Impact of polyethylene microplastics on the clam Ruditapes decussatus (Mollusca: Bivalvia): examination of filtration rate, growth, and immunomodulation.

The present study was conducted to assess, for the first time, the effects of a 14 days experimental exposure to polyethylene (PE) based MPs (40-48 µm) on the clam Ruditapes decussatus. Clams were exposed to three different concentrations of MPs in controlled laboratory conditions: 10 µg/L (low), 100 µg/L (medium), and 1000 µg/L (high). The effects of MPs were assessed using a multi-marker approach, including the filtration rate, growth, and the integrity of immune cells (such as haemocyte numbers, viability, and lysosomal membrane destabilization). The results revealed that as the concentration of PE-MPs increased, the filtration rate decreased, indicating that PE-MPs hindered the clams' ability to filter water. Furthermore, there was a noticeable decrease in the overall weight of the clams, particularly in the group exposed to 1000 µg/L. This decrease could be attributed to the impairment of their nutrient filtration function. In terms of immune system biomarkers, exposure to PE-MPs led to immune system disruption, characterized by a significant increase in the number of haemocytic cells, especially in the group exposed to the high concentration. Additionally, there was a notable reduction in the viability of haemocytes, resulting in the destabilization of their lysosomal membranes, particularly in the groups exposed to medium and high PE-MPs concentrations. The findings of this study indicate that the sensitivity of hemolymph parameter changes and filtration rate in R. decussatus exposed to PE-MPs (100 and 1000 µg/L), surpasses that of growth performance and can serve as reliable indicators to assess habitat conditions and contaminant levels.

Silver nanoparticles' impact on the gene expression of the cytosolic adaptor MyD-88 and the interferon regulatory factor IRF in the gills and digestive gland of mytilus galloprovincialis.

Silver nanoparticles (AgNPs) have been reported as stressors for the bivalves' immune system at different regulatory levels, impacting the detection step and receptors, and other mediators, as well as effector molecules. However, studies on how AgNPs impact the transmission of signals from receptors and whether they have an effect on mediators and transcription factors are still scarce. This study aims to investigate the effect of 12 hours of in vivo exposure to 100 µg/L of AgNPs on the gene expression of the cytosolic adaptor Myeloid, the differentiation protein 88 (MgMyD88-b), and the interferon regulatory factor (Me4-IRF) in the gills and digestive gland of Mytilus galloprovincialis, before and after blocking two major uptake pathways of nanoparticles (clathrin- and caveolae-mediated endocytosis). The results illustrate a tissue-specific gene expression of the MgMyD88-b and the Me4-IRF in the gills and digestive gland of M. galloprovincialis. In the gills, AgNPs did not significantly impact the expression of the two genes. However, blocking the caveolae-mediated endocytosis decreased the expression of Me4-IRF. However, inhibition of clathrin-mediated endocytosis in the digestive gland recorded a significant decrease in the expression of MgMyD88-b. Overall, the inhibition of the AgNPs' uptake routes have highlighted their potential interference with the immune response through the studied mediators' genes, which need to be studied further in future investigations.

Effect of exposure time, particle size and uptake pathways in immune cell lysosomal cytotoxicity of mussels exposed to silver nanoparticles.

Cytotoxicity evaluation of hemocytes (lysosomal membrane stability [LMS] assay) from Mytilus galloprovincialis Lamarck, exposed to a sublethal dose (100 µg/L) of two size of silver nanoparticles (AgNPs: <50 nm and <100 nm) - prior to and after inhibition of potential uptake pathways (i.e., clathrin- and caveolae-mediated endocytosis) within different times of exposure (3, 6, 12 h) - showed that there was a significant cytotoxic effect on immune cells of mussels exposed for different times to either AgNP size (p < 0.01); the greater effect was with the smaller size. However, hemocytes seemed more sensitive to the larger AgNP after clathrin-mediated endocytosis was blocked (p < 0.01); this was not so with inhibition of caveolae-mediated endocytosis. Dimethyl-sulfoxide (DMSO) did not impart a carrier-mediated effect despite an enhanced cytotoxicity when DMSO was present with AgNP. From these results, it is concluded that the immunotoxicity of AgNP in mussels was size-dependent as well as length of exposure-dependent. It was also clear that nanoparticles (NP) internalization mechanisms were a major factor underlying any toxicity.

