DNA Damage In Haemocytes Research Articles

The mechanism of reactive oxygen species generation, DNA damage and apoptosis in hemocytes of Litopenaeus vannamei under ammonia nitrogen exposure

Ammonia-N poses a significant threat to aquatic animals. However, the mechanism of ROS production leading to DNA damage in hemocytes of crustaceans is still unclear. Additionally, the mechanism that cells respond to DNA damage by activating complex signaling networks has not been well studied. Therefore, we exposed shrimp to 0, 2, 10, and 20 mg/L NH4Cl for 0, 3, 6, 12, 24, 48, and 72 h, and explored the alterations in endoplasmic reticulum stress and mitochondrial fission, DNA damage, repair, autophagy and apoptosis. The findings revealed that ammonia exposure led to an increase in plasma ammonia content and neurotransmitter content (DA, 5-HT, ACh), and significant changes in gene expression of PLC and Ca2+ levels. The expression of disulfide bond formation-related genes (PDI, ERO1) and mitochondrial fission-related genes (Drp1, FIS1) were significantly increased, and the unfolded protein response was initiated. Simultaneously, ammonia-N exposure leads to an increase in ROS levels in hemocytes, resulting in DNA damage. DNA repair and autophagy were considerably influenced by ammonia-N exposure, as evidenced by changes in DNA repair and autophagy-related genes in hemocytes. Subsequently, apoptosis was induced by ammonia-N exposure, and this activation was associated with a caspase-dependent pathway and caspase-independent pathway, ultimately leading to a decrease in total hemocytes count. Overall, we hypothesized that neurotransmitters in the plasma of shrimp after ammonia-N exposure bind to receptors on hemocytes membrane, causing endoplasmic reticulum stress through the PLC-IP3R-Ca2+ signaling pathway and leading to mitochondrial fission. Consequently, this process resulted in increased ROS levels, hindered DNA repair, suppressed autophagy, and activated apoptosis. These cascading effects ultimately led to a reduction in total hemocytes count. The present study provides a molecular support for the understanding of the detrimental toxicity of ammonia-N exposure to crustaceans.

Short communication: The boring sponge (Pione vastifica, Hancock, 1849) induces oxidative stress in the Pacific oyster (Magallana gigas, Thunberg, 1793)

Boring sponge infection affects growth, development and reduces the soft tissue weight of oysters. In this study, we investigated the effects of boring sponge on the activity of three antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GP)) in the mantle, and the production of reactive oxygen species (ROS) and potential genotoxicity in hemocytes of the Pacific oyster Magallana gigas. Our results showed a significant increase in ROS production and DNA damage in hemocytes. Notably, the activity of SOD, CAT, and GP in the mantle was not significantly affected by boring sponge infection. Collectively, these results suggest that sponge invasion may cause oxidative stress in Pacific oyster hemocytes through ROS overproduction.

Sublethal Effects of Neonicotinoid Insecticides on the Development, Body Weight and Economic Characteristics of Silkworm.

Silkworm Bombyx mori (L.) (Lepidoptera: Bombycidae) is a critical insect for silk producers, but the inappropriate application of insecticides negatively affects the physiology and behavior of silkworms. This study found that the effects of neonicotinoid insecticides applied using two spraying methods on the growth and development of silkworms were different: the median lethal concentration (LC50) values of two pesticides applied using the leaf-dipping method were 0.33 and 0.83 mg L-1 and those of two pesticides applied using the quantitative spraying method were 0.91 and 1.23 mg kg-1. The concentration of pesticides on the mulberry leaves did not decrease after their application using the quantitative spraying method, and a uniform spraying density was observed after the mulberry leaves were air-dried (no liquid) under realistic conditions. We then treated silkworms with the quantitative spraying method and leaf-dipping method. The treatment of silkworm larvae with imidacloprid and thiamethoxam at sublethal concentrations significantly prolonged the development time and significantly decreased the weight and pupation rate, as well as economic indicators of enamel layers and sputum production. Thiamethoxam treatment significantly increased the activities of carboxylesterase (CarE) and glutathione-S-transferase (GST). The activity of CarE and GST increased, decreased, and then increased, and the highest activity was detected on the 10th and 12th days. Thiamethoxam exposure significantly elevated the transcription levels of CarE-11, GSTe3 and GSTz2 and induced DNA damage in hemocytes. This study confirmed that the quantitative spray method is more stable than the leaf-dipping method. Moreover, imidacloprid and thiamethoxam treatment affected the economy and indexes of silkworms and induced changes in detoxification enzymes and DNA damage in silkworms. These results provide a basis for understanding the mechanism of the sublethal effects of insecticides on silkworms.

