Anesthesia of fish with benzocaine does not interfere with comet assay results

Fish blood erythrocytes are frequently used as sentinels in biomonitoring studies. Usually, fish blood is collected by painful cardiac or caudal vein punctures. Previous anesthesia could decrease animal suffering but it is not known at present whether anesthesia can cause confounding effects. Therefore, using the alkaline single cell gel (SCG)/comet assay with blood erythrocytes of the cichlid fish Nile tilapia, we tested for a possible modulation of induced DNA damage (methyl methanesulfonate; MMS) by the anesthetic benzocaine administered by bath exposure (80mg/l for approximately 10min). Furthermore, benzocaine (80-600mg/l) was tested for its genotoxic potential on fish erythrocytes in vitro and for potential interactions with two known genotoxins (MMS and hydrogen peroxide). Our results did neither indicate a significant increase in the amount of DNA damage (even after a 48h follow-up), nor indicated interactions with MMS-induced DNA damage when fish were exposed to benzocaine in vivo. There was also no increase in DNA damage after in vitro exposure of fish erythrocytes to benzocaine. Clear concentration-related effects were observed for the two genotoxins in vitro, which were not significantly altered by the presence of benzocaine. These results suggest that anesthesia of fish does not confound comet assay results and the use of blood samples from anesthetized fish can be recommended with regard to animal welfare.     

In situ evaluation of the genotoxic potential of the river Nile: II. Detection of DNA strand-breakage and apoptosis in Oreochromis niloticus niloticus (Linnaeus, 1758) and Clarias gariepinus (Burchell, 1822)

This work is part of a wider eco-toxicological study proposed to evaluate the biological impact of contaminants along the whole course of the river Nile, Egypt. Here we present data on the presence of DNA strand-breaks and apoptotic cells assessed by use of comet and diffusion assays in erythrocytes of Nile tilapia (Oreochromis niloticus niloticus) and African catfish (Clarias gariepinus). The results showed high degrees of DNA damage and increased frequencies of apoptotic nuclei in blood of fish collected from downstream compared with those sampled from upstream river Nile. Qualitative analysis revealed a shift in the frequency of DNA-damage classes towards higher damage levels correlating with the increasing pollution gradient. The degree of DNA damage measured by use of comet assay and diffusion assay exhibited seasonal variations. Both fish species showed significant increases in DNA damage during the summer. The results of our study indicated that the alkaline comet assay seems to be a useful technique for in situ genotoxic monitoring. At the same time the diffusion assay is sensitive enough to detect low frequencies of apoptotic nuclei. The results reveal species-specific differences in sensitivities, suggesting that Nile tilapia may serve as a more sensitive test species compared with the African catfish. Based on the outcome of the comet and diffusion assays, it can be concluded that the water quality of the river Nile with respect to the presence of genotoxic compounds needs to be improved, especially in its estuaries. As far as we know this is the first time that the comet and diffusion assays are used for genotoxic monitoring of the river Nile.     

The use of cyprinodont fish, Aphanius fasciatus, as a sentinel organism to detect complex genotoxic mixtures in the coastal lagoon ecosystem

