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Detection of Genotoxic Effects on Cells of Liver and Gills of B. rerio by Means of Single Cell Gel Electrophoresis
... Auch die Angaben über Schadstoffkonzentrationen können nicht genutzt werden, da sie keine Auskunft über ihre Bioverfügbarkeit geben, die von der Interaktion der Organismen untereinander und von Umweltfaktoren abhängt (Ahlf 1995, Liß und Ahlf 1997. Des weiteren bleiben bei reiner chemischer Analytik synergetische und antagonistische Effekte der organischen Substanzen unberücksichtigt (Deventer 1996, Gunkel 1994. Manche Chemikalien werden auch nur in geringen Konzentrationen in einem Fließgewässer nachgewiesen, können aber bei chronischer Exposition schwerwiegende Schäden in Organismen verursachen, wie Untersuchungen an Fischen und Muscheln gezeigt haben (Fent 1996, Schweigert et al. 2001, Vos et al. 2000. ...
... Er eignet sich daher sehr gut als Screeningtest, um das Ausmaß des biologischen Schädigungspotentials von Sedimenten und Schwebstoffen abzuschätzen. Gentoxizitätstest: Die Kontamination von Gewässern mit gentoxischen Substanzen anthropogener Herkunft, ist sowohl aus human-als auch aus ökotoxikologischen Gesichtspunkten bedenklich (Deventer 1996, Helma et al. 1994, Moretti et al. 2002. Gentoxizität kann über Absterben von Gameten und erhöhte Mortalität von Entwicklungsstadien negative Folgen auf die Reproduktion haben, oder durch die Induktion von Mutationen einen starken Einfluss auf die Populationsdynamik und damit auch auf die Stabilität von Ökosystem ausüben (Anderson und Wild 1994, Calmano et al. 1992, Helma et al. 1994. ...
... Gentoxische Belastungen stellen eine Bedrohung für die Überlebensfähigkeit von empfindlichen Arten dar; von Fischen und Muscheln ist bekannt, dass sie wie Säugetiere Karzinome entwickeln können (Stahl 1991). Aus zahlreichen kombinierten Labor-und Feldstudien ist bekannt, dass gentoxische Effekte in vitro mit einer Zunahme an pathologischen Veränderungen korrelieren (Calmano et al. 1992, Deventer 1996, Metcalfe et al. 1990, Stahl 1991 (Hollert und Braunbeck 1997, Helma et al. 1994. Histidinbedürftige (his -) Stämme werden der Testsubstanz ausgesetzt. ...
... In another study, zebrafish were exposed to 0.8-80 lM (88 lg/L-8.8 mg/L) MMS (Deventer 1996). At the highest concentration, the comet assay revealed a significant increase of tail length in liver and gill cells after 8 h of exposure. ...
... Exposure was stopped after 72 h, and subsequent 24 h of recovery in pure water led to a decrease of DNA damage. The highest concentration used by Deventer (1996) is only slightly higher than the second highest concentration used in the present study. On the other hand, a much longer exposure time of two weeks was applied without the possibility to recover from alkylating stress, apparently leading to an accumulation of DNA damage. ...
... Likewise, zebrafish erythrocytes exposed to 1 lM (110 lg/L) MMS for three weeks clearly showed induction of micronuclei . If compared to the comet assay, Deventer (1996) concluded a higher sensitivity for the micronucleus test in zebrafish. Apart from erythrocytes, only few studies have been carried out on the effect of genotoxic agents on micronucleus frequencies in fish gill and liver cells. ...
Since generative tissues are a link between the generations, the detection of genetic damage in testis and ovary of fish is conductive to elucidating the relationship between genotoxicity and impairment of reproduction. In the current study, exposure of zebrafish to methyl methanesulfonate over two weeks caused concentration dependent genotoxic effects in gonads, liver and gills using the alkaline comet assay. Likewise, the micronucleus frequency was elevated in all of these organs. Thus, the comet assay and the micronucleus test proved appropriate for the detection of genotoxicity in primary male and female gonad cells and histological sections of the gonads from zebrafish, respectively.
... Der Comet-Assay findet ein weites Anwendungsfeld in sehr unterschiedlichen Bereichen aus Biologie und Medizin. Exemplarisch zu nennen wären hier Untersuchungen zum Einfluß unterschiedlicher Strahlungsarten auf Zellen und Organismen (Abt et al., 1997;Nocentini, 1995;Plappert et al., 1997;Wojcik et al., 1996), der Einsatz in der Tumorforschung und -diagnostik Olive, 1995;Olive und Banath, 1996), in der Einzelsubstanzprüfung (Andreoli et al., 1997;Ashby et al., 1995;Devaux et al., 1997;Deventer, 1996; sowie neuerdings auch im Umweltmonitoring (Belpaeme et al., , 1998Binkova et al., 1996;Clements et al., 1997;Devaux 1998;Hartmann et al., 1998;Moretti et al., 1996;Pandrangi et al., 1995;Steinert et al., 1998;Weßler et al., 1998). Auch mit Pflanzen wurde der Test in den vergangenen Jahren etabliert, so z. ...
... Die Isolation der Leber-und Kiemenzellen aus dem Zebrabärbling erfolgt nach der für den Zebrabärbling optimierten Modifikation der Immersionsmethode von Pärt (1993), Deventer (1996) und Rahmann (1996 ...
... Die Methode der Einzelzell-Gelelektrophorese oder Comet Assay wurde in der hier angewandten Form von Singh et al. 1988 mit menschlichen Lymphocyten etabliert und verstärkt in der medizinischen Forschung mit menschlichen Zellen oder zumindest Säugerzellen angewendet (Abt et al., 1997;Nocentini, 1995;Olive et al., 1993. Um den Assay zum Nachweis genotoxischer Belastungen in der aquatischen Umwelt mit Fischen und Fischzellen zu nutzen und die dort erarbeiteten Ergebnisse im Vergleich mit schon vorhandenen Studien (Belpaeme et al., , 1998Deventer, 1996;Mitchelmore und Chipman, 1998 a,b;Monod et al., 1998;Pandrangi et al., 1995; ...
In den Untersuchungen zum Comet Assay mit Fischen und Fischzellen stand die Etablierung des Systems mit primären Hepatocyten und Kiemenzellen aus dem Zebrabärbing (Danio rerio), der Vergleich der genotoxischen Wirkung nach in vitro- und in vivo-Exposition sowie die Korrelation der Ergebnisse mit anderen etablierten Testsystemen im Vordergrund. Erste Grundlagenuntersuchungen für den Einsatz mit Fischzellen wurden mit der Dauerzelllinie RTG-2 vorgenommen. Neben bekannten Mutagenen wurden unterschiedlich belastete Wasserproben von Rhein, Elbe und Neckar getestet. Im Gegensatz zu den In vitro-Untersuchungen, mit nur wenigen positiv getesteten Wasserproben, ergaben Freilandstudien in Fischen aus den untersuchten Gewässern gehäuft leichte DNA Schäden und zeigten damit die Grenzen von Kurzzeit-In vitro-Tests auf. Zur Einschätzung der biologischen Relevanz des Endpunkts DNA Strangbruch aus dem Comet Assay, wurden mit den getesteten Mutagenen der Mikrokern Test, histologische und ultrastrukturelle Untersuchungen bei belasteten Fischen und Fischzellen, Early Life Stage Tests mit dem Zebrabärbling sowie vergleichende Freilandstudien mit unterschiedlichen Fischarten durchgeführt. Diese zeigten, daß die mit dem Comet Assay abgebildeten Effekte im Bereich vielfältiger, biologisch faßbarer Wirkungen liegen. Beispielsweise wiesen die belasteten Fischlarven aus den Early Life Stage Untersuchungen deutliche Ähnlichkeiten mit Entwicklungsmutanten aus genetischen Screenings auf. Die ermittelten LC 50-Konzentrationen entsprachen Testkonzentrationen im Comet Assay, die bereits deutliche DNA-Schäden induzierten. Starke Effekte im Comet Assay konnten mit der Induktion apoptotischer Prozesse in RTG-2 Zellen nach dreitägiger Exposition korreliert werden. Abschließend kann der Comet Assay mit Dauer- und Primärzellen aus Fischen als ein wertvolles Werkzeug im Rahmen einer Testbatterie zur Ermittlung genotoxischer Effekte in der aquatischen Umwelt empfohlen werden.
... In total, 62 articles were published using zebrafish as a model system for assessing the MN and other NA induced by pollutants. The first article was published in 1996 (Deventer, 1996), which described MN formation in zebrafish adults exposed to methyl methanesulfonate for 3 days. Between 1996 and 2004, no articles were published in this research area. ...
... Regarding the staining method, it was observed that most studies used Giemsa (n = 30; 48.4%), Acridine orange (n = 7; 11.3%), and 4 ′ ,6diamidino-2-phenylindole (DAPI) (n = 3; 4.8%). The first study found used the acridine orange staining method (Deventer, 1996). Since then, from 2004 to 2009, all studies have used the Giemsa staining method. ...
Nuclear abnormality (NA) assay in fish has been widely applied for toxicity risk assessment under field and laboratory conditions. The zebrafish (Danio rerio) has become a suitable model system for assessing the NA induced by pollutants. Thus, the current study aimed to summarize and discuss the literature concerning micronucleus (MN) and other NA in zebrafish and its applications in toxicity screening and environmental risk assessment. The data concerning the publication year, pollutant type, experimental design, and type of NA induced by pollutants were summarized. Also, molecular mechanisms that cause NA in zebrafish were discussed. Revised data showed that the MN test in zebrafish has been applied since 1996. The MN was the most frequently NA, but 15 other nuclear alterations were reported in zebrafish, such as notched nuclei, blebbed nuclei, binucleated cell, buds, lobed nuclei, bridges, and kidney-shaped. Several pollutants can induce NA in zebrafish, mainly effluents (mixture of pollutants), agrochemicals, and microplastics. The pollutant-induced NA in zebrafish depends on experimental design (i.e., exposure time, concentration, and exposure condition), developmental stages, cell/tissue type, and the type of pollutant. Besides, research gaps and recommendations for future studies are indicated. Overall, the current study showed that zebrafish is a suitable model to assess pollutant-induced mutagenicity.
... Tissue cells suspensions for comet assays were prepared according to the protocol used by Deventer (1996) with some modifications. Briefly, 0.5 g of each sampled tissue was incubated in 2 ml ice-cold PBS-EDTA buffer (10 mM). ...