Genotoxic effects of oxyclozanide on hemocytes of Galleria mellonella (Lepidoptera: Pyralidae) larvae

Oxyclozanide is a salicylanilide derivative anthelmintic drug with a well-known effect on parasites that cause infections in humans and animals. In this study, the effect of oxyclozanide on DNA damage in hemocytes of Galleria mellonella (L.) (Lepidoptera: Pyralidae) larvae, which has been used as a model organism in many fields, was investigated. Hemolymph was collected from the last instar larvae (7th instar) reared on artificial diets containing oxyclozanide at different concentrations (0.003%, 0.03%, 0.3%, and 1.5%) under laboratory conditions and then hemocytes suspension was prepared. Genotoxic damage in hemocytes was determined by the comet assay which enables microscopically detecting DNA damage and is a very sensitive assay in chemical genotoxicity. When compared to the control group, tail length, tail DNA percent, and tail moment values were significantly increased parallel with increasing oxyclozanide concentrations. While the tail length was determined as 5.11 ± 0.46 μm in the control group, it was significantly increased in all tested groups to 13.17 ± 0.53, 27.98 ± 1.08, 98.44 ± 0.77, and 137.67 ± 0.74 μm, respectively. Similarly to tail length, tail DNA percentage and tail moment levels were also significantly increased from 12.86 ± 0.74 to 91.96 ± 0.31 at the highest concentration of oxyclozanide. These results showed that oxyclozanide caused DNA damage in the hemocytes of G. mellonella. It is also known that hemocytes are an important bioindicator in determining the genotoxicity of anthelmintics to be used as insecticides within environmentally friendly limits. It is thought that our results will contribute to the studies in this field.

Single and combined potential of polystyrene microparticles and fluoranthene in the induction of DNA damage in haemocytes of Mediterranean mussel (Mytilus galloprovincialis).

In this study, the possible 'vector effect' within the exposure of Mediterranean mussels (Mytilus galloprovincialis) to polystyrene microplastics with adsorbed fluoranthene was investigated by applying the multibiomarker approach. The major focus was placed on genotoxicological endpoints as to our knowledge there are no literature data on the genotoxicity of polystyrene microparticles alone or with adsorbed fluoranthene in the selected experimental organisms. DNA damage was assessed in haemocytes by comet assay and micronucleus test. For the assessment of neurotoxicity, acetylcholinesterase activity was measured in gills. Glutathione S-transferase was assessed in gills and hepatopancreas since these enzymes are induced for biotransformation and excretion of lipophilic compounds such as hydrocarbons. Finally, differences in physiological response within the exposure to polystyrene particles, fluoranthene, or particles with adsorbed fluoranthene were assessed by the variation of heart rate patterns studied by the noninvasive laser fibre-optic method. The uniform response of individual biomarkers within the exposure groups was not recorded. There was no clear pattern in variation of acetylcholinesterase or glutathione S-transferase activity which could be attributed to the treatment. Exposure to polystyrene increased DNA damage which was detected by the comet assay but was not confirmed by micronucleus formation. Data of genotoxicity assays indicated differential responses among the groups exposed to fluoranthene alone and fluoranthene adsorbed to polystyrene. Change in the heart rate patterns within the studied groups supports the concept of the Trojan horse effect within the exposure to polystyrene particles with adsorbed fluoranthene.

Appraisal of growth inhibitory, biochemical and genotoxic effects of Allyl Isothiocyanate on different developmental stages of Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae)