Histopathological indices and inflammatory response in the digestive gland of the mussel Mytilus galloprovincialis as biomarker of immunotoxicity to silver nanoparticles.

BACKGROUND: Histopathological assessments approaches in bivalves have become an important tool in environmental toxicology. This study seeks to develop a quantitative histopathological index (Ih) and inflammation score as biomarkers in the aim to assess the health status of nanoparticles exposed mussels. METHODS: Digestive gland hematoxylin and eosin (H&E) stained sections from Mytilus galloprovincialis were assessed after in vivo exposure (for 3, 6 and 12 h) to silver nanoparticles (Ag-NPs < 50 nm and Ag-NPs < 100 nm) prior and after manipulating the potential uptake pathways (clathrin- and caveolae-mediated endocytosis) using amantadine and nystatin as blockers. Quantitative models evaluate the impacts of nanoparticles size, as well as their uptake routes within different time of exposure on the inflammation intensity, the digestive tubules histomorphometry and the histopathological indices. RESULTS: Silver nanoparticles clearly induced histopathological alterations in digestive gland (maximum inflammation 2.75 with AgNP < 100 nm [p < 0.05]; significant Ih with AgNP < 50 nm and AgNP < 100 nm at different time-points [p < 0.05]). Significant Ih were recorded after uptake routes were blockade: AgNP < 50 nm + nystatin and AgNP < 100 nm + amantadine; [p < 0.05] all time-points. CONCLUSIONS: Histopathological assessments showed to be promising tool in nanotoxicity which seems to depend on nanoparticles size, exposure time and interestingly to uptake routes. It was not clear: is it the length of exposure or the size of particles is more impactful.

Histopathology and analyses of inflammation intensity in the gills of mussels exposed to silver nanoparticles: role of nanoparticle size, exposure time, and uptake pathways.

Environmentally induced perturbation of health parameters lead to morphological changes associated to the inflammatory response. Hematoxyline and eosin (H&E)-stained gill filaments sections were examined for such changes and inflammation intensity was scored according to a quantitative model in order to evaluate the health status of in vivo exposed (for 3, 6, and 12 h) mussels to silver nanoparticles (Ag-NPs <50 nm and Ag-NPs <100 nm) prior and after the inhibition of two potential uptake pathways (clathrin- and caveolae-mediated endocytosis) with the aid of pharmaceutical inhibitors (amantadine and nystatin). The impacts of the nanoparticles (NPs) size, as well as their uptake routes within different time of exposure on the inflammatory response were assessed. The results showed that Ag-NPs clearly induced morphological changes associated to the inflammatory response in gill tissues (Mann-Whitney p values were <.05). It is also clear that the length of the exposure as well as the NP size highly impacted inflammation intensity (highest histopathological indices recorded with Ag-NPs <100 nm). Also, the routes of NPs entry noticed to be major factor underlying inflammatory response (significant inflammation intensity reported with Ag-NPs <50 nm after blockade of uptake routes; p <.05). Throughout, it was concluded that inflammation intensity was related to NPs size and exposure time. Overall, uptake routes are shown to be the major factor underlying nanotoxicity.

STAT-5 and STAT-6 in Breast Cancer: Potential Crosstalk With Estrogen and Progesterone Receptors Can Affect Cell Proliferation and Metastasis.

