Sex and sex-related differences in gamete development progression impinge on biomarker responsiveness in sentinel mussels.

In marine pollution monitoring, the biomarkers recorded in sentinel organisms are influenced by natural confounding factors that may jeopardise their interpretation. Among these confounding factors, little is known about the influence of sex along the annual reproductive cycle. The present investigation aims at contributing to understand how sex and sex-related differences in gamete development progression impinge on biomarker baseline values and on biomarker responsiveness to pollution in sentinel mussels. Mussels (Mytilus galloprovincialis) were collected from a relatively clean locality and from a chronically polluted site in the Basque Coast (Bay of Biscay) in January, April, August and November. Sex and gametogenesis stages were determined for each mussel. Tissue concentration of metals and PAHs was analysed. A battery of biomarkers was investigated: cytochrome c oxidase, pyruvate kinase and phosphoenolpyruvate carboxykinase enzyme activities; levels of protein carbonyls, malondialdehyde and 4-hydroxy-2-nonenal; lysosomal enlargement and membrane stability; intracellular neutral lipid accumulation; cell type composition and thinning of the digestive gland epithelium; and survival-in-air. Sex- and reproductive stage-related differences were found in bioaccumulation and in the values and responsiveness of most of the biomarkers. However, the patterns of sex-related differences were not consistent across all biomarkers. The differences in the biomarker responses between females and males also depended on the season, reflecting the progression of the gametogenesis cycle. Thus, selecting mussels of one specific sex does not seem to be a crucial requisite to carry out biomarker-based monitoring; yet, it is highly recommended to identify sex condition and gamete developmental stage of each mussel to test for the potentially confounding effects of sex, reproductive status and sex-related variability along the reproductive cycle.

Multi-annual survey of health status disturbance in the Bilbao estuary (Bay of Biscay) based on sediment chemistry and juvenile sole (Solea spp.) histopathology.

The Bilbao estuary (SE Bay of Biscay) is a recovering ecosystem whose sediments are still contaminated. They represent a potential risk for the biota including benthic and demersal species living in direct contact with the sediment. In this context, the present study aims to survey trends of the health status of the Bilbao estuary based on sediment chemistry and sole (Solea spp.) histopathology. Monitoring campaigns were carried out every autumn from 2011 to 2017 along the estuary. Contaminant levels were measured in sediments; liver, gills and gonads of juvenile fish were collected for histopathology. Overall, contaminant levels fluctuated throughout the years, with highest values recorded in the earlier years of the study period. Sole histopathology showed alterations of mild severity. Results permitted to assess the environmental health status of the Bilbao estuary during 7 years, although no clear temporal trend was detected. Longer-term monitoring programmes are necessary to confirm the ecosystem recovery.

Enhanced discrimination of basophilic cells on mussel digestive gland tissue sections by means of toluidine-eosin staining.

Changes in the cell type composition of the digestive gland epithelium constitute a common and recognized biological response to stress in mussels. Usually, these changes are identified as alterations in the relative proportion of basophilic cells, determined in tissue sections stained with hematoxylin-eosin (H&E) and measured in terms of volume density of basophilic cells (VvBAS) after stereological quantification. However, the identification and discrimination of basophilic cells may be a difficult issue, even for a trained operator, especially when, in circumstances of environmental stress, basophilic cells lose their basophilia and the perinuclear area of digestive cells gains basophilia. Thus, the present study was aimed at exploring the best available practices (BAPs) to identify and discriminate basophilic cells on tissue sections of mussel digestive gland. In a first step, a thorough screening of potentially suitable staining methods was carried out; the final selection included several trichrome staining methods and some of their variants, as well as toluidine-based stains. Next, the sample processing (fixation/dehydration steps) was optimized. Toluidine-eosin (T&E) staining after fixation in 4% formaldehyde at 4 °C for 24 h was considered the BAP to identify and discriminate basophilic cells in the digestive gland of mussels. Using the mussel Mytilus galloprovincialis as a target organism, this approach was successfully applied to quantify VvBAS values after automated image analysis and compared with the conventional H&E staining in different field and laboratory tests. It is worth noting that VvBAS values were always higher after T&E staining than after H&E staining, apparently because discrimination of basophilic cells was enhanced. Thus, until more data are available, any comparison with VvBAS values obtained in previous studies using H&E staining must be done cautiously. Finally, the T&E staining was successfully used to discriminate basophilic cells in tissue sections of other marine molluscs of ecotoxicological interest, including Mytilus edulis, Mytilus trossulus, Crassostrea gigas and Littorina littorea.