Allyl isothiocyanate (AITC), a glucosinolates’ hydrolytic product, was studied for its anti-insect potential against an economically important, destructive tephritid pest, Zeugodacus cucurbitae (Coquillett). The first, second and third instar maggots of the pest were fed on artificial diets amended with varied concentrations of AITC viz. 5 ppm, 25 ppm, 50 ppm, 100 ppm, 150 ppm and 200 ppm with DMSO (0.5%) as control. Results revealed high larval mortality, alteration of larval period, prolongation of pupal and total developmental periods in all instars of the maggots treated with AITC as compared to controls. Percent pupation and percent adult emergence decreased in all larval instars. Growth indices viz. Larval Growth Index (LGI) and Total Growth Index (TGI) were negatively affected. Anti-nutritional/post ingestive toxicity of AITC was also revealed by the decrease in Food Assimilation (FA) and Mean Relative Growth rate (MRGR) values with respect to control. Profiles of PO (Phenol oxidase) and other detoxifying enzymes including SOD (Superoxide dismutases), CAT (Catalases), GST (Glutathione-S-transferases), EST (Esterases), AKP (Alkaline phosphatases) and ACP (Acid phosphatases) were also significantly influenced. The genotoxic effect of AITC was also evaluated by conducting comet assays at LC30 and LC50. Significant DNA damage in hemocytes was reflected by increase in Tail length (μm), Percent Tail DNA, Tail Moment (TM) and Olive Tail Moment (OTM) as compared to controls. The results indicated high potential of AITC as biopesticide for pest management.

Application of land snail Helix lucorum for evaluation of genotoxicity of soil pollution

Application of native species as sentinels allows environmental scientists to determine real genotoxic impact of environmental pollutants. The present study aims at investigating the DNA damage in the land snail Helix lucorum as a biomarker of soil pollution. For this reason, the genotoxic impact of contaminated soil on H. lucorum, collected from different polluted areas, was investigated using the comet assay in haemocytes and digestive gland cells. An increase in DNA damage was found in the snails sampled from polluted sites compared with the reference one. Strong correlations between DNA damage in haemocytes and digestive gland cells with the level of contamination indicate pollution-induced genotoxic effects in both tissues. At the same time, the digestive gland was more sensitive towards pollutants compared with haemolymph. A direct relationship between concentrations of Cu, As and Mo in soil and the number of damaged cells for hаemolymph and digestive gland tissue was found. However, the data obtained reflect the total genotoxicity of all pollutants in the studied areas. Significant correlations between the DNA damage measured by the comet assay and metal contents in soil indicate that it is a suitable biomarker in ecotoxicological studies. Our results indicate the effectiveness of H. lucorum in biomonitoring of environmental pollution.

Molecular toxicity and defense mechanisms induced by silver nanoparticles in Drosophila melanogaster

The widely use of silver nanoparticles (AgNPs) as antimicrobial agents gives rise to potential environmental risks. AgNPs exposure have been reported to cause toxicity in animals. Nevertheless, the known mechanisms of AgNPs toxicity are still limited. In this study, we systematically investigated the toxicity of AgNPs exposure using Drosophila melanogaster. We show here that AgNPs significantly decreased Drosophila fecundity, the third-instar larvae weight and rates of pupation and eclosion in a dose-dependent manner. AgNPs reduced fat body cell viability in MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. AgNPs caused DNA damage in hemocytes and S2 cells. Interestingly, the mRNA levels of the entire metallothionein gene family were increased under AgNPs exposure as determined by RNA-seq analysis and validated by qRT-PCR, indicating that Drosophila responded to the metal toxicity of AgNPs by producing metallothioneins for detoxification. These findings provide a better understanding of the mechanisms of AgNPs toxicity and may provide clues to effect on other organisms, including humans.

Effect of Extreme Heatwaves on the Mortality and Cellular Immune Responses of Purplish Bifurcate Mussel Mytilisepta virgata (Wiegmann, 1837) (=Septifer virgatus) in Indoor Mesocosm Experiments

In the rocky intertidal environment, the frequency and duration of heatwaves have increased over the last decade, possibly due to global climate change. Heatwaves often result in lethal or sub-lethal disturbances in benthic animals by changing their metabolic activities. In this study, we investigated the impacts of extreme heatwave stress on the hemocyte functions of Mytilisepta virgata and subsequent mortality to gain a better understanding of the potential causes and consequences of mass mortality events in this mussel during summer. We discriminated three types of hemocytes in the hemolymph, granulocytes, hyalinocytes, and blast-like cells, using flow cytometry and revealed that granulocytes were the major hemocyte involved in cellular defensive activities, such as phagocytosis and reactive oxygen species (ROS) production. For the experiment, mussels were exposed to a 40°C air temperature for 12 h per day over 5 days under laboratory conditions as a simulated semi-diurnal tidal cycle. Mortality began to occur within 3 days after beginning the experiment, and all mussels had died by the end of the experiment. Flow cytometry indicated that the mussels exposed to high air temperatures produced significantly more ROS than did the control mussels within 2 days after the onset of the experiment, which may have caused oxidative stress. Such high levels of ROS in the hemolymph increased DNA damage in hemocytes after 3 days of exposure and decreased the phagocytosis of hemocytes 4 days after the experiment began. The observed mortality and decline in immune capacity suggested that an extreme heat event occurring in the rocky intertidal ecosystem during summer could exert sublethal to lethal impacts on macrobenthic animals.

