Getting fat and stressed: Effects of dietary intake of titanium dioxide nanoparticles in the liver of turbot Scophthalmus maximus.

The extensive use of nanomaterials, including titanium dioxide nanoparticles (TiO2 NPs), raises concerns about their persistence in ecosystems. Protecting aquatic ecosystems and ensuring healthy and safe aquaculture products requires the assessment of the potential impacts of NPs on organisms. Here, we study the effects of a sublethal concentration of citrate-coated TiO2 NPs of two different primary sizes over time in flatfish turbot, Scophthalmus maximus (Linnaeus, 1758). Bioaccumulation, histology and gene expression were assessed in the liver to address morphophysiological responses to citrate-coated TiO2 NPs. Our analyses demonstrated a variable abundance of lipid droplets (LDs) in hepatocytes dependent on TiO2 NPs size, an increase in turbot exposed to smaller TiO2 NPs and a depletion with larger TiO2 NPs. The expression patterns of genes related to oxidative and immune responses and lipid metabolism (nrf2, nfκb1, and cpt1a) were dependent on the presence of TiO2 NPs and time of exposure supporting the variance in hepatic LDs distribution over time with the different NPs. The citrate coating is proposed as the likely catalyst for such effects. Thus, our findings highlight the need to scrutinize the risks associated with exposure to NPs with distinct properties, such as primary size, coatings, and crystalline forms, in aquatic organisms.

Acute Aquatic Toxicity to Zebrafish and Bioaccumulation in Marine Mussels of Antimony Tin Oxide Nanoparticles.

Antimony tin oxide (Sb2O5/SnO2) is effective in the absorption of infrared radiation for applications, such as skylights. As a nanoparticle (NP), it can be incorporated into films or sheets providing infrared radiation attenuation while allowing for a transparent final product. The acute toxicity exerted by commercial Sb2O5/SnO2 (ATO) NPs was studied in adults and embryos of zebrafish (Danio rerio). Our results suggest that these NPs do not induce an acute toxicity in zebrafish, either adults or embryos. However, some sub-lethal parameters were altered: heart rate and spontaneous movements. Finally, the possible bioaccumulation of these NPs in the aquacultured marine mussel Mytilus sp. was studied. A quantitative analysis was performed using single particle inductively coupled plasma mass spectrometry (sp-ICP-MS). The results indicated that, despite being scarce (2.31 × 106 ± 9.05 × 105 NPs/g), there is some accumulation of the ATO NPs in the mussel. In conclusion, commercial ATO NPs seem to be quite innocuous to aquatic organisms; however, the fact that some of the developmental parameters in zebrafish embryos are altered should be considered for further investigation. More in-depth analysis of these NPs transformations in the digestive tract of humans is needed to assess whether their accumulation in mussels presents an actual risk to humans.

Bioaccumulation of titanium dioxide nanoparticles in green (Ulva sp.) and red (Palmaria palmata) seaweed.

A bioaccumulation study in red (Palmaria palmata) and green (Ulva sp.) seaweed has been carried out after exposure to different concentrations of citrate-coated titanium dioxide nanoparticles (5 and 25 nm) for 28 days. The concentration of total titanium and the number and size of accumulated nanoparticles in the seaweeds has been determined throughout the study by inductively coupled plasma mass spectrometry (ICP-MS) and single particle-ICP-MS (SP-ICP-MS), respectively. Ammonia was used as a reaction gas to minimize the effect of the interferences in the 48Ti determination by ICP-MS. Titanium concentrations measured in Ulva sp. were higher than those found in Palmaria palmata for the same exposure conditions. The maximum concentration of titanium (61.96 ± 15.49 µg g-1) was found in Ulva sp. after 28 days of exposure to 1.0 mg L-1 of 5 nm TiO2NPs. The concentration and sizes of TiO2NPs determined by SP-ICP-MS in alkaline seaweed extracts were similar for both seaweeds exposed to 5 and 25 nm TiO2NPs, which indicates that probably the element is accumulated in Ulva sp. mainly as ionic titanium or nanoparticles smaller than the limit of detection in size (27 nm). The implementation of TiO2NPs in Ulva sp. was confirmed by electron microscopy (TEM/STEM) in combination with energy dispersive X-Ray analysis (EDX).