In the present work we aimed to standardise the alkaline comet assay with erythrocytes of the cyprinodont, Mediterranean Killifish, Aphanius fasciatus. The aims of the study were to explore the suitability of this fish to assess biomarkers of genotoxic effects and as a sentinel organism to detect complex genotoxic mixtures in coastal lagoon ecosystems. Following proper optimisation, the application and effectiveness of the comet assay in erythrocytes of A. fasciatus were tested by measuring the tail DNA (%) induced by (a) in vivo exposure of individual fish to X-rays (dose, 3Gy) and (b) following in vitro challenge of erythrocytes with restriction endonucleases Fok-I and Eco-RI, which selectively induce double-strand breaks with cohesive and blunt termini, respectively. Furthermore, in order to evaluate whether circulating fish blood contained actively proliferating cells that could influence the extent of DNA damage in control (untreated) fish, we measured the number of "comets" positive for 5-bromo-2'-deoxyuridine (BrdU) by the use of anti-BrdU antibody and immuno-histochemical methods. Both treatments (i.e. with X-rays and restriction endonucleases) induced statistically significant increases in tail DNA (%) values compared with the relevant untreated controls, indicating the effectiveness of the comet assay in the erythrocytes of A. fasciatus to detect different types of DNA lesions. Results from anti-BrdU antibody labelling of erythrocytes indicated a very low percentage (5%) of "comets" positive for BrdU. Following optimisation and validation of the assay under laboratory conditions, fish were collected in the Orbetello lagoon (Tuscany, Italy), considered to be a significantly polluted site. The results showed statistically significant increases for tail DNA (%) compared with corresponding values observed in erythrocytes of fish caught in the unpolluted reference site "Saline di Tarquinia". The effects of physico-chemical parameters of the water (i.e., salinity, pH and oxygen content) did not significantly influence the induction of DNA damage. These results indicate that the comet assay provides a reliable parameter and that A. fasciatus is a promising "sentinel organism" to detect the genotoxic impact of complex mixtures in coastal lagoon ecosystems.     

Genotoxic effects of Roundup on the fish Prochilodus lineatus

Glyphosate-based herbicides, such as Roundup, represent the most extensively used herbicides worldwide, including Brazil. Despite its extensive use, the genotoxic effects of this herbicide are not completely understood and studies with Roundup show conflicting results with regard to the effects of this product on the genetic material. Thus, the aim of this study was to evaluate the genotoxic effects of acute exposures (6, 24 and 96 h) to 10 mg L(-1) of Roundup on the neotropical fish Prochilodus lineatus. Accordingly, fish erythrocytes were used in the comet assay, micronucleus test and for the analysis of the occurrence of nuclear abnormalities and the comet assay was adjusted for branchial cells. The results showed that Roundup produces genotoxic damage in erythrocytes and gill cells of P. lineatus. The comet scores obtained for P. lineatus erythrocytes after 6 and 96 h of exposure to Roundup were significantly higher than respective negative controls. For branchial cells comet scores were significantly higher than negative controls after 6 and 24 h exposures. The frequencies of micronucleus and other erythrocyte nuclear abnormalities (ENAs) were not significantly different between Roundup exposed fish and their respective negative controls, for all exposure periods. In conclusion, the results of this work showed that Roundup produced genotoxic effects on the fish species P. lineatus. The comet assay with gill cells showed to be an important complementary tool for detecting genotoxicity, given that it revealed DNA damage in periods of exposure that erythrocytes did not. ENAs frequency was not a good indicator of genotoxicity, but further studies are needed to better understand the origin of these abnormalities.     

Effect of safranal, a constituent of Crocus sativus (saffron), on methyl methanesulfonate (MMS)-induced DNA damage in mouse organs: an alkaline single-cell gel electrophoresis (comet) assay

The influence of safranal, a constituent of Crocus sativus L. stigmas, on methyl methanesulfonate (MMS)-induced DNA damage was examined using alkaline single-cell gel electrophoresis (SCGE), or comet, assay in multiple organs of mice (liver, lung, kidney, and spleen). NMRI mice were divided into five groups, each of which contained five mice. The animals in different groups were received the following chemicals: physiological saline (10 mL/kg, ip), safranal (363.75 mg/kg, ip), MMS (120 mg/kg, ip), safranal (72.75 mg/kg, ip) 45 min prior to MMS administration, and safranal (363.75 mg/kg, ip) 45 min prior to MMS administration. Mice were sacrificed about 3 h after the administration of direct mutagen MMS, safranal, or saline, and the alkaline comet assay was used to evaluate the influence of safranal on DNA damage in different mouse organs. Increase in DNA migration was varied between 9.08 times (for spleen) and 22.12 times (for liver) in nuclei of different organs of MMS-treated mice, as compared with those of saline-treated animals (p < 0.001). In control groups, no significant difference was found in the DNA migration between safranal- and saline-pretreated mice. The MMS-induced DNA migration in safranal-pretreated mice (363.75 mg/kg) was reduced between 4.54-fold (kidney) and 7.31-fold (liver) as compared with those of MMS-treated animals alone (p < 0.001). This suppression of DNA damage by safranal was found to be depended on the dose, and pretreatment with safranal (72.75 mg/kg) only reduced DNA damage by 25.29%, 21.58%, 31.32%, and 25.88% in liver, lung, kidney, and spleen, respectively (p < 0.001 as compared with saline-treated group). The results of the present study showed that safranal clearly repressed the genotoxic potency of MMS, as measured by the comet assay, in different mouse organs, but the mechanism of this protection needs to be more investigated using different in vitro system assays and different experimental designs.     

