Effects of sulfamethazine and tetracycline at molecular, cellular and tissue levels in Eisenia fetida earthworms.

Soil contamination by antibiotics is a global issue of great concern that contributes to the rise of bacterial antibiotic resistance and can have toxic effects on non-target organisms. This study evaluated the variations of molecular, cellular, and histological parameters in Eisenia fetida earthworms exposed to sulfamethazine (SMZ) and tetracycline (TC), two antibiotics commonly found in agricultural soils. The earthworms were exposed for 14 days to a series of concentrations (0, 10, 100, and 1000 mg/kg) of both antibiotics. SMZ and TC did not affect the survival of E. fetida, however, other effects at different levels of biological complexity were detected. The two highest concentrations of SMZ reduced the viability of coelomocytes. At the highest TC concentration, there was a noticeable decline in cell viability, acetylcholinesterase activity (neurotoxicity), and the relative presence of mucopolysaccharides in the epidermis (mucous production). Glutathione S-transferase activity decreased in all TC treatments and at the highest SMZ concentration. However, levels of malondialdehyde and protein carbonyls did not change, suggesting an absence of oxidative stress. Tetracycline was neurotoxic to E. fetida and changed the integrity of the epidermis. Both antibiotics altered the intestinal microbiota of E. fetida, leading to a reduction in the relative abundance of bacteria from the phyla Proteobacteria and Bacteroidetes, while causing an increase in the phylum Actinobacteroidota. All observed changes indicate that both SMZ and TC can disrupt the earthworms' immune system and gut microbiome, while fostering the growth of bacteria that harbour antibiotic resistance genes. Finally, both antibiotics exerted additional metabolic and physiological effects that increased the vulnerability of E. fetida to pathogens.

Innovative in vivo and in vitro bioassays for the establishment of toxicity thresholds of pollutants in sediment quality assessment using polychaetes and their immune cells.

Sediment toxicity testing has become a crucial component for assessing the risks posed by contaminated sediments and for the development of sediment quality assessment strategies. Commonly used organisms for bioassays with estuarine sediments include amphipods, Arenicola marina polychaetes and echinoids. Among the latter, the Sea Urchin Embryo test (SET) is the most widely used. However, one relevant limitation of this bioassay is the unavailability of gametes all year-round, particularly outside the natural spawning seasons. Consequently, the establishment of an appropriate and complementary model organism for a continuous assessment of sediment quality is recommended. A reliable assessment of the hazards resulting from pollutants in sediments or pore water, can be achieved with ecologically relevant species of sediment such as the polychaete Hediste diversicolor, which is widespread in estuaries and has the capacity to accumulate pollutants. The aim of this work was to develop reliable in vivo and in vitro bioassays with H. diversicolor and its coelomocytes (immune cells) to determine the toxicity thresholds of different contaminants bounded to sediments or resuspended into water. Polychaetes were exposed to sublethal concentrations of CuCl2 (in vivo) and a non-invasive method for collection of polychaetes coelomocytes was applied for the in vitro bioassay, exposing cells to a series of CuCl2 and AgNPs concentrations. Same reference toxicants were used to expose Paracentrotus lividus following the SET (ICES Nº 51; Beiras et al., 2012) and obtained toxicity thresholds were compared between the two species. In vivo exposure of polychaetes to high concentrations of Cu produced weight loss and histopathological alterations. After in vitro approaches, a significant decrease in coelomocytes viability was recorded for both toxicants, in a monotonic dose-response curve, at very short-exposure times (2 h). The toxicity thresholds obtained with polychaetes were in line with the ones obtained with the SET, concluding that their sensitivity is similar. In conclusion, in vivo and in vitro bioassays developed with H. diversicolor are accurate toxicity screenings of pollutants that could be bounded to sediments or dissolved in the pore water, and may complement the SET outside the spawning period of the echinoderms. The bioassays herein developed could be applied not only to establish the toxicity thresholds of individual compounds or mixtures, but also to assess the toxicity of field collected sediments.

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.

Integrative assessment of the effects produced by Ag nanoparticles at different levels of biological complexity in Eisenia fetida maintained in two standard soils (OECD and LUFA 2.3).

