Genotoxicity of organic pollutants in source of drinking water on microalga Euglena gracilis

The potential toxicities of organic pollutants in the drinking water source at Meiliang Bay of Lake Taihu were investigated by comet assay and antioxidant enzyme approach on microalgae Euglena gracilis. The organic extracts of the water samples could induce DNA damage on microalgae cells. Statistically significant differences (P < 0.05) were observed at groups of 0.3×, 3× and 10× concentrations compared with the control and a solvent control (DMSO). The organic extracts also affected antioxidant enzyme activity and induced lipid peroxidation in the microalga. In the high dose group, there was an obvious increase in SOD content (P < 0.05). The results suggest that the concentrated organics from water sample extracts have adversary effects on E. gracilis and could possibly damage the ecosystem.     

Organic pollutants and ambient severity for the drinking water source of western Taihu Lake

Measurement of the organic compounds found in western Taihu Lake and evaluation of the ambient severity (AS) of the water using multimedia environmental goals (MEG) was conducted. The comet assay and the antioxidant enzyme approach were used to test the potential toxicity of water samples on the microalgae Euglena gracilis. Total concentrations of 25 organic pollutants in samples from two sites were 6.700 and 14.655 μg/l, respectively, with a calculated total ambient severity (TAS) of less than 1 and therefore minimal risk to human and ecological health. Organic extracts from the samples at these two sites was found to induce dose-dependent DNA damage on microalgae cells. DNA damage together with changes in superoxide dismutase (SOD) and peroxidase (POD) activities indicated that the potential pollutant toxicity was far higher at one of the two sites than at the other site. The comet assay combined with the activities of antioxidant enzymes may be of value as a biomarker for presence of organic pollutants in drinking water sources.     

Potential health impact and genotoxicity analysis of drinking source water from Liuxihe Reservoir (P.R. China)

Water from the Liuxihe Reservoir (a source of drinking water for Guangzhou City, P. R. China) was analyzed for semi-volatile organic compounds (SVOCs) and the results were used for a potential health impact assessment and genotoxicity test with the microalgae Euglena gracilis. The SVOCs were tested using USEPA Method 525.2, and the health risk assessment was conducted at a screening level using the hazard quotient (HQ) approach. Alkaline single-cell gel electrophoresis (comet assay) was used to evaluate DNA damage and determine the genotoxicity of the source water. The concentrations of the SVOCs in Liuxihe Reservoir were very low and phthalic acid esters were the main SVOCs present. The mean HQ values of pollutants were all less than one, indicating no risk. However, the lifetime carcinogenic risks (LCRs) were found to be close to the threshold of 1.00E?5. The results show that the water in the Liuxihe Reservoir might pose a potential carcinogenic risk to local residents. The highly concentrated extracts of the water samples could induce DNA damage in the microalgal cells and a dose–effect relationship was identified. These results showed that Liuxihe Reservoir water, as a source of drinking water, could pose a potential LCR to local consumers.     

Evaluating genotoxicity associated with microcystin-LR and its risk to source water safety in Meiliang Bay, Taihu Lake

In recent years cyanobacteria blooms have become a severe problem in Taihu Lake, a large shallow eutrophic lake in China. Microcystins produced by certain genera of cyanobacteria can affect public health in this area because of their acute and chronic toxic effects. In this study, samples of cyanobacteria were collected and extracted by two solvent systems. The extracts were tested with three short-term genotoxicity assays, the ara test, the Ames test, and the SOS/umu test. In addition, temporal variation in the concentrations of microcystin-LR in the water samples was determined and monitored by an ELISA assay. Then the concentration of microcystin-LR in the drinking water was estimated. The risk of microcystin-LR exposure by drinking water was assessed according to tolerable daily intake (TDI). The three genotoxicity assays showed negative results regardless of the solvent system used, and there were clear inconsistencies in the spatiotemporal profiles of genotoxic potential and microcystin concentrations in Taihu Lake. Risk assessment showed that the drinking water from Taihu Lake was not safe from the end of July to the beginning of November because of a high concentration of microcystin-LR. Our study indicated the drinking water from Taihu Lake posed a risk because of the microcystin-LR, although it was neither genotoxic nor associated with genotoxicity of the lake water.     

