Genotoxic effects of glyphosate on Physalaemus tadpoles

Genotoxicity studies have revealed that pesticides bind to genetic material in non-target vertebrates, thereby impairing the genetic integrity of these animals. The main objective of this study was to determine the genotoxic damage in erythrocytes of two native South American amphibian Physalaemus cuvieri and Physalaemus gracilis, both species exposed to a glyphosate-based herbicide. We evaluated the presence of micronuclei (MN) and erythrocyte nuclear abnormalities (ENA) as biomarkers for potential genotoxic compounds. Tadpoles were exposed to doses permitted by Brazilian legislation and concentrations found naturally in Brazilian and Argentinian waters (500, 700 and 1000 μg/L). Glyphosate-based herbicide caused micronuclei formation and several types of erythrocyte nuclear abnormalities in both Physalaemus species. The total frequency of MN and ENA demonstrated the occurrence of cell damage at all tested concentrations. Glyphosate herbicide can be considered a genotoxic that may impact the genetic integrity of native populations of P. cuvieri and P. gracilis.     

Induction of micronuclei and binuclei in blood, gill and liver cells of fishes subchronically exposed to cadmium chloride and copper sulphate.

Common carp (Cyprinus carpio), Prussian carp (Carassius gibelio) and Peppered cory (Corydoras paleatus) were evaluated as target species to perform genotoxicity tests for heavy metals. Fishes were exposed to different doses of cadmium (0.005-0.1 mg/L) and copper (0.01-0.25 mg/L) for 21 days. Hexavalent chromium at a single dose of 5 mg/L was used as a positive control. Frequencies of micronuclei and binuclei were evaluated comparatively in peripheral blood erythrocytes, gill epithelial cells and liver cells. As a result it was observed that, fish species and their tissues showed differential sensitivity to the heavy metal treatment. In general, frequencies of micronucleated and binucleated cells significantly increased following the exposure for 21 days to copper, cadmium and chromium. On the other hand, gill and liver cells showed higher frequencies of micronuclei and binuclei than erythrocytes. Our results indicated the formation of micronuclei and binuclei in fish cells caused by their exposure to cadmium, copper and chromium, thus verifying results obtained earlier on mammals, which indicated that these heavy metals have cytotoxic and genotoxic effects. The suitability of the micronucleus assay in native fish species for the screening of aquatic genotoxicants is highlighted and the importance of target tissue selection in the piscine micronucleus test is emphasized.     

In vivo genotoxicity testing of the amnesic shellfish poison (domoic acid) in piscine erythrocytes using the micronucleus test and the comet assay

Domoic acid (DA) is a neurotoxic amino acid naturally produced in the marine environment by some diatom species belonging to the genus Pseudo-nitzschia. Although the neurotoxic properties of DA have been demonstrated, very little is known about in vivo genotoxicity of DA on aquatic organisms. In the present paper, an in vivo study on the genotoxic effects of domoic acid was carried out on a fish, Oreochromis niloticus, using the micronucleus test and the comet assay. The fish were exposed to three doses of domoic acid (1, 5 and 10 microg/g body weight) by intracoelomic injections. Ethyl methane sulphonate at a single dose of 5mg/l was used as positive control. Analysis of micronuclei, nuclear abnormalities and DNA damage were carried out on peripheral erythrocytes sampled 24, 48 and 72 h post-treatment. Our results revealed significant increases in the frequencies of micronuclei, nuclear abnormalities as well as DNA strand breaks and thus demonstrated the genotoxic potential of DA on fish.     

