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.     

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.     

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.     

Lysosomal responses to a polynuclear aromatic hydrocarbon in a marine snail: Effects of exposure to phenanthrene and recovery

Certain specific aspects of cellular structure-linked functions can be used as rapid and sensitive indicators of cellular responses to environmental stimuli such as chemical contaminants.¹ It is possible to observe structural-functional alterations in lysosomal membrane stability in the cells of some marine organisms at an early stage of such a response to cell injury,^1,2 thus providing an early-warning signal of a potentially deleterious environmental situation. In order to further investigate lysosomal membrane disturbances, mechanisms of injury and capacity for cellular regeneration, the marine snail Littorina littorea was exposed to phenanthrene, which was used as an environmentally widespread and representative polynuclear aromatic hydrocarbon. Lysosomal membrane stability was measured using cytochemical determination of hydrolase latency,² and lysosomal lipofuscin was measured as a possible indicator of enhanced lipid peroxidative damage.³     

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.     

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.     

Effects of nanoparticles in Mytilus edulis gills and hepatopancreas - a new threat to marine life?

Every day new extraordinary properties of nanoparticles (a billionth of a meter) are discovered and worldwide millions are invested into nanotechnology and nanomaterials. Risks to marine organisms are still not fully understood and biomarkers to detect health effects are not implemented, yet. We used the filter feeding blue mussel as a model to analyse uptake and effects of nanoparticles from glass wool, a new absorbent material suggested for use in floating oil spill barriers. In both, gills and hepatopancreas we analysed uptake of nanomaterials by transmission electronmicroscopy (TEM) in unstained ultrathin sections over a period of up to 16 days. Lysosomal stability and lipofuscin content as general indicators of cellular pathology and oxidative stress were also measured. As portals of uptake, diffusion and endocytosis were identified resulting in nanoparticle accumulation in endocytotic vesicles, lysosomes, mitochondria and nuclei. Dramatic decrease of lysosomal membrane stability occurred after 12h of exposure. Lysosomal damage was followed by excessive lipofuscin accumulation indicative of severe oxidative stress. Increased phagocytosis by granulocytes, autophagy and finally apoptosis of epithelial cells of gills and primary and secondary digestive tubules epithelial cells indicated progressive cell death. These pathological responses are regarded as general indices of toxic cell injury and oxidative stress. By the combinational use of biomakers with the ultrastructural localisation of nanoparticle deposition, final evidence of cause-effect relationships is delivered.     

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.     

Field application of lysosomal destabilisation indices in the mussel Mytilus galloprovincialis: biomonitoring and transplantation in the Lagoon of Venice (north-east Italy)

A field study was carried out in the Lagoon of Venice (north-east Italy) with the aim of evaluating the potential use of lysosomal destabilisation as a biomarker of anthropogenic stress in the autochthonous mussel Mytilus galloprovincialis. Two different approaches were adopted in biomonitoring six sites in the Lagoon, evaluating indigenous populations of mussels and organisms transplanted from a reference site and checked at several points in time. Lysosomal membrane stability was investigated by means of two tests: neutral red retention assay (NRRA) for evaluating haemocyte lysosomes and lysosomal latency test (LLT) for digestive cell lysosomes. Results indicate that the lysosomal response measured in haemocytes according to NRRA is a more valuable biomarker of anthropogenic stress in the framework both of passive and active biomonitoring in marine coastal environments.     

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.     

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.     

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.     

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     

Environmental prognostics: is the current use of biomarkers appropriate for environmental risk evaluation?

Environmental Prognostics is proposed as an integrated explanatory framework for adverse changes in whole systems, from cells to animals to ecosystems. The aim is to develop such frameworks for the evaluation of "health of the environment" and prediction of consequences resulting from future environmental events based on integrating the reactions of biomarkers for cellular and physiological processes, through conceptual, statistical and computational modelling. These are urgently needed to synthesise complex information on environmental chemistry and injurious effects of pollutants into predicted harmful impact on health of sentinel animals.     

Stability of lysosomal and cell membranes in haemocytes of the common mussel (Mytilus edulis): effect of low temperatures.