Evaluation of environmental waters using the comet assay in Tilapia rendalli

Testing for environmental pollutants is an ever-growing concern. Various tests in organisms have been utilized for the detection and identification of toxic substances in the air, water and soil. In the present study, we utilized the comet assay in Tilapia rendalli to conduct an environmental assessment of Lake Igapó II, a lake located in the metropolitan area of Londrina, PR-Brazil. The results demonstrated that samples from Lake Igapó II had a significantly greater number of comets, mainly in classes 2 and 3. The results suggest a genotoxicity of the aquatic environment at Lake Igapó II and that the comet assay in T. rendalli provides adequate sensitivity to be utilized as a tool in the monitoring of water pollution and environmental risk assessment.     

Evaluation of cytotoxic potential,oral toxicity,genotoxicity, and mutagenicity of organic extracts of Pityrocarpa moniliformis

The objective of this study was to determine the cytotoxicity of organic extracts of P. moniliformis in vitro and identify the acute toxicity and genotoxicity in vivo. The leaves were extracted using three organic solvents (cyclohexane [EP1], ethyl acetate [EP2], and methanol [EP3]). Phytochemical qualitative analysis was performed by thin layer chromatography (TLC). Cytotoxicity tests were performed on human embryonic kidney (HEK) cells and J774 murine macrophages. Acute toxicity in mice was measured after intraperitoneal (ip) administration of 2000 mg/kg, while evaluation of genotoxicity and mutagenicity were assessed using the comet assay and the micronucleus (MN) test, respectively. The TLC analysis of the extracts revealed the presence of flavonoids, triterpenes, steroids, and saponins. In the cytotoxicity assay, extracts EP1 and EP3 altered proliferation of HEK cells, and all organic extracts increased the viability of J774 cells. In the toxicity tests, no deaths or behavioral alterations were observed in mice exposed to the acute dose of the extracts. Although some extracts led to changes in hematological and histological parameters, these results did not indicate physiological changes. In relation to the MN test and comet assay, no significant changes were detected in the DNA of the animals tested with the extracts EP1, EP2, and EP3. Thus, extracts of P. moniliformis were not considered to be toxic and did not induce formation of MN or damage to cellular DNA in the genotoxicity tests.     

Assessment of RTG-W1, RTL-W1, and PLHC-1 fish cell lines for genotoxicity testing of environmental pollutants by means of a Fpg-modified comet assay

In a context of growing awareness of aquatic pollution impacts, there is an increasing need to develop methods for hazard and risk assessment of pollutants. For this purpose, in vitro models such as fish cell lines warrant to be evaluated as possible alternative to in vivo fish testing, and new toxicity endpoints such as genotoxicity deserve to be considered. This study assesses the interest of the formamido pyrimidine glycosylase (Fpg)-modified comet assay applied to three fish cell lines (RTL-W1, RTG-W1, and PLHC-1) regarding the sensitivity of the system for measuring genotoxicity of various classes of pollutants. Cytochrome P450-dependent EROD activity has also been measured to evaluate the importance of the biotransformation capacity of the cell lines in genotoxicity assessment. For all cell lines and chemicals tested, a concentration dependent genotoxic effect was observed with a 10- to 1000-fold increased sensitivity when using the Fpg-protocol compared to the standard comet assay. Such a modified assay led in particular to improve the detection threshold of oxidative and alkylating DNA damages following exposure at environmentally relevant contaminant concentrations and could partly compensate for the lower sensitivity of cell lines versus whole organism testing often cited as a limit of in vitro testing.     

