Long-term genotoxic effect of monocrotophos in different tissues of freshwater fish Channa punctatus (Bloch) using alkaline single cell gel electrophoresis

Monocrotophos, commonly known as azodrin, is one of the organophosphate pesticides extensively used in agricultural practices throughout the world. However, little information is available on its long-term genotoxic effects in different tissues of fish using genotoxicity biomarkers. The objective of the present study was to detect DNA damage, induced by monocrotophos in freshwater teleost fish Channa punctatus using the comet assay. The LC(50)-96 h value of technical-grade monocrotophos was estimated for the fish species in a semi-static system. On the basis of the LC(50) value, the sublethal and nonlethal concentrations were determined. The DNA damage was measured in the gill, kidney and lymphocytes as the percentage of DNA in comet tails of fish exposed to different sublethal and nonlethal concentrations of monocrotophos. In general, significant effects (P<0.01) from both concentration and time of exposure were observed in exposed fish. It was found that the tissues at all concentrations exhibited the highest DNA damage on day 4, after which there was a decline in percentage tail DNA. The comparison of DNA damage among tissues at different concentrations indicated that the gill cells were more sensitive to the pesticide than the kidney cells and lymphocytes. This study also explored the utility of the comet assay for in vivo laboratory studies using fish for screening the genotoxic potential of various agents.     

Effects of co-exposure to extremely low frequency (50 Hz) magnetic fields and xenobiotics determined in vitro by the alkaline comet assay

In the present study, we used human peripheral blood leukocytes from 4 different donors, to investigate in vitro the possible genotoxic and/or co-genotoxic activity of extremely low frequency magnetic fields (ELF-MF) at 3 mT intensity. Two model mutagens were used to study the possible interaction between ELF-MF and xenobiotics: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 4-nitroquinoline N-oxide (4NQO). Primary DNA damage was evaluated by the alkaline single-cell microgel-electrophoresis ("comet") assay. Control cells (leukocytes not exposed to ELF-MF, nor treated with genotoxins) from the different blood donors showed a comparable level of basal DNA damage, whereas the contribution of individual susceptibility toward ELF-MF and the tested genotoxic compounds led to differences in the extent of DNA damage observed following exposure to the genotoxins, both in the presence and in the absence of an applied ELF-MF. A 3 mT ELF-MF alone was unable to cause direct primary DNA damage. In leukocytes exposed to ELF-MF and genotoxins, the extent of MNNG-induced DNA damage increased with exposure duration compared to sham-exposed cells. The opposite was observed in cells treated with 4NQO. In this case the extent of 4NQO-induced DNA damage was somewhat reduced in leukocytes exposed to ELF-MF compared to sham-exposed cells. Moreover, in cells exposed to ELF-MF an increased concentration of GSH was always observed, compared to sham-exposed cells. Since following GSH conjugation the genotoxic pattern of MNNG and 4NQO is quite different, an influence of ELF-MF on the activity of the enzyme involved in the synthesis of GSH leading to different activation/deactivation of the model mutagens used was hypothesized to explain the different trends observed in MNNG and 4NQO genotoxic activity in the presence of an applied ELF-MF. The possibility that ELF-MF might interfere with the genotoxic activity of xenobiotics has important implications, since human populations are likely to be exposed to a variety of genotoxic agents concomitantly with exposure to this type of physical agent.     

Effect of different organotins on DNA of mollusk (Scapharca inaequivalvis) erythrocytes assessed by the comet assay

The alkaline comet assay, employing a single-cell gel-electrophoresis, is a rapid, simple and sensitive technique for visualizing and measuring DNA damage leading to strand breakage in individual cells. In this study, we report data about the effect of different organotin compounds (MBTC, DBTC and TBTC) on DNA from erythrocytes of the Scapharca inaequivalvis bivalve mollusc. Our results show significant DNA damage after 30 min in vitro incubation with 10microM of organotins. Since TBTC turned out to be the most genotoxic compound, followed by MBTC and DBTC, we exposed the molluscs to 50ppb of TBTC for 11 days. A significant increase of comet parameters was measured in our experimental conditions. The use of the comet test as a high-throughput screening assay to monitor the effect of environmental pollutants on marine organisms has been proposed.     

