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The implications related to the fight against alien organisms often affect different sectors, from the economic to the landscape one or from the public health to the ecological one. This paper is an upgrade of our previous study focused on the evolution of the red palm weevil in a coastal urban area (central Italy); we investigate the evolution of this parasite in the following period (2013-2020), considering both the effectiveness of the chemicals used and their potential effects. With a multidisciplinary approach, on the one hand we carried out a spatio-temporal analysis to analyze the extent and mode of pest spread over time using historical aerial photos, freely available remote sensing images and field surveys, integrated in a GIS environment, and on the other hand we performed an assessment of the chemical risk associated with the substances used to protect the palms from the red weevil. The fight against weevil is now concentrated in specific areas (parks, relevant roads, villas, hotels, farmhouses, nurseries); the applied preventive chemical treatments are very effective in preserving the palms, but they show a toxic potential for all organisms. We discuss current local management of this pest focusing on the several aspects involve into the fight against this beetle in an urban area.

In 2020–2021, a trial to recruit flat oysters was implemented at a longline farm in the central Adriatic, whereby the efficiency recruitment (n. oyster/dm2) of different suspended substrates was evaluated. Two lantern nets (50 cm diameter; 145 cm h) had different substrates composed of 8 mm wide wrinkled ribbon and empty oyster shells positioned in the upper levels of the lanterns. The tumbling evaluation and the presence of mud were also considered. The efficiency recruitment was similar between the wrinkled ribbon and the oyster shell. Recruitment was in the same proportion on the external rough part of the shells as on the internal smooth part of the shells. No significant differences were shown when comparing the different substrates in terms of recruitment efficiency.

In the present study, an organic substrate (coffee silverskin) enriched with spirulina (

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-1β, 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.

The spread of the red palm weevil is now a very severe problem on a global scale. In this paper, we investigate its spread during the 2007–2013 period in a coastal urban area characterized by a high density of palm trees, and discuss the application of a new approach to data collection and processing. While most works on the spread of the red palm weevil are based upon geo-referenced traps, we focused on the health of each palm, considering the surrounding territorial context. To this end, we used historical aerial photos, freely available remote sensing images and field surveys, integrated in a GIS environment to analyze the extent and mode of pest spread over time. Our results show that the exponential increase of new cases for each new year is regulated by this spatial spread model: at the early stage of infestation the red weevil tends to occupy as much space as possible, within the limitations of the distance it can fly; in the following stage, its infestation density increases in recently colonized areas. We discuss current local management of palm trees and the pests, and propose strategies that take into account both the ecology of the red weevil and the territorial context. Finally, we encourage the provision of more resources for early detection and prevention measures, as well as replacement of the dead Phoenix canariensis palms with a greater variety of species, as a varied composition would be less prone to pest-caused depopulation.

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

There is growing concern about the environmentally relevant concentrations of new emerging persistent organic pollutants, such as perfluorinated compounds and pharmaceuticals, which are found to bioaccumulate in aquatic organisms at concentrations suspected to cause reproductive toxicity due to the activation of estrogen receptor (ER) α and β subtypes. Here, we use a combined in silico and in vitro approach to evaluate the impact of perfluorononanoic acid (PFNA) and Enalapril (ENA) on grey mullet (Mugil cephalus) hepatic estrogen signaling pathway. ENA had weak agonist activity on ERα while PFNA showed moderate to high agonist binding to both ERs. According to these effects, hepatocytes incubation for 48 h to PFNA resulted in a concentration-dependent upregulation of ER and vitellogenin gene expression profiles, whereas only a small increase was observed in ERα mRNA levels for the highest ENA concentration. These data suggest a structure–activity relationship between hepatic ERs and these emerging pollutants.

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.

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. 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. 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. 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.