The application of the comet assay to assess the genotoxicity of environmental pollutants in the nematode Caenorhabditis elegans

This study aimed to establish a protocol for cell dissociation from the nematode Caenorhabditis elegans (C. elegans) to assess the genotoxicity of the environmental pollutant benzo[a]pyrene (BaP) using the alkaline version of the single cell electrophoresis assay (comet assay). BaP genotoxicity was assessed in C. elegans (wild-type [WT]; N2, Bristol) after 48 h exposure (0–40 μM). Induction of comets by BaP was concentration-dependent up to 20 μM; comet% tail DNA was ∼30% at 20 μM BaP and ∼10% in controls. Similarly, BaP-induced DNA damage was evaluated in C. elegans mutant strains deficient in DNA repair. In xpa-1 and apn-1 mutants BaP-induced comet formation was diminished to WT background levels suggesting that the damage formed by BaP that is detected in the comet assay is not recognised in cells deficient in nucleotide and base excision repair, respectively. In summary, our study provides a protocol to evaluate DNA damage of environmental pollutants in whole nematodes using the comet assay.     

Oxidative stress and DNA damage in the earthworm <Emphasis Type="Italic">Eisenia fetida</Emphasis> induced by toluene,ethylbenzene and xylene

Superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT), and the comet assay (SCGE) were used as biomarkers to evaluate the oxidative stress and genotoxicity of toluene, ethylbenzene and xylene in earthworms (Eisenia fetida). The results indicated that the exposure of the three pollutants caused a stress response of the three enzymes, an approximate bell-shaped change (a tendency of inducement firstly and then inhibition with increasing concentrations of the pollutants) was mostly found. The three enzymes tested differed in their sensitivity to different pollutants. While the activity of POD was not significantly changed within the concentration range, the concentration thresholds for significant (P < 0.05) responses to toluene based on SOD and CAT were 5 mg kg⁻¹, respectively. Similarly, the concentration thresholds for significant (P < 0.05) responses to ethylbenzene based on CAT and POD were 10 and 5 mg kg⁻¹, respectively, while the activity of SOD was not significantly changed within the concentration range. Significant responses to xylene based on CAT and POD were 5 mg kg⁻¹, respectively, while the activity of SOD was significantly (P < 0.05) induced at 10 mg kg⁻¹. The SCGE assay results showed that these three pollutants could significantly (P < 0.01) induce DNA damage in earthworms and the clear dose-dependent relationships were displayed, indicating potential genotoxic effects of toluene, ethylbenzene, and xylene on E. fetida. The inducement of DNA damage may be attributed to the oxidative attack of toluene, ethylbenzene, and xylene. Toluene seemed to be more genotoxic as it could induce the higher extent of DNA damage than ethylbenzene and xylene. The results suggest that the SCGE assay of earthworms is simple and efficient for diagnosing the genotoxicity of pollutants in terrestrial environment.     

Evaluation of cytotoxicity,genotoxicity and teratogenicity of marine sediments from Qingdao coastal areas using in vitro fish cell assay,comet assay and zebrafish embryo test

Marine sediments are often a final sink for numerous anthropogenic contaminants and may impose serious effects on benthic organisms and ecosystem. An in vitro cell assay using a cell line derived from flounder gill (FG) cells, an in vitro comet assay in FG cells, and an in vitro zebrafish embryo assay were used to evaluate the in vitro cytotoxicity (measured by MTT reduction), genotoxicity and teratogenicity of crude sediment extracts of Li Cang (LC), Zhan Qiao (ZQ) and Olympic Sailing Center (OSC) from Qingdao coastal area. Sediments from the three sites displayed different cytotoxicity, genotoxicity and teratogenicity potencies; however, all three assays yielded similar LOECs (lowest observed effect concentration) for each site, suggesting that the assays were equally sensitive to and suitable for initial screening of the LOECs of marine sediments. The cytotoxicity, genotoxicity and teratogenicity for these three sampling sites were in the same order of LC > ZQ > OSC, indicating different degrees of contamination. Interestingly, trials with the three sediment extracts at the doses inducing a similar cytotoxicity as evaluated with MTT reduction did not produce similar genotoxicity and teratogenicity, with the genotoxic and teratogenic activities of LC and ZQ extracts being markedly higher than those of OSC sediments. These findings indicate that cytotoxicity does not form a fully equivalent toxicity index with that of genotoxicity and teratogenicity. Therefore, in order to assess the true toxic potential of marine sediments, all three assays should be performed. Analysis of 16 EPA (US Environmental Protection Agency) priority PAHs in these three sediment samples showed a clear correlation between PAH concentrations and sediment toxicities, with a higher PAH content corresponding to higher toxicity although PAHs are surely not the only cause.     