The influence of short-term experimental fasting on biomarker responsiveness in oil WAF exposed mussels.

Mussels are widely used in toxicological experimentation; however, experimental setups are not standardized yet. Although there is evidence of changes in biomarker values during food digestion and depending on the mussel nutritive status, the mode of feeding differs among toxicological experiments. Typically, mussels are fed with different diets in different long-term experiments, while fasting is the most common approach for short-term studies. Consequently, comparisons among experiments and reliable interpretations of biomarker results are often unfeasible. The present investigation aimed at determining the influence of fasting (against feeding with Isochrysis galbana) on biomarkers and their responsiveness in mussels exposed for 96 h to the water accommodated fraction (WAF) of a heavy fuel oil (0%, 6.25%, 12.5% and 25% WAF in sea water). PAH tissue levels in digestive gland and a battery of biomarkers were compared. WAF exposure led to decrease of cytochrome-C-oxidase activity, modulated glutathione-S-transferase activity, augmented lipid peroxidation, inhibited acetyl cholinesterase (AChE) activity, and led to lysosomal enlargement (VvLYS and S/VLYS) and membrane destabilisation, lipofuscin accumulation, and histopathological alterations (VvBAS, MLR/MET and CTD ratio) in the digestive gland epithelium; and were integrated as IBR/n (biological response index). Overall, no significant changes were recorded in AChE activity, S/VLYS and CTD ratio in any experimental treatment, while all the other biomarkers showed significant changes depending on the fasting/feeding condition, the exposure to WAF and/or their interaction. As a result, the integrated biomarker index IBR/n was higher at increasing WAF exposure levels both in fasted and fed mussels albeit the response was more marked in the latter. The response profiles were qualitatively similar between fasted and fed mussels but quantitatively more pronounced in fed mussels, especially upon exposure to the highest concentration (25% WAF). Therefore, it is highly recommended that mussels are also supplied with food during short-term, like during long-term toxicological experiments. This practice would avoid the interference of fasting with biological responses elicited by the tested chemicals and allow for reliable comparison with data obtained in long-term experiments and monitoring programmes.

Assessment of ecosystem health disturbance in mangrove-lined Caribbean coastal systems using the oyster Crassostrea rhizophorae as sentinel species.

This investigation was aimed at contributing to develop a suitable multi-biomarker approach for pollution monitoring in mangrove-lined Caribbean coastal systems using as sentinel species, the mangrove cupped oyster, Crassostrea rhizophorae. A pilot field study was carried out in 8 localities (3 in Nicaragua; 5 in Colombia), characterized by different environmental conditions and subjected to different levels and types of pollution. Samples were collected in the rainy and dry seasons of 2012-2013. The biological effects at different levels of biological complexity (Stress-on-Stress response, reproduction, condition index, tissue-level biomarkers and histopathology) were determined as indicators of health disturbance, integrated as IBR/n index, and compared with tissue burdens of contaminants in order to achieve an integrative biomonitoring approach. Though modulated by natural variables and confounding factors, different indicators of oyster health, alone and in combination, were related to the presence of different profiles and levels of contaminants present at low-to-moderate levels. Different mixtures of persistent (As, Cd, PAHs) and emerging chemical pollutants (musk fragrances), in combination with different levels of organic and particulate matter resulting from seasonal oceanographic variability and sewage discharges, and environmental factors (salinity, temperature) elicited a different degree of disturbance in ecosystem health condition, as reflected in sentinel C. rhizophorae. As a result, IBR/n was correlated with pollution indices, even though the levels of biological indicators of health disturbance and pollutants were low-to-moderate, and seasonality and the incidence of confounding factors were remarkable. Our study supports the use of simple methodological approaches to diagnose anomalies in the health status of oysters from different localities and to identify potential causing agents and reflect disturbances in ecosystem health. Consequently, the easy methodological approach used herein is useful for the assessment of health disturbance in a variety of mangrove-lined Caribbean coastal systems using mangrove cupped oysters as sentinel species.