Toxicological effects of graphene on mussel Mytilus galloprovincialis hemocytes after individual and combined exposure with triphenyl phosphate

Graphene nanoparticles are increasingly released into the aquatic environment with the growth of production. However, there are rare investigations focusing on the interaction of nanoparticles with other contaminants. Triphenyl phosphate (TPP) is a frequently detected organophosphate flame retardant in the environment. This study aimed to assess the joint effects of graphene and TPP on Mytilus galloprovincialis hemocytes. Oxidative stress could be induced by graphene and TPP in mussel hemocytes, which could further cause apoptosis, DNA damage and decrease in the lysosomal membrane stability (LMS). Moreover, hemocytes could internalize graphene, thereby resulting in oxidative stress. The oxidative stress and DNA damage in hemocytes were increased in the graphene-exposed group, but significantly reduced after combined exposure of graphene and TPP. The up-regulated genes, including NF-κB, Bcl-2 and Ras, were mainly associated with reduced apoptosis and DNA damage after co-exposure to graphene and TPP.

Thermal stress causes DNA damage and mortality in a tropical insect

Cold tolerance is considered an important factor determining the geographic distribution of insects. We have previously shown that despite its tropical origin, the cockroach Gromphadorinha coquereliana is capable of surviving exposures to cold. However, the freezing tolerance of this species had not yet been examined. Low temperature is known to alter membrane integrity in insects, but whether chilling or freezing compromises DNA integrity remains a matter of speculation. In the present study, we subjected the G. coquereliana adults to freezing to determine their supercooling point (SCP) and evaluated whether the cockroaches were capable of surviving partial and complete freezing. Next, we conducted single cell gel electrophoresis (SCGE) assays to determine whether heat, cold and freezing altered hemocyte DNA integrity. The SCP of this species was high and around -4.76°C, which is within the typical range of freezing-tolerant species. Most cockroaches survived to 1day after partial ice formation (20% mortality), but died progressively in the next few days after cold stress (70% mortality after 4 days). One day after complete freezing, most insects died (70% mortality), and after 4 days, 90% of them had succumbed. The SCGE assays showed substantial levels of DNA damage in hemocytes. When cockroaches were heat-stressed, the level of DNA damage was similar to that observed in the freezing treatment, though all heat-stressed insects survived. The present study shows that G. coquereliana can be considered as moderately freeze-tolerant, and that extreme low temperature stress can affect DNA integrity, suggesting that this cockroach may possess an efficient DNA repair system.

Genotoxic effects of combined multiple stressors on Gammarus locusta haemocytes: Interactions between temperature, pCO2 and the synthetic progestin levonorgestrel

Climate change and pharmaceutical contamination are two priority research topics due to their impacts in the aquatic ecosystems and in the food chain structure. In the bottom of many food chains are the invertebrates, like the amphipods, which are important environmental and ecotoxicological models. In this study, we combined the increase of temperature [ambient and warming temperature], pCO2 [normocapnia and hypercapnia] and the synthetic progestin levonorgestrel (LNG) [environmentally relevant concentration (10 ng L−1) and 100-fold higher (1000 ng L−1)] to evaluate the genotoxic effects on the amphipod Gammarus locusta haemocytes, using the comet assay technique. Additionally, the study examined protective/potentiating effects of the three tested factors against hydrogen peroxide (H2O2)-induced DNA damage in haemocytes after ex vivo exposure. Our data revealed no significant effects of any of the three stressors on DNA damage of G. locusta haemocytes or protection against H2O2-induced DNA damage after twenty-one days exposure. Only a significant effect of the solvent was visible, since it was able to induce higher DNA damage (i.e. strand breaks) on exposed individuals. On the other hand, LNG exposure seemed to induce a slight increase of DNA damage after H2O2 exposure. Our findings suggest that more short-term studies to conclude about the genotoxicity and/or protective effects of the stress factors in G. locusta should be made, attending to the fast turnover rate of repairing cells that could have masked impacts seen only after the end of the experiment.

