Lymphocyte DNA damage in cigarette factory workers measured by the Comet assay.

To investigate whether there were separate and combined effects of occupational exposure to tobacco dust and smoking on lymphocyte DNA damage, 148 workers from a cigarette manufacturing factory (107 occupationally exposed to tobacco dust from the production department and 41 unexposed controls who were managerial workers) were included in the study. The Tail Moment (TM) of Comet assay was used to measure DNA damage. The two groups had similar mean age, mean duration of work and smoking prevalence. The exposed workers had a larger TM than that of the controls (mean+/-S.D.: 43.43+/-13. 77 vs. 38.89+/-8.98, p<0.05). Smokers had significantly larger TM than non-smokers (47.25+/-14.02 vs. 38.90+/-10.75, p<0.001). Analysis of variance after adjustment for age and gender showed that occupational exposure and smoking had a significant and independent effect on Tail Moment (p=0.025 and p=0.002, respectively) and there was a significant positive two way interaction between the two factors (p=0.019). Age and gender had no significant effect on TM. The present study suggests that smoking and tobacco dust exposure can induce lymphocyte DNA damage and there is a synergistic effect of tobacco dust exposure and smoking on DNA damage.     

The Comet assay for the evaluation of genotoxic impact in aquatic environments

This review considers the potential of the Comet assay (or Single Cell Gel Electrophoresis, SCGE) to evaluate the environmental impact of genotoxins in aquatic environments. It focuses on in vivo and in situ studies that have been carried out in various marine and freshwater sentinel species, published in the last 5 years. A large number of the studies reviewed report that the Comet assay is more sensitive when compared with other biomarkers commonly used in genetic ecotoxicology, such as sister chromatid exchanges or micronucleus test. Due to its high sensitivity, the Comet assay is widely influenced by laboratory procedures suggesting that standard protocols are required for both fish and mussel cells. However, there are still a wide variety of personalised Comet procedures evident in the literature reviewed, making comparison between published results often very difficult. Standardization and inter-laboratory calibration of the Comet assay as applied to aquatic species will be required if the Comet assay is to be used routinely by national bodies charged with monitoring water quality.     

A comparison of cell-collecting methods for the Comet assay in urinary bladders of rats

Conducting the single-cell gel electrophoresis (Comet) assay in the urinary bladders of rodents is technically problematic because the bladder is small and thin, which makes it difficult to collect its mucosal cells by scraping. We performed the Comet assay using a simple mincing method in which tissues are minced with scissors. We then compared data obtained with this method with data obtained using the scraping method. Sprague-Dawley rats of both sexes were orally given twice the known carcinogens N-methyl-N-nitrosourea (MNU), ethyl methanesulfonate (EMS), or o-anisidine (OA). Three hours after the second administration, the bladder of each rat was divided into two parts and each part was processed by either the mincing or the scraping method. Both mincing and scraping methods detected DNA damage in MNU-, EMS-, but not OA-treated rats, and thus the mincing method had a sufficient capability to detect DNA damaging agents. The morphological analysis of the prepared cell suspensions revealed that more than 80% of the cells collected by the mincing method were from the epithelium. Because the mincing method requires only one-half of a bladder, the other half remains intact and can be used for histopathological examination. We conclude that the mincing method is easier and more appropriate for the Comet assay in urinary bladder tissue than the scraping method.     

Increases in chromosome aberrations and in abnormal sperm morphology in rubber factory workers

Subjects working at a rubber plant in a chemicals warehouse or in calandering and bambury units were analyzed for both sperm parameters and structural chromosome aberrations in peripheral blood lymphocytes. Sperm analysis war performed in a group of 24 workers for comparison with fertile (n = 24) and infertile (n = 24) control groups. The statistical analyses of semen volume, vitality and sperm count did not show significant differences between exposed and fertile groups but significant differences were found from the infertile group. A significantly lower proportion of normal sperm head shapes was found in exposed subjects when compared to the fertile group (40.1 vs. 57.8). Seven exposed workers were re-analyzed 1 year and their sperm parameters did not change. The cytogenetic analysis showed a significant increase (3.90%) in the percentage of cells with aberrations in bambury workers (n = 11). However, no differences were found between calandering workers (n = 8) and control subjects (n = 10). Workplace air samples taken on the day of tissue sampling did not show any increase above the Cuban maximal allowed concentration for benzo[a]pyrene or toluene.     

