In the present study, we investigated the progressive acclimation of the mussel
Mytilus galloprovincialis to different reduced seawater (SW) salinities and its effect on several biochemical markers ...and biotests. Mussels were purchased from a local mariculture facility during summer (SW temperature 27
°C, salinity 37.5
psu) and winter (13
°C, 37
psu) seasons, and transferred to the laboratory for acclimation to reduced SW salinities (37, 28, 18.5 and 11
psu). At the beginning and at the end of acclimation processes tests of mussel survival in air were provided. After 14 days of acclimation the DNA integrity, p38-MAPK activation, metallothionein induction, oxygen consumption rate, and condition index were measured. Survival in air (SOS test), as a physiological index of mussel's health and vitality, had significantly lower LT50 values (11
psu) in the summer than in the winter, and it seems to be negatively affected by acclimation in comparison to controls (37
psu and mariculture). Condition indexes (CIs) were not significantly different, but mussel's acclimation resulted in decline (i.e., a negative trend), especially of CI-2 and CI-3 calculated on the basis of mussel tissue weight and shell sizes. Oxygen consumption rate (
V
O
2
) of
M. galloprovincialis acclimated to reduced salinities was a concentration-dependent process and increased considerably to about 51 and 65% in lower SW concentrations (28 and 18
psu) compared to control mussels (37
psu). DNA integrity, determined by Fast Micromethod
®, was negatively impacted by salinity acclimation and corresponding physiological stress as well. Some differences in 1D protein expression patterns between control groups and mussels acclimated to 28, 18.5 and 11
psu (SW) were established. Reduced SW salinities (18.5 and 11
psu) resulted in significantly higher p38-MAPK phosphorylation, whereas the SW salinity of 28
psu decreased p-p38 significantly compared to control (37
psu). The concentration of metallothioneins in mussels’ gills was reduced at 28 and 18.5
psu, while it was significantly higher at 11
psu. Results indicated that SW salinity variation (i.e., hypoosmotic stress) in the marine environment can affect all investigated parameters. This investigation expands our understanding of multifactorial effects of the physical marine environment on the specificity of investigated biomarkers and biotests, providing insight into the acclimation, adaptive and stress response processes of mussels. Effects of environmental factors have to be considered in sampling strategies for monitoring programmes to prevent false interpretation of results.
A study performed over 12 months with caged mussels
Mytilus galloprovincialis in the coastal marine zone, which is under urban pressure, reveals a temporal variation of digestive gland mass, which ...causes “biological dilution” of cytosolic metallothionein (MT) and trace metal (Cd, Cu, Zn, Fe, Mn) concentrations. The dilution effect was corrected by expressing the cytosolic MT and metal concentrations as the tissue content. Consequently, the changes of the average digestive gland mass coincide with the changes of MT and trace metal contents. From February to June, MT contents are nearly twice and trace metal contents nearly three times higher than those of the other months. The period of increased average digestive gland mass, of MT and trace metal contents probably overlaps with the sexual maturation of mussels (gametogenesis) and enhanced food availability. Since natural factors contribute more to the MT content than the sublethal levels of Cd, the digestive gland of
M. galloprovincialis is not considered as a tissue of choice for estimating Cd exposure by means of MTs.
Several different procedures were applied to purify metallothionein (MT) isolated from the digestive glands of the mussels (
Mytilus galloprovincialis) exposed to cadmium: heat treatment (at 70 and ...85
°C), solvent precipitation, and gel-filtration. Using electrochemical method, the MT contents were determined. Based on the obtained results each procedure was statistically evaluated. Application of the post-hoc Scheffé test showed that there were significant differences between all the treatments applied. Heat treatment and solvent precipitation effectively remove high molecular weight (HMW) proteins from the samples. In untreated as well as in heat or solvent treated samples MT10 isoform remains unchanged. MT20 isoform is significantly reduced by heat treatment and drastically reduced by solvent precipitation. Due to the fact that MT20 is considered as ‘target’ MT isoform for metal effect and exposure it is recommended to use the purification procedure which less affects MT20 fraction. According to the presented results it is the heat treatment.
