Gene-TEQ--a standardized comparative assessment of effects in the comet assay using genotoxicity equivalents

Existing methods for the comparison of genotoxic effects in the comet assay bear considerable disadvantages such as the problem to link information about concentration dependence and severity of effects. Moreover, given the lack of standardized protocols and the use of various standards, it may be extremely difficult to compare different studies. In order to provide a method for standardized comparative assessment of genotoxic effects, the concept of genotoxicity equivalents (Gene-TEQ) was developed. As potential reference compounds for genotoxic effects, three directly acting (N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl-methanesulfonate, and N-methyl-N-nitrosourea) and three indirectly acting (cyclophosphamide, dimethylnitrosamine, and 4-nitroquinoline-oxide) genotoxic substances were compared with respect to their cytotoxic (neutral red) and genotoxic (comet assay) concentration-response profiles in the permanent fish cell line RTL-W1. For further comparison, two sediment extracts from the upper Danube River were investigated as environmental samples. Based on the results of cytotoxicity and genotoxicity testing, MNNG was selected as the reference compound. At several exposure levels and durations, genotoxic effects of both the other pure substances and the environmental samples were calculated as percentages of the maximum MNNG effect and related to the absolute MNNG effect (EC values). Thus, genotoxicity equivalent factors (Gene-TEQs) relative to MNNG could be calculated. Gene-TEQs can easily be applied to pure substances, mixtures and field samples to provide information about their toxicity relative to the reference compound. Furthermore, the Gene-TEQ concept allows a direct comparison of environmental samples from different laboratories.     

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.     

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.     

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.     

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.     

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.     

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.     

Exposure of Marine Mammals to Genotoxic Environmental Contaminants: Application of the 32P-Postlabeling Assay for Measuring DNA-Xenobiotic Adducts♯

There is little information on exposure of marine mammals to genotoxic environmental contaminants. The 32P-postlabeling assay has been successfully used to assess exposure to genotoxic polycyclic aromatic compounds in fish and humans. In the present study, a preliminary investigation showed that polycyclic aromatic compound-like DNA adducts were present in hepatic tissues of harbor seals (Phoca vitulina richardsi) exposed to petroleum following the Exxon Valdez oil spill. However, for marine mammals, effects from changes in tissue condition on DNA recovery and quality is of concern, because tissue samples are often collected from animals that have been dead for unknown periods of time. To assess the effects of postmortem thermal history on DNA recovery from tissue and on DNA adduct quantitation, samples of harbor porpoise (Phocoena phocoena) hepatic tissue were incubated for up to 10 d at 4 and 30 °C. Only traces (<4 g) of hepatic DNA were recovered from 200 mg of tissue after incubation at 30 °C for 36 h. At 4 °C, DNA (50–130 g) was recovered from tissue incubated for up to 6 d; whereas DNA recovery at 10 d was minimal. Chromatograms of 32P-labeled DNA digests of liver tissue held at 4 and 30 °C and salmon sperm DNA held at 30 °C for 2 d had comparable profiles, suggesting that alteration of DNA bases had occurred during incubation of porpoise liver tissue. Moreover, the chromatograms of DNA extracted from liver tissues of harbor porpoises caught incidentally in a northwest Atlantic fishery, packed in ice and sampled several days later also exhibited similar altered DNA structures. Although, altered DNA structures that can interfere with the DNA adduct quantitation were present in autolyzed tissue, changes in the 32P-postlabeling chromatography conditions can decrease the interference. Moreover, in a study with tissues taken from California sea lions (Zalophus californianus) immediately postmortem and stored at –80 °C until processing, DNA structures associated with tissue breakdown were not observed. The DNA from sea lions, however, had putative age-dependent hepatic DNA modifications, which have a distinctive profile, and must be considered when evaluating exposure of marine mammals to polycyclic aromatic compounds. Overall, the findings showed that with attention to the postmortem thermal history of the tissue samples hepatic DNA adducts, as measured by 32P-postlabeling, have the potential to serve as a biological indicator of exposure of marine mammals to environmental genotoxic compounds.     

