Genotoxicity evaluation of water soil leachates by Ames test, comet assay, and preliminary Tradescantia micronucleus assay

Combining genotoxicity/mutagenicity tests and physico-chemical methodologies can be useful for determining the potential genotoxic contaminants in soil samples. The aim of our study was to evaluate the genotoxicity of soil by applying an integrated physico-chemical-biological approach. Soil samples were collected at six sampling points in a Slovenian industrial and agricultural region where contamination by heavy metals and sulphur dioxide (SO₂) are primarily caused by a nearby power plant. The in vitro alkaline version of the comet assay on water soil leachates was performed with Caco-2 and HepG2 cells. A parallel genotoxicity evaluation of the samples was performed by Ames test using Salmonella typhimurium and the Tradescantia micronucleus test. Pedological analyses, heavy metal content determination, and different physico-chemical analyses, were also performed utilizing standard methodology. Water leachates of soil samples were prepared according to standard methods. Since only a battery of biotests with prokaryotic and eukaryotic organisms or cells can accurately estimate the effects of (geno)toxicants in soil samples and water soil leachates, a combination of three bioassays, with cells or organisms belonging to different trophic levels, was used. Genotoxicity of all six water soil leachates was proven by the comet assay on both human cell lines, however no positive results were detected by bacterial assay, Ames test. The Tradescantia micronucleus assay showed increase in micronuclei formation for three samples. According to these results we can assume that the comet assay was the most sensitive assay, followed by the micronucleus test. The Ames test does not appear to be sensitive enough for water soil leachates genotoxicity evaluations where heavy metal contamination is anticipated.     

A yeast-based cytotoxicity and genotoxicity assay for environmental monitoring using novel portable instrumentation

An assay capable of simultaneously measuring both general toxicity and more subtle genotoxicity, in aqueous environmental samples, is described. The assay uses eukaryotic (yeast) cells, genetically modified to express a green fluorescent protein (GFP) whenever DNA damage, as a result of exposure to genotoxic agents, is repaired. A measure of the reduction in cell proliferation is used to characterise general toxicity producing familiar EC(50) and LOEC data. The assay protocol has been developed for proposed use in the field and hence employs dedicated, portable instrumentation, the development of which is described. A range of environmentally relevant substances has been evaluated using the assay, including solutions of metal ions, solvents and pesticides. Preliminary data comparing the yeast assay's response to that of a standard Daphnia test in the analysis of the toxicity of 34 varied industrial waste effluents are also presented. The sensitivity to a wide range of substances and effluents suggests the assay should be useful for environmental toxicity monitoring.     

Impact of agglomeration and different dispersions of titanium dioxide nanoparticles on the human related in vitro cytotoxicity and genotoxicity

The published results on nanoparticles cytotoxicity and genotoxicity such as titanium dioxide nanoparticles (TiO(2) NPs) are inconsistent, and often conflicting and insufficient. Since different parameters may have impact on the toxicity results, there is need to lay stress on detailed characterization of NPs and the use of different testing conditions for assessment of NPs toxicity. In order to investigate whether dispersion procedures influence NP cytotoxicity and genotoxicity, we compared two protocols giving TiO(2) NP dispersions with different stability and agglomeration states. Detailed primary and secondary characteristics of both TiO(2) NP dispersions in culture media were carried out before toxicological testing; TK6 human lymphoblast cells, EUE human embryonic epithelial cells and Cos-1 monkey kidney fibroblasts were used to assess cytotoxicity (by trypan blue exclusion, proliferation activity and plating efficiency assays) and genotoxicity (by the comet assay). DNA strand breaks were detected by the alkaline comet assay. DNA oxidation lesions (especially 8-oxo-7,8-dihydroguanine, 8-oxoG) were measured with a modified comet assay including incubation with specific repair enzyme formamidopyrimidine DNA glycosylase (FPG). The TiO(2) NPs dispersion with large agglomerates (3 min sonication and no serum in stock solution) induced DNA damage in all three cell lines, while the TiO(2) NPs dispersed with agglomerates less than 200 nm (foetal serum in stock solution and sonication 15 min) had no effect on genotoxicity. An increased level of DNA oxidation lesions detected in Cos-1 and TK6 cells indicates that the leading mechanism by which TiO(2) NPs trigger genotoxicity is most likely oxidative stress. Our results show that the dispersion method used can influence the results of toxicity studies. Therefore at least two different dispersion procedures should be incorporated into assessment of cyto- and genotoxic effects of NPs. It is important, when assessing the hazard associated with NPs, to establish standard testing procedures and thorough strategies to consider the diverse conditions relevant to possible exposures.     

