Hemocytes Of Mussel Mytilus Galloprovincialis Research Articles

ZnO, Ag and ZnO-Ag nanoparticles exhibit differential modes of toxic and oxidative action in hemocytes of mussel Mytilus galloprovincialis

The present study investigates the cytotoxic and oxidative effects of custom-made nanoparticles (NPs) on hemocytes of Mytilus galloprovincialis, utilizing hemolymph serum (HS) as exposure medium. Specifically, hemocyte lysosomal membrane destabilization (in terms of neutral red retention time assay/NRRT), superoxide anion (O2−), nitric oxide (NO, in terms of nitrites) and lipid peroxidation content (in terms of malondialdehyde/MDA equivalents) were determined in cells treated for 1 h with different concentrations (0.1–50 μg mL−1) of ZnO NPs, Ag NPs and ZnO-Ag NPs, as well as AgNO3 and/or ZnCl2 (bulk ions, respectively). According to the results, Ag NPs were more cytotoxic than ZnO-Ag NPs and/or ZnO NPs, while NRRT values observed in AgNO3 treated cells were lower than those of ZnCl2. Furthermore, high levels of both O2− and MDA were detected in cells treated with Ag NPs, ZnO-Ag NPs, and AgNO3 at concentrations lower than 5 μg mL−1, while high NO generation was observed only in cells treated with 5–25 μg mL−1 of ZnO NPs or ZnCl2. Despite the absence of data, regarding the formation of NP-serum protein corona complexes that could mediate NP surface energy and uptake efficiency, the current study firstly revealed that ZnO NPs, probably via their surface charge, particle agglomeration, and NP Zn+ release could promote an immune-related generation of O2− and NO via the respiratory burst stimulation, a process that is questioned in the case of Ag NPs and/or ZnO-Ag NPs. Moreover, ZnO-Ag NP interaction with biological membranes and their oxidative mode of action seemed to be regulated by the release and the antagonistic/synergistic response of its ionic counterparts (ZnO+ and Ag+), but further studies are needed to elucidate the oxidative mode of action of NP metal ions in complex NP mixtures.

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.

Cell cycle alterations in the mussel Mytilus galloprovincialis hemocytes caused by environmental contamination

Environmental contamination includes a mixture of organic substances that can have detrimental effects on marine organisms and should be evaluated in the quality and risk assessment of investigated marine areas. Marine areas selected for this study are a protected area, a mariculture area, a shipyard and an industrial area. Based on the toxicity of the organic seawater extracts these areas were classified as an undisturbed reference area (S1), an area with the low anthropogenic impact (S2), a potentially endangered area (S3) and an area with high anthropogenic impact (S4) respectively. The organic mixtures present in seawater samples collected at the above defined areas were tested for the induction of DNA damage and cell cycle alterations in the mussel Mytilus galloprovincialis hemocytes. Flow cytometric analyses were performed to detect changes in hemocytes DNA content distribution throughout the cell cycle. Organic seawater extracts from sampling sites S2, S3 and S4 induced an increase in the coefficient of variation of the G0/G1 peak and an increase in the number of cells in the G2/M phase reflecting the extent of DNA damage and G2/M arrest, respectively. The G2/M arrest in mussel hemocytes was concentration-dependent upon injection with organic seawater extracts from the S3 site and time dependant for S2, S3 and S4 sampling sites. The time dependence of the induction of the G/M arrest showed a characteristic pattern for each site due to the different quantitative and qualitative composition of the organic seawater extracts. The G2/M arrest was reversible 24 or 72 hours after treatment with organic seawater extracts from S2 or S3, and S4 sites, respectively. This reversibility was time- and site-specific indicating that such DNA damage is repairable to a certain degree according to the organic seawater extract composition. Thus, the hemocytes cell cycle alterations in the mussel Mytilus galloprovincialis caused by organic seawater extracts reliably reflect the extent of organic contamination effects for selected marine areas.