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.     

Plant Bioassays for an in Situ Monitoring of Air Near an Industrial Area and a Municipal Solid Waste – ŽILINA (SLOVAKIA)

The process of a bioindication of genotoxic effects of complex mixtures on the environment using higher plants is very appropriate and effective. We present the results of an in situ indication of the genotoxic effects of polluted environment near Žilina city. For a more complex monitoring we used: the Tradescantia micronucleus (Trad-MCN) assay, the Tradescantia microspore test and an evaluation of the abortivity of the pollen grains of native plant species. We found significant differences in the frequency of the micronuclei when using the Trad-MCN test in local of Dubeň. The Tradescantia pollen abortivity test showed significant differences in the frequency of the abortive pollen grains between the exposed groups and the control group. By using native plant species in the pollen abortivity test we found significant differences in both of the two locations for the four following species during two consecutive years: Artemisia vulgaris, Melilotus albus, Trifolium pratense, Typha latifolia.     

Buccal micronucleus cytome assay of populations under chronic heavy metal and other metal exposure along the Santiago River,Mexico

The Santiago River is one of the most contaminated rivers in Mexico, with heavy metal levels above the allowed limits. Scientific evidence indicates that chronic heavy metal exposure leads to cytogenotoxic effects. The aims of this study were to evaluate the genotoxic and cytotoxic effects of such exposure in buccal mucosa cells by micronucleus (MN) assay and to identify other nuclear abnormalities (NAs), such as nuclear buds (NBUDs), binucleated cells (BNs), pyknotic nuclei (PNs), karyorrhexis (KX), karyolysis (KL), and abnormally condensed chromatin (CC). Assays were performed on samples from four populations located alongside the Santiago River that are under chronic exposure to heavy metals and other metals (HMMs), and the results were compared with those of a population without exposure to HMMs. The exposed group showed increased frequencies of NAs (KX, CC, and KL), which are associated with cytotoxic damage, and NBUDs, which are associated with genotoxic damage. Increased frequencies of NBUDs and CC were observed in subjects from El Salto/Juanacatlán, Ocotlán, and Paso de Guadalupe, and an increase in KX frequency was observed in subjects from El Salto/Juanacatlán. Significant differences in KL frequency were observed in subjects from La Barca, El Salto/Juanacatlán, Paso de Guadalupe, and Ocotlán. Predictors for increased development of MNs and NBUDs were high concentrations of Al, Zn, and Cu. In conclusion, chronic exposure to HMMs, especially Al, Cu, and Zn, in the studied population could be related to increased frequencies of NAs, such as NBUDs, KX, CC, and KL, in the buccal mucosa cells.     

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。     

不同木柴在两种条件下燃烧生成的颗粒物中多环芳烃的排放特性研究

将马尾松、赤桉、甜叶桉树和锦熟黄杨四种木柴,使用普通家用木柴燃烧加热炉在两种条件下进行燃烧,采集燃烧所排放的颗粒物样品并分析,获得了颗粒物中多环芳烃(Polycyclic Aromatic Hydrocarbons简写为PAHs)的排放因子及单个多环芳烃化合物的分布规律。结果显示:在颗粒物中检测出的多环芳烃化合物主要是分子量较大并被称为基因致毒性类的多环芳烃化合物,实验还发现:在四种木柴中,马尾松具有最高的多环芳烃化合物的排放因子,其次是赤桉和锦熟黄杨,最低的是甜叶桉树。两种不同的燃烧条件相比,慢燃烧比快燃烧所产生的总多环芳烃化合物和基因致毒性多环芳烃化合物的量都要高。     

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.     

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.     

