Metallothionein Concentrations Research Articles

Відмінності вмісту металотіонеїну у мозку гризунів за умов постнатального розвитку та кадмієвої інтоксикації

Introduction. Metal-binding metallothionein genes are found in a vast population of organisms. These proteins are non enzymatic and very rich in cysteine residues. The various metallothionein isoforms in different brain regions arguably change over time.The aim of the study – evaluating the distribution of metallothionein in different brain regions of gerbils and Wistar rats at different stages of postnatal development (PND), under standard and low dose Cd-induced conditions.Materials and Methods. 18 Mongolian gerbils and 36 Wistar rats were divided into 6 groups (n=6), by age and condition of experiment: groups 1, 2, 3, 4 – 1, 30, 90 and 180-days old were exposed to standard conditions; group 5 and 6 – 180-days old+0.1 or 1.0 µgµg Cd2+ per animal everyday for 36 days. The metallothionein content in the hippocampus, cerebellum, and thalamus were detected by the ELISA.Results and Discussion. Obtained data was shown the dynamic of metallothionein distribution in different brain regions of gerbils and Wistar rats depending on the stage of postnatal development and functional capacities. The content of metallothionein in the hippocampus continually decreased in both animal types but the cerebella metallothionein distribution pattern was different from that of the hippocampus, but identical in both rodents, rising from day one before decreasing on day 30. The low levels of metallothionein under the influence of Cd were proportional to the doses administered.Conclusions. The level of metallothionein in the brain varies depending on the stage of development of the functional capacities of the brain parts. The significant down-regulation of metallothionein in the investigated brain regions under Cd influence suggests that a decrease in metallothionein levels depends on the dose of Cd and on the time necessary for its accumulation.

Assessment of health status of oysters (Crassostreagigas) exposed to environmentally relevant concentrations of Ag and Cu in brackish waters

Human activities have altered estuarine environments leading to increased presence of different pollutants including metals. Although the implementation of new environmental policies has caused a considerable decrease in trace metal concentrations in estuaries around the Bay of Biscay, some elements such as copper (Cu) and silver (Ag) are still present in relatively high concentrations. Oysters have been widely used in environmental biomonitoring programs as sentinel organisms. Oysters Crassostrea gigas from an uncontaminated estuary were exposed to sublethal, environmentally relevant concentrations of Cu (2000ng Cu/L) and Ag (500ng Ag/L) during 14days in brackish water (S=18). A battery of cell and tissue level (exposure) biomarkers at different levels of biological complexity was applied and integrated into the Integrative Biological Response (IBR) index including: metallothionein contents, intralysosomal metal accumulation, digestive gland atrophy and digestive gland tissue integrity. Condition Index (CI) was incorporated into the IBR index as a complementary parameter that reflects the general physiological condition of oysters (organism level). Results indicated an increase in intralysosomal metal accumulation after 7 and 14days of exposure to Ag together with an increase in the digestive epithelium atrophy and lipofuscin content after 7days of exposure to Ag. The responses detected with the aid of biomarkers integrated in the IBR index showed higher toxicity in oysters exposed to Ag, inducing the clear onset of detoxification processes which also occurred, to a lower extent, in Cu-exposed oysters.

Copper sulfate pretreatment prevents mitochondrial electron transport chain damage and apoptosis against MPP+-induced neurotoxicity

Intrastriatal injection of 1-methyl-4-phenylpyridinium (MPP+) is considered a model to reproduce some biochemical alterations observed in Parkinson's disease (PD) patients. Among those alterations, inhibition of mitochondrial complex I activity, increased free radical production and reduced antioxidant responses have been reported. Copper (Cu) plays an important role in the metabolism and antioxidative responses through its participation as a cofactor in the cytochrome c oxidase enzyme (COX), Cu/Zn-superoxide dismutase (Cu/Zn-SOD), and metallothioneins. We tested the effect of copper sulfate (CuSO4) pretreatment on the mitochondrial electron transport chain (METC) in the striatum after MPP+ toxicity in rats. The results showed that the MPP+ intrastriatal injection reduced mitochondrial complex I, II, IV and V activities; while 10 μmol of CuSO4 pretreatment counteracted this damage. Activities of complexes I, II and IV, were coincident with ATP recovery. Moreover, Cu/Zn-SOD activity was reduced as a consequence of MPP+ damage; however, copper pre-treatment kept the striatal Cu/Zn-SOD activity unchanged in MPP+-damaged animals. We observed that MPP+ also reduced the metallothionein (MT) content and that CuSO4 pretreatment maintained baseline values. CuSO4 pretreatment also reduced the striatal caspase-3 and caspase-9 activities that were increased three days after MPP+-induced damage. The present study provided evidence that copper pretreatment reduced MPP+-induced apoptotic damage, probably through direct action on copper-dependent proteins or indirectly on proteins in the apoptotic pathway.