There is a potential risk to increase the release of silver nanoparticles (Ag NPs) into the environment: For instance. in soils receiving sludge models estimate 0.007 mg Ag NPs kg-1 that will annually increase due to sludge or sludge incineration residues land-disposal. Thus, the concern about the hazards of nanosilver to soils and soil invertebrates is growing. Studies performed up to now have been focused in traditional endpoints, used limit range concentrations and employed different soil types that differ in physico-chemical characteristics. Presently, effects of Ag NPs have been measured at different levels of biological complexity in Eisenia fetida, exposed for 3 and 14 d to high but sublethal (50 mg Ag NPs kg-1) and close to modeled environmental concentrations (0.05 mg Ag NPs kg-1). Since characteristics of the exposure matrix may limit the response of the organisms to these concentrations, experiments were carried out in OECD and LUFA soils, the most used standard soils. High concentrations of Ag NPs increased catalase activity and DNA damage in OECD soils after 14 d while in LUFA 2.3 soils produced earlier effects (weight loss, decrease in cell viability and increase in catalase activity at day 3). At day 14, LUFA 2.3 (low clay and organic matter-OM-) could have provoked starvation of earthworms, masking Ag NPs toxicity. The concentration close to modeled environmental concentrations produced effects uniquely in LUFA 2.3 soil. Accurate physico-chemical characteristics of the standard soils are crucial to assess the toxicity exerted by Ag NPs in E. fetida since low clay and OM contents can be considered toxicity enhancers.

Is there a direct relationship between stress biomarkers in oysters and the amount of metals in the sediments where they inhabit?

The effects exerted by metals in oysters are still a matter of debate and require more detailed studies. In this work we have investigated whether the health status of oysters are affected by the amount of metals present in the sediments of their habitat. Sediments and oysters were collected in the tidal part of the estuary of the Oka River (Basque Country), representative of other mesotidal, well mixed and short estuaries of the European Atlantic coast. The concentrations of 14 elements were determined in all the samples. Several biomarkers were also measured in the soft tissues of oysters. According to the concentrations found, the sediments were classified as non-toxic or slightly toxic. In good agreement, the histological alterations observed in oysters were not severe. Interestingly, in those sampling sites where the sediments showed relatively high metal concentrations, the metallic content in oysters was lower, and vice versa.

Calidad de vida a los 6 meses tras un ictus / Quality of life 6 months after a stroke

Objetivo. Describir la calidad de vida (CV) de los pacientes a los 6 meses de evolución del ictus y su relación con variables sociodemográficas, clínicas y funcionales. Material y métodos. Estudio multicéntrico de cohortes longitudinal (n = 157). Los pacientes incluidos ingresaron en la fase subaguda tras un ictus en 2 Servicios de Rehabilitación. Se recogieron los siguientes datos a los 6 meses postictus: género, edad, riesgo social, comorbilidad, disfagia, afasia, estado cognitivo, depresión y grado de discapacidad evaluado mediante el índice de Barthel modificado. Se evaluó la influencia de estas variables en la CV, para lo que se empleó la escala específica de calidad de vida para el ictus ECVI-38. Resultados. La media de edad fue de 70,93 ± 11,85 años; fue significativamente superior en las mujeres. El paciente tipo presentó una elevada comorbilidad, un bajo riesgo social y dependencia moderada. La puntuación media total de la escala ECVI-38 fue de 35,30 ± 16,17 y los dominios más afectados los referentes a las actividades comunes y básicas de la vida diaria. Las variables que más se relacionan con la CV fueron el género, la afasia, la disfagia, la depresión, el déficit cognitivo y el estado funcional. El dolor de elevada intensidad estuvo presente en un 21% de los pacientes. Conclusión. Son muy diversas las variables que influyen en la CV del paciente con ictus que se deben considerar, para su potencial abordaje, en la planificación de las intervenciones rehabilitadoras (AU)