Use of two population metrics clarifies biodiversity dynamics in large-scale monitoring: the case of trees in Japanese old-growth forests

An alkaline comet assay and a micronucleus test were carried out on erythrocytes of the European chub, Squalius cephalus L., collected in spring and autumn in 2005 and 2006 at three sampling sites in River Sava, near Zagreb, Croatia. The results of comet assay showed the lowest genotoxic influence at the least polluted site, while higher DNA damage was observed at the polluted sites. Although the basal levels of DNA damage were elevated, a clear gradation of DNA damage was found due to pollution intensity in all sampling periods. The lowest cytogenetic damage as revealed by the micronucleus test (MNT) was observed as well at the least polluted site. High variations in MN frequency were observed between sampling periods, although the number of micronucleated erythrocytes was consistently the highest one at the polluted site. The comet assay as a biomarker of genotoxic effect exhibited higher sensitivity in discriminating the genotoxic capacity of studied polluted sites while the MNT was less sensitive. However, both tests should be used together in biomonitoring studies because they can reveal different aspects of DNA damage; comet assay, the early event of genotoxic exposure, and MNT, its final result as a mutagenic potential.     

Genotoxic effects of water pollution on two fish species living in Karasu River,Erzurum, Turkey

Karasu River, which is the only river in the Erzurum plain, is the source of the Euphrates River (Eastern Anatolia of Turkey). The river is in a serious environmental situation as a result of pollution by agricultural and industrial sewage and domestic discharges. The present study aims to evaluate genotoxic effects of toxic metals in chub, Leuciscus cephalus, and transcaucasian barb, Capoeta capoeta, collected from contaminated site of the Karasu River, in comparison with fish from an unpolluted reference site. Heavy metal concentrations in surface water of the river were determined. The condition factor (CF) was taken as a general biomarker of the health of the fish, and genotoxicity assays such as micronucleus (MN) and other nuclear abnormalities (NA) were carried out on the fish species studied. MN and NA such as kidney-shaped nucleus, notched nucleus, binucleated, lobed nucleus, and blebbed nucleus were assessed in peripheral blood erythrocytes, gill epithelial cells, and liver cells of the fish. A significant decrease in CF values associated with a significant elevation in MN and NA frequencies was observed in fish collected from the polluted sites compared with those from the reference site. Results of the current study show the significance of integrating a set of biomarkers to identify the effects of anthropogenic pollution. High concentrations of heavy metals have a potential genotoxic effects, and the toxicity is possibly related to industrial, agricultural, and domestic activities.     

Relative sensitivity of two marine bivalves for detection of genotoxic and cytotoxic effects: a field assessment in the Tamar Estuary, South West England

