Comparative genotoxicity investigation using comet and gammaH2AX assays for screening of genotoxicants in HepG2 human hepatoma cells

As public interest in safety has increased the toxicity evaluation of chemicals become more important. In this study, the DNA-damaging effect of genotoxicants was examined in HepG2 cell line originated from human hepatocellular carcinoma by widely used genotoxicity assays: the comet assay and gammaH2AX immunostaining. Four different direct/indirect genotoxicants were tested in dose-/time-dependent manner. The comet assay and the gammaH2AX immunostaining enables detection of DNA damages in the form of DNA strand breaks with different sensitivity. Therefore, the combination of comet assay and gammaH2AX immunostaining will be complementary tool for evaluation of various forms and degree of DNA damage. Our result also suggested that HepG2 cells could be a suitable model for assessing the genotoxicity of various mutagens and for determining the lowest genotoxic concentration. Further analysis using a larger number of chemicals is warranted to determine the sensitivity and the specificity of HepG2 with in vitro genotoxicity test.     

DNA damage in workers exposed to lead using comet assay

Lead (Pb) is a ubiquitous and toxic metal. Secondary Pb recovery unit workers are prone to possible occupational Pb exposure. Hence, this investigation was conducted to assess the genotoxic effect of Pb exposure in these workers. In the study, 45 workers were monitored for DNA damage in blood leucocytes. Simultaneously 36 subjects were used as control group in this study. All the subjects were estimated for Pb content in whole blood by ICP-MS. The alkali single cell gel electrophoresis assay (comet assay) was adopted for detecting the DNA damage. The air inside the premises of the unit had Pb concentrations of 4.2 microg/m(3). The level of DNA damage was determined as the percentage of cells with comets. The mean Pb content was found to be significantly higher in the study group (248.3 microg/l) when compared with the controls (27.49 microg/l). Significantly more cells with DNA damage (44.58%) were observed in the study group than in the control persons (21.14%). Smoking had a significant effect on DNA damage in the control group whereas an insignificant effect was noticed in the exposed workers. Study as well as the control group failed to show a significant effect on DNA damage with age (P>0.05). Pb content and years of exposure significantly correlated with DNA damage in the study group (r=0.602, r=0.690; P<0.01). The increased levels of DNA damage observed in the exposed workers, justifies the use of the comet assay for the evaluation of genotoxic effects in humans exposed to Pb.     

彗星试验检测7种化合物对外周血淋巴细胞DNA的损伤

目的：检测过氧化氢（Ｈ２Ｏ２）、甲磺酸乙酯（ＥＭＳ）、丝裂霉素Ｃ（ＭＭＣ）、二甲基亚硝胺（ＤＭＮＡ）、苯并（ａ）芘（ＢａＰ）、２－氨基Ｗｕ（２－ＡＦ）和环磷酰胺（ＣＰ）诱发小鼠、大鼠及人外周血淋巴细胞ＤＮＡ单链断裂。方法：体外单细胞微量凝胶碱性电泳试验（慧星试验）。结果：除ＥＭＳ０．９７ｍｍｏｌ·Ｌ＾－１在小鼠淋巴细胞，ＭＭＣ３０μｍｏｌ·Ｌ＾－１在小鼠、人淋巴细胞中呈阴性外，其余均为阳性。最低可     

A comparative study of chemically induced DNA damage in isolated nasal mucosa cells of humans and rats assessed by the alkaline comet assay

