Atrazine promotes biochemical changes and DNA damage in a Neotropical fish species

The effects of Atrazine, an herbicide used worldwide and considered as a potential contaminant in aquatic environments, were assessed on the Neotropical fish Prochilodus lineatus acutely (24 and 48 h) exposed to 2 or 10 μg L⁻¹ of atrazine by using a set of biochemical and genetic biomarkers. The following parameters were measured in the liver: activity of the biotransformation enzymes ethoxyresorufin-O-deethylase (EROD) and glutathione S transferase (GST), antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), content of reduced glutathione (GSH), generation of reactive oxygen species (ROS) and occurrence of lipid peroxidation (LPO); in brain and muscle the activity of acetylcholinesterase (AChE) and DNA damage (comet assay) on erythrocytes, gills and liver cells. A general decreasing trend on the biotransformation and antioxidant enzymes was observed in the liver of P. lineatus exposed to atrazine; except for GR, all the other antioxidant enzymes (SOD, CAT and GPx) and biotransformation enzymes (EROD and GST) showed inhibited activity. Changes in muscle or brain AChE were not detected. DNA damage was observed in the different cell types of fish exposed to the herbicide, and it was probably not from oxidative origin, since no increase in ROS generation and LPO was detected in the liver. These results show that atrazine behaves as enzyme inhibitor, impairing hepatic metabolism, and produces genotoxic damage to different cell types of P. lineatus.     

Effects of methylmercury exposure on glutathione metabolism, oxidative stress, and chromosomal damage in captive-reared common loon (Gavia immer) chicks

We quantified the level of dietary mercury (Hg), delivered as methylmercury chloride (CH₃HgCl), associated with negative effects on organ and plasma biochemistries related to glutathione (GSH) metabolism and oxidative stress, and chromosomal damage in captive-reared common loon (Gavia immer) chicks reared from hatch to 105 days. Mercury-associated effects related to oxidative stress and altered glutathione metabolism occurred at 1.2 μg Hg/g and 0.4 μg Hg/g, an ecologically relevant dietary mercury level, but not at 0.08 μg Hg/g. Among the variables that contributed most to dissimilarities in tissue chemistries between control and treatment groups were increased levels of oxidized glutathione (GSSG), GSH peroxidase, and the ratio of GSSG to GSH in brain tissue; increased levels of hepatic GSH; and decreased levels of hepatic glucose-6-phosphate dehydrogenase (G-6-PDH). Our results also suggest that chronic exposure to environmentally relevant dietary Hg levels did not result in statistically significant somatic chromosomal damage in common loon chicks.     

Cypermethrin has the potential to induce hepatic oxidative stress, DNA damage and apoptosis in adult zebrafish (Danio rerio)

Cypermethrin (CYP), a widely used Type II pyrethroid pesticide, is one of the most common contaminants in the freshwater aquatic system. We studied the effects of CYP exposure on the induction of hepatic oxidative stress, DNA damage and the alteration of gene expression related to apoptosis in adult zebrafish. Hepatic mRNA levels for the genes encoding antioxidant proteins, such as Cu/Zn-Sod, Mn-Sod, Cat, and Gpx, were significantly upregulated when zebrafish were exposed to various concentrations of CYP for 4 or 8 days. In addition, the main genes related to fatty acid β-oxidation and the mitochondrial genes related to respiration and ATP synthesis were also significantly upregulated after exposure to high concentrations (1 and 3 μg L⁻¹) of CYP for 4 or 8 days. Moreover, in a comet assay of zebrafish hepatocytes, tail DNA, tail length, tail moment and Olive tail moment increased in a concentration-dependent manner. The significant induction (p < 0.01) of all four parameters observed with CYP concentrations of 0.3 μg L⁻¹ or higher suggests that heavy DNA damage was induced even at low levels. Furthermore, several apoptosis- related genes, such as p53, Apaf1 and Cas3, were significantly upregulated after CYP exposure, and Bcl2/Bax expression ratio decreased, especially in groups treated with 1 and 3 μg L⁻¹ CYP for 8 days. Taken together, our results suggested that CYP has the potential to induce hepatic oxidative stress, DNA damage and apoptosis in zebrafish. This information will be helpful in fully understanding the mechanism of aquatic toxicology induced by CYP in fish.     

