Frontiers in Genetics (Jun 2015)
The alkaline comet assay used in evaluation of genotoxic damage of drinking water disinfection by-products (bromoform and chloroform)
Abstract
The alkaline comet assay (pH 12.3) is a useful method for monitoring genotoxic effects of environmental pollutants in the root nuclei of Allium cepa and various plants; it allows the detection of single- and double-strand breaks, incomplete excision-repair sites and cross-links. It has been introduced to detect even small changes in DNA structure. It is a technically simple, highly sensitive, fast and economic test which detects in vitro and in vivo genotoxicity (DNA integrity and packing mode) in any cell types examined, and requires just a few cells for its execution (Liman et al., 2011; Yıldız et al., 2009). Chloroform and bromoform are the most important trihalomethanes found in drinking water. Different concentrations of bromoform (25, 50, 75and 100µg/ml) and chloroform (25, 50, 100 and 200 µg/ml) were introduced to onion tuber roots. Distilled water was used as a negative control and methyl methansulfonate (MMS-10 µg/ml) as positive control. All obtained data were subjected to statistical analyses by using SPSS 15.0 for Windows software. For comparison purposes, Duncan multiple range tests using one-way analysis of variance (ANOVA) were employed and p<0.05 was accepted as the test of significance. Comet assay results showed that DNA damage was significant at p <0.05 for the different concentrations of chloroform and bromoform compared to the negative control which has a damage rate equal to 3.5 ± 0.7 and the positive control which has damage rate equal to 13.5 ± 2.12. The exposure of root tip cells to these disinfection by-products increases DNA damage. All concentrations examined in this study of bromoform and chloroform cause significant harm, which could be due to DNA damage induced by oxidative stress. The measurement of DNA damage in the nuclei of higher plant tissues is a new area of study with SCGE. This assay could be incorporated into in situ monitoring of atmosphere, water and soil: the comet assay allows a fast detection without any need to wait for progressing mitosis (Cotelle and Férard, 1999). Based on the results we can conclude that the classical Allium test can give more comprehensive data when done in combination with the comet assay, which is faster, simpler and independent of mitosis (Cotelle and Férad, 1999).
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