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

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

Digestive cell lysosomes as main targets for Ag accumulation and toxicity in marine mussels, Mytilus galloprovincialis, exposed to maltose-stabilised Ag nanoparticles of different sizes.

Bioavailability and toxicity of maltose-stabilised AgNPs of different sizes (20, 40 and 100 nm) in mussels were compared with bulk and aqueous forms of the metal through a two-tier experimental approach. In the first tier, mussels were exposed for 3 d to a range of concentrations (0.75, 75, 750 µg Ag/l) in the form of Ag20-Mal, Ag40-Mal, Ag100-Mal, bulk Ag and aqueous Ag (as AgNO3), as well as to the concentrations of maltose used in the formulation of NPs. Mortality, bioaccumulation, tissue and cell distribution and lysosomal responses were investigated. In the second tier, mussels were exposed for 21 d to Ag20-Mal, Ag100-Mal, bulk Ag and aqueous Ag at the lowest effective concentration selected after Tier 1 (0.75 µg Ag/l), biomarkers and toxicopathic effects were investigated. Aqueous Ag was lethal within 3 d at 75 µg Ag/l; Ag NPs or bulk Ag did not produce significant mortality at 750 µg Ag/l. Ag accumulation was limited and metallothionein gene transcription was not regulated although metal accumulation occurred in digestive, brown and stomach epithelial cells and in gut lumen after exposure to AgNPs and aqueous Ag starting at low concentrations after 1 d. Electrondense particles (<10 nm) in lysosomes and residual bodies after exposure to AgNPs contained Ag and S (X-ray). Intralysosomal metal accumulation and lysosomal membrane destabilisation were enhanced after exposure to all the forms of Ag and more marked after exposure to Ag20-Mal than to larger NPs. 21 d exposure to AgNPs provoked digestive cell loss and loss of digestive gland integrity, resulting in atrophy-necrosis in digestive alveoli and oedema/hyperplasia in gills (Ag NP), vacuolisation in digestive cells (aqueous Ag) and haemocyte infiltration of connective tissue (all treatments). Intralysosomal metal accumulation, lysosomal responses and toxicopathic effects are enhanced at decreasing sizes and appear to be caused by Ag+ ions released from NPs, although the metal was not substantially accumulated.

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

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

Lysosomal responses to heat-shock of seasonal temperature extremes in Cd-exposed mussels.

The present study was aimed at determining the effect of temperature extremes on lysosomal biomarkers in mussels exposed to a model toxic pollutant (Cd) at different seasons. For this purpose, temperature was elevated 10°C (from 12°C to 22°C in winter and from 18°C to 28°C in summer) for a period of 6h (heat-shock) in control and Cd-exposed mussels, and then returned back to initial one. Lysosomal membrane stability and lysosomal structural changes in digestive gland were investigated. In winter, heat-shock reduced the labilisation period (LP) of the lysosomal membrane, especially in Cd-exposed mussels, and provoked transient lysosomal enlargement. LP values recovered after the heat-shock cessation but lysosomal enlargement prevailed in both experimental groups. In summer, heat-shock induced remarkable reduction in LP and lysosomal enlargement (more markedly in Cd-exposed mussels), which recovered within 3 days. Besides, whilst heat-shock effects on LP were practically identical for Cd-exposed mussels in winter and summer, the effects were longer-lasting in summer than in winter for control mussels. Thus, lysosomal responsiveness after heat-shock was higher in summer than in winter but recovery was faster as well, and therefore the consequences of the heat shock seem to be more decisive in winter. In contrast, inter-season differences were attenuated in the presence of Cd. Consequently, mussels seem to be better prepared in summer than in winter to stand short periods of abrupt temperature change; this is, however, compromised when mussels are exposed to pollutants such as Cd.