Monitoring of DNA damage in haemocytes of freshwater mussel Sinanodonta woodiana sampled from the Velika Morava River in Serbia with the comet assay

This study was undertaken to investigate the potential of the freshwater mussel Sinanodonta woodiana for detection of genotoxic pollution of the environment. Study was performed at two sites in the Velika Morava River, from May 2010 to February 2011. The alkaline comet assay on haemocytes was used, and the olive tail moment (OTM) was chosen as a measure of DNA damage. The specimens held on acclimation under controlled laboratory conditions for 10 d were used as a control. Chemical analysis revealed the presence of phosphates and increased concentrations of zinc, copper and nickel at both sites during the entire sampling period. The values of OTM in mussels collected from the environment, significantly correlated with the concentration of zinc (r = 0.6248), temperature (r = 0.7006) and dissolved oxygen (r = 0.7738). Seasonal variations in genotoxic response were observed, with the highest OTM values obtained during summer months. Preliminary results of the in vitro study indicated the effect of water temperature on genotoxic response to zinc and cadmium in S. woodiana suggesting that the presence of genotoxic pollutants during months with lower temperature could be under-estimated. Obtained results indicate that S. woodiana could be a valuable tool for active biomonitoring of aquatic environments and emphasizes the importance of seasonal genotoxic monitoring with this organism.     

Aporrectodea caliginosa, a suitable earthworm species for field based genotoxicity assessment?

There is a growing interest for the application of biomakers to field-collected earthworms. Therefore we have evaluated the usability of native populations of endogeic, widely distributed earthworm Aporrectodea caliginosa in the assessment of soil genotoxicity using the Comet assay. Validation of the Comet assay on earthworm coelomocytes has been established using commercially available Eisenia fetida exposed to copper, cadmium, and pentachlorophenol, along with A. caliginosa exposed to copper in a filter paper contact test. Neutral red retention time (NRRT) assay was conducted on copper exposed and field-collected earthworms. Significant DNA and lysosomal damage was measured using Comet and NRRT assays in native populations of A. caliginosa sampled from the polluted soils in the urban area in comparison to the earthworms from the reference site. The results of this study confirm the employment of A. caliginosa as a suitable species for the in situ soil toxicity and genotoxicity field surveys.     

Cadmium-induced DNA damage and mutations in Arabidopsis plantlet shoots identified by DNA fingerprinting

Random amplified polymorphic DNA (RAPD) test is a feasible method to evaluate the toxicity of environmental pollutants on vegetal organisms. Herein, Arabidopsis thaliana (Arabidopsis) plantlets following Cadmium (Cd) treatment for 26 d were screened for DNA genetic alterations by DNA fingerprinting. Four primers amplified 20-23 mutated RAPD fragments in 0.125-3.0 mg L⁻¹ Cd-treated Arabidopsis plantlets, respectively. Cloning and sequencing analysis of eight randomly selected mutated fragments revealed 99-100% homology with the genes of VARICOSE-Related, SLEEPY1 F-box, 40S ribosomal protein S3, phosphoglucomutase, and noncoding regions in Arabidopsis genome correspondingly. The results show the ability of RAPD analysis to detect significant genetic alterations in Cd-exposed seedlings. Although the exact functional importance of the other mutated bands is unknown, the presence of mutated loci in Cd-treated seedlings, prior to the onset of significant physiological effects, suggests that these altered loci are the early events in Cd-treated Arabidopsis seedlings and would greatly improve environmental risk assessment.     

Effects of organic contaminants in reactive oxygen species, protein carbonylation and DNA damage on digestive gland and haemolymph of land snails

The present study focused on early responses of land snails Eobania vermiculata to organic environmental contaminants, by investigating the use of a newly-established method for the measurement of protein carbonylation as a new biomarker of terrestrial pollution, as well as by measuring the ROS production and the DNA damage. Land snails were exposed to different concentrations of chlorpyrifos, parathion-methyl or PAHs in vivo or in vitro in the laboratory. The susceptibility of exposed snails was increased in relation to oxidative stress induced by contaminants tested. A statistically significant increase in ROS production, protein carbonylation and DNA damage was revealed in the snails treated with pollutants, compared to the untreated ones. The results indicated the effectiveness of measuring ROS production and DNA damage and reinforce the application of the present ELISA method in organic terrestrial pollution biomonitoring studies.     

