Effects of triclosan on acute toxicity, genetic toxicity and oxidative stress in goldfish (<i>Carassius auratus</i>)
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Triclosan (TCS) is used as an antimicrobial agent and has been widely dispersed and detected in the aquatic environment. However, it remains uncertain whether TCS is genotoxic or not. In this study, the acute toxicity of TCS in goldfish (Carassius auratus) was studied. Then, based on the results for acute toxicity, other goldfish were exposed to various concentrations of TCS (control, DMSO control, and 1/4, 1/2, and 1/8 LC50) for 14 days, and the effects on genetic toxicity were evaluated using micronucleus (MN) and nuclear abnormalities (NA) frequencies in peripheral blood and the comet assay in the liver of the goldfish. In addition, malondialdehyde (MDA), reduced glutathione (GSH), catalase (CAT), and total antioxidant capacity (T-AOC) in the liver were assayed to evaluate oxidative stress and the possible mechanism of genotoxicity. The 96 h median lethal concentration of TCS was 1111.9 µg/l. After 14 days of exposure, the MN and NA frequencies were significantly increased in peripheral blood of the TCS-treated groups compared with the solvent control, and the comet tail moment and MDA in the liver in the highest dose of TCS groups were also significantly high. Meanwhile, an evident change in GSH, CAT, and T-AOC of the liver was found as the TCS exposure concentration increased. The results showed that TCS caused oxidative stress and a genotoxic response in goldfish, suggesting that it presents a potential ecotoxicological risk to aquatic ecosystems.Keywords:
Malondialdehyde
Comet Assay
Carassius auratus
Indium Chloride‐induced Micronuclei in In Vivo and In Vitro Experimental Systems: Ryo Takagi, et al. Department of Public Health and Environmental Medicine, Jikei University School of Medicine— Objectives The aim of this study was to investigate the genotoxic effects of indium trichloride (InCl 3 ·4H 2 O; InCl 3 ) using the in vivo bone marrow micronucleus test and the in vitro CHL/IU cell micronucleus test. Method BALB/c mice were administered a single intraperitoneal (i.p.) injection of InCl 3 at a dose 0.625, 1.25, 2.5, 5, or 10 mg/kg b.w. The frequency of micronuclei, the ratio of polychromatic erythrocytes to normochromatic erythrocytes (P/N ratio) and body weight gain were determined 24 h after administration of the InCl In the in vitro micronucleus test, CHL/IU cells were treated continuously for 24, 48, or 72 h in the absence of S9mix (the continuous treatment method) and/or for 6 h with or without S9 mix followed by an 18, 42 or 66 h recovery time (the short time treatment method). The frequency of micronuclei was determined at the end of each culture period. Results The frequency of micronuclei induced by InCl 3 increased in the in vivo erythroblast‐erythrocyte micronucleus test using BALB/c mice at doses of 2.5 and 5 mg/kg b.w. The P/N ratio, a marker of bone marrow toxicity, decreased significantly following the injection of InCl 3 . Body weight gain was also inhibited by InCl 3 . InCl 3 induced micronuclei in the CHL/IU cell micronucleus test in both the continuous treatment method and the short time treatment method, both with and without S9mix. Conclusions These results suggest that InCl 3 has a genotoxic effect on mammalian cells both in vivo and in vitro.
Intraperitoneal injection
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The genotoxic potential of hexachlorocyclohexane (HCH) isomers (α-, β-, and γ-) which are organochlorine pesticides was tested in peripheral blood lymphocyte cultures from two donors by using the cytokinesis-block micronucleus assay. Micronucleus (MN) frequency, binucleated cells with micronucleus (BNMN), and cytokinesis-blocked proliferation index (CBPI) were determined as genotoxic and cytotoxic endpoints. At the concentration ranges tested (12.5-100 μg.L -1), all HCH isomers induced dose-dependent cytotoxic effects, γ-HCH being the most toxic. This isomer was also able to induce significant increase in MN frequency and BNMN cells indicating a genotoxic potential at 50 and 100 μg.L -1. The genotoxic test of β-HCH showed a positive induction of MN and BNMN cells at the highest concentration of 100 μg.L -1 and a significant cytotoxicity at 50 μg.L -1. Under the experimental condition used, α-HCH was unable to induce any significant increase in MN frequency confirming that α-HCH is a non-genotoxic agent.
Binucleated cells
Hexachlorocyclohexane
Clastogen
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The relationship between ionizing radiationinduced cell killing and DNA damage measured by the micronucleus assay was determined in three established cell lines (L929, HL-60, and Chang). Our data revealed a dosedependent increase of cells bearing multiple micronuclei. Cells with the same number of micronuclei were counted separately up to 50 h after irradiation. The counts of these subsets showed a parallel increase and decrease throughout the study. In order to transform the peak of the micronucleus frequency, occurring over only a brief time period into a less time dependent value, we calculated ratios between the different subsets of micronucleated cells. These ratios converged to values which were almost constant beyond 30 h after irradiation. The values showed correlations with cell survival (clonogenic assay) and radiation dose which were comparable with the correlations with the peak of the micronucleus frequency (maximum micronucleus yield) when utilizing the conventional evaluation of the micronucleus assay performed without cytochalasin B. This means that large-scale time kinetics and additional drugs like cytochalasin B can be avoided by changing the evaluation procedure of the conventional micronucleus assay. The modified assay described in this manuscript revealed apoptosisinduced limitations as recently detected for the maximum micronucleus yield assay.
