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.     

In vivo micronucleus test in the assessment of cytogenotoxicity of landfill leachates in three animal models from various ecological habitats

The in vivo micronucleus (MN) test, a standard test for the genotoxicity screening of xenobiotics, was used to evaluate the cytotoxic and genotoxic activities of landfill leachates in Clarias gariepinus, Coturnix coturnix japonica and Rattus norvegicus. These organisms were exposed to various sub-lethal concentrations (1–50 %) of Olusosun and Aba Eku landfill leachates. At post exposure, peripheral erythrocytes from catfish and quail, and bone marrow cells of quail and rat were subjected to MN analysis following standard protocols. The leachates induced significant increase in MN formation and total nuclear abnormalities (NAs) in the peripheral erythrocytes of catfish and quail. NAs occurred in the order; BN > BL > LB > NT in the catfish and BN > BudN > TLN > TN in quail. There was significant increase in MN formation in the bone marrow cells of quail, and micronucleated polychromatic erythrocytes and micronucleated normochromatic erythrocytes formation in the bone marrow of rats. The concentration dependent significant (p < 0.05) decrease in the PCE/NCE ratio in the bone marrow of the leachate treated rats suggest alterations in the bone marrow cell proliferation, leading to the suppression of immature erythrocytes (PCE). MN induction showed positive corrections with leachate concentrations in the test organisms; and it increased with exposure duration in the catfish. Indiscriminate disposal of solid waste generates leachates containing multiple xenobiotics that are capable of increasing genomic instability among vertebrates inhabiting various ecological habitats.     

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.     

Assessment of genotoxic and mutagenic effects of chlorpyrifos in freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis

Chlorpyrifos (CPF) is the single largest selling agrochemical that has been widely detected in surface waters in India. The studies on long-term genotoxic effects of CPF in different tissues of fish using genotoxic biomarkers are limited. Therefore, in the present study DNA damage by CPF in freshwater fish Channapunctatus using micronucleus (MN) and comet assays was investigated. The LC₅₀ – 96 h of CPF was estimated for the fish in a semi-static system. On this basis of LC₅₀ value sublethal and nonlethal concentrations were determined. The DNA damage was measured in lymphocytes and gill cells as the percentage of DNA in comet tails and micronuclei were scored in erythrocytes of fishes exposed to above concentrations of CPF. In general, significant effects for both the concentrations and time of exposure were observed in treated fish. It was found that MN induction in the blood was highest on day 14 at 203.0 μg/l of CPF. The highest DNA damage was observed on day 5, followed by a gradual non-linear decline in the lymphocytes and gill cells. The study indicated MN and comet assays to be sensitive and rapid methods to detect mutagenicity and genotoxicity of CPF and other pollutants in fishes.     

Genotoxic and hematological effects of chlorpyrifos exposure on freshwater fish Labeo rohita

Chlorpyrifos is a commonly used organophosphate insecticide that causes toxicological effects in aquatic organisms especially in fish. This study determined the effects of chlorpyrifos on the genotoxic and hematological parameters of freshwater fish, Labeo rohita. The genotoxic effects of different sublethal concentrations of chlorpyrifos were investigated in the erythrocytes of Labeo rohita (commonly known as Rohu) using the Micronucleus test. Effects of chlorpyrifos on the hematological parameters of the fish were also observed. Fish specimens were exposed to three sublethal concentrations of chlorpyrifos viz., sublethal I (SL-I, 1/6th of LC₅₀?=?～73.8?μg/L), sublethal II (SL-II, 1/4th of LC₅₀?=?～110.7?μg/L) and sublethal III (SL-III, 1/2nd of LC₅₀?=?～221.4?μg/L) for 96?h. Blood samples were collected at every 24?h and were subjected to the Micronucleus assay. The observed micronucleus frequencies were concentration and time-dependent. The MN induction was significantly highest (p?相似文献   

DNA damage caused by extracts of chlorinated drinking water in human derived liver cells (HepG2)

