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.     

Primary DNA damage in chrome-plating workers

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

Evaluation of genotoxic effects in a group of workers exposed to low levels of styrene

Occupational exposure to styrene was studied in a group of workers engaged in the production of fiberglass-reinforced plastics. Sister-chromatid exchanges (SCE), micronuclei (MN), and DNA damage (evaluated by means of comet assay) were measured in peripheral blood cells from the exposed workers and from a control population. Mandelic acid concentration, an indicator of styrene exposure level, was measured in urine samples collected at the end of the work shift. Average estimated values for styrene exposure were slightly below the threshold limit value (TLV) of 20 ppm recommended by the American Conference of Governmental Industrial Hygienists. Significant increases (P< or =0.01) have been found for SCE and MN frequencies and comet tail length among exposed individuals, as well as significant decreases (P< or =0.01) in the proliferation indices, as compared with control population. High correlation has been obtained between endpoints evaluated and exposure length, and increased values of SCE and MN frequencies and comet tail length have been found among smokers only in the exposed population. The high correlation obtained among SCE and MN frequencies and comet tail length, and the increase of these parameters in the exposed group with regard to control group justify the use of these three biomarkers in the evaluation of genotoxic effects in human populations exposed to styrene.     

Assessment of DNA damage in peripheral blood lymphocytes of individuals susceptible to arsenic induced toxicity in West Bengal, India

Assessment of DNA damage was carried out using alkaline comet assay in lymphocytes of 30 individuals exposed to high levels of arsenic (247.12+/-18.93 microg/l) through contaminated groundwater in North 24 Parganas district, West Bengal, India. All of them exhibited high arsenic contents in nail (4.20+/-0.67 microg/g), hair (2.06+/-0.20 microg/g) and urine (259.75+/-33.89 microg/l) samples and manifested various arsenical skin lesions. Unexposed samples were collected from 30 residents of the unaffected East Midnapur district with very little or no exposure to arsenic (7.69+/-0.49 microg/l) in drinking water. The results were evaluated principally by manual analysis of comets and partly by computerized image analysis. Both the analytical methods exhibited a high degree of agreement in results. The exposed participants expressed significantly higher DNA damage (p < 0.01) in their lymphocytes than the unexposed participants. Alkaline comet assay was also combined with formamidopyrimidine-DNA glycosylase enzyme digestion to confirm that arsenic induced oxidative base damage in the lymphocytes. Significant positive trend effects of comet lengths in relation to arsenic levels in water prove that DNA damage can be used as a sensitive biomarker of arsenic exposure. This study demonstrates that arsenic induced significant DNA damage in the exposed participants, which could correspond to a higher susceptibility to arsenic induced toxicity and carcinogenicity.     

Occupational exposure in airport personnel: characterization and evaluation of genotoxic and oxidative effects

Airport personnel can be exposed to several polycyclic aromatic hydrocarbons (PAHs) from jet fuel vapours, jet fuel combustion products and diesel exhaust. The aim of this study was to characterize the exposure and to evaluate genotoxic and oxidative effects in airport personnel (n=41) in comparison with a selected control group (n=31). Environmental monitoring of exposure was carried out analysing 23 PAHs on air samples collected from airport apron, airport building and terminal/office area during 5 working days. The urinary 1-hydroxy-pyrene (1-OHP) following 5 working days, was used as biomarker of exposure. Genotoxic effects and early direct-oxidative DNA damage were evaluated by micronucleus (MN) and Fpg-modified comet assay on lymphocytes and exfoliated buccal cells, and by chromosomal aberrations (CA) and sister chromatid exchange (SCE) analyses. For comet assay, tail moment (the product of comet relative tail intensity and length) values from Fpg-enzyme treated cells (TMenz) and from untreated cells (TM) were used as parameters of oxidative and direct DNA damage, respectively. We found 27,703 microg/m(3) total PAHs in airport apron, 17,275 microg/m(3) in airport building and 9,494 microg/m(3) in terminal/office area. Urinary OH-pyrene did not show differences between exposed and controls. The exposed group showed a higher mean value of SCE frequency in respect to controls (4.6 versus 3.8) and an increase (1.3-fold) of total structural CA in particular breaks (up to 2.0-fold) and fragments (0.32% versus 0.00%), whereas there were no differences of MN frequency in both cellular types. Comet assay evidenced in the exposed group a higher value in respect to controls of mean TM and TMenz in both exfoliated buccal cells (TM 118.87 versus 68.20, p=0.001; TMenz 146.11 versus 78.32, p<0.001) and lymphocytes (TM 43.01 versus 36.01, p=0.136; TMenz 55.86 versus 43.98, p=0.003). An oxidative DNA damage was found, for exfoliated buccal cells in the 9.7% and for lymphocytes in the 14.6% of exposed in respect to the absence in controls. Our findings furnish a useful contribution to the characterization of civil airport exposure and suggest the use of comet assay on exfoliated buccal cells to assess the occupational exposure to mixtures of inhalable pollutants at low doses since these cells represent the target tissue for this exposure and are obtained by non-invasive procedure.     