The impact of expired commercial drugs on non-target marine species: A case study with the use of a battery of biomarkers in hemocytes of mussels

The present study investigated the effects of two expired commercial medicines, like Buscopan Plus and Mesulid, commonly classified as household medical wastes, on hemocytes of mussel Mytilus galloprovincialis. Mussel hemocytes’ lysosomal membrane stability (in terms of neutral red retention assay), superoxide anions (O2·-) and nitric oxides (NO, in terms of nitrites) production, lipid peroxidation (in terms of malondialdehyde/MDA content) and the formation of nuclear abnormalities (using the micronucleus/MN assay) were assessed in hemocytes of mussels treated for 7 days with appropriate amounts of each drug (the concentrations of active substances were considered in each case, due to the absence of data related with the excipients) as well as in hemocytes of post-treated/recovered mussels (7 days post-treatment/recovery period). According to the results, treated mussels showed significantly decreased NRRT values, enhanced O2·-, NO and MDA levels, as well as high frequencies of nuclear abnormalities in both cases. Thοse effects showed a drastic reduction in almost all cases, after the post-treatment/recovery period. Moreover, the “stress on stress” method, commonly performed for estimating mussels’ ability to survive in air, showed significantly reduced LT50 values in challenged mussels, compared to values observed in control mussels. The current findings revealed for the first time that both expired commercial drugs could affect mussels, probably via the formation of active substances bioactivated metabolites, as well as excipients, such as TiO2 and SiO2, at least in case of Buscopan plus. Although further research is needed, the current findings indicate the environmental impact of expired commercial drugs, thus revealing the need for the proper disposal of household medical wastes.

Mediated effect of ultrasound treated Diclofenac on mussel hemocytes: First evidence for the involvement of respiratory burst enzymes in the induction of DCF-mediated unspecific mode of action

The present study investigates the toxic behavior of diclofenac (DCF) before and after its ultrasound (US) treatment, as well as the involvement of intracellular target molecules, such as NADPH oxidase and NO synthase, in the DCF-induced adverse effects on hemocytes of mussel Mytilus galloprovincialis. In this context, appropriate volumes (350 and 500mL) of DCF solutions (at concentrations of 2, 2.5, 5 and 10mgL−1) were treated under different ultrasound operating conditions (frequency at 582 and 862kHz, electric power density at 133 and 167W) for assessing US method efficiency. In parallel, DCF and US DCF-mediated cytotoxic (in terms of cell viability measured with the use of neutral red uptake/NRU method), oxidative (in terms of superoxide anions/.O2−, nitric oxides such as NO2− and lipid peroxidation products, such as malondialdehyde/MDA content) and genotoxic (DNA damage measured by the use of Comet assay method) effects were investigated in hemocytes exposed for 1h to 5, 10 and 100ngL−1 and 1, 10 and 20μgL−1 of DCF. The involvement of NADPH oxidase and NO synthase to the DCF-induced toxicity was further investigated by the use of 10μΜ L-NAME, a NO synthase inhibitor and 10μΜ DPI, a NADPH oxidase inhibitor. According to the results, 350mL of 2mgL−1 DCF showed higher degradation (>50%) under 167W electric power density and frequency at 862kHz for 120min, compared to degradation in all other cases, followed by a significant elimination of its toxicity. Specifically, US DCF-treated hemocytes showed a significant attenuation of DCF-mediated cytotoxic, oxidative and genotoxic effects, which appeared to be caused by NADPH oxidase and NO synthase activation, since their inhibition was followed by a significant elimination of .O2− and NO2− generation and the concomitant oxidative damage within cells. The results of the present study showed for the first time that unspecific mode of action of DCF, associated with the induction of NADPH oxidase and NO synthase in mussel hemocytes, could be significantly diminished after partial US degradation of DCF, at least under optimized operating conditions currently tested.