Genotoxic damage in polychaetes: a study of species and cell-type sensitivities

The marine environment is becoming increasingly contaminated by environmental pollutants with the potential to damage DNA, with marine sediments acting as a sink for many of these contaminants. Understanding genotoxic responses in sediment-dwelling marine organisms, such as polychaetes, is therefore of increasing importance. This study is an exploration of species-specific and cell-specific differences in cell sensitivities to DNA-damaging agents in polychaete worms, aimed at increasing fundamental knowledge of their responses to genotoxic damage. The sensitivities of coelomocytes from three polychaetes species of high ecological relevance, i.e. the lugworm Arenicola marina, the harbour ragworm Nereis diversicolor and the king ragworm Nereis virens to genotoxic damage are compared, and differences in sensitivities of their different coelomic cell types determined by use of the comet assay. A. marina was found to be the most sensitive to genotoxic damage induced by the direct-acting mutagen methyl methanesulfonate (MMS), and showed dose-dependent responses to MMS and the polycyclic aromatic hydrocarbon benzo(a)pyrene. Significant differences in sensitivity were also measured for the different types of coelomocyte. Eleocytes were more sensitive to induction of DNA damage than amoebocytes in both N. virens and N. diversicolor. Spermatozoa from A. marina showed significant DNA damage following in vitro exposure to MMS, but were less sensitive to DNA damage than coelomocytes. This investigation has clearly demonstrated that different cell types within the same species and different species within the polychaetes show significantly different responses to genotoxic insult. These findings are discussed in terms of the relationship between cell function and sensitivity and their implications for the use of polychaetes in environmental genotoxicity studies.     

Effects of temperature on baseline and genotoxicant-induced DNA damage in haemocytes of Dreissena polymorpha

The potential application of the Comet assay for monitoring genotoxicity in the freshwater mussel Dreissena polymorpha was explored and a preliminary investigation was undertaken of the baseline levels of DNA damage in mussel haemocytes of animals kept at different temperatures. In addition, in vitro cell sensitivity against genotoxicants was assessed in relation to increasing temperatures. The mussels were kept at four different constant temperatures (4, 18, 28 and 37 degrees C) for 15 h. The haemocytes withdrawn were treated in vitro with melphalan, as a model genotoxic compound, or sodium hypochlorite, a common water disinfectant capable of producing mutagenic/carcinogenic by-products, at the established temperatures for 1h. The data obtained in vivo, in cells directly withdrawn from the mussels showed a significant (P<0.001, Student's t test) inter-individual variability, probably due to genetic and epigenetic factors and an increasing amount of DNA damage at increasing temperature. Mussel haemocytes showed a clear dose-response effect after in vitro melphalan treatment. Hypochlorite treatment also significantly increased DNA migration: the damage was temperature dependent, with a similar increase at 4 and 28 degrees C and a minimum level at 18 degrees C. This study demonstrates the potential application of the Comet assay to haemocytes of D. polymorpha. However, these findings suggest that temperature could alter both DNA damage baseline levels in untreated animals and cell sensitivity towards environmental pollutants in in vitro conditions. Therefore, more information is needed about seasonal variations and the natural background levels of DNA damage in mussels living in the wild, before they are used for the monitoring of genotoxic effects in aquatic environments.     