A comprehensive evaluation of the environmental quality of a coastal lagoon (Ravenna, Italy): Integrating chemical and physiological analyses in mussels as a biomonitoring strategy

This study aimed at evaluating the environmental quality of a coastal lagoon (Pialassa Piomboni, NW-Adriatic, Italy) by combining analyses of biomarkers of environmental stress and bioaccumulation of contaminants in marine mussels (Mytilus galloprovincialis) transplanted for 28days to six selected sites. Assessed biomarkers encompassed lysosomal endpoints, oxidative stress and detoxification parameters, specific responses to metals, neuro- and genotoxic substances; chemical analyses focused on PAHs, metals, pesticide and pharmaceuticals. Results showed up to a 67-fold bioaccumulation of 4- to 6-ring PAHs, including pyrene, fluoranthene, chrysene and benzo(ghi)perylene in transplanted mussels compared to reference conditions (T0). A 10-fold increase of Fe, Cr and Mn was observed, while pesticides and pharmaceuticals were not or slightly detected. The onset of a significant (p<0.05) general stress syndrome occurred in exposed mussels, as outlined by a 50–57.7% decrease in haemocytes lysosomal membrane stability and an increased lysosomal volume (22.6–26.9%) and neutral lipid storage (18.9–48.8%) observed in digestive gland. Data also revealed a diffuse lipofuscin accumulation (86.5–139.3%; p<0.05) in digestive gland, occasionally associated to a catalase activity inhibition in gill, indicating an increased vulnerability toward pro-oxidant factors. Higher levels of primary DNA damage (258%; p<0.05) and PAH accumulation were found in mussels exposed along the eastern shoreline, hosting a petrochemical settlement. Bioaccumulated metals showed a positive correlation with increased metallothionein content (85–208%; p<0.05) observed in mussels from most sites. Overall, the use of physiological and chemical analyses detected chronic alterations of the mussel health status induced by specific toxicological pathways, proving a suitable approach in the framework of biomonitoring programs of coastal lagoons.

Effects of cadmium on metallothionein and histology in gills of tilapia (Oreochromis niloticus) at different salinities

ABSTRACTThe objectives of this study were to evaluate the effects of sublethal cadmium concentrations on the levels of cadmium, metallothionein (MT) and histological changes in gills of East Java strain tilapia (Oreochromis niloticus) at different salinity levels. The levels of cadmium in control gills were not significantly different at 0, 5 and 10 practical salinity unit (PSU). The cadmium concentrations in gills of cadmium-exposed fish were significantly higher at 0 PSU than at 5, 10 and 15 PSU. The MT concentrations in control gills were not significantly different at 0, 5, and 10 PSU. The MT concentrations of cadmium-exposed fish were significantly higher than those in respective control groups at 0, 5 and 10 PSU. Significant gill damage occurred in fish exposed to cadmium at lower salinity. The epithelial lifting was noted at gills of fish exposed to 2.5 mg/L of cadmium at 0 PSU, and telangiectasis was observed at gills exposed to 5 mg/L of cadmium at 0 PSU. The level of gill damage decreased with increasing salinity of media. The increased MT and histological changes in gills of our findings could be a protective response of animals to toxic effect of cadmium.

Expression, purification of metallothionein genes from freshwater crab (Sinopotamon yangtsekiense) and development of an anti-metallothionein ELISA.

