DNA Damage In Haemocytes Research Articles

In vivo exposure of the marine clam Ruditapes philippinarum to zinc oxide nanoparticles: responses in gills, digestive gland and haemolymph.

Potential nanoparticle (NP) toxicity poses a growing concern in marine coastal environments. Among NPs, zinc oxide nanoparticles (nZnO) are widely used in many common products that ultimately become deposited in coastal habitats from multiple non-point sources. In this study, we evaluated the in vivo effects of nZnO in the clam Ruditapes philippinarum. Animals were exposed to nZnO (1 and 10μg/L) and ZnCl2 (10μg/L) for 7days. ZnCl2 was used to compare the effects of the NPs to those of Zn(2+) and to ascertain whether nZnO toxicity is attributable to the release of ions into the aquatic medium. At differing time intervals during the exposure, several biochemical and cellular responses were evaluated in the clam gills, digestive gland, and haemolymph. The results showed that nZnO, at concentrations close to the predicted environmental levels, significantly affected various parameters in clam tissues. Significant increases in catalase and superoxide dismutase activities and a decreasing trend of glutathione S-transferase activity indicated the involvement of oxidative stress in nZnO toxicity. In clams exposed to ZnCl2, slight variations in antioxidant enzyme activities were detected with respect to nZnO-treated clams. However, no damage to lipids, proteins or DNA was revealed in all exposure conditions, suggesting a protection of antioxidant enzymes in the tissues. Of the various haemolymph parameters measured, haemocyte proliferation increased significantly, in ZnCl2-treated clams in particular. Under nZnO (10μg/L) and ZnCl2 exposure, DNA damage in haemocytes was also revealed, but it was lower in clams exposed to ZnCl2. A decreasing trend in gill AChE activity of treated clams proposed a possible role of zinc ions in nZnO toxicity. However, the dissimilar modulation of the responses in the nZnO- and ZnCl2-exposed clams suggested different mechanisms of action, with nZnO toxicity possibly depending not only on the release of zinc ions but also on NP-specific features. Changes in the biological parameters measured in the clams were consistent with Zn accumulation in their gills and digestive glands.

Biomarkers in the freshwater bivalve Corbicula fluminea confined downstream a domestic landfill leachate discharge.

Landfills represent a severe environmental problem mainly due to the generation of leachates, and this study aimed to evaluate sublethal effects of a domestic landfill leachate in the freshwater bivalve Corbicula fluminea. Clams were submitted to in situ tests along a stream, at three sites, representing increasing distances from the leachate discharge (Pq1, Pq2, and Pq3), for 1, 5, and 15days. The following biomarkers were analyzed in the gills and digestive glands: 7-ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST) activities, multixenobiotic resistance mechanism (MXR), total antioxidant capacity (TAC), reactive oxygen species (ROS), and lipid peroxidation (LPO). Metallothionein (MT) content was determined in the gills and DNA damage in hemocytes. The mortality rate of animals during in situ tests was reduced as the distance from the leachate discharge source increased. On the other hand, biomarker results showed sublethal effects on C. fluminea confined at all sites of PqS. GST, TAC, ROS, and DNA damage were the most significant biomarkers for this species and should be considered for future monitoring and assessment of freshwater environments located in landfill areas.

Genotoxic effects of starvation and dimethoate in haemocytes and midgut gland cells of wolf spider Xerolycosa nemoralis (Lycosidae)

The aim of this study was to assess the genotoxic effects of starvation and dimethoate (organophosphate insecticide) in female and male wolf spiders Xerolycosa nemoralis (Lycosidae) exposed to the stressors under laboratory conditions. DNA damage was measured in haemocytes and midgut gland cells using the comet assay. In response to the two stressing factors, both cell types showed %TDNA, tail length (TL) and OTM values higher in males than in females. Level of DNA damage in haemocytes was greater than in midgut gland cells. In both sexes, the strongest genotoxicity was recorded at single application of dimethoate. After five-time exposure to the pesticide, genotoxic effects of a single dose were sustained in males and reduced to the control level in females. Starvation stress was well tolerated by the females, in which neither cell type was affected by DNA damage. However, in male haemocytes food deprivation induced severe DNA damage, what suggests suppression of the defence potential at prolonged starvation periods.