Single-cell ICP-MS for studying the association of inorganic nanoparticles with cell lines derived from aquaculture species.

The current research deals with the use of single-cell inductively coupled plasma-mass spectrometry (scICP-MS) for the assessment of titanium dioxide nanoparticle (TiO2 NP) and silver nanoparticle (Ag NP) associations in cell lines derived from aquaculture species (sea bass, sea bream, and clams). The optimization studies have considered the avoidance of high dissolved background, multi-cell peak coincidence, and possible spectral interferences. Optimum operating conditions were found when using a dwell time of 50 µs for silver and 100 µs for titanium. The assessment of associated TiO2 NPs by scICP-MS required the use of ammonia as a reaction gas (flow rate at 0.75 mL min-1) for interference-free titanium determinations (measurements at an m/z ratio of 131 from the 48Ti(NH)(NH3)4 adduct). The influence of other parameters such as the number of washing cycles and the cell concentration on accurate determinations by scICP-MS was also fully investigated. Cell exposure trials were performed using PVP-Ag NPs (15 and 100 nm, nominal diameter) and citrate-TiO2 NPs (5, 25, and 45 nm, nominal diameter) at nominal concentrations of 10 and 50 µg mL-1 for citrate-TiO2 NPs and 5.0 and 50 µg mL-1 for PVP-Ag NPs. Results have shown that citrate-TiO2 NPs interact with the outer cell membranes, being quite low in the number of citrate-TiO2 NPs that enters the cells (the high degree of aggregation is the main factor which leads to the aggregates being in the extracellular medium). In contrast, PVP-Ag NPs have been found to enter the cells.

Proteomics reveals multiple effects of titanium dioxide and silver nanoparticles in the metabolism of turbot, Scophthalmus maximus.

Titanium dioxide (TiO2) and silver (Ag) NPs are among the most used engineered inorganic nanoparticles (NPs); however, their potential effects to marine demersal fish species, are not fully understood. Therefore, this study aimed to assess the proteomic alterations induced by sub-lethal concentrations citrate-coated 25 nm ("P25") TiO2 or polyvinylpyrrolidone (PVP) coated 15 nm Ag NPs to turbot, Scophthalmus maximus. Juvenile fish were exposed to the NPs through daily feeding for 14 days. The tested concentrations were 0, 0.75 or 1.5 mg of each NPs per kg of fish per day. The determination of NPs, Titanium and Ag levels (sp-ICP-MS/ICP-MS) and histological alterations (Transmission Electron Microscopy) supported proteomic analysis performed in the liver and kidney. Proteomic sample preparation procedure (SP3) was followed by LC-MS/MS. Label-free MS quantification methods were employed to assess differences in protein expression. Functional analysis was performed using STRING web-tool. KEGG Gene Ontology suggested terms were discussed and potential biomarkers of exposure were proposed. Overall, data shows that liver accumulated more elements than kidney, presented more histological alterations (lipid droplets counts and size) and proteomic alterations. The Differentially Expressed Proteins (DEPs) were higher in Ag NPs trial. The functional analysis revealed that both NPs caused enrichment of proteins related to generic processes (metabolic pathways). Ag NPs also affected protein synthesis and nucleic acid transcription, among other processes. Proteins related to thyroid hormone transport (Serpina7) and calcium ion binding (FAT2) were suggested as biomarkers of TiO2 NPs in liver. For Ag NPs, in kidney (and at a lower degree in liver) proteins related with metabolic activity, metabolism of exogenous substances and oxidative stress (e.g.: NADH dehydrogenase and Cytochrome P450) were suggested as potential biomarkers. Data suggests adverse effects in turbot after medium/long-term exposures and the need for additional studies to validate specific biological applications of these NPs.

Toxicity of oleate-based amino protic ionic liquids towards Escherichia coli, Danio rerio embryos and human skin cells.

