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The toxicity of mycotoxins is well recognized in mammals, but their effects on fish have received less attention. Moreover, in the last years several studies have reported that some mycotoxins may act as endocrine disruptors. The aim of this study was to determine the cytotoxic effects and endocrine activities of three mycotoxins: beauvericin, deoxynivalenol and ochratoxin-A. Cytotoxicity in two fish hepatoma and one mammalian hepatoma cell lines was determined by the AlamarBlue, Neutral Red Uptake and CFDA-AM assays. For the assessment of androgenic, estrogenic and thyroidal agonistic/antagonistic effects three cell lines stably expressing luciferase as reporter gene under the control of hormone receptors were used. Results showed that both fish and mammalian cell lines were very sensitive to the mycotoxins tested. OTA was the least toxic mycotoxin and DON and BEA showed similar acute toxicity. None of the three mycotoxins tested presented agonistic effects at the receptors studied, but all of them showed strong antagonistic effect at the thyroid receptor. BEA showed a weak antagonistic effect at the androgen receptor and OTA produced a biphasic dose-response curve at the estrogen receptor. The data obtained in this work are of high interest for aquaculture industries and for regulators.

The occurrence of bioactive compounds and contaminant-associated effects was assessed by means of in vivo and in vitro assays using different extractable fractions of surface sediments from a contaminated coastal lagoon (Mar Menor, SE Spain). Sediment elutriates and clean seawater, previously exposed to whole sediment, were used for assessing the in vivo toxicity on embryo development of the sea urchin Paracentrotus lividus. Agonist and antagonist activities relating to estrogen and androgen receptors and agonist activities on aryl hydrocarbon receptor (expressed as ethoxyresorufin-O-deethylase (EROD) activities) were investigated in sediment extracts by using HER-Luc, AR-EcoScreenTM and fibroblast-like RTG-2 cell lines. Embryotoxicity effects were greater for sediment elutriates than those incubated in sediment-water interphase, implying that diffusion of bioactive chemicals can occur from sediments to sea water column, favoured by sediment disturbance events. In vitro results show the occurrence in extracts of compounds with estrogen antagonism, androgen antagonism and dioxin-like activities. Multidimensional scaling analysis classified the sampling sites into four sub-clusters according to their chemical-physical and biological similarities, relating in vitro bioactivity with the total organic carbon and known organic chemical load, with particular reference to total sum of PAHs, PCB 180, p,p-DDE and terbuthylazine. Overall, results pointed to the presence of unknown or unanalyzed biologically-active compounds in the sediments, mostly associated with the extracted polar fraction of the Mar Menor lagoon sediments. Our findings provide relevant information to be considered for the environmental management of contaminated coastal lagoons., SI

The use of manure as an agricultural amendment is increasing the release of steroid hormones into the environment. Most research in this field has focused on estrogenic phenomena, with less attention paid to androgenic substances. The present study assessed androgenic activity in broiler manure using in vitro approaches based on cells stably transfected with androgen receptor. Leaching experiments were also performed to observe whether endocrine disruptors present in manure pass through a soil column and potentially reach groundwater. In parallel, an analytical chemistry method was used to determine the contribution of the most important natural androgens to androgenicity. Samplings were performed at 4 farms in 2 seasons. All but 2 samples showed androgen activity. In leakage experiments, however, no androgenic activity was detectable in leachates or in soils after leaching. According to the analytical results, androgenicity can be attributed mainly (but not completely) to androstenedione, and dihydrotestosterone. Similarly to the bioassays, chemical analysis did not reveal the presence of any androgen in leachates or soils. These results point to a rapid degradation of the substances responsible for androgenic activity in soils under the experimental conditions of the present study. However, the long-term effects associated with the constant and intensive application of manure to agricultural land require further attention. Environ Toxicol Chem 2017;36:1746-1754. © 2016 SETAC.

In mammals, numerous reports describe an immunomodulating effect of thyroid-active compounds. In contrast, only few reports have been published on this subject in fish. We previously demonstrated that immune cells of rainbow trout (Oncorhynchus mykiss) possess thyroid hormone receptors (THRs) and that exposure of trout to the thyroid hormone 3,3',5-triiodo-. l-thyronine (T3) or the antithyroid drug propylthiouracil (PTU) alters immune cell transcript levels of THR and several immune genes. The present study aims to further characterize the immunomodulating action of thyroid-active compounds in trout immune cells. We report here the use of a custom-designed 60-mer oligo immune-targeted microarray for rainbow trout to analyze the gene expression profiles induced in the head kidney by T3 and PTU. Morphometric analyses of the thyroid showed that PTU exposure increased the size of the epithelial cells, whereas T3 induced no significant effects. Both T3 and PTU had diverse and partly contrasting effects on immune transcript profiles. The strongest differential effects of T3 and PTU on gene expressions were those targeting the Mitogen Associated Protein Kinase (MAPK), NFkB, Natural Killer (NK) and Toll-Like Receptor (TLR) pathways, a number of multipath genes (MPG) such as those encoding pleiotropic transcription factors (atf1, junb, myc), as well as important pro-inflammatory genes (tnfa, tnf6, il1b) and interferon-related genes (ifng, irf10). With these results we show for the first time in a fish species that the in vivo thyroidal status modulates a diversity of immune genes and pathways. This knowledge provides the basis to investigate both mechanisms and consequences of thyroid hormone- and thyroid disruptor-mediated immunomodulation for the immunocompetence of fish. © 2016 Elsevier B.V.