Genotoxic potential and heart rate disorders in the Mediterranean mussel Mytilus galloprovincialis exposed to Superdispersant-25 and dispersed diesel oil

The effects of ex situ exposure of Mytilus galloprovincialis to Superdispersant-25 (S-25), diesel oil and dispersed diesel oil mixtures were studied by the impact on level of DNA damage in haemocytes (comet assay) and the cardiac activity patterns of mussels. Specimens were exposed for 72 h in a static system to diesel oil (100 μL/L and 1 mL/L), S-25 (5 and 50 μL/L), and dispersed diesel oil mixtures M1 (diesel oil 100 μL/L + S-25 5 μL/L) and M2 (diesel oil 1 mL/L + S-25 50 μL/L). For positive control 40 μM CdCl2 was used. The comet assay results indicated genotoxic potential of S-25 while the effects of diesel oil alone were not observed. The highest response was detected for M1 while the effects of M2 were not detected. The heart rate disorders were recorded for the diesel oil (1 mL/L), S-25 (50 μL/L) and both dispersed diesel oil mixtures.

Development of a comet-FISH assay for the detection of DNA damage in hemocytes of Crassostrea gigas

In this work, the DNA-damaging effect of hydrogen peroxide on the structural integrity of nucleolar organizer regions (NORs) was studied for the first time by comet-FISH in the Pacific oyster Crassostrea gigas. Global DNA damage was assessed in hemocytes using an alkaline version of the comet assay. Next, NOR sensitivity was analyzed by mapping major rDNA repeat unit by fluorescence in situ hybridization (FISH) on the same comet slides. Exposure of hemocytes to 100μM of hydrogen peroxide induced a significant increase in both DNA damage and number of FISH-signals of major ribosomal genes versus the control. Moreover, a significant positive correlation was shown between DNA damage as measured by the comet assay (percentage of DNA in comet tail) and the number of signals present in comet tails. This study demonstrates the potential value of the comet-FISH assay for the study of DNA damage induced by genotoxicant exposure of target genes. It offers a perspective for better understanding the impact of genotoxicity on animal physiology and fitness.

Hemocytes of zebra mussels (Dreissena polymorpha) are relevant cells for the monitoring of environmental genotoxicity by the comet assay.

Event Abstract Back to Event Hemocytes of zebra mussels (Dreissena polymorpha) are relevant cells for the monitoring of environmental genotoxicity by the comet assay. Marc Bonnard1*, Gabrielle Magniez1, Alban Franco1, Laurence Delahaut1, Isabelle Bonnard1, Damien Rioult1, Sandrine Joachim2, Jean-Marc Porcher2 and Alain Geffard1 1 Université de Reims Champagne Ardenne , France 2 INERIS, France The measure of DNA integrity by the single cell gel electrophoresis (SCGE) or comet assay is especially recommended for its sensitivity and its capacity for detecting different types of damages. Therefore, it has been applied in environmental genotoxicity in a variety of organisms. It appears today necessary to define both reference and threshold levels of DNA damage, for their application in in situ biomonitoring. However, little is known about the influence of both biological (sex, reproduction status) or external (temperature…) confounding factors on the measure of DNA damage by the comet assay. These variables need to be taken into account if the robustness of the assay is to be established (Jha, 2008). In the zebra mussel Dreissena polymorpha (recommended as a sentinel species in the evaluation of freshwater quality) the measure of DNA damage by the comet assay is mainly performed on hemocytes, which are circulating cells involved in key physiological functions such as immunity, homeostasis, detoxication…. This communication will present and discuss results from an innovative study about the variability of the baseline level of DNA damage in hemocytes of mussels encaged for one year in the canal de l’Aisne à la Marne (Reims), according to their sex and their reproductive status. The sensitivity and the suitability of hemocytes in the evaluation of environmental genotoxicity will also be discussed, referring to observations during a 6 month-exposure of mussels in mesocosms to environmentally realistic concentrations of carbamazepine. References Jha, AN (2008). Ecotoxicological applications and significance of the comet assay. Mutagenesis, 23(3), 207-221. Keywords: Hemocytes, Zebra mussels, confounding factors, mesocosm, Carbamazepine Conference: ICAW 2015 - 11th International Comet Assay Workshop, Antwerpen, Belgium, 1 Sep - 4 Sep, 2015. Presentation Type: Oral Presentation Topic: Ecogenotoxicology Citation: Bonnard M, Magniez G, Franco A, Delahaut L, Bonnard I, Rioult D, Joachim S, Porcher J and Geffard A (2015). Hemocytes of zebra mussels (Dreissena polymorpha) are relevant cells for the monitoring of environmental genotoxicity by the comet assay.. Front. Genet. Conference Abstract: ICAW 2015 - 11th International Comet Assay Workshop. doi: 10.3389/conf.fgene.2015.01.00065 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 May 2015; Published Online: 23 Jun 2015. * Correspondence: Dr. Marc Bonnard, Université de Reims Champagne Ardenne, Reims, France, marc.bonnard@univ-reims.fr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Marc Bonnard Gabrielle Magniez Alban Franco Laurence Delahaut Isabelle Bonnard Damien Rioult Sandrine Joachim Jean-Marc Porcher Alain Geffard Google Marc Bonnard Gabrielle Magniez Alban Franco Laurence Delahaut Isabelle Bonnard Damien Rioult Sandrine Joachim Jean-Marc Porcher Alain Geffard Google Scholar Marc Bonnard Gabrielle Magniez Alban Franco Laurence Delahaut Isabelle Bonnard Damien Rioult Sandrine Joachim Jean-Marc Porcher Alain Geffard PubMed Marc Bonnard Gabrielle Magniez Alban Franco Laurence Delahaut Isabelle Bonnard Damien Rioult Sandrine Joachim Jean-Marc Porcher Alain Geffard Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