DNA damage in Populus tremuloides clones exposed to elevated O3

The effects of elevated concentrations of atmospheric tropospheric ozone (O₃) on DNA damage in five trembling aspen (Populus tremuloides Michx.) clones growing in a free-air enrichment experiment in the presence and absence of elevated concentrations of carbon dioxide (CO₂) were examined. Growing season mean hourly O₃ concentrations were 36.3 and 47.3 ppb for ambient and elevated O₃ plots, respectively. The 4th highest daily maximum 8-h ambient and elevated O₃ concentrations were 79 and 89 ppb, respectively. Elevated CO₂ averaged 524 ppm (+150 ppm) over the growing season. Exposure to O₃ and CO₂ in combination with O₃ increased DNA damage levels above background as measured by the comet assay. Ozone-tolerant clones 271 and 8L showed the highest levels of DNA damage under elevated O₃ compared with ambient air; whereas less tolerant clone 216 and sensitive clones 42E and 259 had comparably lower levels of DNA damage with no significant differences between elevated O₃ and ambient air. Clone 8L was demonstrated to have the highest level of excision DNA repair. In addition, clone 271 had the highest level of oxidative damage as measured by lipid peroxidation. The results suggest that variation in cellular responses to DNA damage between aspen clones may contribute to O₃ tolerance or sensitivity.     

Phenol tolerance, changes of antioxidative enzymes and cellular damage in transgenic tobacco hairy roots colonized by arbuscular mycorrhizal fungi

Phytoremediation has been recognized as a cheap and eco-friendly technology which could be used for the remediation of organic pollutants, such as phenolic compounds. Besides, the extent to which plants react to environmental pollution might depend on rhizosphere processes such as mycorrhizal symbiosis. In the present work, phenol tolerance of transgenic tobacco hairy roots (HR), namely TPX1, colonized with an arbuscular mycorrhizal fungus (AMF) was studied. However, the question is whether AMF symbiosis can moderate adverse effects of phenol to the plant tissues. Thus, the antioxidative response as well as parameters of oxidative damage, like malondialdehyde (MDA) content, were determined. Antioxidative enzymes such as peroxidase, superoxide dismutase, ascorbate peroxidase were higher in TPX1 HR colonized with AMF, compared to wild type HR colonized by AMF, in the presence of increasing concentrations of the pollutant. Besides, MDA levels remained unaltered in TPX1 HR associated with AMF treated with the xenobiotic. These results, suggested that this culture could tolerate phenol and moreover, it has an efficient protective mechanism against phenol-induced oxidative damage, which is of great importance in the selection of species with remediation capacities. Thus, transgenic HR colonized with AMF could be considered as an interesting model system to study different processes which play a key role in the phytoremediation of organic pollutants.     

Calcium protects roots of Sedum alfredii H. against cadmium-induced oxidative stress

Sedum alfredii is a well-known Cd (cadmium) hyperaccumulator native to China. The impacts of exogenous Ca on Cd-induced oxidative stress and antioxidant systems in roots of S. alfredii were investigated by using cellular and biochemical approaches. Supplementation of the medium with higher Ca levels resulted in alleviated growth inhibition and decreased Cd concentration, as well as increased Ca concentration in roots. Cadmium induced lipid peroxidation and loss of plasma membrane integrity, reactive oxygen species overproduction, as well as ultrastructural changes of root cells were largely reversed by Ca supplementation in the medium. Calcium application significantly altered the Cd effects on antioxidant enzymes and non-enzyme antioxidants (non-protein thiols), and significantly increased glutathione (GSH) biosynthesis. The results suggest that Ca is able to protect the roots of S. alfredii against Cd toxicity by restoration of Cd-displaced Ca, alleviation of the metal induced oxidative stress, as well as promotion of GSH biosynthesis.     