Using the phoA-fusion technology, the recombinant metallothionein (MT) from freshwater crab (Sinopotamon yangtsekiense) has been successfully produced in Escherichia coli. MT purified from the bacterial suspension showed one polypeptide with a molecular weight of 7 kDa by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE). Western-blotting confirmed the polypeptides had a specific reactivity with mouse polyclonal MT anti-serum. Based on the purified MT and MT anti-serum, the reaction parameters for an enzyme-linked immunosorbent assay (ELISA) were developed. The direct coating ELISA showed a higher linear relationship compared to antibody sandwich coating ELISA. The optimal dilution rates of purified MT anti-serum and coating period were shown to be 1:160,000 and 12 hours at 4°C. At 37°C, the appropriate reaction duration of the first antibody and the second antibody were 2 hours and 1 hour, respectively. According to these optimal parameters, the standard linear equation, y = 0.0032x + 0.1769 (R2 = 0.9779, x, y representing MT concentration and OD450 value), was established for the determination of MT concentration with a valid range of 3.9–500 ng/ml. In verification experiments, the mean coefficients of variation of the intra-assay and inter-assay were 3.260% and 3.736%, respectively. According to the result of MT recovery, ELISA with an approaching 100% MT recovery was more reliable and sensitive than the Cd saturation assay. In conclusion, the newly developed ELISA of this study was precise, stable and repeatable, and could be used as a biomarker tool to monitor pollution by heavy metals.

Toxicity effects of di-(2-ethylhexyl) phthalate to Eisenia fetida at enzyme, cellular and genetic levels.

Di-(2-ethylhexyl) phthalate (DEHP) is a dominant phthalic acid ester (PAE) that has aroused public concern due to its resistance to degradation and its toxicity as an endocrine-disrupting compound. Effects of different concentrations of DEHP on Eisenia fetida in spiked natural soil have been studied in the body of the earthworm by means of soil cultivation tests 7, 14, 21 and 28 days after exposure. The results indicated that, in general, superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, metallothionein (MT) content, the expression of heat shock protein 70 (HSP 70) and all the tested geno-toxicity parameters are promoted as time elapses and with increasing concentration of DEHP. However, peroxidase (POD) activity, neutral red retention time (NRRT) and mitochondrial membrane potential difference values were found to decrease even at a low concentration of DEHP of 1 mg kg-1 soil (p<0.05). Clear toxic effects of DEHP on E. fetida have been generally recognized by means of the disturbance of antioxidant enzyme activity/content and critical proteins, cell membrane and organelle disorder and DNA damage estimated by length of tail, tail DNA ratio, and tail moment parameters. A concentration of DEHP of 3 mg kg-1 may be recommended as a precaution against the potential risk of PAEs in soils and for indicating suitable threshold values for other soil animals and soil micro-organisms.

Hepatoprotective effect of blocking N-methyl-d-aspartate receptors in male albino rats exposed to acute and repeated restraint stress.

Stress affects many organs in addition to the brain, including the liver. We assessed the effects on the liver of blocking N-methyl-d-aspartate (NMDA) glutamate receptors with memantine in acute and repeated restraint stress. Forty-two male albino rats were divided into 7 groups; control, acute restraint stress (ARS), ARS + memantine, repeated restraint stress, repeated restraint + memantine, and positive control groups. We measured serum iron, zinc, alanine transferase and aspartame transferase, hepatic malondialdehyde, tumor necrosis factor-α (TNF-α), glutathione peroxidase, superoxide dismutase, metallothionein content, zinc transporter ZRT/IRT-like protein 14 mRNA expression, and hepcidin expression. We conducted a histopathological evaluation via histological staining and immunostaining for glial fibrillary acidic protein and synaptophysin expression, both of which are markers of hepatic stellate cell (HSC) activation. Both ARS and repeated stress increased markers of hepatic cell injury, oxidative stress, and HSC activation. Blocking NMDA with memantine provided a hepatoprotective effect in acute and repeated restraint stress and decreased hepatic cell injury, oxidative stress, and HSC activation.

The effect of ozone on the expression of metallothionein in tissues of rats chronically exposed to cadmium.

Our aims were to evaluate the expression of metallothionein (MT) in an experimental rat model which experienced chronic exposure to cadmium (Cd) and to measure its expression after ozone therapy (OT) or oxygen (Ox) in the same model, as compared to the control group, which was exposed to neither cadmium nor ozone. Forty male Wistar rats were divided into 5 groups: control, Cd, Cd and Ox, Cd and Oz, and Oz. During our research, Cd concentration (ASA) and MT concentration (ELISA) were determined in supernatants of the kidneys, liver and pancreas. SDS-PAGE analyses and immunohistochemical localization were used to evaluate the level of MT expression in the tissue. In rats intoxicated with Cd, the highest concentration of both Cd and MT was observed in the kidneys and liver, with a significantly lower concentration measured in the pancreas. Ozone therapy reduces the accumulation of cadmium in the liver and kidneys, resulting in a reduced expression of metallothionein in those tissues.