Dong (D) lake and the Yangtze (Y) river are the main water supplies of the city of Wuhan, PR China. In the present study, the genotoxic effect of chlorinated drinking water (CDW) processed from raw water of D lake and Y river was evaluated in human HepG2 cells using the Comet assay and the micronucleus test. For that, HepG2 cells were exposed to XAD extracts of CDW corresponding to 0.167, 1.67, 16.7 and 167 ml CDW/ml cell culture. All CDW extracts caused a significant and dose-dependent increase of DNA migration in HepG2 cells. The level of DNA damage varied depending on the sampling time (season) and sampling site. The lowest concentration which caused a significant increase of DNA migration was 1.67 ml CDW/ml culture for water samples collected in August. Water samples collected in March showed their lowest observable effect levels in 167 ml and 16.7 ml CDW/ml culture for Y river and D lake, respectively. Additionally, significant increases of micronuclei (MN) frequencies were found in HepG2 cells after CDW treatment. However, in the MN assay the CDW samples collected in March exhibited higher genotoxicity than the August samples. In conclusion, HepG2 cells provide a useful tool for the detection of genotoxic effects of environmental mixtures.     

In vitro micronuclei tests to evaluate the genotoxicity of surface water under the influence of tanneries

Leather manufacturing has a high potential for environmental pollution due to hides and chemicals that are not completely absorbed during the tanning process. This study aims to investigate the mutagenic potential of surface water samples from Cadeia and Feitoria rivers (RS, Brazil) in areas influenced by tanneries and leather footwear industry. Micronucleus assays using V79 cells and human lymphocytes were used. Cells were exposed to surface water collected bimonthly from three sites for a year, totaling six samples. Significant MN induction in human lymphocytes was shown by 83% of samples from sites FEI001 and CAD001 located downstream from the industrial area, followed by FEI004 (33%), upstream. Only a single sample from site FEI004 showed a positive response for MN in V79 cells. Thirteen discordant and five concordant responses were found between the two in vitro tests. Mutagenic agents were found at the sites where chemical quality was worst, corroborating studies on chronic toxicity, oxidative stress and mutagenicity performed in this area. The assay using human lymphocytes was more sensitive than V79 cells to detect the contaminants from this area, showing that it is an excellent biomarker of environmental genotoxicity.     

Genotoxic evaluation of pirfenidone using erythrocyte rodent micronucleus assay

Pirfenidone is a non-steroidal antifibrotic compound that has been proposed in clinical protocols and experimental studies as a pharmacological treatment for fibroproliferative diseases. The objective of this study was to determine the genotoxicity or cytotoxicity of three doses of pirfenidone using the micronuclei test in peripheral blood erythrocytes of rodent models. Pirfenidone was administered orally to Balb-C mice for 3 days, and also was administered topically to hairless Sprague Dawley rats during the final stage of gestation. Mice were sampled every 24 h over the course of 6 days; pregnant rats were sampled every 24 h during the last 6 days of gestation, and pups were sampled at birth. Blood smears were analyzed and the frequencies of micronucleated erythrocytes (MNEs), micronucleated polychromatic erythrocytes (MNPCEs), and the proportion of polychromatic erythrocytes (PCEs), were recorded in samples from mice, pregnant rats and rat neonates. Increases in MN frequencies (p < 0.03) were noted only in the positive control groups. No genotoxic effects or decreased PCE values were observed neither in newborn rats transplacentally exposed to pirfenidone, or in two adult rodent models when pirfenidone was administered orally or topically.     

Risk assessment of petroleum-contaminated soil using soil enzyme activities and genotoxicity to Vicia faba

Pollution caused by petroleum is one of the most serious problems worldwide. To better understand the toxic effects of petroleum-contaminated soil on the microflora and phytocommunity, we conducted a comprehensive field study on toxic effects of petroleum contaminated soil collected from the city of Daqing, an oil producing region of China. Urease, protease, invertase, and dehydrogenase activity were significantly reduced in microflora exposed to contaminated soils compared to the controls, whereas polyphenol oxidase activity was significantly increased (P < 0.05). Soil pH, electrical conductivity, and organic matter content were correlated with total petroleum hydrocarbons (TPHs) and a correlation (P < 0.01) existed between the C/N ratio and TPHs. Protease, invertase and catalase were correlated with TPHs. The Vicia faba micronucleus (MN) test, chromosome aberrant (CA) analyses, and the mitotic index (MI) were used to detect genotoxicity of water extracts of the soil. Petroleum-contaminated samples indicated serious genotoxicity to plants, including decreased index level of MI, increased frequency of MN and CA. The combination of enzyme activities and genotoxicity test via Vicia faba can be used as an important indicator for assessing the impact of TPH on soil ecosystem.     

