Cd-exposed Mussels Research Articles

Toxicology assessment of deep-sea mining impacts on Gigantidas platifrons: A comparative in situ and laboratory metal exposure study

Deep-sea toxicology is essential for deep-sea environmental impact assessment. Yet most toxicology experiments are conducted solely in laboratory settings, overlooking the complexities of the deep-sea environment. Here we carried out metal exposure experiments in both the laboratory and in situ, to compare and evaluate the response patterns of Gigantidas platifrons to metal exposure (copper [Cu] or cadmium [Cd] at 100 μg/L for 48 h). Metal concentrations, traditional biochemical parameters, and fatty acid composition were assessed in deep-sea mussel gills. The results revealed significant metal accumulation in deep-sea mussel gills in both laboratory and in situ experiments. Metal exposure could induce oxidative stress, neurotoxicity, an immune response, altered energy metabolism, and changes to fatty acid composition in mussel gills. Interestingly, the metal accumulating capability, biochemical response patterns, and fatty acid composition each varied under differing experimental systems. In the laboratory setting, Cd-exposed mussels exhibited a higher value for integrated biomarker response (IBR) while in situ the Cu-exposed mussels instead displayed a higher IBR value. This study emphasizes the importance of performing deep-sea toxicology experiments in situ and contributes valuable data to a standardized workflow for deep-sea toxicology assessment.

Exposure to low environmental concentrations of manganese, lead, and cadmium alters the serotonin system of blue mussels.

Serotonin plays a crucial role in mussel survival and reproduction. Although the serotonin system can be affected by metals, the effects of environmental concentrations of metals such as manganese (Mn), lead (Pd), and cadmium (Cd) have never been studied in blue mussels. The present study aimed to determine the effects of exposure to Mn, Pb, or Cd on serotonin levels, monoamine oxidase (MAO) activity, and serotonin transporter (SERT) levels in the blue mussel Mytilus edulis. Mussels were exposed in vivo to increasing and environmentally relevant doses of Mn (10-1000 nM; 0.5-50 μg/L), Pb (0.01-10 nM; 0.002-2 μg/L), or Cd (0.01-10 nM; 0.001-1 μg/L) for 28 d. Serotonin levels, MAO activity, and SERT expression were analyzed in the mussel mantle. Expression of SERT protein was significantly decreased, by up to 81%, following Mn, Pb, or Cd exposure. The activity of MAO in females was almost 2-fold higher, versus males, in nonexposed control mussels. In mussels exposed to 0.1 nM of Pb (0.02 μg/L), MAO activity was increased in males and decreased in females. In Cd-exposed mussels, a sex-dependent, inverted nonmonotonic pattern of MAO activity was observed. These results clearly indicate that low environmental concentrations of Mn, Pb, and Cd affect the serotonin system in blue mussels. Environ Toxicol Chem 2018;37:192-200. © 2017 SETAC.

Lysosomal responses to heat-shock of seasonal temperature extremes in Cd-exposed mussels

The present study was aimed at determining the effect of temperature extremes on lysosomal biomarkers in mussels exposed to a model toxic pollutant (Cd) at different seasons. For this purpose, temperature was elevated 10°C (from 12°C to 22°C in winter and from 18°C to 28°C in summer) for a period of 6h (heat-shock) in control and Cd-exposed mussels, and then returned back to initial one. Lysosomal membrane stability and lysosomal structural changes in digestive gland were investigated. In winter, heat-shock reduced the labilisation period (LP) of the lysosomal membrane, especially in Cd-exposed mussels, and provoked transient lysosomal enlargement. LP values recovered after the heat-shock cessation but lysosomal enlargement prevailed in both experimental groups. In summer, heat-shock induced remarkable reduction in LP and lysosomal enlargement (more markedly in Cd-exposed mussels), which recovered within 3 days. Besides, whilst heat-shock effects on LP were practically identical for Cd-exposed mussels in winter and summer, the effects were longer-lasting in summer than in winter for control mussels. Thus, lysosomal responsiveness after heat-shock was higher in summer than in winter but recovery was faster as well, and therefore the consequences of the heat shock seem to be more decisive in winter. In contrast, inter-season differences were attenuated in the presence of Cd. Consequently, mussels seem to be better prepared in summer than in winter to stand short periods of abrupt temperature change; this is, however, compromised when mussels are exposed to pollutants such as Cd.

