Extracellular Vesicles in Environmental Toxicological Studies: Association between Urinary Concentrations of Phthalate Metabolites and Exosomal miRNA Expression Profiles.

Phthalates are chemical compounds, mainly used as additives in plastics, which are known to induce harmful impacts to the environment and human health due to their ability to act as hormone-mimics. Few studies have been reported on the relationship between human exposure to phthalates and the level of circulating microRNAs (miRs), especially those miRs encapsulated in extracellular vesicles/exosomes or exosome-like vesicles (ELVs). We examined the relationship of ELV-miR expression patterns and urine of adult men with five phthalate metabolites (i.e., mono isobutyl phthalate, mono-n-butyl phthalate, mono benzyl phthalate, mono-(2-ethyl-5-oxohexyl) phthalate, mono-(2-ethylhexyl) phthalate) to identify potential biomarkers and relevant pathways. We found significant positive associations which were further confirmed by multivariable analysis. Overall, our analyses showed that the Σ phthalate metabolite concentration was associated with a significant increase in the expression level of two miRs found in ELV: miR-202 and miR-543. Different pathways including cancer and immune-related responses were predicted to be involved in this relationship. Analyzing the specific downstream target genes of miR-202 and miR-543, we identified the phosphatase and tensin homolog (PTEN) as the key gene in several converging pathways. In summary, the obtained results demonstrate that exposure to environmental phthalates could be related to altered expression profiles of specific ELV-miRs in adult men, thereby demonstrating the potential of miRs carried by exosomes to act as early effect biomarkers.

Effect of polycyclic aromatic hydrocarbons on homeobox gene expression during embryonic development of cuttlefish, Sepia officinalis.

Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants (POPs) commonly found in marine environments. Their bioaccumulation can cause harm to aquatic organisms, including invertebrates, particularly during the early stages of embryonic development. In this study, we evaluated, for the first time, the patterns of PAH accumulation in both capsule and embryo of common cuttlefish (Sepia officinalis). In addition, we explored the effects of PAHs by analysing the expression profiles of seven homeobox genes [i.e., gastrulation brain homeobox (GBX), paralogy group labial/Hox1 (HOX1), paralogy group Hox3 (HOX3), dorsal root ganglia homeobox (DRGX), visual system homeobox (VSX), aristaless-like homeobox (ARX) and LIM-homeodomain transcription factor (LHX3/4)]. We found that PAH levels in egg capsules were higher than those observed in chorion membranes (35.1 ± 13.3 ng/g vs 16.4 ± 5.9 ng/g). Furthermore, PAHs were also found in perivitellin fluid (11.5 ± 5.0 ng/ml). Naphthalene and acenaphthene were the congeners present at highest concentrations in each analysed egg component suggesting higher bioaccumulation rates. Embryos with high concentrations of PAHs also showed a significant increase in mRNA expression for each of the analysed homeobox genes. In particular, we observed a 15-fold increase in the ARX expression levels. Additionally, the statistically significant variation in homeobox gene expression patterns was accompanied by a concomitant increase in mRNA levels of both aryl hydrocarbon receptor (AhR) and estrogen receptor (ER). These findings suggest that bioaccumulation of PAHs may modulate developmental processes of cuttlefish embryos by targeting homeobox gene-mediated transcriptional outcomes. Mechanisms underlying the upregulation of homeobox genes could be related to the ability of PAHs to directly activate AhR- or ER-related signaling pathways.

Spirulina-enriched Substrate to Rear Black Soldier Fly (Hermetia illucens) Prepupae as Alternative Aquafeed Ingredient for Rainbow Trout (Oncorhynchus mykiss) Diets: Possible Effects on Zootechnical Performances, Gut and Liver Health Status, and Fillet Quality.