DNA damage in haemocytes of Harpalus (Pseudophonus) rufipes (De Geer, 1774) (Coleoptera, Carabidae) as an indicator of sublethal effects of exposure to herbicides

The carabid beetle Harpalus (Pseudophonus) rufipes is one the most common polyphagous species in the agroecosystem and plays an important role in the natural control of pests. The primary focus of the study was to examine whether exposure to herbicides, used for agricultural productivity, can have genotoxic sublethal effects on this epigean non-target species. The neutral comet assay was carried out to provide a quantitative measure of DNA double-strand breaks in haemocytes of adults. Beetles from a conventional potato field treated with herbicides such as metribuzin-, clomazone-, pendimethalin- and cycloxydim-based commercial formulates were compared with specimens from an organic potato field in which weeds were removed mechanically. Analyses showed that the level of DNA damage recorded as tail DNA percentage in adults from the conventional potato field (11.55 ± 0.47) was significantly higher (p = 9.5 × 10−5) than that in beetles from the organic control site (8.93 ± 0.22). Moreover, DNA damage reduction was recorded in beetles from the conventional field sampled about 12 weeks after the field treatment compared with that in beetles sampled about 8 weeks after the treatment. The genotoxic effect of herbicide commercial formulations used in the conventional potato field before and during sampling was determined. This report provides evidence that exposure to herbicides has a detrimental effect on H. rufipes and confirms that the Comet assay may be a useful tool in determining the potential genotoxicity of pollutants on soil arthropods.

Application of Crayfish Astacus Leptodactylus for the Analysis of Water Genotoxicity in the Lake Sevan Basin

Genotoxicity of water pollution at three sites of the Lake Sevan Basin was estimated by using crayfish Astacus leptodactylus as bioindicator. Levels of DNA damage in hemocytes of crayfish were analyzed using the comet assay. A significant rise of the level of DNA damage in terms of parameters of the percentage DNA content in the comet tail and the Olive tail moment was detected in crayfish inhabiting waters near the villages of Artanish and Tsapatakh (Armenia) as compared to the crayfish from the Masrik River Estuary. A significant positive correlation between the parameter of the Olive tail moment in crayfish and the content of Al, Fe, Cu, and Mo in water has been discovered. The obtained data indicate that crayfish Astacus leptodactylus is a sensitive bioindicator for monitoring of aquatic ecosystems.

Cytotoxicity and cellular mechanisms of toxicity of CuO NPs in mussel cells in vitro and comparative sensitivity with human cells

There is a need to assess human and ecosystem health effects of copper oxide nanoparticles (CuO NPs), extensively used in many industrial products. Here, we aimed to determine the cytotoxicity and cellular mechanisms involved in the toxicity of CuO NPs in mussel cells (hemocytes and gill cells) in parallel with exposures to ionic Cu and bulk CuO, and to compare the sensitivity of mussel primary cells with a well-established human cell line (pulmonary TT1 cells). At similar doses, CuO NPs promoted dose-dependent cytotoxicity and increased reactive oxygen species (ROS) production in mussel and human cells. In mussel cells, ionic Cu was more toxic than CuO NPs and the latter more than bulk CuO. Ionic Cu and CuO NPs increased catalase and acid phosphatase activities in both mussel cells and decreased gill cells Na-K-ATPase activity. All Cu forms produced DNA damage in hemocytes, whereas in gill cells only ionic Cu and CuO NPs were genotoxic. Induction of the MXR transport activity was found in gill cells exposed to all forms of Cu and in hemocytes exposed to ionic Cu and CuO NPs. Phagocytosis increased only in hemocytes exposed to CuO NPs, indicating a nanoparticle-specific immunostimulatory effect. In conclusion, toxicity of CuO NPs is driven by ROS in human and mussel cells. Mussel cells respond to CuO NP exposure by triggering an array of defensive mechanisms.