The toxic effects produced by the co-exposure to low- and non-toxic concentrations of zinc oxide (ZnONPs) and copper nanoparticles (CuNPs) was assessed in rainbow trout following the OECD Test Guideline 203. Four groups of trouts were exposed for 96 h to a range of concentrations (0.0425–0.34 mg/L) of CuNPs (50 nm) in combination with a fixed non-toxic concentration (1.25 mg/L) of ZnONPs (25 nm) determined from an independent concentration-response study. One additional group was exposed to the highest concentration of CuNPs alone. Behaviour and mortality were observed during the experiment. After 96 h exposure, accumulated levels of Cu and Zn in the fish were measured by ICP-MS and ICP-OES, respectively. The induction of oxidative stress in liver and gills was evaluated by the glutathione-S-transferase (GST) activity and the reduced glutathione (GSH) / oxidized glutathione (GSSG) ratio. The ethoxyresorufin-O-deethylase (EROD) activity was also assessed. The results showed that CuNPs at the highest tested concentration do not cause acute toxicity, whereas exposure to all mixtures caused mortality, which was inversely proportional to the concentration of CuNPs (from 28% to 86% survival). Accumulated levels of Cu and Zn in the fish increased with the increasing concentrations of CuNPs, suggesting that the presence of CuNPs favours the entry of Zn. In general, the GST activity increased significantly in the gills of co-exposed groups, whereas the GSH/GSSG ratio was altered in the liver. The EROD activity was not modified. In conclusion, the co-exposure to these NPs potentiates their toxicity, observing an alteration of the GST activity and GSH/GSSG ratio in gill and liver, which was more pronounced at the lowest concentration of CuNPs. The lower toxic effect observed with the highest concentrations of CuNPs coincides with a greater internalization of Zn.

The current wide use of manufactured nanomaterials (MNs) is leading to the release of nanoparticles (NPs) to water bodies. Aquatic organisms, including fish, are exposed to low concentrations of NPs for long periods of time being necessary to develop laboratory toxicity tests reflecting realistic conditions. Additionally, today there is a demand of in vitro assays respecting the 3Rs principle. Thus, the main aim of this work was to stablish an in vitro tool for the assessment of long-term NPs ecotoxicity. Considering the key role of liver in detoxification, a rainbow trout liver cell line, RTL-W1, was used. CuO NPs were chosen to validate this tool taking into account their important production level. Cells were exposed for 21 days to 25 or 100 μg CuO NPs/ml. Every seven days cells were split and one fourth of them transferred to a new plate with appropriate concentrations of NPs in culture medium. Lower concentrations of CuO NPs did not cause any deleterious effect, whereas higher concentrations led to significant mortality after 14 days and to the intracellular accumulation of Cu particles. Identical results were observed in cells exposed to CuSO4 at the same Cu concentrations. Therefore, the observed toxic effects might be mainly due to Cu2+ ions.

EROD and BFCOD activities were measured in liver and gills of barbel (Barbus callensis, a native North African species) captured at Beni Haroun lake, the most important water reservoir in Algeria. This lake receives wastewater from different origins. Thus, we assessed the level of pollution through the induction of detoxification activities in tissues of barbel, evaluating simultaneously the suitability of this species to be used as a sentinel. Fish were collected between March 2015 and January 2016 at three locations taking into account the pollution sources and accessibility. In liver, EROD and BFCOD showed the highest induction in October specially in the location of the dam that received pollutants. In gills, only EROD, but not BFCOD, activity was detected. Maximal EROD induction was noted in samples from January. Fish cell lines (RTG-2 and PLHC-1) were exposed to sediments extracts collected at Beni Haroun lake and enzyme activities (EROD and BFCOD, respectively) were measured. Sediment extracts did not induce BFCOD activity. The EROD induction observed in RTG-2 cells was in line with the results observed in fish tissues. Our results suggest that the lake is at risk from pollution and that Barbus callensis is a good sentinel species. © 2017 Elsevier Inc.