The input of anthropogenic contaminants to the aquatic environment is a major concern for scientists, regulators and the public. This is especially relevant in areas such as the Tamar valley in SW England, which has a legacy of contamination from industrial activity in the nineteenth and twentieth centuries. Following on from previous laboratory validation studies, this study aimed to assess the relationship between genotoxic and cytotoxic responses and heavy metal concentrations in two bivalve species sampled from locations along the Tamar estuary. Adult cockles, Cerastoderma edule, and blue mussels, Mytilus edulis, were sampled from five locations in the Tamar and one reference location on the south Devon coast. Bivalve haemocytes were processed for comet and neutral red retention (NRR) assays to determine potential genotoxic and cytotoxic effects, respectively. Sediment and soft tissue samples were analysed for metal content by inductively coupled plasma mass spectrometry. Sediment concentrations were consistent with the physico-chemical nature of the Tamar estuary. A significant correlation (P?=?0.05) was found between total metal concentration in sediment and C. edule soft tissues, but no such correlation was found for M. edulis samples. DNA damage was elevated at the site with highest Cr concentrations for M. edulis and at the site with highest Ni and Pb concentrations for C. edule. Analysis of NRR revealed a slight increase in retention time at one site, in contrast to comet data. We conclude that the comet assay is a reliable indicator of genotoxic damage in the field for both M. edulis and C. edule and discuss reasons for the apparent discrepancy with NRR.     

Genotoxicity screening of the river Rasina in Serbia using the Allium anaphase–telophase test

Evaluation of the presence of genotoxic substances is especially important in rivers that serve as a source of drinking water. Nine water samples collected along the river Rasina in Serbia were analyzed for potential toxic and genotoxic effects using the Allium anaphase-telophase test. Inhibition of root growth relative to the negative control (synthetic water) was observed in all samples. Analysis of the genotoxic potential, through scoring anaphase and telophase aberrations, showed that in seven of the nine samples the level of aberrations was significantly increased relative to the negative control but was lower than that obtained for the positive control (methyl methanesulfonate). Changes in the relation between spindle and chromosome types of aberrations were found in some samples, indicating differences in the potential genotoxic substances present at the analyzed sites. The data, which were obtained from samples collected at the highest level of river water, warn that during periods of low flow the values could reach genotoxic activity. The Allium anaphase-telophase test can be recommended as an monitoring system, that can serve as the first alert for the presence of genotoxic environmental pollutants.     

应用小鼠淋巴细胞彗星试验检测地下水遗传毒性

应用小鼠淋巴细胞彗星试验对长治市5个点位地下水水质作遗传毒性研究。结果表明,各水样有机浓集物均可对小鼠淋巴细胞DNA产生不同程度的遗传损伤,随着剂量的增加,Olive尾矩与阴性对照组(0.72±0.09)相比均有显著性差异(P0.05,P0.01)。在试验浓度范围内,以彗星尾部DNA含量、彗星尾长、尾矩和Olive尾矩所指示的DNA损伤程度随剂量增大而逐渐增加,存在显著的剂量-效应关系,其相关系数0.815。     

Genetic damage induced by lead chloride in different tissues of fresh water climbing perch Anabas testudineus (Bloch)

The present investigation was undertaken to study the induction of DNA damage by lead chloride (PbCl(2)) in freshwater climbing perch Anabas testudineus using alkaline single cell gel electrophoresis (comet assay). Based on the LC(50) values of lead chloride of A. testudineus three different concentrations viz., 0.1, 1.0 and 2.0 mg/L were selected to expose fish. The DNA damage was observed in the gill, kidney and liver tissue as the percentage of DNA in comet tails and comet heads in the tissue of the exposed fish. DNA damage at different concentrations showed sensitivity to particular tissue. The liver tissue exhibited significantly (p < 0.01) higher DNA damage, followed by kidney and gill. However, the DNA damage was found to be dose dependent; at 2 mg/L of PbCl(2) the tail and head DNA of liver tissue were 57.84% and 39.49%, in kidney tissue the values were 52.36% and 44.97% whereas in gill tissue the values were 48.86% and 48.96% respectively. The current study explored the utility of the comet assay for in vivo laboratory studies using A. testudineus species for screening the genotoxic potential of lead chloride.     