Protic ionic liquids (PILs) have been widely employed with the label of "green solvents'' in different sectors of technology and industry. The studied PILs are promising for corrosion inhibition and lubrication applications in industry. Industrial use of the PILs can transform them in wastes, due to accidental spill or drag in water due to washing, that can reach water bodies. In addition, the handling of the product by the workers can expose them to accidental contact. Thus, the aim of this work is to evaluate the toxicity of PILs 2-hydroxyethylammonium oleate (2-HEAOl), N-methyl-2-hydroxyethylammonium oleate (m-2HEAOl) and bis-2-hydroxyethylammonium oleate (BHEAOl) towards Escherichia coli, zebrafish embryos, model organisms that can be present in water, and human skin cells. This is the first work reporting toxicity results for these PILs, which constitutes its novelty. Results showed that the studied PILs did not inhibit E. coli bacterial growth but could cause human skin cells death at the concentrations of use. LC50 values for zebrafish eggs were 40.21 mg/L for 2HEAOl, 12.92 mg/L for BHEAOl and 32.74 mg/L for m-2HEAOl, with sublethal effects at lower concentrations, such as hatching retarding, low heart rate and absence of free swimming.

Detection of Silver Nanoparticles in Seawater Using Surface-Enhanced Raman Scattering.

Nanomaterials significantly contribute to the development of new solutions to improve consumer products properties. Silver nanoparticles (AgNPs) are one of the most used, and as human exposure to such NPs increases, there is a growing need for analytical methods to identify and quantify nanoparticles present in the environment. Here we designed a detection strategy for AgNPs in seawater using surface-enhanced Raman Scattering (SERS). Three commercial AgNPs coated with polyvinylpyrrolidone (PVP) were used to determine the relative impact of size (PVP-15nmAgNPs and PVP-100nmAgNPs) and aggregation degree (predefined Ag aggregates, PVP-50-80nmAgNPs) on the SERS-based detection method. The study of colloidal stability and dissolution of selected AgNPs into seawater was carried out by dynamic light scattering and UV-vis spectroscopy. We showed that PVP-15nmAgNPs and PVP-100nmAgNPs remained colloidally stable, while PVP-50-80nmAgNPs formed bigger aggregates. We demonstrated that the SERS-based method developed here have the capacity to detect and quantify single and aggregates of AgNPs in seawater. The size had almost no effect on the detection limit (2.15 ± 1.22 mg/L for PVP-15nmAgNPs vs. 1.51 ± 0.71 mg/L for PVP-100nmAgNPs), while aggregation caused an increase of 2.9-fold (6.08 ± 1.21 mg/L). Our results demonstrate the importance of understanding NPs transformation in seawater since this can influence the detection method performance.

Biocompatibility and Bioimaging Potential of Fruit-Based Carbon Dots.

Photo-luminescent carbon dots (CD) have become promising nanomaterials and their synthesis from natural products has attracted attention by the possibility of making the most of affordable, sustainable and, readily-available carbon sources. Here, we report on the synthesis, characterization and bioimaging potential of CDs produced from diverse extensively produced fruits: kiwi, avocado and pear. The in vitro cytotoxicity and anticancer potential of those CDs were assessed by comparing human epithelial cells from normal adult kidney and colorectal adenocarcinoma cells. In vivo toxicity was evaluated using zebrafish embryos given their peculiar embryogenesis, with transparent embryos developing ex-utero, allowing a real-time analysis. In vitro and in vivo experiments revealed that the synthesized CD presented toxicity only at concentrations of ≥1.5 mg mL-1. Kiwi CD exhibited the highest toxicity to both cells lines and zebrafish embryos, presenting lower LD50 values. Interestingly, despite inducing lower cytotoxicity in normal cells than the other CDs, black pepper CDs resulted in higher toxicity in vivo. The bio-distribution of CD in zebrafish embryos upon uptake was investigated using fluorescence microscopy. We observed a higher accumulation of CD in the eye and yolk sac, avocado CD being the ones more retained, indicating their potential usefulness in bio-imaging applications. This study shows the action of fruit-based CDs from kiwi, avocado and pear. However the compounds present in these fruit-based CDs and their mechanism of action as a bioimaging agent need to be further explored.

Silencing of PPARαBb mRNA in brown trout primary hepatocytes: effects on molecular and morphological targets under the influence of an estrogen and a PPARα agonist.