Single-cell microgel electrophoresis (comet) assay was used to study genotoxic effects in human nasal mucosa cells and rat nasal and ethmoidal mucosa cells in vitro. Human cells were obtained from tissue samples of 10 patients (3 females/7 males), who underwent surgery (conchotomy) for treatment of nasal airway obstruction. Rat nasal mucosa cells were derived from male Sprague-Dawley rats. Cells were exposed for 1 h to either N-nitrosodiethanolamine (NDELA), epichlorohydrin (EPI), 1,2-epoxybutane (EPB), ethylene dibromide (EDB), or 1,2-dibromo-3-chloropropane (DBCP). Dimethyl sulfoxide (DMSO) was used as negative control. Alkaline comet assay was performed according to a standard protocol and DNA damage was quantified as Olive tail moment using image analysis system. All test substances induced an increase in DNA damage in human and rat cells. The absolute amount of DNA damage in rat nasal mucosa cells was usually higher than in ethmoidal mucosa cells. Human nasal mucosa cells were found to be less sensitive than rat mucosa cells to the genotoxic activities of DBCP (lowest effective concentration in human cells [LEC(human)]: 1.5, in rat cells [LEC(rat)]: 0.01 mM) and NDELA (LEC(human): 25, LEC(rat): 12.5 mM), whereas EPB-treated cells were almost equal (LEC(human) and LEC(rat) 0.78 mM). NDELA induced a marked concomitant cytotoxicity. For EPI (LEC(human) and LEC(rat): 0.097 mM) and EDB (LEC(human): 0.195, LEC(rat): 0.048 mM), pronounced interindividual differences were observed in human samples.     

Detection of DNA damage in peripheral lymphocytes by 7 compounds using comet assay

目的：检测过氧化氢（Ｈ２Ｏ２）、甲磺酸乙酯（ＥＭＳ）、丝裂霉素Ｃ（ＭＭＣ）、二甲基亚硝胺（ＤＭＮＡ）、苯并（ａ）芘（ＢａＰ）、２氨基芴（２ＡＦ）和环磷酰胺（ＣＰ）诱发小鼠、大鼠及人外周血淋巴细胞ＤＮＡ单链断裂．方法：体外单细胞微量凝胶碱性电泳试验（慧星试验）．结果：除ＥＭＳ０９７ｍｍｏｌ·Ｌ－１在小鼠淋巴细胞，ＭＭＣ３０μｍｏｌ·Ｌ－１在小鼠、人淋巴细胞中呈阴性外，其余均为阳性．最低可检测浓度分别为Ｈ２Ｏ２１μｍｏｌ·Ｌ－１，ＥＭＳ０４８ｍｍｏｌ·Ｌ－１，ＢａＰ５０μｍｏｌ·Ｌ－１，ＣＰ２０ｍｍｏｌ·Ｌ－１，ＭＭＣ１０μｍｏｌ·Ｌ－１，ＤＭＮＡ２７３ｍｍｏｌ·Ｌ－１，２ＡＦ６２５μｍｏｌ·Ｌ－１．ＣＰ、ＢａＰ、２ＡＦ需经Ｓ９Ｍｉｘ代谢活化才显示毒性．结论：彗星试验检测出ＭＭＣ诱导大鼠，ＥＭＳ诱导大鼠和人，以及Ｈ２Ｏ２、ＤＭＮＡ、ＢａＰ、ＣＰ和２ＡＦ诱导小鼠、大鼠和人外周血淋巴细胞ＤＮＡ单链断裂损伤．     

Overestimation of nanoparticles-induced DNA damage determined by the comet assay

The increasing use of engineered nanoparticles (NPs) in a wide range of commercial products raises concern about the possible risks that NPs pose to human health. Many aspects of the interaction between living cells and NPs are still unclear, and a reliable assessment of NP genotoxicity would be important. One of the most common tests used for genotoxicity is the comet assay, a sensitive method measuring DNA damage in individual cells. The assay was originally developed for soluble molecules, but it is also used in the assessment of genotoxicity of NPs. However, concerns have been raised recently about the reliability of this test in the case of NPs, but no conclusive results have been presented. Using nuclei isolated from human epithelial cells incubated with NPs, we obtained clear evidence of overestimation of NP genotoxicity by the comet assay in the case of CeO₂, TiO_2, SiO₂, and polystyrene NPs. Removal of the NPs in the cytoplasm was effective in eliminating this genotoxicity overestimation (ex post damage) and determining the actual damage produced by the NPs during incubation with the cells (ex ante damage). This method could improve significantly the determination of NP genotoxicity in eukaryotic cells.     

Evaluation of DNA damage induced by atrazine and atrazine-based herbicide in human lymphocytes in vitro using a comet and DNA diffusion assay.