... This was in agreement with the faster and higher metal accumulation measured in this organ. A similar response pattern was reported for zebrafish exposed to a reference genotoxic agent (Deventer 1996). A clear concentration-response relationship was observed for Cu exposure with the waterborne metal concentration and the tissue accumulation (Pearson p = 0.006). ...
Genotoxic effects of Cd(+2), Cr(+6), and Cu(+2) on the gill and liver of the Argentinean Silverside (Odontesthes bonariensis) were studied using the comet assay and in relation with the metal tissue accumulation. Fish were exposed to three waterborne concentrations of each metal for 2 and 16 days. Genotoxicity was assessed by the single cell gel electrophoresis (comet assay). After 2 days, significant increase of the genetic damage index (GDI) was only observed in the gill of fish exposed to Cr(+6) and Cu(+2), and the LOECs were 2160 nM and 921.1 nM, respectively. The gill LOEC for Cd(+2) by 16 days was 9.4 nM. In the liver, LOECs were obtained only for Cd(+2) and Cr(+6) and were 9.4 and 2160 nM, respectively. The three metals were able to induce genotoxic effects at environmentally relevant concentrations and the gill was the most sensitive organ.
... After direct exposure of turbots (Psetta maximus) to EMS, blood, gill, liver and kidney cells showed a significant increase in DNA damage. Zebra fish (Brachydanio rerio) were exposed to methyl methanesulfonate (MMS) (Deventer, 1996). Isolated cells of gill and liver were observed: genotoxic effects of MMS were displayed with the Comet assay for low concentrations of MMS (80 µM). ...
... This eliminates one problem met with the micronucleus test and allows an increase in the delay of response. Indeed, Deventer (1996) showed that effects of methyl methanesulfonate on micronuclei in the erythrocytes of zebra fish were visible only as late as 6 days after initiation of the treatment, while comets were visible already after 8 hours. ...
The Comet assay, also called the single cell gel electrophoresis (SCGE) assay or microgel electrophoresis (MGE) assay primarily measures DNA strand breakage in single cells. Since the protocol was published by Singh et al. (1988), its use has increased in different topic areas: clinical applications, human monitoring, radiation biology, genetic toxicology and genetic ecotoxicology. This paper is a review of the studies that have involved the alkaline version of the Comet assay in genetic ecotoxicology. It focuses mainly on the type of organisms (plants, worms, molluscs, fish, amphibians and mammalians) but also on the type of cells which have been used for ecotoxicological studies. In the 23 papers published since 1993 and presented here, the original test procedure may have been slightly modified according to the cell type. In vitro and in vivo experiments as well as in situ studies have been carried out in various compartments (water, soil and air). The Comet assay is able to detect genotoxic effects of chemical and physical agents but only chemical substances and environmental complex mixtures will be considered in this review.
... Methyl methanesulfonate (MMS) has frequently been used as a model genotoxicant (Solomon and Faustman 1987;Deventer 1996;Bony et al. 2010;Devaux et al. 2011). Although MMS itself is not relevant in the aquatic environment, it can be considered as a representative of several groups of environmental pollutants such as mucohalic acids (Gómez-Bombarelli et al. 2011) and allylic reagents (Kuehl et al. 1994) due to its alkylating mode of action. ...
... However, it is suggested that different results for the sexes are not due to a mechanism of sex dependence behind genotoxic effects in the present study but to the low level of damage near the detection limit. This is supported by the finding of significant effects in all organs tested from both sexes in the micronucleus test in the current experiment, bearing in mind that the micronucleus test is sensitive to chronic exposure to low-concentrated genotoxicants (Deventer 1996). ...
There is still controversy whether adverse effects by genotoxic anthropogenic pollutants are linked to the decline of fish populations. Further investigations into the relationship between genotoxic stress and detrimental effects on development and reproduction in fish are required. For this end, zebrafish (F0 generation) were exposed in vivo to the alkylating model genotoxin methyl methanesulfonate (MMS) from fertilization to the age of 1 year. F0 fish were mated over 6 months to check for reproductive capacities. F1 fish grew up without exposure in order to allow for regeneration. Mortality of F0 fish depended on MMS concentrations. In MMS-exposed F0 fish, times of first spawning were delayed and fertility was reduced. Using the alkaline comet assay and the micronucleus test, significant genotoxic effects were found in the livers, gills and gonads of either sex in the F0 generation. No detrimental effects on growth were found. In F1 fish with parental exposure, teratogenic effects were increased, and larval survival was reduced. However, fertility capacities of the non-exposed F1 generation had recovered. Development and survival rates further recovered in the F2 generation. Anthropogenic genotoxicants may thus play a considerable role in the decline of wild fish populations.
... After direct exposure of turbots (Psetta maximus) to EMS, blood, gill, liver, and kidney cells showed a significant increase in DNA damage. Zebra fish (Brachydanio rerio) were exposed to methyl methanesulfonate (MMS) [Deventer, 1996]. Isolated cells of gill and liver were observed: genotoxic effects of MMS were displayed with the Comet assay for low concentrations of MMS (80 M). ...
... This eliminates one problem met with the micronucleus test and allows an increase in the delay of response. Indeed, Deventer [1996] showed that effects of methyl methanesulfonate on micronuclei in the erythrocytes of zebra fish were visible only as late as 6 days after initiation of the treatment, whereas comets were already visible after 8 hr. ...
The Comet assay, also called the single cell gel electrophoresis (SCGE) assay or microgel electrophoresis (MGE) assay, primarily measures DNA strand breakage in single cells. Since the protocol was published by Singh et al. [1988], its use has increased in different topic areas: clinical applications, human monitoring, radiation biology, genetic toxicology, and genetic ecotoxicology. This study is a review of the investigations that have involved the alkaline version of the Comet assay in genetic ecotoxicology. It focuses mainly on the type of organisms (plants, worms, molluscs, fish, amphibians, and mammalians) but also on the type of cells that have been used for ecotoxicological studies. In the 23 papers published since 1993 and presented here, the original test procedure may have been slightly modified according to the cell type. In vitro and in vivo experiments as well as in situ studies have been carried out in various environments (water, soil, and air). Although the Comet assay is able to detect genotoxic effects of chemical and physical agents, only chemical substances and environmental complex mixtures will be considered in this review.
... Although comet assay is thought to be more or at least equally sensitive in assessing genotoxicity as MN assay (Deventer, 1996;Mitchelmore and Chipman, 1998;Robbiano et al., 1999;He et al., 2000), it is sometimes noticed that positive results in the MN assay are accompanied by negative ones in the comet assay (Vrzoc and Petras, 1997;Hartmann et al., 2001). Bombail et al. (2001) have detected elevated MN frequencies in butterfish (Pholis gunnellus ) erythrocytes from contaminated sites while, at the same time, comet assay did not reveal any significant differences between animals from polluted and cleaner areas. ...
... DNA damage measured by comet assay appears earlier than MN (Deventer, 1996) that requires cell division. At the same time its longevity is to a great extent shorter, indicating recent pollution status (Bolognesi et al., 1995), than of MN that lasts as long as the cell itself. ...
Assessment of DNA damage is of primary concern when determining the pollution-related stress in living organisms. To monitor genotoxicity of the freshwater environments we used micronucleus (MN) and comet assay on Dreissena polymorpha haemocytes. Caged mussels, collected from the river Drava, were transplanted to four monitoring sites of different pollution intensity in the river Sava. Exposition lasted for a month. The baseline level of MN frequencies in the haemocytes of mussels from reference site (river Drava) was 0.5 per thousand. No increase in MN frequency was found in mussels from the medium-polluted site (Zagreb) in the river Sava while other, more polluted sites showed higher MN frequencies ranging from 2.7 per thousand (Lukavec) and 3.1 per thousand (Oborovo) to 5.2 per thousand (Sisak). Results from comet assay showed concordance with MN assay in indicating intensity of DNA damage. The use of haemocytes from caged, non-indigenous mussels in MN and comet assay proved to be a sensitive tool for the freshwater genotoxicity monitoring.
... The application of comet assays in the assessment of genotoxicity has been reported for many aquatic animals such as fishes and bivalves [38][39][40], but few reports were found in macrocrustaceans such as crabs and shrimps. In the present study, we performed the comet assay in haemocytes successfully to evaluate the genotoxicity of avermectin on E. sinensis. ...
Avermectin is commonly used in aquaculture systems for pest control in recent decades in China. However, no information is provided for the toxic effect to the important commercial species, Chinese mitten crab, Eriocheir sinensis. To investigate the aquatic toxicity of avermectin, an acute toxic test was performed in this study. The results showed that the 48 h- and 96 h- LC50 were 1.663 and 0.954 mg/L, respectively. For further research, crabs were exposed to sublethal concentrations of 0.03, 0.06, 0.12, 0.24 and 0.48 mg/L. Levels of antioxidants, including superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (T-AOC) were significantly (P<0.05) decreased with dose- and time- dependent responses, meanwhile the oxidative products including malondialdehyde (MDA), hydrogen peroxide (H2O2) and protein carbonyl in serum increased significantly (P<0.05) at concentrations of 0.24 and 0.48 mg/L throughout the experiment. A significant (P<0.05) increase of intracellular ROS and decrease of phagocytic activity was observed in high concentration groups, with dose- and time- dependent manners during the exposure. In addition, serious genetic damage was detected, for the significant increase (P<0.05) of both comet ratio and %DNA in tail at each concentration, and micronucleus (MN) frequency at concentrations of 0.12, 0.24 and 0.48 mg/L at 96 h. These results indicated that sublethal concentration exposure of avermectin had a prominent toxic effect on E. sinensis based on the oxidative stress induced by generated ROS, immunological activity inhibition and genotoxicity.
... Depuis la publicationde Singh et al. (1988), le test des comètes a fait l'objet de très nombreuses études dans de nombreux domaines : applications cliniques, surveillance humaine, études des effets des radiations, toxicologie génétique, études des systèmes de réparation de l'ADN, de l'apoptose, etc... . cellules de foie, branchiesDeventer, 1996 truite fario (Salmo trutta fario) érythrocytes Belpaeme et al., 1996a truite arc-en-ciel (Onchorynchus mykiss) Tableau IV : Exemples d'applications du test des comètes à différents organismes D'excellentes revues bibliographiques présentent ces diverses applications ...