The crosstalk between peroxisome proliferator-activated receptor α (PPARα) and estrogenic pathways are shared from fish to humans. Salmonid fish had an additional genome duplication, and two PPARα isoforms (PPARαBa and PPARαBb) were previously identified. Since a negative regulation between estrogen signaling and PPARα was described, a post-transcriptional gene silencing for PPARαBb was designed in primary brown trout hepatocytes. The aims of the study were to: (i) decipher the effects of PPARαBb knock-down on peroxisome morphology and on mRNA expression of potential target genes, and (ii) to assess the cross-interferences caused by an estrogenic compound (17α-ethinylestradiol - EE2) and a PPARα agonist (Wy-14,643 - Wy) using the established knock-down model. A knock-down efficiency of 70% was achieved for PPARαBb and its silencing significantly reduced the volume density of peroxisomes, but did not alter mRNA levels of the studied genes. Exposure to Wy did not change peroxisome morphology or mRNA expression, but under silencing conditions Wy rescued the volume density of peroxisomes to control levels, and increased acyl-coenzyme A oxidase 1-3l (Acox1-3l) mRNA. Exposure to EE2 caused a reduction of peroxisome volume density, but under silencing conditions this effect was abolished and ApoA1 mRNA level was diminished. The morphological alterations of peroxisomes by WY and EE2 demonstrated that obtained results are PPARαBb dependent, and suggest the regulation of unknown downstream targets of PPARαBb. In summary, PPARαBb is involved in the control of peroxisome size and/or number, which opens future opportunities to explore its regulation and molecular targets.

Sex-steroids and hypolipidemic chemicals impacts on brown trout lipid and peroxisome signaling - Molecular, biochemical and morphological insights.

Lipid metabolism involves complex pathways, which are regulated in a similar way across vertebrates. Hormonal and hypolipidemic deregulations cause lipid imbalance from fish to humans, but the underlying mechanisms are far from understood. This study explores the potential of using juvenile brown trout to evaluate the in vivo interferences caused by estrogenic (17α-ethinylestradiol - EE2), androgenic (testosterone - T), and hypolipidemic (clofibrate - CLF) compounds in lipidic and/or peroxisomal pathways. Studied endpoints were from blood/plasma biochemistry, plasma fatty acid profile, ultrastructure of hepatocytes and abundance of their peroxisomes to mRNA expression in the liver. Both T and CLF caused minimal effects when compared to EE2. Estrogenized fish had significantly higher hepatosomatic indexes, increased triglycerides and very-low density lipoproteins (VLDL) in plasma, compared with solvent control. Morphologically, EE2 fish showed increased lipid droplets in hepatocytes, and EE2 and T reduced volume density of peroxisomes in relation to the hepatic parenchyma. Polyunsaturated fatty acids (PUFA) in plasma, namely n-3 PUFA, increased with EE2. EE2 animals had increased mRNA levels of vitellogenin A (VtgA), estrogen receptor alpha (ERα), peroxisome proliferator-activated receptor alpha (PPARα), PPARαBa and acyl-CoA long chain synthetase 1 (Acsl1), while ERß-1, acyl-CoA oxidase 1-3I (Acox1-3I), Acox3, PPARγ, catalase (Cat), urate oxidase (Uox), fatty acid binding protein 1 (Fabp1) and apolipoprotein AI (ApoAI) were down-regulated. In summary, in vivo EE2 exposure altered lipid metabolism and peroxisome dynamics in brown trout, namely by changing the mRNA levels of several genes. Our model can be used to study possible organism-level impacts, viz. in gonadogenesis.

Interest of waiting time for spontaneous early reconnection after cavotricuspid isthmus ablation: A monocentric randomized trial.

INTRODUCTION: The aim of this study was to determine the rate of recurrent atrial flutter (AFl) after isolated cavotricuspid isthmus (CTI) ablation and to evaluate the impact of a waiting period with the search for early resumption of the CTI block on the long-term outcome. METHOD: Three hundred and nineteen consecutive patients referred for typical AFl ablation were randomly assigned to CTI ablation with continuous reevaluation of the CTI block during 30 minutes and early reablation if needed (waiting time [WT] + group, n  =  155) or to CTI ablation with no waiting period after proven bidirectional CTI block (WT - group, n  =  164). All patients were regularly followed-up. RESULT: In the WT+ group, 10 patients (6%) presented a recovery across the CTI (time to recovery: 17 ± 7') and were reablated at the end of the waiting period. After a median follow-up of 21 months, the rate of recurrent AFl was significantly higher in the WT - group as compared to the WT+ group (11.6% [19/164] vs 2.5% [4/155], respectively; P  =  0.007). However, no significant differences in the subsequent rate of AF were observed between the two groups (29% [WT -] vs 32% [WT+], P  =  0.66). During the follow-up, 28 patients from the WT - group underwent a second ablation procedure (16 AFl redo and 12 AF ablation) versus 10 patients form the WT+ group (three AFl redo and seven AF ablation). CONCLUSION: Waiting 30 minutes after CTI ablation to check for early resumption and early reablation allows for decreasing significantly the rate of recurrent atrial flutter.