Assessment of DNA damage in car spray painters exposed to organic solvents by the high-throughput comet assay

Occupational exposure as a painter is associated with DNA damage and development of cancer. Comet assay has been widely adopted as a sensitive and quantitative tool for DNA damage assessment at the individual cell level in populations exposed to genotoxics. The aim of this study was to assess the application of the high-throughput comet assay, to determine the DNA damage in car spray painters. The study population included 52 car spray painters and 52 unexposed subjects. A significant increase in the %TDNA median (p?< 0.001) was observed in the exposed group in comparison to the unexposed group. Neither age (%TDNA: p?= 0.913) nor time of exposure (%TDNA: p?=?0.398) were significantly correlated with DNA damage. The car spray painters who consumed alcohol did not show a significant increase in DNA damage compared to nonalcohol consumers (p >?0.05). The results showed an increase in DNA breaks in car spray painters exposed to organic solvents and paints; furthermore, they demonstrated the application of high-throughput comet assay in an occupational exposure study to genotoxic agents.     

Evaluating the genotoxic effects of workers exposed to lead using micronucleus assay, comet assay and TCR gene mutation test

To evaluate the genotoxic effects of lead (Pb) exposure, 25 workers in a workplace producing storage battery were monitored for three genetic end-points using micronucleus (MN) assay, comet assay and TCR gene mutation test. Twenty-five controls were matched with workers according to age, gender and smoking. The air Pb concentration in the workplace was 1.26 mg/m(3). All subjects were measured for Pb concentration of blood by atom absorption spectrophotometry. The mean Pb concentration of blood in workers (0.32 mg/l) was significantly higher than that in controls (0.02 mg/l). The results of MN test showed that the mean micronuclei rate (MNR) and mean micronucleated cells rate (MCR) in workers were 9.04+/-1.51 per thousand and 7.76+/-1.23 per thousand, respectively, which were significantly higher than those (2.36+/-0.42 per thousand and 1.92+/-0.31 per thousand) in controls (P<0.01). It was found in the comet assay that the mean tail length (MTL) of 25 workers and 25 controls were 2.42+/-0.09 and 1.02+/-0.08 microm, respectively, there was significant difference between workers and controls for MTL (P<0.01), also the difference of the mean tail moment (MTM) between workers (0.85+/-0.05) and controls (0.30+/-0.09) was very significant (P<0.01). However, in TCR gene mutation assay Mfs-TCR of workers and controls were 1.69+/-0.15 x 10(-4) and 1.74+/-0.17 x 10(-4), respectively, there was no significant difference between workers and controls (P>0.05). The results of our study indicated that the genetic damage was detectable in 25 workers occupationally exposed to lead.     

Measuring the effects of pesticides on occupationally exposed humans with the comet assay

In this study we examine the effects of a mixture of pesticides on occupationally exposed agricultural workers. The study was performed on 149 people, 84 agricultural workers and 65 healthy men from the same area, who served as the control group. The exposed group was divided into a subgroup with 65 individuals moderately exposed (39 men and 26 women) and a highly exposed subgroup consisted of 19 men. The statistical analysis of the comet assay results showed that there were no significant differences in basal DNA damage between pesticide-exposed workers and the control group nor between moderately and highly exposed ones. In addition, exposure of peripheral blood lymphocytes to hydrogen peroxide or gamma-irradiation led to a similar degree of DNA damage and subsequent repair for all the studied populations.     

Metabolic activation of herbicide products by Vicia faba detected in human peripheral lymphocytes using alkaline single cell gel electrophoresis.