In situ studies on growth,oxidative stress responses,and gene expression of juvenile bighead carp (Hypophthalmichthys nobilis) to eutrophic lake water dominated by cyanobacterial blooms

Cyanobacterial blooms have received increasing attention as a public biohazard for human and animal health. To assess the effect of cyanobacteria-dominant lake water on juvenile fish, we measured the responses of specific growth rate, condition factor, body weight and body length, oxidative stress, and related gene expression of juvenile bighead carp Hypophthalmichthys nobilis exposed to in situ eutrophic lake (Chl a was around 7.0 μg L⁻¹). Results showed in situ cyanobacteria-dominant lake water had no effect on the growth performance, but significantly elevated the contents of malondialdehyde, the expression of heat shock protein 70, and the activity of superoxide dismutase, indicating that oxidative stress occurred. Meanwhile in situ lake water significantly decreased the expression of catalase and glutathione S-transferase genes. We conclude that in situ cyanobacteria-dominated lake water was harmful to juvenile bighead carp based on the oxidative stress and changes in the related gene expression levels.     

Chromosomal aberrations and DNA damage in human populations exposed to the processing of electronics waste

Background, aim, and scope  It has been known that the pollutants of electronic wastes (E-wastes) can lead to severe pollution to the environment. It has been reported that about 50% to 80% of E-wastes from developed countries are exported to Asia and Africa. It has become a major global environmental problem to deal with ‘E-wastes’. E-waste recycling has remained primitive in Jinghai, China. This not only produces enormous environmental pollution but also can bring about toxic or genotoxic effects on the human body, threatening the health of both current residents and future generations living in the local environment. The concentration of lead in the blood of children in the E-waste polluted area in China is higher than that of the control area. But little is known about the cytogenetic effect to human beings caused by the pollution of E-wastes. In the present study, experiments have been performed to investigate the genetics of permanent residents of three villages with numerous E-waste disposal sites and to analyze the harmful effects of exposure to E-wastes. Materials and methods  In total, 171 villagers (exposed group) were randomly selected from permanent residents of three villages located in Jinghai County of Tianjin, China, where there has been massive disposal of E-wastes. Thirty villagers were selected from the neighboring towns without E-waste disposal sites to serve as controls. Chromosomal aberrations and cytokinesis blocking micronucleus were performed to detect the cytogenetic effect, dic + r (dicentric and ring chromosome), monomer, fragments (acentric fragments, minute chromosomes, and acentric rings), translocation, satellite, quadriradial, total aberrations, and micronuclear rate were scored for each subject. DNA damage was detected using comet assay; the DNA percentage in the comet tail (TDNA%), tail moment (TM), and Olive tail moment (OTM) were recorded to describe DNA damage to lymphocytes. Results  The total chromosome aberration rates (5.50%) and micronuclear rates (16.99%) of the exposure group were significantly higher than in the control group (P = 0.000). The percentage of DNA in the comet tail, tail moment, and Olive tail moment detected by comet assay showed that there was a significant difference in DNA damage in the exposure group (P = 0.000). The chromosome aberration, micronucleus rate, and DNA damage observed in women were significantly higher than those in men. Chromosome aberration and micronuclear rates of both smokers and non-smokers in the exposure group are obviously higher than that in the control group (P = 0.000). Discussion  The use of outdated (and unsafe) ways to deal with E-wastes can lead to exposure to a variety of substances harmful to human health. The components of pollution may enter the human body through the air, drinking water, and food chain to damage human genetic material, resulting in genomic instability. The rates of chromosomal aberration, micronucleus formation, and the degree of DNA damage in women in the group exposed to electronic waste were significantly higher than in men. The reason for this may be concerned with the traditional lifestyle of the local residents or the difference of sensitivity to the exposure to E-wastes or any others. Further investigations are needed to provide evidence to demonstrate this. Conclusions  Here, we report the obviously cytogenetic toxicity to the exposure population by the E-waste pollution for the first time. E-waste pollution may be a potential agent of genetic mutation, and may induce cytogenetic damage within the general population exposed to the pollution. These findings need to be considered, and steps should be taken to protect the current population and future generations from the effects of pollution with E-wastes. Recommendations and perspectives  The above results remind us that the impact of E-waste recycling on environmental quality of Jinghai should be evaluated soon. Moreover, it is urgent for the government to prohibit E-waste import and its processing by outdated ways. The future studies such as pollutant details of drinking water, air, and soil in the area as well as epidemiological investigations on the harmful effect to children must be performed eagerly. All the data available do provide a compelling case for immediate action in both countries to address workplace health and safety and waste management. Qiang Liu and Jia Cao contributed equally to this study and share the first authorship.     

