The role of oxyradicals in intracellular proteolysis and toxicity in mussels

Studies were performed on the common mussel, M. edulis L., to determine whether copper (Cu) exposure can affect the extent to which digestive cell proteins are oxidised and whether such oxidative damage is mediated by free radicals. Three age groups of mussels were exposed for 6 ^-days to environmentally realistic concentrations of Cu and then digestive gland homogenates were examined for evidence of protein carbonyl formation. Significant increases in carbonyls relative to untreated control mussels were seen for the youngest (2–4 year-old) and oldest (≥ 10 year-old) mussels only after exposure for 6 days, followed by recovery from exposure for a further 6 days. Untreated mussels also showed an age-related difference in protein oxidation, with a significantly lower concentration in the youngest animals (2–4 year olds). Copper did not affect the levels of modified tryptophan or tyrosine residues or the extent of total lipid peroxidation in digestive gland homogenate. Significant depletion of total vitamin E (a-tocopherol) was seen only in young and medium-aged mussels following exposure for 6 days. The levels of protein carbonyl groups were increased in digestive cell cytosol and lighter lysosomes but not in heavier lysosomes or digestive gland microsomes following 5 days exposure to Cu. Dihydrohodamine-123 was converted to fluorescent rhodamine-123 following sequestration into digestive cell lysosomes. The results suggest a link between the lysosomal sequestration of copper, a concomitant increase in the production of oxyradicals and the potential for intracellular oxidative damage, as well as an increased capacity for oxidative damage in older animals.     

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     

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.     

Assessment of immune parameters of manila clam <Emphasis Type="Italic">Ruditapes philippinarum</Emphasis> in different physiological conditions using flow cytometry

Cellular and humoral immune parameters are often used as biomarkers to trace environmental and physiological stresses in marine bivalves. In this study, we compared various immune parameters of Manila clams (Ruditapes philippinarum) under normal conditions and under a high level of desiccation, using flow cytometry. The immune parameters analyzed included, total hemocyte count, hemocyte mortality, hemocyte DNA damage, reactive oxygen species (ROS) production, and phagocytosis activity. Total hemocyte count, hemocyte DNA damage, and hemocyte mortality were significantly elevated among clams under high desiccation stress, while phagocytosis activity and spontaneous ROS production were significantly lower compared to those parameters of the control clams (p<0.05). These data suggest that the immune parameters analyzed in this study well reflect the physiological status of clams.     

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.     

Polycyclic aromatic hydrocarbon (PAH) burden of mussels (Mytilus sp.) in different marine environments in relation with sediment PAH contamination, and bioavailability

Sediments and mussels (Mytilus edulis, Mytilus galloprovincialis) were sampled in different European coastal environments (Germany, France, Spain) and analysed for their polycyclic aromatic hydrocarbon (PAH) content by Gas Chromatography/Mass Spectrometry (GC-MS). Bioaccumulation factors of individual compounds from the sediment were calculated and discussed according to the compound solubility. The mussels showed different accumulation patterns according to the pollution source they were exposed to (dissolved fraction of PAHs, particulate fraction, petroleum present in the water column). The exposure source also depends on the geographical location of the mussels. In the Mediterranean Sea, the bivalves were mainly exposed to the dissolved fraction of PAHs, while in the Baltic Sea and in the Atlantic Ocean, the PAHs associated to the particles were significant sources.     

Molluscan lysosomal responses as a diagnostic tool for the detection of a pollution gradient in Tolo Harbour, Hong Kong

Sub-cellular perturbations in the lysosomal compartment of molluscan haemocytes were examined in mussels (Perna viridis), collected along a pollution gradient. The neutral red technique was validated using a well defined contamination gradient among indigenous populations from five stations along Tolo Harbour, Hong Kong. Condition indices (shell length:dry tissue wt) and tissue metal concentrations were also measured in an attempt to identify a relationship between contamination level and adverse physiological effects. Correlations were found between lysosomal retention time and condition along the pollution gradient. There were significant differences between mussels collected from stations on offshore islands and those collected from inner harbour sites (p < 0.05). There was, however, little correlation between metal concentrations and retention time or condition (p > 0.05).     

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.     

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.     

