make a comparison with an acknowledged test system, the Comet assay experiments were run in parallel with an in vivo UDS test on the same hepatocyte suspensions. The results have shown that the in vivo Comet assay is a sensitive assay to detect DNA damage in the tested organs and have demonstrated the major advantage namely, the simultaneous assay of several different tissues f rom the same animal.

In conclusion, a further fully validated genotoxicity assay has been added to the battery of genotoxicity tests available at RCC. Refinement of the technique will continue as experience is accumulated.
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