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.     

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.     

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.     

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.     

Metabolism of a model environmental carcinogen by bivalve molluscs

Bivalve molluscs have been used as monitors of marine pollution because they concentrate xenobiotics of many kinds in their tissues. Marine pollutants often produce stress responses such as reduced scope for growth, lysosomal destabilization, altered immune responsiveness and other sequelae. The ability of bivalves to metabolize organic compounds via mixed-function oxygenases (MFO) was demonstrated by the epoxidation of aldrin¹ and benzo[a]pyrene (BP);² this work has been corroborated and extended in many subsequent studies. Biotransformation of BP and other environmental procarcinogens can generate either activated or detoxified metabolites. In this paper the BP metabolites produced by digestive gland homogenates of the clam (Mercenaria mercenaria) and the oyster (Crassostrea virginica) are described. Control bivalves produced the proximate carcinogen (BP 7,8-dihydrodiol), as well as various minimally reactive BP diols and monohydroxylated metabolites. Seven days after injection of the potent inducer of mammalian MFO, Aroclor 1254, BP 7,8-diol production was augmented in Mercenaria, and atypical quinones and phenols were seen in both species. However, in the Ames bacterial mutagenicity assay, homogenates from Aroclor-treated clams failed to activate benzo[a]pyrene. The biological significance of BP metabolism in bivalves is unknown; however, it may prove to be a useful index of pollution.     

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.     

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.     

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.     

Lysosomal injury and MFO activity in the liver of flounder (Platichthys flesus L.) in relation to histopathology of hepatic degeneration and carcinogenesis

Flounder (Platichthys flesus L.), 18–48 cm total length were sampled in the mouth of the Elbe River during a 3-year interdisciplinary project ‘Fish Diseases in the Wadden Sea’. Lysosomal changes (lysosomal membrane stability) and the activity of biotransforming enzymes (MFOs/EROD) were measured parallel to investigations of ultrastructural changes in liver cells, tissue pathologies and macroscopically visible changes. The aim of the study was to investigate if these cyto- and biochemical parameters were able to reflect contaminant induced biological effects. Interlinking of the results of the MFO activity to the pathological alterations observed at the electron- and light-microscopic levels as well as during macroscopic inspection of identical individuals evidenced that the activity of the biotransformation enzymes (EROD) was not induced in healthy livers, increased considerably with the onset of liver changes and dropped again in those livers with degenerative, preneoplastic and neoplastic lesions. In contrast, concurrent studies of lysosomal membrane stability measured in parallel showed a decrease with the onset and progression of liver lesions from reversible to irreversible, neoplastic change.     

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.     

Uptake of Zn and Mn into body tissues and renal concretions by the Southern Quahog, Mercenaria campechiensis (Gmelin): Effects of elevated phosphate and metal concentrations

The seawater chemistry of potentially toxic metals can affect their availability to marine organisms. Investigation of the relationship between metal chemistry and metal bioavailability has progressed slowly due to difficulties in controlling and measuring metal speciation in uptake media. Recent work with strong metal chelators such as NTA and EDTA has allowed a closer examination of how metal chemistry relates to biological accumulation and toxicity.^1–3 However, the presence of a strong chelator at membrane transport sites and the possible alteration of microenvironments by strong chelators could create unnatural uptake behavior. This study presents another method for stabilizing metal chemistry in accumulation experiments. A cation exchange resin was used to study Mn⁵⁴ accumulation by a small bivalve Donax variabilus. The resin proved an effective method for buffering manganese chemistry in seawater and could provide a useful tool to look for subtle effects present in other metal buffered seawater systems.     

The impact of metals on the reproductive mechanisms of the ascidian Ciona intestinalis

Environmental pollution due to anthropogenic activities may exert an adverse impact on the reproductive mechanisms of animals. In particular, environmental chemicals introduced into seawater are able to disrupt the normal development and function of the reproductive system of marine animals. In this study, we have used the whole‐cell voltage clamp technique to examine the effects of four metals – lead, cadmium, mercury, and zinc – on reproductive mechanisms of the marine ascidian Ciona intestinalis (tunicates). In particular, we measured the effect of metals on plasma membrane electrical properties, the steady‐state conductance, the fertilization current in the mature oocyte, and larval development. Results show that oocyte voltage‐gated sodium currents are significantly reduced by all four metals, steady‐state conductance is affected only by zinc, and post‐fertilization contraction is inhibited only by lead. The fertilization current is suppressed in the presence of zinc and mercury. Embryo development up to larval stage is inhibited by zinc and mercury exposure with a reversible effect; however, a long‐term effect on larval morphology was observed. After exposure to cadmium, fertilization occurs but gives rise to abnormal larval development. These findings highlight the point that exposure to metals represents a significant risk factor for the physiology of reproduction of marine species and suggest a possible role of the C. intestinalis as a bioindicator for marine pollution monitoring.     