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.     

Investigation of histopathological and cytogenetic effects on Lepomis gibbosus (Pisces: Perciformes) in the Cine stream (Aydin/Turkey) with determination of water pollution

Water quality and the distribution of some heavy metals in three different organs of Lepomis gibbosus from the Cine Stream were studied. Also, histopathological changes in gill, liver, and muscle tissue were examined at light microscopical level. Micronucleus (MN) formation in fish erytrocytes, as an indicator of chromosomal damage, has been increasingly used to detect the genotoxic potential of environmental contaminants. The frequency of MN was examined from samples of fish from the Cine Stream and a control group. MN frequency was higher in fish samples caught from the Cine Stream than that in the control group. The chemicals ammonia, nitrite, nitrate, orthophosphate, and sulphate were determined as parameters that possibly affect the gill, liver, and muscle morphology. Zn was the most accumulated metal in tissues as well as in water. Maximum metal accumulation occurred in both liver and gills. For histopathological examinations, samples of gills, liver, and muscle tissues of L. gibbosus were studied by using light microscopy. In this study, a significant decrease in mean length of primary and secondary lamellae were observed. Moreover, cellular proliferation developed with secondary lamellae fusion, ballooning degenerations or club deformation of secondary lamellae, as well as distribution of necrotic, hyperplastic and clavate secondary lamellae. In the liver, altered staining, swollen and ruptured parenchymal cells, loss of cord structure, reduce of glycogen in hepatocytes, and vacuolar structure filled with cellular debris and many dark particles were seen. In muscle tissue, focal necrosis, cellular dissolution, and a decline or loss of striation in muscle fibres were found.     

Evaluation of anti-oxidative,genotoxic and antigenotoxic potency of Codium tomentosum Stackhouse ethanolic extract in human lymphocytes in vitro

The genome is constantly exposed to agents, both exogenous and endogenous, that damage DNA. Consequently, it is very important that determination of this agents and the protective agents. In this work, we evaluated the antigenotoxic/antimutagenic activity of the crude ethanolic extracts of Codium tomentosum Stackhouse (Chlorophyceae) (CTE), collected from The Coast of South East Marmara Sea, in human lymphocytes culture in vitro against genotoxic/mutagenic agents MMC, EMS and H₂O₂ by using chromosome aberration (CA), sister chromatid exchange (SCE) and micronuclei (MN) assays as experimental endpoints. Also, in the present study, we determined total phenolic content and total antioxidant capacity (in soluble lipid and water). In addition, total protein, total carbohydrate, vitamins (A, C and E) and pigments (chlorophyll a, chlorophyll b and carotene) contents were also determined. Results of CA, SCE and MN tests show that CTEs have not shown genotoxic effect. In CTE plus MMC-, EMS- or H₂O₂- treated cultures, CA, SCE and MN frequency which induced by MMC, EMS or H₂O₂ has been decreased significantly (p < 0.05–0.001). This is the first report on genotoxicity/antigenotoxicity and anti-oxidative capacity of Codium tomentosum. Our results have clearly shown that CTE has strong anti-oxidative and antigenotoxic effect.     

Therapeutic efficacies of Coriandrum sativum aqueous extract against metronidazole-induced genotoxicity in Channa punctatus peripheral erythrocytes

Metronidazole (MTZ), a nitroimidazole drug, is primarily used as an anti-protozoan or an anti-bacterial agent in humans, although its genotoxic and carcinogenic effects have been widely reported, particularly in aquatic organisms. MTZ may induce DNA damages through single-strand breaks, modification of bases, DNA–DNA and DNA–protein cross-links, ultimately leading to apoptosis or necrosis. Here, we have assessed the genotoxicity of MTZ in the peripheral erythrocytes of Channa punctatus, using micronucleation (MN) and binucleation (BN) as genotoxicity markers. The therapeutic potential of aqueous extract of Coriandrum sativum against MTZ-induced genotoxicity has also been examined. The results show significant (P < 0.05) increase in both MN and BN formation due to MTZ treatment. Such aberrations were higher in smaller fish samples for a particular dosage of MTZ, as established by correlation analysis between fish body weight and MN/BN count at P < 0.05. However, such degenerative damages were found to be alleviated by a great extent due to treatment with C. sativum leaf extract. Hence, we establish that MTZ can produce considerable degrees of micronucleus and binucleus formation in peripheral erythrocytes of C. punctatus, and such deleterious effect of MTZ treatment can be mitigated by aqueous extract of C. sativum leaves.     