A stock of mussels
Mytilus galloprovincialis was transplanted over 1 year to four sites in a semi-enclosed bay in Croatia which is under the influence of various sources of pollution. The positive ...correlation of metal (Cd, Zn, Cu, Mn, Fe) and metallothionein (MT) tissue contents based on the analysis of heat-treated cytosol of gills with shell mass, as an indicator of mussel age, indicated to accumulation of metals, as well as the increase of MTs with mussel age. The principal component analysis (PCA) revealed that 74% of total variance of obtained results could be explained through two principal components. The first principal component was highly correlated with MT, Cd and Zn indices (metal or MT content/shell mass), and the second one with Cu, Mn, and Fe indices, as well as the gill index. High correlation of MTs with Zn and Cd is consistent with their affinity for binding to MTs.
The influence of the biometric parameters (shell mass, whole soft tissue mass, condition index) on MT and metal levels in the heat-treated cytosol of the whole soft tissue of transplanted mussels was ...studied over the period of one year. The positive correlation of three metals (Cd, Fe, Zn) with the shell mass indicated to time-dependent increase of their contents. Strong correlation of Mn content with the whole soft tissue mass (r = 0.74, p < 0.0001), and almost identical changes of these two parameters over the year make Mn a good indicator of mussel's condition. As opposed to the other metals, Cu content does not exhibit connection with biometry. On the other hand, MTs are highly influenced by biometry. As much as 65% of their variability could be explained by the changes of the shell mass and the whole soft tissue mass. Consequently, it is difficult to distinguish if the obtained positive correlation between Cd and MTs (r = 0.48, p < 0.05) reflects MT induction by Cd, or Cd accumulation as a result of age-dependent increase of MTs. Due to the strong influence of the biometry on MT level, the whole soft tissue is not considered as the best choice for measuring MTs as a biomarker. Better option would be to isolate a specific tissue that shows indisputable connection between MT induction and metal accumulation.
Since fish metalloproteins are still not thoroughly characterized, the aim of this study was to investigate the acidic/basic nature of biomolecules involved in the sequestration of twelve selected ...metals in the soluble hepatic fraction of an important aquatic bioindicator organism, namely the fish species northern pike (Esox lucius). For this purpose, the hyphenated system HPLC-ICP-MS was applied, with chromatographic separation based on anion/cation-exchange principle at physiological pH (7.4). The results indicated predominant acidic nature of metal-binding peptides/proteins in the studied hepatic fraction. More than 90 % of Ag, Cd, Co, Cu, Fe, Mo, and Pb were eluted with negatively charged biomolecules, and >70 % of Bi, Mn, and Zn. Thallium was revealed to bind equally to negatively and positively charged biomolecules, and Cs predominantly to positively charged ones. The majority of acidic (negatively charged) metalloproteins/peptides were coeluted within the elution time range of applied standard proteins, having pIs clustered around 4–6. Furthermore, binding of several metals (Ag, Cd, Cu, Zn) to two MT-isoforms was assumed, with Cd and Zn preferentially bound to MT1 and Ag to MT2, and Cu evenly distributed between the two. The results presented here are the first of their kind for the important bioindicator species, the northern pike, as well as one of the rare comprehensive studies on the acidic/basic nature of metal-binding biomolecules in fish, which can contribute significantly to a better understanding of the behaviour and fate of metals in the fish organism, specifically in liver as main metabolic and detoxification organ.