In vivo and in vitro exposures for the evaluation of the genotoxic effects of lead on the Neotropical freshwater fish Prochilodus lineatus

In the present study, in vivo and in vitro exposures were used to assess the genotoxicity of lead (Pb) to the freshwater fish Prochilodus lineatus. The comet assay using blood, liver and gill cells, and the occurrence of micronuclei (MN) and other erythrocytic nuclear abnormalities (ENA) were used to assess the genotoxic potential of lead in vivo. Metallothionein content (MT) was measured in fish liver in order to evaluate the protection of fish against Pb toxicity. Fish erythrocytes were exposed to Pb in vitro (1, 3 and 6 h) and the number of viable cells, DNA integrity, using the comet assay, and lysosomal membrane stability, measured by the neutral red retention assay (NRRA) were analyzed. The results of the comet assay after in vivo toxicity tests (6, 24 and 96 h) showed that Pb was genotoxic for all the three tissues analyzed after 96 h exposure. A significant increase in liver MT content was observed after 6 and 24 h of Pb exposure. MN frequency did not increase after Pb exposures, but the frequency of the other ENA, such as kidney-shaped nuclei, segmented nuclei and lobed nuclei, showed a significant increase after 24 and 96 h, indicating that ENA is a better biomarker for Pb exposure than MN alone after short-term exposures. The results of the comet assay performed with erythrocytes in vitro exposed to lead confirmed its genotoxic effect and showed that DNA damage increased with increasing exposure time. Moreover, the NRRA clearly indicated that Pb induces a destabilization of the lysosomal membrane. These results demonstrate the potential genotoxicity and cytotoxicity of lead after acute exposures.     

Histopathological changes and erythrocytic nuclear abnormalities in Iberian green frogs (Rana perezi Seoane) from a uranium mine pond

In spite of their sensitivity to anthropogenic stressors, adults of Rana perezi Seoane were found inhabiting effluent ponds from a uranium mine. Due to the presence of such organisms in this environment, it becomes of paramount importance to assess the damages induced by local contamination on these aquatic vertebrates, in order to integrate this information on a site-specific risk assessment that is being carried out in the area. To attain this purpose an ethically and statistically acceptable number of green frogs were captured in the mine pond (M) and in a pristine river (VR), a few kilometres from the mine. Bioaccumulation of metals and histopathological alterations were evaluated in the liver, kidneys, spleen, lungs and testes of the animals. Simultaneously, blood samples were collected for the evaluation of genotoxic damage on erythrocytes. Animals captured in the M pond showed significantly increased levels of Be, Al, Mn, Fe and U in the liver, as well as Pb and U in the kidney. The liver was the main target organ for the bioaccumulation of Be, Al, Fe and U. However, renal histopathologies were more severe than those of liver. The main tissue alterations recorded in animals from the mine were: a slight increase in melanomacrophagic centers (MMC) in liver, lung and kidneys; dilatation of the renal tubules lumen associated with tubular necrosis. A significantly higher number of erythrocytic abnormalities (lobed, notched and kidney shaped nuclei and micronuclei) were recorded in frogs from M than in frogs from VR, along with a significantly lower frequency of immature erythrocytes. Both observations suggested that the removal of abnormal blood cells might be compromised.     

Primary DNA damage in chrome-plating workers

In order to evaluate the primary DNA damage due to occupational exposure to chromium (VI), DNA strand-breaks and apoptosis in peripheral lymphocytes were measured in a group of 19 chrome-plating workers. DNA strand-breaks was assessed by alkaline (pH>13) single-cell microgel electrophoresis ('comet') assay, while apoptosis was measured by flow-cytometry after propidium iodide staining of the cells. Concentrations of chromium in urine, erythrocytes and lymphocytes were investigated as biological indicators of exposure. A group of 18 hospital workers (control group I) and another 20 university personnel (control group II) without exposure to chromium were also studied as controls. The results of the study show that chrome-plating workers have higher levels of chromium in urine, erythrocytes and lymphocytes than unexposed workers. Comet tail moment values, assumed as index of DNA damage, are increased in chromium-exposed workers and results are significantly correlated to chromium lymphocyte concentrations. No difference emerged in the percentage of apoptotic nuclei in exposed and unexposed workers. The study confirms that measurements of chromium in erythrocytes and lymphocytes may provide useful information about recent and past exposure to hexavalent chromium at the workplace. The increase in DNA strand-breaks measured by comet assay suggests this test is valid for the biological monitoring of workers exposed to genotoxic compounds such as chromium (VI).     