La présente étude concerne principalement les effets génotoxiques de différentes matrices complexes sur des plantes supérieures : 2 sols artificiellement contaminés, l'un avec des métaux (SI) et l'autre avec des polluants organiques (SII), 2 sols prélevés sur sites contaminés (SIII et SIV) et 2 boues : une boue digérée de station d'épuration urbaine (B1) et la même boue compostée (B3). Nous avons sélectionné 3 plantes supérieures : tradescantia (une plante herbacée), Vicia faba (la fève) et Allium cepa (l'oignon blanc). Le génome peut être altéré de diverses façons, mais nous nous sommes intéressés aux cassures de brins d'ADN (qui peuvent être détectées par le test des micronoyaux ou par le test des comètes) et aux mutations somatiques. Parallèlement à ces travaux, l'élongation racinaire et l'indice mitotique ont été déterminés sur Vicia et Allium. Tous ces critères ont été systématiquement réalisés sur les lixiviats (rapport solide/liquide = 1/10) des matrices testées. En outre, nous avons comparé 3 modes d'exposition en utilisant Vicia et Allium : un mode direct (phase solide), un mode indirect (lixiviat) et un mode intermédiaire où les olnates ont été mises en contact avec le sol mélangé à un milieu nutritif. Les résultats ont permis de mettre clairement en évidence les avantages de certains tests : la rapidité du test des comètes dont l'applicabilité aux cellules végétales a été démontrée, la plus grande sensibilité de Vicia par rapport aux autres espèces. Une bonne corrélation a été démontrée entre les deux modes d'exposition "alternatifs" à la lixiviation, mais ce procédé demeure le plus efficace pour détecter le potentiel génotoxique des sols contaminés. Enfin, nous avons mis en évidence une génotoxicité des sols SI et SIII et dans une moindre mesure, des sols SII et SIV. Quant à la boue de station d'épuration, elle a induit très peu d'effets génotoxiques sur les plantes étudiées. Ces effets sont d'ailleurs encore moins apparents après compostage
... catalogued the highest TSP level of 221 μg/Ncm, doubly exceeding the long term guideline value of 90 μg/Ncm. The DNA damage measured by the comet assay may reflect not only the actual levels of DNA damage due to current exposure indicative of recent pollution status but even past exposures to the DNA damaging substances (Kopjar et al., 2006;Deventer, 1996;Bonassi et al., 1995). The comet assay measures strand breaks resulting from the complex interaction of two main processes: actual and recent DNA damage (direct scission or alkali labile site and adduct) and repair activation or inhibition (Klobučar et al., 2003), thus the measured damage level is the result of equilibrium between damage infliction and repair. ...
Occupational exposure to vehicular exhaust in Metro Manila, Philippines is a major human health risk concern because of the established DNA damaging potential of some of its components like Polycyclic Aromatic Hydrocarbons (PAHs). Hence, in this study, peripheral blood leucocytes of 50 urban female street sweepers and an equal number of housekeepers and housewives were analyzed for DNA damage utilizing the alkaline single cell gel electrophoresis (SCGE) or comet assay. This study also determined the influence of some demographic characteristics like age, length of fuel exhaust exposure, smoking and alcohol/coffee/tea drinking on DNA damage. Possible association of DNA damage and hematological parameters to include RBC count, WBC count, hematocrit, hemoglobin, lymphocytes and segmenters was also done. Results showed that exposure to vehicular exhaust has caused an increase in tail lengths (8.48±3.41 μm versus 19.35±8.79 μm) and tail moments (1.93±1.43 versus 8.02±5.71) of the leucocytes as demonstrated by the comet assay. Differences in the demographic characteristics of the study population were not significant (p > 0.05) but comet assay results of the smokers, alcohol/coffee/tea drinkers with longer length of exposure to fuel exhaust recorded higher DNA damage compared to the smokers (p < 0.05), alcohol/coffee/tea drinkers of the reference group. Hematological parameters were not affected by fuel exhaust exposure (p > 0.05). Results of the current study suggests of the possibility that constant exposure to fuel exhaust could lead to a transient increase in the levels of damage in the DNA of leucocytes and that the comet assay was a particularly sensitive technique in detecting such effects.
... This test has mostly been applied to erythrocytes, because these cells types can be easily sampled and cell dissociation is not needed (Belpaeme et al., 1996). In addition, gills have also been used (Deventer, 1996), mainly because of their continuous contact with the water. ...
Atrazine (ATZ) is an herbicide extensively used around the world to kill weeds. Due to its applicability and benefits in farming, ATZ can easily reach the aquatic ecosystems and, therefore, represent risks for aquatic biota and human populations. The aim of this study was to evaluate the genotoxic effects of ATZ on Rhamdia quelen through the piscine micronucleus test (MNT) and the comet assay in erythrocytes (ECA) and gill cells (GCA), at three different concentrations (2, 10, and 100 µg L-1) in static (SB) and semi-static bioassays (SSB) during 96 hours. In the SB, we observed an increased frequency of nuclear morphological abnormalities at all concentrations and a dose-dependent effect of ATZ on DNA through the ECA. There was no difference among treatments in gills. In the SSB there was no significant difference in MNT, but the ECA showed an increase of DNA breakages at 10 µg L-1 treatment. GCA showed higher DNA damage on fish exposed to 2 and 100 µg L-1. Our results show a trend to dose-dependent genotoxic effect of ATZ, which causes damage to the DNA of Rhamdia quelen even in a concentration considered safe by regulatory agencies.
... Few other workers have also noticed high DNA fragmentation in liver cells from field studies (Rajaguru et al., 2003;Liney et al., 2006) and from induced studies (Akter et al., 2008;Ahmed et al., 2013). Comet assay is a sensitive biomarker of oxidative damage to cells as it detects recognizable changes in structure and function in tissues earlier than changes revealed by the micronucleus test (Deventer, 1996). This test is also a validation tool to detect oxidative stress in the tissues as reducible heavy metals lead to the formation of hydroxyl radicals leading to base alteration and deoxyribosephosphate backbone breakage inducing increased tail DNA as GSH reduces more and more reducible heavy metals to form free radicals (Li et al., 2010;Fatima et al., 2014a). ...
... Der Mikronukleus-Assay ist eine verbreitete Methode, die sowohl schon häufig im Biomonitoring von Fischen (z. B. Arbeiten von Al-Sabti 1992; De Flora et al. 1993) als auch als in vivo-Test (an Fischen: Rao et al. 1997;Deventer 1996) eingesetzt wurde. In vitro-Anwendungen sind ebenfalls häufig, jedoch seltener an Systemen mit aquatischer Relevanz (Miller et al. 1997;Fischzelllinie BGF: Babich et al. 1990; Wasserproben an Ratten-Hepatozyten: Eckl 1995; Versuche mit RTG-2-Zellen: Rickert 1996). ...
Gewässer sind Lebensgrundlage, jedoch gleichzeitig Schadstoffsenken für eine Vielzahl von Kontaminanten. Biologische Wirkungstests und das Biomonitoring aquatischer Proben sind daher besonders wichtig, um Umwelt-Gefahrenpotenziale erkennen zu können. Der "Comet Assay" (Einzelzell-Gelelektrophorese) ist ein Indikator von DNA-Strangbrüchen und wurde hier als Test auf gentoxische Wirkungen erprobt und angewandt. Mit bekannten, gentoxischen Substanzen wurden Nachweisgrenzen und Dosis-Wirkungs-Beziehungen für die Zelllinien RTG-2 und RTL-W1 (aus der Regenbogenforelle, Oncorhynchus mykiss) in vitro ermittelt und methodische Parameter an die Zellen angepasst. Der Test reagierte sehr sensitiv auf 4-Nitrochinolin-1-oxid. Die Substanz war daher geeignet, um in weiteren Versuchen als Positivkontrolle zu dienen. Zur Bewertung der Messdaten wurde ein geeignetes statistisches Verfahren gefunden, das auch historische Kontrollen mit einbezog. Der zeitliche Verlauf der DNA-Schädigung des Testsystems mit RTG-2-Zellen wurde ermittelt, und durch Inhibition der DNA-Reparatur mit Aphidicolin wurden Zusammenhänge zwischen der Entstehung von DNA-Strangbrüchen, der DNA-Reparaturkapazität sowie der Metabolisierungskapazität untersucht. In einer zweiten Phase wurden unbehandelte Wasserproben aus Rhein, Elbe sowie weitere Oberflächenwasserproben mit dem Comet Assay an RTG-2-Zellen getestet. Bei 15 von 49 Proben zeigten sich gentoxische Effekte. In einer dritten Phase wurden Erythrozyten von freilebenden Döbeln, Leuciscus cephalus, aus der Mosel mit dem Comet Assay untersucht. Die Fische von drei Messstellen zeigten erhöhte Werte von DNA-Schädigungen, gegenüber einer vierten, stromabwärts gelegenen Messstation. Korrelationen mit den Ergebnissen zusätzlicher Biomarker ergaben sich nur teilweise. Chemische Analysen von Wasser- oder Gewebeproben ließen keine Rückschlüsse auf verursachende Kontaminanten zu - gerade dies unterstreicht jedoch die Wichtigkeit biologischer Tests bei komplexen Proben.
... The changes in RAPD band patterns occurred coincidently in gills and liver of fish exposed to aluminum for 6 and 24 h and might reflect the genotoxic effect of Al on liver and gill cells. The fact that no change was detected in the RAPD profiles of both organs after 96 h of exposure to Al would be indicative that DNA damage in these organs was repaired, taking into account that the DNA repair system in fish cells can be effective within 24 h after contact with the contaminant (Deventer 1996). In longer periods of exposure, such as during 15 days, cells of gills and liver could undergo new DNA lesions, which would overcome the capacity of the enzymes responsible for the repair of these damages, leading to the disappearance of some RAPD bands as well the appearance of new bands. ...