Assessment of mutagenic,genotoxic, and cytotoxic potential of water samples of Harike wetland: a Ramsar site in India using different ex vivo biological systems

Harike is a wetland of international importance under the Ramsar Convention. The present study entails the investigation of mutagenic, genotoxic and cytotoxic effect of surface water samples collected from five different areas of the Harike wetland by using the histidine reversion point mutation assay in Salmonella typhimurium (TA98 and TA100) strain with or without S9, bioluminescence mutagenicity assay using Vibrio harveyi (A16) strain, plasmid-nicking assay using pBR322 and 3(4,5-dimethyl-thiazol-2-yl)2,5-diphenyl-tetrazolium bromide assay as well as confocal imaging studies using Chinese hamster ovarian cell line, respectively. It was observed that although, the water sample of all the areas of wetland demonstrated mutagenic, genotoxic as well as cytotoxic activity, the effect was quite significant with the water samples from River Satluj and Khatan area (i.e. reservoir mainly contains Satluj water). The metal analysis of water samples was also conducted with atomic absorption spectrophotometer. The mutagenicity, genotoxicity and cytotoxicity of water samples emerged to be correlated with metal concentration. The source of toxic components seems to be associated with various industrial effluents and agricultural run-off. The results of the present study carry great importance in documenting the water quality monitoring data of the wetland.     

Genotoxic and oxidative stress-inducing effects of deltamethrin in the erythrocytes of a freshwater biomarker fish species, Channa punctata Bloch

Deltamethrin, an alpha-cyano class of pyrethroid insecticide is used in insect pest control and antimalaria programs in several countries including India. Although various toxic manifestations of deltamethrin are reported in mammals, its ecotoxicologic dimensions are not adequately researched in ecologically and commercially important fishes. In this study, we report genotoxic effect of deltamethrin in a biomarker fish Channa punctata (Bloch). Adult fish were exposed to three concentrations of technical grade deltamethrin (0.4, 0.8, and 1.2 microg/L) for 48 and 72 h. Ethyl methane sulfonate was used as a positive control. Fish were analyzed for induction of micronucleus (MN), nuclear abnormalities (NAs), and oxidative stress biomarkers in erythrocytes. Deltamethrin significantly induced MN and NAs accompanied by increased lipid peroxidation. Activity of antioxidant enzyme superoxide dismutase was significantly decreased but an increase was observed in reduced glutathione level after 72 h of exposure. The NAs in exposed fish included blebbed, lobed and notched nuclei, and binucleated erythrocytes. Our findings suggest that oxidative stress may, in part, be contributing to deltamethrin-induced genotoxic damage to erythrocytes. Although MN induction is a nonspecific biomarker, it may provide an indication of pollution load of deltamethrin in the affected fish population when used as part of suite of other biomarkers.     

Exposure to pollutants present in Iguaçu River Southern Brazil affect the health of Oreochromis niloticus (Linnaeus, 1758): Assessment histological,genotoxic and biochemical

Urban sewage is a source of major contamination in aquatic systems and contributes to environmental and human health disturbances. This study investigates the effects of sewage-polluted waters from Iguaçu River on the health of juvenile Oreochromis niloticus. Two hundred four specimens were exposed to riverine water in four groups: no diluted, 25 and 50 % diluted water and a control group without tested water for 72 days. Biological samples were obtained for histopathological, neurotoxicity, antioxidant defenses, genotoxicity, metallothionines expression and polycyclic aromatic hydrocarbons (PAHs) metabolites. The results showed histopathological alterations in liver and gills, genotoxic alteration in erythrocytes, reduction of acetylcholinesterase activity in brain and muscle, activation of antioxidant defenses in the liver, recruitment of metals by metallothionein and the detection of PAHs metabolites in bile. These results demonstrate that juveniles of O. niloticus are susceptible to Iguaçu River exposure water and they can be used as indicator of water quality.     