Assessment of genotoxic effects of chloropyriphos and acephate by the comet assay in mice leucocytes

Two organophosphorus (OP) pesticides (chloropyriphos and acephate) and cyclophosphamide (CP) (positive control) were tested for their ability to induce in vivo genotoxic effect in leucocytes of Swiss albino mice using the single cell gel electrophoresis assay or comet assay. The mice were administered orally with doses ranging from 0.28 to 8.96 mg/kg body weight (b. wt.) of chloropyriphos and 12.25 to 392.00 mg/kg b.wt. of acephate. The assay was performed on whole blood at 24, 48, 72 and 96 h. A significant increase in mean comet tail length indicating DNA damage was observed at 24h post-treatment (P<0.05) with both pesticides in comparison to control. The damage was dose related. The mean comet tail length revealed a clear dose dependent increase. From 48 h post-treatment, a gradual decrease in mean tail length was noted. By 96 h of post-treatment the mean comet tail length reached control levels indicating repair of the damaged DNA. From the study it can be concluded that the comet assay is a sensitive assay for the detection of genotoxicity caused by pesticides.     

Synergistic DNA damaging effects of 4-nitroquinoline-1-oxide and non-effective concentrations of methyl methanesulfonate in human fibroblasts

DNA damage and DNA repair in human fibroblasts induced by the combination mixture of the genotoxic agents methyl methanesulfonate (MMS) and 4-nitroquinoline-1-oxide (4-NQO) were studied using the comet assay and the unscheduled DNA synthesis (UDS), respectively. Cells were simultaneously treated for 1h with the no observed effect concentration (noec) of MMS and increasing concentrations of 4-NQO or vice versa. Different results were obtained with the two types of mixtures. When the noec of 4-NQO was combined with increasing concentrations of MMS, no combination effects were observed. However, in experiments with increasing concentrations of 4-NQO and the noec of MMS, an increase in DNA damage and repair (and an enhancement of cytotoxicity) was demonstrated. Quantitative analysis of the effects by the isobologram method confirmed synergistic responses in both tests. We are proposing interactive actions between 4-NQO and MMS, whereby 4-NQO facilitates the attack of MMS on the DNA bases.     

Genotoxic effects of copper sulphate in freshwater planarian in vivo, studied with the single-cell gel test (comet assay)

The alkaline single-cell gel electrophoresis, or comet assay, was used to evaluate the genotoxic potential of copper sulphate in planarians. Concentration-related increase in DNA damage was induced after 2h and 7 days exposure to CuSO4 dissolved in culture water. To study the influence of copper ions on the persistence of mutagen-induced DNA lesions, planarians were treated with methyl methanesulphonate (MMS), and further incubated in the absence (post-incubation) or presence (post-treatment) of CuSO4. After 2h of post-treatment enhanced persistence of DNA effects in relation to the corresponding post-incubation value was detected, which indicate inhibition of DNA repair by CuSO4. At 4h an increase of DNA migration in relation to the 2h value was observed, which is significant for the post-incubation group. After 24h, DNA damage decreased but was still significantly elevated in relation to the control. From our results, we conclude that planarians are suitable organisms for in vivo detection of copper genotoxicity in the comet assay, and can be used to assess both acute and chronic exposure to this chemical in aquatic ecosystems. The inhibition effect of copper ions on repair of MMS-induced DNA damage suggests that copper could modulate the genotoxic effects associated with complex mixture exposure in the environment.     

Anaerobic bacteria associated with epizootics in grey mullet (Mugil cephalus) and redfish (Sciaenops ocellata) along the Texas Gulf Coast

Anaerobic bacteria tentatively identified as species of Catenabacterium were recovered from brain, liver, kidney and blood of fish involved in a massive epizootic of grey mullet (Mugil cephalus) and redfish (Sciaenops ocellata). Pathogenicity was demonstrated for grey mullet (M. cephalus) and sea catfish (Arius felis) but not for channel catfish (Ictalurus punctatus) or white mice. Diseased fish were disoriented, weak and swimming at the surface of the water. Thioglycolate and salt bovine blood agar containing 40 microg/ml gentamicin were useful as selective culture media.     

