High concentrations of phthalates affect the early development of the sea urchin Paracentrotus lividus.

The toxicity of three phthalates (PAEs) - butylbenzyl phthalate (BBP), diethyl phthalate (DEP), and di-(2-ethylhexyl) phthalate (DEHP) - was tested on the Mediterranean sea urchin Paracentrotus lividus. Fertilized eggs were exposed to environmental and high PAE concentrations for 72 h. The potential toxic effects on larval development and any morphological anomalies were then assessed to estimate PAEs impact. Environmental concentrations never affected development, while high concentrations induced toxic effects in larvae exposed to BBP (EC50: 2.9 ×103 µg/L) and DEHP (EC50: 3.72 ×103 µg/L). High concentrations caused skeletal anomalies, with a slight to moderate impact for DEP/DEHP and BBP, respectively. PAE toxicity was: BBP>DEHP>DEP. In conclusion, the three PAEs at environmental concentrations do not pose a risk to sea urchins. However, PAE concentrations should be further monitored in order not to constitute a concern to marine species, especially at their early developmental stages.

New insights into the impact of leachates from in-field collected plastics on aquatic invertebrates and vertebrates.

The impact of leachates from micronized beached plastics of the Mediterranean Sea and Atlantic Ocean on coastal marine ecosystems was investigated by using a multidisciplinary approach. Chemical analysis and ecotoxicological tests on phylogenetically distant species were performed on leachates from the following plastic categories: bottles, pellets, hard plastic (HP) containers, fishing nets (FN) and rapido trawling rubber (RTR). The bacteria Alivibrio fischeri, the nauplii of the crustaceans Amphibalanus amphitrite and Acartia tonsa, the rotifer Brachionus plicatilis, the embryos of the sea urchin Paracentrotus lividus, the ephyrae of the jellyfish Aurelia sp. and the larvae of the medaka Oryzias latipes were exposed to different concentrations of leachates to evaluate lethal and sub-lethal effects. Thirty-one additives were identified in the plastic leachates; benzophenone, benzyl butyl phthalate and ethylparaben were present in all leachates. Ecotoxicity of leachates varied among plastic categories and areas, being RTR, HP and FN more toxic than plastic bottles and pellets to several marine invertebrates. The ecotoxicological results based on 13 endpoints were elaborated within a quantitative weight of evidence (WOE) model, providing a synthetic hazard index for each data typology, before their integrations in an environmental risk index. The WOE assigned a moderate and slight hazard to organisms exposed to leachates of FN and HP collected in the Mediterranean Sea respectively, and a moderate hazard to leachates of HP from the Atlantic Ocean. No hazard was found for pellet, bottles and RTR. These findings suggest that an integrated approach based on WOE on a large set of bioassays is recommended to get a more reliable assessment of the ecotoxicity of beached-plastic leachates. In addition, the additives leached from FN and HP should be further investigated to reduce high concentrations and additive types that could impact marine ecosystem health.

Nanoplastic uptake temporarily affects the pulsing behavior in ephyrae of the moon jellyfish Aurelia sp.

The aim of this study is to investigate for the first time the uptake and ecotoxicological effects of nanoplastics (NPs) in a marine cnidarian. Ephyrae of the moon jellyfish Aurelia sp. of different ages (0 and 7 days old) were exposed to negatively charged polystyrene NPs for 24 h; then, the uptake was assessed through traditional and novel techniques, namely microscopy and three-dimensional (3D) holotomography. Immobility and behavioral responses (frequency of pulsations) of ephyrae were also investigated to clarify if NP toxicity differed along the first life stages. NP uptake was observed in ephyrae thanks to the 3D technique. Such internalization did not affect survival, but it temporarily impaired the pulsation mode only in 0 day old ephyrae. This may be ascribed to the negative charged NPs, contributing to jellyfish behavioral alteration. These findings promote 3D holotomography as a suitable tool to detect NPs in marine organisms. Moreover, this study recommends the use of cnidarians of different ages to better assess NP ecotoxicological effects in these organisms, key components of the marine food web.

Liver impact of growth hormone (GH) intermittent treatment during the growth period in mice.