In the present study, an organic substrate (coffee silverskin) enriched with spirulina (Arthrospira platensis; 15% w/w), as a source of lipids and bioactive molecules, was used to rear the black soldier fly (Hermetia illucens) prepupae. Three grossly isonitrogenous, isoproteic, isolipidic and isoenergetic experimental diets for rainbow trout (Oncorhynchus mykiss) juveniles were then produced: a control diet (HM0) mostly including fish meal and fish oil, and two other test diets named HM3 and HM20, in which 3 or 20% of the marine ingredients were substituted with full fat black soldier fly prepupae meal (HM), respectively. Experimental diets were provided for 6 weeks, and at the end of the trial the physiological responses and marketable traits of the fish were investigated using a multidisciplinary approach. Generally, all test diets were well accepted, and fish growth, gut and liver health status, and marketable characteristics were not impaired by the experimental diets. However, an increased immuno-related gene expression along with a slight reduction of fillet redness and yellowness was evident in fish from the HM20 group.

Microplastics accumulation in gastrointestinal tracts of Mullus barbatus and Merluccius merluccius is associated with increased cytokine production and signaling.

There is clear evidence that different marine species can be impacted by microplastic (MP) ingestion accumulating such MPs mainly in the gastrointestinal tract. However, there is still limited knowledge on the consequences of MPs' accumulation in the gut. The present study aims to assess MPs and their potential immunotoxic effects in the digestive tract of two species showing different ecological traits: the red mullet (Mullus barbatus) and the European hake (Merluccius merluccius). Infrared spectroscopy (FTIR-ATR), micro-Raman and electron scanning microscope (SEM) were used to accurately identify the main plastic polymers detected in gut contents. In addition, we investigated the association between MP uptake and intestinal inflammation by evaluating expression and secretion of proinflammatory cytokines. MP abundance ranged from 1 to 20 items/individual in red mullet and from 2 to 15 items/individual in European hake. The majority of ingested MPs were fibers, while the dominant colors were black and blue in both species. Chemical characterization indicated polyethylene and polypropylene as the most common polymer types. Moreover, it was observed that MP abundance was highly positive correlated to cytokines (i.e. interleukin-1ß, 10, and interferon) and antioxidant enzyme (i.e. catalase and superoxide dismutase) transcript levels suggesting ROS generation and an infiltration of immune cells in the gut. Our findings provide evidence that the induction of cytokine-dependent signaling pathways is one aspect of the complex mechanism by which MPs affect the gut system in fish.

Organic UV Filters Induce Toll-like-Receptors and Related Signaling Pathways in Peripheral Blood Mononuclear Cells of Juvenile Loggerhead Sea Turtles (Caretta caretta).

Recent evidence suggests that exposure to organic ultraviolet filters (UV filters) is associated with dysregulated neuroendocrine-immune homeostasis. Marine species are likely to be among the most vulnerable to UV filters due to widespread diffusion of these chemicals in the aquatic environment. In the present study, the effects of UV filter bioaccumulation on toll-like-receptors (TLRs) and related signaling pathways were investigated in peripheral blood mononuclear cells (PBMCs) of juvenile loggerhead sea turtles (Caretta caretta). We found that the expression of both TLR1 and TLR2 was significantly increased in UV-filter exposed turtles compared to control animals. Similarly, the signaling pathway downstream of activated TLRs (i.e., Ras-related C3 botulinum toxin substrate 1 (RAC1), Phosphoinositide 3-kinase (PI3K), serine/threonine-protein kinase (AKT3), and nuclear factor κB (NF-κB)) was significantly up-regulated, leading to an enhanced transcription of pro-inflammatory cytokines. In addition, we demonstrated that high levels of plasma UV filters increased lipid peroxidation in sea turtles' PBMCs. Our results indicated that UV filters affected the inflammatory responses of PBMCs via modulation of the TLR/NF-κB signaling pathway and provided a new insight into the link between exposure to sunscreen agents and sea turtle health.

Effects of tributyltin on retinoid X receptor gene expression and global DNA methylation during intracapsular development of the gastropod Tritia mutabilis (Linnaeus, 1758).