Scenario-targeted toxicity assessment through multiple endpoint bioassays in a soil posing unacceptable environmental risk according to regulatory screening values.

Lanestosa is a chronically polluted site (derelict mine) where the soil (Lanestosa (LA) soil) exceeds screening values (SVs) of regulatory policies in force (Basque Country; Europe) for Zn, Pb and Cd. A scenario-targeted toxicity assessment was carried out on the basis of a multi-endpoint bioassay approach. Acute and chronic toxicity bioassays were conducted with selected test species (Vibrio fischeri, Dictyostelium discoideum, Lactuca sativa, Raphanus sativus and Eisenia fetida) in combination with chemical analysis of soils and elutriates and with bioaccumulation studies in earthworms. Besides, the toxicity profile was compared with that of the mine runoff (RO) soil and of a fresh artificially polluted soil (LAAPS) resembling LA soil pollutant profile. Extractability studies in LA soil revealed that Pb, Zn and Cd were highly available for exchange and/or release into the environment. Indeed, Pb and Zn were accumulated in earthworms and LA soil resulted to be toxic. Soil respiration, V. fischeri, vegetative and developmental cycles of D. discoideum and survival and juvenile production of E. fetida were severely affected. These results confirmed that LA soil had unacceptable environmental risk and demanded intervention. In contrast, although Pb and Zn concentrations in RO soil revealed also unacceptable risk, both metal extractability and toxicity were much lower than in LA soil. Thus, within the polluted site, the need for intervention varied between areas that posed dissimilar risk. Besides, since LAAPS, with a high exchangeable metal fraction, was the most toxic, ageing under in situ natural conditions seemingly contributed to attenuate LA soil risk. As a whole, combining multi-endpoint bioassays with scenario-targeted analysis (including leaching and ageing) provides reliable risk assessment in soils posing unacceptable environmental risk according to SVs, which is useful to optimise the required intervention measures.

Season-dependent effects of elevated temperature on stress biomarkers, energy metabolism and gamete development in mussels.

In coastal areas, sessile species can be severely affected by thermal stress associated to climate change. Presently, the effect of elevated temperature on metabolic, cellular and tissue-level responses of mussels was determined to assess whether the responses vary seasonally with seawater temperature and reproductive stage. Mussels were collected in fall, winter and summer, and (a) maintained at 16, 12, and 20 °C respectively or (b) subject to gradual temperature elevation for 8 days (+1 °C per day; from 16 to 24 °C in fall, from 12 to 20 °C in winter and from 20 to 28 °C in summer) and further maintained at 24 °C (fall), 20 °C (winter) and 28 °C (summer) for the following 6 days. Temperature elevation induced membrane destabilization, lysosomal enlargement, and reduced the aerobic scope in fall and summer whereas in winter no significant changes were found. Changes at tissue-level were only evident at 28 °C. Gamete development was impaired irrespective of season. Since the threshold of negative effects of warming was close to ambient temperatures in summer (24 °C or above) studied mussel populations would be vulnerable to the global climate change.

Effects of soil organic matter content on cadmium toxicity in Eisenia fetida: implications for the use of biomarkers and standard toxicity tests.

