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.

Dysfunction of the Brown Adipose Organ in HFD-Obese Rats and Effect of Tart Cherry Supplementation.

Obesity has a great impact on adipose tissue biology, based on its function as a master regulator of energy balance. Brown adipose tissue (BAT) undergoes remodeling, and its activity declines in obese subjects due to a whitening process. The anti-obesity properties of fruit extracts have been reported. The effects of tart cherry against oxidative stress, inflammation, and the whitening process in the BAT of obese rats were investigated. Intrascapular BAT (iBAT) alterations and effects of Prunus cerasus L. were debated in rats fed for 17 weeks with a high-fat diet (DIO), in DIO supplemented with seed powder (DS), and with seed powder plus the juice (DJS) of tart cherry compared to CHOW rats fed with a normo-caloric diet. iBAT histologic observations revealed a whitening process in DIO rats that was reduced in the DS and DJS groups. A modulation of uncoupling protein-1 (UCP-1) protein and gene expression specifically were detected in the obese phenotype. An upregulation of UCP-1 and related thermogenic genes after tart cherry intake was detected compared to the DIO group. Metabolic adjustment, endoplasmic reticulum stress, protein carbonylation, and the inflammatory microenvironment in the iBAT were reported in DIO rats. The analysis demonstrated an iBAT modulation that tart cherry promoted. In addition to our previous results, these data confirm the protective impact of tart cherry consumption on obesity.

Effects of Chronic Sleep Restriction on Transcriptional Sirtuin 1 Signaling Regulation in Male Mice White Adipose Tissue.

Chronic sleep restriction (CSR) is a prevalent issue in modern society that is associated with several pathological states, ranging from neuropsychiatric to metabolic diseases. Despite its known impact on metabolism, the specific effects of CSR on the molecular mechanisms involved in maintaining metabolic homeostasis at the level of white adipose tissue (WAT) remain poorly understood. Therefore, this study aimed to investigate the influence of CSR on sirtuin 1 (SIRT1) and the peroxisome proliferator-activated receptor γ (PPARγ) signaling pathway in the WAT of young male mice. Both genes interact with specific targets involved in multiple metabolic processes, including adipocyte differentiation, browning, and lipid metabolism. The quantitative PCR (qPCR) results demonstrated a significant upregulation of SIRT-1 and some of its target genes associated with the transcriptional regulation of lipid homeostasis (i.e., PPARα, PPARγ, PGC-1α, and SREBF) and adipose tissue development (i.e., leptin, adiponectin) in CSR mice. On the contrary, DNA-binding transcription factors (i.e., CEBP-ß and C-myc), which play a pivotal function during the adipogenesis process, were found to be down-regulated. Our results also suggest that the induction of SIRT1-dependent molecular pathways prevents weight gain. Overall, these findings offer new, valuable insights into the molecular adaptations of WAT to CSR, in order to support increased energy demand due to sleep loss.

Multiple Aspects of the Fight against the Red Palm Weevil in an Urban Area: Study Case, San Benedetto del Tronto (Central Italy).

The fight against alien invasive insect pests of plants in the urban environment often affects varied sectors of the economy, landscape gardening, public health, and ecology. This paper focuses on the evolution of the red palm weevil in San Benedetto del Tronto, a coastal urban area in central Italy. We investigated the evolution of this insect pest of palm trees in the 2013-2020 period, considering both the effectiveness of the chemicals used and their potentially harmful effects. With a multidisciplinary approach, we carried out a spatio-temporal analysis of 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. We also assessed the toxicity risk associated with the chemicals used to protect the palms from the red weevil. The fight against this weevil is now concentrated in specific areas such as parks, roads, villas, hotels, farmhouses, and nurseries. The preventive chemical treatments applied 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 several aspects involved in the fight against this beetle in an urban area.

Endurance-dependent urinary extracellular vesicle signature: shape, metabolic miRNAs, and purine content distinguish triathletes from inactive people.

Extracellular vesicles (EVs) enriched with bioactive molecules have gained considerable attention in nanotechnology because they are critical to intercellular communication while maintaining low immunological impact. Among biological matrices, urine has emerged as a noninvasive source of extracellular-contained liquid biopsy, currently of interest as a readout for physiological adaptations. Therefore, we aimed to evaluate chronic adaptations of endurance sport practice in terms of urinary EV parameters and evaluated by food consumption assessment. Two balanced groups of 13 inactive controls vs. triathlon athletes were enrolled; their urinary EVs were obtained by differential ultracentrifugation and analyzed by dynamic light scattering and transmission electron and atomic force microscopy. The cargo was analyzed by means of purine and miRNA content through HPLC-UV and qRT-PCR. Specific urinary EV signatures differentiated inactive versus endurance-trained in terms of peculiar shape. Particularly, a spheroid shape, smaller size, and lower roughness characterize EVs from triathletes. Metabolic and regulatory miRNAs often associated with skeletal muscle (i.e., miR378a-5p, miR27a-3p, miR133a, and miR206) also accounted for a differential signature. These miRNAs and guanosine in urinary EVs can be used as a readout for metabolic status along with the shape and roughness of EVs, novel informative parameters that are rarely considered. The network models allow scholars to entangle nutritional and exercise factors related to EVs' miRNA and purine content to depict metabolic signatures. All in all, multiplex biophysical and molecular analyses of urinary EVs may serve as promising prospects for research in exercise physiology.

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.

