Ionan Marigómez

Pollutant-specific and general lysosomal responses in digestive cells of mussels exposed to model organic chemicals

The present study was carried out to elucidate whether lysosomal size reduction in digestive cells of mussels Mytilus galloprovincialis constitutes a selective response against a particular group of organic chemical compounds, in contrast to the lysosomal enlargement characteristic of general stress response. Mussels were treated with di(2-ethylhexyl) phthalate (DEHP), benzo(a)pyrene (B[a]P), and the water accommodated fraction (WAF) of a lubricant oil, which were daily applied by either injection through the adductor muscle for 7 days or water-exposure for 21 days. Control mussels were either kept untreated in clean sea water, or treated with acetone (injection and water-exposure), vehicle used for DEHP and B[a]P. A third set of controls consisted of mussels with pierced shell kept in clean seawater. Digestive glands were excised at various treatment days and beta-glucuronidase activity was demonstrated in 8-microm cryotome sections. Lysosomal volume, surface and numerical densities, and surface-to-volume ratio were quantified by image analysis. Other sections were stained with oil red 0 to demonstrate neutral lipids and changes in lipid levels were quantified by image analysis. Neutral lipid accumulation in digestive cells was used as a complementary indication of exposure to organic chemicals. It resulted to be a very prompt and all-or-nothing response, which reached a plateau before 1 day of treatment with WAF, DEHP and B[a]P after both injection and water-exposure. DEHP-treatment induced a general stress response characterised by lysosomal enlargement in digestive cells, which was already induced after 1 day. Treatment with either WAF or B[a]P elicited a comparable biphasic response. A transient lysosomal enlargement, shorter with WAF than with B[a]P treatment, was evidenced after both injection and water-exposure. Further, under water-exposure conditions, WAF reduced the endo-lysosomal system in size more markedly than B[a]P. Such lysosomal size reduction constitutes a transient response after exposure to diverse organic xenobiotics (acetone, WAF and B[a]P). In addition, this lysosomal size reduction might be followed by a further lysosomal enlargement, which later might yet again give rise to an apparent lysosomal size reduction under chronic exposure conditions. As a whole, the lysosomal response is intricate and cannot be simply interpreted in environmental pollution monitoring programmes. Nevertheless, it still constitutes a powerful and sensitive biomarker extremely useful as early-warning signal.

Structural changes in the digestive lysosomal system of sentinel mussels as biomarkers of environmental stress in mussel-watch programmes

Abstract A field study has been carried out to validate the measure of structural changes in the digestive lysosomal system of sentinel mussels as biomarkers of environmental stress. Previous laboratory studies demonstrated that the digestive lysosomal system of molluscs reponds to a variety of pollutants and to different stress situations by exhibiting significant changes in its structure. Mussels were collected monthly over 1 year at the Abra estuary (Bizkaia, Biscay Bay) from six sites with different degrees of pollution. The changes in the structure of the digestive lysosomes were quantified on cryostat sections of the digestive gland by means of automated image analysis. Four stereological parameters were recorded: lysosomal volume density, surface density, surface-to-volume ratio and numerical density. A seasonal pattern in the structure of the digestive lysosomes was evidenced with reduced volume, surface, size and numbers of lysosomes in winter-spring; increased volume, surface, size and numbers in summer and an intermediate situation in autumn. The structure of digestive lysosomes was also dissimilar among sites, the most significant differences being found between Plentzia (nonpolluted site) and Galea (polluted site). The digestive lysosomes of mussels collected from Galea were smaller and more abundant than in Plentzias mussels in most sampling times. The basis of these differences are discussed to conclude that organic chemical pollution might be the cause for these specific changes which are different from the enlargement of digestive lysosomes described as a result of various sources of environmental stress. It is concluded that structural changes in the digestive lysosomes of sentinel mussels are sensitive to pollution-induced environmental stress even in the complex situation of the field where many factors may interact to affect the structure of the digestive lysosomal system.

Digestive lysosome enlargement induced by experimental exposure to metals (Cu, Cd, and Zn) in mussels collected from a zinc-polluted site

This investigation was carried out to quantify changes in the structure of the digestive lysosomes of mussels, Mytilus galloprovincialis, exposed to sublethal concentrations of metals, in both field and laboratory conditions. Digestive lysosomes were automatically measured in an image analysis system to calculate several stereological parameters (volume density, surface density, surface to volume ratio, and numerical density) after the histochemical demonstration of β-glucuronidase activity on cryotome sections. The field study revealed that increasing environmental levels of bioavailable zinc (Zn) are associated with enlarged digestive lysosomes in mussels living in their natural habitat. When these Zn-polluted mussels were depurated in clean seawater for short time periods (1-week acclimation plus 6 experimental days), the digestive lysosomal size was reduced significantly. After longer depuration periods (over 20 experimental days), the stress due to laboratory handling provoked reduced numbers of large digestive lysosomes. On the other hand, the subsequent laboratory exposure to Zn and to other metals (copper, Cu, and cadmium, Cd) caused an enlargement in the digestive lysosomes of Zn-polluted mussels beyond that produced by Zn-pollution in the field. In summary, the enlargement of digestive lysosomes reactive to β-glucuronidase is a sensitive biomarker for non-specific environmental stress induced by sublethal concentrations of bioavailable metals. In addition, we propose the four studied stereological parameters, calculated by means of automated image analysis, as quick, reliable, and useful measurements of such lysosomal enlargement.