DNA damage in haemocytes and midgut gland cells of Steatoda grossa (Theridiidae) spiders exposed to food contaminated with cadmium

The aim of this study was to assess the genotoxic effects of Cd on haemocytes and midgut gland cells of web-building spiders, Steatoda grossa (Theridiidae), exposed to the metal under laboratory conditions. Analyzes were conducted on adult females and males, fed for four weeks with cadmium-contaminated Drosophila hydei flies, grown on a medium suplemented with 0.25mM CdCl2. The comet assay, providing a quantitative measure of DNA strand breaks, was used to evaluate the DNA damage caused by the metal. Cadmium content was measured in whole spider bodies by the AAS method. Metal body burden was significantly lower in females (0.25µgg−1 dry weight) than in males (3.03µgg−1 dry weight), suggesting that females may have more effective mechanisms controlling the uptake of metal, via the digestive tract, or its elimination from the body. Irrespectively of sex, spiders fed prey contaminated with cadmium showed significantly higher values of comet parameters: tail DNA (TDNA), tail length (TL) and olive tail moment (OTM), in comparison with the control. In midgut gland cells, the level of DNA damage was higher for males than females, while in haemocytes the genotoxic effect of cadmium was greater in females. The obtained results indicate that in spiders cadmium displays strong genotoxic effects and may cause DNA damage even at low concentrations, however the severity of damage seems to be sex- and internal organ-dependent.The comet assay can be considered a sensitive tool for measuring the deleterious effect of cadmium on DNA integrity in spiders.

The impact of in vivo and in vitro exposure to base analogue 5-FU on the level of DNA damage in haemocytes of freshwater mussels Unio pictorum and Unio tumidus

The impact of in vivo and in vitro exposure to anticancer drug 5-Fluorouracil (5-FU) on the level of DNA damage was evaluated using comet assay on haemocytes of freshwater mussels Unio pictorum and Unio tumidus. For the in vivo experiment, the groups of 5 mussels per concentration were exposed for 72 h to 5-FU (0.04, 0.4, 4, 40 and 100 μM) with 0.4 μM being the lowest concentration to induce significant DNA damage. For the in vitro experiment, the primary cultures of haemocytes were treated with 0.04, 0.4, 4 and 40 μM 5-FU for 22 h and the treatment with CdCl2 was used as a positive control. In contrast to in vivo exposure, 5-FU did not induce significant increase of DNA damage in vitro, possibly because of the absence of haemocytes proliferation in primary cultures.