Ametryn and metribuzin S-triazines derivatives and EPTC thiocarbamate are herbicides used extensively in Mexican agriculture, for example in crops such as corn, sugar cane, tomato, wheat, and beans. The present study evaluated the DNA damage and cytotoxic effects of three herbicides after metabolism by Vicia faba roots in human peripheral lymphocytes using akaline single cell gel electrophoresis. Three parameters were scored as indicators of DNA damage: tail length, percentage of cells with DNA damage (with comet), and level DNA damage. The lymphocytes were treated for 2 h with 0.5-5.0 microg/ml ametryn or metribuzin and 1.5-10 microg/ml EPTC. Lymphocytes also were coincubated for 2 h with 20 microl V. faba roots extracts that had been treated for 4 h with 50-500 mg/l of the two triazines or with the thiocarbamate herbicide or with ethanol (3600 mg/l), as positive control. The lymphocytes treated with three pesticides without in vivo metabolic activation by V. faba root did not show significant differences in the mean values between genotoxic parameters compared with negative control. But when human cells were exposed to three herbicides after they had been metabolized the frequency of cell comet, tail length and level DNA damage all increased. At highest concentrations of the three herbicides produced severe DNA damage compared with S10 fraction and negative control. The linear regression analysis of the tail length values of three herbicides indicated that there was genotoxic effect concentration-response relationship with ametryn and ametribuzin but no EPTC. The ethanol induced major increase DNA damage compared with S10 fraction and the three pesticides. There were not effects in cell viability with treatment EPTC and metribuzin whether or not it had been metabolized. High concentrations of ametryn alone and after it had been metabolized decreased cell viability compared with the negative control. The results demonstrated that the three herbicides needed to be activated by the V. faba root metabolism to produce DNA damage in human peripheral lymphocyte. The alkaline comet technique is a rapid and sensitive assay, to quickly evaluate DNA damage the metabolic activation of herbicide products by V. faba root in human cells in vitro.     

Comparative evaluation of genotoxicity of captan in amphibian larvae (Xenopus laevis and Pleurodeles waltl) using the comet assay and the micronucleus test

Captan (N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide) is a fungicide used to inhibit the growth of many types of fungi on plants used as foodstuffs. The toxic and genotoxic potentials of captan were evaluated with the micronucleus test (MNT; AFNOR,2000) and the comet assay (CA) using amphibian larvae (Xenopus laevis and Pleurodeles waltl). Acute toxicity results showed that captan was toxic (1) to Xenopus larvae exposed to from 2 mg/L to 125 or 62.5 microg/L, depending on the nature of the water [reconstituted water containing mineral salts or mineral water (MW; Volvic, Danone, France)] and (2) to Pleurodeles exposed to from 2 mg/L to 125 microg/L in both types of water. The MNT results obtained in MW showed that captan (62.5 microg/L) was genotoxic to Xenopus but not genotoxic to Pleurodeles at all concentrations tested. CA established that the genotoxicity of captan to Xenopus and Pleurodeles larvae depended on the concentration, the exposure times, and the comet parameters (tail DNA, TEM, OTM, and TL). The CA and MNT results were compared for their ability to detect DNA damage at the concentrations of captan and the exposure times applied. CA showed captan to be genotoxic from the first day of exposure. In amphibians, CA appears to be a sensitive and suitable method for detecting genotoxicity such as that caused by captan.     

Genotoxic effects in C57Bl/6J mice chronically exposed to arsenate in drinking water and modulation of the effects by low-selenium diet

In C57Bl/6J mice chronically exposed to arsenate in drinking water at 50, 200, or 500 microg As/L, genotoxic effects in bone-marrow cells using micronucleus test and in peripheral blood leukocytes using the comet assay were determined after 3, 6 or 12 mo. To assess the modulating role of selenium in development of the effects, the animals were fed a specially prepared low-selenium diet and were supplemented with sodium selenite (200 microg Se/L) in drinking water (supplemented groups) or were without Se supplementation (nonsupplemented groups). Measurements of glutathione peroxidase activity in erythrocytes and plasma as well as selenium concentration in plasma were performed after 3, 6, and 12 mo and showed a marked decrease in values in animals in non-Se supplemented compared to Se-supplemented groups. After 3 mo of arsenic exposure in the Se-supplemented animals the level of DNA fragmentation (without Endo III and Fpg enzymes) did not differ from the control; however, increased oxidative damage of purine and pyrimidine bases was observed. In groups not supplemented with Se, an increase of DNA fragmentation was observed; however, the levels of oxidative DNA damage in these groups did not differ from the control. None of the positive effects observed in the comet assay after 3 mo was related to arsenate concentration. The levels of DNA damage after 6 and 12 mo of exposure to arsenic as well as the frequency of micronuclei after 3, 6, and 12 mo did not differ significantly between exposed and control animals, irrespective of Se supplementation status.     

