Lysosomal reaction to xenobiotics in mussel hemocytes using BODIPY-FL-verapamil

Many cellular and sub-cellular biomarkers associated with mussel (Mytilus edulis) digestive gland and kidney have been characterised. The lysosomal compartment of these tissues have been recognised as being particularly sensitive, exhibiting pollutant induced responses which could be potentially used as a ‘biomarker’. However, relatively few studies have investigated the lysosomal response within molluscan hemocytes. This study was conducted to test whether lysosomal reactions, in live hemocytes isolated from mussels, can be used as a biomarker of pollutant exposure and deleterious effect. Lysosomal responses to a number of hydrocarbons, including anthracene and phenanthrene, and to the amphiphilic heterocylic chemical, chlorpromazine, were examined. The supravital dye neutral red (NR) was used to examine lysosomal membrane fragility, following xenobiotic exposure. NR was also used to verify the lysosomal compartment as the reported accumulation site of a new molecular probe, BODIPY-FL-verapamil (BFLV). The use of BFLV, with confocal laser microscopy and image analysis enabled visualisation and quantification of lysosomal distribution and perturbation. BFLV showed that exposure of molluscan hemocytes to xenobiotics (20 ppb–10 ppm) induced the formation of pathologically enlarged lysosomes. The internal trafficking of lysosomes was shown to be severely compromised after exposure to chlorpromazine. Exposed molluscan hemocytes exhibited significantly reduced lysosomal retention times, for neutral red. Preliminary data is presented demonstrating the opportunity for these non-destructive biomarker techniques to detect pollution gradients in situ.     

Biomarkers and trace metals in the digestive gland of indigenous and transplanted mussels, Mytilus galloprovincialis, in Venice Lagoon, Italy.

The aim of this study was to investigate the cellular and biochemical response of mussels, Mytilus galloprovincialis, transplanted from a relatively pristine site to a polluted one and vice versa in the Lagoon of Venice (northeast of Italy) and to apply auto-metallography, a rapid and sensitive histochemical technique, to determine the bio-available fraction of heavy metals accumulated in the body tissues of organisms. Animal digestive glands have been used for morphological analyses (lysosomal volume, surface and numerical density, mean epithelial thickness, mean diverticular and luminar radius), autometallographical black silver deposits quantification and biochemical assays (superoxide dismutase and catalase activity). Furthermore, heavy metal content was determined by atomic absorption spectrophotometry (AAS) using standard procedures. The overall results indicate a direct influence of the environment in the thinning of the digestive cells and in the increasing number of lysosomes in mussels from the more polluted site. These data are in agreement with the metal content in digestive cell lysosomes as determined by autometallography, whereas AAS measurements show less significant differences.     

Spatial and temporal variation of biomarkers in mussels (Mytilus galloprovincialis) from the Lagoon of Venice, Italy

A multiple biomarker approach was adopted in a seasonal study carried out in the Lagoon of Venice, with the double aim of evaluating the natural and anthropic stresses influencing the biological responses of mussel (Mytilus galloprovincialis) and of assessing the effects due to spatial rather than temporal variations. Biochemical (aldehyde dehydrogenase and catalase activities), cellular (neutral red retention time) and physiological (survival in air and condition index) biomarkers were determined in mussels collected in four differently impacted lagoon areas. Multivariate analysis showed that samples were distributed mainly according to temperature and four seasonal groups were identified. The combination of spatial and temporal information enabled us to distinguish physiological variations due to natural causes from those due to anthropic stress, and to identify the sampling period when several biomarkers are less influenced by both natural and endogenous factors.     