Liver impact of prolonged GH-treatment given to non-GH-deficient growing mice between the third and eighth week of life was evaluated in both sexes. Tissues were collected 6 h after last dose or four weeks later. Somatometric, biochemical, histological, immunohistochemical, RT-qPCR and immunoblotting determinations were performed. Five-week GH intermittent administration induced body weight gain and body and bone length increase, augmented organ weight, higher hepatocellular size and proliferation, and increased liver IGF1 gene expression. Phosphorylation of signaling mediators and expression of GH-induced proliferation-related genes was decreased in GH-treated mice liver 6h after last injection, reflecting active sensitization/desensitization cycles. In females, GH elicited EGFR expression, associated to higher EGF-induced STAT3/5 phosphorylation. Four weeks after treatment, increased organ weight concomitant to body weight gain was still observed, whereas hepatocyte enlargement reverted. However, basal signaling for critical mediators was lower in GH-treated animals and in male controls compared to female ones, suggesting signaling declination.

Cold storage effects on lethal and sublethal responses of Amphibalanus amphitrite Nauplii.

Bioassays are extensively used in ecotoxicology and there is a constant need for even more sensitive, reliable and easy to rear and obtain model organisms. Larvae of the crustacean Amphibalanus amphitrite are a good ecotoxicological model, for their high sensitivity to a wide range of toxicants and emerging contaminants. A standardized protocol for this toxicity bioassay has been recently proposed. Nevertheless, a limit of this model organism is the lack of resting stages and the need to use larvae immediately after their release from adults, thus increasing laboratory efforts related to the maintenance of adults. The aim of this work is to verify if short-term cold storage of A. amphitrite larvae prior to use in ecotoxicological tests may affect the ecotoxicological responses of these organisms. Three end-points (mortality, immobilization and swimming speed alteration) were measured on nauplii after storing them at 4 ± 1 °C for different times (24, 72 and 120 h) before bioassay set-up. Bioassays were set up using: (i) clean filtered natural sea water (0.22 µm FNSW), (ii) a reference toxicant (Cadmium Nitrate) and (iii) an environmental matrix (sediment elutriate). Results show that mortality, differently from the other two endpoints, was not affected by cold-storage. Even after 5 days of larvae storage at 4 ± 1 °C before bioassay set up, mortality data were comparable to those obtained for non-cold-stored organisms. Moreover, larval sensitivity to the reference toxicant and sediment elutriate did not change. Regarding the other two end points, low cadmium concentrations significantly changed immobility and swimming activity in cold-stored nauplii compared to larvae used immediately after larval release. In conclusion, short-term cold storage of A. amphitrite nauplii before bioassay set up is an appropriate procedure in ecotoxicological testing if mortality is the endpoint to be considered for final evaluation.

Insulin signaling in the heart is impaired by growth hormone: a direct and early event.

Growth hormone (GH) exerts major actions in cardiac growth and metabolism. Considering the important role of insulin in the heart and the well-established anti-insulin effects of GH, cardiac insulin resistance may play a role in the cardiopathology observed in acromegalic patients. As conditions of prolonged exposure to GH are associated with a concomitant increase of circulating GH, IGF1 and insulin levels, to dissect the direct effects of GH, in this study, we evaluated the activation of insulin signaling in the heart using four different models: (i) transgenic mice overexpressing GH, with chronically elevated GH, IGF1 and insulin circulating levels; (ii) liver IGF1-deficient mice, with chronically elevated GH and insulin but decreased IGF1 circulating levels; (iii) mice treated with GH for a short period of time; (iv) primary culture of rat cardiomyocytes incubated with GH. Despite the differences in the development of cardiomegaly and in the metabolic alterations among the three experimental mouse models analyzed, exposure to GH was consistently associated with a decreased response to acute insulin stimulation in the heart at the receptor level and through the PI3K/AKT pathway. Moreover, a blunted response to insulin stimulation of this signaling pathway was also observed in cultured cardiomyocytes of neonatal rats incubated with GH. Therefore, the key novel finding of this work is that impairment of insulin signaling in the heart is a direct and early event observed as a consequence of exposure to GH, which may play a major role in the development of cardiac pathology.

Synthesis and Antifouling Activity Evaluation of Analogs of Bromosphaerol, a Brominated Diterpene Isolated from the Red Alga Sphaerococcus coronopifolius.