Applying an integrated approach using the Comet, micronucleus (MN), and random amplified polymorphic DNA (RAPD) assays, occurrence of erythrocytic nuclear abnormalities (ENAs) and the liver activity of antioxidants enzymes (catalase and glutathione-S-transferase (GST)) was carried out to evaluate the effects of acute (6, 24, and 96 h) and subchronic (15 days) exposures to aluminum on fish Prochilodus lineatus. The Comet assay showed that fish erythrocytes exhibited significantly higher DNA damage after 6 and 96 h of Al exposure. MN frequencies were very low and did not increase significantly after Al exposures, while ENAs frequency increased significantly after all exposure periods. RAPD profiles obtained with DNA from fish fins collected before the toxicity tests were compared to the profiles with DNA from gills and liver of the same fish sampled after Al exposures. Alterations in RAPD profiles, including appearance and disappearance of bands, after 6 h, 24 h, and 15 days of Al exposure were detected. Fish exposed to Al for 6 and 24 h also showed significant increases in GST and catalase activities. These results indicated that Al exposure was genotoxic to P. lineatus, inducing DNA damage in peripheral erythrocytes. The induction of antioxidant enzymes might be an indication that Al causes oxidative damage to DNA, while the very low frequency of MN suggests that Al does not produce clastogenic or aneugenic effects. Genotoxic effects after 15 days of Al exposure was revealed only by RAPD, showing that this assay represents a sensitive method to detect genotoxic damage, occasionally not detected by other genotoxic tests used in toxicological genetics studies.
... Alkylating agents represent an abundant class of chemicals that cause DNA damage, and may be produced endogenously (Rydberg & Lindahl, 1982;Taverna & Sedgwick, 1996), encountered in the environment (Ballschmiter, 2003;Hamilton et al., 2003;Hecht, 1999), or be introduced during medical procedures such as cancer therapies (Christmann et al., 2007). The wellcharacterized simple monofunctional alkylating agent methyl methanesulfonate (MMS) is used as a model genotoxicant (Bony et al., 2010;Devaux et al., 2011;Deventer, 1996;Solomon & Faustman, 1987). MMS modifies DNA via the N3 or N7 positions of the adenosine or guanosine moiety (Kim & LeBreton, 1994) and causes random lesions that result in single strand (Fortini et al., 2000) or double strand breaks (Pascucci et al., 2005) at random positions in the genome. ...
Whole genome transcriptomic studies are powerful for characterizing the molecular mechanisms underlying the physiological effects of chemicals, and are informative for environmental health risk assessment. Alkylating agents are an abundant class of chemicals that can damage DNA in the environment, and are used for anticancer treatments. Currently, little is known regarding the molecular mechanisms of toxic alkylating agents in zebrafish cell lines. In this study, RNA-sequencing was used to investigate the transcriptomic responses of zebrafish ZF4 cells following exposure to the model genotoxicant methyl methanesulfonate (MMS). The half-maximal inhibitory concentration (IC50 ) of MMS was 639.16 ± 61.8 µm, and apoptosis was induced within 24 h of exposure. RNA sequencing identified 3601 differentially expressed genes (DEGs) that were upregulated and 3037 that were downregulated. Gene ontology enrichment analysis revealed that most DEGs belonged to synthesis and metabolism categories. RNA-associated processes were the most upregulated, while cell cycle and adhesion were the most repressed processes, and neuron-related processes were the most downregulated developmental process. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis identified DNA damage repair, cell cycle, apoptosis and spliceosome as overrepresented terms. Six types of alternative splicing were detected. In total, 1156 alternative splicing DEGs were specifically expressed following MMS treatment, many of which belonged to metabolism and catabolic process categories. Cluster analysis of orthologs was able to extrapolate toxicotranscriptomic data between zebrafish and yeast. These results provide insight into the genome-wide response of ZF4 cells following exposure to MMS, and this knowledge will inform future toxicogenomic data analysis and environmental health risk assessment. Copyright © 2015 John Wiley & Sons, Ltd.
Copyright © 2015 John Wiley & Sons, Ltd.
... The action of parental compounds or their metabolites may damage membrane lipids, DNA, proteins and carbohydrates directly, and the generation of ROS may result in the same damage indirectly (Oliveira et al. 2009). SCGE, which is widely used to evaluate the genotoxicity of chemicals in the environment, was used to assess the toxic effects on zebrafish embryos and liver (Deventer 1996;Sun et al., 2004;Kosmehl et al. 2008;Seitz et al. 2008). Therefore, we use SCGE as an index to evaluate the genotoxicity of chlorpyrifos and TCP. ...
Chlorpyrifos is a broad-spectrum organophosphorus insecticide (O,O-diethyl -O-3,5,6-trichloro-2-pyridyl phosphorothioate) that is used in numerous agricultural and urban pest controls. The primary metabolite of chlorpyrifos is 3,5,6-trichloro pyridine-2-phenol (TCP). Because of its strong water solubility and mobility, this harmful metabolite exists in the environment in a large amount. Although TCP has potentially harmful effects on organisms in the environment, few studies have addressed TCP pollution. Therefore, this study was undertaken to investigate the effect of chlorpyrifos and TCP on the microsomal cytochrome P450 content in the liver, on the activity of NADPH-P450 reductase and antioxidative enzymes [catalase (CAT) and superoxide dismutase (SOD)], and on reactive oxygen species (ROS) generation and DNA damage in zebrafish. Male and female zebrafish were separated and exposed to a control solution and three concentrations of chlorpyrifos (0.01, 0.1, 1 mg L(-1)) and TCP (0.01, 0.1, 0.5 mg L(-1)), respectively, sampled after 5, 10, 15, 20 and 25 days. The results indicated that the P450 content and the NADPH-P450 reductase and antioxidative enzyme (CAT and SOD) activities could be induced by chlorpyrifos and TCP. DNA damage of zebrafish was enhanced with increasing chlorpyrifos and TCP concentrations. Meanwhile, chlorpyrifos and TCP induced a significant increase of ROS generation in the zebrafish hepatopancreas. In conclusion, this study proved that chlorpyrifos (0.01-1 mg L(-1)) and TCP (0.01-0.5 mg L(-1)) are both highly toxic to zebrafish.
... Similarly, other investigators have also reported high DNA fragmentation in liver cells from natural water studies (Rajaguru et al. 2003;Liney et al. 2006) and from induced studies (Ahmed et al. 2013;Akter et al. 2009). Comet assay is a sensitive biomarker of oxidative damage to cells because it induces changes in tissue structure and function earlier than those induced by MNT (Deventer 1996). This test is also an indirect tool to detect oxidative stress in tissues because reducible heavy metals lead to the formation of hydroxyl radicals, which in turn leads to base alteration and deoxyribosephosphate backbone breakage. ...
The aim of the study was to evaluate the effect of heavy-metal contamination on two fish species (Channa striatus and Heteropneustes fossilis) inhabiting a small freshwater body of northern India. After being captured, each specimen was weighed, measured, and analyzed for heavy metals (chromium [Cr], nickel [Ni], and lead [Pb]). Accumulation of heavy metals was found to be significantly greater (p < 0.05) in different tissues (gill, liver, kidney, and muscle) of fishes captured from the reservoir than from the reference site. Levels of heavy-metal contamination in Shah jamal water was Cr (1.51 mg/l) > Ni (1.22 mg/l) > Pb (0.38 mg/l), which is significantly greater than World Health Organization standards. Bioaccumulation factor was calculated, and it was observed that Pb was most detrimental heavy metal. Condition factor was also influenced. Micronucleus test of fish erythrocytes and comet assay of liver cells confirmed genotoxicity induced by heavy-metal contamination in fishes. Heavy metals (Cr, Ni, and Pb) were increased in both fish species as determined using recommended values of Federal Environmental Protection Agency for edible fishes. This raises a serious concern because these fishes are consumed by the local populations and hence would ultimately affect human health.
... Induction kinetics of primary DNA damage has already been described in many cell types of aquatic organisms after in vivo exposure to genotoxicants exhibiting various modes of action (Deventer, 1996;Rank and Jensen, 2003;Bony et al., 2008;Canty et al., 2009;Lacaze et al., 2010). In the present work, the initial induction time described as the latent period required before observing a significant biological response arises after 6 days of MMS exposure. ...
... The observation, that % tail DNA values remained elevated after 14 and 21 days and that the values in fish exposed to the contaminated sediments remained consistently higher than those exposed to sediments from the reference site at Ballymacoda, suggest that beyond a 7-day exposure, their genotoxic response was not time-related. This seems to support the findings of Deventer (1996) who reported that DNA damage in isolated blood cells of Brachydanio rerio, exposed to methyl methane sulphonate, increased initially and leveled off at around 96 h. Furthermore, Belpaeme et al. (1998) reported significant DNA damage in turbot liver cell preparations exposed to ethyl methane sulphonate occurring only after 7 days exposure. ...
... Although genotoxic potency of this compound (acting as a direct alkylating agent) has been mainly described in vitro, our results are in accordance with some in vivo studies demonstrating its genotoxicity (or of the parent compound EMS) in fish using the micronucleus or comet assay in erythrocytes and liver cells through water exposure. Thus, a significant increase in micronuclei frequency was demonstrated in zebrafish erythrocytes exposed for 6 to 12 days to micromolar concentrations of MMS and a significant increase in strand breaks formation in liver cells after 4 days [11]. Moreover, water exposure for 14 days of the fish Clarias lazera and for one day of Oreochromis niloticus to micromolar concentrations of EMS and MMS respectively, led to a significant increase in DNA damage measured in erythrocytes using either the micronucleus or the comet assay [12,13]. ...
This study deals with the use of a chronic exposure scenario of zebrafish (Danio rerio) in laboratory conditions to evaluate the genotoxic potential of diuron and azoxystrobin, two pesticides intensively used in vineyard agriculture. Adult male zebrafish were exposed during three weeks in the semi static mode in four 20 L aquaria. Treatment allowed to each aquarium was: negative control (untreated), positive control (methyl methane sulphonate 1 µM), diuron 4.3 nM and azoxystrobin 1.2 nM. Once per week, genotoxicity was assessed (6 fish/treatment) by the use of two complementary biomarkers: the primary DNA damages evaluated in somatic (liver) and germ (spermatozoa) cells by the alkaline version of the Comet assay and the micronucleus formation assessed in erythrocytes. Very low basal DNA damages were obtained with both biomarkers in negative control during the three consecutive weeks and a significant genotoxic response was obtained in 1 µM MMS exposed fish, both in liver and germ cells with the Comet assay and in erythrocytes with the micronucleus test, respectively starting after one and three weeks. With this chronic exposure scenario, both diuron and azoxystrobine revealed a genotoxic potential at realistic environmental concentrations and a significant response was obtained in all cell types investigated and with both biomarkers used, mainly after 7 or 14 days, thus stressing the interest of long-term exposure scenario. Further studies will be undertaken in order to evaluate whether DNA damage observed in spermatozoa of fish exposed to environmental concentration of pesticides could lead to subsequent reproductive disorders.