•Important new contribution to better understanding of metal behaviour/fate in fish•Predominant acidic nature of cytosolic metalloproteins in the northern pike liver•Tl and Cs notably bound to basic biomolecules•Ag, Cd, Cu, and Zn presumably bound to two MT-isoforms•Cd/Zn preferentially bound to MT1, Ag to MT2, Cu evenly distributed between the two
Iron plays vital roles in important biological processes in fish, but can be toxic in high concentrations. The information on metalloproteins that participate in maintenance of Fe homeostasis in an ...esocid fish, the northern pike, as an important freshwater bioindicator species, are rather scarce. The aim of this study was to identify main cytosolic constituents that sequester Fe in the northern pike liver. The method applied consisted of two-dimensional HPLC separation of Fe-binding biomolecules, based on anion-exchange followed by size-exclusion fractionation. Apparent molecular masses of two main Fe-metalloproteins isolated by this procedure were ~360 kDa and ~50 kDa, with the former having more acidic pI, and indicated presence of ferritin and hemoglobin, respectively. MALDI-TOF-MS provided confirmation of ferritin subunit with a m/z peak at 20.65 kDa, and hemoglobin with spectra containing main m/z peak at 16.1 kDa, and smaller peaks at 32.1, 48.2, and 7.95 kDa (single-charged Hb-monomer, dimer, and trimer, and double-charged monomer, respectively). LC-MS/MS with subsequent MASCOT database search confirmed the presence of Hb-β subunits and pointed to close relation between esocid and salmonid fishes. Further efforts should be directed towards optimization of the conditions for metalloprotein analysis by mass spectrometry, to extend the knowledge on intracellular metal-handling mechanisms.Iron plays vital roles in important biological processes in fish, but can be toxic in high concentrations. The information on metalloproteins that participate in maintenance of Fe homeostasis in an esocid fish, the northern pike, as an important freshwater bioindicator species, are rather scarce. The aim of this study was to identify main cytosolic constituents that sequester Fe in the northern pike liver. The method applied consisted of two-dimensional HPLC separation of Fe-binding biomolecules, based on anion-exchange followed by size-exclusion fractionation. Apparent molecular masses of two main Fe-metalloproteins isolated by this procedure were ~360 kDa and ~50 kDa, with the former having more acidic pI, and indicated presence of ferritin and hemoglobin, respectively. MALDI-TOF-MS provided confirmation of ferritin subunit with a m/z peak at 20.65 kDa, and hemoglobin with spectra containing main m/z peak at 16.1 kDa, and smaller peaks at 32.1, 48.2, and 7.95 kDa (single-charged Hb-monomer, dimer, and trimer, and double-charged monomer, respectively). LC-MS/MS with subsequent MASCOT database search confirmed the presence of Hb-β subunits and pointed to close relation between esocid and salmonid fishes. Further efforts should be directed towards optimization of the conditions for metalloprotein analysis by mass spectrometry, to extend the knowledge on intracellular metal-handling mechanisms.
The possible influence of historical contamination of water/sediments on the metal(loid) bioaccumulation in the digestive gland of mussel Unio crassus Philipsson, 1788, from two differently ...contaminated sites at the Mrežnica River was studied in three seasons. The first data for this species on total/cytosolic concentrations of 27 (non)essential elements were obtained by HR ICP-MS. Higher bioaccumulation was observed at the historically contaminated site, with several nonessential elements (Bi, Cs, Pb, Sb, Tl, U) found in 5–6 times higher concentrations compared to the reference site. Although both total and cytosolic levels revealed the influence of water/sediment contamination, the latter showed association between bioaccumulation and exposure for larger number of studied elements. At the reference site, several elements (Ba, Ca, Cd, Cr, Mn, Sr) were also found in 2–10 times higher concentrations compared to contaminated one, but it was attributed to background levels characteristic for karst rivers (for Ca and Cd), and to coaccumulation due to chemical similarity (for Ba, Cr, Mn, Sr). The seasonal variability was also observed, with generally highest metal(loid) concentrations in mussel digestive glands found in autumn which was associated to mussels reproductive period. Our results confirmed that sediment-dwelling mussels, specifically U. crassus, represent a good bioindicators for detection of historical pollution due to their direct contact/exposure to contaminants stored in sediments, with concurrent consideration of physiological/chemical factors. Historical contamination potentially can have serious impact on freshwater environment even long time after its cessation, and, therefore, a careful continuous monitoring is recommended.
