Genotoxicity assessment of acute exposure of chlorpyrifos to freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis

Chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridylphosphorothioate) is one of the organophosphate pesticides widely used in agricultural practices throughout world and irreversible inhibitor of cholinesterase in all animal species. Limited efforts have been made to study acute genotoxic effects of chlorpyrifos (CPF) in different tissues of fish using genotoxic biomarkers. Therefore, the present investigation was aimed to study the induction of DNA damage by CPF in freshwater teleost fish Channapunctatus using micronucleus assay (MN assay) and alkaline single-cell gel electrophoresis (comet assay). The value of LC(50) - 96 h of CPF was determined as 811.98 microgl(-1) for C. punctatus, in a semi-static system and on the basis of LC(50) value three acute concentrations viz., 203, 406 and 609 microgl(-1) were determined. The fishes were exposed to the different concentrations of CPF for 96 h and samplings were done at regular intervals for assessment of the MN frequencies and DNA damage. In general, significant effects (P<0.01) from both concentrations and time of exposure were observed in exposed fishes. It was found that the micronucleus induction was highest on 96 h at all concentrations in the peripheral blood. Similar trend was observed for the DNA damage measured in terms of the percentage of tail DNA in the lymphocyte and gill cells. This study explored the combined use of micronucleus assay and comet assay for in vivo laboratory studies using fresh water fish for screening the genotoxic potential of xenobiotics.     

DNA damage effect of cyprodinil and thiacloprid in adult zebrafish gills

Cyprodinil and thiacloprid are two of the most commonly used pesticides in Turkey. It is more likely to reach humans or animals due to their widespread use. This study aims to investigate whether there is a DNA damage risk due to cyprodinil and thiacloprid exposure. Zebrafish, which is used as a model organism in health and environmental research, and comet assay were chosen to demonstrate this damage. Ten zebrafish per group were exposed to 2 different concentrations for each pesticides (0.31 and 0.155 mg/L for cyprodinil and 1.64 and 0.82 mg/L for thiacloprid) for 21 days. After, gills were excised and comet assay was performed. Photos of an average of 50 cells per slide were taken and were analyzed with visual evaluation program. DNA damage was found to be increased in the 0.31 mg/L cyprodinil, 0.82 mg/L thiacloprid, and 1.64 mg/L thiacloprid treatment groups when compared to the control group (p < 0.001). Average tail DNA percentage parameter values were 9.45 ± 0.51, 10.30 ± 0.34, 11.17 ± 0.33, and 2.47 ± 0.06 respectively. Cyprodinil and thiacloprid were identified as genotoxic agents that should be investigated further.

     

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.     

Assessment of genotoxic effects of lead in occupationally exposed workers

The genotoxicological effects in 200 lead acid storage battery recycling and manufacturing industry workers in Hyderabad along with matched 200 controls were studied. The genetic damage was determined by comet, micronucleus (MN), and chromosomal aberration (CA) test in peripheral blood lymphocytes (PBL). The MN test was also carried out in buccal epithelial cells (BECs). Pb in ambient air, blood Pb (B-Pb) concentrations, and hematological parameters were measured. The superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidase (GPx), and malondialdehyde (MDA) formed were also studied. The results of the present study showed that there was a statistically significant (P?P?GPx, and CAT levels were significantly decreased while GSH and MDA levels increased in exposed group when compared to control group. The present study suggests that environmental health standards should be enforced to control Pb contamination from battery industries to reduce human health risk.     

Toxicological responses of earthworm (Eisenia fetida) exposed to metal-contaminated soils

The aim of this study was to evaluate the toxicological responses of earthworm (Eisenia fetida) induced by field-contaminated, metal-polluted soils. Biochemical responses and DNA damage of earthworm exposed to two multi-metal-contaminated soils in a steel industry park and a natural reference soil in Zijin Mountain for 2, 7, 14, and 28 days were studied. Results showed that three enzyme activities, including superoxide dismutase (SOD), acetylcholinesterase (AChE), and cellulase, in earthworm in metal-contaminated soils were significantly different from those of the reference soil. Cellulase and AChE were more sensitive than SOD to soil contamination. The Olive tail moment of the comet assay after 2-day exposure increased 56.5 and 552.0 % in two contaminated soils, respectively, compared to the reference soil. Our findings show that cellulase and DNA damage levels can be used as potential biomarkers for exposure of earthworm to metal-polluted soils.     

