Comet Assay Protocol Research Articles

DNA damage in zebrafish larvae induced by exposure to low-dose rate γ-radiation: detection by the alkaline comet assay

This study has determined the sensitivity of the alkaline comet assay for the detection of strand breaks in the DNA of cells taken from a whole organism rather than a single cell type as in previously reported studies. The assay has been performed on cells from whole zebrafish larvae irradiated for 1 or 24 h at dose rates of 0.4, 1.2 or 7.2 mGy/h. Zebrafish larvae exposed to only 1.2 mGy/h of γ-radiation for 1 h showed a statistically significant increase in DNA damage compared to controls. This represents a high sensitivity of this animal model for DNA damage and of the comet assay protocol used for detecting such damage. Increasing the exposure time from 1 to 24 h caused significant increases in DNA damage in zebrafish larvae, although the modest size of these increases in damage for the relatively large increases (24 times) in total absorbed dose indicates that dose rate may be the major factor in determining the level of DNA damage observed under the conditions of these experiments.

Comparison of DNA damage detected by plant comet assay in roadside and non-roadside environments

Although mixed air pollutants generated from traffic are suspected as one of the causes of DNA damage in living species, effects of the combination of these pollutants and other micro-environmental factors on urban biota have not been clarified yet. Thus, this study mainly aimed to detect the genetic damages in selected plant biomonitors, which were ginkgo ( Ginkgo biloba), pohtos ( Epipremnum aureum), and periwinkle ( Vinca rosea), setting in roadside and non-roadside environments. Two monitoring positions were selected in the Hongo campus of the University of Tokyo, Japan. This area was categorized as the urban residential zone. Both roadside and non-roadside samples were analyzed by using comet assay protocol. Different distribution changes in DNA migration ratios of all species could be observed and further interpreted as percentages of DNA damage. For all test species, in the final stage of experiment, roadside samples showed significantly higher degrees of DNA damage than non-roadside one. Time-dependent response pattern of each species to the overall environmental stresses was performed. Increase in the percentages of DNA damage could be expressed by regression equations. In addition, ratio of percentage of DNA damage between roadside and non-roadside species (R/N ratio) was introduced in order to clarify the additional genetic effect caused by roadside air pollutants. Series of response phase of plant under stresses, including cell destabilization, damage intensification, and re-stabilization, were explained. This study might be applied as a preliminary method in urban air quality assessment for detecting the existing effects of air pollutants and micro-environmental stress in an urban ecosystem.

DNA damage and repair in Arabidopsis thaliana as measured by the comet assay after treatment with different classes of genotoxins

The three protocols of the comet assay A/N, A/A and N/N were for the first time applied to the plant species Arabidopsis thaliana. The purpose of the experiments was to establish conditions for genotoxic exposure causing DNA damage in Arabidopsis nuclei. This is required for comprehensive gene expression profiling with the intention to screen for genes involved in response of Arabidopsis cells to genotoxic stress. Five chemicals belonging to different classes of mutagens (the monofunctional alkylating agents N-methyl- N-nitrosourea and methyl methanesulfonate, the polyfunctional alkylating agent mitomycin C, the radiomimetic bleomycin and the herbicide maleic hydrazide) were tested. Except for maleic hydrazide, dose-dependent increases in DNA damage were found using the A/N comet assay protocol. While a rapid repair of bleomycin-mediated SSBs and DSBs was found, no significant reduction of DNA migration was observed up to 48 h after treatment with the monofunctional alkylating agents.

Analysis of chromate-induced DNA-protein crosslinks with the comet assay

Modifications of the comet assay have been introduced to measure crosslinks by determining the reduction of induced DNA migration. Our previous results indicated that the modified protocol of the alkaline comet assay is a sensitive tool for the detection of formaldehyde-induced DNA-protein crosslinks. But results for mitomycin C and cisplatin suggested that the modified protocol is not well suited for the evaluation of DNA–DNA crosslinkers. We now used the comet assay to investigate in V79 cells the effect of potassium chromate (K 2CrO 4), another DNA-protein crosslinker, to see whether the results obtained for formaldehyde can be generalized. However, chromate did not reduce spontaneous or radiation-induced DNA migration in the alkaline (pH 13) comet assay but led to a small but significant induction of DNA migration. A crosslinking effect of chromate could also not be detected with the alkaline comet assay after postincubation of cells in normal medium after chromate treatment to enable repair of other (migration-inducing) lesions that might mask the crosslinking effect. Exposure of slides to proteinase K further increased DNA migration of chromate-treated cells, thus indicating the presence of DNA-protein crosslinks. In contrast to the alkaline comet assay, a “neutral” version at pH 9 was suited to demonstrate reduced induction of DNA migration after γ-irradiation of chromate-treated cells. The crosslinking effect was seen immediately at the end of the chromate treatment as well as after a 3 h postincubation period. Using the “neutral” protocol in combination with proteinase K, we were able to demonstrate the presence of DNA-protein crosslinks as the probable cause for the migration-reducing effect. Further investigations will have to show whether this protocol can be recommended as a universal approach for the detection of DNA-protein crosslinks and also of DNA–DNA crosslinks with the comet assay.