The tributyltin (TBT)-mediated induction of imposex in marine snails is considered a common mechanism of endocrine disruption through the retinoid X receptor (RXR)-dependent pathway. However, there is evidence that regulation of RXR also relates to metabolic processes, differentiation, apoptosis, and embryonic development, playing a key role in molluscan neuronal differentiation and organogenesis. In this regard, very little is known about the gastropod Tritia mutabilis especially in relation to the effects of TBT exposure during intracapsular embryonic development. In this study, we have investigated the RXR expression fold changes of T. mutabilis encapsulated embryos exposed to different concentrations (10-10 to 10-12 M) of TBT up to 10 days of treatment. We demonstrate that RXR is sequentially expressed during development and that exposure to the lowest and highest TBT doses induces time-dependent changes in RXR gene transcription. We also show that TBT treatment is associated with global DNA demethylation and reduced DNA-methyltransferase I (DNMT1) expression and activity levels. Overall, our data indicate that RXR has important functions during the early stages of T. mutabilis embryo development and is involved in mediating the potential epigenetic alterations induced by TBT exposure.

Tart cherry (Prunus cerasus L.) dietary supplement modulates visceral adipose tissue CB1 mRNA levels along with other adipogenesis-related genes in rat models of diet-induced obesity.

PURPOSE: There is increasing evidence for the involvement of dietary bioactive compounds in the cross-talk modulation of endocannabinoid system and some of the key regulators of transcriptional control for adipogenesis. METHODS: We aimed to characterize the expression of cannabinoid CB1/CB2 receptors and fatty acid amide hydrolase (FAAH) along with selected adipogenesis-related genes (PPARγ, SREBP-1c and PREF-1), adipocyte-secreted factors (leptin and adiponectin), mitochondrial bioenergetic modulators (PGC-1A and UCP-2), and transient receptor potential vanilloid subtype 1 (TRPV1) and 2 (TRPV2) channels in visceral adipose tissue of rats fed with a high-fat diet (HFD) containing either tart cherry seeds alone or tart cherry seeds and juice for 17 weeks. The visceral adipose tissue was weighed and checked the expression of different markers by qRT-PCR, Western blot and immunohistochemistry. RESULTS: Tart cherry supplements were able to downregulate the HFD-induced mRNA expression of CB1 receptor, SREBP-1c, PPARγ, leptin, TRPV1 and TRPV2 resulting in potential anti-adipogenic effects. CONCLUSION: The present study points out that the intake of bioactive constituents of tart cherry may attenuate the effect of adipogenesis by acting directly on the adipose tissue and modulating the interplay between CB1, PPARγ and TRPV channel gene transcription.

Microplastics and their associated organic pollutants from the coastal waters of the central Adriatic Sea (Italy): Investigation of adipogenic effects in vitro.

Even though microplastic (MP) pollution in aquatic environment is nowadays widely studied, a huge gap of knowledge exists on their actual biological effects. In this study we first reported environmental baseline data on the occurrence and characterization of floating MPs in Italian coastal waters of the Central Adriatic Sea by using a standardized monitoring protocol. Further, we analyzed the concentrations of MP-associated chemicals and evaluated their potential adipogenic effects using 3T3-L1 preadipocytes. MPs were found in each sampling stations showing the highest abundance (1.88 ± 1.78 items/m3) in the sites more distant from the coast with fragments as the most common shape category. All targeted organic pollutants (i.e. polychlorinated biphenyls - PCBs, polycyclic aromatic hydrocarbons -PAHs, organophosphorus - OP, and organochlorine - OC pesticides) have been detected on the surface of the collected MPs. The highest concentrations of PAHs were found on MPs from inshore (i.e. <1.5 NM) surface waters with low-ring PAHs as dominant components. Similarly, MPs from inshore waters had higher ΣPCB concentrations (64.72 ng/g plastic) than those found in offshore (i.e. >6 NM) waters (10.37 ng/g plastic). Among pesticides, all measured OPs were detected in each sample analyzed with pirimiphos-methyl as the most representative compound. For OCs, the sum of all concentrations of congeners was higher in coastal with respect to offshore waters. Moreover, in vitro 3T3-L1 screening of MP extracts indicated potential metabolic effects resulting in both adipogenesis and lipid uptake/storage.

Transcriptional Alteration of Gene Biomarkers in Hemocytes of Wild Ostrea edulis with Molecular Evidence of Infections with Bonamia spp. and/or Marteilia refringens Parasites.