Objective. To describe quality of life (QoL) in patients at 6 months poststroke and its relationship with sociodemographic, clinical and functional characteristics. Material and method. This multicenter longitudinal cohort study (n = 157) included patients from 2 hospital rehabilitation services who were admitted for stroke in the subacute phase. The following data were gathered at 6 months poststroke: gender, age, social risk, comorbidity, dysphagia, aphasia, cognitive status, depression, and disability measured by the modified Barthel Index. The influence of these variables on QoL was evaluated using the stroke-specific quality of life scale, ECVI-38. Results. The mean age was 70.93 ± 11.85 years and was significantly higher in women. Most of the patients had high comorbidity, low social risk and moderate dependence. The mean total score on the ECVI-38 scale was 35.30 ± 16.17 and the most affected domains were those concerning common and basic activities of daily living. The variables associated with QoL were gender, aphasia, dysphagia, depression, cognitive impairment, and functional status. High-intensity pain was present in 21% of patients. Conclusion. Numerous variables influence QoL in patients with stroke and should be considered in the planning of rehabilitation interventions (AU)

Atención subaguda al paciente con ictus. Satisfacción y resultados / Subacute care of stroke-affected patients. Satisfaction and results

Objetivo: Conocer la satisfacción de los pacientes con ictus y sus cuidadores durante su hospitalización en rehabilitación y analizar la efectividad del tratamiento, el riesgo social y el destino del paciente al alta. Material y método: Estudio multicéntrico de cohorte longitudinal prospectivo. Se valoró el riesgo social (escala Gijón), la comorbilidad (índice de Charlson), la discapacidad (índice de Barthel), la efectividad del tratamiento rehabilitador, la satisfacción con la atención (cuestionario de Pound) y el destino al alta en 241 pacientes. De 119 cuidadores evaluados a los 6 meses postictus se recogieron la edad, el parentesco, las horas diarias dedicadas al cuidado y la satisfacción con la información/formación y con la accesibilidad del equipo rehabilitador. Resultados: El perfil del paciente era el de un varón de 71 años, con riesgo social bajo/intermedio, comorbilidad alta y dependencia total/severa. Un 27,1% vivían solos. El 96,6% referían estar satisfechos/muy satisfechos con el tratamiento, siendo menor la satisfación con la recuperación (80,3%). La efectividad fue de 32,5 ± 20,4. El 81,7% de los pacientes regresaron a su domicilio. La media de edad de los cuidadores fue de 58,8 ± 12,3 años y el 73,9% eran mujeres. La dedicación a los cuidados era superior a 6 h diarias en el 62%. El 89,9% de los cuidadores estaban satisfechos/muy satisfechos con la información recibida. Conclusiones: Los pacientes ingresados para tratamiento rehabilitador tras un ictus obtienen una ganancia funcional significativa durante su hospitalización y regresan a su domicilio en la mayoría de los casos. La satisfacción con el tratamiento rehabilitador y la información es elevada. Un campo especialmente mejorable es la formación del cuidador (AU)

Objective: To determine the satisfaction of the stroke inpatients and their caregivers in Rehabilitation Service and to analyze the effectiveness, social risk, and discharge destination. Material and method: Prospective longitudinal cohort multicenter study. An analysis was made of the social risk (Gijón Scale), co-morbidity (Charlson Index), disability (Barthel Index), effectiveness of the rehabilitation treatment, satisfaction (Pound Questionnaire) and discharge destination of 241 patients. An evaluation was also made on 119 caregivers 6 months post-stroke, recording age, family relationship, time care-giving, satisfaction with the information/training, and accessibility to the rehabilitation team. Results: The patient profile is a 71 year-old male, with low/intermediate social risk, high comorbidity and total/severe dependence, with 27.1% living alone. Almost all (96.6%) of the patients claimed to be satisfied/very satisfied with the treatment, with satisfaction with the recovery being lower (80.3%). The effectiveness was 32.5 ± 20.4. Home was the discharge destination of 81.7% of the patients.The average age of the caregivers was 58.8 ± 12.3 years, and 73.9% were women. The time dedicated to care-giving was over 6 hours per day in the 62% of the cases. Being satisfied/very satisfied with the received information was recorded by 89.9% of the caregivers. Conclusions: Los pacientes ingresados para tratamiento rehabilitador tras un ictus obtienen una ganancia funcional significativa durante su hospitalización y regresan a su domicilio en la mayoría de casos. Patients admitted for stroke rehabilitation achieve significant functional gain during hospitalization and return to their homes in most cases. The satisfaction with the rehabilitation treatment and received information is high. The training of the caregiver is an aspect that needs improving

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.