Bioavailability is affected by soil physicochemical characteristics such as pH and organic matter (OM) content. In addition, OM constitutes the energy source of Eisenia fetida, a well established model species for soil toxicity assessment. The present work aimed at assessing the effects of changes in OM content on the toxicity of Cd in E. fetida through the measurement of neutral red uptake (NRU) and mortality, growth, and reproduction (Organisation for Economic Co-operation and Development [OECD] Nos. 207 and 222). Complementarily, metallothionein (MT) and catalase transcription levels were measured. To decrease variability inherent to natural soils, artificial soils (Organization for Economic Cooperation and Development 1984) with different OM content (6, 10, and 14%) and spiked with Cd solutions at increasing concentrations were used. Low OM in soil decreased soil ingestion and Cd bioaccumulation but also increased Cd toxicity causing lower NRU of coelomocytes, 100 % mortality, and stronger reproduction impairment, probably due to the lack of energy to maintain protection mechanisms (production of MT).Cd bioaccumulation did not reflect toxicity, and OM played a pivotal role in Cd toxicity. Thus, OM content should be taken into account when using E. fetida in in vivo exposures for soil health assessment.

Health status assessment through an integrative biomarker approach in mussels of different ages with a different history of exposure to the Prestige oil spill.

A battery of cell and tissue-level biomarkers was applied in mussels of 6 size-classes collected from Galicia and the Basque coast in summer 2007 in an attempt to examine the health status of individuals affected as adults (mature before 2003), affected during their developmental or juvenile stages (2003-2004 offspring), or not directly affected by the Prestige oil spill (POS) exposure (presumably 2005-2006 offspring). This battery of biomarkers was akin to those formerly applied on mussels of 3.5-4.5 cm shell length for which there exist biomarker reference values in the studied geographical areas. The cause-effect relationship between biological responses and the different history of exposure to POS fuel oil was intricate for different reasons: (a) growth rate was dissimilar in mussels of the two studied localities and much lower than expected, (b) a chronological basis could not be directly associated to POS events (all mussels except the smallest from Galicia had been subjected to the direct POS impact at one or another stage of their life-cycle); and (c) some biomarkers and histopathology seemingly depended on size/age irrespectively of the locality and the POS chronology. As a whole, the present study gives a very useful set of reference values of biomarkers obtained for Mytilus galloprovincialis of different size-classes. Finally, it is recommended that Mussel Watch programmes should be designed by standardising the age of the sentinel mussels rather than their size, especially if the programme covers large or diverse geographical areas, if long-term trends are relevant or if significant pollution effects on growth are expected.

Toxicity assessment through multiple endpoint bioassays in soils posing environmental risk according to regulatory screening values.

Toxicity profiles of two soils (a brownfield in Legazpi and an abandoned iron mine in Zugaztieta; Basque Country) contaminated with several metals (As, Zn, Pb and Cu in Legazpi; Zn, Pb, Cd and Cu in Zugaztieta) and petroleum hydrocarbons (in Legazpi) were determined using a multi-endpoint bioassay approach. Investigated soils exceeded screening values (SVs) of regulatory policies in force (Basque Country; Europe). Acute and chronic toxicity bioassays were conducted with a selected set of test species (Vibrio fischeri, Dictyostelium discoideum, Lactuca sativa, Raphanus sativus and Eisenia fetida) in combination with chemical analysis of soils and elutriates, as well as with bioaccumulation studies in earthworms. The sensitivity of the test species and the toxicity endpoints varied depending on the soil. It was concluded that whilst Zugaztieta soil showed very little or no toxicity, Legazpi soil was toxic according to almost all the toxicity tests (solid phase Microtox, D. discoideum inhibition of fruiting body formation and developmental cycle solid phase assays, lettuce seed germination and root elongation test, earthworm acute toxicity and reproduction tests, D. discoideum cell viability and replication elutriate assays). Thus, albeit both soils had similar SVs, their ecotoxicological risk, and therefore the need for intervening, was different for each soil as unveiled after toxicity profiling based on multiple endpoint bioassays. Such a toxicity profiling approach is suitable to be applied for scenario-targeted soil risk assessment in those cases where applicable national/regional soil legislation based on SVs demands further toxicity assessment.