Assessment of DNA damage in peripheral blood of heavy smokers with the comet assay and the micronucleus test

The comet assay (single-cell gel electrophoresis, SCG) is being increasingly used in human biomonitoring for the detection of genotoxic exposures. Cigarette smoking is a well-documented source of a variety of potentially mutagenic and carcinogenic compounds. Therefore, smoking should represent a relevant mutagenic exposure and lead to genotoxic effects in exposed cells. However, our previous investigations as well as several other published studies on human biomonitoring failed to show an effect of smoking on DNA migration in the comet assay, while some other studies did indicate such an effect. Although many factors can contribute to the generation of discrepant results in such studies, clear effects should be obtained after high exposure. We therefore performed a comparative study with healthy male heavy smokers (>20 cigarettes per day) and non-smokers (n=12 in each group). We measured the baseline comet assay effects in fresh whole blood samples and isolated lymphocytes. In addition, the amount of 'formamidopyrimidine DNA-glycosylase (FPG)-sensitive sites' was determined by a combination of the standard comet assay with the bacterial FPG protein. Furthermore, the influence of a repair inhibitor (aphidicolin, APC) on baseline DNA damage was comparatively analysed. Duplicate slides from each sample were processed and analysed separately. In all experiments, a reference standard (untreated V79 cells) was included to correct for assay variability. Finally, to compare the comet assay results with another genetic endpoint, all blood samples were investigated in parallel by the micronucleus test (MNT). Baseline and gamma radiation-induced micronucleus frequencies were determined. None of these approaches revealed a significant difference between heavy smokers and non-smokers with regard to a genotoxic effect in peripheral blood cells.     

Possible repair action of Vitamin C on DNA damage induced by methyl methanesulfonate, cyclophosphamide, FeSO4 and CuSO4 in mouse blood cells in vivo

Interaction between Vitamin C (VitC) and transition metals can induce the formation of reactive oxygen species (ROS). VitC may also act as an ROS scavenger and as a metal chelant. To examine these possibilities, we tested in vivo the effect of two doses of VitC (1 and 30 mg/kg of mouse body weight) on the genotoxicity of known mutagens and transition metals. We used the alkaline version of the comet assay to assess DNA damage in peripheral white blood cells of mice. Animals were orally given either water (control), cyclophosphamide (CP), methyl methanesulfonate (MMS), cupric sulfate or ferrous sulfate. A single treatment with each VitC dose was administered after treatment with the mutagens or the metal sulfates. Both doses of VitC enhanced DNA damage caused by the metal sulfates. DNA damage caused by MMS was significantly reduced by the lower dose, but not by the higher dose of VitC. For CP, neither post-treatment dose of VitC affected the DNA damage level. These results indicate a modulatory role of Vitamin C in the genotoxicity/repair effect of these compounds. Single treatment with either dose of VitC showed genotoxic effects after 24 h but not after 48 h, indicating repair. Double treatment with VitC (at 0 and 24 h) induced a cumulative genotoxic response at 48 h, more intense for the higher dose. The results suggest that VitC can be either genotoxic or a repair stimulant, since the alkaline version of the comet assay does not differentiate "effective" strand breaks from those generated as an intermediate step in excision repair (incomplete excision repair sites). Further data is needed to shed light upon the beneficial/noxious effects of VitC.     

In vivo genotoxicity of the pyrethroid pesticide beta-cyfluthrin using the comet assay in the fish Bryconamericus iheringii

Environmental pollution by pesticide residues is a major environmental concern due to the extensive use of these substances in agriculture. The insecticide beta-cyfluthrin is a synthetic pyrethroid widely used in agricultural and other domestic activities. The aim of the present study was to assess the genotoxic effects of a sublethal exposure of the fish Bryconamericus iheringii (Characidae) to a commercial formulation of beta-cyfluthrin using the comet assay. Fish were exposed to sublethal concentrations (4.2 and 5.6 microg/L) of beta-cyfluthrin under static conditions during 24- and 48-h exposure periods. Fish in tap water were used as negative controls. Results obtained by the comet assay revealed genotoxic effects of the pyrethroid in the higher concentration and at the longer exposure period. The mean DNA damage index of fish exposed to 5.6 microg/L beta-cyfluthrin for 48 h was significantly higher (145.9 +/- 51.8) than in the control group (69.3 +/- 39.5). These findings indicate that native fish species might be at risk for genotoxic damage in waters contaminated with beta-cyfluthrin.     