Testosterone-induced modulation of peroxisomal morphology and peroxisome-related gene expression in brown trout (Salmo trutta f. fario) primary hepatocytes.

Disruption of androgenic signaling has been linked to possible cross-modulation with other hormone-mediated pathways. Therefore, our objective was to explore effects caused by testosterone - T (1, 10 and 50µM) in peroxisomal signaling of brown trout hepatocytes. To study the underlying paths involved, several co-exposure conditions were tested, with flutamide - F (anti-androgen) and ICI 182,780 - ICI (anti-estrogen). Molecular and morphological approaches were both evaluated. Peroxisome proliferator-activated receptor alpha (PPARα), catalase and urate oxidase were the selected targets for gene expression analysis. The vitellogenin A gene was also included as a biomarker of estrogenicity. Peroxisome relative volumes were estimated by immunofluorescence, and transmission electron microscopy was used for qualitative morphological control. The single exposures of T caused a significant down-regulation of urate oxidase (10 and 50µM) and a general up-regulation of vitellogenin. A significant reduction of peroxisome relative volumes and smaller peroxisome profiles were observed at 50µM. Co-administration of T and ICI reversed the morphological modifications and vitellogenin levels. The simultaneous exposure of T and F caused a significant and concentration-dependent diminishing in vitellogenin expression. Together, the findings suggest that in the tested model, T acted via both androgen and estrogen receptors to shape the peroxisomal related targets.

Balanço de massa no tratamento de resíduos sólidos orgânicos provenientes de restaurantes em biorreator / Mass balance in the treatment of organic wastes from restaurants in bioreactor

RESUMO O tratamento de resíduos sólidos orgânicos por digestão anaeróbia é realizado por um consórcio de micro-organismos, no qual as archaea metanogênicas são as limitantes do processo, por serem mais sensíveis às mudanças nas condições do meio e possuírem crescimento lento. Para acompanhar a evolução do tratamento, algumas variáveis do processo de digestão anaeróbia são monitoradas, dentre elas, a demanda química de oxigênio (DQO), geralmente utilizada para estimar a matéria degradável e passível de ser convertida em biogás. Com o objetivo de avaliar a eficiência do processo de conversão de biomassa em metano, este artigo se baseou no balanço de massa adaptado da literatura, utilizando valores de DQO e volume de biogás gerado no reator anaeróbio, aqui chamado de biorreator. A produção de biogás foi monitorada diariamente utilizando o método de deslocamento de água, com o auxílio de um contador eletrônico. Com base no balanço de massa, o tratamento mostrou-se viável, visto que 50% da concentração de DQO que entrou no sistema foi convertida em gás metano. Comparando-se aos valores descritos na literatura, que se encontram na faixa de 50 a 70%, a eficiência do tratamento poderá ser elevada com ajustes nos parâmetros de controle que influenciam o processo de digestão anaeróbia, tais como manter a temperatura constante em 37°C e o pH e a alcalinidade equilibrados, o que poderá melhorar as condições do meio em todas as etapas de degradação da matéria orgânica e aumentar a conversão em gás metano.

ABSTRACT The treatment of organic solid waste by anaerobic digestion is carried out by a consortium of microorganisms, in which the methanogenic archaea bacteria are the limiting of the process because they are more sensitive to changes in environmental conditions and have slow growth. To monitor the treatment, some variables of anaerobic digester process are monitored, among them, the chemical oxygen demand (COD), usually used to estimate the degradable material and that can be converted into biogas. In order to evaluate the efficiency of biomass conversion process into methane, this article is based on adapted mass balance of the literature using COD values ​​and volume of biogas generated in anaerobic reactor, here called the bioreactor. Biogas production was monitored daily using the water displacement method, with the aid of an electronic counter.The treatment was satisfactory, based on mass balance, which showed that 50% of the amount of COD entered into the system was completely converted into methane gas. Comparing to the values described in the literature that are in the range of 50 to 70%, the treatment efficiency can be elevated with adjustments to control parameters which influence the process of anaerobic digestion, such as keep the temperature constant at 37°C and balanced pH and alkalinity, which can improve the environmental conditions at all stages of degradation of organic matter and increase conversion into methane gas.

