Mussel Digestive Cells Research Articles

Lysosomal responses to different gold forms (nanoparticles, aqueous, bulk) in mussel digestive cells: a trade-off between the toxicity of the capping agent and form, size and exposure concentration

Gold nanoparticles (NPs) are increasingly used in technological materials and consumer products and may have toxicological characteristics distinct from bulk and aqueous gold. The aim of this work was to understand the effects of Au NPs especially, how the form, the size and the coating influence bioaccumulation/biodistribution and toxicity of NPs in mussels, Mytilus galloprovincialis. Mussels were exposed for 3 d to concentrations of Au (0.75, 75 and 750 μg Au/l) supplied as Au-Cit NPs (5 and 40 nm; Au5-Cit and Au40-Cit), bulk and aqueous Au (HAu(III)Cl4), and to the capping agent (Na-citrate) in doses used in the formulation of NPs (0.005, 0.5, 5 mg/l). Citrate-stabilised NPs formed stable suspensions of aggregates in seawater (SW) available for mussels. Au accumulation in soft tissues was similar in Au40-Cit and aqueous Au exposed mussels, lower in Au5-Cit and negligible after bulk exposure. Au NPs were identified (X-ray microanalysis) in different compartments of the endolysosomal system in digestive cells, and small size NPs (5 nm) were more accumulated than 40 nm NPs, aqueous and bulk. The degree of lysosomal membrane destabilisation was related with intralysosomal metal accumulation and depended on the form, NP size (Au5-Cit > Au40-Cit > aqueous > bulk) and concentration. Citrate alone provoked extreme reduction in lysosomal membrane stability. Toxicopathic alterations were recorded in digestive gland cells (vacuolisation, swollen RER, connective tissue disruption and cell death) especially in mussels exposed to 40 nm NPs. Deleterious effects resulted from digestive tract obliteration (agglomerates) and digestion malfunction. The toxic effect of Au-Cit NPs was influenced both by NP size, capping agent composition and the dose of capping agent carried by NPs, which was size dependent.

Nanoparticle size and combined toxicity of TiO2 and DSLS (surfactant) contribute to lysosomal responses in digestive cells of mussels exposed to TiO2 nanoparticles

The aim of this investigation was to understand the bioaccumulation, cell and tissue distribution and biological effects of disodium laureth sulfosuccinate (DSLS)-stabilised TiO2 nanoparticles (NPs) in marine mussels, Mytilus galloprovincialis. Mussels were exposed in vivo to 0.1, 1 and 10 mg Ti/L either as TiO2 NPs (60 and 180 nm) or bulk TiO2, as well as to DSLS alone. A significant Ti accumulation was observed in mussels exposed to TiO2 NPs, which were localised in endosomes, lysosomes and residual bodies of digestive cells, and in the lumen of digestive tubules, as demonstrated by ultrastructural observations and electron probe X-ray microanalysis. TiO2 NPs of 60 nm were internalised within digestive cell lysosomes to a higher extent than TiO2 NPs of 180 nm, as confirmed by the quantification of black silver deposits after autometallography. The latter were localised mainly forming large aggregates in the lumen of the gut. Consequently, lysosomal membrane stability (LMS) was significantly reduced upon exposure to both TiO2 NPs although more markedly after exposure to TiO2-60 NPs. Exposure to bulk TiO2 and to DSLS also affected the stability of the lysosomal membrane. Thus, effects on the lysosomal membrane depended on the nanoparticle size and on the combined biological effects of TiO2 and DSLS.

Lysosomal enlargement and lysosomal membrane destabilisation in mussel digestive cells measured by an integrative index

Lysosomal responses (enlargement and membrane destabilisation) in mussel digestive cells are well-known environmental stress biomarkers in pollution effects monitoring in marine ecosystems. Presently, in laboratory and field studies, both responses were measured separately (in terms of lysosomal volume density – Vv – and labilisation period –LP) and combined (lysosomal response index – LRI) in order to contribute to their understanding and to develop an index useful for decisions makers. LRI integrates Vv and LP, which are not necessarily dependent lysosomal responses. It is unbiased and more sensitive than Vv and LP alone and diminishes background due to confounding factors. LRI provides a simple numerical index (consensus reference = 0; critical threshold = 1) directly related to the pollution impact degree. Moreover, LRI can be represented in a way that allows the interpretation of lysosomal responses, which is useful for environmental scientists.