Expanding industrial activities in the Arctic require an urgent assessment of the toxicity of chemicals at low temperatures. Organisms acclimatized to low temperature exhibit specific adaptations. For example, the amount of unsaturated lipids is increased to maintain the fluidity of the cell membranes. It has been hypothesized that such temperature-induced alterations in membrane lipid composition may affect the stability of lysosomal and cell membranes in the common mussel, Mytilus edulis, an organism exposed to seasonal temperature extremes. As mussels may be exposed to petroleum compounds along industrialized coastlines, we tested the combined effects of exposure to low temperature and the petroleum compound, phenanthrene, on haemocyte membrane stability. Test animals, acclimated to either 0 or 10 degrees C, were exposed to phenanthrene (0 = control or 500 micrograms l-1) and haemocytes were examined using the neutral red retention assay (lysosomal stability) and a fluorescence assay (cell membrane stability). At 0 degree C, lysosomal and cell membranes from uncontaminated mussels were destabilized compared with 10 degrees C (P = 0.0005). No significant effects (P > 0.05) of phenanthrene were detected at either temperature. Possible mechanisms underlying membrane destabilization include a weaker physical resistance of the membrane due to a higher amount of unsaturated lipids, a potentially higher level of reactive oxygen radicals at low temperature and the higher susceptibility of unsaturated lipids to oxidative stress. More work is required to better understand the consequences of this membrane destabilization at low temperature on the susceptibility of the organism to pollutants.     

Impact of the Sea Empress oil spill on lysosomal stability in mussel blood cells.

Coastal zones are among the most productive and vulnerable areas on the planet. An example of impact on these fragile environments was shown in the case of the "Sea Empress" oil tanker, which ran aground in the Bristol Channel in 1996, spilling 72,000 tons of "Forties" crude oil. The objective was to investigate the sub-lethal cellular pathology and tissue hydrocarbon contamination in marine mussel populations, 4 months after the initial spill, using the neutral red retention (NRR) assay for lysosomal stability in blood cells. NRR was reduced in mussels, and indicative of cell injury, from the two sites closest to the spill in comparison with more distant and reference sites. Lysosomal stability was inversely correlated with polycyclic aromatic hydrocarbon concentrations in mussel tissues. Reduced lysosomal stability has previously been shown to contribute to impaired immunocompetence and to autophagic loss of body tissues. The use of this type of technique is discussed in the context of cost-effective, ecotoxicological tools for Integrated Coastal Zone Management.     

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.     

Linkages between cellular biomarker responses and reproductive success in oysters--Crassostrea virginica

The purpose of these studies was to evaluate if there were relationships between lysosomal destabilization or glutathione concentrations and gamete viability of oysters, Crassostrea virginica. Oysters were collected from field sites during the peak spawning period (May-June) during 2001 and 2002. Lysosomal destabilization rates and glutathione concentrations of hepatopancreas tissues (e.g. digestive gland) were determined. Eggs and sperm from the same adults were also used to conduct embryo development assays with reference seawater collected from a clean site, site water, and also a range of Cd concentrations (the Cd exposures were used to determine if there were differences in susceptibility to pollutants). Baseline embryo development success (e.g. percent normal development when the assays were conducted with reference seawater or site water) was related to lysosomal destabilization, but not glutathione status. However, the susceptibility of embryos to metal toxicity was related to glutathione status, i.e., sensitivity to Cd exposures increased with decreasing glutathione levels. These studies support the hypotheses that there are linkages between embryo development success and susceptibility to pollutant stress and cellular biomarker responses. These kinds of effects on reproductive success could lead to subtle but significant long-term effects on recruitment and viability of oyster populations.     

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.     

Linkages between cellular biomarker responses and reproductive success in oysters – Crassostrea virginica

The purpose of these studies was to evaluate if there were relationships between lysosomal destabilization or glutathione concentrations and gamete viability of oysters, Crassostrea virginica. Oysters were collected from field sites during the peak spawning period (May–June) during 2001 and 2002. Lysosomal destabilization rates and glutathione concentrations of hepatopancreas tissues (e.g. digestive gland) were determined. Eggs and sperm from the same adults were also used to conduct embryo development assays with reference seawater collected from a clean site, site water, and also a range of Cd concentrations (the Cd exposures were used to determine if there were differences in susceptibility to pollutants). Baseline embryo development success (e.g. percent normal development when the assays were conducted with reference seawater or site water) was related to lysosomal destabilization, but not glutathione status. However, the susceptibility of embryos to metal toxicity was related to glutathione status, i.e., sensitivity to Cd exposures increased with decreasing glutathione levels. These studies support the hypotheses that there are linkages between embryo development success and susceptibility to pollutant stress and cellular biomarker responses. These kinds of effects on reproductive success could lead to subtle but significant long-term effects on recruitment and viability of oyster populations.