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.     

Acute toxicity and genotoxicity of two novel pesticides on amphibian, Rana N. Hallowell

Imidacloprid [1-(6-chloro-3-pyridylmethyl)-N-nitro-imidazolidin-2-ylideneamine] and RH-5849 [2'-benzoyl-l'-tert-butylbenzoylhydrazinel] are two pesticides used in China since 1992. In the present study we conducted acute toxicity test, micronucleus (MN) test and comet assay of the two pesticides on amphibian, Rana N. Hallowell, a sensitive organism suitable for acting as the bio-indicator of aquatic and agricultural ecosystems. The values of LC50-48 h of imidacloprid were found to be 165 mg l(-1) for tadpoles of Rana limnocharis and 219 mg l(-1) for tadpoles of Rana N. Hallowell. On the other hand, RH-5849 showed no acute toxicity to tadpoles during the 96 h exposure even it was saturated in the test solutions. There were significant differences in the MN frequencies between the negative controls and the treated groups at the dose of 8 mg l(-1) for imidacloprid (p < 0.05) and 40 mg l(-1) for RH-5849 (p < 0.01). Comet assay found significant differences (p < 0.01) in the distributions of DNA damage grades between the negative controls and groups treated in vitro with 0.05, 0.1, 0.2 and 0.5 mg l(-1) of imidacloprid and 5, 25, 50 and 100 mg l(-1) of RH-5849, respectively. DNA damage scores increased with the exposure levels of the two pesticides and dose-effect relationships were observed for both imidacloprid (r2 = 0.92) and RH-5849 (r2 = 0.98). The MN test and comet assay revealed potential adverse effects of the two pesticides on DNA in the erythrocytes of amphibians in aquatic and agricultural ecosystems.     

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.     

Use of sensitive methods for detection of DNA damage on human lymphocytes exposed to p,p'-DDT: Comet assay and new criteria for scoring micronucleus test

Wide distribution, stability and long persistence in the environment of dichlorodiphenyltrichloroethane (DDT), probably the best-known and most useful insecticide in the world, imposes the need for further examination of the effect of this chemical on human health and especially on the human genome. In this study, peripheral blood human lymphocytes from a healthy donor were exposed to 0.025 mg/L concentration of p,p'-DDT at different time periods (1, 2, 24 and 48 h). For the assessment of genotoxic effect, the new criteria for scoring micronucleus test and alkaline comet assay were used. Both methods showed that p,p'-DDT induces DNA damage in low concentration used in this research. Results of micronucleus test showed a statistically significant (p < 0.05) genotoxic effect of p,p'-DDT on human lymphocytes compared with corresponding control and a different exposure time. A comet assay also showed increased DNA damage caused in p,p'-DDT-exposed human lymphocytes than in corresponding control cells for the tail length. Results obtained by measuring the level of DNA migration and incidence of micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) indicate the sensitivity of these tests and their application in detection of primary genome damage after long-term exposure to establish the effect of p,p'-DDT on human genome.     