Biomonitoring of the genotoxic potential of aqueous extracts of soils and bottom ash resulting from municipal solid waste incineration, using the comet and micronucleus tests on amphibian (Xenopus laevis) larvae and bacterial assays (Mutatox and Ames tests)

The management of contaminated soils and wastes is a matter of considerable human concern. The present study evaluates the genotoxic potential of aqueous extracts of two soils (leachates) and of bottom ash resulting from municipal solid waste incineration (MSWIBA percolate), using amphibian larvae (Xenopus laevis). Soil A was contaminated by residues of solvents and metals and Soil B by polycyclic aromatic hydrocarbons and metals. MSWIBA was predominantly contaminated by metals. Two genotoxic endpoints were analysed in circulating erythrocytes taken from larvae: clastogenic and/or aneugenic effects (micronucleus induction) after 12 days of exposure and DNA-strand-breaking potency (comet assay) after 1 and 12 days of exposure. In addition, in vitro bacterial assays (Mutatox and Ames tests) were carried out and the results were compared with those of the amphibian test. Physicochemical analyses were also taken into account. Results obtained with the amphibians established the genotoxicity of the aqueous extracts and the comet assay revealed that they were genotoxic from the first day of exposure. The latter test could thus be considered as a genotoxicity-screening tool. Although genotoxicity persisted after 12 days' exposure, DNA damage decreased overall between days 1 and 12 in the MSWIBA percolate, in contrast to the soil leachates. Bacterial tests detected genotoxicity only for the leachate of soil A (Mutatox). The results confirm the ecotoxicological relevance of the amphibian model and underscore the importance of bioassays, as a complement to physico-chemical data, for risk evaluation.     

Assessment of DNA damage in coal open-cast mining workers using the cytokinesis-blocked micronucleus test and the comet assay

Coal mining is one of the most important causes of environmental pollution, as large quantities of coal dust particles are emitted. Colombia-South America has large natural coal reserves and “El Cerrejón” is the world's largest open-cast mine located in the northern department of Guajira. The aim of the present study was to evaluate genotoxic effects in a population exposed to coal residues from the open-cast mine “El Cerrejón”. 100 exposed workers and 100 non-exposed control individuals were included in this study. The exposed group was divided according to different mining area activities: (i). Transport of extracted coal, (ii). Equipment field maintenance, (iii). Coal stripping and, (iv). Coal embarking. Blood samples were taken to investigate biomarkers of genotoxicity, specifically, primary DNA damage as damage index (DI), tail length and% of tail DNA using the Comet assay (alkaline version) and chromosome damage as micronucleus (MN) frequency in lymphocytes. Both biomarkers showed statistically significantly higher values in the exposed group compared to the non-exposed control group. No difference was observed between the exposed groups executing different mining activities. These results indicate that exposure to coal mining residues may result in an increased genotoxic exposure in coal mining workers. We did not find a correlation between age, alcohol consumption and service time with the biomarkers of genotoxicity. Our results are the first data of genotoxic effects induced by coal mining exposure in Colombia, and thus, contribute to the exploration of test batteries use for monitoring of exposed populations and may stimulate designing control, hygiene and prevention strategies for occupational health risk assessment in developing countries.     

In vitro toxicity of surface water disinfected by different sequential treatments

The in vitro toxicity of extracts of Hanjiang water disinfected by different sequential treatments was evaluated. Hanjiang water was disinfected using ozone, chloride dioxide or chlorine as the primary disinfectant followed by chlorine as the secondary disinfectant. HepG₂ cells were exposed to extracts corresponding to concentrations of 0.2, 1, 5, 25 and 125 mL water/mL medium. Compared with control, HepG₂ cells exposed to extracts of raw water and all disinfected water for 24 h increased oxidative stress level, DNA damage and micronuclei frequency, and decreased cell viability. Water disinfected by Cl₂ + Cl₂ had the highest DNA double-strand breaks. All disinfected water and raw water increased micronuclei frequency via clastogenic and aneugenic effects. Oxidative stress induced DNA strand breaks and micronuclei frequency and therefore reduced cell viability either in disinfected water or raw water. Compared with raw water, water after disinfection increased DNA strand breaks, decreased cell viability and changed oxidative stress potential. Compared with chlorination, sequential treatment using O₃ or ClO₂ as primary disinfectant followed by chlorine disinfection reduced chlorinated by-products, DNA double-strand breaks and cell viability, but did not decrease micronuclei frequency and other DNA damage such as DNA single-strand break, alkali liable sites and incomplete excision sites. Sequential treatments did not significantly reduce in vivo toxicity of disinfected Hanjiang water.     