The Comet Assay with Multiple Mouse Organs: Comparison of Comet Assay Results and Carcinogenicity with 208 Chemicals Selected from the IARC Monographs and U.S. NTP Carcinogenicity Database

ABSTRACTThe comet assay is a microgel electrophoresis technique for detecting DNA damage at the level of the single cell. When this technique is applied to detect genotoxicity in experimental animals, the most important advantage is that DNA lesions can be measured in any organ, regardless of the extent of mitotic activity. The purpose of this article is to summarize the in vivo genotoxicity in eight organs of the mouse of 208 chemicals selected from International Agency for Research on Cancer (IARC) Groups 1, 2A, 2B, 3, and 4, and from the U.S. National Toxicology Program (NTP) Carcinogenicity Database, and to discuss the utility of the comet assay in genetic toxicology.Alkylating agents, amides, aromatic amines, azo compounds, cyclic nitro compounds, hydrazines, halides having reactive halogens, and polycyclic aromatic hydrocarbons were chemicals showing high positive effects in this assay. The responses detected reflected the ability of this assay to detect the fragmentation of DNA molecules produced by DNA single strand breaks induced chemically and those derived from alkali-labile sites developed from alkylated bases and bulky base adducts. The mouse or rat organs exhibiting increased levels of DNA damage were not necessarily the target organs for carcinogenicity. It was rare, in contrast, for the target organs not to show DNA damage. Therefore, organspecific genotoxicity was necessary but not sufficient for the prediction of organ-specific carcinogenicity. It would be expected that DNA crosslinkers would be difficult to detect by this assay, because of the resulting inhibition of DNA unwinding. The proportion of 10 DNA crosslinkers that was positive, however, was high in the gastrointestinal mucosa, stomach, and colon, but less than 50% in the liver and lung. It was interesting that the genotoxicity of DNA crosslinkers could be detected in the gastrointestinal organs even though the agents were administered intraperitoneally.Chemical carcinogens can be classified as genotoxic (Ames test-positive) and putative nongenotoxic (Ames test-negative) carcinogens. The Ames test is generally used as a first screening method to assess chemical genotoxicity and has provided extensive information on DNA reactivity. Out of 208 chemicals studied, 117 are Ames test-positive rodent carcinogens, 43 are Ames test-negative rodent carcinogens, and 30 are rodent noncarcinogens (which include both Ames test-positive and negative noncarcinogens). High positive response ratio (110/117) for rodent genotoxic carcinogens and a high negative response ratio (6/30) for rodent noncarcinogens were shown in the comet assay. For Ames test-negative rodent carcinogens, less than 50% were positive in the comet assay, suggesting that the assay, which detects DNA lesions, is not suitable for identifying nongenotoxic carcinogens. In the safety evaluation of chemicals, it is important to demonstrate that Ames test-positive agents are not genotoxic in vivo. This assay had a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic noncarcinogens, suggesting that the comet assay can be used to evaluate the in vivo genotoxicity of in vitro genotoxic chemicals. For chemicals whose in vivo genotoxicity has been tested in multiple organs by the comet assay, published data are summarized with unpublished data and compared with relevant genotoxicity and carcinogenicity data.Because it is clear that no single test is capable of detecting all relevant genotoxic agents, the usual approach should be to carry out a battery of in vitro and in vivo tests for genotoxicity. The conventional micronucleus test in the hematopoietic system is a simple method to assess in vivo clastogenicity of chemicals. Its performance is related to whether a chemical reaches the hematopoietic system. Among 208 chemicals studied (including 165 rodent carcinogens), 54 rodents carcinogens do not induce micronuclei in mouse hematopoietic system despite the positive finding with one or two in vitro tests. Forty-nine of 54 rodent carcinogens that do not induce micronuclei were positive in the comet assay, suggesting that the comet assay can be used as a further in vivo test apart from the cytogenetic assays in hematopoietic cells. In this review, we provide one recommendation for the in vivo comet assay protocol based on our own data.