Background: Signal transducers and activators of transcription 5a and 6 (STAT5a and STAT6) play a critical role in tumorigenesis of mammary glands. Based on previous studies, the breast cancer is largely dependent on hormone receptors. Consequently, it is very interesting to decipher the relationship between the STAT5a and STAT6 expression and the molecular distribution of estrogen receptors (ERs) and progesterone receptors (PRs) in mammary tumors. Methods: Our study analyzed the expression of STAT5a and STAT6, ERα, ERß and PR in 40 breast tumor tissues using quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, the Ki-67 and HER2 status were detected using immunohistochemistry. Results: STAT5a and STAT6 were retained in the majority of the cases studied. Increasing of STAT5a and STAT6 is significantly associated with ERs and PR. The coexpression of both STAT5a and STAT6 with ERs and PR is associated with high tumor grades. Moreover, the coexpression of STAT5a and STAT6 with ERα and PR is associated with a high proliferation index. In addition, (STAT6 + ERß+) and (STAT6 + PR+) breast cancer subgroups are associated with lymph node infiltration (P = 0.001 and P = 0.03, respectively). Conclusions: Our study results provide an interaction between STAT5a and STAT6 with ERs and PR inducing cell proliferation. Coexpression of STAT5a and STAT6 with ERs and PR can predict sensibility to hormonal therapy.

Effect of silver nanoparticles on gene transcription of land snail Helix aspersa.

Silver nanoparticles (Ag-NPs) are extremely useful in a diverse range of consumer goods. However, their impact on the environment is still under research, especially regarding the mechanisms involved in their effect. Aiming to provide some insight, the present work analyzes the transcriptional activity of six genes (Hsp83, Hsp17.2, Hsp19.8, SOD Cu-Zn, Mn-SOD, and BPI) in the terrestrial snail Helix aspersa in the presence of different concentrations of Ag-NPs. The animals were exposed for seven days to Lactuca sativa soaked for one hour in different concentrations of Ag-NPs (20, 50, 100 mg/L). The results revealed that the highest concentration tested of Ag-NPs (100 mg/L) led to a statistically significant induction of the Hsp83 and BPI expression in the digestive gland compared to the control group. However, a trend to upregulation with no statistical significance was observed for all the genes in the digestive gland and the foot, while in the hemolymph, the trend was to downregulation. Ag-NPs affected the stress response and immunity under the tested conditions, although the impact was weak. It is necessary to explore longer exposure times to confirm that the effect can be maintained and impact on health. Our results highlight the usefulness of the terrestrial snail Helix aspersa as a bioindicator organism for silver nanoparticle pollution biomonitoring and, in particular, the use of molecular biomarkers of pollutant effect as candidates to be included in a multi-biomarker strategy.

Redox proteomic insights into involvement of clathrin-mediated endocytosis in silver nanoparticles toxicity to Mytilus galloprovincialis.

Clathrin-mediated endocytosis is a major mode of nanoparticle (NP) internalization into cells. However, influence of internalization routes on nanoparticle toxicity is poorly understood. Here, we assess the impact of blocking clathrin-mediated endocytosis upon silver NP (AgNP) toxicity to gills and digestive glands of the mussel Mytilusgalloprovincialisusing the uptake inhibitor, amantadine. Animals were exposed for 12h to AgNP (< 50 nm) in the presence and absence of amantadine. Labeling of oxidative protein modifications, either thiol oxidation, carbonyl formation or both in two-dimensional electrophoresis separations revealed 16 differentially affected abundance spots. Amongst these, twelve hypothetical proteins were successfully identified by peptide mass fingerprinting (MALDI TOF-MS/MS). The proteins identified are involved in buffering redox status or in cytoprotection. We conclude that blockade of clathrin-mediated endocytosis protected against NP toxicity, suggesting this uptake pathway facilitates toxicity. Lysosomal degradation and autophagy are major mechanisms that might be induced to mitigate NP toxicity.

Role of endocytotic uptake routes in impacting the ROS-related toxicity of silver nanoparticles to Mytilus galloprovincialis: A redox proteomic investigation.

Oxidative stress is often implicated in nanoparticle toxicity. Several studies have highlighted the role of internalization routes in determining nanotoxicity. Here, we investigate how two endocytotic mechanisms (clathrin- and caveolae-mediated) impact on redox balance in gill and digestive gland of the mussel, Mytilus galloprovincialis. Animals were exposed (for 3, 6 and 12 h) to two sizes of silver nanoparticles (AgNP: <50 nm and <100 nm) prior to and after blockade of two endocytic pathways (amantadine blocks clathrin-mediated endocytosis while nystatin blocks caveolae-mediated endocytosis). Redox-proteomic tools were used to determine effects. Our results demonstrate the ability of both sizes of AgNP (<50 and <100 nm) to cause protein thiol oxidation and/or protein carbonylation. However, blockade of endocytotic routes mitigated AgNP toxicity. Differential ROS-related toxicity of AgNP to mussel tissues seemed to be linked to tissue-specific mode of action requirements. Cell uptake mechanism strongly influences toxicity of AgNPs in this filter-feeder.