The in vitro genotoxicity of ethanol and acetaldehyde

Ability of ethanol to produce chromosomal changes has been controversial in past many years; nevertheless many recent studies have shown that ethanol itself produces genotoxic effects like acetaldehyde. This study was carried out to evaluate the ability of ethanol and its metabolite acetaldehyde to induce chromosomal changes using in vitro CBMN assay (Cytokinesis Blocked Micronucleus assay) in conjunction with immunofluorescent labeling of kinetochores. Kinetochore staining was used with a view to differentiate, between the genotoxic effects of both chemicals, and ascertain the mechanisms of genotoxicity induction by ethanol and acetaldehyde. Both ethanol and acetaldehyde produced statistically significant (P < 0.05) dose dependent increase in MN induction as compared with the controls over the dose range tested. Kinetochore analysis proved that the MN induced in ethanol were originated by an aneugenic mechanism, whereas in the case of acetaldehyde most of the MN had originated by a clastogenic mechanism. This not only confirms the ability of ethanol to produce DNA damage in vitro but it also establishes the efficacy of CBMN assay to detect and differentiate between the genotoxic effects of different genotoxins. Here we report that ethanol is itself genotoxic, at least in vitro, and produces genotoxic effects mainly through an aneugenic mechanism whereas its metabolite acetaldehyde is a clastogen.     

Microalga Euglena as a bioindicator for testing genotoxic potentials of organic pollutants in Taihu Lake,China

The microalga Euglena was selected as a bioindicator for determining genotoxicity potencies of organic pollutants in Meiliang Bay of Taihu Lake, Jiangsu, China among seasons in 2008. Several methods, including the comet assay to determine breaks in DNA and quantification of antioxidant enzymes were applied to characterize genotoxic effects of organic extracts of water from Taihu Lake on the flagellated, microalga Euglena gracilis. Contents of photosynthetic pigments, including Chl a, Chl b and carotenoid pigments were inversely proportion to concentrations of organic extracts to which E. gracilis was exposed. Organic extracts of Taihu Lake water also affected activities of superoxide dismutase (SOD) and peroxidase (POD) of E. gracilis. There were no statistically significant differences in SOD activities among seasons except in June but significant differences in POD activities were observed among all seasons. The metrics of DNA fragmentation in the alkaline unwinding assay (Comet assay), olive tail moment (OTM) and tail moment (TM), used as measurement endpoints during the genotoxicity assay were both greater when E. gracilis was exposed to organic of water collected from Taihu Lake among four seasons. It is indicated that the comet assay was useful for determining effects of constituents of organic extracts of water on E. gracilis and this assay was effective as an early warning to organic pollutants.     

Genotoxic effects of monocrotophos,an organophosphorous pesticide,on an estuarine bivalve, Meretrix ovum

Genotoxicity studies evaluate the effects of pollutants on organisms and consequently, their implications on human health. Meretrix ovum was exposed to different concentrations of monocrotophos, viz. 5.5, 11.0 and 16.5 mg/L continuously for four different time periods viz. 2, 3, 7 and 14 days. Gills of these animals were collected immediately after exposure at the above time intervals and analyzed for genotoxic effect employing micronucleus test and effect on somatic growth by estimating the total RNA/DNA ratio. Data were analyzed employing Student’s ‘t’ test and ANOVA. Significant increase of micronuclei observed in the present study in a dose dependant manner indicates the possible chromosomal damage induced by monocrotophos in this species and thereby reveals its genotoxic potency. Significant reduction of the total RNA/DNA ratio observed in a time dependant manner indicates a considerable retardation of somatic growth in monocrotophos treated mussels. Results of the present study indicate that monocrotophos is genotoxic on M. ovum and also induces a pollution stress related retardation of somatic growth of this mussel. Further, this study indicated the possibility of using MN test in bivalves as a marker for screening/monitoring the genotoxic potential of pollutants in aquatic ecosystems.     

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.