Marine biofouling is an epibiotic biological process that affects almost any kind of submerged surface, causing globally significant economic problems mainly for the shipping industry and aquaculture companies, and its prevention so far has been associated with adverse environmental effects for non-target organisms. Previously, we have identified bromosphaerol (1), a brominated diterpene isolated from the red alga Sphaerococcus coronopifolius, as a promising agent with significant antifouling activity, exerting strong anti-settlement activity against larvae of Amphibalanus (Balanus) amphitrite and very low toxicity. The significant antifouling activity and low toxicity of bromosphaerol (1) motivated us to explore its chemistry, aiming to optimize its antifouling potential through the preparation of a number of analogs. Following different synthetic routes, we successfully synthesized 15 structural analogs (2-16) of bromosphaerol (1), decorated with different functional groups. The anti-settlement activity (EC50) and the degree of toxicity (LC50) of the bromosphaerol derivatives were evaluated using cyprids and nauplii of the cirriped crustacean A. amphitrite as a model organism. Derivatives 2, 4, and 6-16 showed diverse levels of antifouling activity. Among them, compounds 9 and 13 can be considered as well-performing antifoulants, exerting their activity through a non-toxic mechanism.

Ocean warming affected faunal dynamics of benthic invertebrate assemblages across the Toarcian Oceanic Anoxic Event in the Iberian Basin (Spain).

The Toarcian Oceanic Anoxic Event (TOAE; Early Jurassic, ca. 182 Ma ago) represents one of the major environmental disturbances of the Mesozoic and is associated with global warming, widespread anoxia, and a severe perturbation of the global carbon cycle. Warming-related dysoxia-anoxia has long been considered the main cause of elevated marine extinction rates, although extinctions have been recorded also in environments without evidence for deoxygenation. We addressed the role of warming and disturbance of the carbon cycle in an oxygenated habitat in the Iberian Basin, Spain, by correlating high resolution quantitative faunal occurrences of early Toarcian benthic marine invertebrates with geochemical proxy data (δ18O and δ13C). We find that temperature, as derived from the δ18O record of shells, is significantly correlated with taxonomic and functional diversity and ecological composition, whereas we find no evidence to link carbon cycle variations to the faunal patterns. The local faunal assemblages before and after the TOAE are taxonomically and ecologically distinct. Most ecological change occurred at the onset of the TOAE, synchronous with an increase in water temperatures, and involved declines in multiple diversity metrics, abundance, and biomass. The TOAE interval experienced a complete turnover of brachiopods and a predominance of opportunistic species, which underscores the generality of this pattern recorded elsewhere in the western Tethys Ocean. Ecological instability during the TOAE is indicated by distinct fluctuations in diversity and in the relative abundance of individual modes of life. Local recovery to ecologically stable and diverse post-TOAE faunal assemblages occurred rapidly at the end of the TOAE, synchronous with decreasing water temperatures. Because oxygen-depleted conditions prevailed in many other regions during the TOAE, this study demonstrates that multiple mechanisms can be operating simultaneously with different relative contributions in different parts of the ocean.

Exposure to growth hormone is associated with hepatic up-regulation of cPLA2α and COX.

Continuously elevated levels of growth hormone (GH) during life in mice are associated with hepatomegaly due to hepatocytes hypertrophy and hyperplasia, chronic liver inflammation, elevated levels of arachidonic acid (AA) at young ages and liver tumors development at old ages. In this work, the hepatic expression of enzymes involved in AA metabolism, cPLA2α, COX1 and COX2 enzymes, was evaluated in young and old GH-transgenic mice. Mice overexpressing GH exhibited higher hepatic expression of cPLA2α, COX1 and COX2 in comparison to controls at young and old ages and in both sexes. In old mice, when tumoral and non-tumoral tissue were compared, elevated expression of COX2 was observed in tumors. In contrast, exposure to continuous lower levels of hormone for a short period affected COX1 expression only in males. Considering the role of inflammation during liver tumorigenesis, these findings support a role of alterations in AA metabolism in GH-driven liver tumorigenesis.

Temperature-related body size change of marine benthic macroinvertebrates across the Early Toarcian Anoxic Event.