Combined effects of thermal stress and Cd on lysosomal biomarkers and transcription of genes encoding lysosomal enzymes and HSP70 in mussels, Mytilus galloprovincialis

In estuaries and coastal areas, intertidal organisms may be subject to thermal stress resulting from global warming, together with pollution. In the present study, the combined effects of thermal stress and exposure to Cd were investigated in the endo-lysosomal system of digestive cells in mussels, Mytilus galloprovincialis. Mussels were maintained for 24h at 18°C and 26°C seawater temperature in absence and presence of 50μg Cd/L seawater. Cadmium accumulation in digestive gland tissue, lysosomal structural changes and membrane stability were determined. Semi-quantitative PCR was applied to reveal the changes elicited by the different experimental conditions in hexosaminidase (hex), β-glucuronidase (gusb), cathepsin L (ctsl) and heat shock protein 70 (hsp70) gene transcription levels. Thermal stress provoked lysosomal enlargement whilst Cd-exposure led to fusion of lysosomes. Both thermal stress and Cd-exposure caused lysosomal membrane destabilisation. hex, gusb and ctsl genes but not hsp70 gene were transcriptionally up-regulated as a result of thermal stress. In contrast, all the studied genes were transcriptionally down-regulated in response to Cd-exposure. Cd bioaccumulation was comparable at 18°C and 26°C seawater temperatures but interactions between thermal stress and Cd-exposure were remarkable both in lysosomal biomarkers and in gene transcription. hex, gusb and ctsl genes, reacted to elevated temperature in absence of Cd but not in Cd-exposed mussels. Therefore, thermal stress resulting from global warming might influence the use and interpretation of lysosomal biomarkers in marine pollution monitoring programmes and, vice versa, the presence of pollutants may condition the capacity of mussels to respond against thermal stress in a climate change scenario.

Waterborne cadmium impacts immunocytotoxic and cytogenotoxic endpoints in green-lipped mussel, Perna canaliculus

Mussels are sentinel species that can be used to monitor coastal metal pollution through the application of biomarkers. Among the several important metal toxicants in coastal settings, cadmium (Cd) is of particular concern, being a non-essential metal that is known to cause harmful impacts in aquatic organisms at low concentrations. The aim of the present study was to examine the immunocytotoxic and cytogenotoxic effects of Cd on the green-lipped mussel, Perna canaliculus, under laboratory conditions. The acute (96h; 0, 2000 and 4000μg CdL−1) and subchronic (28d; 0, 200 and 2000μg CdL−1) toxic effects of waterborne Cd were measured in haemocytes and gill cells using differential haemocyte cell count, the micronucleus test and the comet assay. During subchronic exposure to Cd the relative counts of eosinophils and hyalinocytes increased significantly in Cd-exposed mussels while the proportion of basophils decreased. All of these effects were time- and concentration-dependent. Conversely, the relative numbers of basophils and eosinophils increased significantly during acute Cd exposure. Nuclear aberrations such as the formation of micronuclei, nuclear buds, fragmented-apoptotic cells and binuclei were observed in gill cells of Cd-exposed mussels. All of these parameters increased significantly at 2000μg CdL−1 during subchronic exposure to Cd, and all showed a strong and significant correlation to gill Cd accumulation. Comet assay results demonstrated a significant increase in DNA damage in the haemocytes of mussels exposed to subchronic Cd concentrations. The results indicate that Cd has the capacity to induce immune system and genotoxic damage in green-lipped mussels, an impact that may have implications such as reduced disease resistance and compromised survival. These data also suggest that immunocytotoxic and cytogenotoxic biomarkers would be a valuable addition to environmental monitoring programmes using green-lipped mussels.