Application of the comet assay and detection of DNA damage in haemocytes of medicinal leech affected by aluminium pollution: A case study

This report describes an investigation of genotoxic effects in medicinal leech (Hirudo verbana) exposed to water and sediment of Lake Njivice (Krk Island, Croatia) contaminated by aluminium compounds. The levels of primary DNA damage in leech haemocytes and loss of DNA integrity caused by acute and chronic exposure to contaminated water and sediment were investigated using the alkaline comet assay. Genotoxic effects induced by acute exposure to contaminants were evaluated on leech haemocytes and blood cells of fish and mouse treated ex vivo. The effects of chronic exposure were assessed on haemocytes sampled from an animal kept under laboratory conditions on contaminated water and sediment for 180 days. The results indicate the DNA damaging potential of aluminium compounds present in an excess amount in tested samples.     

Contamination assessment of a coastal lagoon (Ria de Aveiro, Portugal) using defence and damage biochemical indicators in gill of Liza aurata - An integrated biomarker approach

Fish gill importance in toxicants uptake, bioconcentration and excretion allied to meagre knowledge on branchial damage/protection responses substantiate this study. Five critical sites in Ria de Aveiro (Portugal) were assessed in comparison with a reference site (Torreira), focusing on Liza aurata gill antioxidant defences versus damage (oxidative and genetic). Only in Barra fish displayed damage (lipid peroxidation) though no differences were found in antioxidants. In all other sites, except Rio, antioxidant alterations were found. Thus, fish from Gafanha, Laranjo and Vagos showed higher total glutathione, glutathione peroxidase and catalase. Higher glutathione reductase and glutathione S-transferase activity was also found in the first and the last sites, respectively. In Laranjo, metallothionein levels were higher though lower in Gafanha and Vagos. In general, damage was not accompanied by defences weakening confirming that predicting damage based on antioxidants depletion is not straightforward. The integrated biomarker response index ranked sites as: Gafanha > Barra > Laranjo > Vagos > Rio > Torreira.     

Influence of arsenic stress on synthesis and localization of low-molecular-weight thiols in Pteris vittata

The roles of low-molecular-weight thiols (LMWTs), such as glutathione and phytochelatins, in arsenic (As) tolerance and hyperaccumulation in Pteris vittata an As-hyperaccumulator fern remain to be better understood. This study aimed to thoroughly characterize LMWT synthesis in P. vittata to understand the roles played by LMWTs in As tolerance and hyperaccumulation. LMWT synthesis in P. vittata was induced directly by As, and not by As-mediated oxidative stress. Expression of PvECS2, one of the putative genes of γ-glutamylcysteine synthetase (γECS), increases in P. vittata shoots at 48 h after the onset of As exposure, almost corresponding to the increase in the concentrations of γ-glutamylcysteine and glutathione. Furthermore, localization of As showed similar trends to those of LMWTs in fronds at both whole-frond and cellular levels. This study thus indicates the specific contribution of LMWTs to As tolerance in P. vittata. γECS may be responsible for the As-induced enhancement of LMWT synthesis.     

Heavy metal exposure in large game from a lead mining area: Effects on oxidative stress and fatty acid composition in liver

The Pb mining area of the valley of Alcudia and the Sierra Madrona mountains (S. Spain) has been exploited intermittently for over 2100 years, since Roman occupation and up until the late 1900s. Red deer (n = 168) and wild boar (n = 58) liver and bone (metacarpus) were analyzed for Pb, Zn, Cu, Cd, As and Se. Lipid peroxidation, total and oxidized glutathione (GSH), superoxide dismutase (SOD), GSH peroxidase (GPX) and fatty acid composition were studied in liver of red deer. The concentrations of Pb in liver and bone of red deer and wild boar were higher in the mining area than in the control area, and higher in the wild boar than red deer, but well below the level associated with clinical signs of Pb poisoning. Liver levels of Cu, Cd and Se were also higher in red deer from the mining area. Red deer from the mining area had 39% less total GSH than in the control area. The percentage of docosahexaenoic acid in liver of red deer from the mining area was 16% lower than in the control area.     