Genome specific PPARαB duplicates in salmonids and insights into estrogenic regulation in brown trout.

Peroxisome proliferator-activated receptors (PPARs) are key regulators of many processes in vertebrates, such as carbohydrate and lipid metabolism. PPARα, a member of the PPAR nuclear receptor gene subfamily (NR1C1), is involved in fatty acid metabolism, namely in peroxisomal ß-oxidation. Two gene paralogues, pparαA and pparαB, were described in several teleost species with their origin dating back to the teleost-specific genome duplication (3R). Given the additional salmonid-specific genome duplication (4R), four genes could be theoretically anticipated for this gene subfamily. In this work, we examined the pparα gene repertoire in brown trout, Salmo trutta f. fario. Data disclosed two pparα-like sequences in brown trout. Phylogenetic analyses further revealed that the isolated genes are most likely genome pparαB duplicates, pparαBa and pparαBb, while pparαA is apparently absent in salmonids. Both genes showed a ubiquitous mRNA expression across a panel of 11 different organs. In vitro exposed primary brown trout hepatocytes strongly suggest that pparα gene paralogues are differently regulated by ethinylestradiol (EE2). PparαBb mRNA expression significantly decreased with dosage, reaching significance after exposure to 50µM EE2, while pparαBa mRNA increased, significant at 1µM EE2. The present data enhances the understanding of pparα function and evolution in teleost, and reinforces the evidence of a potential crosstalk between estrogenic and pparα signaling pathways.

Cross-interference of two model peroxisome proliferators in peroxisomal and estrogenic pathways in brown trout hepatocytes.

Peroxisome proliferators cause species-specific effects, which seem to be primarily transduced by peroxisome proliferator-activated receptor alpha (PPARα). Interestingly, PPARα has a close interrelationship with estrogenic signaling, and this latter has already been promptly activated in brown trout primary hepatocytes. Thus, and further exploring this model, we assess here the reactivity of two PPARα agonists in direct peroxisomal routes and, in parallel the cross-interferences in estrogen receptor (ER) mediated paths. To achieve these goals, three independent in vitro studies were performed using single exposures to clofibrate - CLF (50, 500 and 1000µM), Wy-14,643 - Wy (50 and 150µM), GW6471 - GW (1 and 10µM), and mixtures, including PPARα agonist or antagonist plus an ER agonist or antagonist. Endpoints included gene expression analysis of peroxisome/lipidic related genes (encoding apolipoprotein AI - ApoAI, fatty acid binding protein 1 - Fabp1, catalase - Cat, 17 beta-hydroxysteroid dehydrogenase 4 - 17ß-HSD4, peroxin 11 alpha - Pex11α, PPARαBb, PPARαBa and urate oxidase - Uox) and those encoding estrogenic targets (ERα, ERß-1 and vitellogenin A - VtgA). A quantitative morphological approach by using a pre-validated catalase immunofluorescence technique allowed checking possible changes in peroxisomes. Our results show a low responsiveness of trout hepatocytes to model PPARα agonists in direct target receptor pathways. Additionally, we unveiled interferences in estrogenic signaling caused by Wy, leading to an up-regulation VtgA and ERα at 150µM; these effects seem counteracted with a co-exposure to an ER antagonist. The present data stress the potential of this in vitro model for further exploring the physiological/toxicological implications related with this nuclear receptor cross-regulation.

Peroxisome proliferator-activated receptor gamma (PPARγ) in brown trout: Interference of estrogenic and androgenic inputs in primary hepatocytes.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a pivotal regulator of lipid and glucose metabolism in vertebrates. Here, we isolated and characterized for the first time the PPARγ gene from brown trout (Salmo trutta f. fario). Hormones have been reported to interfere with the regulatory function of PPARγ in various organisms, albeit with little focus on fish. Thus, primary hepatocytes isolated from juveniles of brown trout were exposed to 1, 10 and 50µM of ethinylestradiol (EE2) or testosterone (T). A significant (3 fold) decrease was obtained in response to 50µM of EE2 and to 10 and 50µM of T (13 and 14 folds), while a 3 fold increase was observed at 1µM of EE2. Therefore, trout PPARγ seems a target for natural/synthetic compounds with estrogenic or androgenic properties and so, we advocate considering PPARγ as another alert sensor gene when assessing the effects of sex-steroid endocrine disruptors.