[Subacute care of stroke-affected patients. Satisfaction and results]. / Atención subaguda al paciente con ictus. Satisfacción y resultados.

OBJECTIVE: To determine the satisfaction of the stroke inpatients and their caregivers in Rehabilitation Service and to analyze the effectiveness, social risk, and discharge destination. MATERIAL AND METHOD: Prospective longitudinal cohort multicenter study. An analysis was made of the social risk (Gijón Scale), co-morbidity (Charlson Index), disability (Barthel Index), effectiveness of the rehabilitation treatment, satisfaction (Pound Questionnaire) and discharge destination of 241 patients. An evaluation was also made on 119 caregivers 6 months post-stroke, recording age, family relationship, time care-giving, satisfaction with the information/training, and accessibility to the rehabilitation team. RESULTS: The patient profile is a 71 year-old male, with low/intermediate social risk, high co-morbidity and total/severe dependence, with 27.1% living alone. Almost all (96.6%) of the patients claimed to be satisfied/very satisfied with the treatment, with satisfaction with the recovery being lower (80.3%). The effectiveness was 32.5 ± 20.4. Home was the discharge destination of 81.7% of the patients.The average age of the caregivers was 58.8 ± 12.3 years, and 73.9% were women. The time dedicated to care-giving was over 6 hours per day in the 62% of the cases. Being satisfied/very satisfied with the received information was recorded by 89.9% of the caregivers. CONCLUSIONS: Patients admitted for stroke rehabilitation achieve significant functional gain during hospitalization and return to their homes in most cases. The satisfaction with the rehabilitation treatment and received information is high. The training of the caregiver is an aspect that needs improving.

Rehabilitation efficiency and destination on discharge after stroke.

BACKGROUND: Predicting functional recovery of patients is key for setting the objectives of the rehabilitation programme and making decisions on their destination on discharge. Many variables have an impact on disability and quality of life after stroke, including patient age, comorbidity, severity of neurological deficit, state of mind and social risk. Accordingly, it is also essential from a care perspective to optimise the functional recovery and efficiency of rehabilitation programmes, exploring their relationship with these variables. AIM: To analyze the efficiency of post-stroke rehabilitation, identifying factors that most strongly influence functional recovery and destination on discharge. DESIGN: Multicentre prospective cohort study SETTING/POPULATION: All patients admitted to the Rehabilitation Units of the two hospitals after stroke over eight months METHODS: Collected data on sociodemographic characteristics, social risk (Gijón Scale), comorbidity (Charlson Index), neurological severity (National Institute of Health Stroke Scale), daily living functioning scale (Barthel Index), length of hospital stay and destination on discharge. RESULTS: We included 241 patients, with a mean stay of 35±22 days, 81.5% returning home on discharge. On admission 45.2% were totally dependent, and this figure fell to 12.8% on discharge, the mean Barthel Index score increasing by 32.5 points. Neurological severity, hemiparetic severity, impairment of deep sensation and trunk control on admission were the mayor variables influence on rehabilitation efficiency (P<0.001). Destination on discharge was most closely associated with civil status, social risk and Barthel Index score (P<0.001). The likelihood of transferring to residential care is 3- and 2.71-fold higher among patients with total dependence and high comorbidity scores, respectively. CONCLUSION: Many variables influence on outcomes of stroke inpatient rehabilitation. CLINICAL REHABILITATION IMPACT: Comprehensive assessments are required to predict patient recovery, efficiency and plan for discharge.

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.

Reversible alterations in epithelial cell turnover in digestive gland of winkles (Littorina littorea) exposed to cadmium and their implications for biomarker measurements.