Oxidative stress and related biomarkers in cupric and cuprous chloride-treated rainbow trout.

We examined the time-course stress responses in the liver of rainbow trout exposed to cuprous chloride (CuCl) and cupric chloride (CuCl2). The treatment groups received a single intraperitoneal injection of CuCl or CuCl2 (both at a dose of 0.01 and 0.05mg/kg); the control group received only the physiologic solution vehicle. Liver tissue samples were analyzed for total copper, superoxide dismutase, catalase, glutathione peroxidases, glutathione reductase, glutathione S-transferase, glyoxalases, and lactate dehydrogenase at 3, 6, and 9days post-injection. Total glutathione, metallothionein, and malondialdehyde levels were also measured. The time course of metal accumulation differed between the groups; no dose-response relationship for metal load was found. Both copper species elicited significant changes in oxidative stress markers and in metal trapping. Copper underwent adaptive shifts in glutathione and metallothionein concentrations. The defense strategy primarily versus CuCl2 first involved glutathione, with a peak in metallothionein levels at day 6 for CuCl2 (at both doses) and for CuCl (0.05mg/kg). Early stimulation of lipid peroxidation was noted after treatment with the higher copper dose and at day 9 after treatment with the lower dose of both CuCl and CuCl2. Antioxidant enzyme activity was impaired due to a more or a less severe oxidative stress condition in relation to the copper species and exposure time. Copper dynamics, in terms of metal accumulation and homeostatic regulation, is noticeably complex. The present findings may advance our understanding of the effects of both copper species on the antioxidant response of rainbow trout.

Alterations of tissue metallothionein and vitellogenin concentrations in tropical cup oysters (Saccostrea sp.) following short-term (96 h) exposure to cadmium

Metallothioneins and vitellogenins are low molecular weight proteins that have been used widely in environmental monitoring as biomarkers of exposure and damage to metals and estrogenic compounds, respectively. In the present study, the responses of metallothionein and vitellogenin tissue concentrations were measured following acute (96h) aqueous exposures to cadmium in Saccostrea sp., a tropical cup oyster native to the Western Pacific Ocean that has recently established itself in the Caribbean Sea. Adult oysters (1.5–5.0cm shell length) collected from the municipal marina of Santa Marta, Colombia (Caribbean Sea) and acclimated for 5days in the laboratory, were exposed to Cd at five concentrations (0, 1, 10, 100 and 1000μg/L) and their tissues (gills, digestive gland and adductor muscle) were analyzed in pools of 5 individuals (3 replicates per concentration). Metallothioneins in digestive glands of oysters exposed to Cd concentrations≥100μg/L showed a significant increase, from 8.0 to 14.8μg MT/mg total protein, whereas metallothionein concentrations in gills increased to lesser extent, and no differences were observed in adductor muscle. Metallothionein concentrations in digestive gland and gills correlated directly with whole soft tissue Cd concentrations (ranging from 2 to 297μg/g dw Cd). Vitellogenin in homogenates of oyster gonad tissue, after 96h of exposure to 1000μg/L Cd, were significantly lower (0.04mg P/g gonad) compared to control oysters (0.68mg P/g gonad), suggestive of an anti-estrogenic effect of Cd at high concentrations, whereas no significant changes in vitellogenin concentrations were observed at intermediate Cd exposure concentrations. This study confirms acute responses of metallothionein and vitellogenin concentrations in tissues of Saccostrea sp. exposed to high concentrations of cadmium (Cd≥100μg/L, 96h). The present results are first step towards validating the use of these two proteins as biomarkers of metal exposure in this species.

Endocrine and cellular stress effects of zinc oxide nanoparticles and nifedipine in marsh frogs Pelophylax ridibundus