Combined use of native and caged mussels to assess biological effects of pollution through the integrative biomarker approach.

Native and caged mussels were used in combination for the monitoring of pollution biological effects through an integrative biomarker approach. Mussels (Mytilus galloprovincialis) were deployed in cages in two well-known model localities with different pollution levels in the Basque coast. After 3 weeks caged and native mussels were collected from each site and a suite of effect and exposure biomarkers (from molecular/cellular to organism level) was applied and chemical contaminants (metals, PAHs, PCBs, phthalates and nonylphenol ethoxylates) were analytically determined. Integrative biomarker indices and pollutant indices of tissues were calculated. Several biomarkers used herein responded similarly in native and caged mussels, whereas others exhibited significant differences. Overall, biomarkers in-a-suite depicted site-specific profiles useful for the diagnostic of mussel health status and therefore for ecosystem health assessment in marine pollution biomonitoring. On the other hand, biomarkers and bioaccumulation exhibited different response times, which was especially evident when comparing biomarker and pollutant indices of tissues. The suite of biomarkers was more sensitive after caging (short-term response), whereas tissue pollutant concentrations were more sensitive in native mussels (long-term response). Thus, the combination of native and caged mussels is highly recommended to monitor biological effects of pollution in mussels through the integrative biomarker approach, especially in chronically polluted sites.

Effects of exposure to prestige-like heavy fuel oil and to perfluorooctane sulfonate on conventional biomarkers and target gene transcription in the thicklip grey mullet Chelon labrosus.

Thicklip grey mullets Chelon labrosus inhabit coastal and estuarine areas where they can be chronically exposed to commonly released pollutants such as polycyclic aromatic hydrocarbons (PAHs) and perfluorinated compounds. These pollutants can also originate from accidental spills, such as the Prestige oil spill in 2002, which resulted in the release of a heavy fuel oil that affected coastal ecosystems in the Bay of Biscay. Peroxisome proliferation (PP), induced biotransformation metabolism, immunosuppression and endocrine disruption are some of the possible biological effects caused by such chemicals. With the aim of studying the effects of organic toxic chemicals on such biological processes at the transcriptional and at the cell/tissue level, juvenile mullets were exposed to the typical mammalian peroxisome proliferator perfluorooctane sulfonate (PFOS), and to fresh (F) and weathered (WF) Prestige-like heavy fuel oil for 2 and 16 days. First, fragments of genes relevant to biotransformation, immune/inflammatory and endocrine disruption processes were cloned using degenerate primers. Fuel oil elicited a significant PP response as proved by the transcriptional upregulation of palmitoyl-CoA oxidase (aox1), peroxisome proliferator activated receptor alpha (pparalpha) and retinoic X receptor, by the AOX1 activity induction and by the increased peroxisomal volume density. PFOS only elicited a significant induction of AOX1 activity at day 2 and of PPARalpha mRNA expression at day 16. All treatments significantly increased catalase mRNA expression at day 16 in liver and at day 2 in gill. Cyp1a transcription (liver and gill) and EROD activity were induced in fuel oil treated organisms. In the case of phase II metabolism only hepatic glutathione S-transferase mRNA was overexpressed in mullets exposed to WF for 16 days. Functionally, this response was reflected in a significant accumulation of bile PAH metabolites. WF treated fish accumulated mainly high molecular weight metabolites while F exposure resulted in accumulation of mainly low molecular ones. Fuel oil significantly regulated immune response related complement component C3 and hepcidin transcription followed by a significant regulation of inflammatory response related apolipoprotein-A1 and fatty acid binding protein mRNAs at day 16. These responses were accompanied by a significant hepatic inflammatory response with lymphocyte accumulations (IRLA) and accumulation of melanomacrophage centers (MMC). PFOS did not elicit any transcriptional response in the studied biotransformation and immune related genes, although histologically significant effects were recorded in IRLA and MMC. A significant reduction of lysosomal membrane stability was observed in all exposed animals. No endocrine disruption effects were observed in liver while brain aromatase mRNA was overexpressed after all treatments at day 2 and estrogen receptor alpha was downregulated under WF exposure at day 16. These results show new molecular and cellular biomarkers of exposure to organic chemicals and demonstrate that in mullets PP could be regulated through molecular mechanisms similar to those in rodents, although the typical mammalian peroxisome proliferator PFOS and heavy fuel oil follow divergent mechanisms of action.

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.