The Toarcian Oceanic Anoxic Event (TOAE, Early Jurassic, ~182 Ma ago) was characterised by severe environmental perturbations which led to habitat degradation and extinction of marine species. Warming-induced anoxia is usually identified as main driver, but because marine life was also affected in oxygenated environments the role of raised temperature and its effects on marine life need to be addressed. Body size is a fundamental characteristic of organisms and is expected to decrease as a response to heat stress. We present quantitative size data of bivalves and brachiopods across the TOAE from oxygenated habitats in the Iberian Basin, integrated with geochemical proxy data (δ13C and δ18O), to investigate the relationship between changes in temperature and body size. We find a strong negative correlation between the mean shell size of bivalve communities and isotope-derived temperature estimates, suggesting heat stress as a main cause of body size reduction. While within-species size changes were minor, we identify changes in the abundance of differently sized species as the dominant mechanism of reduced community shell size during the TOAE. Brachiopods experienced a wholesale turnover across the early warming phase and were replaced by a virtually monotypic assemblage of a smaller-sized, opportunistic species.

Microplastics ingestion in the ephyra stage of Aurelia sp. triggers acute and behavioral responses.

For the first time, we report a correspondence between microplastics (MP) ingestion and ecotoxicological effects in gelatinous zooplankton (Cnidarian jellyfish). The ephyra stage of the jellyfish Aurelia sp. was exposed to both environmental and high concentrations of fluorescent 1-4 µm polyethylene MP (0.01-10 mg/L). After 24 and 48 h, MP accumulation, acute (Immobility) and behavioral (Frequency pulsation) endpoints were investigated. MP were detected by confocal and tomographic investigations on gelatinous body and mouth, either attached on the surface or ingested. This interaction was responsible for impairing ephyrae survival and behavior at all tested concentrations after 24 h. Acute and behavioral effects were also related to mechanical disturbance, caused by MP, triggering a loss of radial symmetry. Contaminated ephyrae exposed to clean seawater showed full recovery after 72 h highlighting the organisms without the microspheres, attached on body jellyfish surface around the mouth and lappets. In conclusion, short-term exposure to MP affects ephyrae jellyfish health, impairing both their survival and behavior. Polyethylene MP temporarily affect both Immobility and Frequency of pulsation of Aurelia sp. jellyfish. This study provides a first step towards understanding and clarifying the potential impacts of MP contamination in gelatinous zooplankton.

Microplastics do not affect standard ecotoxicological endpoints in marine unicellular organisms.

In this study, the acute toxicity of microplastics (MPs) on unicellular organisms as marine decomposers and microalgae was assessed, by evaluating standards endpoints included in International Standard Organization (ISO) protocols. The bacteria Vibrio fischeri and the diatom Phaeodactylum tricornutum were exposed to different sizes (1-500 µm) of polyethylene MPs in order to evaluate bioluminescence inhibition and microalgal growth. No acute toxicity was found on bacteria or microalgae in an order of magnitude above environmentally relevant concentrations, suggesting that tested MPs did not affect the investigated biological processes. In conclusion, standard ecotoxicological endpoints are not sufficiently sensitive to assess the potential effects of MPs on decomposers and primary producers, conversely to nanoplastics. These findings highlight that the current approach for MP risk assessment in unicellular species should be revised, by providing alternative endpoints to be included in standardized protocols, able to monitor the fate and biological effects of MPs.

Growth hormone upregulates the pro-tumorigenic galectin 1 in mouse liver.

Transgenic mice overexpressing growth hormone (GH) spontaneously develop liver tumors, including hepatocellular carcinoma (HCC), within a year. The preneoplastic liver pathology in these mice recapitulates that observed in humans at high risk of developing hepatic cancer. Although increased expression of galectin 1 (GAL1) in liver tissue is associated with HCC aggressiveness, a link between this glycan-binding protein and hormone-related tumor development has not yet been explored. In this study, we investigated GAL1 expression during liver tumor progression in mice continuously exposed to high levels of GH. GAL1 expression was determined by Western blotting, RT-qPCR and immunohistochemistry in the liver of transgenic mice overexpressing GH. Animals of representative ages at different stages of liver pathology were studied. GAL1 expression was upregulated in the liver of GH-transgenic mice. This effect was observed at early ages, when animals displayed no signs of liver disease or minimal histopathological alterations and was also detected in young adults with preneoplastic liver pathology. Remarkably, GAL1 upregulation was sustained during aging and its expression was particularly enhanced in liver tumors. GH also induced hepatic GAL1 expression in mice that were treated with this hormone for a short period. Moreover, GH triggered a rapid increment in GAL1 protein expression in human HCC cells, denoting a direct effect of the hormone on hepatocytes. Therefore, our results indicate that GH upregulates GAL1 expression in mouse liver, which may have critical implications in tumorigenesis. These findings suggest that this lectin could be implicated in hormone-driven liver carcinogenesis.