... The changes in RAPD band patterns occurred coincidently in gills and liver of fish exposed to aluminum for 6 and 24 h and might reflect the genotoxic effect of Al on liver and gill cells. The fact that no change was detected in the RAPD profiles of both organs after 96 h of exposure to Al would be indicative that DNA damage in these organs was repaired, taking into account that the DNA repair system in fish cells can be effective within 24 h after contact with the contaminant (Deventer 1996). In longer periods of exposure, such as during 15 days, cells of gills and liver could undergo new DNA lesions, which would overcome the capacity of the enzymes responsible for the repair of these damages, leading to the disappearance of some RAPD bands as well the appearance of new bands. ...
Applying an integrated approach using the Comet, micronucleus (MN), and random amplified polymorphic DNA (RAPD) assays, occurrence
of erythrocytic nuclear abnormalities (ENAs) and the liver activity of antioxidants enzymes (catalase and glutathione-S-transferase (GST)) was carried out to evaluate the effects of acute (6, 24, and 96h) and subchronic (15days) exposures
to aluminum on fish Prochilodus lineatus. The Comet assay showed that fish erythrocytes exhibited significantly higher DNA damage after 6 and 96h of Al exposure.
MN frequencies were very low and did not increase significantly after Al exposures, while ENAs frequency increased significantly
after all exposure periods. RAPD profiles obtained with DNA from fish fins collected before the toxicity tests were compared
to the profiles with DNA from gills and liver of the same fish sampled after Al exposures. Alterations in RAPD profiles, including
appearance and disappearance of bands, after 6h, 24h, and 15days of Al exposure were detected. Fish exposed to Al for 6
and 24h also showed significant increases in GST and catalase activities. These results indicated that Al exposure was genotoxic
to P. lineatus, inducing DNA damage in peripheral erythrocytes. The induction of antioxidant enzymes might be an indication that Al causes
oxidative damage to DNA, while the very low frequency of MN suggests that Al does not produce clastogenic or aneugenic effects.
Genotoxic effects after 15days of Al exposure was revealed only by RAPD, showing that this assay represents a sensitive method
to detect genotoxic damage, occasionally not detected by other genotoxic tests used in toxicological genetics studies.
KeywordsAquatic environment-Genotoxicity-Comet assay-RAPD-Micronucleus-Oxidative stress
... The accumulation of ROS may be implicated in the reduction in the detoxifying capabilities of GST. The comet assay, which is widely used to evaluate the genotoxicity of chemicals in the environment, was used to assess the toxic effects on zebrafish embryos and liver (Deventer, 1996;Sun et al., 2004;Kosmehl et al., 2008;Seitz et al., 2008). Our results indicated that the DNA damage in zebrafish hepatopancreas increased with increasing atrazine concentration, indicating that atrazine-induced DNA damage in zebrafish and the levels of DNA damage imposed by atrazine could be quantified by comet assay. ...
This study was undertaken to investigate the protective effect of atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-S-triazine) on the activity of glutathione-S-transferase (GST) and DNA damage in males and females of adult zebrafish (Danio rerio). Zebrafish were exposed to control and three treatments (0.01, 0.1, and 1 mg/L) of atrazine for 5, 10, 15, 20, and 25 days. The results indicated that, for males, the GST activity at lower atrazine concentrations (0.01 and 0.1 mg/L) was markedly higher than that of the controls throughout the duration of the experiment while there was a significant inhibition of the GST activity at 1 mg/L atrazine at days 5 and 20. For females, a significant increase was detected at 0.1 mg/L on the days 5 and 15 and at 0.01 mg/L on day 20. The DNA damage in zebrafish was evaluated using the comet assay; the olive tail moments obtained for hepatopancreas were enhanced after treatment with different concentrations of atrazine on days 5, 10, 15, 20, and 25. The DNA damage increased with increasing atrazine concentrations, indicating that genotoxicity of atrazine and significant differences was found compared to the controls. In conclusion, these findings provide further evidence of the effects of atrazine on aquatic ecosystems.
... O teste do cometa é geralmente aplicado em eritrócitos periféricos de peixes, anfíbios e répteis, e em linfócitos do sangue periférico de mamíferos por serem facilmente amostrados e a dissociação celular não é necessária, entretanto há estudos que utilizaram brânquias e fígado. (Deventer, 1996e Nacci et al, 1996. ...
Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Animal, 2009. A preocupação dos efeitos de cianobactérias na saúde humana cresce em muitos países. Casos de florações de cianobactérias em reservatórios e lagos criam problemas para o abastecimento de água, além de envenenamento, tanto animal como humano, se tornando uma preocupação para a saúde pública. As toxinas das cianobactérias são um grupo de toxinas naturais do ponto de vista químico e toxicológico. Podem ser neurotoxinas, hepatotoxinas, citotoxinas, toxinas irritantes e gastrintestinais. Microcystis aeruginosa é a espécie mais comum em florações de água doce de cianobactérias nocivas. Este trabalho teve como objetivo a avaliação da toxicidade e da genotoxicidade do extrato de uma floração de Microcystis spp contendo microcistina em peixes. Utilizaram-se duas espécies de peixes, sendo uma exótica (Oreochromis niloticus) e outra nativa do Brasil (Astyanax bimaculatus). Para a determinação da toxicidade aguda, utilizouse o método Trimed Spearman-Karber, e para a genotoxicidade aplicaram-se os testes de micronúcleo, do cometa e necrose versus apoptose com eritrócitos de sangue periférico coletados dos peixes. Estes foram expostos por duas vias (exposição corpórea e intraperitoneal) em diferentes concentrações. Verificou-se que em O. niloticus a CL50 (72 h) foi maior que 103,725 μg/L e a DL50 (72h) maior que 138 μg/100g de peso corpóreo. E para A. bimaculatus a CL50 (72h) calculada foi de 242,81 com intervalo de confiança de 152,74 a 386,00 μg/L e a DL50 (72h) igual a 491,9 com intervalo de confiança de 385,8 a 627,3 μg/100g de peso corpóreo. A maior média de micronúcleos observada em O. niloticus foi através de exposição corpórea à concentração de 103,725μg/L. Em A. bimaculatus a maior média foi na concentração 368,8μg/100g de peso expostos via i.p., seguida pela média da concentração 103,725μg/L e pela média da concentração 245,86μg/100g de peso via i.p. No teste do cometa os resultados demonstraram, tanto para O. niloticus como para A. bimaculatus, que em ambas as vias de exposição e concentrações mais elevadas, observaram-se valores significativos. No teste de necrose versus apoptose, observou-se, em O. niloticus, um maior índice de células em necrose com a maior dose e um maior índice de apoptoses na dose mais baixa. Em A. bimaculatus observaram-se células em necrose e em apoptose na dose mais alta e células em apoptoses com a dose mais baixa. O extrato da floração apresentou ação tóxica apenas para A. bimaculatus e ação genotóxica nas duas espécies de peixes. Em concentrações maiores, o extrato provocou necrose e em doses baixas provocou apoptose. _______________________________________________________________________________ ABSTRACT The concern with the effects of cyanobateria in the humans health raise in many countries. Cases of cyanobacteria bloom in reservoir and lakes turn out to be a problem for the water supply besides poisoning animals as human being becoming a source of issue for the public health. The cyanotoxins are a group of natural toxins form the toxicological and chemist point of view. They can be neurotoxin, hepatotoxin, citotoxin, irritating and gastrointestinal toxin. Microcystis aeruginosa is the most ordinary specie in fresh water bloom of harmful cyanobacteria. The objective of this project was the valuation of toxicity and the genotoxicity in fishes of the extract of a bloom of Microcystis spp containing microcystin. Two species were used, one of them is exotic (Oreochromis niloticus) and the other is native from Brazil (Astyanax bimaculatus). To determine the toxicity the method Trimed Spearman- Karber was used, and for the genotoxicity the micronucleus test, the comet assay and necrosis versus apoptosis test were applied using erythrocytes of peripheral blood from fishes. These were exposed by two ways (body exposure and via intraperitoneal (i.p.)) in different concentrations. One has checked that in O. niloticus. the LC50 (72 h) surpassed 103,725 μg/L and the LD50 (72h) surpassed 138 μg/100g of body weight. For A. bimaculatus the LC50 (72h) calculated was the 242,81 with confidence interval of 152,74 at 386,00 μg/L and the LD50 (72h) equal to 491,9 with confidence interval of 385,8 at 627,3 μg/100g of body weight. The largest average of the micronucleus observed in O. niloticus was given by the body exposition to the concentration of 103,725μg/L. In A. bimaculatus the largest average was in the concentration 368,8μg/100g of exposed weights via i.p., followed by the concentrated average 103,725μg/L and by the concentrated average 245,86μg/100g of weight via i.p. In the comet assay, for both O. niloticus and A. bimaculatus, the results show that in both ways of exposition and the highest concentrations, significant values were observed. In the necrosis versus apoptosis test one has observed a larger incidence of cells in necrosis with the highest dose and a higher incidence of apoptosis in the lowest dose in O. niloticus. And one has observed cells in necrosis and in apoptosis in the highest dose and cells in apoptosis with the lowest dose in A. bimaculatus. The extract of bloom has shown toxic action only for the A. bimaculatus. In higher concentrations the extract causing necrosis and in low doses the extract caused apoptosis.
... The comet assay has been previously employed on several aquatic organisms with varying degrees of success and was found to be suitable as a non-specific biomarker for genotoxicity in fish and in a few aquatic invertebrates (Pandrangi et al., 1995;Deventer, 1996;Steinert, 1996;Clements et al., 1997;Devaux et al., 1997;Mitchelmore and Chipman, 1998;Mitchelmore et al., 1998a,b;Monod at el., 1998;Steinert et al., 1998;Wilson et al., 1998;Lee et al., 2000). ...
