Comet Percentage Research Articles

DNA damage of tumor-associated lymphocytes and total antioxidant capacity in cancerous patients

The purpose of this study was to assess DNA damage of tumor-associated lymphocytes (TALs) in malignant pleural effusion (MPE), the total antioxidant capacity (TAC) of plasma and MPE from patients with carcinoma, and DNA repair effect of melatonin. TAC of plasma was measured in 28 cancer patients with MPE and in 33 healthy persons, and also TAC of MPE supernatant was measured in these patients. DNA damages of peripheral blood mononuclear cells (PBMCs) and of TALs were assessed using comet assay. The TAC of plasma was remarkably lower in cancer patients (8.41±1.78 U/ml) than that in healthy persons (10.52±1.64 U/ml, P<0.001). The TAC of MPE supernatant (6.34±1.57 U/ml) was significantly lower than that of plasma in cancer patients (8.41±1.78 U/ml, P<0.001). The comet percentage of PBMCs was higher in cancer patients (16.8±7.9) than that in healthy persons (10.4±4.9, P<0.01). Within cancer patients, the comet percentage of TALs (41.9±11.7) was significantly higher than that of PBMCs (16.8±7.9, P<0.001). A negative correlation was observed between the TAC of MPE supernatant and the comet percentage of TALs in patients ( r=−0.538, P<0.01). After treatment with melatonin, comet percentage of TALs declined significantly from 42.6±12.8 to 27.1±9.9 ( P<0.001). These data show that lower TAC of MPE supernatant may be related to higher degree of DNA damage of TALs and that melatonin may facilitate the repair of the damaged DNA.

Genotoxicity of the Kishon River, Israel: the application of an in vitro cellular assay

The alkaline comet assay, a sensitive method for DNA strand breaks and alkali labile site detection in individual cells, was employed here as an ecotoxicological monitoring tool for evaluation of genotoxicity in the Kishon River, Israel. This river, the most polluted river in Israel, has recently elicited major public concern with regard to cancer incidences in people who have dived there over many years. Five water samples were collected every odd month throughout the year 2001, from four localities. The comet assay was employed on fish hepatoma cell line RTH-149. Cells were exposed for 2 h, in triplicate, to Kishon water: medium (1:1) samples that were pre-adjusted for pH and salinity percentage levels. Three DNA damage parameters (comet percentages, score of damage, and cumulative tail lengths of the comet), revealed significantly higher genotoxic values in Kishon water-treated cells as compared with the controls (up to 2.4, 3.2, and 3.6-fold, respectively). Part of the sampling sites revealed higher genotoxicity than other polluted sites. The results of this study demonstrate that the comet assay with RTH-149 cells can be successfully applied to a variety of aquatic samples revealing freshwater, marine and estuary conditions. The method found to be fast, sensitive, and suitable for monitoring programs.

In vitro application of the comet assay for aquatic genotoxicity: considering a primary culture versus a cell line

The comet assay, one of the most widely used techniques for the evaluation and detection of DNA strand breaks, is frequently employed in vivo. In vitro assays are usually performed with mammalian cell lines, clearly not the best choice for tests on aquatic genotoxicity. Here we evaluated a fish hepatoma cell line (RTH-149) and a primary blood cell culture from the intertidal colonial tunicate Botryllus schlosseri as possible model targets for comet assays using the genotoxic agent H 2O 2. We found that DNA strand break levels in RTH-149 fitted dose-dependent responses better than the tunicate cells. Moreover, in B. schlosseri controls, 34% of the cells were already ranked as severely damaged. Assays were then performed on water samples from the polluted Kishon river (Israel) on three different dates, using RTH-149 cells (50% dilutions, 2-h exposures). In all cases, high genotoxicity of the river water was revealed by evaluating comet percentages, average tail lengths and DNA damage levels. This assay was found to be fast and sensitive, appropriate to be employed as a part of a monitoring program. The use of B. schlosseri blood cells should be validated in additional work.

Detection of DNA damage in response to cooling injury in equine spermatozoa using single-cell gel electrophoresis.

Single-cell gel electrophoresis (SCGE), or comet assay, has the ability to detect damage at the single cell level and has not been reported for equine sperm. The ability to detect nuclear damage at the single cell level could aid in the advancement of protocols for optimal semen preservation. The goals of these experiments were to adapt this assay for use with equine sperm and to utilize the assay for determining the integrity of equine sperm DNA following treatments with storage at various decreased temperatures (-20 degrees C and 5 degrees C). Results from experiments in which sperm were frozen (-20 degrees C) in the absence of cryoprotectants revealed that significantly more cells with fragmented tails of DNA, or comets, occurred among those exposed to 1, 3, and 5 freeze-thaw cycles (65% +/- 6%, 76% +/- 11%, 92% +/- 6%, respectively) compared with fresh, untreated sperm (19% +/- 16%, P < .05). In addition DNA damage was different (P < .05) between the three freeze-thaw treatments. Sensitivity of SCGE on equine sperm was further tested with known ratios of frozen-thawed and fresh cells. The amount of detectable DNA damage was positively correlated with the percentage of cryo-damaged cells in each treatment (r2 = 0.92, P < .05). Potential damage as a result of cooled storage was also investigated and results revealed that sperm stored for 48 hours (at 5 degrees C) had a higher percentage of comets than that of fresh sperm (63% +/- 13.9% and 28% +/- 15.6%, respectively, P < .05). The percentage of viable sperm also decreased linearly over time and was inversely correlated with percent of comets (r2 = 0.805, P < .001). Detection of sublethal and/or uncompensable fertility factors in semen, such as DNA fragmentation, could be useful for detecting male differences in semen for cooling or cryopreservation potential and could provide a tool for monitoring and preserving fertility for individual stallions.

Susceptibility of Human Sperm toin SituDNA Denaturation Is Strongly Correlated with DNA Strand Breaks Identified by Single-Cell Electrophoresis

Susceptibility of mammalian sperm DNA to low pH- or heat-induced denaturationin situhas shown very strong dose–response relationships with animal and human exposure to chemical and physical toxicants and also fertility potential. In this study, 23 human semen samples representing a wide range in percentage (7–86%) of sperm exhibiting abnormally high susceptibility of DNAin situto denaturation were studied for the integrity of their DNA using alkaline comet assay (single-cell microgel electrophoresis, pH 10.0). The percentage of comets observed for these samples ranged from 5 to 95%; these data correlated strongly with the percentage of sperm with increased DNA denaturability (r= 0.973;P< 0.001). Labeling of 3′ ends of nicked DNA sites with 5-bromo-2′-deoxyuridine 5′-triphosphate (BrdUTP) followed by tagging with FITC–BrdUTP monoclonal antibody and flow cytometry also indicated significantly strong correlations of BrdUTP incorporation with both abnormal susceptibility of DNA to denaturation (r= 0.859,P< 0.001) and comet assay (r= 0.812,P< 0.001). The relationship among susceptibility of sperm chromatin to acid denaturationin situ,BrdUTP incorporation, and formation of comets suggests that DNA fragmentation monitored by these assays may have important physiological relevance in terms of sperm quality and fertility potential.