Investigation into possible DNA damaging effects of ultrasound in occupationally exposed medical personnel--the alkaline comet assay study

In the present paper the possible DNA damaging effects of ultrasound in occupationally exposed medical personnel were investigated using the alkaline comet assay. The extent of DNA migration in peripheral blood leucocytes was measured. Parameters of the comet assay were studied in 30 medical workers occupationally exposed to ultrasound and in 30 corresponding unexposed control subjects. It was found that the subjects who were occupationally exposed to ultrasound for various periods of time showed a highly significant increase in levels of DNA damage compared with the control. The results obtained have confirmed the usefulness of the alkaline comet assay as a sensitive biodosimetric method, reflecting the current level of DNA damage and/or repair in peripheral blood leucocytes of ultrasound-exposed subjects. In spite of their limitations, the results of the present investigation indicate that individuals occupationally exposed to ultrasound may experience an increased genotoxic risk, emphasizing the need for more research into the nature and extent of the biological consequences to medical personnel working with ultrasonic equipment.     

Genotoxic damage in hospital workers exposed to ionizing radiation and metabolic gene polymorphisms

Of all workers exposed globally to synthetic sources of radiation, medical personnel represent the largest group, but receive relatively low doses. Accidental or therapeutic acute radiation exposure of humans was observed to induce various forms of cytogenetic damage, including the possibility of increasing the incidence of micronuclei (MN) and chromosomal aberrations (CA). The aim of this study was to assess occupationally induced chromosomal damage in a large population of hospital workers exposed to low doses of ionizing radiation (IR). The cytokinesis-block MN and comet assays were used to examine peripheral blood lymphocytes (PBL) of 31 exposed workers to IR and 33 control subjects corresponding in gender, age, and smoking. Glutathione S-transferases (GSTM1, GSTT1, and GSTP1) are postulated to be involved in the detoxification of endogenous and exogenous genotoxicants. The association between these biomarkers and polymorphic genes of xenobiotic metabolizing enzymes was thus also assessed. MN frequency was significantly higher in the exposed subjects compared controls. Comet assay results showed a significant increase of tail length in workers exposed to IR. Data obtained suggest that GSTM1, GSTT1, and GSTP1 polymorphism do not modify significantly the genotoxic potential of IR. Therefore, the exposed medical personnel need to carefully apply radiation protection procedures and minimize, as low as possible, IR exposure to avoid possible genotoxic effects.     

Cytotoxicity and DNA-damage in human lung epithelial cells exposed to respirable alpha-quartz.

Occupational exposure to respirable crystalline silica is associated with the development of silicosis, lung cancer and airways diseases. In order to assess cytotoxic effects and direct-oxidative DNA damage induced by short-term exposure to different doses of respirable alpha-quartz (NIST SRM1878a), we conducted a study using A549 cells. The cells were exposed to alpha-quartz at 25, 50, 100 microg/ml for 4 h and analysed by scanning electron microscope (SEM) and LDH release assay for cytotoxic effect evaluation. Cells were also exposed to 10, 25, 50, 100 microg/ml of alpha-quartz for 2 h and 4 h and analysed by Fpg comet test to evaluate direct and oxidative DNA damage. SEM observations of treated cells showed bleb development at lower doses and alterations of microvilli morphology at the highest dose. A slight LDH release was found only at 100 microg/ml. Fpg comet test showed a dose-related oxidative DNA damage in cells exposed for 2 h to quartz. Cells exposed for 4h at the same concentrations showed a dose-related direct DNA damage and the presence of oxidative DNA damage at lower doses. The bleb induction on cell surface evidenced by SEM at lower doses correlates with the presence of oxidative DNA damage at 4 h. The cell surface modifications observed by SEM at 100 microg/ml indicate that high doses of quartz induce more evident cytotoxic effects confirmed by LDH analysis and correlate with the genotoxicity showed by comet assay.     