The comet assay, one of the most widely used techniques for the evaluation and detection of DNA strand breaks, is frequently employed in vivo. In vitro assays are usually performed with mammalian cell lines, clearly not the best choice for tests on aquatic genotoxicity. Here we evaluated a fish hepatoma cell line (RTH-149) and a primary blood cell culture from the intertidal colonial tunicate Botryllus schlosseri as possible model targets for comet assays using the genotoxic agent H2O2. We found that DNA strand break levels in RTH-149 fitted dose-dependent responses better than the tunicate cells. Moreover, in B. schlosseri controls, 34% of the cells were already ranked as severely damaged. Assays were then performed on water samples from the polluted Kishon river (Israel) on three different dates, using RTH-149 cells (50% dilutions, 2-h exposures). In all cases, high genotoxicity of the river water was revealed by evaluating comet percentages, average tail lengths and DNA damage levels. This assay was found to be fast and sensitive, appropriate to be employed as a part of a monitoring program. The use of B. schlosseri blood cells should be validated in additional work.
... For the other tissues sampled the genotoxic response appeared to stabilize after 7 days. This finding is in contrast with those of Belpaeme et al. [1998] and Deventer [1996], who found clear time-related genotoxic effects from exposure to EMS and methyl methanesulfonate, respectively. In this study it is most likely that DNA excision-repair processes were taking place after the initial exposure period preventing further DNA damage/instability from accumulating in the exposed tissues. ...
Sediments frequently cause damage to biota due to the accumulation of toxic compounds and the bioavailability of sediment-bound contaminants. Damage can be assessed using biomarkers, such as the degree of genotoxic impact following in vivo exposure to pollutants. Genotoxic damage, expressed as single-strand DNA breaks, was measured in cells isolated from haemolymph, gill and digestive gland from the clam Tapes semidecussatus, using the single cell gel electrophoresis (Comet assay). Clams were exposed for three weeks to sediment samples collected from a polluted site and a 'clean' reference site. The level of DNA damage was assessed using an image analysis package and expressed as Tail Moment. Throughout the study, significant differences in DNA damage were recorded for each tissue type between clams exposed to the two sediment samples. We conclude that the Comet assay is a useful tool for the detection of DNA damage in clams chronically exposed to polluted sediments.
... The results of the comet assay after various aquatic vertebrate and invertebrates animals were exposed to a variety of genotoxicants are presented in Table 1 [17,20,21,23,35,37,38,55,[67][68][69][70][71][72][73][74][75][76]. For most studies, genotoxic compounds, dissolved in solvent or water, were added to water in aquaria containing the test animal. ...
The comet assay is a rapid, sensitive and inexpensive method for measuring DNA strand breaks. The comet assay has advantages over other DNA damage methods, such as sister chromatid exchange, alkali elution and micronucleus assay, because of its high sensitivity and that DNA strand breaks are determined in individual cells. This review describes a number of studies that used the comet assay to determine DNA strand breaks in aquatic animals exposed to genotoxicants both in vitro and in vivo, including assessment of DNA damage in aquatic animals collected from contaminated sites. One difficulty of using the comet assay in environmental work is that of comparing results from studies that used different methods, such as empirical scoring or comet tail lengths. There seems to be a consensus in more recent studies to use both the intensity of the tail and the length of the tail, i.e. DNA tail moment, percentage of DNA in the tail. The comet assay has been used to assess DNA repair and apoptosis in aquatic animals and modifications of the comet assay have allowed the detection of specific DNA lesions. There have been some recent studies to link DNA strand breaks in aquatic animals to effects on the immune system, reproduction, growth, and population dynamics. Further work is required before the comet assay can be used as a standard bio-indicator in aquatic environments, including standardization of methods (such as ASTM method E2186-02a) and measurements.
... For the other tissues sampled the genotoxic response appeared to stabilize after 7 days. This finding is in contrast with those of Belpaeme et al. [1998] and Deventer [1996], who found clear time-related genotoxic effects from exposure to EMS and methyl methanesulfonate, respectively. In this study it is most likely that DNA excision-repair processes were taking place after the initial exposure period preventing further DNA damage/instability from accumulating in the exposed tissues. ...
The alkaline single cell gel electrophoresis (SCGE) or Comet assay was employed to test the potential of surficial sediment collected from Cork Harbor, Ireland, to induce DNA damage in turbot (Scophthalmus maximus L.) in a laboratory exposure experiment. Turbot were exposed for 21 days to field-collected sediment from Cork Harbor and from a relatively clean reference site at Ballymacoda and sampled at 0, 7, 14, and 21 days. As a positive control for the sediment exposure experiment, a subsample of the turbot was exposed to cadmium chloride-spiked seawater. DNA damage analysis was performed on epidermal, gill, spleen, liver, and whole blood cell preparations. Liver, gill, and blood were the most sensitive tissues while a lower level of damage was detected in the epidermis and spleen. The blood was determined to be a suitable predictor of DNA damage in the whole organism. Chemical analysis of the sediment indicated that polycyclic aromatic hydrocarbons formed the bulk of the contaminants, with the harbor sites having almost double the levels of those from the reference site. The data indicated that turbot exposed to sediments from Cork Harbor elicited a significant increase in DNA damage in comparison with those exposed to sediment from the reference site and that exposure to the contaminated sediments caused a multi-organ genotoxic response. Results from the study indicate a relationship between the presence of genotoxicants in sediment and DNA damage. This finding was encouraging with regard to the potential use of the Comet assay as part of a marine biomonitoring strategy.
INTRODUCTION
Rifampicin conjugated (R-CP), and rifampicin -isoniazid dual conjugated (RI-CP) norbornene-derived nanocarriers are newly designed for pH stimuli-responsive delivery of tuberculosis (TB) drugs. Its biosafety level is yet to be well established.
OBJECTIVES
To assess the impacts of the nanocarriers on liver cells using zebrafish animal model and human liver cell line model (HepG2).
METHODS
Initially, lethal dose concentration for the norbornene-derived nanocarrier systems in zebrafish was determined. The toxic effects were analysed at the sub-lethal drug concentration by histopathological study, total GSH level, gene expression and DNA damage in zebrafish liver cells. Fish erythrocyte nuclear abnormalities were also evaluated. Cell viability and oxidative stress level (ROS generation) after exposure to the nanoconjugates was determined using HepG2 cell in the in vitro study.
RESULTS
In vivo studies of both R-CP and RI-CP showed 100% mortality at 96 hours for exposure concentration >100mg/l and showed toxic changes in zebrafish liver histology, GSH, and DNA damage levels. A noticeable upregulated PXR, CYP3A and cyp2p6 genes was observed in RI-CP exposure than in RIF or R-CP molecules. The in vitro study revealed a dose-dependent effect on cell viability and ROS generation for RIF, R-CP and RI-CP exposures in HepG2 cells.
CONCLUSION
The current study reports that the rifampicin conjugated (R-CP) and rifampicin-isoniazid conjugated (RI-CP) norbornene derived nanocarriers exhibit enhanced toxic responses in both adult zebrafish and HepG2 cells. The pH-sensitive norbornene derived nanocarriers on conjugation with different drugs exhibited varied impacts on hepatic cells. Hence the present investigation recommends a complete metabolomics analysis and norbornene carrier-drug interaction study to be performed for each drug conjugated norbornene nanocarrier to ensure its biosafety.
Abamectin (ABM) has been extensively used in Chinese aquaculture systems for parasite control, but no information is available regarding its effects on the important freshwater commercial fish species Schizothorax prenanti. We performed an acute toxicity test to determine the effects of ABM on S. prenanti, and the 48- and 96-h median lethal concentration values were 33.32 and 15.98 μg/L, respectively. In a second test, animals were exposed to sublethal concentrations of ABM (0.5, 2 or 8 μg/L) for 8 days, and various cytological and biochemical parameters were measured. ABM caused DNA damage in hepatocytes, with significant increases in Olive Tail Moment values and 8-hydroxy-2'-deoxyguanosine levels. Hepatocytic apoptosis occurred following all treatments, and was accompanied by an increase in reactive oxygen species (ROS) generation and caspase activity in a dose- and time-dependent manner. In addition, there were significant decreases in glutathione peroxidase levels and superoxide dismutase and catalase activity and increases in malonaldehyde levels. ABM-induced hepatocytic apoptosis in S. prenanti was probably triggered by ROS generation following a cascade reaction of caspases in mitochondrial or death receptor pathways, which caused antioxidant inhibition, oxidative product accumulation, and DNA damage in the liver.
In the present study, an acute toxic test was performed to assess the oxidative stress and genotoxic effects of the herbicide on the freshwater shrimp Macrobrachium nipponensis. The results showed that the 48-h and 96-h LC50 values of Roundup to M. nipponensis were 57.684 mg/L and 11.237 mg/L, respectively. For further investigation, the shrimps were exposed to sublethal concentrations of 0.35, 0.70, 1.40, 2.80 and 5.60 mg/L for 96 h. A significant decrease in total haemocytes count (THC) was observed at concentration of 5.60 mg/L throughout the experiment. The level of superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (T-AOC) in all the treatments decreased in a dose- and time-dependent manner except for the concentration group of 0.35 mg/L. The malondialdehyde (MDA), hydrogen peroxide (H2O2) and protein carbonyl in serum increased significantly at concentrations of 2.80 mg/L and 5.60 mg/L. A significant decrease in acetylcholinesterase (AChE) activity was observed at each concentration (P＜0.05). In addition, the micronucleus (MN) frequency of haemocytes significantly increased (P＜0.05) at concentrations of 1.40, 2.80 and 5.60 mg/L, whereas the comet ratio and %DNA in the tails exhibited a clear time- and dose-dependent response during the exposure. The analysis of the integrated biomarker response (IBR) showed the induction of oxidative stress biomarkers and the inhibition of antioxidants, and this dose-dependent relation suggests the sensitivity and availability of all the biomarkers. These results revealed that Roundup had a prominent toxic effect on M. nipponensis based on the antioxidative response inhibition and genotoxicity.