Spatial and temporal variation of biomarkers in mussels (Mytilus galloprovincialis) from the Lagoon of Venice, Italy

A multiple biomarker approach was adopted in a seasonal study carried out in the Lagoon of Venice, with the double aim of evaluating the natural and anthropic stresses influencing the biological responses of mussel (Mytilus galloprovincialis) and of assessing the effects due to spatial rather than temporal variations.Biochemical (aldehyde dehydrogenase and catalase activities), cellular (neutral red retention time) and physiological (survival in air and condition index) biomarkers were determined in mussels collected in four differently impacted lagoon areas.Multivariate analysis showed that samples were distributed mainly according to temperature and four seasonal groups were identified. The combination of spatial and temporal information enabled us to distinguish physiological variations due to natural causes from those due to anthropic stress, and to identify the sampling period when several biomarkers are less influenced by both natural and endogenous factors.     

Measuring lysosomal stability as an effective tool for marine coastal environmental monitoring

The use of lysosomal stability in the mussel, Mytilus galloprovincialis, as a potential biomarker of environmental contamination has been evaluated along the Portuguese coast. To this end, the neutral red retention (NRR) time was measured in mussel haemocytes gathered from nine different locations reflecting different degrees of anthropogenic contamination. Mussels collected in the vicinity of industrial and urban areas showed the lowest lysosomal stability. Additionally, no significant seasonal variability (winter-spring/summer) for NRR time was observed. In order to further support the usefulness of this method as an integrated tool for monitoring marine coastal environments, we compared the levels of xenobiotics in mussel tissues with the obtained NRR values. The results highlighted a consistent pattern, with the lowest lysosomal stability intimately correlated with the higher contaminant concentrations. In summary, this integrated approach further demonstrated that the NRR assay can provide useful and objective indications of the real health status of organisms subjected to different stress agents, being a valid option for environmental monitoring.     

Environmental prognostics: an integrated model supporting lysosomal stress responses as predictive biomarkers of animal health status

The potential prognostic use of lysosomal reactions to environmental pollutants is explored in relation to predicting animal health in marine mussels, based on diagnostic biomarker data. Cellular lysosomes are already known to accumulate many metals and organic xenobiotics and the lysosomal accumulation of the carcinogenic polycyclic aromatic hydrocarbon 3-methylcholanthrene (3-MC) is demonstrated here in the hepatopancreatic digestive cells and ovarian oocytes of the blue mussel. Lysosomal membrane integrity or stability appears to be a generic indicator of cellular well-being in eukaryotes; and in bivalve molluscs it is correlated with total oxygen and nitrogen radical scavenging capacity (TOSC), protein synthesis, scope for growth and larval viability; and inversely correlated with DNA damage (micronuclei), as well as lysosomal swelling (volume density), lipidosis and lipofuscinosis, which are all characteristic of failed or incomplete autophagy. Integration of multiple biomarker data is achieved using multivariate statistics and then mapped onto "health status space" by using lysosomal membrane stability as a measure of cellular well-being. This is viewed as a crucial step towards the derivation of explanatory frameworks for prediction of pollutant impact on animal health; and has facilitated the development of a conceptual mechanistic model linking lysosomal damage and autophagic dysfunction with injury to cells, tissues and the whole animal. This model has also complemented the creation and use of a cell-based bioenergetic computational model of molluscan hepatopancreatic cells that simulates lysosomal and cellular reactions to pollutants. More speculatively, the use of coupled empirical measurements of biomarker reactions and modelling is proposed as a practical approach to the development of an operational toolbox for predicting the health of the environment.     

Disorder and recovery of environmental health monitored by means of lysosomal stability in liver of European flounder (Platichthys flesus L.)

From 1995 to 2000 biological effects were studied in liver of flounder (Platichtysflesus L.) from the German Bight. During the study period deleterious consequences of acute discharges of DDT and PCBs in early spring 1996 and after 1998 due to remobilization of contaminants from riverbed deepening of the River Elbe became evident. As core biomarker which reflects toxically induced liver pathologies and integrates effects of various classes of pollutants we measured the integrity of lysosomal membranes in individual flounder liver. During the study period, twice statistically significant disturbances of lysosomal function was detected in fish from the River Elbe: in summer 1996 and in spring 1999. Yet, the detrimental contaminant effects were not only restricted to individuals from the Elbe but expanded to those flounder inhabiting formerly less polluted reference areas. In contrast to flounder of the Elbe, their ability to recover from the lysosomal disorders were limited. While in autumn 2000 Elbe individuals showed clear signs of recovery, those fish caught in areas more distant to the source of toxicant input still maintained significantly decreased lysosomal membrane integrity. It can be speculated that fish populations which are not continuously exposed to chronic anthropogenic stress may have a lower potential or need a longer period to recover from the effects of pollution.     