Genotoxic and mutagenic effects of passive smoking and urban air pollutants in buccal mucosa cells of children enrolled in public school

Nuclear abnormalities (micronuclei and meta-nuclear changes) have been used as biomarkers to identify cell damages. As children are more vulnerable to the adverse effects of pollution when compared to adults, assessing genetic damage caused by environmental influences is of great interest. As such, the objective was to determine metanuclear (karyolysis, pycnosis, karyorrhexis, binucleated cells, chromosome bridges and micronuclei) in cells from the oral mucosa of children associated with the school environment, gender, exposure to cigarette smoke and vehicular traffic. Analyses of nuclear abnormalities were performed in exfoliated buccal cells of children from two public schools located in Dourados – MS. The data were analyzed through Kruskal–Wallis test considering a significance level of 5% (p?相似文献   

Assessment of micronuclei and sister chromatid exchange frequency in the petroleum industry workers in province of Vojvodina,Republic of Serbia

Persons who work with petroleum and petroleum derivatives (PPD) are potentially at risk of developing cancer mostly due to the carcinogenity of benzene. Therefore, the aim of this study was to determine in which degree occupational exposure of workers to PPD causes damage to DNA by analysis of micronuclei (MN), sister chromatid exchanges (SCE) and proliferation index (PI). 30 workers of refinery in Novi Sad, participated in the study as exposed and 30 volunteers as control group. Workers exposed to PPD had significantly higher values of MN and SCE in comparison to controls. Exposition time to PPD and type of working place have also significantly effects to DNA damage. The influence of confounding factor such as smoking and age were also evaluated.     

Sublethal effects of treated liquid effluent from a petroleum refinery. V. Reproduction of Daphnia pulex and overall evaluation

The 48-h LC₅₀ of treated refinery effluent for 2-day-old Daphnia pulex was 76% effluent. The 14-day LC₅₀ was 6.4% effluent and this was a threshold value for mortality. For reproductive failure, the 14-day EC₅₀ was 3.1% effluent, and the EC₅ of 0.52% effluent was considered to approximate the threshold of sublethal effect.Daphnia reproduction was the most sensitive response in a series of studies that included fish growth, reproduction, locomotion, and respiration. Results are considered representative for a well-treated effluent from a petroleum refinery. The 48-h lethal test with D. pulex would be a useful tool for monitoring or assessing such effluents, since it is simple, small-scale, quick, and about 2.6 times as sensitive as a lethal test with trout.     

Detection of micronucleus and abnormal nucleus in erythrocytes from the gill and kidney of Labeo bata cultivated in sewage-fed fish farms.

Determination of genotoxic effect in fish, micronucleus test as well as the study of the abnormal shape of nuclei is a suitable measure, in which the presence or absence of genotoxins can be detected in water. In the present study, micronuclei and abnormal nuclei frequencies were scored in the gill and kidney erythrocytes of the fish Labeo bata grown in the sewage-fed fish farms of East Calcutta wetlands. Three experimental sites were chosen, namely, Bantala, Chowbaga and Chingrihata (basically these sites have sewage-fed fishponds), which were compared with fishponds of no sewage influence as the control area. Highly significant differences (P<0.001) were noticed for micronucleus frequencies in the gill and kidney erythrocytes of experimental fishes, where kidney erythrocytes showed an increased value than gill erythrocytes without any statistical differences. The frequencies of nuclear abnormalities such as necrotic cells, apoptotic cells, notch nucleated cells and binucleated cells were also counted separately for gill and kidney erythrocytes, in which significantly (P<0.001, P<0.01, P<0.05) increased values were obtained in comparison to control populations. These genotoxicity results confirmed that the sewage-fed ponds contain genotoxic metals such as Cr, Zn, Cu, Pb, Mn, Fe through wastewater and sludge because of the direct use of sewage water without pretreatment which may lead to health risks among humans through chronic consumption of fish from these experimental fish ponds. Other vertebrates grown in sewage-fed ponds may also suffer a certain amount of genotoxic substances.     