Inactivation of cadmium induced immunotoxicological alterations in rats by Tunisian montmorillonite clay.

Cadmium (Cd(2+)) is a heavy metal that is dispersed throughout the modern environment mainly as a result of pollution from a variety of sources. The aims of the current study were to investigate the efficacy of purified Tunisian montmorillonite clay (TMC) to adsorb Cd, to test the stability of the resulting complex under different conditions in vitro, and to utilize the rat bioassay as an in vivo model to evaluate the protective role of TMC against Cd-induced toxicity and immunodysfunction. In the in vitro study, three concentrations of TMC (0.5, 1.0 and 1.5 g/l aqueous solution) and three concentrations of CdCl(2) (25, 50 and 100 ppm) were tested. The results of the in vitro study showed that TMC had a high capacity of adsorbing Cd at different concentrations tested. The adsorption ranged from 95.7-100% of the available CdCl(2) in aqueous solutions. The complex TMC-Cd was stable at different pHs at 37 degrees C. The in vivo results indicated that treatment with CdCl(2) (2.5 mg/kg BW) for 2 weeks resulted in a significant decrease in triglycerides, total protein, creatinine, creatine kinase, immunoglobulin profile (Ig A and Ig G) and T-cell sub-types (CD3(+), CD4(+), CD8(+) and CD56(+)). Whereas, it significantly increase serum level of AST, ALT, LDH and induced degenerative changes in pro-inflammatory cytokines (TNF-alpha and IL-1). Rats treated with TMC alone (400, 600 and 800 mg/kg BW) were comparable to the control regarding all the tested parameters. The combined treatment of CdCl(2) and TMC at the lowest dose (400 mg/kg BW) showed a significant improvement of all tested parameters. It could be concluded that TMC was effective to protect against Cd hazards at a dose as low as 400 mg/kg BW. These results supported our hypothesis that TMC tightly-bind and immobilized Cd resulted in reduction of metal bioavailability in the gastrointestinal tract.

Impact of exposure time, particle size and uptake pathway on silver nanoparticle effects on circulating immune cells in mytilus galloprovincialis.

Nanomaterials have increasingly emerged as potential pollutants to aquatic organisms. Nanomaterials are known to be taken up by hemocytes of marine invertebrates including Mytilus galloprovincialis. Indeed, assessments of hemocyte-related parameters are a valuable tool in the determination of potentials for nanoparticle (NP) toxicity. The present study assessed the effects from two size types of silver nanoparticles (AgNP: <50 nm and <100 nm) on the frequency of hemocytes subpopulations as immunomodulation biomarkers exposed in a mollusk host. Studies were performed using exposures prior to and after inhibition of potential NP uptake pathways (i.e. clathrin- and caveolae-mediated endocytosis) and over different durations of exposure (3, 6 and 12 h). Differential hemocyte counts (DHC) revealed significant variations in frequency of different immune cells in mussels exposed for 3 hr to either AgNP size. However, as exposure duration progressed cell levels were subsequently differentially altered depending on particle size (i.e. no significant effects after 3 h with larger AgNP). AgNP effects were also delayed/varied after blockade of either clathrin- or caveolae-mediated endocytosis. The results also noted significant negative correlations between changes in levels hyalinocytes and acidophils or in levels basophils and acidophils as a result of AgNP exposure. From these results, we concluded AgNP effects on mussels were size and duration of exposure dependent. This study highlighted how not only was NP size important, but that differing internalization mechanisms could be key factors impacting on the potential for NP in the environment to induce immunomodulation in a model/test sentinel host like M. galloprovincialis.