Mixtures of tritiated water, zinc and dissolved organic carbon: Assessing interactive bioaccumulation and genotoxic effects in marine mussels, Mytilus galloprovincialis

Release of tritium (3H) in the marine environment is of concern with respect to its potential bioaccumulation and detrimental impact on the biota. Previous studies have investigated the uptake and toxicity of this radionuclide in marine mussels, and the interaction of 3H with dissolved organic ligands and elevated temperature. However, despite the well-established view that toxicity is partly governed by chemical speciation, and that toxic effects of mixture of contaminants are not always additive, there have been no studies linking the prevailing chemistry of exposure waters with observed biological effects and tissue specific accumulation of 3H in combination with other constituents commonly found in natural waters. This study exposed the marine mussel Mytilus galloprovincialis for 14 days to mixtures of 3H (as tritiated water, HTO) and zinc (Zn) at 5 Mbq L−1, and 383, 1913 and 3825 nM Zn, respectively, to investigate (a) 3H and Zn partitioning in soft tissues of mussels, and (b) DNA damage in haemocytes, determined using the single cell gel electrophoresis or the comet assay. Additionally, the extent of association of 3H with dissolved organic carbon (DOC, added as humic acid) over the exposure period was investigated in order to aid the interpretation of biological uptake and effects. Results concluded a clear antagonistic effect of Zn on 3H-induced DNA damage at all Zn concentrations used, likely explained by the importance of Zn in DNA repair enzymes. The interaction of DOC with 3H was variable, with strong 3H-DOC associations observed in the first 3 d of the experiment. The secretion of 3H-binding ligands by the mussels is suggested as a possible mechanism for early biological control of 3H toxicity. The results suggest risk assessments for radionuclides in the environment require consideration of potential mixture effects.

Transcriptional and cellular effects of paracetamol in the oyster Crassostrea gigas

Acetaminophen (paracetamol) (PAR) is one of the most popular non-steroidal anti-inflammatory drugs (NSAIDs) with analgesic and antipyretic properties consumed worldwide and often detected in the aquatic environment. Due to the fact that PAR induces oxidative stress in mammals, the aim of this study was to evaluate if similar effects were observed in oysters Crassostrea gigas, given their economic and ecological importance and worldwide distribution. Oysters were exposed for 1, 4 and 7 days to two different sublethal PAR concentrations (0, 1 and 100μgL−1). Cell viability, DNA damage in hemocytes and enzymatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx), glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH) and glutathione S-transferases (GST) were evaluated in oyster gills. In addition, changes at transcriptional level of Cu/Zn superoxide dismutase (SOD), catalase-like (CAT-like), cytochrome P450 genes (CYP30C1, CYP2AU2, CYP3071A1, CYP356A1), glutathione S-transferase isoforms (GST-ω and GST-π-like), cyclooxygenase (COX), fatty acid binding proteins-like (FABP-like), and caspase genes were evaluated in oyster gills and digestive gland. No changes in cell viability and DNA damage were observed in oysters exposed to both PAR concentrations. Similarly, no significant changes were detected in the major antioxidant enzymes (except for auxiliary enzyme GR) in oyster gills, suggesting that changes in GR activity are enough to counteract a potential oxidative stress in C. gigas gills under these experimental conditions. Furthermore, changes at transcriptional level are concentration and tissue dependent. PAR elicited an inhibition of CYP30C1, CYP3071A1 and FABP-like transcripts highlighting their role in drug metabolism, transport and detoxification of PAR in the gills. GST transcript levels were type, tissue and concentration-dependent. GST-π-like was down-regulated in oyster gills exposed to the lowest PAR concentration and up-regulated in the digestive gland of oysters exposed to the highest PAR concentration. However, GST-ω transcript levels were lower only in oysters digestive gland exposed to the lowest PAR concentration. Therefore, changes at transcriptional level were more sensitive to assess the exposure to PAR at environmental relevant concentrations.

Effect of substituted phenylamine antioxidants on three life stages of the freshwater mussel Lampsilis siliquoidea