Evaluation of the impact of bioaccumulation of PAH from the marine environment on DNA integrity and oxidative stress in marine rock oyster (<Emphasis Type="Italic">Saccostrea cucullata</Emphasis>) along the Arabian sea coast

Marine pollution due to oil spills is of great concern globally for their impact on the health of marine ecosystems. We assessed the genotoxic effects and oxidative stress due to genotoxic pollutants accumulated from the ambient marine environment in the tissues of marine rock oyster, Saccostrea cucullata along the Arabian Sea coast around Goa, India. The extent of DNA damage in S. cucullata was determined by comet assay as variation of comet parameter: mean % tail DNA along the coast with respect to that at the reference site (Tiracol, Goa, India). In addition, the oxidative stress responses of rock oysters exposed to marine pollutants such as polycyclic aromatic hydrocarbons (PAHs) were assessed as a function of variation in antioxidant enzyme activities such as glutathione-s-transferase (GST), catalase (CAT) and superoxide dismutase (SOD) along the coast. Spearman correlation analysis showed significant correlation between different components of PAHs (viz., 2–3-PAH, 4–6-PAH and oxy-PAH) in the tissues of the rock oysters and the antioxidant enzyme activities. The antioxidant enzyme activities in S. cucullata increased with increasing concentrations of PAHs in tissues in the following order of sampling sites: Tiracol?<?Arambol?<?Betul?<?Velsao. Among the PAHs, oxy-PAH was found to be most predominant in causing DNA damage in S. cucullata. These results provide an insight into environmental genotoxicity and oxidative stress induced by PAHs along the Arabian Sea coast, India.     

体内彗星试验在药物遗传毒性评价中的研究方法

体内彗星试验是采用单细胞凝胶电泳的方法检测体内DNA损伤的技术。具有灵敏度高、操作简便、经济快速等优点。随着遗传毒性研究的发展,体内彗星试验已经成为重要的药物遗传毒性评价方法。对动物试验阶段和彗星试验阶段各操作步骤进行了归纳总结,以期对相关方法学建立提供参考,并提出采用简化和标准化的研究方法将有利于体内彗星试验在药物遗传毒性评价的应用。     

Ecotoxicity evaluation of a liquid detergent using the automatic biotest ECOTOX

Synthetic detergents are common pollutants reaching aquatic environments in different ways after usage at homes, institutions and industries. In this study a liquid detergent, used for dish washing, was evaluated for its toxicity during long- and short-term tests using the automatic biotest ECOTOX. Different parameters of Euglena gracilis like motility, swimming velocity, gravitactic orientation, cell compactness and cell growth were used as end points. In short-term experiments, the maximum adverse effects on motility, velocity, cell shape and gravitaxis were observed after 1 h of exposure. With further increase in exposure time to the detergent a slight recovery of these parameters was observed. In long-term experiments, the detergent caused severe disturbances to E. gracilis. Motility, cell growth and cell compactness (shape) with EC₅₀ values of 0.064, 0.18 and 2.05 %, respectively, were found as the most sensitive parameters to detergent stress. There was a slight positive effect on gravitactic orientation at the lowest two concentrations; at higher concentrations of the detergent cells orientation was highly impaired giving EC₅₀ values of 1.75 and 2.52 % for upward swimming and r-value, respectively.     