Marine ecosystem health status assessment through integrative biomarker indices: a comparative study after the Prestige oil spill “Mussel Watch”

Five integrative biomarker indices are compared: Bioeffects Assessment Index (BAI), Health Status Index (HSI), integrated biological response (IBR), ecosystem health condition chart (EHCC) and Integrative Biomarker Index (IBI). They were calculated on the basis of selected biomarker data collected in the framework of the Prestige oil spill (POS) Mussel Watch monitoring (2003–2006) carried out in Galicia and the Bay of Biscay. According to the BAI, the health status of mussels was severely affected by POS and signals of recovery were evidenced in Galicia after April-04 and in Biscay Bay after April-05. The HSI (computed by an expert system) revealed high levels of environmental stress in 2003 and a recovery trend from April-04 to April-05. In July-05, the health status of mussels worsened but in October-05 and April-06 healthy condition was again recorded in almost all localities. IBR/n and IBI indicated that mussel health was severely affected in 2003 and improved from 2004 onwards. EHCC reflected a deleterious environmental condition in 2003 and a recovery trend after April-04, although a healthy ecosystem condition was not achieved in April-06 yet. Whereas BAI and HSI provide a basic indication of the ecosystem health status, star plots accompanying IBR/n and IBI provide complementary information concerning the mechanisms of biological response to environmental insult. Overall, although the integrative indices based on biomarkers show different sensitivity, resolution and informative output, all of them provide coherent information, useful to simplify the interpretation of biological effects of pollution in marine pollution monitoring. Each others’ advantages, disadvantages and applicability for ecosystem health assessment are discussed.

Use of polyclonal antibodies for the detection of changes induced by cadmium in lysosomes of aquatic organisms.

Lysosomal responses are widely accepted cellular effect biomarkers of general stress. Up to now, these biomarkers have been analysed by means of conventional techniques based on enzyme histochemical methods, where lysosomal enzymes such as acid phosphatase and beta-glucuronidase (beta-GUS) have been employed as markers of lysosomes. The aim of the present work was to develop more advanced and sensitive methods based on the use of polyclonal antibodies to measure lysosomal enzymes in different sentinel organisms. For this purpose, we have studied the cross-reactivity of two commercial polyclonal antibodies against the lysosomal enzymes acid phosphatase and beta-GUS with molluscan digestive gland by means of immunoblotting and immunohistochemistry. The antibody against acid phosphatase cross-reacted specifically with the lysosomal fraction of the digestive gland, while unspecific immunoreaction occurred with digestive gland whole homogenates and tissue sections. The antibody against beta-GUS cross-reacted specifically with digestive gland whole homogenates and tissue sections. The cross-reactivity of this antibody was tested also in crab hepatopancreas and mullet liver where the same successful results were obtained. The second aim of the present study was to test if the immuno-based approach was sensitive enough to detect lysosomal alterations provoked by contaminants. For this purpose two experiments were carried out with mussels treated with cadmium in two ways: in vivo treatment by injection and in vitro treatment using digestive gland explants. Afterwards immunoblotting studies with the antibody against beta-GUS were applied and immunoreactive bands were quantified by means of a gel analysis programme. We found that beta-GUS protein levels were higher in treated mussels when compared with controls in either in vivo or in vitro treatments. All these data suggest that the polyclonal antibody against beta-GUS is adequate to be used in immuno-based approaches to detect contaminant-induced lysosomal alterations.

Changes in digestive cell lysosomal structure in mussels as biomarkers of environmental stress in the Urdaibai Estuary (Biscay coast, Iberian Peninsula)

In order to validate the use of digestive cell lysosomal changes in mussels as biomarkers of water quality in coastal and estuarine areas, the structure of the digestive cell lysosomal system was quantified in mussels from the Urdaibai Estuary (Biscay coast, Iberian Peninsula) by automated image analysis. Mussels were collected bimonthly from September 1991 to September 1992 and cryostat sections of the digestive gland were processed for cytochemical demonstration of the lysosomal marker enzyme, β-glucuronidase. Four stereological parameters were calculated: lysosomal volume density, surface density, surface to volume ratio and numerical density. Seasonal differences in the lysosomal structure were observed, with higher values of lysosomal volume, surface, size and numbers in the summer. Additionally, the stereological parameters revealed differences between sampling locations except in winter-spring. In autumn, the water quality was lower in the sites on the west side of the estuary affected by urban and industrial activities, whereas in summer the sites on the east also showed signs of poor water quality. It is concluded that the structure of digestive cell lysosomes can be used as sensitive biomarkers of coastal and estuarine water quality.

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 (VvBSD). Significantly higher values were recorded in Cd-exposed mussels compared with controls at all exposure times. However, VvBSD values were lower at days 7 and 21 than at day 1. This relative decrease in VvBSD reflected another (and confounding) response elicited by Cd-exposure in the digestive epithelium: the volume density of basophilic cells (VvBAS) increased significantly as exposure progressed. Due to this cell-type replacement, the net accumulative capacity of the digestive epithelium decreases at long exposure times.