Aqueous phenanthrene toxicity after high-frequency ultrasound degradation

Given that polycyclic aromatic hydrocarbons (PAHs), such as phenanthrene (PH), possess a potent risk for aquatic biota, a great attempt to develop and apply advanced oxidation processes, such as ultrasound (US), is of great concern nowadays. However, because US PAH-derived toxic intermediates are difficult to detect, the present study investigates aqueous PH toxicity before and after high-frequency US degradation, in hemocytes of mussel Mytilus galloprovincialis. Specifically, cell viability (with the use of neutral red uptake/NRU method), and oxidative-stress indices in terms of superoxide anions, (O2−), nitric oxides (NO, in terms of nitrites), lipid peroxidation products (in terms of malondialdehyde/MDA content) and DNA damage (with the use of Comet assay method) were investigated in mussel hemocytes exposed to environmentally relevant concentrations of PH (0.01, 0.1, 1 and 10μgL−1), before and after US treatment for 120min (at a frequency of 582kHz). According to the results, the NRU method showed a significant attenuation of PH-induced mortality in US PH-treated hemocytes in all cases. Moreover, the increased levels of O2− and NO generation, as well as MDA content measured in PH-treated hemocytes, were drastically decreased after US degradation in any case. Similarly, the disturbance of DNA integrity (in terms of % DNA in tail, OM and TM), was negligible in case of US PH-treated hemocytes. Although further in vitro and in vivo studies are needed, the present study showed for the first time that high frequency US could be applied as a highly efficient and “environmentally friendly” process for degrading low molecular weight PAH, such as PH.

Carbamazepine-mediated pro-oxidant effects on the unicellular marine algal species Dunaliella tertiolecta and the hemocytes of mussel Mytilus galloprovincialis

This study investigates the pro-oxidant behavior of the antiepileptic drug carbamazepine (CBZ) on the marine algal species Dunaliella tertiolecta and the immune defense-related hemocytes of mussel Mytilus galloprovincialis. A phytotoxicity test, performed in a first step, showed a significant inhibition of the growth rate and the chlorophyll alpha (Chl-α) content in algae after exposure for 24 h to different concentrations of CBZ (1-200 mg L(-1)). On the other hand, the increased levels of lipid peroxidation products, such as MDA, measured in 24 h CBZ-treated cells were attenuated with time (48-96 h), followed by a significant recovery of both the algal growth rate and the Chl-α content in all cases. The latter could be related to the concomitant enhancement of total carotenoids in CBZ-treated algae with time, which in turn could protect algal growth and survival against CBZ-induced oxidative stress. On the other hand, the increased levels of cell death, superoxide anions ((·)O2 (-)), nitric oxides (NO, in terms of nitrites, NO2 (-)) and MDA content observed in mussel hemocytes exposed to environmentally relevant (0.01-1 μg L(-1)) and/or higher (10 and 100 μg L(-1)) concentrations of the drug, clearly indicate the ability of CBZ to induce oxidative effects on cells of non-target species, such as mussels, affecting thus their overall health status. The significant relationships occurred among the tested biological parameters in both bioassays, further reinforce CBZ-mediated pro-oxidant effects on species, widely used in ecotoxicological and toxicological studies and provide a more comprehensive view on its environmental fate and ecotoxicological risk evaluation.

Antioxidant and pro-oxidant challenge of tannic acid in mussel hemocytes exposed to cadmium

The present study investigates the antioxidant and pro-oxidant behavior of tannic acid (TA) in hemocytes of mussel Mytilus galloprovincialis, in the presence or the absence of cadmium (Cd). TA at concentrations up to 20 μM, primarily found to be no toxic (in terms of cell viability, superoxide anions, nitric oxide and lipid peroxidation products currently estimated), significantly diminished the cytotoxic and oxidative effects induced by the metal (50 and/or 100 μM) in all cases. On the other hand, higher concentrations of TA (40 and 60 μM) were toxic, thus enhancing Cd-mediated cytotoxic and oxidative effects. The present study showed TA beneficiary properties in hemocytes of mussels, at least at low concentrations, while TA at concentrations higher than 20 μM could serve as an excellent oxidized substrate, thus enhancing toxic effects either alone or with the presence of micromolar concentrations of non transition metals, such as Cd.