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•First report on total/cytosolic metal(loid) accumulation for Unio crassus mussels.•Mussel digestive gland – reliable detection of historical freshwater contamination.•Cytosolic levels reveal metal(loid) bioavailability better than total tissue loads.•Importance of considering metal coaccumulation due to element chemical similarity.•Seasonal changes of metal loads in digestive gland caused by reproductive cycle.
•Testosterone significantly increases MN frequencies in L929 cells.•Nuclear division index is higher after 48h than 24h of incubation with testosterone.•A benchmark doses for MN and nuclear division ...index differ.
Data on the possible genotoxic effects of testosterone are limited: in particular, the potential clastogenic and/or aneugenic effects have not yet been properly investigated. An in vitro micronucleus (MN) assay was performed on L929 cells exposed to testosterone at doses of 10, 15, 20, 30, and 40μg/mL. Significantly increased MN frequencies were detected at doses of 20, 30, and 40μg/mL after 24h and 48h of incubation. The nuclear division index was higher after 48h than 24h of incubation. A benchmark dose (BMD) calculation was used to estimate the 1% extra risk level for MN and increased tetranucleated cells. The calculated 1% extra risk level for MN at 24h was 12.01μg/mL, with a 95% lower confidence limit (BMDL) at 8.98μg/mL; the corresponding BMD and BMDL at 48h were 17.35μg/mL and 10.69μg/mL, respectively. The BMD for tetranucleated cells at 24h was 14.86μg/mL, with a BMDL of 7.75μg/mL; the corresponding values at 48h were 0.50μg/mL for BMD and 0.87μg/mL for BMD. These findings suggest that the intensity of the mitogenic effect of testosterone increases upon prolonged exposure. The results of our study show that testosterone acts both as a mitogenic and genotoxic agent in L929 cells.
Prussian carp (Carassius gibelio) is an invasive freshwater fish known for its high tolerance to aquatic pollution. Our aim was to try to clarify its tolerance to increased exposure to ...metals/nonmetals, by determining their cytosolic distributions among peptides/proteins of different molecular masses (MM), which form a part of the fish protective mechanisms. The applied approach consisted of fractionation of gill and hepatic cytosols of Prussian carp from the Croatian river Ilova by size-exclusion high performance liquid chromatography, whereas Cd, Cu, Zn, Fe, Mo, and Se analyses were done by high resolution inductively coupled plasma mass spectrometry. The results indicated high detoxification of Cd by its binding to metallothioneins (MTs) in both fish organs. In addition, binding to MTs was observed for Cu in both organs and for Zn in the liver, whereas clear Zn binding to MTs in the gills was not recorded. Zinc in the gills was predominantly bound to proteins of higher MM (50–250 kDa) and to biomolecules of MM below 2 kDa. Predominant Fe binding to proteins of MM of ~400 kDa (presumably storage protein ferritin) was observed in the liver, whereas in the gills Fe was mainly associated to proteins of MM of ~15–65 kDa (presumably hemoglobin oligomers). Maximum Mo and Se elutions in the liver were noted at 235 kDa and 141 kDa, respectively, and in the gills below 10 kDa. The striking difference was observed between two organs of Prussian carp, with predominant metal/nonmetal binding to high MM proteins (e.g., enzymes, storage proteins) in the liver, and to very low MM biomolecules (<10 kDa) in the gills (e.g., antioxidants, metallochaperones, nonprotein cofactors). Such metal/nonmetal distributions within the gills, as the first site of defense, as well as association of several metals to MTs, indicated highly developed defense mechanisms in some organs of Prussian carp.
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•Distribution profiles of Cd, Cu, Zn, Fe, Mo, and Se by SEC-HPLC and HR ICP-MS•Strong association of Cd to metallothioneins in both liver and gills•Metals/Se bound to proteins of molecular mass > 100 kDa in liver, <10 kDa in gills.•Organ- and species-specific differences in metal/nonmetal cytosolic distributions
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