Atrazine is one of the most widely used herbicides in the world. When applied, it is not used as a pure active ingredient but in the form of commercial formulations. Besides atrazine, these formulations contain other substances that might represent a risk to human health due to their mutual interactions. We evaluated the genotoxicity, apoptosis and necrosis induction of atrazine as an active ingredient, the commercial formulation Gesaprim, and a Gesaprim adjuvant mixture without atrazine by comet and DNA diffusion assay, respectively. Human lymphocytes were treated for 0.5, 1, 3, 5, and 8 h with 0.047 microg/ml, 0.47 microg/ml, 4.7 microg/ml of substances tested both in the presence and in the absence of an exogenous metabolic activator. Atrazine did not appear to be genotoxic or to be capable of inducing apoptosis or necrosis. Unlike atrazine, Gesaprime and the adjuvant mixture increased DNA damage in lymphocytes. After 5 h of treatment, it also increased the number of apoptotic cells. Metabolic activation additionally enhanced the DNA-damaging potential of Gesaprim and the adjuvant mixture but did not affect atrazine genotoxicity. Thus, both assay endpoints differed significantly for the active ingredient and formulation. To evaluate the potential health risk of simultaneous exposure to adjuvants and an active ingredient, further efforts using a biomonitoring approach should be made.     

DNA damage measured by comet assay and 8-OH-dG formation related to blood chemical analyses in aged rats

To evaluate the effects of aging on DNA damage, spontaneous and chemical-induced DNA damage and its repair were examined using comet assays at pH 9, 12.1 and 13, and an 8-OH-dG assay in the liver and kidney of young (9-week-old) and aged (20-month-old) rats. Additionally, blood chemistry was examined to investigate any correlation between vital functions and age-dependent DNA damage. DNA migration at pH 13 and 8-OH-dG levels increased in the liver and/or kidney of aged rats, but DNA migration did not increase at pH 9 or 12.1; that is, alkali-labile sites and 8-OH-dG were concomitantly accumulated in aged rats. These results suggest that 8-OH-dG production caused by reactive oxygen species exceeded glycosylation and that the glycosylation activity is far more than the AP endonucleation in aged rats. Methyl methanesulfonate (MMS, 80 mg/kg, i.p.) increased DNA migration at pH 12.1 and 13 in the liver and kidney at 3 and 24 hr after treatment in young and aged rats. The DNA damage in aged rats was less and decreased more slowly compared with young rats. The pictures of MMS-induced DNA migrations at pH 12.1 and 13 were very similar to each other. These results suggest that the adduct glycosylation and repair of the single-strand breaks (SSBs) of aged rats are less than those of young rats, although AP endonucleation is sufficient to remove the AP sites. N-nitrosodiethylamine (160 mg/kg, i.p.) increased DNA migration at pH 12.1 and 13 in the liver and kidney at 3 and 24 hr in young rats and at pH 12.1 and 13 in the kidney at 24 hr in aged rats. These results showed that SSBs were predominantly detected as chemical-induced DNA damage and DNA repairs such as N-glycosylase, DNA polymerase and DNA ligase, and that the metabolic activation declined in aged rats. Aspartate aminotransferase, alanine aminotransferase, total bilirubin, total cholesterol, total protein, globulin, creatinine and chloride age-dependently increased and alkaline phosphates, albumin/globulin ratio, inorganic phosphorus and potassium age-dependently decreased, and these changes were correlated with the DNA migration at pH 13 and/or 8-OH-dG. These results suggest that the activity of DNA repair and metabolic activation enzymes declines in aged rats and that the accumulation of spontaneous DNA damage may affect vital functions.     

彗星试验在检测甲氨蝶呤对小鼠精子DNA损伤中的应用

精子DNA损伤对人类生殖及其下一代健康的影响已越来越引起人们的重视,引起精子DNA损伤的因素很多,例如放射线、H2O2、化学药物等。甲氨蝶呤(Methotrexate,MTX)是一种二氢叶酸还原酶(dihydrofolate reductase,DHFR)抑制剂,常用于肿瘤的化疗,临床上长期低剂量应用MTX可引起月经不     

Detection of DNA damage in two cell lines from rainbow trout, RTG-2 and RTL-W1, using the comet assay