Biomonitoring of the cytogenetic effect of antitumor therapy by means of micronucleus assay in exfoliated epithelial cells

Data are presented concerning the possibility of using the micronuclei (MN) level as a biomarker for cytogenetic effects in exfoliated epithelial cells of cancer patients under therapy. The number of MN in buccal cells of cancer patients under chemotherapy are very contradictory. A significant dose-dependent increment of MN in tumor and normal epithelial cells due to radiotherapy was shown in most investigations. Evaluation of MN induced by radiotherapy in exfoliated tumor cells can potentially identify the radiosensitivity of tumors and the outcome of treatment after the first fractions of irradiation. This technique is almost completely noninvasive and easily done in accessible primary cancers (oral cavity and uterine cervix). The text was submitted by the author in English.     

Comet assay for assessment of DNA damage in greenhouse workers exposed to pesticides

Purpose: The main goal of the present study was to determine DNA damage in pesticide-exposed greenhouse workers and pesticides non-exposed controls.

Materials and methods: The DNA damage was measured by alkaline comet assay method (pH?>?13) in 41 greenhouse workers and 45 non-exposed individuals as the control. Pesticide exposure was assessed by duration of working in the greenhouse and pesticide application in the greenhouse time. DNA damage was estimated by arbitrary unit and damage frequency.

Results: Arbitrary unit and damage frequency were consistently significantly higher in greenhouse workers than those of the controls (p?=?0.001). In terms of gender in greenhouse, DNA damage of female workers was significantly higher than those in male workers (p?<?0.05). We found significant correlation between DNA damage and working hours spent. Multiple linear regression analysis showed that working hours in the greenhouse as an indication of pesticide exposure were significantly associated with the DNA damage, which can be attributed to the genotoxic potential of the pesticide mixture.

Conclusions: The comet assay is sensitive to detect the damage exposed to chronic effect of pesticides in greenhouse workers. Significant DNA damage was obtained for the exposed group, which was associated with the pesticide exposure.     

The in vitro micronucleus assay for detection of cytogenetic effects induced by mutagen-carcinogens: comparison with the in vitro sister-chromatid exchange assay

The sensitivity of a cytogenetic assay, as expressed by the in vitro induction of micronuclei (MN), was compared to the in vitro induction of sister-chromatid exchanges (SCEs). Chinese hamster lung (V79) cells were exposed to 3 known alkylating agents: methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and to 5 newly synthesized naphthofurans: 2-nitro-7-methoxynaphtho[2,1-b]furan (A), 2-nitro-8-methoxynaphtho[2,1-b]furan (B), 2-nitronaphtho[2,1-b]furan (C), 2-nitro-7-bromonaphtho[2,1-b]furan (D) and 7-methoxynaphtho[2,1-b]furan (E). The induction of MN only was also analysed after exposure of the cells to 4 alcohols: ethanol, methanol, butanol and propanol. The lowest dose at which a significant effect could be observed was determined. In both assays, MNNG, MMS and EMS were equally active with the following order of potency: MNNG greater than MMS greater than EMS, the latter being a very weak inducer of MN and SCE. Compounds A and B were also very effective in both assays. Compound C was a more active inducer of SCE than MN. Compounds D and E were not active in either assay. None of the 4 alcohols induced MN. Our results are compared with the previously published data on in vitro and in vivo induction of SCE and MN. We conclude that the MN in vitro assay which detects clastogens as well as agents affecting the spindle apparatus, is a good indicator of genotoxicity, though slightly less sensitive than the in vitro SCE test. It could provide a rapid, simple and inexpensive complementary short-term test for the evaluation of potentially mutagenic chemicals.     

A cytogenetic study of nuclear power plant workers using the micronucleus-centromere assay.