Substituted phenylamines (SPAs) are incorporated into a variety of consumer products (e.g., polymers, lubricants) in order to increase the lifespan of the products by acting as a primary antioxidant. Based on their physicochemical properties, if SPAs were to enter the aquatic environment, they would likely partition into sediment. No studies to date have investigated the effect of sediment-associated SPAs on aquatic organisms. The current study examined the effect of four SPAs (diphenylamine (DPA); N-phenyl-1-napthylamine (PNA); N-(1,3-dimethylbutyl)-N’-phenyl-1,4-phenylenediamine (DPPDA); 4,4’-methylene-bis[N-sec-butylaniline] (MBA)) on three different life stages of the freshwater mussel, Lampsilis siliquoidea. The viability of larvae (glochidia) of L. siliquoidea and Lampsilis fasciola was assessed after 48 h of exposure to SPAs in water. The 48-h EC50s for glochidia viability of L. siliquoidea were 5951, 606, 439, and 258 μg/L for DPA, PNA, DPPDA, and MBA, respectively, and 7946, 591, 137, and 47 μg/L, respectively, for L. fasciola. Juvenile (7–15 months) and adult L. siliquoidea were exposed to sediment-associated SPAs for 28 d. LC50s for juvenile mussels were 18, 55, 62, and 109 μg/g dry weight (dw) of sediment for DPA, PNA, DPPDA, and MBA, respectively. Adult mussels were exposed to sub-lethal concentrations of sediment-associated SPAs in order to investigate reactive oxygen species (ROS), lipid peroxidation and total glutathione in the gill, gonad, and digestive gland tissue, and viability and DNA damage in hemocytes. No significant concentration-dependent trend in any of these biochemical and cellular endpoints relative to the concentration of sediment-associated SPAs was observed in any tissues. Investigations into the concentration of SPAs in the aquatic environment are required before a conclusion can be made on whether these compounds pose a hazard to the different life stages of freshwater mussels.

Arsenic-contaminated freshwater: assessing arsenate and arsenite toxicity and low-dose genotoxicity in Gammarus elvirae (Crustacea; Amphipoda)

Arsenic (As) contamination of freshwater is largely due to geogenic processes, but As is also released into the environment because of improper anthropic activities. The European regulatory limits in drinking water are of 10 μg L-1 As. However, knowledge of the genotoxic effects induced by low doses of As in freshwater environments is still scanty. This study was designed to investigate arsenate (As(V)) and arsenite (As(III)) toxicity and low-dose genotoxicity in Gammarus elvirae, which has proved to be a useful organism for genotoxicity assays in freshwater. As(V) and As(III) toxicity was assessed on the basis of the median lethal concentration, LC(50), while estimates of DNA damage were based on the Comet assay. The G. elvirae LC (50-240 h) value we calculated was 1.55 mg L-1 for As(V) and 1.72 mg L-1 for As(III). Arsenic exposure (240 h) at 5, 10, and 50 µg L-1 of As in assays with either arsenate or arsenite-induced DNA damage in hemocytes of G. elvirae in a concentration-dependent manner. Our study provides a basis for future genotoxic research on exposure to freshwater that contains low levels of arsenic.

Indirect effects of climate changes on cadmium bioavailability and biological effects in the Mediterranean mussel Mytilus galloprovincialis

Despite the great interest in the consequences of climate change on the physiological functioning of marine organisms, indirect and interactive effects of rising temperature and pCO2 on bioaccumulation and responsiveness to environmental pollutants are still poorly explored, particularly in terms of cellular mechanisms. According to future projections of temperature and pH/pCO2, this study investigated the main cellular pathways involved in metal detoxification and oxidative homeostasis in Mediterranean mussels, Mytilus galloprovincialis, exposed for 4 weeks to various combinations of two levels of pH/pCO2 (8.2/∼400 μatm and 7.4/∼3000 μatm), temperature (20 and 25 °C), and cadmium addition (0 and 20 μg/L). Bioaccumulation was increased in metal exposed organisms but it was not further modulated by different temperature and pH/pCO2 combinations. However, interactions between temperature, pH and cadmium had significant effects on induction of metallothioneins, responses of the antioxidant system and the onset of oxidative damages, which was tissue dependent. Multiple stressors increased metallothioneins concentrations in the digestive gland revealing different oxidative effects: while temperature and cadmium enhanced glutathione-dependent antioxidant protection and capability to neutralize peroxyl radicals, the metal increased the accumulation of lipid peroxidation products under acidified conditions. Gills did not reveal specific effects for different combinations of factors, but a general stress condition was observed in this tissue after various treatments. Significant variations of immune system were mainly caused by increased temperature and low pH, while co-exposure to acidification and cadmium enhanced metal genotoxicity and the onset of permanent DNA damage in haemocytes. Elaboration of the whole biomarker data in a cellular hazard index, corroborated the synergistic effects of temperature and acidification which increased the toxicological effects of cadmium. The overall results confirmed that climate change could influence ecotoxicological effects of environmental contaminants, highlighting the importance of a better knowledge of cellular mechanisms to understand and predict responsiveness of marine organisms to such multiple stressors.