In vivo effects of n-TiO2 on digestive gland and immune function of the marine bivalve Mytilus galloprovincialis.

Due to the increasing production of nanoparticles (NPs) and their potential release in the aquatic environment, evaluation of their biological impact on aquatic organisms represents a major concern. Suspension feeding invertebrates, in particular bivalve mollusks, may play a role in NP biotransformation and transfer through food webs and may represent a significant target for NP toxicity. In this work, the in vivo effects of titanium dioxide (n-TiO2), one of the most widespread NPs in use, were investigated in the bivalve Mytilus galloprovincialis, largely utilised as a sentinel for marine contamination. Mussels were exposed for 96h to different concentrations of n-TiO2 suspensions (1, 10 and 100µgL(-1)) and multiple responses were evaluated in the digestive gland and immune cells, the haemocytes. In the digestive gland, n-TiO2 affected lysosomal and oxidative stress biomarkers and decreased transcription of antioxidant and immune-related genes. In the haemocytes, n-TiO2 decreased lysosomal membrane stability-LMS and phagocytosis, increased oxyradical production and transcription of antimicrobial peptides; moreover, pre-apoptotic processes were observed. The effects of n-TiO2 on digestive gland and haemocytes were distinct, also depending on the endpoint and on nominal NP concentrations, with many significant responses elicited by the lowest concentrations tested. The results show that n-TiO2, at concentrations close to predicted environmental levels, significantly affected different functional and molecular parameters of mussel digestive gland and immune cells. In particular, the observed changes in immune parameters that represent significant biomarkers of exposure at the organism level suggest that exposure to n-TiO2 may pose a serious risk to mussel health.

Direct effects of Bisphenol A on lipid homeostasis in rat hepatoma cells.

Bisphenol A (BPA), used in the manufacture of polycarbonate plastic and epoxy resin, is one of the most abundant endocrine disruptors in the environment, considered as a xenoestrogen. BPA has recently become of additional public health concern because of increasing evidence of deleterious effects on metabolism. Dietary intake seems the most important route for BPA exposure, followed by rapid biotransformation in the gut and liver and elimination in the urine. Although hepatocytes can represent a significant target for this compound, little is known on the direct effects and mechanisms of action of BPA on lipid homeostasis at the cellular level. In this work, the effects of BPA (0.3-3-30-300 ng mL(-1), 24 h) were investigated in rat FaO hepatoma, a well differentiated liver cell line. At both 30 and 300 ng mL(-1), BPA significantly increased intracellular triglyceride (TAG) content and lipid accumulation in lipid droplets (LDs), without affecting cell viability. The effects of BPA were associated with decreased mRNA levels of the transcription factors Peroxisome Proliferator-Activated Receptor (PPAR) isoforms α and ßδ, as well as of their downstream genes acyl-CoA oxidase (AOX) and carnitine palmitoyl transferase (CPT1) involved in lipid oxidation. No increase in transcription of lipogenic genes was observed. BPA also decreased mRNA levels of ApolipoproteinB (apoB) and the extracellular TAG content, indicating alterations in lipid secretion. FaO cells did not express Estrogen Receptor α (ERα and showed a very low expression of ERß compared to rat liver. All the effects of BPA were prevented by cell pretreatment with Wortmannin, indicating the involvement of phosphatidyl inositol-3 kinase activation. The results demonstrate a direct action of BPA on lipid homeostasis in FaO cells through interference with lipid oxidation and secretion, and add further information on the cellular pathways that can be perturbed by this compound.

Effects of sublethal, environmentally relevant concentrations of hexavalent chromium in the gills of Mytilus galloprovincialis.

Hexavalent chromium Cr(VI) is an important contaminant released from both domestic and industrial effluents, and represents the predominant chemical form of the metal in aquatic ecosystems. In the marine bivalve Mytilus galloprovincialis exposure to non-toxic, environmentally relevant concentrations of Cr(VI) was shown to modulate functional parameters and gene expression in both the digestive gland and hemocytes. In this work, the effects of exposure to Cr(VI) (0.1-1-10 µg L(-1) animal(-1) for 96 h) in mussel gills were investigated. Gill morphology and immunolocalization of GSH-transferase (GST), of components involved in cholinergic (AChE and ChAT), adrenergic (TH) and serotoninergic (5-HT(3) receptor) systems, regulating gill motility, were evaluated. Total glutathione content, activities of GSH-related enzymes (glutathione reductase - GSR, GST), of catalase, and of key glycolytic enzymes (phosphofructokinase - PFK and pyruvate kinase - PK) were determined. Moreover, mRNA expression of selected Mytilus genes (GST-π, metallothionein isoforms MT10 and MT20, HSP70 and 5-HT receptor) was assessed by RT-q-PCR. Cr(VI) exposure induced progressive changes in gill morphology and in immunoreactivity to components involved in neurotransmission that were particularly evident at the highest concentration tested, and associated with large metal accumulation. Cr(VI) increased the activities of GST and GSR, and total glutathione content to a different extent at different metal concentrations, this suggesting Cr(VI) detoxication/reduction at the site of metal entry. Cr(VI) exposure also increased the activity of glycolytic enzymes, indicating modulation of carbohydrate metabolism. Significant changes in transcription of different genes were observed. In particular, the mRNA level for the 5-HTR was increased, whereas both decreases and increases were observed for GST-π, MT10, MT20 and HSP70 mRNAs, showing sex- and concentration-related differences. The results demonstrate that Cr(VI) significantly affected functional and molecular parameters in mussel gills, and indicate that this tissue represents the major target of exposure to environmentally relevant concentrations of the metal.