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.     

Immune inhibition in marine mussels by polycyclic aromatic hydrocarbons

Polycyclic aromatic pollutants produced by a range of industrial processes and the combustion of fossil fuels. They eventually enter the marine environment, and are bioaccumulated in the tissues of sessile, filter-feeding bivalves, causing a variety of sublethal effects. Few studies, however, have examined immune inhibition in molluscs by these xenobiotics. Here, the immunotoxic effects of PAHs were investigated in marine mussels (Mytilus edulis) by means of in vivo exposure experiments. Animals were exposed to a PAH cocktail of anthracene, fluoranthene and phenanthrene for 2 or 4 weeks. The haemolymph was then extracted, and phagocytosis and lysosomal neutral red retention assays performed to examine the effects of PAHs on particle uptake by haemocytes and lysosome integrity, respectively. PAHs were found to inhibit phagocytosis and damage lysosomes. It is hypothesized that PAHs exert their toxicity directly on the lysosomes. A possible consequence of such lysosomal damage will be immune impairment in mussels leading to reduced resistance to infectious diseases.     

Use of colloidal iron as a tracer to measure the ingestion rates of free-living marine nematodes

Ingestion rates of free-living marine nematodes were measured using colloidal iron as a tracer. Detritus labeled with colloidal iron was fed to the dominant nematode species and cultured. The iron ingested by the animals was quantified using a scanning electron microscope equipped with differential X-ray energy analyzer (EDX). Average ingestion rates (μgC ind.⁻¹day⁻¹) measured were 0.28 for Symplocostoma sp., 0.29 for Polygastrophora sp., 0.73 for Mesacanthion sp. and 0.15 for Metachromadora sp. These values were higher than the values determined using radioactive organic materials as tracers, though the present method is considered to provide conservative values. This result thus strongly suggests that the ingestion rates of nematodes measured so far were underestimated, and the use of colloidal iron has advantages over the use of radioactive organic matter as a tracer.     

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.     

细菌源3-羟基脂肪酸作为环境变化代用指标的研究进展

细菌源3-羟基脂肪酸(C₁₀–C₁₈)作为环境变化指示指标具有良好的应用前景,但相关研究还很不系统,在海洋环境中的应用刚刚起步。3-羟基脂肪酸主要用于环境中pH和温度的重建,通过其支链比(异构和反异构3-羟基脂肪酸之和/正构3-羟基脂肪酸之和)与pH的显著正相关关系反演环境中的pH,通过其C₁₅和C₁₇同系物的反异构/正构比(RAN₁₅和RAN₁₇)与大气年均温的显著负相关关系反演环境中的温度,相比基于GDGT或其他生物标志物的环境代用指标具有明显的优势。然而,陆地生态系统中基于3-羟基脂肪酸的环境指标不适用于海洋环境,最新研究提出了基于3-羟基脂肪酸的新的海洋温度指标(RAN₁₃),而3-羟基脂肪酸作为海洋环境中pH替代指标的成功应用尚未见报道。3-羟基脂肪酸与特定细菌群落的空间耦合或菌株培养实验显示含有3-羟基脂肪酸的细菌可能主要是变形菌、蓝细菌等。分析表明,3-羟基脂肪酸作为全球环境演变有效的替代指标需要更多的数据和证据支持,未来可从海洋适用性、新指标体系和微生物来源几个方面展开继续研究。     

Bivalve molluscs as a unique target group for nanoparticle toxicity

Due to the continuous development and production of manufactured nanomaterials or nanoparticles (NPs), their uptake and effects in the aquatic biota represent a major concern. Estuarine and coastal environments are expected to represent the ultimate sink for NPs, where their chemical behavior (aggregation/agglomeration) and consequent fate may be critical in determining the biological impact.Bivalve mollusks are abundant from freshwater to marine ecosystems, where they are widely utilized in biomonitoring of environmental perturbations. As suspension-feeders, they have highly developed processes for cellular internalization of nano- and micro-scale particles (endo- and phagocytosis), integral to key physiological functions such as intra-cellular digestion and cellular immunity.Here we will summarise available information on the effects of different types of NPs in different bivalve species, in particular Mytilus spp. Data on the effects and modes of action of different NPs on mussel hemocytes in vitro demonstrate that cell-mediated immunity represents a significant target for NPs. Moreover, in vivo exposure to NPs indicates that, due to the physiological mechanisms involved in the feeding process, NP agglomerates/aggregates taken up by the gills are directed to the digestive gland, where intra-cellular uptake of nanosized materials induces lysosomal perturbations and oxidative stress. Overall, bivalves represent a particularly suitable model for investigating the effects and mechanisms of action underlying the potential toxicity of NPs in marine invertebrates.