Assessing the genotoxic potentials of arsenic in tilapia (Oreochromis mossambicus) using alkaline comet assay and micronucleus test

This experiment was conducted to study the genotoxic potentials of sodium arsenite (NaAsO₂) in freshwater fish Oreochromis mossambicus by using alkaline comet assay and micronucleus (MN) test. Fish were exposed to three different concentrations (3 ppm, 28 ppm and 56 ppm) of arsenic and gill, liver and blood tissue samples were collected after 48 h, 96 h and 192 h of exposure. Arsenic exposure induced DNA damage in all tissues examined in a concentration dependent manner. A significant (p < 0.05) increase in the comet tail DNA (%) of the exposed fish liver, gill, and blood was observed after 48 h and 96 h of exposure, but a decline in DNA damage was recorded in all the tissues at all the three concentrations studied after 192 h of exposure. Liver tissue exhibited significantly (p < 0.05) higher DNA damage at all the concentrations examined, followed by gill and blood. Higher liver tail DNA (51.38 ± 0.21%) refers that it is more prone to injury to arsenic toxicity than the gill and blood. In blood samples arsenic induced micronucleus formation in a concentration dependent manner and highest (5.8 ± 0.46%) value was recorded in 56 ppm after 96 h of exposure, whereas, it was decreased after 192 h of exposure at all the three concentrations of NaAsO₂ examined which refers to the DNA repairing ability of fish to arsenic toxicity. The results of this study depict the genotoxic potentials of arsenic to fish which in turns provide insight on advanced study in aquatic toxicology.     

AHH activity, tissue dose and DNA damage in different tissues of the scallop Chlamys farreri exposed to benzo[a]pyrene

A collaborative study was performed on scallops (Chlamys farreri) exposed to 0.5, 3 and 10 microg/L benzo[a]pyrene (B[a]P) for 20 days. The levels of aryl hydrocarbon hydroxylase (AHH) activity, B[a]P accumulation and DNA strand break were assayed in the gill and digestive gland. Results showed that AHH activity and B[a]P accumulation were significantly related to B[a]P dose. AHH activity was induced and then became stable gradually. B[a]P accumulation increased first and showed an incoming plateau. DNA strand break levels in the 0.5 and 3 microg/L B[a]P groups remained high and significantly different from control values until day 6, followed by a reduction in the gill and no recovery in the digestive gland. The 10 microg/L B[a]P groups remained significantly lower than control until the end. These results suggested that the application of comprehensive indices may give information on the actual exposure of organisms to pollutants, and also information on toxic effects.     

Influence of fly ash aided phytostabilisation of Pb, Cd and Zn highly contaminated soils on Lolium perenne and Trifolium repens metal transfer and physiological stress

Due to anthropogenic activities, large extends of soils are highly contaminated by Metal Trace Element (MTE). Aided phytostabilisation aims to establish a vegetation cover in order to promote in situ immobilisation of trace elements by combining the use of metal-tolerant plants and inexpensive mineral or organic soil amendments. Eight years after Coal Fly Ash (CFA) soil amendment, MTE bioavailability and uptake by two plants, Lolium perenne and Trifolium repens, were evaluated, as some biological markers reflecting physiological stress. Results showed that the two plant species under study were suitable to reduce the mobility and the availability of these elements. Moreover, the plant growth was better on CFA amended MTE-contaminated soils, and the plant sensitivity to MTE-induced physiological stress, as studied through photosynthetic pigment contents and oxidative damage was lower or similar. In conclusion, these results supported the usefulness of aided phytostabilisation of MTE-highly contaminated soils.     

Environmental ozone exposure and oxidative DNA damage in adult residents of Florence, Italy

In 71 adults residing in Florence, Italy, enrolled in a prospective study, we investigated the correlation between individual levels of oxidative DNA damage detected by the Comet assay in circulating lymphocytes, and a specific ozone exposure score calculated in 10 different time-windows (0-5 to 0-90 days) before blood drawing, based on daily measurements provided by the local environmental monitoring system.Overall, statistically significant positive correlations between average ozone concentrations and DNA damage emerged in almost all time-windows considered; correlations were more evident among males, non-smokers, and traffic-exposed workers. Multivariate regression analyses taking into account selected individual characteristics, showed an independent effect on DNA damage of average ozone concentrations in the last 60-90 days before blood drawing. Local residents showed a divergent pattern with correlations restricted to shorter time-windows.Our results suggest that ozone concentrations at ground levels modulate oxidative DNA damage in circulating lymphocytes of residents of polluted areas.