In this study, comet assay (single-cell gel electrophoresis), real-time quantitative PCR (qPCR) and proteomics approach were used to comprehensively assess toxicity elicited by roxarsone exposure in C. auratus at 50, 150 and 300 μg/L for 7, 14 and 21 days. Results of comet assay showed that DNA were seriously damaged under the pressure of roxarsone, especially the concentration of 50 μg/L that always maintained a sustained and increased damage effect to fish liver cell during the 21 days experiment. The expressions of biomarker genes showed that hsp70 gene expressions raised significantly and the group of 50 μg/L also showed a continued increased response effect, whereas mt gene was only slightly increased. Results of proteomics for the concentration of 300 μg/L found that thirty six significantly changed proteins were identified by MALDI-TOF/TOF-MS. They are involved in many important processes including energy producing, cytoskeleton stabilization, substance metabolism and stress response. Among these metabolites, carbohydrate metabolism (mainly occurred during day 1-14) and cytoskeleton proteins (mainly occurred during day 14-21) were the most identified proteins. These results revealed that the low levels of 50 μg/L probably led to a continuous damage than the higher groups during the experiment time. Furthermore, proteomics results might implied that though cell system expected to mobilize almost all the functional proteins to quickly establish a new homeostasis together when facing the roxarsone at first, but in the end the destroyed cell cytoskeleton structure might burst the bubble.
The oxidative stress and genotoxic effect of deltamethrin on the Chinese mitten crab Eriocheir sinensis were assessed using several commonly used biomarkers in this study. The results showed that the 48 h and 96 h LC50 values of deltamethrin to E. sinensis were 2.319 and 1.164 μg/L, respectively, and the safe concentration was 0.293 μg/L. According to these results, deltamethrin was applied at concentrations of 1/16, 1/8, 1/4, 1/2 and 1/1 96 h LC50 for 8 d in an exposure experiment. The activity of superoxide dismutase (SOD) increased remarkably at 1 d, but decreased at 4 d in concentration group of 1/4, 1/2 and 1/1 LC50, whereas catalase (CAT) activity decreased during the exposure. The total antioxidant capacity (T-AOC) at the concentrations of 1/4, 1/8 and 1/16 LC50 increased significantly at 1 d or 2 d respectively, whereas it decreased gradually until the end of the experiment under the concentrations above 1/4 LC50. The oxidative stress products malondialdehyde (MDA) and hydrogen peroxide (H2O2) in serum increased significantly at each concentration except H2O2 at concentration of 1/16 LC50. Additionally, the micronucleus (MN) frequency of haemocytes increased at the concentrations of 1/4, 1/2 and 1/1 LC50 throughout the exposure, similar trend was observed in the comet ratio and percentage of tail-DNA (%DNA) in haemocytes. These results revealed that deltamethrin had a prominent toxic effect on E. sinensis based on antioxidative response inhibition and genotoxicity that was possibly due to lipid peroxidation (LPO) induced by oxidative products, and the accumulation of peroxide, even under a sublethal concentration.
As a broad-spectrum organophosphorus herbicide, glyphosate is widely utilized around the world. The toxic effects of glyphosate on Chinese mitten crab, Eriocheir sinensis, were assessed using immunotoxicity and genotoxicity biomarkers in this study. The results showed that 24 h and 96 h LC50 values of glyphosate for E. sinensis were estimated as 461.54 and 97.89 mg/L, respectively, and the safe concentration was 4.4 mg/L. According to the results above, glyphosate was applied at concentrations of 0, 4.4, 9.8, 44 and 98 mg/L, for 96 h in the exposure experiment. Total haemocyte count (THC) and percentage of granulocytes decreased significantly following 6 h exposure to each concentration of glyphosate and tended to gradually stabilize after 12 h except in 4.4 mg/L, which rapidly recovered to a normal level in 12 h. Phagocytic activity in all treatments decreased dramatically at 6 h and maintained stability until the 96-h mark. Comet tail has been observed early at 24 h in each treatment, and the comet ratio and percentage of DNA (% DNA) in the tail increased as the exposure experiment progressed. Immune-related enzyme activity varied during the experiment. Acid phosphatase (ACP) and alkaline phosphatase (AKP) activities in 44 and 98 mg/L treatments decreased significantly after 48 h exposure, while AKP activities in all concentrations increased markedly at the beginning of exposure. The superoxide dismutase (SOD) and peroxidase (POD) activities increased significantly after 6 h exposure to 44 and 98 mg/L of glyphosates but decreased at 24 h. In addition, the β-glucuronidase (β-GD) activities in the 9.8, 44 and 98 mg/L groups, increased after 6-h exposure and were significantly higher than those in the control at 96 h. These results indicated that glyphosate has evident toxic effect on E. sinensis by immune inhibition that is possibly due to the haemocyte DNA damage and a sharp decline in haemocyte numbers, which subsequently induced changes in activities of immune-related enzymes and haemocyte phagocytosis.
Zahlreiche Umweltchemikalien, die aus den unterschiedlichsten Quellen in die Gewässer gelangen, werden der Wasserphase zunächst dadurch entzogen, daβ sie sich den Schwebstoffen und Sedimenten anlagern (z.B. Schwermetallverbindungen, Pestizide, halogenierte Kohlenwasserstoffe, polyclische aromatische Kohlenwasserstoffe). Unter dem Aspekt der ursprünglich wirksamen Schadstoffkonzentration wirkt dieser Prozeβ für die Organismen in der Wassersäule entgiftend. Der Prozess ist teilweise reversibel und Sedimente fungieren als Quellen für Schadstoffe, die auf aquatische Organismen wirken. Darüberhinaus werden Benthosorganismen ständig diesen sedimentgebundenen Chemikalien ausgesetzt. Sedimenttoxizität ist im weitesten Sinn definiert als ökologische und biologische Änderung, die durch kontaminiertes Sediment verursacht wird. Sie kann aber auch pragmatisch bestimmt werden, als negative Wirkung an einem Testorganismus, der einem belasteten Sediment ausgesetzt wurde. Bei der Differenzierung von toxischen Effekten sedimentassoziierter Schadstoffe müssen jedoch sowohl die Sensitivitäten von Testorganismen als auch die möglichen Aufnahmewege der Chemikalien berücksichtigt werden. Dieses Kapitel beschäftigt sich mit der Diskussion moglicher Prozesse, durch die sedimentgebundene Chemikalien biologisch verfügbar werden und nachteilige Wirkungen erzeugen können.
After the original description by Berry and Friend in 1969 of the high yield isolation of rat liver parenchymal cells by collagenase perfusion [1], the use of isolated hepatocytes was soon established as an ideal system for the study of many aspects of hepatic metabolism [2]. In 1976, Birnbaum and colleagues [3] adapted the technique of hepatocyte isolation for studies of hormone-stimulated glycogenolysis in goldfish cells. Since then, isolated fish hepatocytes have been used intensively in fresh suspensions and in primary culture as a powerful, yet simple tool for the investigation of piscine hepatic functions. Cultured hepatocytes have proved to be well suited for applications not only in basic fish physiology, but also in the fields of pharmacology and environmental toxicology [4–7].
A recent biochemical technique, the alkaline single‐cell microgel‐electrophoresis ("comet") assay, allows the evaluation of damage occurred at the DNA level in individual cells associated with exposure to genotoxic compounds. The extent of DNA damage may be directly evaluated by the use of simple visual techniques. However, the development of computerized image analysis systems specifically devoted to this assay has made possible an easier and more detailed measure. In a biological monitoring approach aimed to assess the genotoxic hazard following rubber processing, peripheral blood leukocytes of 19 male workers from the rubber industry and of age‐matched controls living in the same area were analyzed for the presence of DNA damage using the “comet”; assay. In this paper we report results obtained after a re‐analysis by a computerized system of photomicrographs previously used for the direct manual measurement of comet length. Considering tail parameters such as tail intensity and tail moment, a statistically significant higher extent of DNA damage was demonstrated in exposed subjects, as compared to controls. On the contrary, visual analysis gave negative results.
This study has determined the sensitivity of the alkaline comet assay for the detection of strand breaks in the DNA of cells taken from a whole organism rather than a single cell type as in previously reported studies. The assay has been performed on cells from whole zebrafish larvae irradiated for 1 or 24 h at dose rates of 0.4, 1.2 or 7.2 mGy/h. Zebrafish larvae exposed to only 1.2 mGy/h of gamma-radiation for 1h showed a statistically significant increase in DNA damage compared to controls. This represents a high sensitivity of this animal model for DNA damage and of the comet assay protocol used for detecting such damage. Increasing the exposure time from 1 to 24 h caused significant increases in DNA damage in zebrafish larvae, although the modest size of these increases in damage for the relatively large increases (24 times) in total absorbed dose indicates that dose rate may be the major factor in determining the level of DNA damage observed under the conditions of these experiments.
In this paper, we examine a trader's order choice between market and limit orders using a sample of orders submitted through NYSE SuperDot. We find that traders place more limit orders relative to market orders when: (1) the spread is large, (2) the order size is large, and (3) they expect high transitory price volatility. A rise in informational volatility appears neither to increase nor decrease the placement of limit orders. We also find that a rise in lagged price volatility decreases the size of spread, which is driven by the increase in the placement of limit orders.
Existing methods for the comparison of genotoxic effects in the comet assay bear considerable disadvantages such as the problem to link information about concentration dependence and severity of effects. Moreover, given the lack of standardized protocols and the use of various standards, it may be extremely difficult to compare different studies. In order to provide a method for standardized comparative assessment of genotoxic effects, the concept of genotoxicity equivalents (Gene-TEQ) was developed. As potential reference compounds for genotoxic effects, three directly acting (N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl-methanesulfonate, and N-methyl-N-nitrosourea) and three indirectly acting (cyclophosphamide, dimethylnitrosamine, and 4-nitroquinoline-oxide) genotoxic substances were compared with respect to their cytotoxic (neutral red) and genotoxic (comet assay) concentration-response profiles in the permanent fish cell line RTL-W1. For further comparison, two sediment extracts from the upper Danube River were investigated as environmental samples. Based on the results of cytotoxicity and genotoxicity testing, MNNG was selected as the reference compound. At several exposure levels and durations, genotoxic effects of both the other pure substances and the environmental samples were calculated as percentages of the maximum MNNG effect and related to the absolute MNNG effect (EC values). Thus, genotoxicity equivalent factors (Gene-TEQs) relative to MNNG could be calculated. Gene-TEQs can easily be applied to pure substances, mixtures and field samples to provide information about their toxicity relative to the reference compound. Furthermore, the Gene-TEQ concept allows a direct comparison of environmental samples from different laboratories.