Monitoring of DNA damage in haemocytes of freshwater mussel Sinanodonta woodiana sampled from the Velika Morava River in Serbia with the comet assay

This study was undertaken to investigate the potential of the freshwater mussel Sinanodonta woodiana for detection of genotoxic pollution of the environment. Study was performed at two sites in the Velika Morava River, from May 2010 to February 2011. The alkaline comet assay on haemocytes was used, and the olive tail moment (OTM) was chosen as a measure of DNA damage. The specimens held on acclimation under controlled laboratory conditions for 10d were used as a control. Chemical analysis revealed the presence of phosphates and increased concentrations of zinc, copper and nickel at both sites during the entire sampling period. The values of OTM in mussels collected from the environment, significantly correlated with the concentration of zinc (r=0.6248), temperature (r=0.7006) and dissolved oxygen (r=0.7738). Seasonal variations in genotoxic response were observed, with the highest OTM values obtained during summer months. Preliminary results of the in vitro study indicated the effect of water temperature on genotoxic response to zinc and cadmium in S. woodiana suggesting that the presence of genotoxic pollutants during months with lower temperature could be under-estimated. Obtained results indicate that S. woodiana could be a valuable tool for active biomonitoring of aquatic environments and emphasizes the importance of seasonal genotoxic monitoring with this organism.

Genotoxicity monitoring of freshwater environments using caged crayfish (Astacus leptodactylus)

Genotoxicity of freshwater pollution was assessed by measuring DNA damage in haemocytes of caged freshwater crayfish Astacus leptodactylus by the means of Comet assay and micronucleus test, integrated with the measurements of physiological (total protein concentration) and immunological (total haemocyte count) haemolymph parameters as biomarkers of undergone stress. Crayfish were collected at the reference site (River Mrežnica) and exposed in cages for 1week at three polluted sites along the Sava River (Zagreb, Sisak, Krapje). The long term pollution status of these locations was confirmed by chemical analyses of sediments. Statistically significant increase in DNA damage measured by the Comet assay was observed at all three polluted sites comparing to the crayfish from reference site. In addition, native crayfish from the mildly polluted site (Krapje) cage-exposed on another polluted site (Zagreb) showed lower DNA damage than crayfish from the reference site exposed at the same location indicating adaptation and acclimatisation of crayfish to lower levels of pollution. Micronuclei induction showed similar gradient of DNA damage as Comet assay, but did not reach the statistical significance. Observed increase in total haemocyte count and total protein content in crayfish from polluted environments in the Sava River also confirmed stress caused by exposure to pollution. The results of this study have proved the applicability of caging exposure of freshwater crayfish A. leptodactylus in environmental genotoxicity monitoring using Comet assay and micronucleus test.

Antioxidant deficit in gills of Pacific oyster ( Crassostrea gigas) exposed to chlorodinitrobenzene increases menadione toxicity

Disturbances in antioxidant defenses decrease cellular protection against oxidative stress and jeopardize cellular homeostasis. To knock down the antioxidant defenses of Pacific oyster Crassostrea gigas, animals were pre-treated with 1-chloro-2,4-dinitrobenzene (CDNB) and further challenged with pro-oxidant menadione (MEN). CDNB pre-treatment (10 μM for 18 h) was able to consume cellular thiols in gills, decreasing GSH (53%) and decrease protein thiols (25%). CDNB pre-treatment also disrupted glutathione reductase and thioredoxin reductase activity in the gills, but likewise strongly induced glutathione S-transferase activity (270% increase). Surprisingly, hemocyte viability was greatly affected 24 h after CDNB removal, indicating a possible vulnerability of the oyster immune system to electrophilic attack. New in vivo approaches were established, allowing the identification of higher rates of GSH–CDNB conjugate export to the seawater and enabling the measurement of the organic peroxide consumption rate. CDNB-induced impairment in antioxidant defenses decreased the peroxide removal rate from seawater. After showing that CDNB decreased gill antioxidant defenses and increased DNA damage in hemocytes, oysters were further challenged with 1 mM MEN over 24 h. MEN treatment did not affect thiol homeostasis in gills, while CDNB pre-treated animals recovered GSH and PSH to the control level after 24 h of depuration. Interestingly, MEN intensified GSH and PSH loss and mortality in CDNB-pre-treated animals, showing a clear synergistic effect. The superoxide-generating one-electron reduction of MEN was predominant in gills and may have contributed to MEN toxicity. These results support the idea that antioxidant-depleted animals are more susceptible to oxidative attack, which can compromise survival. Data also corroborate the idea that gills are an important detoxifying organ, able to dispose of organic peroxides, induce phase II enzymes, and efficiently export GSH–CDNB conjugates.