Biochemical biomarker responses of green-lipped mussel, Perna canaliculus, to acute and subchronic waterborne cadmium toxicity

The biochemical responses of the green-lipped mussel, Perna canaliculus, to waterborne cadmium (Cd) were investigated in order to delineate toxic mechanisms, and the impacts of exposure dose and duration, of this important toxicant in a potential sentinel species. Mussels were exposed for either 96h (acute: 0, 2000, 4000μgL−1 Cd) or for 28d (subchronic: 0, 200, 2000μgL−1 Cd), and the digestive gland, gill and haemolymph were examined for impacts. Biochemical responses measured included those associated with metal detoxification (metallothionein-like protein; MTLP), oxidative stress (catalase, lipid peroxidation), cellular homeostasis (alkaline phosphatase, Na+, K+-ATPase; NKA), and energy utilisation (glycogen, haemolymph protein). Following acute exposure, digestive gland glycogen and gill NKA activity were significantly altered by Cd exposure relative to levels in mussels exposed to Cd-free seawater. Subchronic Cd exposure resulted in a significant increase in MTLP levels in both the gill and the digestive gland. This increase was correlated strongly with the levels of Cd accumulation measured in these tissues (R=0.957 for gill, 0.964 for digestive gland). Catalase activity followed a similar pattern, although the correlation with tissue Cd accumulation was not as strong (R=0.907 for gill, 0.708 for digestive gland) as that for MTLP. Lipid peroxidation increased in the digestive gland at Days 7 and 14 at both subchronic Cd levels tested, but this effect had largely dissipated by Days 21 and 28 (with the exception of the 2000μgL−1 group at Day 28). Alkaline phosphatase activity decreased significantly with Cd exposure in both tissues. This effect was observed at both tested concentrations in the gill, but only at the highest concentration for digestive gland. A decrease in digestive gland glycogen levels was observed in Cd-exposed mussels (Days 14 and 21 at 2000μgL−1), while haemolymph protein levels increased as a result of subchronic Cd exposure. These findings indicated that biochemical responses in Cd-exposed mussels were tissue-specific, dose- and time-dependent, with duration of exposure being the predominant effect. This study shows that biochemical changes in Cd-exposed green-lipped mussels can be linked to tissue metal accumulation and are consistent with previously reported physiological effects. It also suggests that green-lipped mussels are amenable to a multiple biomarker approach and may be of use as a bioindicator species for monitoring coastal metal pollution.

Toxic Effects of Copper, Cadmium, and Methoxychlor Shown by Neutral Red Retention Assay in Two Species of Freshwater Molluscs

We used neutral red retention assay in lysosomes of digestive gland cells as an indicator for stress effects by the environmental contaminants Cu, Cd, and the pesticide methoxychlor in two freshwater molluscs, the unionid mussel, Elliptio complanata (Lightfoot) and the ramshorn snail Helisoma trivolvis (Say). Mussels and snails were exposed for 7 and 14 days to Cu and Cd each at nominal concentrations of 2.5 µg/L, 5.0 µg/L, and 10.0 µg/L, and to methoxychlor concentrations of 1.0 µg/L, 10.0µg/L, and 100.0 µg/L. Both mussels and snails exposed to Cu showed a significant increase in the percent of destabilized lysosomes compared with lab control and freshly-collected (field control) mussels at both 7 and 14 days exposure for all concentrations. Cd-exposed mussels did not show a significant difference with either of the controls at 7 days, but at 14 days exposure, Cd significantly increased the percent of destabilized lysosomes at all concentrations compared to field control mussels. Compared to laboratory controls, Cd increased lysosomal destabilization at 5.0 µg/L and 10.0 µg/L. Snails exposed to Cd for 7 days had a significantly higher percentage of lysosomal destabilization than both lab and field controls but at 14 days, significant differences were only seen at the two highest Cd concentrations. Methoxychlor-exposed mussels showed no significant lysosomal destabilization at 7 days compared to controls. But at 14 days exposure, the pesticide increased the percentage of lysosomal destabilization at 10.0µg/L compared to lab controls, and increased at both 10.0µg/L and 100.0µg/L compared to field control mussels. Methoxychlor-exposed snails had a higher percentage of lysosomal destabilization at 7 and 14 days at all concentrations compared to both controls with the exception of the 1.0 µg/L -7 day exposure group. Snails were more sensitive to Cd and to methoxychlor than were mussels possibly because they lack an operculum and are thus completely exposed to the environment. The lowest observed effect concentration (LOEC) for Cd was 2.5 µg/L (14 days) for Elliptio and 2.5 µg/L (7 and 14 days) for Helisoma. For methoxychlor, the LOEC was 10.0 µg/L (14 days) for Elliptio and 10.0 and 1.0 µg/L (7 and 14 days, respectively) for Helisoma. The LOEC for Cu was 2.5 µg/L (7 and 14 day exposure) for both Elliptio and Helisoma. These results show that lysosomal destabilization as indicated by neutral red retention is a reliable indicator of stress from heavy metals and a pesticide in freshwater molluscs, including a taxon that is endangered or threatened in North America.