A cytogenetic study was performed in 215 nuclear power plant workers occupationally exposed to radiation using the micronucleus-centromere assay for peripheral blood lymphocytes. As control population served administrative staff with yearly doses below 1 mSv. The increase of the micronucleus frequency with age, observed in the non-smoking control population, is mainly due to an enhanced number of centromere-positive micronuclei, pointing to an increased chromosome loss. No differences in the number of micronuclei, centromere-positive and centromere-negative micronuclei between smokers and non-smokers are observed. An analysis of the micronucleus data vs. the dose accumulated over the 10 years preceding the venepuncture shows no significant clastogenic or aneuploidogenic effects of the exposure in the studied population which is representative for workers in the nuclear industry at present. According to the linear fits to our data an increase of the micronucleus frequency pro rata 0.5 per 1000 binucleated cells per year, related to the centromere-negative micronuclei, may be expected for workers with the maximal tolerable dose of 20 mSv/year.     

Comparing the genotoxic sensitivities of human peripheral blood lymphocytes and mucosa cells of the upper aerodigestive tract using the Comet assay

Carcinogenesis in the upper aerodigestive tract is influenced by multiple factors. Besides tobacco and alcohol consumption, specific pollutants such as phthalates, nitrosamines, and polycyclic aromatic carbohydrates may be important in tumor initiation. Genetic factors related to mutagen sensitivity and DNA repair capacity also play a role. The aim of this study was to investigate whether human peripheral blood lymphocytes and mucosal epithelium of the upper aerodigestive tract, the target for volatile and liquid xenobiotics, are equally sensitive to genotoxic agents.The Comet assay was used to detect for DNA damage induced by genotoxic agents in mucosal epithelial cells and peripheral blood lymphocytes of 60 volunteers. Mucosa was harvested from larynx, oropharynx, and inferior nasal turbinates. Xenobiotics investigated were dibutylphthalate (DBP), diisobutylphthalate (DiBP), N'-nitrosodiethylamine (NDELA), benzo[a]pyrene (B[a]P), and N'-methyl-N'-nitro-N-nitrosoguanidine (MNNG). DBP, DiBP, B[a]P, NDELA and MNNG induced a significant increase in DNA migration in both cell populations. Peripheral blood lymphocytes were more sensitive than mucosal cells to DBP and DiBP, but not to NDELA and B[a]P. The correlation, in terms of DNA migration, between lymphocytes and mucosal cells among volunteers was relatively poor. Based on the poor correlation in response between the two cell types, the sensitivity of peripheral blood lymphocytes to genotoxic agents appears to be a poor predictor of sensitivity in the target cells of the upper aerodigestive tract. Further attention should be focused on intra-individual mutagen sensitivities and inter-individual genetic differences as regards susceptibility to upper aerodigestive tract cancer.     

Evaluation of DNA damage by the Comet assay in shoe workers exposed to toluene and other organic solvents

The alkaline single-cell gel electrophoresis (or Comet) assay was applied to evaluate DNA damage in cryopreserved peripheral blood mononuclear leukocytes from 34 female shoe workers exposed to organic solvents and a group of 19 non-exposed women. We also investigated whether the polymorphisms of glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) genes affect individual level of DNA damage possibly induced by the solvent exposure. Chemical measurements of workplace air in the two factories studied showed that the workers were exposed to acetone, gasoline, and toluene in both factories and to ethylacetate and diisocyanate in one factory. In the exposed workers, the average level of blood hemoglobin was lower and that of urinary hippuric acid higher than in the non-exposed individuals. However, the occupational exposure to organic solvents did not affect the Comet values. Neither did age, smoking, or the GSTM1 genotype have any effect on the outcome of this assay. The low prevalence of the GSTT1-null genotype precluded conclusions on the influence of GSTT1 polymorphism.     

Comparative study with the alkaline Comet assay and the chromosome aberration test

The alkaline Comet assay is becoming a useful tool for early genotoxicity testing of new pharmaceutical drug candidates. The aim of this study was to elucidate the predictive value of Comet assay results for the outcome of the chromosome aberration (CA) test. For this purpose, a validation exercise with 13 drug candidates was carried out utilizing V79 Chinese hamster cells and human lymphocytes. The study demonstrates that results of the Comet assay and the chromosome aberration test show a high degree of agreement, irrespective of the cell type used. In the Comet assay, seven compounds were positive and six were negative, while in the CA test, six were positive and seven were negative. The only discrepancy was found with one compound that was positive in the Comet assay with V79 cells, negative in the Comet assay with human lymphocytes and clearly negative in the CA test with human lymphocytes. For the selection of concentrations for testing in the Comet assay, cytotoxicity by means of cell count after incubation or viability by means of Trypan-blue dye exclusion (TBDE) were used. The results show that either parameter led to analysis of a concentration range in the Comet assay similar to that chosen in the CA test, in which cell count (when using V79 cells) or mitotic index (in case of lymphocytes) were used. However, since cell count after incubation of cells is much more labour-intensive, viability was preferred as the parameter to assess cytotoxicity and for selecting concentrations for analysis in the Comet assay. The data presented in this study may contribute the regulatory acceptance of the Comet assay, e.g. for mechanistic studies.     