Orientadora : Marta Margarete Cestari Co-orientador : Marcos Vinicius M. Ferraro Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Genética. Defesa: Curitiba, 2007 Inclui bibliografia
Tetraploid yeast cells (Saccharomyces cerevisiae) were used in the comet assay with the intention of developing a new, fast and easy assay for detecting environmental genotoxic agents without using higher organisms. Two DNA-damaging chemicals, H(2)O(2) and acrylamide, together with wastewater from three municipal treatment plants were tested for their effect on the yeast-cell DNA. The main problem with using yeast in the comet assay is the necessity to degrade the cell wall. This was achieved by using Zymolase 100 T twice during the procedure, since Zymolase 20 T did not open the cell wall. Analytical problems that arose due to the small amount of DNA in the yeast nuclei in haploid and diploid cells, which contain 13 Mbp and 26 Mbp DNA per cell, respectively, were solved by using tetraploid yeast cells (52 Mbp) instead. DNA damage was shown after exposure to H(2)O(2) and acrylamide. The lowest dose causing significant DNA damage was 20 microM for H(2)O(2) and 200mg/l for acrylamide. Tertiary-treated wastewater from the outlets of three municipal wastewater-treatment plants was tested, but did not cause DNA damage. Even though it is possible to produce comets with tetraploid yeast cells, the amount of DNA is likely too small for a proper comet assay.
Although it is well known that micronuclei may arise from either DNA breakage leading to acentric chromosome fragments or from chromosome/chromatid lagging in anaphase, the ratio between the amount of DNA breakage induced and the frequency of micronuclei expressed in the following interphase is unclear. With the development of the alkaline single cell gel electrophoresis assay, which measures single strand and/or double strand breaks in a cell by cell approach, it is new possible to address this question at the cellular level. We therefore compared the genotoxic potential of pure cobalt powder (Co) and a cobalt-containing alloy, cobalt-tungsten carbide (WC-Co), involved in specific lung disorders, in parallel with the alkaline single cell gel electrophoresis (SCGE) assay (comet assay) and the cytokinesis-blocked micronucleus (MN) test, both carried out in vitro on isolated human leukocytes. The comet assay indicated that the WC-Co mixture produced a higher level of DNA damage than Co alone; WC alone was not able to induce a dose-dependent DNA breakage effect as was seen for Co and WC-Co. Results from the MN test confirmed these observations. It was clear that the clastogenic property of Co-containing dust is significantly enhanced when the Co metal is mixed with WC and suggested that their physicochemical characteristics may act as one of the important parameters responsible for the increased incidence of lung cancers observed in the population of hard metal workers. In agreement with data obtained in the same laboratory on liposoluble chemicals (PCBs and chlorinated aliphatic hydrocarbons) and from the literature, the results indicate that both the comet assay and the micronucleus test were able to detect differences in the genotoxic potential of the compounds studied. Although the micronucleus test seemed to be less sensitive to assess a synergistic DNA damaging potential of the mixture involved, it detects chromosomal aberrations (chromosome/genome mutations) and not just repairable DNA breakage or alkali-labile sites. Combination of the comet assay and the in vitro MN test might therefore be recommended for genotoxins to understand the mechanisms underlying mutagenicity and to assess the lowest efficient dose.
The assessment of genotoxic potential in surface water requires test methods, among which are those that detect initial DNA damage in organisms of aquatic biocenosis. The microgel electrophoresis (MGE) "comet assay" was applied to a ubiquitous unicellular green alga (Chlamydomonas reinhardtii) to detect DNA damage caused by genotoxins. For this, the test protocol described by Singh NP et al. [Exp Cell Res 175: 184-191, 1988] was modified. Major modifications were the use of alkaline lysis buffer with ionic detergents and the reduction of preincubation and electrophoresis times. Short-time exposure of Chlamydomonas to the well-known genotoxicants 4-nitroquinoline-1-oxide (4-NQO), N-nitrosodimethylamine, and hydrogen peroxide led to dose-dependent DNA damage. Chlamydomonas responded very sensitively to treatment with increasing doses of 4-NQO. At a concentration of 25 nM, significant DNA damage was observed. At higher 4-NQO doses (> 100 nM), DNA damage was visible as complete DNA fragmentation into fine granules. N-Nitrosodimethylamine caused genotoxic effects at a concentration range from 0.014 to 0.14 mM without producing complete DNA fragmentation at the concentrations tested (highest dose, 140 mM). To evaluate the influence of illumination conditions during exposure, cells were incubated with increasing doses of H2O2 (0.25-1.0 mM) in darkness and in light. Our results indicate that incubation in light enables Chlamydomonas to cope with oxidative stress more efficiently than under dark conditions. To a certain extent, cytotoxic as well as genotoxic effects of H2O2 depend on the illumination condition or repair and anti-oxidative protection mechanisms activated by light, respectively.
Biomonitoring is an important subject within environmental sciences. Biomonitoring tests are required to be quick, relatively inexpensive, accurate, and reproducible. No genetic test currently fulfils all of these requirements. The chromosome aberration and sister chromatid exchange tests are very time consuming, the DNA adduct technique is rather expensive, and the micronucleus test has not inconclusively proven its use as a reliable monitoring tool. This work is focused on the validation of the comet assay as a candidate for monitoring marine ecosystems. For the comet assay, this work deals with the effectiveness of tissue dissociation, storage of cells in lysing buffer and in liquid nitrogen, different electrophoretic conditions, neutralisation and fixation of slides, interindividual variation between samples, and responsiveness of four tissue types to ethyl methanesulphonate (EMS). The main conclusions are: (i) dissociation of solid tissues in a phosphate buffer supplemented with 200 mM N-t-butyl-alpha-phenylnitrone provides cells with an acceptable background DNA damage; (ii) freezing of cells or tissues in liquid nitrogen generally leads to an increase in DNA breakage, especially for liver, gill and kidney tissue; (iii) storage of slides in the lysing solution for up to one week gives minor changes in comet tails; (iv) differences in protocols for neutralisation and fixation may influence the results; (v) high intra- and interindividual variations in comets (length and DNA content) may obscure the interpretation of comet results; (vi) blood, gill, liver and kidney all showed a statistically significant increase of DNA damage after exposure to 50 mg EMS/l; (vii) electrophoresis at low voltage for longer periods is to be preferred to high voltage and short electrophoresis times. The simplicity and sensitivity of the comet assay make it an adequate test system for biomonitoring of chronic low level exposure. However, protocols and experimental conditions have to be chosen carefully.
In aquatic toxicology, cytotoxicity tests using continuous fish cell lines have been suggested as a tool for (1) screening or toxicity ranking of anthropogenic chemicals, compound mixtures and environmental samples, (2) establishment of structure-activity relationships, and (3) replacement or supplementation of in vivo animal tests. Due to the small sample volumes necessary for cytotoxicity tests, they appear to be particularly suited for use in chemical fractionation studies. The present contribution reviews the existing literature on cytotoxicity studies with fish cells and considers the influence of cell line and cytotoxicity endpoint selection on the test results. Furthermore, in vitro/in vivo correlations between fish cell lines and intact fish are discussed.
During recent years, fish cell lines have been increasingly used for purposes beyond their meanwhile established role for cytotoxicity measurements. They have been successfully introduced for detection of genotoxic effects, and cell lines are now applied for investigations on toxic mechanisms and on biomarkers such as cytochrome P4501A. The development of recombinant fish cell lines may further support their role as a bioanalytical tool in environmental diagnostics.
The zebrafish (Danio rerio) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. The zebrafish genome will be completely sequenced within the next 1-2 years. Together with the substantial historical database regarding basic developmental biology, toxicology, and gene transfer, the rich foundation of molecular genetic and genomic data makes zebrafish a powerful model system for clarifying mechanisms in toxicity. In contrast to the highly advanced knowledge base on molecular developmental genetics in zebrafish, our database regarding infectious and noninfectious diseases and pathologic lesions in zebrafish lags far behind the information available on most other domestic mammalian and avian species, particularly rodents. Currently, minimal data are available regarding spontaneous neoplasm rates or spontaneous aging lesions in any of the commonly used wild-type or mutant lines of zebrafish. Therefore, to fully utilize the potential of zebrafish as an animal model for understanding human development, disease, and toxicology we must greatly advance our knowledge on zebrafish diseases and pathology.
The aim of the present paper was to assess the biological damage caused by exposure of the test organism (Gambusia holbrooki: Cyprinodontiformes, Poecilidae) to various mutagenic agents present in the polluted waters of the Sarno River. For this purpose, we performed a micronuclei (MN) test and single cell gel electrophoresis (the Comet assay), testing DNA migration in an electrophoretic field using erythrocytes of G. holbrooki specimens both from the Sarno River and from the waters of the crater of the Astroni natural reserve as negative controls. The results indicate statistically higher values for both MN and DNA migration in the samples from the Sarno River compared with those from Astroni and point to a strong genotoxic action of the mixture of pollutants present in the Sarno River. These data were compared with the values found in the G. holbrooki specimens from the Sarno River kept under laboratory conditions for 100 days in clean water.
Genotoxicity assays were developed in embryonic stages of two species of fish, striped bass (Morone saxitilis) and sheepshead minnow (Cyprinodon variegatus), and in the adults of Fundulus heteroclitus using metaphase chromosome aberrations as the endpoint. Dose-dependent responses were obtained with several chemical mutagens, including 9-aminoacridine, ethylmethane sulphonate, cyclophosphamide and n-methyl-n-nitro-n-nitroguanidine, added to estuarine water at doses spanning several orders of magnitude in compound concentration. Exposures ranged from one to four days; however, two-day exposure time was found to be optimal in eggs and larvae. Tissues from the gills, kidney and intestinal tract of adult Fundulus were found to be responsive to mutagen exposure; however, the intestine gave the best responses. The results of these experiments suggest that these assays are sufficiently sensitive to be used in the field as well as in laboratory tests.
Organic concentrates were recovered using XAD-2/8 resin adsorption from the leachates of municipal solid waste landfills and their mutagenic activities were tested for 8 months using the Ames Salmonella/microsome assay. Highly polluted leachates (COD and BOD > or = 40 mg/l) generally had equal or higher mutagenic activities than lightly polluted leachates (COD and BOD < 40 mg/l). But there was no clear difference in mutagenicity per amount of concentrate between the two leachates. These results suggest that the mutagenic activity of landfill leachate is decided to some degree by the organic concentration in the leachate. The mutagenic activities detected even in lightly polluted leachates were not so low as those of various kind of surface waters ever reported. It is suggested that it is important to investigate the mutagenic activity of the leachate for evaluation of the impact of landfill leachate on the environment.