Pyruvate Kinase, Phosphoenolpyruvate Carboxykinase, Cytochrome C Oxidase and Catalase Activities in Cadmium ExposedPerna viridisSubjected to Anoxic and Aerobic Conditions

ABSTRACT This work concerns the effects of cadmium exposure on the anaerobic and oxidative metabolism in tissues of the hypoxia-tolerant green mussel Perna viridis from Venezuelan coastal waters. The mussels were exposed to different Cd concentrations (0, 20, 40 and 60 µg/L Cd) for 7 days, and were subsequently exposed to anoxia. Prior to anoxia exposure, Cd concentration was determined in the digestive gland and adductor muscle. After 24 h of anoxia, the activities of pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (PEPCK), and cytochrome c oxidase (CCO), and catalase (CAT) were measured in the digestive gland and adductor muscle from Cd-treated mussels. Also, effects of the Cd treatments on the median survival time (LT50) during anoxia were determined. The short-term Cd-exposed mussels augmented the metal uptake in the tested tissues in a dose-dependent manner, associated with a concomitant reduction in the ability to withstand anoxia. During normoxic and anoxic conditions, the Cd-cxposed indivi...

Cadmium induction of metallothionein isoforms in juvenile and adult mussel (Mytilus edulis).

Isoforms of metallothionein in the digestive gland of control and experimentally Cd-exposed mussels (Mytilus edulis) (200 microg L(-1) Cd2+ and 400 microg L(-1) Cd2+; 20 days) were studied using the reverse transcriptase-polymerase chain reaction. In addition, glutathione S-transferase (GSTpi) primers were designed to evaluate the reduction in the antioxidant defense systems (glutathione) accompanying the aging process in the same organisms. Following experimental exposure, an accumulation of Cd was observed in the digestive gland of exposed mussels, both adults and juveniles, up to 500 times higher than in the control. An induction of the dimeric form MT20 II was detected in 400 microg L(-1) exposed mussels, as well as a visible inhibition of the monomeric form MT10 IV. After 20 days of exposure juveniles expressed increased GSTpi compared with adults. Results reveal individual variation of both metallothioneins and GSTpi expression among control and Cd2+-exposed mussels of different ages. The ecotoxicological significance of MT utilization in biomonitoring of seawater for trace metals has been considered in light of these results.

Multiple forms of metallothionein from the digestive gland of naturally occurring and cadmium-exposed mussels, Mytilus galloprovincialis

Polymorphism of metallothioneins in the digestive gland of naturally occurring (control) and experimentally Cd-exposed mussels Mytilus galloprovincialis (200 µg Cd l–1; 14 days) was studied by applying the conventional methods of Sephadex column liquid chromatography (G-75 and DEAE A-25), and by an electrochemical method (DPASV) for determination of Cd, Zn and Cu concentrations in chromatographic fractions. In both control and Cd-exposed mussels, two distinct molecular mass components of the metallothioneins, monomeric (MT-10) and dimeric (MT-20), were resolved by Sephadex G-75 gel filtration chromatography. In control mussels, the MT-10 component was predominantly expressed as containing markedly higher constitutive levels of Zn (100×) and Cu (10×) than of Cd. Each of these two molecular mass components was further resolved into seven metal-rich peaks by anion-exchange chromatography. In Cd-exposed mussels the larger proportion of Cd was bound to the MT-20 than to the MT-10 component, suggesting that the dimeric component may be considered as a primarily inducible metallothionein. The elution positions of metal-binding maxima of Cd-exposed and control mussels on the respective DEAE chromatographic profiles were comparable. A great similarity in elution positions of Cd maxima between the composite and single-specimen samples was also observed. Our study confirms a high multiplicity of MT forms in mussels from the Mytilus genus not only under the laboratory high-level metal exposure conditions, but also at a natural seawater metal exposure level. The ecotoxicological significance of dimeric and monomeric MT forms, as well as their possible application in the biomonitoring of seawater for trace metals, has been considered.