Morphometric and meristic variation in ricefishes,genusOryzias: a comparison with cytogenetic data

Morphometric and meristic data from type and other material of ricefishes of the genusOryzias, particularly onO. latipes andO. curvinotus, were retaken to compare relationships of ricefishes indicated by morphological data with the relationships indicated by the cytogenetic analyses (Uwa, 1986). Along with morphometric and meristic characters traditionally used, two morphometric characters are useful for the classification of this genus: the relative length of the abdominal part and the relative position of the dorsal-fin origin. External anatomical data mostly agree with a classification of ricefishes based on karyology; although, like the cytogenetic data, they are not sufficient to distinguish all species, or to define monophyletic groups.Oryzias latipes is widespread in eastern Asia.Oryzias curvinotus, a name applied to ricefish from Hainan Island, is distributed more widely in southern China, including Hainan, Guangdong, Hong Kong, and Hanoi.     

Comet assay: a reliable tool for the assessment of DNA damage in different models

New chemicals are being added each year to the existing burden of toxic substances in the environment. This has led to increased pollution of ecosystems as well as deterioration of the air, water, and soil quality. Excessive agricultural and industrial activities adversely affect biodiversity, threatening the survival of species in a particular habitat as well as posing disease risks to humans. Some of the chemicals, e.g., pesticides and heavy metals, may be genotoxic to the sentinel species and/or to non-target species, causing deleterious effects in somatic or germ cells. Test systems which help in hazard prediction and risk assessment are important to assess the genotoxic potential of chemicals before their release into the environment or commercial use as well as DNA damage in flora and fauna affected by contaminated/polluted habitats. The Comet assay has been widely accepted as a simple, sensitive, and rapid tool for assessing DNA damage and repair in individual eukaryotic as well as some prokaryotic cells, and has increasingly found application in diverse fields ranging from genetic toxicology to human epidemiology. This review is an attempt to comprehensively encase the use of Comet assay in different models from bacteria to man, employing diverse cell types to assess the DNA-damaging potential of chemicals and/or environmental conditions. Sentinel species are the first to be affected by adverse changes in their environment. Determination of DNA damage using the Comet assay in these indicator organisms would thus provide information about the genotoxic potential of their habitat at an early stage. This would allow for intervention strategies to be implemented for prevention or reduction of deleterious health effects in the sentinel species as well as in humans. IITR Communication No. 2656     

Comet assay and analysis of micronucleus formation in patients with rheumatoid arthritis

Oxidants play a significant role in causing oxidative stress, which underlies the pathogenesis of rheumatoid arthritis (RA). Genetic factors that predispose individuals to RA are considered to play an important role in the development of the disease. The aim of this study was to determine, by use of the comet assay and the micronucleus (MN) test, whether DNA damage has an effect on the pathogenesis of RA. Furthermore, our aim was to show if there is an association between oxidative stress and DNA damage in RA. This study was conducted between January and June 2010 in the Erzurum Training and Research Hospital. We analyzed lymphocytes from patients with RA (12 in active and 31 in inactive periods) and 30 healthy controls for effects in the comet assay and the MN test. In addition, the levels of malondialdehyde (MDA) and superoxide dismutase (SOD), the activity of glutathione peroxidase (GSH-Px), the erythrocyte sedimentation rate (ESR) and the high-sensitivity C-reactive protein (hs-CRP) rate were determined in all the subjects. The comet-tail length, the MN frequencies and the MDA levels were significantly higher in patients--both in the active and the inactive period--than in the controls. In contrast, the SOD and GSH-Px levels were significantly lower in both patient groups than in the controls. Our results suggest that an increased plasma MDA level and decreased plasma GSH-Px and SOD levels reflect the higher degree of oxidative stress in RA patients, a situation that may impair genetic stability in those patients. Thus, the results suggest that increased DNA damage may play an important role in the pathogenesis of RA.     