Evidence for phosphatidylinositol-3-OH-kinase (PI3-kinase) involvement in Cd-mediated oxidative effects on hemocytes of mussels

This study investigated phosphatidylinositol-3-OH-kinase (PI3-kinase) involvement in the induction of cadmium-mediated oxidative effects on hemocytes of mussel Mytilus galloprovincialis. PI3-kinase was investigated with the use of wortmannin, a specific covalent inhibitor of PI3-kinase. Moreover, phorbol-myristate acetate (PMA), a well-known protein kinase C (PKC)-mediated NADPH oxidase and nitric oxide (NO) synthase stimulator, was also used for elucidating PI3-kinase involvement during the respiratory burst process in challenge hemocytes. According to the results, cells pre-treated with non-toxic concentrations of wortmannin (1 and/or 50nM, as revealed by neutral red retention assay) for 15min, showed a significant attenuation of cadmium ability (at concentration of 50μM) to promote cell death, superoxide anion (O2−) production, NO generation and lipid peroxidation (in terms of malondialdehyde equivalents). On the other hand, wortmannin-treated cells showed a significant attenuation of PMA ability to induce NO generation but not O2− production. These findings reveal that PI3-kinase could lead to a PKC-independent induction of NO synthase activity in cells faced with pro-oxidants, such as cadmium, while its activation could be fundamental for the regulation of NAPDH oxidase activity, probably through a PKC-dependent signaling pathway.

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.

The role of selenium-dependent glutathione peroxidase (Se-GPx) against oxidative and genotoxic effects of mercury in haemocytes of mussel Mytilus galloprovincialis (Lmk.)

This study investigated whether mercury (Hg) oxidative and genotoxic effects are related with its ability to inhibit selenium-dependent glutathione peroxidase (Se-GPx) activity in haemocytes of mussel Mytilus galloprovincialis. Se-GPx activity was measured both in Se-free cells’ cytosolic fraction and in Se-treated cells, pre-treated with 4 μg/l of Se (as sodium selenite), before the exposure to the metal. Hg at concentrations ranged within 10 or 20 μg/l, thus representing the onset of Hg toxic effects, showed to inhibit Se-GPx activity in Se-free cells, followed by increased levels of superoxide anions ( O 2 - ) and nitric oxide (NO) generation, lipid peroxidation and DNA damage as well. On the other hand, increased enzymatic activity and a significant attenuation of Hg toxicity were measured in Se-treated cells exposed to Hg in all cases. The results of the present study showed that inhibition of Se-GPx activity by Hg could promote a shift in the balance between oxidants and antioxidants in favor of oxidants, resulted in the enhancement of Hg-induced oxidative and genotoxic effects. In addition, Se bioavailability within phagocytic cells, such as haemocytes, could regulate the antioxidant role of Se-GPx, thus reinforcing haemocytes’ immune system against toxic effects induced by pro-oxidants, such as Hg.

The role of signalling molecules on actin glutathionylation and protein carbonylation induced by cadmium in haemocytes of musselMytilus galloprovincialis(Lmk)