Subcellular distribution of cadmium and its cellular ligands in mussel digestive gland cells as revealed by combined autometallography and X-ray microprobe analysis.

Autometallography (AMG) and electron probe X-ray microanalysis (EPXMA) were applied in combination to determine the subcellular distribution of Cd and its subcellular ligands in the digestive gland cells of Cd-exposed mussels Mytilus galloprovincialis. Black silver deposits (BSD), which reveal the presence of metals when AMG is applied, were only localized in digestive cell lysosomes. Digestive cell cytoplasm and basophilic cells were devoid of BSD. EPXMA (static probe and X-ray mapping) indicated that Cd, S (possibly associated with metallothioneins or metallothionein-like proteins) and autometallographical Ag ions are co-localized within digestive cell lysosomes. In addition, Cd and S co-occur in the absence of Ag in the cytosol of digestive cells. AMG does not reveal the presence of the Cd 'pool' strongly bound to cytosolic Cd-metallothionein complexes; only 'free' Cd or Cd supposedly loosely bound to (semi)digested metallothionein within lysosomes was revealed. The levels of lysosomal Cd were indirectly quantified by stereology as the volume density of BSD (V(v)BSD). Significantly higher values were recorded in Cd-exposed mussels compared with controls at all exposure times. However, V(v)BSD values were lower at days 7 and 21 than at day 1. This relative decrease in V(v)BSD reflected another (and confounding) response elicited by Cd-exposure in the digestive epithelium: the volume density of basophilic cells (V(v)BAS) increased significantly as exposure progressed. Due to this cell-type replacement, the net accumulative capacity of the digestive epithelium decreases at long exposure times.

Effect of Cd on accumulation and loss of 210 Po in the mussel Mytilus edulis

As a result of 210Po's previously identified association with sulphur-rich proteins, metallothioneins could have a significant effect on the behaviour and fate of 210Po in molluscs. Starved control and cadmium-exposed mussels, Mytilus edulis, were fed 210Po-labelled algae (Isochrysis galbana) for 5 d and then allowed to depurate in clean sea water. Cadmium-exposed M. edulis accumulated less 210Po in the digestive gland and the remainder of the tissue than control mussels, although this was due to a decrease in tissue weight. More than 40% of 210Po was identified as being associated with high molecular weight and heat-treated cytosol proteins in M. edulis. Mussels in a starved state are known to recycle as much as 90% of their amino acids. It is proposed that 210Po associated with these and other proteins is recycled, explaining why no significant loss of 210Po was observed from the remainder of the tissue in either control or Cd-exposed mussels. Cadmium-induced metallothioneins had no effect on the distribution of 210Po in M. edulis; <5% associated with the cytosolic fraction was considered to principally contain metallothioneins. It is suggested that 210Po's apparent relationship with metallothioneins is coincidental rather than connected with its role in the regulation of metals.