Comparative in vitro and in vivo assessment of genotoxic effects of etoposide and chlorothalonil by the comet assay.

The alkaline single cell gel electrophoresis (comet) assay was used to assess in vitro and in vivo genotoxicity of etoposide, a topoisomerase II inhibitor known to induce DNA strand breaks, and chlorothalonil, a fungicide widely used in agriculture. For in vivo studies, rats were sacrificed at various times after treatment and the induction of DNA strand breaks was assessed in whole blood, bone marrow, thymus, liver, kidney cortex and in the distal part of the intestine. One hour after injection, etoposide induced DNA damage in all organs studied except kidney, especially in bone marrow, thymus (presence of HDC) and whole blood. As observed during in vitro comet assay on Chinese hamster ovary (CHO) cells, dose- and time-dependent DNA effects occurred in vivo with a complete disappearance of damage 24 h after administration. Even though apoptotic cells were detected in vitro 48 h after cell exposure to etoposide, such a result was not found in vivo. After chlorothalonil treatment, no DNA strand breaks were observed in rat organs whereas a clear dose-related DNA damage was observed in vitro. The discrepancy between in vivo and in vitro models could be explained by metabolic and mechanistic reasons. Our results show that the in vivo comet assay is able to detect the target organs of etoposide and suggest that chlorothalonil is devoid of appreciable in vivo genotoxic activity under the protocol used.     

Assessment of the genotoxicity in toad Bufo raddei exposed to petrochemical contaminants in Lanzhou Region, China

Single cell gel electrophoresis or comet assay, micronucleus (MN) test and global DNA methylation detection were used to assess the genotoxicity in toad Bufo raddei exposed to the petrochemical (mainly oil and phenol) polluted area in Lanzhou Region (LZR) comparing with a relatively unpolluted area in Liujiaxia Region (LJXR). The results from the present study indicated that DNA damage and MN frequency in toad from LZR were significantly higher than those from LJXR at the same sampling month, whereas the degree of global DNA methylation was lower, which implies that the petrochemical contaminants at environmental level in LZR were genotoxic to B. raddei. The degree of genotoxic damage was obviously related with the extent of pollution among the three sampling months in LZR. The significantly positive correlations between DNA damage and concentrations of oil and/or phenol existed in liver cells but erythrocytes, implying that liver is more suitable as a sentinel tissue for the assessment of genotoxic impact of low-level contamination. The results from both comet assay and global DNA methylation detection on liver cells showed that the genotoxicity varied significantly with oil and/or phenol concentrations, suggesting that these two methods are relatively sensitive and suitable for monitoring the genotoxicity of petrochemical pollutants on amphibians.     

Genotoxic effects in the Eastern mudminnow (Umbra pygmaea L.) after exposure to Rhine water, as assessed by use of the SCE and Comet assays: a comparison between 1978 and 2005

Surface water used for drinking-water preparation requires continuous monitoring for the presence of toxic compounds. For monitoring of genotoxic compounds fish models have been developed, such as the Eastern mudminnow (Umbra pygmaea L.) because of its clearly visible 22 meta-centric chromosomes. It was demonstrated in the late seventies that Rhine water was able to induce chromosome aberrations and sister chromatid exchange in this fish species. Although in vitro mutagenicity studies of the RIWA (Rhine Water Works, The Netherlands) have shown that the genotoxicity of the river Rhine steadily decreased during the last decades, there is still concern about the presence of some residual mutagenicity. In addition, in most studies the water samples have been tested only in in vitro test systems such as the Salmonella-microsome test. For this reason, and in order to be able to make a comparison with the water quality 27 years ago, a study was performed with the same experimental design as before in order to measure the effect of Rhine water on the induction of SCE in the Eastern mudminnow. As a new test system the single cell gel electrophoresis assay (Comet assay) was performed. Fish were exposed to Rhine water or to groundwater for 3 and 11 days in flow-through aquaria. Fish exposed for 11 days to Rhine water had a significantly higher number of SCE and an increased comet tail-length compared with control fish exposed to groundwater. After exposure for three days to Rhine water there was no difference in SCE and a slightly increased comet tail-length compared with the control. It was concluded that genotoxins are still present in the river Rhine, but that the genotoxic potential has markedly decreased compared with 27 years ago. Furthermore, the Comet assay appears to be a sensitive assay to measure the genotoxic potential of surface waters in fish.     