Freshwater organisms including amphibians experience increasing exposures to emerging pollutants such as nanoparticles and pharmaceuticals, which can affect their fitness and performance. We studied the effects of two common pollutants extensively used in industry, pharmaceutical and personal care products, nano-zinc oxide (nZnO) and a Ca-channel blocker nifedipine (Nfd), on endocrine status and cellular stress markers of the marsh frog Pelophylax ridibundus. Males were exposed for 14days to nZnO (3.1μM), Zn2+ (3.1μM, as a positive control for nZnO exposures), Nfd (10μM), and combination of nZnO and Nfd (nZnO+Nfd). Exposure to nZnO and Zn2+ led to an increase in Zn burdens, elevated concentrations of the metal-bound metallothioneins (MT-Me) in the liver and increased vitellogenin in the serum, whereas exposures to Nfd and nZnO+Nfd resulted in the metal release from MTs and a significant increase in the ratio of total to metal-bound MTs. This likely reflects oxidative stress caused by Nfd exposures as manifested in the elevated levels of oxyradical production, upregulation of superoxide dismutase activity (SOD) and increase in the total and oxidized glutathione concentrations in Nfd-exposed frogs. Zn-containing exposures upregulated activity of deiodinase (in nZnO and nZnO+Nfd exposures) and serum thyrotropin level (in the case of Zn2+). All exposures caused an increase in DNA fragmentation, lipofuscin accumulation as well as upregulation of caspase-3 and CYP450 levels reflecting cytotoxicity of the studied compounds in the liver. Across all experimental treatments, nZnO exposures in the absence of Nfd had the least impact on the cellular stress traits or redox status in frogs. This indicates that at the low environmentally relevant levels of pollution, pharmaceuticals such as Nfd and free metals (such as Zn2+) may represent a stronger threat to the health of the frogs than nZnO particles.

Variations of biomarkers response in mussels Mytilus galloprovincialis to low, moderate and high concentrations of organic chemicals and metals

The changes of acetylcholinesterase activity (AChE), metallothioneins content (MTs), catalase activity (CAT) and lipid peroxidation (LPO) were assessed after 4 days exposure of mussels Mytilus galloprovincialis to a wide range of sublethal concentrations of chlorpyrifos (CHP, 0.03–100 μg/L), benzo(a)pyrene (B(a)P, 0.01–100 μg/L), cadmium (Cd, 0.2–200 μg/L) and copper (Cu, 0.2–100 μg/L). The activity of AChE in the gills decreased after exposure to CHP and Cu, whereas no change of activity was detected after exposure to B(a)P and Cd. Both induction and decrease of MTs content in digestive gland occurred after exposure to CHP and B(a)P, while a marked increase was evident at highest exposure concentrations of Cd. The content of MTs progressively decreased of MTs with increasing concentration of Cu. CAT activity and LPO in the gills did not change after exposure to any of the chemicals. The results demonstrate different response profile in relation to the type of chemical compound, and highlight the potential implications for evaluation of biological effect of contaminants in marine environment. Furthermore, the AChE activity in the gills and MTs content in the digestive gland could be modulated by CHP and Cu at environmentally relevant concentrations indicating the potential risks of short-term transient mussels exposure that may occur due to run-off from land or accidental releases.

Tolerance and stress response of sclerotiogenic Aspergillus oryzae G15 to copper and lead.

Aspergillus oryzae G15 was cultured on Czapek yeast extract agar medium containing different concentrations of copper and lead to investigate the mechanisms sustaining metal tolerance. The effects of heavy metals on biomass, metal accumulation, metallothionein (MT), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were evaluated. Cu and Pb treatment remarkably delayed sclerotial maturation and inhibited mycelial growth, indicating the toxic effects of the metals. Cu decreased sclerotial biomass, whereas Pb led to an increase in sclerotial biomass. G15 bioadsorbed most Cu and Pb ions on the cell surface, revealing the involvement of the extracellular mechanism. Cu treatment significantly elevated MT level in mycelia, and Pb treatment at concentrations of 50-100mg/L also caused an increase in MT content in mycelia. Both metals significantly increased MDA level in sclerotia. The variations in MT and MDA levels revealed the appearance of heavy metal-induced oxidative stress. The activities of SOD, CAT, and POD varied with heavy metal concentrations, which demonstrated that tolerance of G15 to Cu and Pb was associated with an efficient antioxidant defense system. In sum, the santioxidative detoxification system allowed the strain to survive in high concentrations of Cu and Pb. G15 depended mostly on sclerotial differentiation to defend against Pb stress.

Effects of Zinc Glycinate on Productive and Reproductive Performance, Zinc Concentration and Antioxidant Status in Broiler Breeders.