Genotoxicity of two novel pesticides for the earthworm, Eisenia fetida

In this paper, several studies were conducted to evaluate the genotoxicity of two pesticides, Imidacloprid and RH-5849, for earthworm (Eisenia fetida). Earthworms were exposed in different exposure systems to evaluate their acute toxicity and the genotoxicity of the two pesticides was evaluated by using the method of sperm deformity assessment, micronucleus test of root tip cells in Vicia faba, a mouse bone-marrow micronucleus test, and comet assay. LC(50) (interpolated concentration at which 50% mortality of test population occurs) for earthworms varied in different exposure systems. The results indicated that Imidacloprid was consistently more toxic than RH-5849 in all exposure systems. In this study, sperm deformity test was used to detect the potential adverse influences of pesticides on the reproduction of earthworms. The results demonstrated that significant induction of sperm deformity (p<0.01) and a dose-effect relationship displayed at Imidacloprid concentrations higher than 0.5 mg/kg dry soil. However, the sperm deformity frequency of groups exposed to RH-5849 did not show significant difference (p>0.05) from the control until the dose reached 100 mg/kg dry soil. The results of the V. faba micronucleus tests showed that micronuclei frequency of the exposed group did not show significant difference (p>0.05) from the control until the concentration of Imidacloprid and RH-5849 reached 100 mg/ml. The results of the mouse bone-marrow micronuclei test also indicate that two pesticides did not show significant effects (p>0.05) on the micronuclei frequency in mice bone-marrow cells until the dose reached 100 mg/kg for Imidacloprid and 300 mg/kg for RH-5849 (2/3 LD(50)). Although no genotoxicity was detected by using the micronucleus tests, the results of the comet assay showed that the two pesticides induce significant DNA damage (p<0.01) in earthworms and dose-effect relationships were displayed. The 'earthworm comet assay' is a rapid and sensitive way to screen chemicals or terrestrial environments for their DNA-damaging properties.     

Evaluating the potential genotoxicity of phthalates esters (PAEs) in perfumes using in vitro assays

We previously reported high levels of phthalate esters (PAEs) added as solvents or fixatives in 47 brands of perfumes. Diethyl phthalate was the most abundant compound (0.232–23,649 ppm), and 83.3% of the perfumes had levels >1 ppm, the threshold limit cited by a Greenpeace investigation. All samples had dimethyl phthalate levels higher than its threshold limit of 0.1 ppm, and 88, 38, and 7% of the perfumes had benzyl butyl phthalate, di(2-ethylhexyl) phthalate, and dibutyl phthalate levels, respectively, above their threshold limits. The role of PAEs as endocrine disruptors has been well documented, but their effect on genotoxic behavior has received little attention. We used in vitro single-cell gel electrophoresis (comet) and micronucleus (MN) assays with human lymphoblastoid TK6 cells to evaluate the genotoxic potency of 42 of the same perfumes and to determine its association with PAEs. All perfumes induced more DNA damage than a negative control (NEG), ≥ 90% of the samples caused more damage than cells treated with the vehicles possibly used in perfume’s preparations such as methanol (ME) and ethanol (ET), and 11.6% of the perfumes caused more DNA damage than a positive control (hydrogen peroxide). Chromosome breakage expressed as MN frequency was higher in cells treated with 71.4, 64.3, 57.1, and 4.8% of the perfumes than in NEG, cells treated with ME or ET, and another positive control (x-rays), respectively. The genotoxic responses in the comet and MN assays were not correlated. The comet assay indicated that the damage in TK6 cells treated with five PAEs at concentrations of 0.05 and 0.2 ppm either individually or as a mixture did not differ significantly from the damage in cells treated with the perfumes. Unlike the comet assay, the sensitivity of the MN assay to PAEs was weak at both low and high concentrations, and MN frequencies were generally low. This study demonstrates for the first time the possible contribution of PAEs in perfumes to DNA damage and suggests that their use as solvents or fixatives should be regulated. Other ingredients with mutagenic/genotoxic properties, however, may also have contributed to the DNA damage. Future studies should focus on applying a series of assays that use different cellular models with various endpoints to identify the spectrum of genotoxic mechanisms involved.

     

Mammalian cell-line based toxicological evaluation of paper mill black liquor treated in a soil microcosm by indigenous alkalo-tolerant Bacillus sp

Organic pollutants present in the soil of a microcosm containing pulp and paper mill black liquor were extracted with hexane/acetone (1:1 v/v) to study the biodegradation and detoxification potential of a Bacillus sp. gas chromatography-mass spectroscopic (GC-MS) analysis performed after biodegradation showed formation of simpler compounds like p-hydroxyhydrocinnamic acid (retention time [RT] 19.3 min), homovanillic acid methyl ester (RT 21.6 min) and 3,5-dimethoxy-p-coumaric alcohol (RT 24.7 min). The methyltetrazolium (MTT) assay for cytotoxicity, 7-ethoxyresorufin-O-deethylase (EROD) assay for dioxin-like behavior and alkaline comet assay for genotoxicity were carried out in the human hepatocarcinoma cell line HuH-7 before and after bacterial treatment. Bioremediation for 15 days reduced toxicity, as shown by a 139-fold increase in black liquor’s LC₅₀ value, a 343-fold reduction in benzo(a)pyrene equivalent value and a 5-fold reduction in olive tail moment. The EROD assay positively correlated with both the MTT and comet assays in post biodegradation toxicity evaluation.     

