Use of the comet assay for studying environmental genotoxicity: Comparisons between visual and image analyses

Abstract

In order to evaluate the applicability of different measurement parameters employed in the comet assay for analyzing environmental samples, fish hepatoma (RTH-149) cells were exposed to concentrations of the model genotoxic agent hydrogen peroxide (H(2)O(2); 1, 5, and 10 microM) and to five water samples from sites along the Kishon River, the most polluted river in Israel. DNA damage was scored in parallel by visual and computer-image (Viscomet) analyses using 12 different parameters. Each parameter exhibited a different profile of responses. The four visual parameters were highly sensitive to the lowest (1 microM) H(2)O(2) concentration (1.8-7.0-fold of the control). At 10 microM H(2)O(2) exposure, the visual parameter, percentage severe damage, showed the highest (40.3-fold) response while four other parameters, tail area, tail extent moment (Viscomet), mean actual tail length and cumulative tail length (visual analysis), also had substantially elevated responses (8-11-fold). We found that the DNA damage induced by field samples was similar in magnitude to the damage induced by 1 microM H(2)O(2), with only some of the parameters being highly sensitive to the damage. Only about one-half of the parameters could distinguish four significant levels of genotoxicity among the five sampling sites, while the remaining parameters detected only three levels. It is concluded that the choice of parameters for analyzing genotoxicity in ecotoxicological studies should be made in accordance with the characteristics of each parameter.

Dopamine alleviation of diaphragm contractile dysfunction and reduction of deoxyribonucleic acid damage in rats

“…41 Since the introduction of the alkaline modification using this assay, 42,43 there have been studies using this method to visualize DNA degradation. 44, 45 Singh et al 43 found that the comet moments are the most sensitive indices of DNA damage.…”

“…The comet assay, a gel electrophoresis technique, is a sensitive technique for evaluating DNA damage. 41 Since the introduction of the alkaline modification using this assay, 42,43 there have been studies using this method to visualize DNA degradation. 44,45 Myonuclei DNA damage also was assessed using a gel electrophoresis assay (comet).…”

<i>IN-VIVO</i> EVALUATION OF HEXAVALENT CHROMIUM INDUCED DNA DAMAGE BY ALKALINE COMET ASSAY AND OXIDATIVE STRESS IN <i>CATLA CATLA</i>

“…After electrophoresis, the slides were washed three times with neutralization buffer (0.4M Tris, pH 7.5). All preparative steps were conducted in yellow light to prevent occurance of additional DNA damage (Avishai et al, 2003;Tice et al, 2000;Velma and Tchounwou, 2010).…”

UV incites diverse levels of DNA breaks in different cellular compartments of a branching coral species

“…The cell images were projected onto a high resolution Heper-HAD TM (Sony, Tokyo, Japan) CCD camera [8 bits (Applitec, Holon, Israel; LIS-700)] and analyzed with Viscomet image analysis software using the MV Delta frame grabber (Matrix Vision, Oppenweiler, Germany). DNA damage in each treatment was measured in duplicates of 50 cells each, using two highly informative parameters (Avishai et al, 2003): tail extent (sum of all distances of each horizontal scan line from the first signal pixel to the last signal pixel divided by number of scan lines) and tail extent moment (tail length ϫ percentage tail DNA). Controls (algal-free cells, animal cells, holobionts) were handled as experimental cells, but without UVR treatment.…”

“…The coral species used in this study is the Indo-Pacific, shallow water branching form, Stylophora pistillata. UV-induced DNA damage and DNA repair levels were evaluated by the single cell gel electrophoresis assay, also known as the comet assay, one of the most reliable and sensitive methods for evaluating DNA damage induced in individual cells by various agents (Mitchelmore and Chipman, 1998;Avishai et al, 2003;Reinhardt et al, 2003).…”