In marine molluscs, the epithelium of the digestive gland is composed of two cell types, namely, digestive and basophilic cells. Under normal physiological conditions digestive cells outnumber basophilic cells, but under different stress situations the composition of the epithelium changes, basophilic cells apparently replace digestive cell. Winkles, Littorina littorea, were exposed to 1.25mg/l Cd for 20 days to provoke cell type replacement. Then, animals were depurated in clean seawater for 10 days to determine whether cell type replacement was reversible. Digestive glands were fixed in Carnoy and paraffin embedded for histological analysis. The volume densities of basophilic cells (Vv(BAS)) and digestive cells (Vv(DIG)) were calculated by stereology on hematoxylin-eosin stained sections. Vv(BAS) increased and Vv(DIG) decreased in Cd-exposed animals. After estimation of cell size and absolute cell numbers, these changes were attributed to digestive cell loss and concomitant basophilic cell hypertrophy but not to increased numbers of basophilic cells. Cell type composition and cell size almost fully returned to normal values after 10-day depuration. Accordingly, PCNA immunohistochemistry demonstrated that proliferating digestive cells were more abundant in winkles exposed to Cd and after 10-day depuration than in control specimens, suggesting that net digestive cell loss was accompanied by increased digestive cell proliferation. Thus, Cd-exposure seems to provoke an enhanced digestive cell turnover in order to cope with Cd detoxification. Intralysosomal accumulation of metals (autometallographied black silver deposits; BSD) was used as a biomarker of exposure to Cd and lysosomal structural changes as an effect biomarker to see whether cell type composition might have any effect on these endpoints. BSD formed around Cd ions, in digestive cell lysosomes of Cd-exposed winkles whereas basophilic cells appeared devoid of them. After depuration, BSD were less conspicuous. Enlarged lysosomes were observed in Cd-exposed winkles, lysosome size returning to control levels after 10-day depuration. Changes in digestive cell proliferation, digestive cell loss and basophilic cell hypertrophy did not apparently affect the biomarkers investigated herein.

Circatidal variation in epithelial cell proliferation in the mussel digestive gland and stomach.

Epithelial cell renewal in mussel ( Mytilus galloprovincialis, Lmk) digestive gland and stomach was investigated by bromodeoxyuridine (BrdU) immunohistochemistry. Mussels were exposed to 4 mg BrdU/l seawater continuously. Starting at 6 h after treatment, samples were collected every 2 h for 2 days and BrdU labelling was estimated by direct counting at the light microscope, with values being noted per thousand BrdU-positive cells. BrdU-positive reaction was observed in the nuclei of digestive, basophilic, duct and stomach cells, and in haemocytes. Cell renewal in digestive diverticula was synchronised following a circatidal pattern: BrdU labelling increased during low tide and decreased during high tide. Clearcut mitotic figures were identified in digestive cells, thereby confirming that mature cell types proliferate, in agreement with results from immunohistochemistry for proliferating cell nuclear antigen and BrdU. Epithelial cell renewal in the stomach also appeared to be synchronised.

Subcellular distribution of cadmium and its cellular ligands in mussel digestive gland cells as revealed by combined autometallography and X-ray microprobe analysis.

Autometallography (AMG) and electron probe X-ray microanalysis (EPXMA) were applied in combination to determine the subcellular distribution of Cd and its subcellular ligands in the digestive gland cells of Cd-exposed mussels Mytilus galloprovincialis. Black silver deposits (BSD), which reveal the presence of metals when AMG is applied, were only localized in digestive cell lysosomes. Digestive cell cytoplasm and basophilic cells were devoid of BSD. EPXMA (static probe and X-ray mapping) indicated that Cd, S (possibly associated with metallothioneins or metallothionein-like proteins) and autometallographical Ag ions are co-localized within digestive cell lysosomes. In addition, Cd and S co-occur in the absence of Ag in the cytosol of digestive cells. AMG does not reveal the presence of the Cd 'pool' strongly bound to cytosolic Cd-metallothionein complexes; only 'free' Cd or Cd supposedly loosely bound to (semi)digested metallothionein within lysosomes was revealed. The levels of lysosomal Cd were indirectly quantified by stereology as the volume density of BSD (V(v)BSD). Significantly higher values were recorded in Cd-exposed mussels compared with controls at all exposure times. However, V(v)BSD values were lower at days 7 and 21 than at day 1. This relative decrease in V(v)BSD reflected another (and confounding) response elicited by Cd-exposure in the digestive epithelium: the volume density of basophilic cells (V(v)BAS) increased significantly as exposure progressed. Due to this cell-type replacement, the net accumulative capacity of the digestive epithelium decreases at long exposure times.