Screening methods to indicate the genotoxic potential of individual chemicals or environmental mixtures rely mainly on short-term bacterial tests. Differences in the genotoxic response of prokaryotic and eukaryotic cells necessitate the development of nonbacterial screening assays. A promising approach for this purpose could be the comet (single-cell gel electrophoresis) assay performed with fish cells in vitro. In the present study, we evaluated the comet assay with two different fish cell lines from rainbow trout (Oncorhyhnchus mykiss), the fibroblast-like RTG-2 cell line established from gonad tissue, and the epitheloid RTL-W1 cell line established from liver tissue. The cells were exposed in vitro during 2 hr to the genotoxins, 4-nitroquinoline-1-oxide (NQO), and benzo(a)pyrene (BaP), as well as to environmental samples. The LOEC values for NQO were similar in both cell lines, whereas for BaP, the RTL-W1 cells were found to be more sensitive than the RTG-2 cells. The slopes of the concentration-response curves of the two test compounds differed between the two cell lines, with RTG-2 cells showing a steeper slope for NQO, and RTL-W1 cells showing a steeper slope for BaP. When exposed to environmental samples from a remediation site, the RTL-W1 cell line, but not the RTG-2 cell line, indicated a genotoxic potential of the samples. The differences in the genotoxic response pattern of the two cell lines could be only partly explained in relation to metabolic enzymes, cytochrome P4501A, glutathione-S-transferase, and xenobiotic reductase. The findings of this study demonstrate that the comet assay with fish cell lines is suitable as in vitro screening assay in environmental genotoxicity testing, but the choice of test cell line may be critical.     

Ochratoxin A reduces aflatoxin B1 induced DNA damage detected by the comet assay in Hep G2 cells

Mycotoxins aflatoxin B1 (AFB1) and ochratoxin A (OTA) can be present together in food commodities. These food contaminants are considered to be genotoxins, acting by different mechanisms. The aim of this work was to characterize combined genotoxic in vitro effects of both mycotoxins in Hep G2 cells. For this purpose, cytotoxicity was first determined in isolated and combined treatments in order to determine the dose range of genotoxicity studies. Co-exposure of cells to OTA + AFB1 for 24 h resulted in additive effects. Genotoxicity was determined in Hep G2 cells by the modified comet assay with restriction enzymes (endo III and FPG). Significant reactive oxygen species formation was detected in both single and combined treatments. AFB1 was genotoxic after 3 h with external metabolic activation (S9 mix) and after 24 h without metabolic activation. Co-exposure to OTA significantly decreased DNA damage induced by AFB1, not only in breaks and apurinic sites but also in FPG-sensitive sites. The apparent contradiction between additive cytotoxic effects and antagonic genotoxic effects may be explained if AFB1 and OTA compete for the same CYPs, yielding more ROS but less AFB1 adducts.     

Leucocytes DNA damage in mice exposed to JS-118 by the comet assay

JS-118 is an extensively used insecticide in China. The present study investigated the genotoxic effect of JS-118 on whole blood at 24, 48, 72 and 96 h by using alkaline comet assay. Male Kunming mice were given 6.25, 12.5, 25, 50 and 100 mg/kg BW of JS-118 intraperitoneally. A statistically significant increase in all comet parameters indicating DNA damage was observed at 24 h post-treatment (p < 0.05). A clear concentration-dependent increase of DNA damage was revealed as evident by the OTM (arbitrary units), tail length (μm) and tail DNA (%). From 48 h post-treatment, a gradual decrease in mean comet parameters was noted. By 96 h of post-treatment, the mean comet tail length reached control levels indicating repair of damaged DNA. This study on mice showed different DNA damage depending on the concentration of JS-118 and the period of treatment. The present study provided further information of the potential risk of the genetic damage caused by JS-118.     

Assessment of DNA damage in C6 glioma cells after antidepressant treatment using an alkaline comet assay

The effects of acute (24 h) exposure to the antidepressants amitriptyline, imipramine (both tricyclics), fluoxetine (a selective serotonin re-uptake inhibitor) and tranylcypromine (a monoamine oxidase inhibitor) on DNA damage in cultured C6 rat glioma cells were determined using an alkaline comet assay. The effects of manipulation of intracellular cyclic AMP by pretreatment with dibutyryl cyclic AMP (dBcAMP) and 3-isobutyl-1-methylxanthine (IBMX) were also studied. For fluoxetine, the effects of addition of exogenous glutathione (GSH) and pretreatment with L-buthionine sulfoximine (BSO) were also assessed. There were increases in DNA damage with increasing concentrations of antidepressants. IBMX pretreatment protected against antidepressant-induced DNA damage in C6 cells pretreated with dBcAMP. Addition of exogenous reduced GSH and BSO increased DNA damage after fluoxetine exposure. The data show that the antidepressants induce significant amounts DNA damage in C6 cells.     