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.     

Evaluation of multi-organ DNA damage by comet assay from 28 days repeated dose oral toxicity test in mice: A practical approach for test integration in regulatory toxicity testing

The use of comet assay is not new in the evaluation of genotoxic potential of different agents; however, its broad use in product safety for regulatory testing is a relatively new approach. The present study was aimed to integrate genotoxicity tests (micronucleus and comet assay) in 28 days repeated dose oral toxicity of methotrexate (MTX) in mice. MTX was administered at the dose of 0.5, 1 and 2 mg/kg per oral repeatedly for 28 days in mice. The endpoints of evaluation for routine toxicity testing included body weight, organ weight, food intake, water intake, hematology and histology, while for the genotoxicity testing micronucleus and comet assay were used. There were no significant changes in food intake, water intake and organ weight; however, the body weight significantly decreased at the highest dose of MTX treatment as compared to control group. Histological data revealed the morphological alterations in the liver and lung cells at the highest dose of MTX treatment. Micronucleus assay results indicated that the highest dose of MTX led to significant increase in MNERTs/1000ERTs (P < 0.001) as compared to control group. Further, percentage of reticulocytes (% RETs) was significantly decreased at the highest dose of MTX as compared to control group. Comet assay results indicate significant DNA damage in different organs induced by MTX as compared to control group. The results of the present study successfully demonstrates the integration of genotoxicity tests using comet and micronucleus assay in 28 days repeated dose oral toxicity test. Integration of genotoxicity test with routine toxicity test would reduce the cost of additional animals, test item and provide further information at an early stage of product development.     

Genotoxicity testing in vitro – Development of a higher throughput analysis method based on the comet assay

Higher throughput methods, high content analysis and automated screening methods are of highest demand in drug development today. In toxicology, these strategies are becoming increasingly important, as well. Therefore, an integrated higher throughput method for the comet assay is addressed by the development presented here.The sensitivity, specificity and relevance of the comet assay as a method for determination of DNA damage in vivo and in vitro have been highlighted in many studies. Actually, efforts are made to include it in a panel of genotoxicity tests for regulatory purposes. However, the standard comet assay is a time consuming procedure due to the specific methods needed. The improvements presented here lead to a faster and easier slide-production, a smaller amount of cells needed, a higher amount of comets quantified, a fully automated analysis of comets including reanalysis, storing, visualisation and documentation possibilities using standard comet quantification models such as tail length or tail moment, and – by introduction of clearly definable selection criteria based on image analysis algorithms – clearly improve objectivity and standardization of the analysis procedure.Results prove the high reproducibility, flexibility, efficiency and suitability of the procedure as a fully automated analysis method in higher throughput genotoxicity testing in vitro.     

Chemical investigation of different crude extracts from Teucrium ramosissimum leaves. Correlation with their antigenotoxic and antioxidant properties

The effect of extracts obtained from Teucrium ramosissimum leaves on genotoxicity and SOS response induced by aflatoxin B₁ (0.5 μg/assay) as well as nitrofurantoin (5 μg/assay) was investigated in a bacterial assay system, i.e., the SOS chromotest with Escherichia coli PQ37. The T. ramosissimum tested extracts exhibited no genotoxicity either with or without the external S9 activation mixture. However, all the extracts, particularly the total oligomers flavonoids (TOF) extract significantly decreased the genotoxicity induced by aflatoxin B₁ and nitrofurantoin. Antioxidant capacity of the tested extracts was evaluated using the enzymatic (xanthine/xanthine oxidase assay) (X/XOD) and the non-enzymatic (NBT/Riboflavine assay) systems. TOF extract was the most effective one in inhibiting both xanthine oxidase activity and NBT reduction. Our findings emphasize the potential of T. ramosissimum to prevent mutations and also its antioxidant effect.     