Variability of the hemocyte parameters of cultivated mussel Mytilus galloprovincialis (Lmk 1819) in Sabaudia (Latina,Italy) coastal lagoon

The Sabaudia's lake consists of a protected coastal lagoon, located in the central Italy, historically characterized by recurrent mortality events of marine fauna during warmer months. A field study was monthly conducted on mussels Mytilus galloprovincialis cultivated inside the lagoon, measuring hemocyte parameters as total circulating count (THC), viability (HV), spreading and oxidative response to in vitro phagocytosis stimulation. A depression of the immune response was observed during the spring season, as indicated by higher values of hemocyte circularity and lower luminescence levels related to respiratory burst, also associated to modulation of THC and HV. The water temperature and the oxygen concentration appeared as the major environmental factors having influence on the phagocytosis activity. Therefore, the hemocyte variations have been intended as early danger signal to evaluate the immunodepression induced by the environmental stressors which could reveal in advance the development of critical situations for mussel survival.     

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.     

Responses of the mussel Mytilus edulis to copper and phenanthrene: Interactive effects

Most investigations of the responses of marine organisms to xenobiotics have concentrated on single contaminants and little is known of possible interactive effects of different classes of xenobiotics. As these latter seldom occur in environmental isolation, it is important to understand any interactions (synergistic or antagonistic) which may occur. This problem has been approached in the mussel Mytilus edulis by exposing estuarine mussels to copper (20 μg litre⁻¹) and phenanthrene (100 μg litre⁻¹) both individually and in combination, and measuring cytochemical subcellular and physiological responses after 3 days exposure and 3 days and 12 days recovery period. Results showed that mussels accumulated both xenobiotics during 3 days exposure. Depuration of copper was complete in 3 days recovery period, while loss of phenanthrene ranged from 30% to 70% of the concentration reached after 3 days exposure. There were no interactive effects on depuration.Both copper and phenanthrene reduced lysosomal hydrolase latency in digestive cells, and copper appeared to have a synergistic effect in preventing recovery of latency of lysosomal N-acetyl-β-hexosaminidase during the recovery period. There was evidence, in the digestive cells, of an antagonistic effect of copper on stimulation of activity of the microsomal respiratory chain (measured as NADPH-neotetrazolium reductase) by phenanthrene. Stimulation of this system by phenanthrene persisted after 12 days recovery period. There was a synergistic interaction of copper and phenanthrene on elevation of oxygen consumption and ammonium excretion. Clearance rates and scope for growth (physiological condition) were depressed by copper but not by phenanthrene after 3 days exposure.These findings are discussed in terms of known effects of copper and phenanthrene and the interactions are considered in terms of environmental effects measurements.     

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.     

Implications from a field study regarding the relationship between polycyclic aromatic hydrocarbons and glutathione S-transferase activity in mussels

An aluminium smelter on the west coast of Scotland discharges an aqueous effluent containing polycyclic aromatic hydrocarbons (PAHs) at the head of Loch Leven. The loch also supports two mussel (Mytilus edulis) farms. Data are presented on burdens of PAHs in the soft tissues of mussels and the effect of these contaminants on glutathione S-transferase (GST) activity in mussel hepatopancreas. GST activity is shown to be correlated with total PAH burden and also with the concentrations of certain individual PAHs. These field data show that high molecular weight PAHs are closely correlated to GST activity, whereas low molecular weight PAHs are not. This suggests that 5- and 6-ring PAHs have a more pronounced role than 2- to 4-ring compounds in inducing GST activity in mussels from Loch Leven. It is proposed that it may be more appropriate to link GST activity with 5- and 6-ring compounds only, rather than with the total PAH burden.     

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.     

Enhancement of polycyclic aromatic hydrocarbons in estuarine invertebrates by surface runoff at a decommissioned military fuel depot

Accumulation of polycyclic aromatic hydrocarbons (PAHs) was determined in blue mussels (Mytilus spp.) and shore crabs (Hemigrapsus sp.) at a recently closed military fuel depot in central San Francisco Bay, California. In April 1996, during a period of above average precipitation, specimens were collected at the depot, near the depot, and at sites 10 and 20 km south of the depot. Four weeks after the rains ended, blue mussels were again collected at the depot, and at two additional sites in the central Bay region. In April, total PAHs in mussels from the depot were significantly higher only than that in mussels collected 20 km from the depot; however, seven specific, substituted PAHs were higher at the depot than at all other sites. In June, only two of the 38 PAHs common in mussels in April were detected at the depot; these concentrations were comparable to ambient concentrations in mussels at the Bay. It seemed that bioavailability of PAHs at the depot was enhanced by rainfall, probably due to the mobilization of PAHs via groundwater into the Bay. Concentrations in mussels from chronically contaminated sites were about five times higher than mussels collected from the depot. Low PAH concentrations were detected in shore crabs near the depot, and the highest levels were not associated with the depot. Observed PAH concentrations are discussed in relation to upper trophic organisms.     