Assessing real-time signaling and agonist-induced CRHR1 internalization by optical methods.

The development of live-cell sensors for real-time measurement of signaling responses, with improved spatial and temporal resolution with respect to classical biochemical methods, has changed our understanding of cellular signaling. Examination of cAMP generation downstream activated GPCRs has shown that signaling responses can be short-lived (generated from the cell surface) or prolonged after receptor internalization. Class B secretin-like Corticotropin-releasing hormone receptor 1 (CRHR1) is a key player in stress pathophysiology. By monitoring real-time signaling in living cells, we uncovered cell context-dependent temporal characteristics of CRHR1-elicited cAMP responses and disclosed a specific link between cAMP generation and receptor signaling from internal compartments. We describe technical aspects and elaborate the protocols for cell line expression of Förster resonance energy transfer (FRET)-based biosensors to study the dynamics of cAMP and calcium signaling responses downstream activated CRHR1, live-cell imaging and analysis, and fluorescence flow cytometry to determine receptor levels at the cell surface.

A new approach to testing potential leaching toxicity of fouling release coatings (FRCs).

Fouling release coatings (FRCs) are today the main environment-friendly alternative to traditional self-polishing coatings, that continuously release biocides and/or heavy metals into water. FRCs are available on the market as environmentally friendly AF paints and most of them do not contain bioactive agents, however no complete and reliable assessment of their environmental impact has yet been carried out. Only few literature data proving their AF efficacy combined with a demonstrated lack of toxicological effects are available. Ecotoxicological bioassays are commonly used to predict the potential environmental impact of traditional AF paints. Standardized methodologies to obtain leaching products from biocide-based paints are available, while few studies propose experimental methods to assess the potential effects of biocide-free FRCs leachates on non-target organisms. The aim of this work is to propose an experimental protocol to obtain leaching products from biocide-free FRCs in order to evaluate the potential release of substances having toxic effects, by means of an ecotoxicological bioassay. Two ecotoxicological end-points with different sensitivity levels were considered (multi-end-point approach). Five silicone-based commercial coatings were used: their leaching products were collected after different immersion times following the developed experimental method and then two ecotoxicological end-points were evaluated on II stage nauplii of the crustacean Amphibalanus amphitrite as model organism. Moreover, chemical analyses were performed on leachates collected after each immersion time, focusing on the presence of metals in leaching products. From the results obtained from the bioassay, even if not indicative of the real environmental impact of FRCs, a release of toxic substances was observed from tested coatings during early immersion stages, likely to affect the exposed model organism. The potential leaching toxicity of the five tested products was compared. No clear correspondence could be identified between the concentrations of metals detected in leachates and the obtained ecotoxicological data, thus suggesting that other active components might be released by FRCs responsible for the toxic effects pointed out on A. amphitrite larvae.

Ecotoxicological effects of polystyrene microbeads in a battery of marine organisms belonging to different trophic levels.

The aim of this study was to detect ecotoxicological effects of 0.1 µm polystyrene microbeads in marine organisms belonging to different trophic levels. MP build up, lethal and sub-lethal responses were investigated in the bacterium Vibrio anguillarum (culturability), in the green microalga Dunaliella tertiolecta (growth inhibition), in the rotifer Brachionus plicatilis (mortality and swimming speed alteration) and in the sea urchin Paracentrotus lividus (immobility and swimming speed alteration) exposed to a wide range of microplastic (MP) concentrations (from 0.001 to 10 mg L-1). Survival was not affected in all organisms up to 10 mg L-1, while algal growth inhibition, rotifer and sea urchin larvae swimming behaviour alterations were observed after exposure to MPs. Ingestion was only observed in rotifers and it was directly correlated with sub-lethal effects. These results account for the ecotoxicological risk associated to the polystyrene microbeads, which are able to affect different endpoints in primary producers and consumers (rotifers and sea urchins) since no effects were observed in decomposers. This study points out the importance of using a battery of marine organisms belonging to different trophic levels by studying acute toxicity of MPs at low and high contamination levels, and investigating sub-lethal responses. Further investigations aimed at studying the transfer of these materials through the web are particularly recommended.

Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies.

Mas receptor (MasR) is a G protein-coupled receptor proposed as a candidate for mediating the angiotensin (Ang)-converting enzyme 2-Ang (1-7) protective axis of renin-angiotensin system. Because the role of this receptor is not definitively clarified, determination of MasR tissue distribution and expression levels constitutes a critical knowledge to fully understanding its function. Commercially available antibodies have been widely employed for MasR protein localization and quantification, but they have not been adequately validated. In this study, we carried on an exhaustive evaluation of four commercial MasR antibodies, following previously established criteria. Western Blotting (WB) and immunohistochemistry studies starting from hearts and kidneys from wild type (WT) mice revealed that antibodies raised against different MasR domains yielded different patterns of reactivity. Furthermore, staining patterns appeared identical in samples from MasR knockout (MasR-KO) mice. We verified by polymerase chain reaction analysis that the MasR-KO mice used were truly deficient in this receptor as MAS transcripts were undetectable in either heart or kidney from this animal model. In addition, we evaluated the ability of the antibodies to detect the human c-myc-tagged MasR overexpressed in human embryonic kidney cells. Three antibodies were capable of detecting the MasR either by WB or by immunofluorescence, reproducing the patterns obtained with an anti c-myc antibody. In conclusion, although three of the selected antibodies were able to detect MasR protein at high expression levels observed in a transfected cell line, they failed to detect this receptor in mice tissues at physiological expression levels. As a consequence, validated antibodies that can recognize and detect the MasR at physiological levels are still lacking.

Effects of polystyrene microbeads in marine planktonic crustaceans.

Plastic debris accumulates in the marine environment, fragmenting into microplastics (MP), causing concern about their potential toxic effects when ingested by marine organisms. The aim of this study was to verify whether 0.1µm polystyrene beads are likely to trigger lethal and sub-lethal responses in marine planktonic crustaceans. MP build-up, mortality, swimming speed alteration and enzyme activity (cholinesterases, catalase) were investigated in the larval stages of Amphibalanus amphitrite barnacle and of Artemia franciscana brine shrimp exposed to a wide range of MP concentrations (from 0.001 to 10mgL-1) for 24 and 48h. The results show that MP were accumulated in crustaceans, without affecting mortality. Swimming activity was significantly altered in crustaceans exposed to high MP concentrations (> 1mgL-1) after 48h. Enzyme activities were significantly affected in all organisms exposed to all the above MP concentrations, indicating that neurotoxic effects and oxidative stress were induced after MP treatment. These findings provide new insight into sub-lethal MP effects on marine crustaceans.

Analysis of Different Approaches for the Selection of Reference Genes in RT-qPCR Experiments: A Case Study in Skeletal Muscle of Growing Mice.

The reliability of reverse transcription-quantitative PCR (RT-qPCR) results in gene expression studies depends on the approaches used to account for non-biological variations. In order to find a proper normalization strategy for the study of genes related to growth hormone signaling in skeletal muscle of growing mice, nine unrelated genes were evaluated as internal controls. According to the most used algorithms-geNorm, the Comparative ΔCq method, NormFinder and BestKeeper-GSK3B, YWHAZ, RPL13A and RN18S were found as the most stable. However, the relative expression levels of eight of the potential reference genes assessed decreased with age in cDNA samples obtained from the same amount of total RNA. In a different approach to analyze this apparent discrepancy, experiments were performed with cDNA obtained from equal amounts of purified mRNA. Since the decline was still observed, the hypothesis of an age-related change in mRNA to total RNA ratio that could account for the systematic decrease was rejected. Differences among experimental groups could be due to a substantial increase with age in highly expressed mRNAs, which would bias the quantitation of the remaining genes. Consequently, those reference genes reflecting this dilution effect, which would have been discarded considering their variable relative expression levels, arose as suitable internal controls.