Time-course study of the early lysosomal responses to pollutants in mussel digestive cells using acid phosphatase as lysosomal marker enzyme

Lysosomal biomarkers are early warning signals of the biological effects caused by environmental pollutants but the promptness of lysosomal responses to pollutants has not been investigated yet. This work is aimed to determine the response-time of digestive cell lysosomes in mussels exposed to metals and hydrocarbons. Mussels, Mytilus galloprovincialis, were exposed, under laboratory conditions to Cd and to the water-accommodated fraction of a lubricant oil. One mussel per experimental group was sacrificed and processed every hour from 0 h to 30 h. Changes in AcP activity, immunoreactivity and LMS test based on AcP histochemistry, discriminates significantly control and exposed mussels within 5 h exposure. The present results suggested that after 15–20 h exposure digestive cell loss might be accompanied by increased AcP activity (extralysosomal) without a parallel increase in the levels of immunoreactive AcP protein, especially after Cd-exposure. The reduced labilisation period of lysosomal membrane constitute a cost effective early warning signal that, however, is not necessarily correlated with the exposure time. The routine application of immunochemical techniques deserves more research efforts before its implementation although, these techniques are very valuable to understand and interpret correctly lysosomal responses to pollutants.

Natural variability in size and membrane stability of lysosomes in mussel digestive cells: seasonal and tidal zonation

Lysosomal responses in mussel digestive cells are widely accepted as effective biomark- ers that respond to pollutant exposure, as well as to natural factors (tide, food, temperature). The effects of tide and season on digestive cell lysosomes in mussels were determined in different tran- sect and transplant experiments on mussels at different tide levels and in winter and summer. We measured lysosomal enlargement through the lysosomal structural changes (LSC) test after β-glu- curonidase (β-Gus) histochemistry and membrane destabilisation through the lysosomal membrane stability (LMS) test after hexosaminidase (Hex) histochemistry. A marked gradient along the inter- tidal zone, which was more prominent in winter than in summer, was found in lysosomal size and membrane stability. A transplant experiment revealed that digestive cell lysosomes respond rapidly to immersion of mussels. The study of the time-course changes in mussels from high tidemark level indicated that lysosomes respond very promptly to air exposure conditions. We suggest that the lyso- somal changes observed are the result of the digestive process in parallel with tide. Since such natural variability might mask the effects of pollution, we conclude that a standardised sampling protocol — which should include the use of mussels from low intertidal levels — is needed.

β-Glucuronidase and hexosaminidase are marker enzymes for different compartments of the endo-lysosomal system in mussel digestive cells

In environmental toxicology, the most commonly used techniques used to visualise lysosomes in order to determine their responses to pollutants (LSC test: lysosomal structural changes test; LMS test: lysosomal membrane stability test) are based on the histochemical application of lysosomal marker enzymes. In mussel digestive cells, the marker enzymes used are beta-glucuronidase (beta-Gus) and hexosaminidase (Hex). The present work has been aimed at determining the distribution of these lysosomal marker enzymes in the various compartments of the endo-lysosomal system (ELS) of mussel digestive cells and at exploring whether intercellular transfer of lysosomal enzymes occurs between digestive and basophilic cells. Immunogold cytochemistry has allowed us to conclude that beta-Gus is present in every compartment of the digestive cell ELS, whereas Hex is not so widely distributed. Moreover, Hex is intimately linked to the lysosomal membrane, whereas beta-Gus appears to be not necessarily membrane-bound. Therefore, two populations of heterolysosomes with different enzyme load and membrane stability have been distinguished in the digestive cell. In addition, heterolysosomes of different electron density have been commonly observed merging together by contact; we suggest that some might act as storage granules for lysosomal enzymes. On the other hand, beta-Gus seems to be released to the digestive alveolar lumen in secretory lysosomes produced by basophilic cells and endocytosed by digestive cells. Regarding the implications of the present study on the interpretation of lysosomal biomarkers, we conclude that beta-Gus, but not Hex, histochemistry provides an appropriate marker for the LSC test and that, although both lysosomal marker enzymes can be employed in the LMS test, different values would be obtained depending on the marker enzyme employed.