Induction of micronuclei and nuclear lesions in Channa punctatus following exposure to carbosulfan,glyphosate and atrazine

Abstract

The genotoxic effects of commonly used agricultural pesticides viz., carbosulfan, glyphosate, and atrazine, were evaluated in Channa punctatus (Pisces, Perciformes) using micronucleus (MN) test and induction of nuclear lesions (NL). The 96?h LC₅₀ value were estimated by probit analysis as 0.27, 32.0 and 42.0?mg?L^?1, respectively, for carbosulfan, glyphosate, and atrazine using semi-static bioassays. Based on these values, three sublethal test concentrations of carbosulfan (0.07, 0.13, 0.20?mg?L^?1), glyphosate (8.1, 16.3, 24.4?mg?L^?1) and atrazine (10.6, 21.2, 31.8?mg?L^?1) corresponding to ¼, ½ and ¾ of the LC₅₀ of the pesticides respectively, were selected for exposure for 96?h. Peripheral blood samplings were taken at intervals of 24?h for assessment of MN and NL frequencies. Considerably higher genotoxic damage was induced by carbosulfan as compared to glyphosate and atrazine. There were significant effects (p?<?0.01) of concentrations in all the treated groups. The induction of MN and NL was highest at 96?h pesticide exposure at all test concentrations. The nuclear abnormalities recorded in this study, such as blebbed-, lobed-, notched- and bi-nuclei, other than micronuclei, are indicators of genotoxic damage.     

Sublethal effects of treated liquid effluent from a petroleum refinery. IV. Respiratory movements and coughing of rainbow trout

Rainbow trout increased the frequency of gill irrigation in linear relation to the concentration of treated refinery effluent. Full-strength effluent resulted in a significantly higher frequency than in clean water, but 50% and lower did not, because of variation in response. Coughing rate increased sharply and was a less variable measure of response. A concentration of 50% effluent caused significantly more coughs than in clean water, while 25% did not. The eight samples of effluent caused little lethality at full strength, and average physico-chemical characteristics were near or below Canadian regulatory limits, except for elevated oil and grease. Coughing rate shows promise as a rapid initial method for sublethal screening or monitoring of refinery effluents.     

Genotoxic and Cytotoxic Effects in Exfoliated Buccal and Nasal Cells of Chromium and Cobalt Exposed Electroplaters

ABSTRACT

Results of a number of studies indicate that electroplaters have increased cancer risks as a consequence of exposure to genotoxic metals such as chromium (VI) and nickel. These effects may be due to induction of damage of the genetic material which plays a key role in the etiology of cancer, and it was found that workers in galvanization factories exhibited increased levels of DNA damage. The aim of the present study was to investigate genetic stability in workers of a bright plating factory who are exposed to chromium (Cr) and cobalt (Co). Exfoliated cells were collected from the buccal and nasal mucosa of workers (n = 42) and matched controls (n = 43) and analyzed for induction of micronuclei (MN) which are formed as a consequence of chromosomal aberrations. In addition, other nuclear anomalies namely nuclear buds (Nbuds) which are formed as a consequence of gene amplification and markers indicating different stages of cell death (condensed chromatin, karyorrhexis, karyolysis, and pyknosis) were also assessed. No evidence was noted for induction of MN, but significantly increased rates of Nbuds in cells from both, buccal and nasal mucosa, were found. Parameters which are indicative for cytotoxic effects were more pronounced in nasal cells and rose with duration of employment period. Overall, our findings indicated that no apparent chromosomal damage occurred in bright electroplaters. However, data demonstrated that acute cytotoxic effects may lead to inflammations and/or lesions in epithelia of the respiratory tract of the workers.     

In vivo genotoxicity of mercury chloride and lead acetate: Micronucleus test on acridine orange stained fish cells.