The European flat Ostrea edulis is highly susceptible to intracellular parasitic infections, particularly bonamiosis and marteiliosis. The defensive response of oyster to both bonamiosis and marteiliosis is typically mediated by hemocytes, which play a pivotal role in immune system homeostasis. In the present study, we first used a DNA-based tool in order to rapidly and specifically detect the presence of parasites in oysters from natural banks in the middle Adriatic Sea. In a second step, we used qRT-PCR to analyze the mRNA levels of a set of genes (i.e., superoxide dismutase (SOD), glutathione S-transferase (GST), metallothionein (MT), heat shock protein (HSP) 70 and 90, inhibitor of apoptosis (IAP), fas ligand (FAS), galectin (GAL) and extracellular superoxide dismutase (Ec-SOD)) expressed by hemocytes of flat oysters infected by the parasites, present singularly or in combination, compared to hemocytes from non-infected specimens. The results indicate that the presence of parasite DNA may be associated to a general upregulation of host genes related to apoptosis, detoxification and oxidative stress protection, with the exception of Ec-SOD, whose trend to a downregulation might reflect a mechanism for parasite escape before internalization.

Sunscreen active ingredients in loggerhead turtles (Caretta caretta) and their relation to molecular markers of inflammation, oxidative stress and hormonal activity in wild populations.

The present study sought to determine bioaccumulation and potential molecular effects of four of the most environmentally significant sunscreen agents in juvenile loggerhead sea turtles (Caretta caretta) from the Italian coasts of the central Adriatic Sea. Each of these sunscreen agents were found in most of the plasma samples analyzed, with benzophenone-3 as the dominant species. Total concentrations of the sunscreen agents ranged from not detected (

Changes in expression of microRNA potentially targeting key regulators of lipid metabolism in primary gilthead sea bream hepatocytes exposed to phthalates or flame retardants.

Metabolism disrupting chemicals (MDCs) belong to the group of endocrine-disrupting chemicals (EDCs) and are known to affect endocrine and metabolic functions of liver. There is growing evidence that MDCs may also act modulating the expression levels of micro ribonucleic acids (miRNAs) and thus affecting post-transcriptional expression of hundreds of target genes. Herein, we used a gilthead sea bream in vitro hepatocyte model for analyzing the effects of an exposure to phthalates (i.e. DiDP) or flame retardants (i.e.TMCP) on the expression levels of three miRNAs (i.e. MiR133, MiR29 and MiR199a) selected on the basis of their regulatory roles in signaling pathways related to lipid metabolism. Following computational identification of genes that are regulated by the selected miRNAs, we identified six miRNA targets to be tested in differential gene expression analysis. To determine whether lipid metabolism was altered we have also measured the intracellular total cholesterol and triglyceride levels. The results of our study show that DiDP/TMCP exposure leads to a general decrease in the expression profiles of each miRNA leading to a corresponding upregulation of almost all their putative targets. In addition, these findings were also associated to a corresponding increased hepatocellular lipid content. The present study thus contributes to support the importance of these small molecules in regulating MDC-induced expression of genes associated with hepatic lipid metabolism and highlights the need for more toxicological studies examining miRNAs transcriptional regulatory networks controlling metabolic alterations in fish.

Gene expression profiles of putative biomarkers in juvenile loggerhead sea turtles (Caretta caretta) exposed to polycyclic aromatic hydrocarbons.

There is evidence that polycyclic aromatic hydrocarbons (PAHs) are consistently the predominant organic contaminants in concentration found in loggerhead sea turtles (Caretta caretta) from the North and Central Adriatic Sea. Hence this study investigates the PAH toxicity to loggerheads by using a particular set of genes [i.e. CYP1B, CAT, GPX, GSTT1, SOD3, DNMT1, Epoxide hydrolase 1 (EPHX1), Poly (ADP-ribose) polymerase 1 (PARP1), Lamin-A/C isoform 3 (LMNA), Talin 1 (TLN1), Annexin A1 (ANXA1)] whose altered expression is potentially dependent on and specific for the PAH-related mechanism of action. Twenty healthy juvenile loggerheads were thus divided into high and low exposure groups (mean of ΣPAHs: 80.34 ng mL-1 vs. 8.84 ng mL-1, P < 0.0001) according to the median split of ΣPAHs. Interestingly, we found that the whole blood mRNA levels of each gene biomarker tested were significantly increased in high PAH-exposed turtles thus proving to be useful for the biological monitoring of PAH toxicity and hematotoxicity in sea turtles.