Glycosylation and sorting pathways of lysosomal enzymes in mussel digestive cells

Our aim was to contribute to the understanding of the synthesis, maturation and activation of lysosomal enzymes in an invertebrate cellular model: the endo-lysosomal system (ELS) of mussel digestive cells. The activities of 5'-nucleotidase (AMPase), arylsulphatase (ASase) and acid phosphatase (AcPase), which are transported towards acidic compartments as membrane proteins, were localised by enzyme cytochemistry. AcPase activity was found within large heterolysosomes and residual bodies. ASase was located in endosomes, endolysosomes and heterolysosomes. AcPase and ASase activities were recorded within small vesicles and cisterns of the trans-Golgi network. Conversely, AMPase activity was primarily found in microvilli and apical vesicles and, less conspicuously, in lysosomes and the cis-side of the Golgi and the cis-Golgi network (CGN). In order to understand the processes of synthesis and maturation of these lysosomal enzymes, selected glycoconjugates were localised after lectin cytochemistry. N-acetylglucosamine, mannose and fucose residues were almost ubiquitous in the ELS, as were galactose residues, which were apparently less abundant. N-acetylglucosamine residues occurred in the inner membrane co-localised with mannose residues within the lysosomal and pre-lysosomal acidic compartments. Based on these results, glycosylation and sorting pathways are proposed for both soluble and membrane enzymes. Unlike in mammalian cells, O-glycosylation is fully completed in the CGN, mannose addition in N-glycosylation extends beyond the CGN and galactose addition is fully achieved at the intermediate side. Sorting of soluble lysosomal enzymes, as in crustaceans, is mediated by the indirect transport of membrane-linked proteins with GlcNAc1-P6Man residues that are removed in endolysosomes and heterolysosomes.

Comparison of cytochemical procedures to estimate lysosomal biomarkers in mussel digestive cells

Enlargement and membrane destabilisation in digestive cell lysosomes of mussels are biomarkers of pollution effect. Cytochemical methods are currently applied to determine lysosomal membrane stability (LMS) and lysosomal structural changes (LSC). LMS, determined after grading N-acetyl-β-hexosaminidase activity on cryotome sections of digestive gland, is measured as labilisation period (LP). LSC, determined after image analysis of cryotome sections where β-glucuronidase activity is revealed, are measured as lysosomal volume (Vv), surface (Sv), numerical (Nv) densities and surface-to-volume ratio (S/V). Both methods have now been compared in a field study. Mussels were collected from Biscay Bay (Plentzia, reference; Muskiz, moderately polluted) and North Aegean Sea (Olympiada, reference; Limani, heavily polluted). Higher Vv and Sv and lower S/V and LP were recorded in polluted sites than in reference sites. Significant correlations with LP were found for Vv and S/V. The cost/effectiveness and environmental significance of both methods are discussed.

Immunoelectron microscope analysis of epidermal growth factor receptor (EGFR) in isolated Mytilus galloprovincialis (Lam.) digestive gland cells: evidence for ligand-induced changes in EGFR intracellular distribution.

In mammalian cells, the binding of epidermal growth factor (EGF) to its receptor (EGFR), a glycoprotein with intrinsic tyrosine kinase activity, leads to the pleiotropic responses to EGF. Among these, a negative feedback response by stimulation of receptor internalization and lysosomal degradation, this attenuating signal transduction. In this work, data are reported on the identification of specific EGFRs in isolated digestive gland cells from the marine mussel (Mytilus galloprovincialis Lam.) By immunoelectron microscopy. In control digestive cells, EGFR immunoreactivity was mainly associated with cytoplasmic membrane structures and, to a lesser extent, the cell membrane. The presence of EGFR-like receptors was confirmed by Western blotting of digestive gland cell extracts with two different monoclonal antibodies that recognize either intracellular or extracellular epitopes. The addition of mammalian EGF resulted in significant time and temperature-dependent changes in EGFR subcellular distribution in mussel cells. In cells exposed to EGF for 0-15 min at 4 degrees C, the distribution of EGFR was not significantly different from that of the control cells. On the other hand, at 18 degrees C, an increased labelling along the cell membrane was observed after 5-10 min after EGF addition, with a concomitant decrease in the cytoplasmic signal. Moreover, after 20 min of exposure to EGF, ligand binding apparently resulted in EGFR compartmentation within the lysosomes. These observations were confirmed by quantitative analysis of EGFR labelling at different times of EGF exposure. Similar results were obtained utilizing the two different monoclonal antibodies. The results indicate that, in mussel digestive cells, the binding of heterologous EGF to specific receptors induces a negative feedback response by stimulating the lysosomal degradation of EGFR, thus suggesting the presence of mechanisms responsible for receptor downregulation similar to those observed in mammalian cells.