Effects of heavy metal ions (Cu2+, Pb2+ and Cd2+) on DNA damage of the gills, hemocytes and hepatopancreas of marine crab, Charybdis japonica

There are rising concerns about the hazardous effects of heavy metals on the environment. In this study, comet assay and DNA alkaline unwinding assay were conducted on the tissues (gills, hepatopancreas, and hemocytes) of Charybdis japonica in order to illustrate genotoxicity of three heavy metal ions (Cu2+, Pb2+, and Cd2+) on the marine crabs C. japonica. The crabs were exposed to Cu2+ (10, 50, and 100 μg.L−1), Pb2+ (50, 250, and 500 μg L−1) and Cd2+ (5, 25, and 50 μg L−1), and the tissues were sampled at days 0.5, 1, 3, 6, 9, and 15. DNA alkaline unwinding assay was used for testing the DNA single strand break in gills and hepatopancreas and comet assay was employed for testing the DNA damage in hemocytes. The results showed that the DNA damage (F-value) of gills in the crabs exposed to the three heavy metals was decreased gradually during the exposure periods and there was a dose-time response relationship in certain time, suggesting that the levels of DNA single strand break in all the experimental groups increased significantly compared to the controls. Changes of F-value in hepatopancreas of the crabs exposed to the three heavy metals were similar to those in gills except that the peak values were found in the 500 μg L−1 Pb2+ treatment group at day 3 and the 50 μg L−1 Cd2+ treatment group at day 9. The ranks of DNA damage in gills and hepatopancreas induced by the three heavy metal ions (50 μg L−1, day 15) were Cd2+ >Pb2+ >Cu2+ and Pb2+ >Cu2+ >Cd2+. The levels of DNA damage in gills were higher than those in hepatopancreas in the same experimental group. It can be concluded that indices of DNA damage can be used as the potential biomarkers of heavy metal pollution in marine environment.

Assessment of genotoxic potency of sulfate-rich surface waters on medicinal leech and human leukocytes using different versions of the Comet assay

The aim of the present study was to investigate how exposure to sulfate-rich surface waters affects the level of primary DNA damage in hemocytes of leech Hirudo medicinalis. Samples of surface water were collected at two sites near a gypsum factory (Knin, Croatia) and two reference sites. In the laboratory, samples were subjected to detailed chemical analysis and used in toxicity testing. For that purpose, previously acclimatized individuals of H. medicinalis were sub-chronically exposed (for 28 days) to tested water samples. Levels of primary DNA damage were evaluated using the alkaline Comet assay in hemocytes collected on days 7, 14, 21 and 28 of exposure and compared with their baseline values. Genotoxic potency of the water sample with the highest sulfate concentration was further evaluated using the alkaline, neutral and hOGG1-modified Comet assay on human peripheral blood leukocytes exposed ex vivo for 30 min. The purpose was to explore which mechanisms are responsible for DNA damage. Chemical analysis revealed that sulfate concentrations in two water samples collected in Mali Kukar Lake (1630 mg/L SO 4) and Kosovčica River (823.3 mg/L SO 4) exceeded the WHO and US EPA defined limits for sulfate in drinking water. Increased levels of metals were found only in the water sample collected in Mali Kukar Lake. However, of the 65 elements analyzed, only nickel and titanium exceed the value legally accepted in Croatia for drinking water. The levels of DNA damage, estimated by the alkaline Comet assay in hemocytes of medicinal leech, increased with the duration of exposure to two sulfate-rich water samples. Since hemocytes responded sensitively to treatment, they could be used for biomonitoring purposes. As observed on treated human peripheral blood leukocytes, all versions of the Comet assay were effective in detecting DNA damage, which was measured in samples with sulfate concentrations equal to or higher than the legally accepted levels for drinking water. Based on the obtained results, it can be assumed that genotoxicity was a consequence both of direct (single- and double-strand DNA breaks) and indirect effects (oxidative damage) caused by the combined effects of all contaminants present in the tested water samples. Our results indicate the need for in situ monitoring and purification of gypsum mine water prior to its release in the natural environment.