The impact of long-term exposure to space environment on adult mammalian organisms: a study on mouse thyroid and testis.

Hormonal changes in humans during spaceflight have been demonstrated but the underlying mechanisms are still unknown. To clarify this point thyroid and testis/epididymis, both regulated by anterior pituitary gland, have been analyzed on long-term space-exposed male C57BL/10 mice, either wild type or pleiotrophin transgenic, overexpressing osteoblast stimulating factor-1. Glands were submitted to morphological and functional analysis.In thyroids, volumetric ratios between thyrocytes and colloid were measured. cAMP production in 10(-7)M and 10(-8)M thyrotropin-treated samples was studied. Thyrotropin receptor and caveolin-1 were quantitized by immunoblotting and localized by immunofluorescence. In space-exposed animals, both basal and thyrotropin-stimulated cAMP production were always higher. Also, the structure of thyroid follicles appeared more organized, while thyrotropin receptor and caveolin-1 were overexpressed. Unlike the control samples, in the space samples thyrotropin receptor and caveolin-1 were both observed at the intracellular junctions, suggesting their interaction in specific cell membrane microdomains.In testes, immunofluorescent reaction for 3ß- steroid dehydrogenase was performed and the relative expressions of hormone receptors and interleukin-1ß were quantified by RT-PCR. Epididymal sperm number was counted. In space-exposed animals, the presence of 3ß and 17ß steroid dehydrogenase was reduced. Also, the expression of androgen and follicle stimulating hormone receptors increased while lutenizing hormone receptor levels were not affected. The interleukin 1 ß expression was upregulated. The tubular architecture was altered and the sperm cell number was significantly reduced in spaceflight mouse epididymis (approx. -90% vs. laboratory and ground controls), indicating that the space environment may lead to degenerative changes in seminiferous tubules.Space-induced changes of structure and function of thyroid and testis/epididymis could be responsible for variations of hormone levels in human during space missions. More research, hopefully a reflight of MDS, would be needed to establish whether the space environment acts directly on the peripheral glands or induces changes in the hypotalamus-pituitary-glandular axis.

The organophosphate Chlorpyrifos interferes with the responses to 17ß-estradiol in the digestive gland of the marine mussel Mytilus galloprovincialis.

BACKGROUND: Many pesticides have been shown to act as endocrine disrupters. Although the potencies of currently used pesticides as hormone agonists/antagonists are low compared with those of natural ligands, their ability to act via multiple mechanisms might enhance the biological effect. The organophosphate Chlorpyrifos (CHP) has been shown to be weakly estrogenic and cause adverse neurodevelopmental effects in mammals. However, no information is available on the endocrine effects of CHP in aquatic organisms. In the digestive gland of the bivalve Mytilus galloprovincialis, a target tissue of both estrogens and pesticides, the possible effects of CHP on the responses to the natural estrogen 17ß-estradiol (E(2)) were investigated. METHODOLOGY/PRINCIPAL FINDINGS: Mussels were exposed to CHP (4.5 mg/l, 72 hrs) and subsequently injected with E(2) (6.75 ng/g dw). Responses were evaluated in CHP, E(2) and CHP/E(2) treatment groups at 24 h p.i. by a biomarker/transcriptomic approach. CHP and E(2) induced additive, synergistic, and antagonistic effects on lysosomal biomarkers (lysosomal membrane stability, lysosome/cytoplasm volume ratio, lipofuscin and neutral lipid accumulation). Additive and synergistic effects were also observed on the expression of estrogen-responsive genes (GSTπ, catalase, 5-HTR) evaluated by RT-Q-PCR. The use of a 1.7K cDNA Mytilus microarray showed that CHP, E(2) and CHP/E(2), induced 81, 44, and 65 Differentially Expressed Genes (DEGs), respectively. 24 genes were exclusively shared between CHP and CHP/E(2), only 2 genes between E(2) and CHP/E(2). Moreover, 36 genes were uniquely modulated by CHP/E(2). Gene ontology annotation was used to elucidate the putative mechanisms involved in the responses elicited by different treatments. CONCLUSIONS: The results show complex interactions between CHP and E(2) in the digestive gland, indicating that the combination of certain pesticides and hormones may give rise to unexpected effects at the molecular/cellular level. Overall, these data demonstrate that CHP can interfere with the mussel responses to natural estrogens.