The effect of shore location on biomarker expression in wild Mytilus spp. and its comparison with long line cultivated mussels

Biomarkers are a common tool in the assessment of potential effects of contaminants in aquatic organisms. In order to identify the effects of anthropogenic pollution it is essential to identify background levels and to know the range of natural variability in the biomarker response. In this study, we examined various biomarkers of stress (glutathione S-transferase and metallothionein), damage (lipid peroxidation (LPO) and DNA damage (DNA)) and reproduction (vitellin-like proteins) in marine mussels (Mytilus spp.) from four locations along a vertical transect from high to low shore and compared them with cultivated long line mussels. High shore and cultivated mussels showed significantly higher LPO and DNA damage expression than the low shore mussels indicating a level of oxidative stress resulting from mussel location. Significant effects in physiological endpoints were also found. This study highlights the need to consider the diversity of natural environmental stress factors when using biomarkers in environmental assessment.     

Acute sublethal toxicity of MV Rena contaminants (heavy fuel oil,oil dispersant and cryolite) to finfish and rock lobster

ABSTRACT

The wreck of the MV Rena in October 2011 discharged large quantities of potentially toxic materials in the coastal marine environment of the Bay of Plenty, New Zealand. We evaluated sublethal effects of the water soluble fractions of physically and chemically dispersed heavy fuel oil (HFO) and the water soluble fraction of cryolite on the haematology of subadult or adult snapper (Chrysophrys auratus), spotted wrasse (Notolabrus celidotus) and red rock lobster (Jasus edwardsii). Small but transient effects occurred, the most significant of which were changes in circulating immune cell populations in fish and lobster that recovered by the conclusion of a 96-h exposure period. No post-exposure effects were observed during a 10-day recovery in toxicant-free seawater. Corexit 9500 oil dispersant increased polycyclic aromatic hydrocarbon (PAH) uptake from HFO in fish and lobster by around two-fold and four-fold, respectively, and caused a larger change in immune cell abundance in snapper than exposure to HFO alone. While depuration of PAHs was more rapid in fish than lobster, significantly elevated PAH levels were still evident after 10 days of depuration.     

Correlation of CYP1A1 induction, as measured by the P450 RGS biomarker assay, with high molecular weight PAHs in mussels deployed at various sites in San Diego Bay in 1993 and 1995

In 1993 collections of marine mussels (Mytilus galloprovincialis) were deployed 1 m from the water surface at six sites in San Diego Bay for 88 days. A similar mussel deployment was conducted in 1995, except the animals were deployed 1 m off the bottom and only for 32 days. After recovery from the sites, tissue was extracted with dichloromethane and the solvent extracts analyzed for chemical contaminant content and the ability to produce CYP1A1 induction in a transgenic cell line (TV101L cells). The cells used in the assay (P450 RGS) are stably transfected with a plasmid containing firefly luciferase linked to human CYP1A1 promoter sequences. Induction (fold increase compared to control) was determined by luminometry 16 h after application of small volumes (2–10 μl) of solvent extracts to cultured cells. Small mussels deployed in the Naval Station (NAV) in 1993 exhibited very high bioaccumulation of polycyclic aromatic hydrocarbons (PAHs; 52 μg/g) and polychlorinated biphenyls (PCBs), in addition to very strong induction of CYP1A1 measured by reporter gene system (RGS) responses. Large mussels deployed at the NAV station in 1993 and intermediate-sized animals placed at three stations within the NAV station in 1995 accumulated 13–29 μg PAH/g and exhibited relatively high RGS responses. Correlation of RGS responses for all mussel samples to the measured PAH concentrations was 0.85 (r²). When the concentrations of seven specific PAHs found in the samples are converted to benzo[a]pyrene equivalents, from previously derived toxic equivalency factors (TEFs) for this test system, and compared to measured RGS responses, the correlations are approximately 0.9. The results of these studies indicate that the RGS biomarker can be used as a screening tool for detection of CYP1A1-inducing compounds in tissues, and an estimate of potential human health or ecological risk from ingestion of contaminated organisms. Positive RGS responses can be followed by detailed chemical analyses of PAHs and coplanar PCBs using the same extract.     

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