An experiment was conducted to investigate the effects of zinc glycinate (Zn-Gly) supplementation as an alternative for zinc sulphate (ZnSO4) on productive and reproductive performance, zinc (Zn) concentration and antioxidant status in broiler breeders. Six hundred 39-week-old Lingnan Yellow broiler breeders were randomly assigned to 6 groups consisting of 4 replicates with 25 birds each. Breeders were fed a basal diet (control group, 24mg Zn/kg diet), basal diet supplemented with 80mg Zn/kg diet from ZnSO4 or basal diet supplemented with 20, 40, 60 and 80mg Zn/kg diet from Zn-Gly. The experiment lasted for 8weeks after a 4-week pre-test with the basal diet, respectively. Results showed that Zn supplementation, regardless of sources, improved (P < 0.05) the feed conversion ratio (kilogram of feed/kilogram of egg) and decreased broken egg rate, and elevated (P < 0.05) the qualified chick rate. Compared with the ZnSO4 group, the 80mg Zn/kg Zn-Gly group significantly increased (P < 0.05) average egg weight, fertility, hatchability and qualified chick rate, whereas it decreased (P < 0.05) broken egg rate. The Zn concentrations in liver and muscle were significantly higher (P < 0.05) in 80mg Zn/kg Zn-Gly group than that in ZnSO4 group. Compared with ZnSO4 group, 80mg Zn/kg Zn-Gly group significantly elevated (P < 0.05) the mRNA abundances of metallothionein (MT) and copper-zinc superoxide (Cu-Zn SOD), as well as the Cu-Zn SOD activity and MT concentration in liver. Moreover, the 80mg Zn/kg Zn-Gly group had higher (P < 0.05) serum T-SOD and Cu-Zn SOD activities than that in the ZnSO4 group. This study indicated that supplementation of Zn in basal diet improved productive and reproductive performance, Zn concentration and antioxidant status in broiler breeders, and the 80mg Zn/kg from Zn-Gly was the optimum choice for broiler breeders compared with other levels of Zn from Zn-Gly and 80mg/kg Zn from ZnSO4.

Metal Accumulation and Metallothionein Response in Fucus Spiralis

Seaweeds are established sentinels for metal contamination and are utilised for biomonitoring. Metallothionein (MT) is a protein that is induced by metal exposure, and has been widely used as a biomarker for metal pollution. MT has not been reported in spiral wrack (Fucus spiralis), but has been identified in bladder wrack (Fucus vesiculosus), where it has been suggested as a protective mechanism against metal exposure. This study aimed to evaluate the potential use of MT in F. spiralis as a biomarker for metal pollution for the first time. Samples were collected from Poole Harbour, UK, over a year-long period, from January to October 2015. MT and metal concentrations were quantified during winter, spring, summer, and autumn seasons. Linear regression analysis showed few relationships between MT and metal concentrations, apart from in summer. During summer, significant positive relationships existed between MT concentrations and iron (R 2 = 0.631), nickel (R 2 = 0.486), tin (R 2 = 0.579), and lead (R 2 = 0.415). It is possible that for most of the year, metal concentrations in Poole Harbour are not high enough to elicit a MT response in F. spiralis, as it is a metal tolerant species. However, during summer, rates of photosynthesis and growth increase, which may increase metal toxicity, due to the inhibition of photosynthesis and growth. Thus, MT may be induced in order to prevent disruption. This study suggests that the use of MT as a biomarker for metal pollution in F. spiralis may not be a sensitive biomarker at low levels of metal pollution. However, MT concentrations in F. spiralis may respond to metal exposure when natural processes are vulnerable to pollution. The potential for MT to be used as a biomarker in Fucus spp. has been highlighted, warranting further research to develop a promising cosmopolitan bioindicator for metal pollution.

Early and efficient induction of antioxidant defense system in Mytilus galloprovincialis embryos exposed to metals and heat stress