Use of sensitive methods for detection of DNA damage on human lymphocytes exposed to p,p′-DDT: Comet assay and new criteria for scoring micronucleus test

Wide distribution, stability and long persistence in the environment of dichlorodiphenyltrichloroethane (DDT), probably the best-known and most useful insecticide in the world, imposes the need for further examination of the effect of this chemical on human health and especially on the human genome. In this study, peripheral blood human lymphocytes from a healthy donor were exposed to 0.025 mg/L concentration of p,p′-DDT at different time periods (1, 2, 24 and 48 h). For the assessment of genotoxic effect, the new criteria for scoring micronucleus test and alkaline comet assay were used. Both methods showed that p,p′-DDT induces DNA damage in low concentration used in this research. Results of micronucleus test showed a statistically significant (p < 0.05) genotoxic effect of p,p′-DDT on human lymphocytes compared with corresponding control and a different exposure time. A comet assay also showed increased DNA damage caused in p,p′-DDT-exposed human lymphocytes than in corresponding control cells for the tail length. Results obtained by measuring the level of DNA migration and incidence of micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) indicate the sensitivity of these tests and their application in detection of primary genome damage after long-term exposure to establish the effect of p,p′-DDT on human genome.     

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.     

Application of the comet and micronucleus assays to butterfish (Pholis gunnellus) erythrocytes from the Firth of Forth, Scotland.

This report describes an investigation of genotoxic effects in an inter-tidal fish species sampled along a pollution gradient in the Firth of Forth, Scotland, UK. The comet assay is an electrophoretic technique for measuring DNA breakage in nuclei from individual cells and has only recently been applied to field investigations of genotoxicity. The measurement of nuclear anomalies (NA), such as the presence of micronuclei (MN) and 'lobes', has been successfully utilised in many field studies of genotoxic effects of contaminated sediments. These two techniques were applied to nucleated red blood cells (RBC) from the butterfish, Pholis gunnellus. The comet assay was adapted and validated for use in this species. Fish were sampled from the inner Firth of Forth, which has a legacy of industrial contamination and the outer Firth of Forth which is comparatively clean. The analysis of DNA strand breakage using this technique did not reveal any significant differences between animals sampled from inner and outer zones of the Firth. In contrast, MN and NA frequencies were elevated in the inner polluted zone of the Firth compared to the outer zone. This study suggests: (1) there are genotoxic effects associated with contaminants in the inner Firth of Forth, and (2) the comet assay may not be a suitable genotoxicity biomarker in fish.     

Toxicity and genotoxic evaluations in African catfish Clarias gariepinus (Burchell 1822) exposed to Act Force Gold®, Butaforce®, and Atraforce®

Act Force Gold^®, Butaforce^®, and Atraforce^® are among the most commonly used pesticides in Nigeria. The lethal concentrations and the respective toxic units for the three pesticides were determined. The genotoxic effects of the three pesticides were investigated in the red blood cells of Clarias gariepinus using micronucleus (MN) assay. The 96 h LC₅₀ was 4.75, 4.84, and 54.74 mg L⁻¹ for Act Force Gold^®, Butaforce^®, and Atraforce^®, respectively. The toxic units in ascending order of toxicity were 1.83, 20.66, and 21.05 for Act Force Gold^®, Butaforce^®, and Atraforce^® respectively. The estimated safe levels based on NAS/NAE varied from 4.75 × 10⁻¹–4.75 × 10⁻⁵ in Act Force Gold^® through 4.64 × 10⁻¹–4.85 × 10⁻⁵ in Butaforce^® to 5.74–5.74 × 10⁻⁵ in Atraforce^®. Fish specimens were exposed to the pesticides and sampling was done at regular intervals at days 1, 7, 14, and 21 and after another 7-day recovery period. The results obtained indicated concentration- and duration-dependent increase in % MN formation with maximum values of 3.40 ± 0.25 for Act Force Gold^® on day 14 and 3.05 ± 0.36 and 2.35 ± 0.14 for Butaforce^® and Atraforce^® respectively on day 7 of exposure. The 7-day recovery period could not reverse the trend as the % MN values obtained were significantly different from the control. The results further support the use of MN assay in assessing the toxicity of aquatic pollutants and can be used in the monitoring of aquatic ecosystems.