In this study, we examined the seasonal association between Polycyclic Aromatic Hydrocarbon (PAH) concentrations and mRNA expression profiles of some antioxidant genes (i.e. CAT, GST and SOD), as well as lipid peroxidation (LPO), in muscle of sexually inactive females of red mullet (Mullus barbatus). Fish were captured in a fishery area of the Northern Adriatic Sea during both winter and summer. We found significantly (p < 0.05) higher ?HMW-PAHs concentrations in muscle of specimens caught during winter than summer. On the basis of sampling season, red mullets exhibited different gene expression profiles of antioxidant enzymes showing lower levels of both CAT and GST in winter than in summer. Accordingly, CAT was found to be negatively associated with ?PAH concentrations, especially ?LMW-PAH, in individuals collected during winter. Seasonal-related downregulation of some oxidative stress biomarker expression is suggestive of greater susceptibility of red mullets to PAHs during winter.

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.

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.

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 (LOD) to 28.43 μg mL

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. 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.

Vitellogenin (Vtg) has proven to be a sensitive and simple biomarker in determining sex, sexual maturity, and xenoestrogenic effects in fish. Thus, our investigation has been focused on identification, partial characterization, and quantification of grey mullet (Mugil cephalus) Vtg through the use of a variety of biochemical and immunological analytical techniques. Mullet is considered both a promising aquaculture candidate and an important species for improving sediment quality in polyculture systems. In the first part of this work, grey mullet Vtg was purified from plasma of 17β-estradiol (E2)-induced male fish by a one-step chromatographic protocol, and partially characterized. Specific polyclonal antibodies were then raised against the mullet Vtg, and both an indirect ELISA and an optical immunosensor were set up and validated to quantify plasma Vtg. The indirect ELISA and the optical immunosensor assay developed showed linear measuring in the range 56.8–1047.1 ng mL−1 and 70–739 ng mL−1 Vtg concentrations in standard solutions, respectively. The results obtained suggest that the indirect ELISA allows Vtg detection over a wide dynamic range, thus resulting more suitable for rapid and sensitive sample screening. Therefore, we suggest that the direct immunosensor is a promising tool which needs more investigation to improve the sensitivity.

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.

The interactions between tri-m-cresyl phosphate (TMCP; an organophosphate flame retardant) and peroxisome proliferator activated receptors (PPARs) or liver X receptor α (LXRα) were investigated in seabream hepatocytes. The study was designed to characterize the binding of TMCP to PPARα, PPARγ and LXRα by computational modeling (docking) and transcriptional regulation of signaling pathways. TMCP mainly established a non-polar interaction with each receptor. These findings reflect the hydrophobic nature of this binding site, with fish LXRα showing the highest binding efficiency. Further, we have investigated the ability of TMCP to activate PPAR and LXR controlled transcriptional processes involved in lipid/cholesterol metabolism. TMCP induced the expression of all the target genes measured. All target genes were up-regulated at all exposure doses, except for fatty acid binding protein 7 (FABP7) and carnitine palmitoyltransferase 1B. Collectively, our data indicate that TMCP can affect fatty acid synthesis/uptake and cholesterol metabolism through LXRα and PPARs, together with interactions between these transcription factors in seabream liver.

Liver X receptor is a ligand-activated transcription factor, which is mainly involved in cholesterol homeostasis, bile acid and triglycerides metabolism, and, as recently discovered, in the glucose metabolism by direct regulation of liver glucokinase. Its modulation by exogenous factors, such as drugs, industrial by-products, and chemicals is documented. Owing to the abundance of these synthetic molecules in the environment, and to the established target role of this receptor, a number of representative compounds of phthalate, organophosphate and fibrate classes were tested as ligands/modulators of human liver X receptor, using an integrated approach, combining an in silico molecular docking technique with an optical SPR biosensor binding study. The compounds of interest were predicted and proved to target the oxysterols-binding site of human LXRα with measurable binding kinetic constants and with affinities ranging between 4.3 × 10(-7) and 4.3 × 10(-8)M. Additionally, non-cytotoxic concentration of these chemicals induced relevant changes in the LXRα gene expression levels and other target genes (SREBP-1c and LGK) in human liver hepatocellular carcinoma cell line (HepG2), as demonstrated by q-RT-PCR.

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).

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 β (Cebpβ) 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.

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.