The genotoxic effects of mercury chloride and lead acetate were evaluated in vivo using the micronucleus (MN) assay on acridine-orange (AO) stained peripheral blood erythrocytes, gill and fin epithelial cells of Carassius auratus auratus. Fish were exposed to three different concentrations of mercury chloride (MC) (1 microg/, 5 microg/L and 10 microg/L) and lead acetate (LA) (10 microg/L, 50 microg/L and 100 microg/L) for 2, 4 and 6 days. A single dose of 5 mg/L cyclophosphamide was used as a positive control. In addition to micronuclei, nuclear buds (NBs) were assessed in the erythrocytes. The ratio of polychromatic and normochromatic erythrocytes (PCE/NCE) in peripheral blood was also evaluated to assess cytotoxicity. MN frequencies in all three tissues were elevated in fish exposed to both LA and MC. However, NBs showed different sensitivity to metal treatments. MN frequencies in both control and treated fish were highest in gill cells and generally lower in erythrocytes and fin cells. PCE/NCE rations decreased in relation to MC and LA treatments. The results of this study indicate that LA and MC have genotoxic and cytotoxic damage in fish and confirmed that AO staining is a suitable technique for in vivo MN test in fish.     

Fish hepatic micronuclei as an indication of exposure to genotoxic environmental contaminants

An In vivo assay protocol for detecting hepatic micronuclei in fish was performed to evaluate whether this genotoxic response could serve as a bioindicator of environmental exposure to genotoxic substances. The incidence of hepatic micronuclei was compared in brown bullheads (Ameiurus nebulosus) with external lesions collected from a contaminated site and fish showing no externally visible pathologies collected from reference sites. Laboratory experiments were performed by exposing hatchery-raised rainbow trout (Onchorhynchus mykiss) to a pulp mill effluent extract (XAD-4-NaOH) with known genotoxic activity. Both bullhead and trout were injected with allyl formate to induce hepatic necrosis and regenerative proliferation of hepatocytes prior to examining the liver tissue for micronuclei. An elevated incidence of hepatic micronuclei was observed in Hamilton Harbour bullheads showing visible lesions relative to the micronucleus incidence in bullheads from reference sites with no external pathologies. Similarly, rainbow trout exposed to pulp mill effluent extracts exhibited an elevated incidence of hepatic micronuclei compared to controls. These results suggest that the hepatic micronucleus assay could serve as a valuable biomonitoring tool for assessing the impacts of genotoxic environmental contaminants on fish. ©1997 by John Wiley & Sons, Inc. Environ Toxicol Water Qual 12 : 217–222, 1997     

Assessment of micronuclei induction in peripheral erythrocytes of fish exposed to xenobiotics under controlled conditions

The aim of the present study was to standardize and to assess the predictive value of the cytogenetic analysis by MN test in fish erythrocytes as a biomarker for marine environmental contamination. MN frequency baseline in erythrocytes was evaluated in a number of fish species from a reference area (S. Teresa, La Spezia Gulf) and genotoxic potential of a number of common chemical contaminants and mixtures was determined in fish experimentally exposed in aquarium under controlled conditions. Fish (Scophthalmus maximus) were exposed for 3 weeks to 50 ppb of single chemicals (dialkyl phthalate, bisphenol A, tetrabromodiphenyl ether), 30 ppb nonylphenol and mixtures (North Sea oil and North Sea oil with alkylated phenols). Chromosomal damage was determined as micronuclei (MN) frequency in fish erythrocytes. Nuclear anomalies such as blebbed, notched and lobed nuclei were also recorded. Significant increase in MN frequency was observed in erythrocytes of fish exposed to bisphenol A and tetrabromodiphenylether. Chemical mixture North Sea oil+alkylated phenols induced the highest MN frequency (2.95 micronucleated cells/1000 cells compared to 1 MNcell/1000 cells in control animals). The study results revealed that micronucleus test, as an index of cumulative exposure, appears to be a sensitive model to evaluate genotoxic compounds in fish under controlled conditions.     