A Season-dependent Variation of Genotoxicity of Surface Water Samples from Taihu Lake, Yangzte Delta

The present study is to assess a year round change of genotoxicity of surface water in Taihu Lake, Yangtze delta. The water samples were collected in spring, summer, autumn and winter of 2001, respectively. The bacterial L-arabinose resistance forward mutation test (ara test), along with the Ames test, was performed for the evaluation of time profile of mutagenicity of the surface water samples. The strong genotoxic potentials were detected in the near-shore area, especially in the vicinity to the major city. All the samples collected from the lake center and areas distant from dense human activity showed genotoxicity-free in year round time. Intensified human activity along the lake may play an important role for the genotoxic pollution. By comparison of the samples from year round, the winter samples showed the strongest mutagenic potential of all seasons, which indicates that meteorological and hydrological factors also affect the water quality. The impact of dense human activity, improper management and season change should be put into consideration for designing an effective remediation project in the lake basin area.     

Recovery of a freshwater wetland from chemical contamination after an oil spill

In March 2009, a cargo ship spilled 250 tons of heavy fuel oil off the Queensland coast of Australia. The pristine National Park Moreton Island, seven nautical miles to the east of the spill site, was most affected by the oil slick. Contamination of the island's shoreline was widespread, with freshwater wetlands particularly slow to recover as clean-up needed to be carefully managed to avoid damage to this sensitive ecosystem. During the clean-up process on Moreton Island a monitoring program was initiated using traditional chemical analysis in combination with bioanalytical techniques to assess the extent and variability in contamination at sites on the shoreline and freshwater wetlands. Water accommodated fractions (WAF) of oil residues from samples taken directly after the spill on the shoreline showed the same level of toxic potency as samples from the wetland while baseline-toxicity equivalent concentrations (baseline-TEQ) and 2,3,7,8-tetrachlorodibenzodioxin equivalent concentrations (TCDDEQ) were much lower in oil collected from the sandy beach. The umuC assay for genotoxicity and the E-SCREEN assay for estrogenic effects indicated the extracts were not genotoxic or estrogenic. PAH concentrations and toxicity in grab water samples were below detectable levels, however, extracts from time integrated silicone passive samplers deployed for several weeks at the contaminated sites gave measurable responses in the bioassays with TCDDEQ levels increased relative to the control site. The low levels of baseline-TEQ and TCDDEQ present after 8 months had further decreased 6 months later indicating satisfactory recovery of this pristine ecosystem after an oil spill.     

Cytotoxic and genotoxic effects of silver nanoparticle/carboxymethyl cellulose on <Emphasis Type="Italic">Allium cepa</Emphasis>

Several mutagenic agents may be present in substances released in the environment, which may cause serious environmental impacts. Among these substances, there is a special concern regarding the widespread use of silver nanoparticles (AgNP) in several products due to their widely known bactericidal properties, including in the medical field and the food industry (e.g., active packaging). The assessment of the effects of AgNP released in the environment, having different concentrations, sizes, and being associated or not to other types of materials, including polymers, is therefore essential. In this research, the objective was to evaluate the genotoxic and cytotoxic effects of AgNP (size range between 2 and 8 nm) on root meristematic cells of Allium cepa (A. cepa). Tests were carried out in the presence of colloidal solution of AgNP and AgNP mixed with carboxymethylcellulose (CMC), using distinct concentrations of AgNP. As a result, when compared to control samples, AgNP induced a mitotic index decrease and an increase of chromosomal aberration number for two studied concentrations. When AgNP was in the presence of CMC, no cytotoxic potential was verified, but only the genotoxic potential for AgNP dispersion having concentration of 12.4 ppm.     

Evaluation of lot-to-lot repeatability and effect of assay media choice in the recombinant Factor C assay

