Use of the alkaline comet assay for industrial genotoxicity screening
Andreas HartmannAzeddine ElhajoujiEvangelos KiskinisFranziska PoetterHansjeorg MartusAnn FjällmanWilfried FrieauffWilli Suter
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Abstract The Berdan River, which empties into the Mediterranean Sea on the east coast of Turkey, receives discharges of industrial and municipal waste. In the present study, the in vivo piscine micronucleus (MN) test was used to evaluate the genotoxicity of water samples collected from different locations along the Berdan River. Nile tilapia ( Oreochromis niloticus ) were exposed in the laboratory for 2, 4, and 6 days, and micronuclei were evaluated in peripheral blood erythrocytes, gill cells, and caudal fin epithelial cells. A single dose of 5 mg/L cyclophosphamide was used as a positive control. In addition to micronuclei, nuclear abnormalities (NAs), such as binucleated cells and blebbed, notched, and lobed nuclei, were assessed in the erythrocytes, and chemical analyses were carried out to determine the amount of heavy metals in the water samples. MN and NA frequencies were significantly elevated (up to 2‐ to 3‐fold) in fish exposed to river water samples taken downstream of potential discharges, and the elevated responses in gill and fin cells were related to the concentration of heavy metals in the water. MN frequencies (expressed as micronucleated cells/1,000 cells), in both treated and untreated fish, were greatest in gill cells (range: 0.80–3.70), and generally lower in erythrocytes (range: 0.50–2.80), and fin cells (range: 0.45–1.70). The results of this study indicate that the Berdan River is contaminated with genotoxic pollutants and that the genotoxicity is related to the discharge of wastes into the river water. Environ. Mol. Mutagen., 2007. © 2007 Wiley‐Liss, Inc.
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The genotoxicity effects of different doses of triazophos on Hylarana guentheri tadpoles were evaluated by micronucleus test and single cell gel electrophoresis (SCGE, Comet assay). Results showed that exposure to triazophos at the doses as low as 0.2mg·L -1 for 72h and 1.2mg·L -1 for 24h resulted in strong significant increases in micronucleus frequency and nuclear anomalies frequency in the tadpoles. When the exposure time was fixed, the frequencies of micronucleus and nuclear anomalies increased with the increase of triazophos concentrations. When the concentration was fixed, the micronucleus frequencies increased with exposure time in groups treated with lower concentration of triazophos(≤ 0.4mg·L-1) while in groups treated with higher concentration of triazophos (≥1.2mg·L-1), the micronucleus frequencies first increased and then decreased. The results of the comet assay showed that after exposure to triazophos solution for 24h, the indices of the comet assay, such as Tail Length, Comet Length, Tail Moment, and Olive Tail Moment, increased significantly with the increase of triazophos concentrations, There were strong linear correlations between these indices and the concentrations of triazophos. Triazophos at concentration as low as 0.2mg·L-1 resulted in significant increases in Comet Assay Tail Factor of DNA damage in the tadpoles, compared with the control group. The results of this study indicated that triazophos could cause DNA damage in amphibian's tadpoles in a dose -dependent manner, and that the micronucleus assay and the Comet assay may be useful tools for measuring genotoxicity in tadpoles exposed to pollutants.
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Objective To compare the sensitivity of comet assay and cell-blocked micronucleus for studying the genotoxicity of extractable matter in air particulate matters. Methods Particulate matters (TSP, PM10)were sampled in urban Guangzhou and the DNA damage potential of extractable organics were study by both comet assay and cell-blocked micronucleus (CBMN) on human peripheral blood lymphocyte. Results At doses 0, 4, 12 and 20 m3 eq/ml significant dose-response effects were observed for comet assay while no dose-response effects were observed for the CBMN tests at same doses. Conclusion The results indicated that comet assay was much more sensitive than CBMN.
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The genotoxicity and interaction of CHCI_(3) and CCI_(4) were analyzed by micronucleus test in Vicia Faba root tip cells. CHCI_(3)(0.503-50.302μmol/L) and CCI_(4) 0.0195-1.9501μmol/L)significantly induced micronuclei in the Vicia Faba cells(P<0.01),respectively. A significant dose-response was found. A significant effects on micronucleus induction and interaction were observed when the solution containing 2 chemicals was used to treat the Vicia Faba cells. 2 chemicals showed some antagonistic effect. The results indicated that CHCI_(3) and CCI_(4) possess significant genotoxicity. The safety of maximum acceptable concentraton of CHCI_(3) and CCI_(4) in drinking water,was also discussed.
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In this paper, the genotoxicity of (1-(4-chlorophenyl)-3-(2,6-difluoro-benzoyl)urea) is investigated by a micronuclear test on Danio rerio, as a standard test object, at concentrations of 0.5, 1 and 2 mg/l. As a result of the work, a significant increase in the frequency of occurrence of micronuclei (0.73%) was found, while other nuclear anomalies in the maximum concentrations of erythrocytes were also significant. It was found that the frequency of micronuclei in concentrations of 0.5 and 1 mg/l on the fifth day of the experiment was the maximum, while at the maximum concentration (2 mg/l) the level of micronuclei was lower, which is probably due to toxic effects. An increase in the level of micronuclei may be associated with the genotoxic effect of DFB decay products. The genotoxicity results obtained using the micronucleus test method were contradictory. For this reason, it is necessary to conduct additional studies using the comet method or experiments on cell cultures.
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Comet assay and micronucleus test have been used increasingly to evaluate the genotoxicity of many metals and their organic compounds in aquatic ecosystems. The use of endemic aquatic organisms as biological sentinels has proved useful in environmental monitoring. In this study, the genetic damage caused by methylmercury (MeHg) in Aequidens tetramerus (commonly called acará-sela) was assessed using the comet assay and by testing for micronucleus and other nuclear abnormalities.Specimens were acclimatized in individual aquariums to laboratory conditions and then exposed to 2 mg l(-1)MeHg. Peripheral blood samples were obtained from specimens of A. tetramerus and subjected to the comet and micronucleus assays.The comet assay showed a significant increase of tailed nucleoids in the erythrocytes of fish treated with MeHg (p<0.0001). Our results in the micronucleus test also indicated that MeHg is potentially mutagenic (p<0.003), with more nuclear morphological alterations than typical micronuclei.The combination of both assays - comet and micronucleus - is adequate and advantageous for genotoxicity evaluation. The level of damage detected by comet assay was higher than that found in the micronucleus test and the damage index increased with a longer exposure of fish to this xenobiotic; such damage is often not inherited by future cellular generations and, for this reason, cannot be detected by the micronucleus assay. Our results also demonstrated that A. tetramerus is an adequate model for biological studies that evaluate genotoxic effects in aquatic environments.
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The genotoxicity of single-walled carbon nanotubes (SWCNTs) was determined using a battery of genotoxicity assays, comprising a bacterial reverse mutation test, an in vitro mammalian chromosomal aberration test and a mammalian erythrocytes micronucleus test. SWCNTs had no mutagenicity in S. typhimurium TA98, TA100, TA1535 or TA1537, or in E. coli WP2uvrA, in the absence or presence of metabolic activation. SWCNTs did not increase the number of structural or numerical chromosomal aberrations after short-term or continuous exposure. In the micronucleus test using CD-1 mice, SWCNTs did not affect the proportion of immature erythrocytes, the total proportion of erythrocytes or the number of micronuclei in immature erythrocytes. SWCNTs appear not to pose a genotoxic risk.
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