Comparative evaluation of the genotoxic properties of potassium bromate and potassium superoxide in V79 Chinese hamster cells

The genotoxic potential of two oxidizing compounds, potassium bromate and potassium superoxide, was comparatively tested in various genotoxicity tests with V79 Chinese hamster cells. Both substances clearly induced cytotoxicity, chromosome aberrations and increased DNA migration in the alkaline comet assay. Using a modified comet assay protocol with FPG protein, a DNA repair enzyme which specifically nicks DNA at sites of 8-oxoguanines and formamidopyrimidines, we detected oxidative DNA base damage only after potassium bromate treatment. HPLC analysis also revealed significantly increased levels of 8-oxodeoxyguanosine after potassium bromate treatment but not after potassium superoxide treatment. Furthermore, potassium bromate clearly induced gene mutations at the HPRT locus while potassium superoxide only had a small effect on HPRT mutant frequencies. Molecular analysis of potassium bromate-induced mutations indicated a high portion of deletion mutations. Three out of four point mutations were G to T transversions which typically arise after replication of 8-oxoguanine. Our results suggest that the two oxidizing compounds induce specific patterns of genotoxic effects that reflect the types of DNA alterations induced by different reactive oxygen species (ROS).

Detection of crosslinks with the comet assay in relationship to genotoxicity and cytotoxicity.

The alkaline comet assay is a sensitive test for the detection of a variety of DNA lesions. However, crosslinks are not readily detected under standard test conditions. Recently, modifications have been introduced measuring crosslinks by determining the reduction of induced DNA migration. We used the comet assay to comparatively investigate in V79 cells the effect of three different crosslinkers: formaldehyde (FA), which predominantly induces DNA-protein crosslinks, cisplatin (DDP), which mainly produces DNA-DNA-intrastrand crosslinks, and mitomycin C (MMC), which mainly leads to DNA-DNA-interstrand crosslinks. In the standard alkaline comet assay, only MMC induced a slight increase in DNA migration at high toxic concentrations. FA and DDP did not induce any DNA migration under the test conditions used. In the modified comet assay, all three crosslinkers led to a clear reduction of gamma-ray-induced DNA migration. This reduction was seen in the case of FA parallel to the induction of cytotoxicity and SCE, while for MMC and DDP induction of cytotoxicity, SCE and HPRT gene mutations occurred at much lower concentrations than the effects in the comet assay. The DNA-DNA crosslinkers caused a reduction of induced DNA migration only at cytotoxic concentrations. Our results indicate that the modified comet assay protocol is a sensitive test for the detection of DNA-protein crosslinks. However, the results for MMC and DDP suggest that the modified protocol is not well suited for the evaluation of DNA-DNA crosslinkers. Furthermore, the relationship between crosslinking and genotoxicity seems to be very different for the three different types of crosslinking substances.

Evaluation of the genotoxic properties of paraquat in V79 Chinese hamster cells

The genotoxic potential of the herbicide paraquat (PQ), an intracellular generator of superoxide, was comparatively tested in various genotoxicity tests with V79 Chinese hamster cells. PQ clearly induced cytotoxicity and chromosome aberrations but did not induce gene mutations at the HPRT locus or increased DNA migration in the comet assay under the same treatment conditions. Using a modified comet assay protocol with formamidopyrimidine–DNA glycosylase (FPG) protein, a DNA repair enzyme which specifically nicks DNA at sites of 8- oxo-guanines and formamidopyrimidines, we could not detect oxidative DNA base damage after PQ treatment. When cells were treated directly on the slides after lysis (i.e., after the cell membrane barrier was eliminated), increased DNA migration was observed after treatment with high PQ-concentrations. Our results suggest that PQ does not significantly induce DNA lesions relevant for HPRT gene mutations in cultivated V79 cells. Since PQ-induced chromosome aberrations only occur after treatment with high concentrations which totally prevent cell survival and are not preceded by an induction of DNA strand breakage in intact cells, their biological significance has to be questioned.

A new method for the enrichment of single renal proximal tubular cells and their first use in the comet assay

A protocol was developed to isolate and enrich single renal proximal tubular cells, performing the following steps: in situ kidney perfusion; isolation of renal tissue pieces by collagenase digestion; selective enrichment of proximal tubular fragments by Percoll gradient centrifugation; and isolation of single proximal tubular cells by digestion of proximal tubular fragments with trypsin. The mean enrichment rate, determined by the glucose-6-phosphatase staining method, was 78.9% with a mean cell viability of 93.8%. After modification of the comet assay protocol, genotoxicity in proximal tubular cells could be investigated. A dose-dependent genotoxic effect of ethyl methanesulphonate in these cells was proven.