The objective of the present study was to evaluate the modulation of acute stress response by dietary nucleotides (NT) in sole, Solea solea. A basal diet was supplemented with levels of 0 (normal diet), or 0.4 g NT/kg dry diet for 8 weeks. At the end of feeding trial, fish fed the normal and NT-supplemented diet were subjected to a standardized protocol of disturbance and sampled over a 24h recovery after the stressor exposure. Modulatory effects of NT on acute stress response (cortisol and glucose), proopiomelanocortin (POMC) and cannabinoid receptor 1 splice variants (CB1A and CB1B) mRNA levels were studied. Both plasma cortisol and glucose levels of fish fed NT-supplemented diet were significantly lower than fish fed the control diet at 1 and 4h post-stress time-points. There are no significant effects of dietary NT on POMC and HSP70 mRNA levels. In our study, both CB1A and CB1B trascript levels were induced in fish fed the normal diet at 1 and 4h post-stress intervals. Collectively, the results obtained suggest that dietary NT modulates the CB1-like receptor mRNA expressions leading to attenuation in stressor-induced plasma cortisol level in sole.

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.

Based on pharmacological, behavioral and neuroanatomical studies, the endocannabinoids appear to be pivotal in some important neuroendocrine regulations of both vertebrates and invertebrates. Interestingly, a well developed endocannabinoid system was recently demonstrated by us in different bonyfish brain areas which control reproduction, energy balance and stress. Fish in particular are very sensitive to different types of stressors which can heavily affect their reproductive activity and negatively reverberate on aquaculture. Since recent new data have been reported on endocrine disruptors (EDs) impact on zebrafish receptor CB1 expression, in the present research we have investigated the response of the endocannabinoid system to acute treatment with an environmental stressor such as the xenoestrogen nonylphenol (4NP) in the brain and peripheral tissues of the goldfish Carassius auratus . First of all the estrogenic effects induced by 4NP were demonstrated by a dose-dependent increase of plasma levels and gene expression of the biomarker vitellogenin, then changes in cannabinoid receptors and anandamide degradative enzyme, the fatty acid amide hydrolase (FAAH), were analysed by means of Real Time PCR. As the exposure to EDs may lead to an activation of estrogen receptors and affects the Aromatase (AROB) transcription, changes in mRNA levels for ER subtypes and AROB were also evaluated. Our results confirm in goldfish the effect of 4NP on ERα and ERβ1 receptors and point out a different sensitivity of CB1 and CB2 for this compound, suggesting distinct roles of these cannabinoid receptors in some adaptive processes to contrast stress induced by xenoestrogen exposure.

Endogenous cannabinoids, through the CB1 receptor, are involved in the control of several functions including stress responses. The aim of this study was to investigate the presence of cannabinoid receptor CB1 in the sole ovary by partial cloning of brain CB1 cDNA; in a stress paradigm of disturbance by handling, which consisted in catching, netting and hand-sorting, changes of CB1 mRNA were related with those of proopiomelanocortin (POMC) mRNA; the trend and timing of stress responses and adaptation were monitored by measuring plasma cortisol levels. We characterized two forms of CB1-like receptor, termed CB1A and CB1B. The two sole CB1 (both 799bp) share 76% identity in their cDNAs, and the deduced amino acid sequences are 80% identical. The handling stress induced a sustained increase in plasma cortisol levels 1h after the handling began and decreased to low levels 12h after initiation of handling, showing the same trend of ovarian POMC mRNA expression. In addition, while CB1A mRNA did not show any significant changes during handling stress, significantly lower levels of CB1B mRNA were found in stressed fish 1h after the beginning of handling, with CB1 expression increased 24h after stress induction, both in the ovary and brain. It can be concluded that endocannabinoid system is involved in the modulation of adaptive responses to environmental conditions.