Use of native species Crenicichla menezesi (Ariidae) as a model for in situ evaluation of genotoxicity in surface water

The aim of this work was to analyze the genotoxicity in surface water from the Pitimbú River (Natal, Brazil) using a neotropical species Crenicichla menezesi as bioindicator for in situ evaluation. The genotoxicity was analyzed using the micronucleus (MN) and comet assays in erythrocytes. The animals were collected from five sites of the river. A significant increase of MN frequency and comet class L1 to L4 were observed only in specimens collected from three sampling sites (SS3, SS4 and SS5) located into urban areas. These same sites also presented high metals concentration. Correlation of DNA damage and concentrations of Cu (Spearman r = 0.8080), Cr (Spearman r = 0.9772), and Ni (Spearman r = 0.9909) were observed. These data indicate the presence of genotoxic agents. Moreover the C. menezesi species showed to be a good indicator for prompt analyses since this species presents sedentary and carnivore habits.     

Genotoxic effects of Israeli industrial pollutants on residents of Bruqeen village (Salfit district,Palestine)

Many industrial byproducts are genotoxic agents that induce cytogenetic changes and DNA damage. Bruqeen, a Palestinian village in Salfit district in the northern West bank, is subject to industrial waste products coming from Barqan Israeli industrial settlement. We evaluated the extent of chromosomal breaks (Csb) and DNA damage induced to human cells using whole blood samples from both test and control sites. Csb were assessed by routine cytogenetic methods and DNA damage was assessed via the Comet assay which is also called single cell gel electrophoresis. Cytogenetic analysis for Bruqeen residents’ sample (n?=?30) showed 133 Premature centromere separation (PCS), 43 Csb, 40 chromatid breaks (Ctb) and 26 dicentrics. In total, the findings showed an average of 4.08% chromosomal aberrations (CA) and 3.81% cells with CA. The results of controls (from Bethlehem area n?=?8) showed 21 PCS, 2 Csb, 5 Ctb and 2 dicentric, and in total the average showed 1.97 CA and 1.91% cells with CA. Statistical analysis showed that there were no significant differences between exposed and control subjects for PCS, Ctb and Dicentric (p-value?>?0.05), but there was a statistically significant difference for CA frequency for cells that have CA and Csb (p-value?p-value?相似文献   

Impact of ozonation on the genotoxic activity of tertiary treated municipal wastewater

Ozonation is an emerging technology for the removal of micropollutants from treated wastewater. Aim of the present study was to investigate the impact of ozone treatment on genotoxic and acute toxic effects of tertiary treated municipal wastewater. It is known that DNA-damaging chemicals cause adverse effects in the environment and that exposure to humans leads to cancer and other diseases. Toxicity was tested in organisms from three trophic levels namely in bacteria (Salmonella/microsome assays) which enable the detection of gene mutations, in a plant bioassay (micronucleus assay with root tip cells of Allium cepa) which reflects clastogenic and aneugenic effects and in single cell gel electrophoresis (SCGE) tests with mammalian cells which detect DNA migration caused by single-, double strand breaks and alkali labile sites. In the bacterial tests negative results were obtained with untreated samples but after concentration with C₁₈ cartridges a positive result was found in strains TA1537 and TA98 which are sensitive to frameshift mutagens while no mutations were induced in other tester strains (TA100, TA102 and YG1024). Ozone treatment led to a decrease of the mutagenic activity of the samples. In the SCGE experiments, DNA migration was detected with the unconcentrated effluent of the treatment plant and ozonation led to a substantial decrease of this effect. In the plant bioassays, negative results were obtained with the effluent and ozone treatment did not cause an alteration of the micronucleus frequencies. Also acute toxic effects were monitored in the different indicator organisms under all experimental conditions. The bacteriocidal/bacteriostatic effects which were seen with the concentrated samples were reduced by ozonation. In the experiments with the eukaryotic (plant and animal) cells no acute toxicity was seen with the effluents and ozonation had no impact on their viability. In conclusion findings of this study indicate that ozonation of tertiary effluents of a municipal treatment plant reduces the adverse effects caused by release of mutagens in aquatic ecosystems and does not decrease the viability of bacteria and eukaryotic cells. However, future research is required to find out if, and to which extent these findings can be generalized and which mechanisms account for the detoxification of the wastewater.     