Age-related differences in the recovery of lysosomes from stress-induced pathological reactions in marine mussels

This study was conducted to investigate the adaptability of marine mussels Mytilus edulis of increasing age to induced stress and subsequent recovery. Lysosomes, present in large numbers in the molluscan digestive gland, play a major role in intracellular digestion, and the stability of their membranes provides a sensitive biomarker for generalised cell injury which is correlated with the stress response of the whole animal. Lysosomal stability was measured in mussels of three age groups (2–4, 6–8 and ≥ 10 years) during exposure to hypoxia/hyperthermia and, in a separate experiment, to copper (50 ppb: where billion = 10⁹). The lysosomal reactions of all three age groups to both experimental stressors were similar. However, recovery from the induced pathological reactions was most pronounced in the youngest animals and least apparent in the oldest group. These findings indicate that the stress reaction is independent of age but that the potential for recovery of lysosomal integrity is age-related.     

Networking and expert-system analysis: next frontier in biomonitoring

The extensive use of biomarkers in environment biomonitoring programmes has raised the problem of data management and intercomparison. A research project (Pollution Effect Network, PEN) is proposed here, consisting of the realisation of an on-line warehouse for biomarker data (http://www.muf.unipmn.it/pen). The web site will contain repository sections and expert system procedures able to integrate information from different biomarkers and provide ranking of the organism health status in terms of synthetic stress syndrome indexes. Researchers accessing the site will be able to submit and process their own data. This will allow common criteria in the evaluation of the biological effects of pollutants, and an intercomparison of biomonitoring data among different geographic areas and sentinel species.     

Integration of biochemical, histochemical and toxicogenomic indices for the assessment of health status of mussels from the Tamar Estuary, U.K

The aim of this study was to examine whether a combination of biochemical, histopathological and toxicogenomic data could be used as a valuable tool for the assessment of biological risk associated with pollutants within the Tamar River and Estuary, S.W. England, U.K. Accordingly, biochemical and histopathological biomarkers (protein carbonyls, lipofuscin, neutral lipids, lysosomal stability [N-acetyl-β-hexosaminidase and neutral red], lysosomal volume, ferric reducing antioxidant power [FRAP] and malonaldehyde [MDA]) and gene expression profiles were assessed in 5 sites from the Tamar River and Estuary (Neal Point, Town Quay, Wilcove, Cremyll Ferry and Whitsand; and a reference site, Trebarwith Strand, N. Cornwall). PAHs were measured in mussel tissue and sediment and metals were measured in mussel tissue only. Data from the biomarkers was integrated into a Mussel Expert System (MES) model to produce a simple assessment of mussel stress. Clear gradients of mussel toxicity were identified by the biomarkers (with the exception of neutral lipids) with the highest impacted animals found furthest up the Tamar, whilst the MES was unable to identify a gradient of effect. Gene expression profiles also indicated a gradient of stress with the greatest number of significantly up- or down- regulated genes found at the uppermost 2 sites. The MES did, however, determine that mussels from all sites, except the reference site, were highly stressed; a conclusion that could not be inferred from the biomarker data alone. It is concluded that the MES is a valuable tool that permits integration and interpretation of complex sets of biomarker data by identifying the biological meaning of biomarker changes.     