Zonation in the digestive tract of Eisenia fetida: implications in biomarker measurements for toxicity assessment.

Eisenia fetida is a model species for soil health assessment and different biomarkers that detect either the presence of bioavailable contaminants or their biological effect have been developed. These parameters are performed in a target tissue or whole earthworm, without considering the marked zonation in histological organisation, enzyme activities and gene expression pattern existing along the body. Thus, the present work was aimed at (a) characterising the morphofunctional heterogeneity along the digestive tract of E. fetida in tissue morphology and turnover, lysosomal enzyme markers (ß-glucuronidase, ß-GUS; hexosaminidase, HEX), lipofuscin contents (LPF) and metallothionein (MT) and catalase (CAT) gene expression; and (b) determining whether the responsiveness to Cd exposure varies among tissues and along the digestive tract. HEX and ß-GUS exhibited a heterogeneous distribution pattern along and across the digestive tract and Cd exposure caused a marked decrease of HEX and an increase of ß-GUS activity. Likewise, the significant decrease of cell turnover and the induction of MT transcription were was zone-dependent. Therefore, in was concluded that the consideration of the zonation when applying biomarker for toxicity assessment would reduce the intrinsic variability that results from overlooking the marked morphofunctional heterogeneity that exists in annelids along their body axis.

Effects of exposure to Prestige-like heavy fuel oil and to perfluorooctane sulfonate on conventional biomarkers and target gene transcription in the thicklip grey mullet Chelon labrosus.

Thicklip grey mullets Chelon labrosus inhabit coastal and estuarine areas where they can be chronically exposed to commonly released pollutants such as polycyclic aromatic hydrocarbons (PAHs) and perfluorinated compounds. These pollutants can also originate from accidental spills, such as the Prestige oil spill in 2002, which resulted in the release of a heavy fuel oil that affected coastal ecosystems in the Bay of Biscay. Peroxisome proliferation (PP), induced biotransformation metabolism, immunosuppression and endocrine disruption are some of the possible biological effects caused by such chemicals. With the aim of studying the effects of organic toxic chemicals on such biological processes at the transcriptional and at the cell/tissue level, juvenile mullets were exposed to the typical mammalian peroxisome proliferator perfluorooctane sulfonate (PFOS), and to fresh (F) and weathered (WF) Prestige-like heavy fuel oil for 2 and 16 days. First, fragments of genes relevant to biotransformation, immune/inflammatory and endocrine disruption processes were cloned using degenerate primers. Fuel oil elicited a significant PP response as proved by the transcriptional upregulation of palmitoyl-CoA oxidase (aox1), peroxisome proliferator activated receptor alpha (pparalpha) and retinoic X receptor, by the AOX1 activity induction and by the increased peroxisomal volume density. PFOS only elicited a significant induction of AOX1 activity at day 2 and of PPARalpha mRNA expression at day 16. All treatments significantly increased catalase mRNA expression at day 16 in liver and at day 2 in gill. Cyp1a transcription (liver and gill) and EROD activity were induced in fuel oil treated organisms. In the case of phase II metabolism only hepatic glutathione S-transferase mRNA was overexpressed in mullets exposed to WF for 16 days. Functionally, this response was reflected in a significant accumulation of bile PAH metabolites. WF treated fish accumulated mainly high molecular weight metabolites while F exposure resulted in accumulation of mainly low molecular ones. Fuel oil significantly regulated immune response related complement component C3 and hepcidin transcription followed by a significant regulation of inflammatory response related apolipoprotein-A1 and fatty acid binding protein mRNAs at day 16. These responses were accompanied by a significant hepatic inflammatory response with lymphocyte accumulations (IRLA) and accumulation of melanomacrophage centers (MMC). PFOS did not elicit any transcriptional response in the studied biotransformation and immune related genes, although histologically significant effects were recorded in IRLA and MMC. A significant reduction of lysosomal membrane stability was observed in all exposed animals. No endocrine disruption effects were observed in liver while brain aromatase mRNA was overexpressed after all treatments at day 2 and estrogen receptor alpha was downregulated under WF exposure at day 16. These results show new molecular and cellular biomarkers of exposure to organic chemicals and demonstrate that in mullets PP could be regulated through molecular mechanisms similar to those in rodents, although the typical mammalian peroxisome proliferator PFOS and heavy fuel oil follow divergent mechanisms of action.