Evaluation of chlorite and chlorate genotoxicity using plant bioassays and in vitro DNA damage tests

In the last few years chlorine dioxide has been increasingly used for disinfecting drinking water in many countries. Although it does not react with humic substances, chlorine dioxide added to water is reduced primarily to chlorite and chlorate ions, compounds that are under investigation for their potential adverse effects on human health. The aim of this research was to study the genotoxicity of chlorite and chlorate and their mixtures. The end-points included two plant tests (chromosomal aberration test in Allium cepa and micronucleus assay in Tradescantia, carried out at different times of exposure) and two genotoxicity tests in human HepG2 cells (comet assay and cytokinesis-blocked micronucleus test). Preliminary toxicity tests were carried out for both plant and HepG2 assays. The results showed that chlorite and chlorate are able to induce chromosomal damage to plant systems, particularly chromosomal aberrations in A. cepa root tip cells, even at concentrations lower than the limit established by Italian normative law and WHO guidelines. In HepG2 cells increased DNA damage was only observed for chlorate at the lowest concentration. No increase in micronuclei frequency was detected in any of the samples tested in human HepG2 cells.     

Cytotoxic and genotoxic potential of drinking water: a comparison between two different concentration methods

The level of exposure to hazardous compounds through drinking water is low but it is maintained throughout life, therefore representing a risk factor for human health. The use of techniques averaging the consumer's exposure over time could be more useful than relying on intermittent grab samples that may misrepresent average tap water concentrations due to short-term temporal variability. In this study, we compared the induction of in vitro cytotoxic and genotoxic effects (DNA damage by the comet assay) in relation to different sampling methods, i.e. exposure over time (semipermeable membrane devices, SPMDs, exposed for 30 days) or intermittent grab samples (5 weekly water sampling, C18 concentration). Waters with different chemical characteristics were sampled to test the sensitivity of the two methods. We did not found any positive correlation between the biological findings and water chemical parameters. SPMD extracts induced a significantly greater DNA damage than C18. The different behaviour was specially found for the water samples with a low level of organic compounds and when C18 extracts were highly cytotoxic. Our findings suggest that SPMD could be of a great interest in assessing genotoxic contaminants in both raw and drinking water, with great suitability for continuous monitoring. Furthermore, the results of this study have confirmed the great importance of the biological assays in evaluating the effects of a complex mixture such as water in addition to the conventional chemical examination of water quality.     

Genotoxicity of recycling electronic waste in Idhna,Hebron District,Palestine

Most Electronic waste (e-waste) ends up in landfills while some is recycled. A major site for e-waste recycling in Palestine is the village of Idhna in the Hebron District and most of this waste originates from Israel. The objective of this study was to evaluate the effects of e-waste on human DNA damage and chromosome breaks. The test sample was 46 non-smoker individuals with direct exposure to e-waste, either employed in the workshops or resident in Idhna. Genotoxicity data were compared with a control sample of sixteen unexposed individuals from Bethlehem and Al-Aizariya (Bethany). DNA damage was evaluated using the Comet assay while chromosome aberrations were tested by using conventional cytogenetic techniques. We noted an average of 4.83 aberration/cell/subject in test samples while in controls the average was 0.75. Chromosome aberration frequency was statistically different between exposed and control samples for total aberrations, for chromatid and chromosome breaks, and for formation of rings but not for dicenterics and tetraploidy. The Comet assay likewise showed that there was significant difference between exposed and control samples for DNA damage (p < 0.05). We therefore recommend measures to mitigate the health impact of e-waste recycling.     

Reference values of tests for the assessment of mutagenic activity.

The determination of mutagenic activity in biological media aims at detecting the exposure to mutagenic chemicals, or to chemicals transformed by the organism into mutagenic metabolites. Mutagenic activity is detected by various short-term tests which rely upon the interaction of the chemical with the DNA of, bacteria, Drosophila or mammalian cells. The urinary fluctuation test has been particularly useful in determining mutagenic activity in the urine of subjects exposed to low concentrations of suspected genotoxic chemicals. The assay procedure itself is relatively simple, the data, however, should be carefully evaluated in relation to the attributes of the donor, bearing in mind the confounding variables related to life-style, diet, occupation and drug intake.     

Pet dogs as sentinels for environmental contamination.