Propidium iodide-based methods for monitoring drug action in the kinetoplastidae: comparison with the Alamar Blue assay

The urgent need for new drug development for African trypanosomiasis is widely recognized. This requires reliable and informative high-throughput assays. Currently, drug action is determined with a fluorimetric/colorimetric assay based on the metabolism of the dye Alamar Blue (resazurin) by live cells. However, this assay does not easily distinguish between cell death and growth arrest, or supply information about the rate at which test compounds affect these parameters. We report here an alternative fluorimetric assay, based on the interaction of propidium iodide with DNA, that allows either real-time monitoring of cell viability or the generation of EC₅₀ values at a predetermined time-point. The assay is highly sensitive and fluorescence readings easily correlate to numbers of parasites or DNA content. The EC₅₀ values were highly similar to those obtained with the standard Alamar Blue assay. The procedure lends itself readily to applications in drug development or resistance monitoring.     

Validation of a testosterone and dihydrotestosterone liquid chromatography tandem mass spectrometry assay: Interference and comparison with established methods

Testosterone (T) and its metabolite dihydrotestosterone (DHT) are androgens with different biologic profiles. T and DHT measurements are required for assessment of patients with ambiguous genitalia, hirsutism, during 5 alpha reductase treatment of prostate disorders, and new androgen formulations. Our laboratory has developed and validated a method to simultaneously measure serum T and DHT with liquid chromatography tandem mass spectrometry (LC-MS/MS) for use in a clinical chemistry laboratory. Analysis of sera from blood collected in tubes containing clot activator gave results of T that were fourfold higher than blood collected in plain tubes. Changing the ion pair selected for monitoring eliminated this interference by clot activators. Blood collected in fluoride-coated tubes gave serum T and DHT levels that were 20 and 15% lower, respectively than levels measured in blood collected in plain tubes (no additives). Addition of T enanthate to blood collected in plain tubes caused a dose related increase serum T levels due to the action of non-specific esterases in the red cells. This esterase activity could be avoided by using fluoride tubes for blood collection. Serum DHT levels were consistently lower when measured by LC-MS/MS versus radioimmunoassay. The differences were concentration dependent and the variance for the difference was large when serum DHT concentration was low. Celite chromatograph prior to radioimmunoassay reduced the differences between the two methods, thus confirming that higher levels of DHT obtained by immunoassays were probably due to interfering substances which were partially removed by Celite chromatography.     

Comet assay using mullet (Mugil sp.) and sea catfish (Netuma sp.) erythrocytes for the detection of genotoxic pollutants in aquatic environment

The development of comet assay for aquatic organisms is of particular relevance in light of the importance of coastal fisheries to several countries around the world. Two of the most common fish species native to southern Brazil are the gray mullet (Mugil sp.) and sea catfish (Netuma sp.) for which we have produced a standardized comet assay using whole erythrocytes taken from samples of these fish. We investigated the potential of the comet assay for monitoring genotoxicity in mullet and sea catfish and made a preliminary investigation of the baseline levels of DNA damage in the erythrocytes of samples of these fish from non-polluted areas as well as assessing the in vitro sensitivity of erythrocyte exposed to 2, 4 and 8 x 10(-5) M of methyl methanesulfonate (MMS) for 1, 2, 6 and 24h at 25 and 37 degrees C. Our results show that there was an increase in baseline DNA damage at higher temperatures and that the amount of MMS-induced DNA damage also increased at higher temperatures and that there was a clear dose/time response to treatment with MMS. To assess the possibility of using fish for environmental biomonitoring we also used the comet assay to investigate the in vitro genotoxic effect of MMS on whole blood cells from human donors and found a clear concentration-related effect at all exposure times, findings which agree with those of other workers. This study demonstrates the potential application of the comet assay to erythrocytes of mullets and sea catfish. However, these findings also suggest that temperature could alter both baseline DNA damage in untreated animals and in vitro cell sensitivity towards genotoxic pollutants.