This study investigated the role of Na(+)/H(+) exchanger (NHE) and signalling molecules, such as cAMP, PKC, PI 3-kinase, and immune defence enzymes, NADPH oxidase and nitric oxide synthase, in the induction of protein glutathionylation and carbonylation in cadmium-treated haemocytes of mussel Mytilus galloprovincialis. Glutathionylation was detected by western blot analysis and showed actin as its main target. A significant increase of both actin glutathionylation and protein carbonylation, were observed in haemocytes exposed to micromolar concentration of cadmium chloride (5 micromol l(-1)). Cadmium seems to cause actin polymerization that may lead to its increased glutathionylation, probably to protect it from cadmium-induced oxidative stress. It is therefore possible that polymerization of actin plays a signalling role in the induction of both glutathionylation and carbonylation processes. NHE seems to play a regulatory role in the induction of oxidative damage and actin glutathionylation, since its inhibition by 2 micromol l(-1) cariporide, significantly diminished cadmium effects in each case. Similarly, attenuation of cadmium effects were observed in cells pre-treated with either 11 micromol l(-1) GF-109203X, a potent inhibitor of PKC, 50 nmol l(-1) wortmannin, an inhibitor of PI 3-kinase, 0.01 mmol l(-1) forskolin, an adenylyl cyclase activator, 10 micromol l(-1) DPI, a NADPH oxidase inhibitor, or 10 micromol l(-1) L-NAME, a nitric oxide synthase inhibitor, suggesting a possible role of PKC, PI 3-kinase and cAMP, as well as NADPH oxidase and nitric oxide synthase in the enhancement of cadmium effects on both actin glutathionylation and protein carbonylation.

Production of superoxides and nitric oxide generation in haemocytes of mussel Mytilus galloprovincialis (Lmk.) after exposure to cadmium: A possible involvement of Na +/H + exchanger in the induction of cadmium toxic effects

The present study investigates cadmium (Cd) ability to enhance superoxides ( O 2 −) and nitric oxide (NO) production (as nitrites) in haemocytes of mussel Mytilus galloprovincialis as well as the possible involvement of Na +/H + exchanger (NHE) in the induction of NADPH oxidase and NO synthase activity. PMA, a well-known PKC-mediated NADPH oxidase as well as NO synthase stimulator was also used, in order to verify Cd effects on both O 2 − and NO generation. According to the results of the present study, micromolar concentrations of Cd (0.05, 5, 10 and 50 μM) seemed to enhance O 2 − and NO generation in haemocytes of mussels. Moreover, O 2 − and NO generation in haemocytes exposed to Cd could be enhanced by its ability to induce reactive oxygen species (ROS) but respiratory burst activation as well. Inhibition of NO synthase with 10 μM l-NAME, significantly attenuated Cd ability to enhance O 2 − production and diminished NO generation, thus leading to the suggestion that Cd toxic effects, started at concentration of 50 μM, could enhance NADPH oxidase and NO synthase stimulation in haemocytes of mussels. NHE seems to play a regulatory role in the induction of either O 2 − or NO generation in haemocytes exposed to the metal, since its inhibition with the use of 10 μM EIPA significantly decrease both O 2 − and NO production. The involvement of NHE in the induction of O 2 − and NO generation, probably via PKC-mediated NADPH oxidase and NO synthase activation, is likely to be crucial to haemocytes exposed to heavy metals, such as Cd.

The influence of Zn on signaling pathways and attachment of Mytilus galloprovincialis haemocytes to extracellular matrix proteins

The present study investigates the cytotoxic mechanisms induced by zinc (Zn) in haemocytes of mussel Mytilus galloprovincialis. Haemocytes play a key role in the immune defence of mussels. Micromolar concentration of Zn (50 μM) play an important role in the elevation of pHi and increase in Na + influx in haemocytes. The observed effects were inhibited by the Na +/H + exchanger (NHE) inhibitor, ethyl- N-isopropyl-amiloride (EIPA). Furthermore, our results showed that Zn caused an increase in O 2 − production that was reversed after NHE inhibition. Phorbol ester (PMA) caused a significant rise both in pHi and Na + influx as well as in O 2 − production. These effects were reversed by calphostin C. Our results indicated that Zn also enhanced haemocyte attachment to both BSA and laminin which was reversed by EIPA and calphostin C. The enhancement of haemocytes attachment to both BSA and laminin after Zn suggests that it is likely to play a signal role in cytoskeleton-dependent process of cell growth and migration in mussel M. galloprovincialis haemocytes . We conclude that Zn induces a signaling pathway with the involvement of NHE, PKC, O 2 − and α 1- and β-adrenergic receptors.