Beta-glucuronidase and hexosaminidase are marker enzymes for different compartments of the endo-lysosomal system in mussel digestive cells.

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

Changes in cell-type composition in digestive gland of slugs and its influence in biomarkers following transplantation between a relatively unpolluted and a chronically metal-polluted site.

Changes in cell-type composition (CCTC) is a general phenomenon that takes place in the digestive gland epithelium of stressed molluscs. The aim of the present work was to determine whether CCTC is a reversible process in the digestive gland of sentinel slugs chronically exposed to metal pollution and how CCTC affects metal accumulation parameters and different cell and tissue biomarkers of exposure and effect. Slugs (Arion ater) from an abandoned zinc mine were transferred to a relatively unpolluted site and the other way around for 3, 10 and 28 d. The volume density of black silver deposits (Vv(BSD)) after autometallography, and metallothionein (MT) levels were used as biomarkers of exposure to metals and CCTC and lysosomal responses were selected as effect biomarkers. Results indicated that slugs were sensitive to recent metal pollution; however, slugs chronically exposed to metals presented some characteristic features and were less responsive to pollution cessation without signs of CCTC reversal.

Epithelial cell renewal in the digestive gland and stomach of mussels: season, age and tidal regime related variations.

The natural variability in cell proliferation activity in the epithelium of the digestive gland and stomach was investigated in mussels, Mytilus galloprovincialis (Lmk), of different age and tidal level at different seasons. After treating mussels with the thymidine analogue bromodeoxyuridine (BrdU) for 6 hours, BrdU immunohistochemistry was performed every 2 hours for the next 36. The relative proportion of BrdU positive cells was quantified as BrdU labelling (per thousand). Marked seasonal differences were recorded in BrdU labelling, with much higher proliferating activity in summer than in autumn and winter. Cell proliferation seemed not to be significantly dissimilar between mussels of different age (size). In contrast, the digestive gland epithelium of mussels from intertidal and subtidal populations differed not only in the levels but also in the pattern of variation of BrdU labelling, which in intertidal mussels appeared to be modulated by photoperiod and tide, unlike in subtidal mussels, in which variations followed a circatidal pattern.

Digestive cell turnover in digestive gland epithelium of slugs experimentally exposed to a mixture of cadmium and kerosene.

Slugs, Arion ater (L), have been proposed as sentinel organisms to assess soil health. In slugs under the influence of pollutants, digestive cell loss and the concomitant increase of excretory cells of the digestive gland have been described. The aim of the present work was to determine up to what extent digestive cell loss affects biomarkers and whether the affectation is reversible after exposure to a mixture of metal and organic pollutants. Slugs were dosed with a mixture of cadmium and kerosene in the food for 27 days. Apart from chemical analyses, the volume density of black silver deposits (Vv(BSD)) after autometallography, and acyl-CoA oxidase (AOX) activity were used as biomarkers of exposure to metals and organic compounds, respectively. As effect biomarkers, changes in the volume density of the cell types that constitute the digestive gland epithelium were calculated. Proliferating cells were identified by means of bromodeoxyuridine (BrdU) immunohistochemistry. Results revealed that the mixture of pollutants provoked an increase in Vv(BSD) and AOX activity and a decrease in the number of digestive cells. These changes had no effect in the digestive gland accumulation capacity or in the effect and exposure biomarkers employed. BrdU-labelling showed that exposure to pollutants provoked an enhanced digestive cell proliferation.