Environmental estrogen-like compounds (i.e. xenoestrogens) are a variety of pollutants, ranging from synthetic to natural occurring molecules, that are found in surface and waste waters over a wide range of concentrations. In aquatic environment, the overall estrogenic activity is often due to the presence of a mixture of chemicals and their degraded products which can induce synergistic effects. Current strategies for monitoring estrogen-like chemicals are based on the use of a battery of in vivo and in vitro ecotoxicological tests. In this regard, the aim of the present work was to carry out a bio-monitoring study for testing estrogenicity of the Chienti river (Marche Region, Italy) by using both an E-screen and a vitellogenin (Vtg) induction assay in juvenile goldfish. Three sites were used for analysis, localized at the mouth (sampling point 1), in the middle (sampling point 2) and at the origin (sampling point 3) of Chienti river. For most of the water samples (i.e. samples collected at sampling points 2 and 3), clear estrogenic activity was detected in the E-screen assay suggesting different proliferation activities in function of the collecting site. In contrast, the Vtg ELISA demonstrated that water samples collected from each sampling point were estrogenic. Overall, we showed for the first time that the estrogenic activities in water samples from the Chienti river were significant in both in vivo and in vitro; we also observed a different sensitivity between bioassays.

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, ERβ1, ERβ2, 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 ERβ, 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.

Investigation of the ability of TMCP to activate PPAR and LXR controlled transcriptional processes in seabream hepatocytes.

Evidence that endocrine-disrupting chemicals (EDCs) may target metabolic disturbances, beyond interference with the functions of the endocrine systems has recently accumulated. Among EDCs, phthalate plasticizers like the diisodecyl phthalate (DiDP) are commonly found contaminants of aquatic environments and have been suggested to function as obesogens by activating peroxisome proliferator activated receptors (PPARs), a subset of nuclear receptors (NRs) that act as metabolic sensors, playing pivotal roles in lipid homeostasis. However, little is known about the modulation of PPAR signaling pathways by DiDP in fish. In this study, we have first investigated the ligand binding efficiency of DiDP to the ligand binding domains of PPARs and retinoid-X-receptor-α (RXRα) proteins in fish using a molecular docking approach. Furthermore, in silico predictions were integrated by in vitro experiments to show possible dose-relationship effects of DiDP on PPAR:RXR-dependent gene expression pathways using sea bream hepatocytes. We observed that DiDP shows high binding efficiency with piscine PPARs demonstrating a greater preference for RXRα. Our studies also demonstrated the coordinate increased expression of PPARs and RXRα, as well as their downstream target genes in vitro. Principal component analysis (PCA) showed the strength of relationship between transcription of most genes involved in fatty acid metabolism and PPAR mRNA levels. In particular, fatty acid binding protein (FABP) was highly correlated to all PPARs. The results of this study suggest that DiDP can be considered an environmental stressor that activates PPAR:RXR signaling to promote long-term changes in lipid homeostasis leading to potential deleterious physiological consequences in teleost fish.

Many synthetic chemicals, termed xenoestrogens, have been shown to interact as agonists with the estrogen receptor (ER) to elicit biological responses similar to those of natural hormones. To date, the regulation of vitellogenesis in oviparous vertebrates has been widely used for evaluation of estrogenic effects. Therefore, Carassius auratus juveniles were chosen as a fish model for studying the effects of estradiol-17beta and different concentrations (10(-6) and 10(-7) M) of 4-nonylphenol (4-NP) on the expression of liver ERbeta-1 subtype; plasma vitellogenin and sex steroids (androgens and estradiol-17beta) were also evaluated together with the bioaccumulation process, through mass-spectrometry. C. auratus is a species widespread in the aquatic environment and, on the toxicological point of view, can be considered a good "sentinel" species. Juveniles of goldfish were maintained in tanks with only tap water or water with different concentrations (10(-6) and 10(-7) M) of 4-nonylphenol (4-NP), or 10(-7) M of estradiol-17beta. After 3 weeks of treatment, animals were anesthetized within 5 min after capture, and blood was immediately collected into heparinized syringes by cardiac puncture and stored at -70 degrees C; the gonads were fixed, then frozen and stored at -70 degrees C; the whole fish, liver, and muscle tissues were harvested and immediately stored at -70 degrees C for molecular biology experiments and bioaccumulation measurements. The estrogenic effects of 4-NP were evidenced by the presence of plasma vitellogenin in juveniles exposed both to estradiol-17beta and the two doses of 4-NP; moreover, exposure to 4-NP also increased aromatization of androgens, as suggested by decreasing androgens and increasing estradiol-17beta plasma levels. The changes of these parameters were in agreement with the increasing transcriptional rate of ERbeta-1 mRNA in the liver, demonstrating that both estradiol-17beta and 4-NP modulate the vitellogenin rate through interaction with the ERbeta-1 subtype. The present study also suggests that 4-NP at the concentration of 10(-6) M bioaccumulates in the liver.