Genotoxicity of three pesticides used in Costa Rican banana plantations

The in vitro genotoxicity of imazalil and thiabendazole fungicides and the insecticide chlorpyrifos, compounds used in Costa Rican banana plantations, was evaluated with the single-cell gel electrophoresis technique (comet assay). The comet assay is a simple, rapid and low cost technique for quantification of DNA damage. This assay detects DNA single-strand breaks and alkali-labile sites in individual cells. The effects were analyzed by using human lymphocytes exposed to doses of 0, 25, 50, 75 and 100 microg/ml of each pesticide for 30 min at 37 degrees C. The cells were embedded in agarose, lysed, subjected to alkaline electrophoresis (pH >13) for 20 min at 25V, neutralized and dehydrated to be stained with a fluorescent dye and later comets visualization with the epifluorescence microscope. Chlorpyrifos and imazalil induced significant DNA damage in a dose-dependent manner. Chlorpyrifos was the major inductor of DNA breaks. These results indicate that both are genotoxic compounds in vitro. Thiabendazole fungicide did not induced DNA damage using the comet assay for all concentrations tested.     

Profenofos induced DNA damage in freshwater fish, Channa punctatus (Bloch) using alkaline single cell gel electrophoresis

The aim of the present study was to evaluate the induced genotoxicity (DNA damage) due to organophosphate pesticide profenofos (PFF) in gill cells of freshwater fish Channa punctatus using single cell gel electrophoresis (SCGE)/Comet assay. The 96h LC(50) value of PFF (50% EC) was estimated for the fish species in a semistatic system and then three sub-lethal of LC(50) concentrations viz the sub-lethal 1, sub-lethal 2 and sub-lethal 3 concentrations were determined as 0.58ppb, 1.16ppb and 1.74ppb, respectively. The fish specimens were exposed to these concentrations of the pesticide and the gill tissue samplings were done on 24h, 48h, 72h and 96h post exposure for assessment of DNA damage in terms of percentage of DNA in comet tails. In general, a concentration dependent response was observed in the gill cells with induction of maximum DNA damage at the highest concentration of PFF. The results of the present investigation indicated that PFF could potentially induce genotoxic effect in fish, even in sub-lethal concentrations and SCGE as a sensitive and reliable tool for in vivo assessment of DNA damage caused by the genotoxic agents.     

DNA damage in frog erythrocytes after in vitro exposure to a high peak-power pulsed electromagnetic field

Till the present time, the genotoxic effects of high peak-power pulsed electromagnetic fields (HPPP EMF) on cultured cells have not been studied. We investigated possible genotoxic effects of HPPP EMF (8.8 GHz, 180 ns pulse width, peak power 65 kW, repetition rate 50 Hz) on erythrocytes of the frog Xenopus laevis. We used the alkaline comet assay, which is a highly sensitive method to assess DNA single-strand breaks and alkali-labile lesions. Blood samples were exposed to HPPP EMF for 40 min in rectangular wave guide. The specific absorption rate (SAR) calculated from temperature kinetics was about 1.6 kW/kg (peak SAR was about 300 MW/kg). The temperature rise in the blood samples at steady state was 3.5 +/- 0.1 degrees C. The data show that the increase in DNA damage after exposure of erythrocytes to HPPP EMF was induced by the rise in temperature in the exposed cell suspension. This was confirmed in experiments in which cells were incubated for 40 min under the corresponding temperature conditions. The results allow us to conclude that HPPP EMF-exposure at the given modality did not cause any a-thermal genotoxic effect on frog erythrocytes in vitro.