Measurement of environmental endotoxin exposures is complicated by variability encountered using current biological assay methods arising in part from lot-to-lot variability of the Limulus-amebocyte lysate (LAL) reagents. Therefore, we investigated the lot-to-lot repeatability of commercially available recombinant Factor C (rFC) kits as an alternative to LAL. Specifically, we compared endotoxin estimates obtained from rFC assay of twenty indoor dust samples, using four different extraction and assay media, to endotoxin estimates previously obtained by Limulus amebocyte lysate (LAL) assay and amounts of 3-hydroxy fatty acids (3-OHFA) in lipopolysaccharide (LPS) using gas-chromatography mass spectroscopy (GC-MS). We found that lot-to-lot variability of the rFC assay kits does not significantly alter endotoxin estimates in house dust samples when performed using three of the four assay media tested and that choice of assay media significantly altered endotoxin estimates obtained by rFC assay of house dust samples. Our findings demonstrate lot-to-lot reproducibility of rFC assay of environmental samples and suggest that use of rFC assay performed with Tris buffer or water as the extraction and assay medium for measurement of endotoxin in dust samples may be a suitable choice for developing a standardized methodology.     

饮用水消毒副产物基因毒性与致癌性研究进展

简述了饮用水消毒副产物（DBPs）的基因毒性与致癌性的研究进展。从Ames试验、SOS/umu试验、彗星试验、微核试验及一些新颖的致突变试验结果对DBPs基因的毒性,以及从毒理学实验、流行病学研究和致癌风险评估3个方面对DBPs的致癌性进行了分析和总结,以期为今后饮用水DBPs毒性效应及其致毒机理研究提供参考,进而促进饮用水质量管理与立法的发展。     

Genotoxicity assessment of soils from wastewater irrigation areas and bioremediation sites using the Vicia faba root tip micronucleus assay

Genotoxicity potential of soils taken from wastewater irrigation areas and bioremediation sites was assessed using the Vicia faba root tip micronucleus assay. Twenty five soils were tested, of which 8 were uncontaminated soils and taken as the control to examine the influence of soil properties; 6 soils were obtained from paddy rice fields with a history of long-term wastewater irrigation; 6 soils were obtained from bioremediation sites to examine effects of bioremediation; and 5 PAH-contaminated soils were used to examine methodological effects between direct soil exposure and exposure to aqueous soil extracts on micronuclei (MN) frequency ( per thousand) in the V. faba root tips. Results indicate that soil properties had no significant influences on MN frequencies (p > 0.05) when soil pH varied between 3.4 to 7.6 and organic carbon between 0.4% and 18.6%. The MN frequency measured in these control soils ranged from 1.6 per thousand to 5.8 per thousand. MN frequencies in soils from wastewater irrigation areas showed 2- to 48-fold increase as compared with the control. Soils from bioremediation sites showed a mixed picture: MN frequencies in some soils decreased after bioremediation, possibly due to detoxification; whereas in other cases remediated soils induced higher MN frequencies, suggesting that genotoxic substances might be produced during bioremediation. Exposure to aqueous soil extracts gave a higher MN frequency than direct exposure in 3 soils. However, the opposite was observed in the other two soils, suggesting that both exposure routes should be tested in case of negative results from one route. Data obtained from this study indicate that the MN assay is a sensitive assay suitable for evaluating genotoxicity of soils.     

Genotoxic effects of the herbicide Roundup® in the fish Corydoras paleatus (Jenyns 1842) after short-term, environmentally low concentration exposure

The glyphosate-based herbicide, Roundup^®, is one of the most used pesticides worldwide. In concert with the advent of transgenic crops resistant to glyphosate, the use of this pesticide has led to an increase in agricultural yields. The objective of this study was to evaluate the genotoxic effect that the herbicide Roundup^® (at a concentration of 6.67 μg/L, corresponding to 3.20 μg/L glyphosate) can have on the fish Corydoras paleatus. Treatment groups were exposed for 3, 6, and 9 days, and effects were analyzed using the piscine micronucleus test (PMT) and comet assay. A group subjected to filtered water only was used as a negative control. The PMT did not show differences between the control and exposed groups for any of the treatment times. In contrast, the comet assay showed a high rate of DNA damage in group exposed to Roundup^® for all treatment times, both for blood and hepatic cells. We conclude that for the low concentration used in this research, the herbicide shows potential genotoxic effects. Future research will be important in evaluating the effects of this substance, whose presence in the environment is ever-increasing.     