Simplified greywater treatment systems: Slow filters of sand and slate waste followed by granular activated carbon.

One of the main actions of sustainability that is applicable to residential, commercial, and public buildings is the rational use of water that contemplates the reuse of greywater as one of the main options for reducing the consumption of drinking water. Therefore, this research aimed to study the efficiencies of simplified treatments for greywater reuse using slow sand and slow slate waste filtration, both followed by granular activated carbon filters. The system monitoring was conducted over 28 weeks, using analyses of the following parameters: pH, turbidity, apparent color, biochemical oxygen demand (BOD), chemical oxygen demand (COD), surfactants, total coliforms, and thermotolerant coliforms. The system was run at two different filtration rates: 6 and 2 m(3)/m(2)/day. Statistical analyses showed no significant differences in the majority of the results when filtration rate changed from 6 to 2 m(3)/m(2)/day. The average removal efficiencies with regard to the turbidity, apparent color, COD and BOD were 61, 54, 56, and 56%, respectively, for the sand filter, and 66, 61, 60, and 51%, respectively, for the slate waste filter. Both systems showed good efficiencies in removing surfactants, around 70%, while the pH reached values of around 7.80. The average removal efficiencies of the total and thermotolerant coliforms were of 61 and 90%, respectively, for the sand filter, and 67 and 80%, respectively, for the slate waste filter. The statistical analysis found no significant differences between the responses of the two systems, which attest to the fact that the slate waste can be a substitute for sand. The maximum levels of efficiency were high, indicating the potential of the systems, and suggesting their optimization in order to achieve much higher average efficiencies.

Estrogenic and anti-estrogenic influences in cultured brown trout hepatocytes: Focus on the expression of some estrogen and peroxisomal related genes and linked phenotypic anchors.

Estrogens, estrogenic mimics and anti-estrogenic compounds are known to target estrogen receptors (ER) that can modulate other nuclear receptor signaling pathways, such as those controlled by the peroxisome proliferator-activated receptor (PPAR), and alter organelle (inc. peroxisome) morphodynamics. By using primary isolated brown trout (Salmo trutta f. fario) hepatocytes after 72 and 96h of exposure we evaluated some effects in selected molecular targets and in peroxisomal morphological features caused by: (1) an ER agonist (ethinylestradiol-EE2) at 1, 10 and 50µM; (2) an ER antagonist (ICI 182,780) at 10 and 50µM; and (3) mixtures of both (Mix I-10µM EE2 and 50µM ICI; Mix II-1µM EE2 and 10µM ICI and Mix III-1µM EE2 and 50µM ICI). The mRNA levels of the estrogenic targets (ERα, ERß-1 and vitellogenin A-VtgA) and the peroxisome structure/function related genes (catalase, urate oxidase-Uox, 17ß-hydroxysteroid dehydrogenase 4-17ß-HSD4, peroxin 11α-Pex11α and PPARα) were analyzed by real-time polymerase chain reaction (RT-PCR). Stereology combined with catalase immunofluorescence revealed a significant reduction in peroxisome volume densities at 50µM of EE2 exposure. Concomitantly, at the same concentration, electron microscopy showed smaller peroxisome profiles, exacerbated proliferation of rough endoplasmic reticulum, and a generalized cytoplasmic vacuolization of hepatocytes. Catalase and Uox mRNA levels decreased in all estrogenic stimuli conditions. VtgA and ERα mRNA increased after all EE2 treatments, while ERß-1 had an inverse pattern. The EE2 action was reversed by ICI 182,780 in a concentration-dependent manner, for VtgA, ERα and Uox. Overall, our data show the great value of primary brown trout hepatocytes to study the effects of estrogenic/anti-estrogenic inputs in peroxisome kinetics and in ER and PPARα signaling, backing the still open hypothesis of crosstalk interactions between these pathways and calling for more mechanistic experiments.