Use of artificial neural networks to identify the origin of green macroalgae

This study demonstrates application of artificial neural networks (ANNs) for identifying the origin of green macroalgae (Enteromorpha sp. and Cladophora sp.) according to their concentrations of Cd, Cu, Ni, Zn, Mn, Pb, Na, Ca, K and Mg. Earlier studies confirmed that algae can be used for biomonitoring surveys of metal contaminants in coastal areas of the Southern Baltic. The same data sets were classified with the use of different structures of radial basis function (RBF) and multilayer perceptron (MLP) networks. The selected networks were able to classify the samples according to their geographical origin, i.e. Southern Baltic, Gulf of Gdańsk and Vistula Lagoon. Additionally in the case of macroalgae from the Gulf of Gdańsk, the networks enabled the discrimination of samples according to areas of contrasting levels of pollution. Hence this study shows that artificial neural networks can be a valuable tool in biomonitoring studies.     

Pathological reactions and recovery of hepatopancreatic digestive cells from the marine snail Littorina littorea following exposure to a polycyclic aromatic hydrocarbon

The aim of this study was to investigate the cellular pathological responses of hepatopancreatic digestive cells from the periwinkle Littorina littorea exposed to the polycyclic aromatic hydrocarbon (PAH) fluoranthene and to ascertain whether any injurious effects were reversible within the experimental time scale. A secondary objective was to establish the relationship of the various reactions to animal health status, using lysosomal stability as an index of well-being. Exposure of snails to a concentration of 335 microgl(-1) (1.7 microM) fluoranthene (seawater renewed and spiked daily with fluoranthene) for 5 days resulted in a reduction in lysosomal stability (neutral red retention) and endocytosis; and an increase in smooth endoplasmic reticulum (ER) and 7-ethoxycoumarin-o-deethylase (ECOD; measured as cyano-ECOD) activity measured in isolated live digestive cells. Exposed snails treated with clean seawater for a further 8 days resulted in a return to control levels of lysosomal stability, ECOD and ER; endocytosis showed only a partial recovery. Multi-variate and uni-variate analysis showed that there were strong correlations between the various cellular biomarker responses. These findings are interpretable within the current framework of molluscan biomarker responses to PAHs. Principal component analysis was used to derive the first principal component for endocytosis, ER and ECOD reactions and these were plotted against lysosomal stability as a measure of cellular well-being. The resulting significant regression represents the mapping of the individual biomarkers within health status space for a gradient of fluoranthene toxicity. From this analysis, we concluded that endocytosis is an indicator of healthy snails while proliferation of ER and to a lesser extent induced ECOD are indicative of dysfunction and reduced health. Finally, the results indicate that stress induced by chronic exposure to a PAH is reversible.     

Critical evaluation of an intercalibration exercise undertaken in the framework of the MED POL biomonitoring program.

The results of an intercalibration exercise among the laboratories participating in the MED POL program for monitoring biological effects of pollutants along the Mediterranean coasts are presented. Three established biomarkers, i.e. lysosomal membrane stability, metallothionein concentration and ethoxyresorufin-O-deethylase (EROD) activity, were intercalibrated. The stability of lysosomal membranes in mussels (Mytilus galloprovincialis Lam.) was assessed with a cytochemical method. The four participating laboratories were able to discriminate between control animals (membrane labilization times ranging from 21 to 35 min) and Cu-exposed animals (40 micrograms/l Cu for 3 days) (labilization times ranging from 4.5 to 7.4 min). The metallothionein concentration was evaluated in digestive gland homogenates of control mussels and of animals exposed to 200 micrograms/l Cd for 7 days. The eight participating laboratories were able to discriminate between controls and treated samples using a spectrophotometric method. The EROD activity was evaluated by 11 laboratories. All laboratories were able to discriminate between liver microsomal preparations obtained from control and from benzo-a-pyrene exposed fish (Dicentrarchus labrax), with values ranging from 0.5 to 15.88 pmol/min/mg protein in controls and from 5.41 to 165.13 pmol/min/mg protein in treated animals. Using S9 fractions, it was possible to correctly identify control and treated fish, with a variation similar to that found using microsomal fractions, albeit with an inevitable difference in specific activity. As a corollary, all laboratories involved produced comparable data and were able to identify pollutant-induced stress syndromes in sentinel organisms. Thus, intercalibration enables the use of biomarkers in large biomonitoring programs.     