Plasticizers used in food-contact materials affect adipogenesis in 3T3-L1 cells.

Recent studies suggest that exposure to some plasticizers, such as Bisphenol A (BPA), play a role in endocrine/metabolic dispruption and can affect lipid accumulation in adipocytes. Here, we investigated the adipogenic activity and nuclear receptor interactions of four plasticizers approved for the manufacturing of food-contact materials (FCMs) and currently considered safer alternatives. Differentiating 3T3-L1 mouse preadipocytes were exposed to scalar concentrations (0.01-25 µM) of DiNP (Di-iso-nonyl-phthalate), DiDP (Di-iso-decyl-phthalate), DEGDB (Diethylene glycol dibenzoate), or TMCP (Tri-m-cresyl phosphate). Rosiglitazone, a well-known pro-adipogenic peroxisome proliferator activated receptor gamma (PPARγ) agonist, and the plasticizer BPA were included as reference compounds. All concentrations of plasticizers were able to enhance lipid accumulation, with TMCP being the most effective one. Accordingly, when comparing in silico the ligand binding efficiencies to the nuclear receptors PPARγ and retinoid-X-receptor-alpha (RXRα), TMPC displayed the highest affinity to both receptors. Differently from BPA, the four plasticizers were most effective in enhancing lipid accumulation when added in the mid-late phase of differentiation, thus suggesting the involvement of different intracellular signalling pathways. In line with this, TMCP, DiDP, DiNP and DEGDB were able to activate PPARγ in transient transfection assays, while previous studies demonstrated that BPA acts mainly through other nuclear receptors. qRT-PCR studies showed that all plasticizers were able to increase the expression of CCAAT/enhancer binding protein ß (Cebpß) in the early steps of adipogenesis, and the adipogenesis master gene Pparγ2 in the middle phase, with very similar efficacy to that of Rosiglitazone. In addition, TMCP was able to modulate the expression of both Fatty Acid Binding Protein 4/Adipocyte Protein 2 (Fabp4/Ap2) and Lipoprotein Lipase (Lpl) transcripts in the late phase of adipogenesis. DEGDB increased the expression of Lpl only, while the phthalate DiDP did not change the expression of either late-phase marker genes Fabp4 and Lpl. Taken together, our results suggest that exposure to low, environmentally relevant doses of the plasticizers DiNP, DiDP, DEGDB and TMCP increase lipid accumulation in 3T3-L1 adipocytes, an effect likely mediated through activation of PPARγ and interference at different levels with the transcriptional cascade driving adipogenesis.

Investigating the potential impact of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) on gene biomarker expression and global DNA methylation in loggerhead sea turtles (Caretta caretta) from the Adriatic Sea.

Polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) are priority contaminants that bioaccumulate through the food webs and affect the biology of a variety of resident and migratory species, including sea turtles. Few studies have evaluated toxicological biomarkers of exposure to PAHs and PCBs in these animals. The present paper reports the results of an initial field study to quantify the association between plasma concentrations of PAHs/PCBs and whole blood cell expression of gene biomarkers in juvenile loggerhead sea turtles (Caretta caretta) rescued along the Italian coasts of the northern and central areas of the Adriatic Sea. While detectable levels of PAHs were found in all plasma samples examined, only three PCB congeners (PCB52, PCB95, and PCB149) were noted, with detection percentages ranging between 48% and 57%. A significant correlation was found between 3 of the 6 gene biomarkers assessed (HSP60, CYP1A and ERα) and plasma levels of some PAH congeners. In contrast, no significant association between PCB burden and gene expression was observed. The global DNA methylation levels were significantly and positively correlated with the concentrations of most of the PAHs and only one of the PCB congeners (PCB52). The relation between PAH concentration and gene expression in whole blood cells suggests that these genes may respond to environmental contaminant exposure and are promising candidates for the development of biomarkers for monitoring sea turtle exposure to persistent organic pollutants (POPs).