Assessment of seasonal relationship between polycyclic aromatic hydrocarbon accumulation and expression patterns of oxidative stress-related genes in muscle tissues of red mullet (M. barbatus) from the Northern Adriatic Sea.

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.

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.

Seasonal Expression of NGF and Its Cognate Receptors in the Ovaries of Grey Squirrels (Sciurus carolinensis).

The grey squirrel is an invasive alien species that seriously threatens the conservation of the native red squirrel species. With the aim of characterizing the reproductive physiology of this species due to its great reproductive success, the function of the ovarian nerve growth factor (NGF) system was analyzed in a grey squirrel population living in central Italy. During the breeding and nonbreeding seasons, the ovarian presence, distribution, and gene expression of NGF, neurotrophic tyrosine kinase receptor 1 (NTRK1), and nerve growth factor receptor (NGFR), as well as NGF plasma concentrations, were evaluated in female grey squirrels. NGF was found in the luteal cells and in the thecal and granulosa cells of follicles, while NTRK1 and NGFR were only observed in follicular thecal and granulosa cells. NGF and NGFR transcripts were almost two-fold greater during the breeding season, while no seasonal differences were observed in NTRK1 gene expression. During the breeding season, NGFR was more expressed than NTRK1. Moreover, no changes were observed in NGF plasma levels during the reproductive cycle. The NGF system seems to be involved in regulating the ovarian cycle mainly via local modulation of NGF/NGFR, thus playing a role in the reproductive physiology of this grey squirrel population.

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 (

Effect of chelating and antioxidant agents on morphology and DNA methylation in freeze-drying rabbit (Oryctolagus cuniculus) spermatozoa.

Freeze-drying (FD) has been exhaustively tried in several mammalian species as an alternative technique to sperm cryopreservation, but few studies have been done in rabbits (Oryctolagus cuniculus). The main objective of this study was to compare the protective effect of various antioxidants added to EDTA medium on structural and functional components of FD rabbit spermatozoa and on their status of global DNA methylation. FD media used were composed of basic FD medium (10 mM Tris-HCl buffer and 50 mM NaCl) supplemented with either 50 mM EDTA alone (EDTA) or added with 105 µM of rosmarinic acid (RA, EDTA-RA) or 10 µM of melatonin (MLT, EDTA-MLT). The effect of each medium on the preservation of FD spermatozoon structure was evaluated with light and scanning electron microscopy (SEM). Global DNA methylation was quantified in all FD sperm samples as well as in fresh spermatozoa. Morphologically, fracture points were evidenced in the neck, mid and principal piece of the spermatozoon tail. No differences in spermatozoon fracture points were evidenced among FD treatments: intact spermatozoa were the largest (p < .01) category, whereas the most frequent (p < .01) injury was the neck fracture, resulting in tailless heads. At SEM, the head of spermatozoa showed a well-conserved shape and intact membrane in all treatments. DNA methylation status was the same in all FD treatments. In conclusion, supplementation of EDTA, EDTA-RA and EDTA-MLT during FD preserved rabbit sperm morphological integrity and methylation status as well. Therefore, the difficulty of getting viable offspring using FD semen is likely unrelated to the impact of the lyophilization process on DNA methylation and morphology of lyophilized spermatozoa.

Apelin system detection in the reproductive apparatus of ewes grazing on semi-natural pasture.

Apelin (APLN) is an adipokine with pleiotropic effects involved in the regulation of metabolic, cardiovascular, immune, and electrolyte balance function. Recent studies demonstrated a pivotal role in the regulation of male and female reproduction. APLN and its receptor (APLNR) were found in the hypothalamic-pituitary-gonad axis tissues, regulating gonadotropin release and steroidogenesis. However, to date, there are no studies that describe APLN system in the reproductive apparatus of the sheep. The study was performed on 10 Comisana x Appenninica adult dry ewes reared in a semi-natural pasture. Organ samples were collected from five animals in the two pasture functional phases: after maximum pasture flowering (Group 1) and after maximum pasture dryness (Group 2). Experiments were devised to characterize the gene expression and protein localization of the APLN/APLNR system in ewe reproductive apparatus; in addition, the concentration of plasma APLN was evaluated during the trial. Through immunohistochemical analysis, a positive staining for APLN was observed in the large luteal cells, in the epithelial cell coat of the ampulla, in the uterus epithelial lining and in the uterine glands. APLNR was observed in the granulosa cells, in the large luteal cells, in the secreting cells of the ampulla, in the uterus epithelial lining and uterine glands. The transcripts for APLN and APLNR were evidenced in all organ tissues examined. The highest level of APLN mRNA was detected in the Group 2 ewes in the luteal phase of the ovarian cycle compared to Group 1 ewes in the anestrous one. The relative content of APLN transcript was respectively twofold higher in the ovary (P < 0.05) and uterus (P < 0.05) and threefold higher in the ampulla (P < 0.05) in the Group 2 vs Group 1. The same trend of APLN transcript was evaluated for APLNR mRNA in uterus (P < 0.05) and ovary (P < 0.05). No difference was evidenced between Group 1 and Group 2 for APLNR mRNA levels. The plasma APLN level was fairly constant during the trial period. In conclusion, the present data suggest that the apelinergic system is involved in the reproduction function of ewes, being differentially distributed and expressed in the organs of the reproductive apparatus of ewes; these variations could be related to the sexual cycle and to the cyclic activity of the reproductive apparatus.

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.