The influence of environmental contaminants on lysosomal activity in the digestive cells of mussels ( Mytilus galloprovincialis) from the Venice Lagoon

Lysosomes are subcellular organelles bounded by a semipermeable lipoprotein membrane that contain a battery of hydrolytic enzymes that are collectively capable of degrading all classes of indogenous and exogenous macromolecules. Lysosomes accumulate a diverse range of chemical contaminants which can lead to membrane damage resulting in leakage of their contents into the cytosol and damage to cells. Total lysosomal activity for two acid hydrolases, N-acetyl- β- d-hexosaminidase and β-glucuronidase, with different substrate specificities was determined histochemically in digestive gland sections of mussels, Mytilus galloprovincialis from a series of sites in the Venice Lagoon and the Adriatic Sea and correlated, using multi-stepwise regression analysis, with tissue contaminant burdens in order to explore causality. The results indicated that whilst activity of N-acetyl- β- d-hexosaminidase correlated with body burdens of mercury, β-glucuronidase, by contrast, correlated with DDT, Arochlor 1254 and eight PCB congeners in combination with iron or zinc.

Lysosomes as cellular markers of environmental pollution: Time‐ and dose‐dependent responses of the digestive lysosomal system of mussels after petroleum hydrocarbon exposure

AbstractLysosomes are cell organelles containing hydrolytic enzymes and involved in intracellular digestion. Numerous environmental contaminants can interfere with lysosomes, and the purpose of the present work was to quantify changes induced by petroleum hydrocarbons on the structure of the lysosomal system of mussel digestive cells. Mussels, Mytilus galloprovincialis Lmk., were exposed for three months to the water accommodated fraction (WAF) of two crude oils (URAL and MAYA) and of a lubricant oil. Three different exposure doses (0.6, 6, and 40% WAF) were used for each type of hydrocarbon and mussels were sampled at different time intervals (days 21, 49, and 91). A stereological study, using an automated image analysis system, was made on sections stained for demonstration of β‐glucuronidase activity and four parameters were calculated: lysosomal volume density, surface density, surface to volume ratio, and numerical density. The results indicate that the effect of the exposure dose on lysosomal structure is variable and there is no linear relationship of lysosomal changes with dose. Regarding the effect of the exposure time, two different responses were evidenced: (a) a short‐term response (at day 21) with a decrease in size and numbers of lysosomes and (b) a long‐term response (at days 49 and 91) with an increase in lysosomal size and a decrease in their numbers. The short‐term response may be due to a disintegration process of the digestive cells with subsequent loss of lysosomes and could represent an adaptative response to hydrocarbon exposure. At long exposure times, lysosomal changes, apparently caused by fusion processes giving rise to enlarged lysosomes, correspond to a “general stress response”. © by John Wiley & Sons, Inc.

Contaminant induced lysosomal membrane damage in marine mussel digestive cells: an in vitro study

Damage to lysosomes in isolated molluscan digestive cells was measured in vitro, following prior experimental in vivo exposure of mussels to the model polycyclic aromatic hydrocarbon fluoranthene for 7 days. Damage was assessed using the retention of the cationic diazine probe neutral red in the lysosomal compartment as a determinant of effect. The results showed that probe retention time was significantly reduced ( P < 0.001) in the lysosomes of cells isolated from exposed mussels as compared to those from mussels exposed to the solvent vehicle alone. These findings indicate that the functional integrity of the lysosomal membrane is impaired following hydrocarbon exposure and further illustrates that lysosomes are the target of toxic action of pollutants. Corroborative evidence for fluoranthene-induced cell injury was apparent from increased accumulation of lipid and lysosomal hydrolase activity in tissue sections of digestive gland from the same experimental material.

Haemolymph volume, changes in the biochemical composition of the blood, and cytological responses of the digestive cells inMytilus californianus Conrad, induced by nutritional, thermal and exposure stress

1. The volume of the extracellular space inMytilus californianus is 46% of the wet weight of the tissues (excluding the shell). 2. Fluid lost through bleeding is replaced within four hours, and both the dissolved and the cellular components (haemocytes) of the haemolymph are quickly restored. 3. The level of ammonia in the plasma increases when starved mussels begin to feed. This is a result of the specific dynamic action (SDA) which is associated with protein metabolism during feeding. 4. Ammonia accumulates in the plasma when mussels are exposed to air, which indicates that catabolism of nitrogenous substrates continues when the animal is exposed at low tide. 5. There are few changes in plasma carbohydrate, lipid and protein levels which are attributable to exposure, ration or increased temperature, suggesting good short-term regulation. 6. Lysosomal intracellular digestion continues in the digestive cells of mussels exposed to air. During exposure, there is also an increase in the numbers of circulating haemocytes, which may be associated with the continuation of the digestion process. 7. At high ration and elevated temperature, the digestive epithelium thickens, and there is some evidence that the rate of lysosomal intracellular digestion increases. During periods of starvation the epithelium becomes thinner and there is a loss of cytoplasm, probably by autophagy.