Cytochalasin B
Cytochalasin
Clonogenic assay
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Cytochalasine B is an indicator in the CBMN assay, but can also be geno- or cytotoxic. The aim of the study was to determine the optimal dose of cytochalasine B for the Cytokinesis-Block Micronucleus (CBMN) assay for the domestic cat. The results revealed a surprisingly high number of micronuclei (MN) in relation to the doses used. It was found that a concentration of 2.5 μg/ml is enough to obtain binuclear cells, with a minimal toxic effect on the lymphocytes of the domestic cat. In the material analysed, three forms of chromatin structure abnormalities were found. The most prevalent abnormalities were isolated micronuclei, with the mean number of binucleated cells with one micronucleus (BNCs + 1 MN) equal to 39.96 ± 9.02. Moreover, the numbers of binucleated cells with one micronucleus, nuclear buds and nucleoplasmic bridges did not differ significantly according to the sex or age of European cats.
Binucleated cells
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We assessed the in vitro cytogenetic effects of extracts of the commonly used medicinal plants Equiseti herba, Ononidis radix, and Uva ursi on irradiated human blood lymphocytes. We examined the acquired micronucleus formation in unirradiated and irradiated samples of cultured blood lymphocytes using the cytochalasin block micronucleus test (CBMN). Centromere-positive micronuclei were identified by fluorescence in situ hybridization using a DNA probe labeled with a-satellite digogsigenin. Equiseti herba had weak clastogenic properties, increasing the yield of micronuclei in unirradiated samples and reducing the level of radiation-induced micronuclei in a concentration-dependent manner. In the control, unirradiated samples, 36.8% of micronuclei were centromerepositive (MNC+), while in the irradiated ones the percentage of MNC+ ranged from 10.8-15.3%, indicating a clastogenic mechanism for the micronuclei formation. Ononidis radix was a strong clastogen and radiosensitizer, rapidly increasing the yield of micronuclei in unirradiated samples up to 5-fold and potentiating the yield of radiation-induced micronuclei up to 1.7-fold. In cultures treated with Ononidis radix, the percentage of MNC+ micronuclei ranged from 18.8 to 23.8%, indicating that micronuclei originated by a clastogenic mechanism. Uva ursi did not affect the yield of micronuclei either in unirradiated or in irradiated samples. The micronucleus formation assay is a reliable screen for plant extracts and purified compounds, for the identification of compounds that might either inhibit clastogenesis or potentiate radiotherapy for malignancy.
Clastogen
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Clastogen
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Our current study demonstrates the usefulness of the micronucleus as a biological marker of exposure and the resulting damage to the genetic material. Studies were conducted in rat lung fibroblasts and dog blood lymphocytes exposed to chemicals, ethyl nitrosourea (ENU), and methyl methanesulfonate (MMS), and radiation. Micronuclei are formed when fragments of chromosomes or whole aberrant chromosomes are produced as the result of an interaction between chemicals or radiation, and the nuclear material. The number and distribution of micronuclei were evaluated both in vitro and in vivo and provided a useful biological model of the chemical mutagens ENU and MMS showed a significant increase in the number of micronuclei in their lung fibroblasts. Primary rat lung fibroblasts exposed to radon in vitro showed a remarkable increase in the number of micronuclei. The micronucleus frequency in vitro was used as a first estimate of the dose to lung fibroblasts following in vivo inhalation exposure to 320 WLM of radon. In a dog blood lymphocyte model, the frequency of micronuclei increased as a function of x-ray exposure. Using the frequency of the sensitivity of the cells to subsequent x-ray-induced micronuclei. These studies demonstrate that micronuclei are very useful biomarkers of exposure, dose,more » and alterations in cell sensitivity.« less
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To analyze the genomic instability induced by γ-rays, cytokinesis-block MN assay was employed to investigate the gene damage of cells and their progeny cells exposed to radiation. The micronucleus frequency were related to the irradiation dose and decreased as the culture time lengthened. There was a correlation between the micronucleus frequency and the initial dose after the second irradiation. The micronucleus frequency may be causally related to radiation-induced genomic instability which is expressed as chromosomal damage leading to micronuclei.
Chromosome instability
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Measurement of micronuclei in peripheral blood is usually used to detect chromosome damage and mutagenic action of environmental chemicals in toxicology. The cytokinesis-block micronucleus(CBMN) assay is the most widely used method for measuring micronuclei in human lymphocytes,which has been considered as a useful biomarker to detect chromosome damage and gene stability. In this paper,the formation mechanism,endpoint,and the influencing factors of CBMN and the advantage and disadvantage of CBMN assay is reviewed to boost the use of cytokinesis-block micronucleus in protecting workers exposed to environmental chemicals.
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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.
Comet Assay
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