Early genotoxic effects in gill cells and haemocytes of Dreissena polymorpha exposed to cadmium, B[ a]P and a combination of B[ a]P and Cd

The aim of this study was to assess the genotoxic potential of environmentally relevant concentrations of Cd on the zebra mussel, an important freshwater sentinel organism, and to determine the stability of DNA damage in gill cells and haemocytes. The oxidative DNA damage and the co-genotoxicity of Cd in combination with B[ a]P were investigated. We measured DNA damage in haemocytes and gill cells of zebra mussels exposed for 11 days to a constant concentration of Cd (10 μg/L), B[ a]P (10 μg/L) or the two combined chemicals (10 μg/L + 1 μg/L). Enzymatic dissociation of gills with dispase gave the lower percentage DNA in tail, compared with collagenase/dispase or collagenase. Bioaccumulation of cadmium in the soft tissues of mussels exposed to CdCl 2 or CdCl 2 + B[ a]P increased in a time-dependent manner indicating that both exposures were effective. Cd (10 μg/L) is genotoxic only during the first 3 days of exposure in gill cells, while in haemocytes the genotoxicity of Cd was observed later. B[ a]P (10 μg/L) induced an early increase of DNA damage in gill cells (after 10 h and 1 day), while in both gill cells and haemocytes, B[ a]P caused a marked increase of DNA damage after 3 days of exposure. The Cd + B[ a]P mixture decreased the DNA-damaging effect of Cd and B[ a]P in both cell types. Cd induced an increase of DNA damage in Fpg-treated slides, indicating that Cd contributed to oxidative DNA damage. Cadmium induced a cytogenetic effect in gill cells, assessed by the number of micronuclei, throughout the duration of the exposure, while B[ a]P did not induce any cytogenetic effect. B[ a]P, Cd and Cd + B[ a]P induced a transient increase in the number of bi-nucleated cells. Our data clearly show that gills are more sensitive to Cd and B[ a]P, which makes them more suitable for future bio-monitoring studies.

Selenium in water enhances antioxidant defenses and protects against copper-induced DNA damage in the blue mussel Mytilus edulis

Selenium and copper are naturally occurring elements in the environment that have important roles in cellular function. Selenium is known for its role in antioxidant defense, whereas copper is a redox-active metal capable of acting as a pro-oxidant. We investigated the effects of short term selenium (Na 2SeO 3) supplementation (4 μg/L for 3 days) on antioxidant parameters of the blue mussel, Mytilus edulis, and its possible protective effects against a subsequent copper (CuSO 4) exposure (56 μg/L for 3 days). Selenium supplementation caused a 4-fold increase in glutathione levels in gills. The activity of selenium-dependent glutathione peroxidase was modulated by selenium in gills (2-fold increase) and also in cell-free haemolymph (40% increase). Copper exposure produced decreases in protein thiol levels (35%) and in thioredoxin reductase activity (60%) in gills and induced an increase in DNA damage in haemocytes (70% increase in % tail DNA observed using the comet assay). The decrease in thioredoxin reductase activity may constitute a mechanism of copper toxicity in bivalves, warranting further investigation. Pre-treatment with selenium largely prevented these deleterious effects of copper on protein thiols, thioredoxin reductase activity and DNA damage. The results suggest that induction of key antioxidant defenses such as glutathione and selenium-dependent glutathione peroxidase, as a result of selenium supplementation, may play an important role in protection of aquatic organisms against oxidative stress.

Involvement of Na +/H + exchanger and respiratory burst enzymes NADPH oxidase and NO synthase, in Cd-induced lipid peroxidation and DNA damage in haemocytes of mussels

This study investigated cadmium-induced oxidative and genotoxic effects, such as lipid peroxidation and disturbance of DNA integrity (DNA damage) in haemocytes of mussel Mytilus galloprovincialis and the possible involvement of Na +/H + exchanger (NHE), and/or the main enzymes of respiratory burst, NADPH oxidase and nitric oxide (NO) synthase, in the induction of Cd toxic effects . In order to verify the role of either NHE, or NADPH oxidase and NO synthase in Cd-mediated toxicity, inhibitors such as ethyl-N-isopropyl-amiloride (EIPA), diphenyleneiodonium chloride (DPI) and N G-nitro- l-arginine methyl ester (L-NAME) were used in each case. Moreover, phorbol-myristate acetate (PMA), a well-known protein kinase C (PKC)-mediated NADPH oxidase and NO synthase stimulator, as well as hydrogen peroxide (H 2O 2), a well-known genotoxic agent, was also used for elucidating the modulation of signaling molecules within cells, thus leading to the induction of lipid peroxidation and DNA damage. The results of the present study showed that micromolar concentrations of Cd (0.05–50 μΜ) could enhance both lipid peroxidation and DNA damage, possible via a PKC-mediated signaling pathway with the involvement of NHE, thus leading to the induction of NADPH oxidase and NO synthase activity, since inhibition of either NHE, or NADPH oxidase and NO synthase activity, significantly attenuates Cd-induced toxic effects in each case.