Biomarker responses in Macoma nasuta (Bivalvia) exposed to sediments from northern San Francisco Bay

Our study investigates biomarker responses and survival of Macoma nasuta exposed to sediments collected from six locations in northern San Francisco Bay. Biomarkers analyzed were stress proteins (hsp70) in gill, mantle and digestive gland, lysosomal membrane damage and histopathologic lesions. Sediments and clam tissues were analyzed for a comprehensive suite of heavy metals and trace organic pollutants. Sediment grain size and organic carbon content were determined. Clams accumulated metals, polyaromatic hydrocarbons (PAHs) and organochlorine pesticides (aldrin and p,p(')-DDT and its metabolites p,p(')-DDD and p,p(')-DDE). Pearson and Spearman correlation analysis revealed that mortality, hsp70 in gill and histopathologic lesion scores in gonads, and lysosomal membrane damage were significantly correlated with tissue concentrations of DDT and/or its metabolites. Tissue concentrations of metals, in particular nickel, chromium, and copper, were associated with macrophage aggregates in digestive gland and germ cell necrosis. Cadmium was linked to mortality and lysosomal membrane damage.     

Concepts for the implementation of biomarkers in environmental monitoring

Biomarkers are promising tools for biomonitoring, both in the marine and freshwater environment. It is however clear that much more information is needed about the exact relation between biomarker responses and the health and fitness of organisms, and even more so between biomarker responses and risks for the ecosystem. In order to address these questions, it is important to realise that the possibilities for the application of a biomarker depend on the concept that is chosen for environmental monitoring. The purpose of monitoring programs varies from simple screening to risk characterisation on the ecosystem level. This paper compares a number of these concepts with respect to how biomarkers can be used and with respect to the specific requirements for further implementation of biomarkers in environmental monitoring.     

A multibiomarker approach in the clam Ruditapes decussatus to assess the impact of pollution in the Ria Formosa lagoon,South Coast of Portugal

The Ria Formosa lagoon is an ecosystem whose water quality reflects the anthropogenic influence upon the surrounding areas. In this lagoon, the clam Ruditapes decussatus has a great economical importance and has been widely used as a biomonitor. A multibiomarker approach (δ-aminolevulinic acid dehydratase, metallothionein, lipid peroxidation, acetylcholinesterase, alkali-labile phosphates, DNA damage) was applied to assess the environmental quality of this ecosystem and the accumulation of contaminants and their potential adverse effects on clams. Clams were sampled in different shellfish beds in the period between July 2007 and December 2008 and abiotic parameters (temperature, salinity, pH and dissolved oxygen of seawater and organic matter in the sediment), condition index, metals (Cd, Cu, Zn, Ni, Pb), TBTs and PAHs concentrations were measured in clam tissues. Data was integrated using Principal Component Analyses and biomarker indices: IBR (Integrated Biomarker Response) and HSI (Health Status Index). This multibiomarker approach enabled discrimination of a time and space trend between sites with different degrees of anthropogenic contamination, identifying one of them (site 2) as the most stressful and summer months as the most critical period for clams due to an increase of environmental stress (anthropogenic pressure along with extreme environmental conditions, e.g. temperature, dissolved oxygen, organic matter in the sediments, etc). The selected biomarkers provided an integrated response to assess the environmental quality of the system, proving to be a useful approach when complex mixtures of contaminants occur.     

