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.     

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     

The alkaline comet assay: towards validation in biomonitoring of DNA damaging exposures

Generation of DNA damage is considered to be an important initial event in carcinogenesis. The single cell gel electrophoresis (comet) assay is a technically simple and fast method that detects genotoxicity in virtually any mammalian cell type without requirement for cell culture. This review discusses the strength of the comet assay in biomonitoring at its present state of validation. The simple version of the alkaline comet assay detects DNA migration caused by strand breaks, alkaline labile sites, and transient repair sites. By incubation with bacterial glycosylase/endonuclease enzymes, broad classes of oxidative DNA damage, alkylations, and ultraviolet light-induced photoproducts are detected as additional DNA migration. The most widely measured enzyme sensitive sites have been those detected by formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (ENDOIII). Reports from biomonitoring studies show that the basal level of DNA damage in leukocytes is influenced be a variety of lifestyle and environmental exposures, including exercise, air pollution, sunlight, and diet. Although not all types of carcinogenic exposures should be expected to damage DNA in leukocytes, the comet assay is a valuable method for detection of genotoxic exposure in humans. However, the predictive value of the comet assay is unknown because it has not been investigated in prospective cohort studies. Also, it is important that the performance of the assay is investigated in multi-laboratory validation trials. As a tool in risk assessment the comet assay can be used in characterization of hazards.     

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.     

应用彗星试验检测乌头类生物碱致细胞DNA损伤作用

目的:研究乌头类生物碱对细胞DNA的损伤作用,以期在分子水平进一步阐明乌头类生物碱的毒效作用特征及其分子机制.方法:以500、100、50和10μg·mL(-1)乌头碱、次乌头碱或新乌头碱分别染毒HepG2细胞1.5 h,应用24孔板彗星试验检测细胞DNA损伤.结果:乌头类生物碱作用组细胞平均拖尾长度和拖尾细胞率与生物碱浓度存在剂量反应关系,且100和50μg·mL(-1)作用组细胞平均拖尾长度与阴性对照组之间差异具有统计学意义(P<0.05).结论:乌头类生物碱具有细胞DNA损伤作用.     

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．     

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.     

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     

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.     

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.     

Detection of DNA damage in mouse peripheral blood leukocytes by the comet assay after oral administration of monocrotophos

The objective of this study was to determine if single/double strand DNA breaks could be induced by monocrotophos (organophosphorus pesticide) in mice in vivo using the comet assay. Mice were dosed orally with 0.046, 0.093, 0.186, 0.373 and 0.746 mg/kg body weight of monocrotophos, and the assay was performed on whole blood after 24, 48 and 72 h. A significant increase in mean comet tail length indicating DNA damage was observed at 24 and 48 h post-treatment with monocrotophos when compared to controls. A decrease in the mean tail length was observed at 72 h post-treatment indicating repair of the damaged DNA. The mean tail length showed a dose-related increase and time dependent decrease. The study reveals that comet assay is a sensitive and rapid method to detect genotoxicity of monocrotophos.     

Effect of cigarette smoking on DNA damage of human cumulus cells analyzed by comet assay

Cigarette smoking has been reported to induce intrafollicular oxidative stress that may lead to DNA damage. The purpose of this study was to determine damage in DNA in human cumulus cells caused by tobacco smoke in females who had received in vitro fertilization. The level of DNA damage in freshly isolated cumulus cells was determined by comet assay. Statistically significant increase (p<0.05) was observed in damaged nuclear DNA in smokers, both at basal level and after oxidative stress induced by hydrogen-peroxide. Since cumulus cells have an important role in oocyte maturation, ovulation and fertilization, this method could be used both as a test for the evaluation of the biological potential of the female reproductive system and as a direct means to measure certain toxic effects.     

The essential comet assay: a comprehensive guide to measuring DNA damage and repair

The comet assay (single cell gel electrophoresis) is the most common method for measuring DNA damage in eukaryotic cells or disaggregated tissues. The assay depends on the relaxation of supercoiled DNA in agarose-embedded nucleoids (the residual bodies remaining after lysis of cells with detergent and high salt), which allows the DNA to be drawn out towards the anode under electrophoresis, forming comet-like images as seen under fluorescence microscopy. The relative amount of DNA in the comet tail indicates DNA break frequency. The assay has been modified to detect various base alterations, by including digestion of nucleoids with a lesion-specific endonuclease. We describe here recent technical developments, theoretical aspects, limitations as well as advantages of the assay, and modifications to measure cellular antioxidant status and different types of DNA repair. We briefly describe the applications of this method in genotoxicity testing, human biomonitoring, and ecogenotoxicology.     

Micronucleus test and comet assay for the evaluation of zebrafish genomic damage induced by erythromycin and lincomycin

An enormous quantity of pharmacologically active principles are currently being introduced into the environment, with consequent escalation of environmental problems, but only a small number of studies are focusing on an assessment of their genotoxic effects. The aim of this article is to assess the genotoxic effects of erythromycin, lincomycin, and of a combination of these two antibiotics on the genome of the zebrafish. The genotoxicity of the two antibiotics was assessed by applying the micronucleus test to erythrocytes and performing a Comet assay on erythrocytes and hepatocytes. The fish were exposed to antibiotics at different concentrations and times of exposure, under standard laboratory conditions. Depending on the different experimental conditions, erythromycin and lincomycin induced a significant increase in DNA migration (tail moment) and a significant increase in micronuleus frequency. We also conducted an analysis on the activation of repair mechanisms when the genotoxic agent was removed. Only a few of the cells displayed a decrease in damage under these test conditions.     

DNA alkylation lesions and their repair in human cells: modification of the comet assay with 3-methyladenine DNA glycosylase (AlkD)

3-Methyladenine DNA glycosylase (AlkD) belongs to a new family of DNA glycosylases; it initiates repair of cytotoxic and promutagenic alkylated bases (its main substrates being 3-methyladenine and 7-methylguanine). The modification of the comet assay (single cell gel electrophoresis) using AlkD enzyme thus allows assessment of specific DNA alkylation lesions. The resulting baseless sugars are alkali-labile, and under the conditions of the alkaline comet assay they appear as DNA strand breaks. The alkylating agent methyl methanesulfonate (MMS) was used to induce alkylation lesions and to optimize conditions for the modified comet assay method with AlkD on human lymphoblastoid (TK6) cells. We also studied cellular and in vitro DNA repair of alkylated bases in DNA in TK6 cells after treatment with MMS. Results from cellular repair indicate that 50% of DNA alkylation is repaired in the first 60 min. The in vitro repair assay shows that while AlkD recognises most alkylation lesions after 60 min, a cell extract from TK6 cells recognises most of the MMS-induced DNA adducts already in the first 15 min of incubation, with maximum detection of lesions after 60 min’ incubation. Additionally, we tested the in vitro repair capacity of human lymphocyte extracts from 5 individuals and found them to be able to incise DNA alkylations in the same range as AlkD. The modification of the comet assay with AlkD can be useful for in vitro and in vivo genotoxicity studies to detect alkylation damage and repair and also for human biomonitoring and molecular epidemiology studies.     

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.     

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.     

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.     

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.