Pleiotropic effects of hexavalent chromium (CrVI) in Mytilus galloprovincialis digestive gland.

Hexavalent Chromium Cr(VI) is an important contaminant considered as a model oxidative toxicant released from both domestic and industrial effluents, and represents the predominant chemical form of the metal in aquatic ecosystems. On the other hand, in mammals the reduced form Cr(III) is considered an essential microelement, involved in regulation of lipid and carbohydrate metabolism; moreover, recent evidence suggests that Cr may have endocrine effects. In this work, the effects of Cr(VI) were investigated in the digestive gland of the marine bivalve Mytilus galloprovincialis. Mussels were exposed to 0.1-1-10-100 µg Cr(VI) L(-1) animal(-1) for 96 h. At 100 µg L(-1), a large increase in total Cr tissue content was observed; in these conditions, the lysosomal membranes were completely destabilized, whereas other lysosomal biomarkers (neutral lipids-NL and lipofuscin-LF), as well as different enzyme activities and gene expression were unaffected, this indicating severe stress conditions in the tissue. On the other hand, at lower concentrations, changes in other histochemical, biochemical and molecular endpoints were observed. In particular, at both 1 and 10 µg L(-1), lysosomal destabilization was associated with significant NL and LF accumulation; however, no changes in catalase and GSH transferase (GST) activities were observed. At the same concentrations, GSSG reductase (GSR) activity was significantly increased, this probably reflecting the recycling of GSSG produced in the GSH-mediated intracellular reduction of Cr(VI). Increased activities of the key glycolytic enzymes PFK (phosphofructokinase) and PK (pyruvate kinase) were also observed, indicating that Cr(VI) could affect carbohydrate metabolism. Cr(VI) induced downregulation or no effects on the expression of metallothioneins MT10 and MT20, except for an increase in MT20 transcription in males. Moreover, significant up-regulation of the Mytilus estrogen receptor MeER2 and serotonin receptor (5-HTR) were observed in both sexes. The results demonstrate that exposure to Cr(VI) in the low ppb range did not result in strong toxicity or oxidative stress conditions in mussel digestive gland. On the other hand, our data support the hypothesis that low concentrations of the metal can exert pleiotropic effects on mussel physiology, from modulation of lipid and carbohydrate metabolism, to effects on the expression of estrogen-responsive genes.

Effects of vibrio challenge on digestive gland biomarkers and antioxidant gene expression in Mytilus galloprovincialis.

In bivalve molluscs, responses to bacterial infection have been largely characterized in terms of both functional responses and gene expression in the immune cells, the hemocytes. The effects of bacterial challenge at the tissue level, where bacterial infection may cause stressful conditions, have not been so far specifically investigated. Biomarkers are widely utilised to evaluate the health status of bivalves, from the molecular to the organism level, in response to both natural and anthropogenic stressors. In this work, the effects of in vivo challenge with heat-killed vibrio species, Vibrio splendidus LGP32 and Vibrio anguillarum (ATCC19264), on different biomarkers in the digestive gland of the marine bivalve Mytilus galloprovincialis were investigated. Mussels were injected with either vibrio and tissues sampled at 3, 6 and 24 h post injection (p.i.). Lysosomal biomarkers, such as lysosomal membrane stability (LMS) and lipofuscin accumulation, as well as specific activities of antioxidant enzymes (catalase and glutathione transferase-GST) were evaluated. Moreover, the expression of antioxidant molecules (catalase, GST-pi and metallothioneins MT10 and MT20) was determined by quantitative RT-PCR. Both V. splendidus and V. anguillarum significantly affected all parameters measured, to a different extent and at different times p.i. Interestingly, whereas both vibrios induced lysosomal membrane destabilisation and increases in the activities of antioxidant enzymes, distinct responses were observed in terms of lysosomal lipofuscin accumulation and expression of antioxidant molecules. In particular, V. splendidus induced a general increase in the transcription of antioxidant genes, indicating that Mytilus digestive gland can mount an efficient antioxidant response towards this vibrio species. On the other hand, a general down-regulation or no effect was observed with V. anguillarum. The lack of this response was reflected in stronger oxidative stress conditions in the digestive gland of mussels challenged with V. anguillarum, as indicated by higher levels of lysosomal lipofuscin observed at longer times p.i. Overall, these data indicate that lysosomal and oxidative stress biomarkers could be usefully applied in order to monitor early changes in the health status of bivalves induced by bacteria. Moreover, the results support the hypothesis that host responses to bacteria may be taken into account when interpreting biomarker data in ecotoxicological studies.