Assessment of lysosomal membrane stability and peroxisome proliferation in the head kidney of Atlantic cod (Gadus morhua) following long-term exposure to produced water components

There is a need for sensitive biological effect methods by which to detect impacts of chronic exposure to low concentrations of contaminants. Two methods shown to be potentially useful for monitoring purposes in fish include lysosomal membrane stability and peroxisome proliferation. These biological endpoints were assessed in Atlantic cod (Gadus morhua) head kidney following exposure to a mixture of produced water components including polycyclic aromatic hydrocarbons, phenol, and alkylphenols. Lysosomal damage of head kidney cells occurred within the first two weeks and did not recover during the entire exposure period (32 weeks). Lysosomal membrane stability was not affected by gender and was responsive at low concentrations of contamination, indicating that lysosomal membrane stability measured in the head kidney could be a useful biomarker for effects of offshore pollution. Peroxisome proliferation, measured as acyl-CoA oxidase activity in the head kidney, appeared to be a potential biomarker in male cod exposed less than 16 weeks.     

Significance of metallothioneins and lysosomes in cadmium toxicity and homeostasis in the digestive gland cells of mussels exposed to the metal in presence or absence of phenanthrene

Metallothioneins and lysosomes are known to be involved in cellular detoxication and sequestration of certain metals^1–3 and both have been identified in this role in elimination of copper from marine mussels (Mytilus edulis/galloprovincialis).³ Cadmium (Cd), however, has been shown to persist in the cells of the digestive gland for long periods with only minimal elimination. An experiment was designed to test the effects of Cd on the fragility of lysosomal membranes in the digestive cells as a measure of cellular injury,^4,5 metallothionein content of the digestive gland and cadmium concentration in this organ. Phenanthrene was used also to destabilise lysosomal membranes⁶ in order to test if increased lysosomal fragility interfered with cadmium metabolism and detoxication. The results demonstrated that Cd induced metallothionein synthesis and that elimination of Cd was minimal after 28 days in clear seawater. Lysosomal fragility was initially increased but this effect was soon reversed, even with continued exposure to Cd. The lysosomal destabiliser, phenanthrene, did not appear to affect accumulation of Cd or levels of metallothionein.     

Effects of polynuclear aromatic hydrocarbons on lysosomal membranes in molluscs

Lysosomal sequestration of polynuclear aromatic hydrocarbons (PNAHs), a major class of environmental contaminant, is a well-established phenomenon;¹ considerably less is known about their pathological effects on lysosomes. Marine molluscs contain a number of lysosome-rich tissues and PNAHs are known to induce deleterious alterations in lysosomal structure and latency of lysosomal enzymes.² The latter are presumed to involve destabilisation of the lysosomal membrane, resulting in increased permeability and reduced enzyme latency. If lysosomal injury involves derangement of membrane-lipid structure due to the interaction of PNAHs then it would be expected that membrane damage would be closely linked to the structural characteristics of the intruding molecule. Our results show that the effects of the isomeric PNAHs phenanthrene and anthracene on digestive cell lysosomal stability were markedly different in the marine mussel (Mytilus edulis) over the same range of tissue concentrations. Lysosomal membrane stability was determined using a cytochemical test for enzyme latency.³     

The fine structure of lysosomal membranes and endoplasmic reticulum in the digestive cells of Mytilus edulis exposed to anthracene and phenanthrene

The stability of lysosomes in the digestive cells of Mytilus edulis is affected by changes in both the chemical and physical conditions of the environment.¹ This type of response must reflect, at least in part, some change in the organisation of the lysosomal membrane, which is a structure which can be resolved by the electron microscope. However, conventional methods of tissue preparation have produced low contrast images of the digestive gland and damage to the lysosomal membranes. Recently a cryopreparation technique² has produced lysosomal membrane preparations in the digestive cells of the digestive gland of Mytilus which have high contrast and structural integrity (Fig. 1) in control animals. This method has been used to demonstrate pathological alterations in the lysosomal membrane induced by the polynuclear aromatic hydrocarbon (PNAH) phenanthrene. Anthracene, an isomeric PNAH, had no effect on the morphology of the lysosomal membrane. Phenanthrene also induced apparent proliferation of smooth endoplasmic reticulum in the digestive cells.