The presence of environmental contaminants in air, water and food may pose significant health risks to the exposed human population. However, problems associated with assessing chronic exposure to low doses of environmental chemicals, multiple exposure routes, diseases with long latency periods, and non-specific health outcomes make it difficult to conduct the appropriate human epidemiologic studies. It may be useful to complement human epidemiology with animal studies. Animals monitored or evaluated in situ for the appropriate suite of endpoints can provide information about both exposure levels and potential adverse health effects. Animals have served as sentinel indicators for health effects associated with a number of environmental exposures, including pesticides and asbestos. Pet dogs may be particularly valuable sentinels because they share the human environment. In addition, dogs respond to many toxic insults in ways analogous to humans, they have physiologically compressed life spans, and they are free from some important lifestyle risk factors for disease. An example of how pet dogs may be used as sentinels for potential human health hazards involves a study of the genotoxic effects resulting from exposure to a mixture of chemicals from nearby Superfund sites. We conducted a cross-sectional study of exposed dogs (living in the community with the Superfund sites) and controls (living in a nearby community). The pet owners completed a questionnaire, and we collected a blood sample from each dog. The blood samples were analyzed for standard clinical parameters and assays for possible genotoxic effects (peripheral blood lymphocyte micronucleus frequency and lymphocyte subtyping). Pet dogs living near the Superfund sites had a higher micronucleus frequency than control animals, suggesting that the dogs may have been exposed to environmental contaminants from these sites.     

Delayed effects of low level acute irradiation and chronic environmental radioactive contamination on DNA lymphocytes of people living in Dolon, a settlement located in the vicinity of the Semipalatinsk nuclear test site (Kazakhstan)

During 42 years several hundred nuclear tests were performed by the former USSR at the Semipalatinsk Test Site (STS, Kazakhstan), of which more than 100 were done in the atmosphere. We report here the late genetic damage of external exposure to radiation and environmental radioactive contamination in people living in Dolon, a small settlement situated in the vicinity of the STS. The comet assay was applied on DNA lymphocytes of 20 exposed women and 32 non-exposed women living at 500 km from the STS. We observed a statistically significant difference between the exposed and control groups for mean tail moment (MTM) and DNA% in the tail. The mean values of all comet assay parameters (MTM, DNA% in the tail and score) were higher in the group of women born before 1949 as compared to those born after 1950, which could reflect an effect of external irradiation in 1949 due to the most contaminating explosion. These results suggest that people exposed 50 years ago to relatively small doses of external irradiation and/or still living in an environment contaminated by small amounts of long life radionuclides, still present DNA damage which is in agreement with other cytogenetical studies performed at the same site, on the same population.     

Urban benzene exposure and oxidative DNA damage: influence of genetic polymorphisms in metabolism genes

Benzene has been implicated as an environmental risk factor in leukaemia and other haematological diseases. Relationships between urban benzene exposure, oxidative DNA damage and polymorphisms in metabolism enzymes were examined in 40 volunteers living and working in Copenhagen. Personal exposures to benzene, toluene and methyl tert-butyl ether (MTBE) were monitored during a 5-day period. DNA damage was measured by 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) in lymphocyte DNA and urine and by comet assay with use of fapyguanine glycosylase (FPG) and endonuclease III (ENDO). Excretion of the benzene metabolites trans,trans-muconic acid (ttMA) and S-phenylmercapturic acid (S-PMA) were measured in urine. Polymorphisms in glutathione-S-transferases T1 (GSTT1), M1 (GSTM1) and P1 (GSTP1) and NAD(P)H:quinone oxidoreductase (NQO) were determined. Median exposures to benzene, toluene and MTBE were 2.5, 18.7 and 0.86 microg/m(3). No significant correlations between external benzene exposure and any of the biomarkers were found. However, a significant correlation between S-PMA excretion and 8-oxodG in lymphocytes was found (R(s)=0.39). Men were found to excrete significantly more ttMA than the women did and ttMA excretion in men was found to be significantly associated with external benzene exposure (R=0.53, P=0.025). In addition, ttMA and S-PMA excretion was significantly higher in subjects with the NQO+/-genotype compared with subjects with the wild type (P=0.004 and P=0.011, respectively). Even though there are some limitations in this study due to the low range of benzene exposure and biomarker concentrations as well as a small number of subjects, these results could suggest that even at ambient concentrations exposure to benzene could have genotoxic effects in susceptible individuals.     