Several environmental chemicals are known to interact with functions related with the neuroendocrine system, and many of the endocrine disruptors present in the aquatic environment have estrogenic activity and can be responsible for feminization processes in wild fish and amphibians. The estrogenic effects of such disruptors include several levels of investigation : biological significance 'in vivo', and 'in vitro' by use of estrogenic biomarkers, and effects on the brain-pituitary-gonadal axis. Amphibians spend a great part of their life in the water, and can be considered 'sentinel species'; therefore, the effects of an estrogenic compound, 4-nonylphenol, were studied by 'in vivo' and 'in vitro' experiments. As a biomarker of estrogenic effects, vitellogenin changes were assayed in male frogs and newts; the plasma sex steroid trend was evaluated, and brain aromatase-like-immunoreactivity was described. 4-Nonylphenol treatment was able to induce plasma vitellogenin in male frogs and newts, changes in the secondary sexual characteristics, and increase of androgen plasma levels in the treated male newts. Moreover, a reduction in both the number of ARO-LI containing neurons and their immunolabeling intensity was seemingly observed in the more caudal cluster of aromatase containing cells or ventral infundibular nucleus. In conclusion, 4-nonylphenol seems to affect different targets variously involved in amphibian reproduction.

In the view of an urgent necessity to improve the quantity and the quality of farmed fish species, there is a strong need to improve our basic knowledge on the effects of first feeding during the developmental stages of fish larvae. High mortality, mainly due to food deprivation or inappropriate food quality, has been observed in many larval fish species, but knowledge about the morphological, biochemical and molecular processes related to this topic is still poorly understood. The understanding of the early larval ontogeny as well as the larval nutritional requirements and the molecular and cellular mechanisms elicited by fish larvae during food deprivation and starvation are thus of primary importance. At this regard, this study investigates, in Dover sole larvae, the effects of starvation and starving/re-feeding procedures at a morphological, histological, biochemical and molecular level. The results evidenced that starved larvae progressively decrease in growth, lipid content, affected their gastrointestinal tract and muscle development and increased cortisol and heat shock protein 70 levels. On the contrary, starved and re-fed larvae showed, after the restoration of a favourable feeding condition, a compensatory growth. In conclusion, this is the first study analysing through a multidisciplinary approach the effects of food deprivation on the development of an important economic species, the Dover sole.

The purpose of this study was to develop and evaluate a delivery system comprising a thermosensitive gel for the sustained release of steroidal hormones in fish, over an extended period of time after a single intramuscular (i.m.) injection and for the improved reproductive performance in fish. Controlled delivery systems based on thermosensitive gels are easy to prepare, low cost and high versatility dosage forms, which have been shown to be effective in several animal species for sustained release of hormones. In this work, a thermosensitive gel system based on poloxamer 407 in water:ethanol medium, able to work as a prolonged release carrier for 17β-estradiol (E2), has been developed. Such a system was able to solubilize the lipophilic E2 and to gel at the required water temperature for fish rearing (20 °C). Moreover, the system exhibited the best injection condition at temperatures below 15 °C when the system behaved as a low viscosity Newtonian liquid. The thermosensitive gel system was tested in vivo in the fish model, Carassius auratus, and the results compared with a single i.m. injection of E2 dissolved in corn-oil and other relevant control systems not containing E2. The results were particularly interesting, since fish injected with the E2 thermosensitive gel formulation, showed significantly higher levels of the circulating hormone than corn oil-E2 treated animals at 72 and 96 h after injection. In addition, the thermogel system was able to sustain the plasma level of E2 for about 11 days. The increased plasma levels of E2 were also accompanied by maintained higher values of plasma vitellogenin (VTG), thus suggesting that the thermosensitive polymer based delivery system could prevent rapid hepatic clearance of E2, resulting in prolonged stimulation of estrogen receptor-mediated pathways in goldfish.