Genotoxicity of environmental agents assessed by the alkaline comet assay

Generation of DNA damage is considered to be an important initial event in carcinogenesis. A considerable battery of assays exists for the detection of different genotoxic effects of compounds in experimental systems, or for investigations of exposure to genotoxic agents in environmental or occupational settings. Some of the tests may have limited use because of complicated technical setup or because they only are applicable to a few cell types. The single cell gel electrophoresis (comet) assay is technically simple, relatively fast, cheap, and DNA damage can be investigated in virtually all mammalian cell types without requirement for cell culture. The aim of this thesis was to evaluate the comet assay as a genotoxicity test in genetic toxicology of environmental agents, encompassing both experimental animal models and biomonitoring. The comet assay detects strand breaks (SB). The cells are embedded in agarose and lysed, generating nucleus-like structures in the gel (referred to as nucleoids). Following alkaline electrophoresis, the DNA strands migrate toward the anode, and the extent of migration depends on the number of SB in the nucleoid. The migration is visualized and scored in a fluorescence microscope after staining. Broad classes of oxidative DNA damage can be detected as additional SB if nucleoids are incubated with bacterial DNA glycosylase/endonuclease enzymes. Oxidized pyrimidines and purines can be detected by incubation with endonuclease III and formamidopyrimidine DNA glycosylase, respectively. The animal experimental studies indicated that the comet assay was able to detect genotoxic effects of diesel exhaust particles in lung tissue, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced DNA damage in colon epithelial cells and liver tissue, and benzene-induced damage in bone marrow and liver cells. The strength of the comet assay was further outlined by application of repair enzymes, indicating no oxidative DNA base damage following IQ treatment. High levels of oxidative DNA lesions were detected after exposure to benzene or X-ray irradiation. The comet assay did not detect DNA damage in colon or liver following ingestion of diets containing of high contents of animal fat or sucrose, although other indices of DNA damage were found. Determined from the results of a large Japanese study, the discrimination between carcinogens and non-carcinogens appears to be similar between the comet assay and alkaline elution, which also detects SB. This suggests that the comet assay is a reliable genotoxicity test in animal experimental systems. In the biomonitoring studies, we investigated the effect of common exposures and lifestyle factors (rather than effects of known carcinogens) on the level of oxidative DNA damage in mononuclear blood cells of humans. In the first study, based on repeated measurements, it was shown that interindividual variation and seasonal variation were major determinants for the basal level of SB, whereas no effect of age, exercise, or antioxidant intake could be detected. The effect of exercise was further investigated under both normoxic and hypoxic circumstances, showing a strong effect of hypoxia, and only effect of exercise in terms of SB in hypoxia. In a placebo-controlled parallel dietary fruit and vegetable (or the corresponding amount of antioxidants) intervention study, no effects of the level of oxidative DNA damage or sensitivity to hydrogen peroxide were observed. Although this may seem in contrast to other antioxidant intervention studies, a critical literature survey of antioxidant intervention studies on oxidative DNA damage suggested that well-controlled studies tended to show no effect of antioxidant supplementation. In summary, the aggregated data from the publications included in this thesis, and other publications encompassing the comet assay, indicate that the comet assay is a reliable method for detection of DNA damage in tissues of experimental animals. Although not all types of genotoxic exposures should be expected to result in DNA damage in mononuclear blood cells, the comet assay seems to be a valuable tool for detection of genotoxic exposure in humans. The comet assay indicates that DNA damage is abundant in mammalian cells and affected by lifestyle and many environmental exposures, including diet, exercise, hypoxia, and sunlight.     

Evaluation of DNA damage in jewellery workers occupationally exposed to nitric oxide

Nitric oxide is a metastable radical, reacts with oxygen to produce toxic nitrogen oxides (N(2)O(3), ONOO(-)) which damage DNA. Occupational exposure to nitric oxide leads to increased frequency of chromosomal aberrations in humans. In the present study the DNA damage among the jewellery workers occupationally exposed to nitric oxide was analyzed using buccal cell comet assay. The result of this study shows increased levels of DNA damage among jewellery workers. The habit of cigarette smoking among the jewellery workers has a synergistic effect on inducing DNA damage.     