Comet and micronucleus assays in zebra mussel cells for genotoxicity assessment of surface drinking water treated with three different disinfectants

The aim of this research was to study the influence of classic (sodium hypochlorite and chlorine dioxide) and alternative (peracetic acid [PAA]) disinfectants on the formation of mutagens in surface waters used for human consumption. For this proposal, in vivo genotoxicity tests (Comet and micronucleus assay) were performed in an experimental pilot plant set up near Lake Trasimeno (Central Italy). The effects were detected in different tissues (haemocytes for the Comet assay and gills for the micronucleus test [MN]) of Dreissena polymorpha exposed in experimental basins supplied with lake water with/without the different disinfectants. Specimen collection was performed before disinfectant input for both tests and after the start of disinfection (3 h and 20 days for the Comet assay and 10 and 20 days for micronucleus test, respectively) to assess short- and long- term exposure effects during three sampling campaigns (October 2000, February 2001, and June 2001). Seasonal differences in baseline levels of DNA migration and micronucleus frequency were observed. Raw water quality modulation on disinfection by-product formation was shown. The results of the micronucleus and Comet assays on zebra mussel cells after in situ exposure to water disinfected with the two chlorinated compounds clearly indicate DNA/by-product interaction. PAA did not induce either clastogenic/aneugenic effects or DNA damage on this bioindicator.     

Comparative assessment of genotoxicity of mineral water packed in polyethylene terephthalate (PET) and glass bottles

The potential migration of genotoxic compounds into mineral water stored in polyethylene terephthalate (PET) bottles was evaluated by an integrated chemical/biological approach using short-term toxicity/genotoxicity tests and chemical analysis. Six commercial brands of still and carbonated mineral water bottled in PET and in glass were stored at 40 °C for 10 days in a stove according to the standard EEC total migration test (82/711/EEC), or at room temperature in the dark. After treatment, the samples were analysed using gas-chromatography/mass spectrometry (GC/MS) to detect volatile and non-volatile compounds, the Microtox^® test to evaluate potential toxicity of the samples, and three mutagenicity tests - Tradescantia and Allium cepa micronucleus tests and the Comet assay on human leukocytes - to detect their genotoxic activity.GC/MS analysis did not detect phthalates or acetaldehyde in the water samples. The Microtox^® test found no toxic effects. Mutagenicity tests detected genotoxic properties of some samples in both PET and glass bottles. Statistical analyses showed a positive association between mineral content and mutagenicity (micronuclei in A. cepa and DNA damage in human leukocytes). No clear effect of treatment and PET bottle was found. These results suggest the absence of toxic compounds migrating from PET regardless of time and conditions of storage. In conclusion, bottle material and stove treatment were not associated with the genotoxic properties of the water; the genotoxic effects detected in bottled water may be related to the characteristics of the water (minerals and CO₂ content).     

Antimutagenic effect of neem leaves extract in freshwater fish, Channa punctatus evaluated by cytogenetic tests

Neem (Azadirachta indica), an indigenous plant commonly grown in India and its sub-continent is a multipurpose plant well known for its insecticidal and biomedical properties, however, its antimutagenic effects in vertebrate organisms are lacking. The present work is therefore, focused on possible antimutagenic potential of ethanolic extract of neem leaves evaluated on the clastogenicity induced by Pentachlorophenol (PCP) and 2, 4-dichlorophenoxyacetic acid (2,4-D) in freshwater fish, Channa punctatus used as a vertebrate model, by cytogenetic endpoints: chromosome aberration (CA) and micronucleus (MN) test. In the first set of experiment, fish were exposed by medium treatment to a single treatment of each chemical (PCP, 0.6 ppm; 2,4-D, 75 ppm; neem extract, 3 ppm) along with the controls. The chromosome preparations were made after processing kidney cells and micronucleus slides were prepared from peripheral blood at multiple duration (48, 72 and 96 h). PCP and 2,4-D when used alone, induced significant CA and MN in a time dependent manner. Neem extract did not show genotoxic potential in both assays. The maximum frequency of CA were recorded as 18.58% and 15.17%, while frequency of MN reached to 8.08% and 4.62% by PCP and 2,4-D respectively, after 96 h exposure. In the second set of experiment, three concentrations of neem extract (1, 2 and 3 ppm) were run simultaneously with the same concentration of PCP (0.6 ppm) and 2,4-D (75 ppm) for antimutagenicity estimates. In mixed treatment, neem extract significantly reduced the frequency of CA and MN. The reduction in the frequency of CA ranged from 40-75% and 45.4-83.3% and similar values for MN were 40.2-75.3% and 44.1-65.8% for PCP and 2,4-D respectively. Although the reductions were significant but not dependent on concentration and time intervals employed. Results suggested that under present experimental conditions, neem extract exhibit strong antimutagenic activity in this fish model, which could further contribute to study its benefit in humans.