     

Cytogenetic status of human lymphocytes after exposure to low concentrations of p,p'-DDT, and its metabolites (p,p'-DDE, and p,p'-DDD) in vitro

Despite that the use of DDT has been restricted for more than 40 years to malaria affected areas, low doses of this pesticide and its metabolites DDE and DDD can be found in the environment around the world. Although it has been shown that these pollutants induce cell and DNA damage, the mechanisms of their cytogenotoxic activity remains largely unknown. This study looks into their possible genotoxic effects, at doses that can be found in body fluids, on human lymphocytes using the cytokinesis-block micronucleus assay and the comet assay. After exposure for 1, 6, and 24 h compounds p,p′-DDT (0.1 μg mL⁻¹), p,p′-DDE (4.1 μg mL⁻¹), and p,p′-DDD (3.9 μg mL⁻¹) showed increase in DNA damage. The most significant results were observed at exposure period of 24 h where number of micronucleated cells increased from control 2.5 ± 0.71 to 23.5 ± 3.54, 13.5 ± 0.71, and 16.5 ± 6.36 for DDT, DDE, and DDD, respectively. Similar effect was observed using comet test where the percentage of DNA in comets tail increased from control 1.81 ± 0.16 to 17.24 ± 0.55, 11.21 ± 0.56 and 9.28 ± 0.50 for each compound, respectively. At the same time Fpg-comet assay failed to report induction of oxidative DNA damage of these pollutants. Additionally, the type of cell death was determined using diffusion assay and necrosis dominated. Our findings suggest that even at low concentrations, these pesticides could induce cytogenetic damage to human peripheral blood lymphocytes and in that manner have the impact on human health as well.     

Nitrogen dioxide inhalation induces genotoxicity in rats

Nitrogen dioxide (NO₂) is a ubiquitous reactive free-radical gas, which has been associated with momentary and chronic health effects. In the present study, comet, micronucleus (MN) and DNA–protein crosslinks (DPC) assays were used to investigate the genotoxicity following in vivo inhalation exposure of rats to NO₂. The results show that inhalation exposure of rats to NO₂ induced DNA strand breakage and the formation of DPC in the cells from various internal organs (brain, lung, liver, spleen, kidney and heart), as well as resulted in obvious increase of MN frequency in the bone marrow cells of rats. Furthermore, above genotoxic responses showed significant linear dose-dependent manners. These results implicate that NO₂ is a genotoxic agent and these observations are informative for understanding the mechanisms of adverse effects of nitrogen dioxide.     

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.     

DNA integrity of onion root cells under catechol influence

Catechol is a highly toxic organic pollutant, usually abundant in the waste effluents of industrial processes and agricultural activities. The environmental sources of catechol include pesticides, wood preservatives, tanning lotion, cosmetic creams, dyes, and synthetic intermediates. Genotoxicity of catechol at a concentration range 5?×?10^?1–5 mM was evaluated by applying random amplified polymorphic DNA (RAPD) and time-lapse DNA laddering tests using onion (Allium cepa) root cells as the assay system. RAPD analysis revealed polymorphisms in the nucleotidic sequence of DNA that reflected the genotoxic potential of catechol to provoke point mutations, or deletions, or chromosomal rearrangements. Time-lapse DNA laddering test provided evidence that catechol provoked DNA necrosis and apoptosis. Acridine orange/ethidium bromide staining could distinguish apoptotic from necrotic cells in root cells of A. cepa.