Genetic damage in the bivalve mollusk Corbicula fluminea induced by the water-soluble fraction of gasoline

Although gasoline is an important contaminant of aquatic ecosystems, information concerning the potential effects of this petroleum derivative on the DNA of aquatic biota is lacking. The present study aimed to evaluate the genotoxic and mutagenic effects of acute exposures (6, 24, and 96 h) to gasoline water-soluble fraction (GWSF), diluted to 5%, on the Asian clam Corbicula fluminea. The comet assay and the micronucleus (MN) test were performed on hemocytes and gill cells of C. fluminea. For the three different times tested, the comet assay indicated DNA damage in hemocytes and gill cells of C. fluminea exposed to GWSF. The MN test detected significant damage in the genetic material of the hemocytes only after 96 h of exposure to GWSF. The recovery capacity of organisms previously exposed for 6 h to GWSF was also evaluated with the comet assay. The results revealed a great capacity of this species to repair DNA damage; following 6 h of recovery, the comet score returned to that of the control groups. Overall, our findings indicate that GWSF has genotoxic and mutagenic effects on C. fluminea. In addition, the present results confirm the sensitivity of C. fluminea to damage caused by exposure to environmental contaminants, and, therefore, its suitability for use in environmental monitoring studies.

DNA damage in hemocytes of Schistocerca gregaria (Orthoptera: Acrididae) exposed to contaminated food with cadmium and lead

We measured in a comet assay the damage of DNA in the hemocytes of various stages of the grasshopper Schistocerca gregaria after ex-posing them to various doses of Cd and Pb in the food. The mechanisms of Cd and Pb toxicity for grasshopper are discussed. The accumula-tion of heavy metals and stage of the insect may play important roles in causing the DNA damage. S. gregaria may be considered a valuable bio-indicator for evaluation the genotoxicity of en-vironmental pollutants.

Assessment of genotoxicity in polluted freshwaters using caged painter’s mussel, Unio pictorum

This study was undertaken to evaluate the applicability of caged painter's mussel, Unio pictorum for freshwater environmental genotoxicity assessment. Mussels in cages were exposed for 3 weeks in 2002-2004 to polluted sites in two large rivers in the Croatia, the Sava and Drava, and on the respective reference sites. DNA damage was assessed in haemocytes of the exposed mussels by the comet and micronucleus assays. Both assays provided good discriminative power between polluted and control sites and showed the same gradation of sites according to their genotoxic properties, with high concordance between investigated years. Background levels of the DNA damage in haemocytes of painter's mussels are defined for both assays for easier detection of contamination-related genotoxicity. U. pictorum is found to be a very suitable sentinel species, sufficiently sensitive to the impact of pollution but at the same time unsusceptible to stress caused by translocation or cage exposure.

Application of the comet assay and detection of DNA damage in haemocytes of medicinal leech affected by aluminium pollution: A case study

This report describes an investigation of genotoxic effects in medicinal leech ( Hirudo verbana) exposed to water and sediment of Lake Njivice (Krk Island, Croatia) contaminated by aluminium compounds. The levels of primary DNA damage in leech haemocytes and loss of DNA integrity caused by acute and chronic exposure to contaminated water and sediment were investigated using the alkaline comet assay. Genotoxic effects induced by acute exposure to contaminants were evaluated on leech haemocytes and blood cells of fish and mouse treated ex vivo. The effects of chronic exposure were assessed on haemocytes sampled from an animal kept under laboratory conditions on contaminated water and sediment for 180 days. The results indicate the DNA damaging potential of aluminium compounds present in an excess amount in tested samples.