DNA damage as a biomarker of genotoxic contamination in Mytilus galloprovincialis from the south coast of Portugal

DNA damage was evaluated in the haemolymph of Mytilus galloprovincialis from nine sites along the south coast of Portugal using the comet assay. DNA damage was low, in the same range of sites considered to suffer low impact from genotoxic contaminants. Even so, differences between sites, seasons and genders were found. Highest values were in mussels from the main estuaries and the fishery harbour, reflecting higher genotoxin levels, whereas the lowest values can be used as a baseline for future work. Non-contaminant related factors (e.g. temperature and oxygen) were also shown to influence DNA damage. Between seasons, highest values were in summer related not only to the increase of tourism in this region (～10-fold), but also to temperature. Between genders, males were found to be more sensitive. The condition index was also generally higher in summer. Lipid peroxidation, another damage biomarker, was measured in gills to assess if there is any association between the responses of both biomarkers and if they are similarly affected by the same environmental conditions. LPO like DNA damage was higher in summer. This work confirms that DNA damage is a sensitive biomarker to discriminate genotoxic contamination, even in areas considered to suffer low impact from genotoxins.     

Genotoxic effects and gene expression in <Emphasis Type="Italic">Danio rerio</Emphasis> (Hamilton 1822) (Cypriniformes: Cyprinidae) exposed to mining-impacted tributaries in Manizales,Colombia

The zebrafish (Danio rerio) is one of the most studied aquatic organisms for water biomonitoring, due to its sensitivity to environmental degradation and resistance to toxic substances. This study determined the presence of micronuclei and nuclear abnormalities in peripheral blood erythrocytes, and assessed the gene expression of caspase-3 (CASP-3) and metallothionein 1 (MT-1) in the gills and liver of D. rerio. The study fish (n = 45) were exposed to water collected from two stations with mining impact (E2 and E3) and a reference station without evident mining contamination (E1), all located in La Elvira stream (Manizales-Colombia). In addition, a positive control (PC) with HgCl₂ (50 μg/L) and negative control (NC) with tap water were included. The fish from the PC and E2 and E3 treatments displayed genotoxic effects and changes in gene expression, with significant differences in micronuclei formation and the presence of blebbed nuclei. The cytochrome oxidase subunit I (COI) gene was used as reference and proved to be stable compared to the β-actin and 28S ribosomal RNA (28S) genes. In gills, CASP-3 expression was higher in the PC, and MT-1 expression was higher in the PC and E3 treatment. In liver, CASP-3 was expressed in the E2 treatment, and MT-1 expression was low. These results show that the genotoxic effects and differential gene expression observed in fish exposed to water from La Elvira stream could also be affecting the organisms present in this habitat.     

Cytotoxicity and genotoxicity of Guaribas river water (Piauí, Brazil), influenced by anthropogenic action

In general, tropical rivers have a great impact on human activities. Bioaccumulation of toxins is a worldwide problem nowadays and has been, historically, overlooked by the supervisory authorities. This study evaluated cytogenotoxic effects of Guaribas river (a Brazilian river) water during dry and rainy seasons of 2014 by using the Allium cepa test system. The toxicogenetic variables, including root growth, mitotic index, and chromosomal aberrations, were analyzed in meristematic cells of A. cepa exposed to water samples taken from the up-, within, and downstream of the city Picos (state: Piauí). The physical-chemical parameters were also analyzed to explain water quality and possible anthropogenic action. Additionally, the presence of heavy metals was also analyzed to explain water quality and possible damaging effects on eukaryotic cells. The results suggest that the river water exerted cytotoxic, mutagenic, and genotoxic effects, regardless of the seasons. In addition, Guaribas river presented physico-chemical values outside the Brazilian laws, which can be a characteristic of human pollution (domestic sewage, industrial, and local agriculture). The genetic damage was positively correlated with higher levels of heavy metals. The pollution of the Guaribas river water may link to the chemical contamination, including the action of heavy metals and their impacts on genetic instability in the aquatic ecosystem. In conclusion, necessary steps should be taken into account for further toxicogenetic studies of the Guaribas river water, as it has an influence in human health of the same region of Brazil.     

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.