Oxidative stress and genetic damage among workers exposed primarily to organophosphate and pyrethroid pesticides

The indiscriminate use of pesticides in agriculture and public health campaigns has been associated with an increase of oxidative stress and DNA damage, resulting in health outcomes. Some defense mechanisms against free radical‐induced oxidative damage include the antioxidant enzyme systems. The aim of this study was to determine the levels of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and the relationship of antioxidant enzyme levels with DNA damage among sprayers (workers) occupationally exposed to pesticides. The determinations of MDA and antioxidant enzymes were performed spectrophotometrically. The genotoxic effects were evaluated using the comet assay. The results showed a marginally significant decrease in SOD and CAT activities in the high exposure group compared to the control group. For MDA, statistically significant differences were found among people working long term vs. those working temporarily (P = 0.02) as sprayers. In the moderate exposure group, a positive correlation was observed between MDA levels and GPx activity. In the high exposure group, a negative correlation was observed between GR and CAT activities, and between MDA levels and GPx activities. Furthermore, in the high exposure group, a positive correlation between DNA damage parameters and MDA levels was observed. The results suggest an important role of antioxidant enzymes for the protection of DNA damage caused by occupational exposure to pesticides.     

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

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

Genotoxic damage in pathology anatomy laboratory workers exposed to formaldehyde

Formaldehyde (FA) is a chemical traditionally used in pathology and anatomy laboratories as a tissue preservative. Several epidemiological studies of occupational exposure to FA have indicated an increased risk of nasopharyngeal cancers in industrial workers, embalmers and pathology anatomists. There is also a clear evidence of nasal squamous cell carcinomas from inhalation studies in the rat. The postulated mode of action for nasal tumours in rats was considered biologically plausible and considered likely to be relevant to humans. Based on the available data IARC, the International Agency for Research on Cancer, has recently classified FA as a human carcinogen. Although the in vitro genotoxic as well as the in vivo carcinogenic potentials of FA are well documented in mammalian cells and in rodents, evidence for genotoxic effects and carcinogenic properties in humans is insufficient and conflicting thus remains to be more documented. To evaluate the genetic effects of long-term occupational exposure to FA a group of 30 Pathological Anatomy laboratory workers was tested for a variety of biological endpoints, cytogenetic tests (micronuclei, MN; sister chromatid exchange, SCE) and comet assay. The level of exposure to FA was evaluated near the breathing zone of workers, time weighted average of exposure was calculated for each subject. The association between the biomarkers and polymorphic genes of xenobiotic metabolising and DNA repair enzymes was also assessed. The mean level of exposure was 0.44+/-0.08ppm (0.04-1.58ppm). MN frequency was significantly higher (p=0.003) in the exposed subjects (5.47+/-0.76) when compared with controls (3.27+/-0.69). SCE mean value was significantly higher (p<0.05) among the exposed group (6.13+/-0.29) compared with control group (4.49+/-0.16). Comet assay data showed a significant increase (p<0.05) of TL in FA-exposed workers (60.00+/-2.31) with respect to the control group (41.85+/-1.97). A positive correlation was found between FA exposure levels and MN frequency (r=0.384, p=0.001) and TL (r=0.333, p=0.005). Regarding the genetic polymorphisms studied, no significant effect was found on the genotoxic endpoints. The results of the present biomonitoring study emphasize the need to develop safety programs.     

Genotoxic effects of the fungicide thiophanate-methyl on Podarcis sicula assessed by micronucleus test, comet assay and chromosome analysis

The increasing use of pesticides in modern agriculture has raised the need to evaluate their potential threat to animal and human health. In the present study, the genotoxic effects of environmentally relevant exposure to the fungicide thiophanate-methyl (TM) were assessed in the lizard Podarcis sicula (Reptilia, Lacertidae) using micronucleus test, chromosome aberration analysis and single-cell gel electrophoresis (comet) assay. The number of micronuclei increased significantly with exposure time in lizard specimens exposed to 1.5% TM for 30–40 days. In situ hybridization with the specific HindIII centromeric satellite was positive in 18.7% of the micronuclei observed, suggesting an aneugenic effect of TM during mitosis. DNA damage, evaluated by the comet assay, documented a significant gain in comet length in relation to exposure time that was paralleled by a reduction in head size. Finally, cytogenetic analysis showed a significant increase in chromosome aberrations in exposed animals compared with controls. Our data suggest that long-term TM exposure induces a genomic damage that is positively correlated to exposure time. If such genotoxic effects arise so clearly in an ectothermal vertebrate, such as P. sicula, prolonged exposure TM must be considered as a cytogenetic hazard.