A comet assay study reveals that aluminium induces DNA damage and inhibits the repair of radiation-induced lesions in human peripheral blood lymphocytes

Although it is known that many metals induce DNA damage and inhibit DNA repair, information regarding aluminium (Al) is scarce. The aim of this study was to analyze the level of DNA damage in human peripheral blood lymphocytes treated with Al and the impact of Al on the repair of DNA damage induced by ionizing radiation. Cells were treated with different doses of aluminium chloride (1, 2, 5, 10 and 25 microg/ml AlCl(3)) for 72 h. The level of DNA damage and of apoptosis was determined by the comet assay. The level of oxidative damage was determined by the application of endonuclease III and formamidopyrimidine DNA glycosylase. The results on apoptosis were confirmed by flow cytometry. Based on the fluorescence intensity, cells were divided into cohorts of different relative DNA content that corresponds to G(1), S and G(2) phases of the cell cycle. Our results revealed that Al induces DNA damage in a dose-dependent manner, however, at the dose of 25 microg/ml the level of damage declined. This decline was accompanied by a high level of apoptosis indicating selective elimination of damaged cells. Cells pre-treated with Al showed a decreased repair capacity indicating that Al inhibits DNA repair. The possible mechanisms by which Al induces DNA damage and inhibits the repair are discussed.     

DNA damage and repair in human leukocytes exposed to styrene-7,8-oxide measured by the comet assay.

Styrene-7,8-oxide (SO) is produced by cytochrome p450 monooxygenases as the main mammalian metabolite of styrene, an important industrial chemical present at high concentrations in the ambient air of fiberglass-reinforced plastic plants. Previous studies have shown positive results for SO in the induction of several cytogenetic endpoints in vitro. In this work we have evaluated, by means of the comet assay, the potential of SO to act as a DNA damaging agent in human peripheral leukocytes and the ability of white blood cells to repair the DNA damage induced by this compound. Our results show that SO induces DNA damage at concentrations higher than 50 microM in a dose-dependent manner, and that the lesions produced by SO are efficiently removed within a few hours after the end of treatment.     

Comparative evaluation of DNA damage by genotoxicants in primary rat cells applying the comet assay

Various compounds known to cause DNA damage (hydrogen peroxide, visible light-excited methylene blue, N-nitrosomorpholine and benzo[a]pyrene) were tested with different primary rat cells (lymphocytes, testicular cells, type II pneumocytes and hepatocytes) to determine the range of induced DNA damage applying the comet assay. A dose-dependent increase of DNA breaks was observed after treatment with hydrogen peroxide in all cell types studied. The most prominent effect was observed in lymphocytes, whereas only a slight increase of DNA breaks was observed in hepatocytes. Visible light-excited methylene blue caused significant oxidative DNA damage, which did not significantly differ between the cell types used with the exception of hepatocytes, for which a lower level of DNA damage was observed. N-Nitrosomorpholine and benzo[a]pyrene induced a moderate but significant increase of DNA strand breaks in pneumocytes and hepatocytes while in lymphocytes no effect was observed. Our results clearly demonstrate that due to their differential function which is also expressed by the level of drug metabolizing and/or antioxidant enzymes, freshly isolated rat cells (lymphocytes, testicular cells, type II pneumocytes and hepatocytes) respond differently to the exposure to genotoxic agents as detected by comet assay.     

Detection of xenobiotic-induced DNA damage by the comet assay applied to human and rat precision-cut liver slices.