Pregnane X receptor (PXR) signaling in seabream primary hepatocytes exposed to extracts of seawater samples collected from polycyclic aromatic hydrocarbons (PAHs)-contaminated coastal areas.

Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants damaging to the marine environment and the wildlife. Herein, we investigated the effects of extracts from coastal seawaters (central Adriatic sea, Italy), showing high concentrations of PAHs, on pregnane X receptor (PXR)-transcriptional regulation of the cytochrome P450 3A (CYP3A) gene using seabream primary hepatocytes. The results show that concentrated extracts of seawater with original ΣPAH concentrations above the putative threshold of 30 ng L-1 increased expression of PXR and its main target gene, CYP3A. Similar results were observed for LXR and its target gene SREBP-1c suggesting pathway cross-talk. These data are further supported by the finding of multiple PXR and LXR response elements in the putative promoters of their target genes. Overall, our data indicate the capacity of seawater extracts, containing environmentally relevant levels of PAHs, to affect multiple pathways, including lipid and cholesterol metabolism.

Differential tissue regulation of peroxisome proliferator-activated receptor α (PPARα) and cannabinoid receptor 1 (CB1) gene transcription pathways by diethylene glycol dibenzoate (DEGB): preliminary observations in a seabream (Sparus aurata) in vivo model.

Today a variety of endocrine disrupting chemicals (EDCs) are recognized in the group of metabolic disruptors, a wide range of environmental contaminants that alter energy balance regulation by affecting the peroxisome proliferator-activated receptor (PPAR)/retinoid X receptor (RXR) pathway. Herein, we investigated the effect of diethylene glycol dibenzoate (DEGB), a dibenzoate-based plasticizer used as alternative to phthalates, on the expression of key genes involved in lipid metabolism and energy balance by using Sparus aurata juveniles as models. We also evaluated the correlation between cannabinoid receptor 1 (CB1) and PPARα transcriptional patterns in both liver and brain tissues. Exposure to the highest DEGB concentration differentially modulated PPARα/CB1 transcriptional pathways in liver/brain tissues of seabream. We hypothesize that, at peripheral level (i.e. liver), DEGB acts as PPARα agonist resulting in a potential stimulation of key lipolytic genes and a concomitant down-regulation of endocannabinoid metabolic enzyme genes.

Effects of endocrine disrupting chemicals on estrogen receptor alpha and heat shock protein 60 gene expression in primary cultures of loggerhead sea turtle (Caretta caretta) erythrocytes.

The loggerhead turtle (Caretta caretta) can be considered a good indicator species for studying the ecological impact of endocrine disrupting chemicals (EDCs) on wildlife. However, the effect of these environmental pollutants on nuclear steroid hormone signaling has not yet been addressed in sea turtles mainly due to the legal constraints of their endangered status. Here we describe the use of primary erythrocyte cell cultures as in vitro models for evaluating the effects of different EDCs on the expression of estrogen receptor α (ERα). In addition, we evaluated erythrocyte toxicity caused by EDCs using Alamar Blue assay and heat shock proteins 60 (HSP60) expression. Primary cultures of erythrocytes were exposed to increasing concentrations of 4-nonylphenol (4NP), Diisodecyl phthalate (DiDP), Tri-m-cresyl phosphate (TMCP) and Tributyltin (TBT) for 48h. Alamar Blue demonstrated that exposure of erythrocytes to each contaminant for up to 48h led to a significant impairment of cellular metabolic activity at 100µM, with the exception of TBT. Moreover, our data indicate that loggerhead erythrocytes constitutively express ERα and HSP60 at the transcript level and respond to EDCs by up-regulating their expression. In this regard, ERα was up-regulated in a dose-dependent manner after 48h exposure to both 4NP and TMCP. Interestingly, the dosage-dependent effects of DiDP on ERα expression were opposite in comparison to that obtained following exposure to the other tested compounds. This work provides the first indication regarding the potential of primary erythrocytes as study models for evaluating the effects of EDCs on sea turtles.