Genotoxic evaluation of the insecticide endosulfan based on the induced GADD153-GFP reporter gene expression

Endosulfan is one of the few organochlorine insecticides still in use in China for protecting crops from a variety of insects. Endosulfan is toxic in fishes and rodents in the in vivo assays, but its genotoxicity in mammalian cells has not been well tested. In this work, a genotoxic testing system has been developed based on the induction of a HepG2/GADD153-GFP reporter gene expression in response to the DNA-damaging agents. Methyl methanesulfonate, a known carcinogenic and genotoxic agent, was used to test the effects of damage dose and post-treatment incubation time on GADD153-GFP expression. Subsequently, the system was applied to the genotoxicity evaluation of endosulfan. Endosulfan was able to cause the increase of GADD153-GFP expression at a sublethal dose (0.02?C20 mg/L). In particular, it induced a maximum green fluorescent protein expression at the tested concentration of 0.2 mg/L, with 4.07-fold inflorescence relative to untreated cells. The results suggest that endosulfan has the potential genotoxicity for HepG2 cell line by inducing DNA damage. The study also confirms that the induced GADD153-GFP expression system is an appropriate and sensitive method for the assessment of genotoxicity from a broad range of pesticides with the DNA-damaging potential.     

Genotoxic evaluation of different doses of inorganic lead (PbII) in Hoplias malabaricus

Different genetic biomarkers have been used to evaluate the pollution effects of mutagenic agents such as metals and also a great variety of chemicals delivered on the environment by human activities. This way, the aim of the present report was to evaluate the effects of inorganic lead in fishes through the frequency of piscine micronuclei and nuclear morphological alterations in peripheral cells, chromosomal aberration frequency and comet assays in blood and kidney cells. Specimens of Hoplias malabaricus received different doses of lead by intra-peritoneal injections at time of 96 h. There was not a significant difference between control and treated groups for the piscine micronucleus and chromosomal aberration assays. In the comet assays there was a significant difference between control and contaminated groups. However, a significant difference between the applied doses was not observed. The results obtained with the comet assays also show that blood presented a higher sensibility than the kidney tissue, possibly due to the acute contamination. Although the results showed the genotoxic potential of lead at the 21 and 63 microg Pb(2 + )/g doses for both tissues, the lowest dose is considered more appropriate for future bioassays.     

鱼血微核试验评价长江江苏段水污染遗传毒性

采用原位监测野生鲫鱼外周血红细胞的微核发生率评价长江江苏段水污染的遗传毒性,并与实验室内的Cr(Ⅵ)诱变试验结果进行对比分析。结果表明,常熟望虞河口、江阴滨江村调查点的水污染对鲫鱼的遗传毒性最高,海门江心沙农场其次,江浦、江宁、靖江、镇江调查点的毒性最低。     

Combining passive sampling and toxicity testing for evaluation of mixtures of polar organic chemicals in sewage treatment plant effluent

Effluent from sewage treatment plants has been associated with a range of pollutant effects. Depending on the influent composition and treatment processes the effluent may contain a myriad of different chemicals which makes monitoring very complex. In this study we aimed to monitor relatively polar organic pollutant mixtures using a combination of passive sampling techniques and a set of biochemistry based assays covering acute bacterial toxicity (Microtox), phytotoxicity (Max-I-PAM assay) and genotoxicity (umuC assay). The study showed that all of the assays were able to detect effects in the samples and allowed a comparison of the two plants as well as a comparison between the two sampling periods. Distinct improvements in water quality were observed in one of the plants as result of an upgrade to a UV disinfection system, which improved from 24x sample enrichment required to induce a 50% response in the Microtox assay to 84x, from 30x sample enrichment to induce a 50% reduction in photosynthetic yield to 125x, and the genotoxicity observed in the first sampling period was eliminated. Thus we propose that biochemical assay techniques in combination with time integrated passive sampling can substantially contribute to the monitoring of polar organic toxicants in STP effluents.