The comet assay is a simple and sensitive method for measuring DNA damage at the level of individual cells and is extensively used in genotoxicity studies. It is commonly applied to cultured cells. The aim of this study was to apply the comet assay for use in fresh liver tissue, where metabolic activity, all cell types and tissue architecture are preserved. The response of liver slices to genotoxic agents was tested with the reactive oxygen species generating tert-butyl hydroperoxide (tBOOH, 0.1-2 mM), [corrected] and the pro-carcinogens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ, 0.5-2 mM) and benzo(a)pyrene (BaP, 10-100 microM). Dose-dependent DNA damage was observed and compared to HepG2 cells. At non-cytotoxic concentrations of carcinogens, human liver slices were more sensitive to tBOOH than rat liver slices, while no significant difference was found for BaP and IQ. Human liver slices were more sensitive to IQ than HepG2 cells, equally sensitive to BaP and less to tBOOH. Control slices showed low levels of DNA damage, which did not increase during 24 h preservation (0 degrees C) or 48 h culturing (37 degrees C). In conclusion, the comet assay that we applied for measuring DNA damage in precision-cut liver slices is an useful tool to study genotoxic effects induced by various potential genotoxicants, allowing for detection of species differences in susceptibility to carcinogens.     

Assessment of DNA damage in workers occupationally exposed to pesticide mixtures by the alkaline comet assay

The potential genetic hazard of pesticides to human beings is of great concern in occupational and environmental settings because of the widespread use of these chemicals for domestic and industrial applications. Various studies have revealed a significantly elevated risk for particular tumours in humans exposed to some pesticides. Results from the biological monitoring or cytogenetic methods for the detection of health risks to pesticides have given both positive and negative results of mutagenicity. In this study DNA damage in peripheral lymphocytes of 33 pesticide-exposed workers employed in the municipality of Ankara (Turkey) for at least 1 year was examined by alkaline single-cell gel electrophoresis, the 'comet' technique. Results were compared with those from 33 controls of comparable age, sex and smoking habits, which were not occupationally exposed to pesticides. Work characteristics of the exposed workers and the use of personnel protective measures were also investigated. The DNA damage observed in lymphocytes of the workers was significantly higher than that in the controls ( P<0.001). The observed DNA damage was found to be significantly lower ( P<0.001) in workers applying some of the necessary individual safety protections during their work. Cigarette smoking was not related to increases in DNA damage; also, no significant association was found between the duration of occupational exposure to pesticides and the degree of DNA damage.     

Genotoxicity study with special reference to DNA damage by comet assay in fission yeast, Schizosaccharomyces pombe exposed to drinking water.

The objective of this study was to investigate genotoxicity, especially DNA damage, in drinking water samples collected from tap by using fission yeast Schizosaccharomyces pombe as a model organism. Generally raw water potabolization is done by treatment with polymeric coagulant, alum, chlorine, etc. In the comet test, highly significant (P<0.001) effects of DNA damage were detected in treated water (tap water) when compared to negative control (raw water) as well as laboratory control (distilled water) samples for both 1 h and 2 h exposure. In the water treatment plant, raw water treatment is done by the process of prechlorination, alum and polymeric coagulant (CatflocT) dosing, postchlorination, filtration and final discharge for consumption. In conclusion it can be stated from the results that chlorinated disinfectant, alum and polymeric coagulant (CatflocT) mixture used in drinking water has a potent cumulative genotoxic effect in the eukaryotic cells and may pose potential genotoxic risk for human health following long-term consumption.     

Assessment of DNA damage in car spray painters exposed to organic solvents by the high-throughput comet assay

Occupational exposure as a painter is associated with DNA damage and development of cancer. Comet assay has been widely adopted as a sensitive and quantitative tool for DNA damage assessment at the individual cell level in populations exposed to genotoxics. The aim of this study was to assess the application of the high-throughput comet assay, to determine the DNA damage in car spray painters. The study population included 52 car spray painters and 52 unexposed subjects. A significant increase in the %TDNA median (p?< 0.001) was observed in the exposed group in comparison to the unexposed group. Neither age (%TDNA: p?= 0.913) nor time of exposure (%TDNA: p?=?0.398) were significantly correlated with DNA damage. The car spray painters who consumed alcohol did not show a significant increase in DNA damage compared to nonalcohol consumers (p >?0.05). The results showed an increase in DNA breaks in car spray painters exposed to organic solvents and paints; furthermore, they demonstrated the application of high-throughput comet assay in an occupational exposure study to genotoxic agents.