The present study aims to elucidate the stress response of early life stages of Mytilus galloprovincialis to the combine effects of selected metals and elevated temperature.For this purpose, we investigated the response of a large panel of oxidative stress markers such as catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (GST) activities and lipid peroxidation (thiobarbituric acid reactive substrates (TBARS) concentration) and metallothionein accumulation (MT) as well as selected gene transcription level and metal accumulation in mussels larvae exposed to a sub-lethal concentration of Cu (9.54µg/L), Ag (2.55µg/L) and mixture of the two metals (Cu (6.67µg/L)+Ag (1.47µg/L)) along with a temperature gradient (18, 20 and 22°C) for 48h.Cu and Ag applied as single or mixture were differentially accumulated in mussel larvae according to the exposure temperature. Sod, cat, gst and mt-10 gene transcription levels showed an important increase in larvae exposed to Cu, Ag or to the mix compared to the control condition at 18°C. The same pattern but with higher induction levels was recorded in larvae co-exposed to metals at 20°C. At 22°C, a significant decrease in mRNA abundance of cat, gst and sod and a significant up-regulation of mts targets (mt10 and mt20) were observed. Resultssuggest that co-exposure to metals and moderate elevated temperature (20 and 22°C) significantly increased the antioxidant enzyme activities of catalase (CAT), and glutathione-S-transferase (GST) and caused an increase of metal and metallothionein concentrations. In contrast, no significant change in lipid peroxidation products measured as TBARS content was observed indicating a protective response of anti-oxidative system.This study provides first evidences of the early and efficient protective response of antioxidant defense mechanisms in mussel's early life stages facing in multi stressors situations.

Blood cell responses and metallothionein in the liver, kidney and muscles of bullfrog tadpoles, Lithobates catesbeianus, following exposure to different metals

The hematological parameters and metallothionein (MT) levels in the liver, kidney and muscles were measured in bullfrog tadpoles, Lithobates catesbeianus, following exposures to 1 μg L−1 of zinc (Zn), copper (Cu) and cadmium (Cd) alone or in combination (1:1 and 1:1:1) for 2 and 16 days. Metal accumulation occurred in all organs, with the highest values found in the kidney, followed by the muscles and liver. After exposure to isolated metals, the accumulation was in the following order: Cd > Zn > Cu in the liver and muscles and Cd > Cu > Zn in the kidney. Exposure to combined metals (Zn + Cu, Zn + Cd, Cu + Cd and Zn + Cu + Cd) revealed complex responses, such as metal accumulation increased or decreased over the exposure periods, suggesting possible competion at the uptake sites and/or metabolization and elimination processes in each organ. The MT concentration increased in the organs of tadpoles following metal exposure alone, mainly in the liver, for both periods. After the combined exposures, the MT levels were higher in the liver and muscles at 16 days, suggesting that the interaction between metals was additive, and the level was decreased in the kidney after 2 and 16 days of exposure. The whole blood hemoglobin content (Hb), red blood cell count (RBCs) and mean corpuscular hemoglobin (MCH) differed from the control groups after 2 and 16 days of exposure, showing changes in the improvement of oxygen transport. The number of lymphocytes increased, and the levels of neutrophils, eosinophils, basophils and monocytes were reduced after exposure to the metals. The changes in blood cells suggested that tadpoles have a mechanism to improve oxygen transport probably because of the increased oxygen demand and a general reduction in defense cells. The exposure of L. catesbeianus to metals during the larval phase can generate long-term dysfunction to a degree, which could lead to alterations in their health status.

Metallothionein and Glutathione Content as Biomarkers of Metal Pollution in Mussels and Local Fishermen in Abu Qir Bay, Egypt.

Background.When heavy metals accumulate in air, soil, and water, the risk of human exposure increases among industrial workers, as well as in people living near polluted areas. Heavy metals adversely affect a variety of bodily systems such as the cardiovascular, respiratory, endocrine, immune, and reproductive systems. In addition, long-term exposure and accumulation of heavy metals in the body may disturb oxidative stress genes and thus increase the susceptibility to various diseases.Objectives.The aim of this study is to estimate the metallothionein concentration in both mussel samples from Abu Qir Bay, Egypt and the blood of local fishermen as a biomarker of exposure to metal pollution.Methods.Levels of metallothionein and heavy metals were measured in mussels. Blood levels of metallothionein and heavy metals of local fishermen were measured and compared with a control group. The effect of heavy metal exposure on oxidative stress status was investigated through the determination of malondialdehyde (MDA), catalase and glutathione content.Results.The results of this study showed high concentrations of metallothionein in mussels and in fishermen's blood, accompanied by high concentrations of metals such as cadmium (Cd), copper (Cu), lead (Pb), chromium (Cr), and zinc (Zn). At the same time, a significant decrease in glutathione content and catalase enzyme activity was associated with a significant increase in the malondialdehyde concentrations in sera of fishermen.Conclusions.The present study found that the El Maadiya region is polluted with heavy metals, inducing oxidative stress in fishermen in the vicinity. These results reveal the necessity of further environmental monitoring in the study area in order to evaluate other types of pollutants and their effects on human health.

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.