Transcriptional variations in biomarkers of Mytilus galloprovincialis sampled from Central Adriatic coastal waters (Marche region, Italy).

OBJECTIVE: this work evaluates the expression of several biomarker genes (i.e. CAT, HSPs, MT10, p53, GSTpi, ER1/2) in Mytilus galloprovincialis native specimens to identify pollution related transcriptional changes. METHODS: gene expression levels were analysed in the gills of mussels from five sites along a coastal area of the central Adriatic Sea, using qPCR. RESULTS: we observed higher expression levels of biomarker genes in mussels from the local harbour or river mouths than in mussels from the reference site and offshore platform. CONCLUSIONS: the variations in gene expression observed indicated high sensitivity to pollutants, and showed differences among the sampling sites.

Alterations of gene expression indicating effects on estrogen signaling and lipid homeostasis in seabream hepatocytes exposed to extracts of seawater sampled from a coastal area of the central Adriatic Sea (Italy).

Recent evidences suggest that the toxicological effects of endocrine disrupting chemicals (EDCs) involve multiple nuclear receptor-mediated pathways, including estrogen receptor (ER) and peroxisome proliferator-activated receptor (PPAR) signaling systems. Thus, our objective in this study was to detect the summated endocrine effects of EDCs with metabolic activity in coastal waters of the central Adriatic Sea by means of a toxicogenomic approach using seabream hepatocytes. Gene expression patterns were also correlated with seawater levels of polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs). We found that seawater extracts taken at certain areas induced gene expression profiles of ERα/vitellogenin, PPARα/Stearoyl-CoA desaturase 1A, cytochrome P4501A (CYP1A) and metallothionein. These increased levels of biomarkers responses correlated with spatial distribution of PAHs/PCBs concentrations observed by chemical analysis in the different study areas. Collectively, our data give a snapshot of the presence of complex EDC mixtures that are able to perturb metabolic signaling in coastal marine waters.

In silico prediction and in vivo analysis of antiestrogenic potential of 2-isopropylthioxanthone (2-ITX) in juvenile goldfish (Carassius auratus).

Previous studies have shown both anti-estrogenic and anti-androgenic activities of 2-isopropylthioxanthone (2-ITX), a well known food contaminant, in in vitro assays. However, no data are available on the anti-estrogenic potentials and risks of 2-ITX in aquatic organisms. This work evaluated the potential endocrine disrupting effects of 2-ITX at the level of estrogen receptor (ER) signaling cascade using juvenile goldfish (Carassius auratus) as model. Firstly, we investigated the ligand binding efficiency of 2-ITX to the ligand binding domains (LBD) of goldfish ER subtypes using a molecular docking approach. Secondly, we assessed the effects of 2-ITX on E2-induced hepatic expression of ERα1, ERß1, ERß2, and vitellogenin (VTG) in vivo. Crosstalk between ER-VTG and aryl hydrocarbon receptor 2 (AhR2)-cytochrome P4501A (CYP1A) was also investigated. Fish were injected with increasing doses of 2-ITX ranging from 2 to 10µg/g BW, and results were compared to the effect of tamoxifen, a well-known ER modulator. We observed that compared to ERß, the interaction potentials of 2-ITX to goldfish ERα1 LBD was more stable in the inactive receptor conformation. The in silico docking simulation analysis also revealed that 2-ITX acted as agonist for the goldfish AhR2 LBDs suggesting the ability of this compound to activate the cross-talk between the ERα- and AhR-signaling pathways. In vivo experiments confirm in silico simulation predictions demonstrating that 2-ITX reduced the estrogenicity of E2 at both transcriptional and post-transcriptional levels, indicating a clear anti-estrogenic effect. Co-exposure of E2 and 2-ITX also resulted in a significant decrease of CYP1A gene expression with respect to 2-ITX alone. Results from these studies collectively revealed that the antiestrogenic property of 2-ITX can be ascribed to a combination of effects on multiple signaling pathways suggesting the potential for this environmental contaminant to affect the hormonal control of reproductive processes in fish.