BSD Extent, an Index for Metal Pollution Screening Based on the Metal Content within Digestive Cell Lysosomes of Mussels as Determined by Autometallography

The extent of autometallographical black silver deposits (BSD) has been semiquantified at the light microscope in the gills and digestive gland of either control mussels or Zn-polluted mussels after depuration and on exposure to sublethal concentrations of Cu, Zn, and Cd. The BSD extent in the gills and digestive gland of control mussels was much reduced compared to that in other experimental mussels. The extent of BSD in the gills of depurating mussels was reduced at short depuration times due to decreased levels in the abfrontal cells while in the digestive gland it did not change with the depuration period. The extent of BSD in digestive lysosomes of Cu- and Zn-exposed mussels followed a logarithmic pattern in relation to metal concentration increasing with metal concentrations in the digestive gland. However, a reduced extent of BSD was related to the presence of high metal concentrations under Cd-exposure conditions. This is because the great extent of BSD present in the lumen of the digestive tubules was not taken into account to carry out semiquantification, but, however, the chemical analysis measured the Cd content of these BSD. As such, the extent of BSD in digestive lysosomes followed a logarithmic pattern with total metal concentrations in the digestive gland of Cd-exposed mussels. Therefore, the semiquantitative estimation of BSD in the digestive lysosomes could be considered a reliable index to reflect changes in metal bioavailability in sea water.

Tissue and cell distribution of copper, zinc and cadmium in the mussel, Mytilus galloprovincialis, determined by autometallography

The localization of metals in selected tissues of metal-exposed mussels was investigated by means of autometallography. Mussels collected from a Zn-polluted site were (a) depurated or, alternatively, (b) exposed to either Cu, Zn or Cd for 41 d. Mussels collected from a clean site were used as experimental reference. Autometallographically demonstrated black silver deposits (BSD), indicating the presence of metals, were observed in gills, (a) in frontal cells and haemocytes of Cu-exposed mussels, (b) in secretory postlateral and abfrontal cells and in endothelial cells of Zn-exposed mussels, and (c) in frontal, postlateral and endothelial cells but mainly in abfrontal cells and haemocytes of Cd-exposed mussels. Autometallography also revealed the presence of BSD in connective tissue brown cells underlying the mantle. Additionally, adipogranular cells of the connective tissue surrounding the gonad follicles were positively stained but no BSD was found in gonad tissue. Scarce BSD were found in the cytoplasmic granules of the stomach wall of control mussels whilst depurating and metal-exposed mussels exhibited BSD lining the apex of the stomach epithelial cells. Contrary to the results previously obtained with marine gastropod molluscs, BSD were not found in the basal lamina of digestive tubules, stomach, and ducts of mussels. Highly conspicuous BSD were observed in digestive cell lysosomes of depurating and metal-exposed mussels. Basophilic cells were always devoid of BSD. Finally, BSD were also found in nephrocyte lysosomes and mineralized concretions.

Metallothioneins and lysosomes in metal toxicity and accumulation in marine mussels: the effect of cadmium in the presence and absence of phenanthrene

It has been confirmed that metallothioneins play an important role in the accumulation of cadmium (Cd) in the digestive gland cells of mussels (Mytilus galloprovincialis Lam.). The content of Cd in the tissue of mussels exposed for 9 d to the metal (estimated dosage of 180 μg Cd mussel-1 d-1) was 66.2 ppm. This value is about the same as the metal content found in the digestive gland of Cd-exposed mussels kept in clean water for a recovery period of 28 d. At the end of the recovery period, however, the Cd bound to thionein had increased by approximately 250%. Our data demonstrate that the stability of lysosomes, a biological parameter adopted as a cellular stress index, is extremely low in mussels exposed to Cd for 9 d, but returns to control values in the digestive gland cells of mussels allowed to recover for 28 d in uncontaminated sea water. At this point most of the Cd present in the cytosol is bound to thionein. These data demonstrate the importance of metallothionein induction in the reduction of the cytotoxic effects exerted by high levels of Cd accumulation. The results of tests designed to clarify the reasons for the long biological half-life of Cd demonstrated that, in the digestive gland of mussels, the lysosomes are not able to eliminate Cd either bound to insoluble thionein polymers or to lipid peroxidation products such as lysosomal lipofuscin, both of which are apparently involved in the elimination of copper. The absence of these two mechanisms of metal sequestration and elimination via excretion of residual bodies (tertiary lysosomes) is in agreement with the persistence of cadmium in the digestive gland of mussels. Finally, the results also demonstrate that simultaneous exposure of mussels to Cd and phenanthrene, an established lysosomal membrane destabilizer, did not significantly alter the accumulation of Cd or the kinetics of the metal in mussels.