The use of ex vivo human skin tissue for genotoxicity testing

As a result of the chemical legislation concerning the registration, evaluation, authorization and restriction of chemicals (REACH), and the Seventh Amendment to the Cosmetics Directive, which prohibits animal testing in Europe for cosmetics, alternative methods for safety evaluation of chemicals are urgently needed. Current in vitro genotoxicity assays are not sufficiently predictive for the in vivo situation, resulting in an unacceptably high number of misleading positives. For many chemicals and ingredients of personal care products the skin is the first site of contact, but there are no in vitro genotoxicity assays available in the skin for additional evaluation of positive or equivocal responses observed in regulatory in vitro genotoxicity assays. In the present study ex vivo human skin tissue obtained from surgery was used for genotoxicity evaluation of chemicals by using the comet assay. Fresh ex vivo human skin tissue was cultured in an air-liquid interface and topically exposed to 20 chemicals, including true positive, misleading positive and true negative genotoxins. Based on the results obtained in the present study, the sensitivity, specificity and accuracy of the ex vivo skin comet assay to predict in vivo genotoxicity were 89%, 90% and 89%, respectively. Donor and experimental variability were mainly reflected in the magnitude of the response and not the difference between the presence and absence of a genotoxic response. The present study indicates that human skin obtained from surgery is a promising and robust model for safety evaluation of chemicals that are in direct contact with the skin.     

Genotoxicity in Oreochromis niloticus (Cichlidae) induced by Microcystis spp bloom extract containing microcystins

Studies of genotoxicity in fish caused by cyanobacterial extracts containing microcystins (MCs) can be useful in determining their carcinogenic risk due to a genotoxic mechanism. An extract of cyanobacterial Microcystis ssp, containing MC-LR and -LA from a bloom collected in a eutrophic lake, showed genotoxicity to Oreochromis niloticus. DNA damage (comet assay) was significantly induced in peripheral erythrocytes with both tested concentrations of 6.90 μg kg⁻¹ bw and 13.80 μg kg⁻¹ bw through intraperitoneal injection (ip). There was no micronucleus induction after ip injection at concentrations of 6.90 μg kg⁻¹ bw and 13.80 μg kg⁻¹ bw. Body exposure resulted in micronucleus induction and DNA damage only at the highest tested concentrations of 103.72 μg L⁻¹. Thus, comet assay and ip injection revealed the highest levels of the genotoxicity of MCs. Apoptosis-necrosis test carried out at concentrations of 6.90 μg kg⁻¹ bw and 13.80 μg kg⁻¹ bw revealed that at low concentrations more apoptosis than necrosis occurred. At higher concentrations more necrosis than apoptosis occurred.     

Contamination by metals and pharmaceuticals in northern Taihu Lake (China) and its relation to integrated biomarker response in fish

Taihu Lake is the largest shallow freshwater lake in eastern China and is suffering not only from an increasingly serious threat of eutrophication but also potential ecological risk due to the input of emerging contaminants. Active biomonitoring was conducted in Taihu Lake using transplanted goldfish (Carassius auratus) to determine the contamination by pharmaceuticals and metals and to assess the potential ecological risk. A suite of biomarkers including acetylcholinesterase, ethoxyresorufin O-deethylase, glutathione S-transferase, glutathione peroxidase and superoxide dismutase activities in fish after 7, 14, 21 and 28 days of exposure in situ, as well as pharmaceuticals and metals in water, were determined during the field exposure period. The results indicate that pharmaceuticals exist mainly in Zhushan Bay and Meiliang Bay, while metals are present mainly in Gong Bay. An integrated biomarker response (IBR) was calculated and used to evaluate the ecological risk of the polluted area of Taihu Lake. It was found that Zhushan Bay might present higher risk to fish, followed by Meiliang Bay. IBR values were in good agreement with copper and caffeine concentrations.