Genotoxicity of sludges, wastewater and effluents from three different industries

Many surface waters in Europe, Asia and South America have been reported to be contaminated with genotoxic substances. Therefore, it is important to establish strategies for identification of the most critical sources. In this study, we used a battery of four genotoxicity assays namely chromosomal aberration, DNA strand break, DNA laddering and P53 accumulation tests in mononuclear blood cells. Before cleaning of wastewater high levels of genotoxic contamination could be observed. For instance, we observed an increase in chromosomal aberrations from 2.6 ± 1.1 (aberrant cells in %; control), to 33.6 ± 6.6 in a petrochemical plant, 29.4 ± 3.3 in a petroleum refinery and 14.4 ± 1.8 in a coke plant of steel industry. A good correlation between the four assays was found. The most sensitive and reproducible results were obtained with the chromosomal aberration assay. Interestingly, clear differences in the efficiency of wastewater cleaning in three different treatment plants were observed. The first and second treatment plants in petrochemical industry and coke plant of steel industry completely eliminated genotoxicity of the wastewater. However, the third plant in petroleum refinery could achieve a reduction in genotoxicity but significant genotoxic contaminations were still present. In conclusion, our battery of genotoxicity tests allows the identification of critical sources contributing to contamination of surface waters. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Micronuclei Induced by Imipramine and Desipramine in Mice: A Subchronic Study

Abstract: Depression is a common disease that may cause severe damage to human health. Imipramine (IMI) and desipramine (DES) are medicaments used for treatment, yet studies on their genotoxic potential have given controversial results. Therefore, we designed the present assay to determine their effect as inducers of micronucleated polychromatic erythrocytes (MNPE) and micronucleated normochromatic erythrocytes (MNNE) in mice. The study was carried out in animals administered daily with the compounds for 4 weeks, and the determination of micronuclei was done each week. We also evaluated the bone marrow cytotoxicity induced by the chemicals. Besides, the same determinations were carried out in the following 4 consecutive weeks, but in this period the animals were not treated with the tested compounds. Our results showed a significant increase in both MNPE and MNNE induced by both compounds from the first week of administration. At the fourth week, IMI increased three times the control level, while the effect of DES was about seven times such level. In the second, 4‐week phase, we observed a reduction in the rate of micronuclei approaching the control level. We also detected a bone marrow‐mitotic division decrease by the evaluated chemicals. Our results point to the need for cautiousness in the clinical use of the compounds as well as for testing the effect in patients under treatment.     

Detection and quantification of genotoxicity in wastewater-treated Tetrahymena thermophila using the comet assay

In the present study, the comet, or single-cell, gel electrophoresis assay was adapted for use with the ubiquitous unicellular protozoan Tetrahymena thermophila, and the method was evaluated for its ability to detect DNA damage induced by known genotoxins and wastewater samples. The original comet assay protocol was substantially modified (e.g., lower concentrations of detergents were used in the lysis buffer; electrophoresis time was reduced). Using the modified method, T. thermophila were subjected to short exposures of phenol, hydrogen peroxide, and formaldehyde, leading to concentration-dependent increases in DNA damage. The genotoxic potential of influent and effluent water samples from a local municipal wastewater treatment plant was evaluated. The results indicated that the influent wastewater was genotoxic and that the genotoxicity in the effluent water was substantially reduced. We assume employing T. thermophila in the use of the comet assay may become a cost-effective and reliable tool for genotoxicity screening and monitoring of wastewater and similar systems.     

Evaluation of genotoxicity from Nilufer Stream (Bursa/Turkey) water using piscine micronucleus test

In the present study, the in vivo micronucleus (MN) test in fish erythrocytes was used to evaluate the genotoxic potentials of water samples collected from four different sites along the Nilufer Stream which empties into the Marmara Sea on the north-west of Turkey. Nilufer Stream receives discharges of industrial and domestic wastes resulting from industrialization and urbanization activities in Bursa city. Nile tilapias (Oreochromis niloticus) were exposed to collected water samples under laboratory conditions for 3 and 6 days. Micronuclei analyses were carried out in peripheral blood erythrocytes. In addition to micronuclei, other nuclear abnormalities (NAs) such as bi-nucleated cells and binuclei with nucleoplasmic bridge and cells with blebbed, notched and lobbed nuclei, were assessed in the erythrocytes. Chemical analyses were also carried out in the water samples to assess the presence of major pollutants. MN and NA frequencies were significantly elevated in fishes exposed to water from polluted areas compared to those exposed to clean water sample. The results of this study indicate that Nilufer Stream is contaminated with potential genotoxic chemicals and the genotoxicity is possibly related with the industrial, agricultural and domestic activities.