An ecotoxicological survey of soils that were polluted with wastes from lindane (γ-HCH) production assessed the effects of organochlorine compounds on the metabolism of microbial communities and the toxicity of these compounds to a native earthworm (Allolobophora chlorotica). Furthermore, the bioremediation role of earthworms as facilitators of soil washing and the microbial degradation of these organic pollutants were also studied. Soil samples that presented the highest concentrations of ε-HCH, 2,4,6-trichlorophenol, pentachlorobenzene and γ-HCH were extremely toxic to earthworms in the short term, causing the death of almost half of the population. In addition, these soils inhibited the heterotrophic metabolic activity of the microbial community. These highly polluted samples also presented substances that were able to activate cellular detoxification mechanisms (measured as EROD and BFCOD activities), as well as compounds that were able to cause endocrine disruption. A few days of earthworm activity increased the extractability of HCH isomers (e.g.;γ-HCH), facilitating the biodegradation of organochlorine compounds and reducing the intensity of endocrine disruption in soils that had low or medium contamination levels. © 2017 Elsevier Inc.

Made available in DSpace on 2018-12-11T16:45:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) European Regional Development Fund Chemical substances with potential to disrupt endocrine systems have been detected in aquatic environments worldwide, making necessary the investigation about water treatments able to inhibit such potential. The present work aimed to assess the efficiency for removing endocrine disruptors (with estrogenic and androgenic activity) of three simple and inexpensive substrates that could be potentially used in sectors or regions with limited resources: powdered activated carbon (PAC), powdered natural zeolite (ZEO) (both at a concentration of 500�mg�L−1) and natural aquatic humic substances (AHS) (at 30�mg�L−1). MilliQ-water and mature water from fish facilities (aquarium water, AW), were artificially spiked with 17β-estradiol (E2), 17α-ethinylestradiol and dihydrotestosterone. Moreover, effluent samples from waste water treatment plants (WWTP) were also submitted to the remediation treatments. Estrogenic and androgenic activities were assessed with two cell lines permanently transfected with luciferase as reporter gene under the control of hormone receptors: AR-EcoScreen containing the human androgen receptor and HER-LUC transfected with the sea bass estrogen receptor. PAC was efficiently removing the estrogenic and androgenic compounds added to milliQ and AW. However, androgenic activity detected in WWTP effluents was only reduced after treatment with ZEO. The higher surface area of PAC could have facilitated the removal of spiked hormones in clean waters. However, it is possible that the substances responsible of the hormonal activity in WWTP have adsorbed to micro and nanoparticles present in suspension that would have been retained with higher efficiency by ZEO that show pores of several microns in size. Department of Environment Spanish National Institute for Agricultural and Food Research and Technology (INIA), Ctra. De la Coru�a Km 7.5 S�o Paulo State University “J�lio de Mesquita Filho” (UNESP), Sorocaba Campus, Avenida Tr�s de Mar�o, 511, Bairro: Alto da Boa Vista S�o Paulo State University “J�lio de Mesquita Filho” (UNESP), Sorocaba Campus, Avenida Tr�s de Mar�o, 511, Bairro: Alto da Boa Vista FAPESP: 2012/14583-5 European Regional Development Fund: RTA2012-00053-00-00

We used small-interference RNA (siRNA) to explore the mechanisms of some vasoactive intestinal peptide (VIP) actions on human breast cancer cells. Transfection of estrogen-dependent (T47D) and estrogen-independent (MDA-MB-468) breast cancer cells with VPAC(1)-receptor siRNA completely abolished VIP stimulatory effect on secretion of the main angiogenic factor, vascular endothelial growth factor (VEGF), and transactivation of epidermal growth factor receptor (EGFR or HER1) and HER2, two members of HER family of tyrosine-kinase receptors. The silencing procedure suggested the involvement of EGFR and HER2 transactivation in VIP-stimulated VEGF secretion. It was further supported by blocking tyrosine kinase activity by the selective HER inhibitors AG-1478 (EGFR) and AG-825 (HER2). Results give value to the specific signaling of VIP through VPAC(1) receptor in human breast cancer cells and support the potential use of VPAC(1)-receptor antagonists in combined targeted therapies for breast cancer. Molecular therapies involving RNA interference of VPAC(1)-receptor expression could also be considered.

Vasoactive intestinal peptide (VIP) and its receptors (VPACs) are involved in proliferation, survival, and differentiation in human breast cancer cells. Its mechanism of action is traditionally thought to be through specific plasma membrane receptors. There is compelling evidence for a novel intracrine mode of genomic regulation by G-protein-coupled receptors (GPCRs) that implies both endocytosis and nuclear translocation of peripheral GPCR and/or the activation of nuclear-located GPCRs by endogenously-produced, non-secreted ligands. Regarding to VPAC receptors, which are GPCRs, there is only a report suggesting them as a dynamic system for signaling from plasma membrane and nuclear membrane complex. In this study, we show that VPAC(1) receptor is localized in cell nuclear fraction whereas VPAC(2) receptor presents an extranuclear localization and its protein expression is lower than that of VPAC(1) receptor in human breast tissue samples. Both receptors as well as VIP are overexpressed in breast cancer as compared to non-tumor tissue. Moreover, we report the markedly nuclear localization of VPAC(1) receptors in estrogen-dependent (T47D) and independent (MDA-MB-468) human breast cancer cell lines. VPAC(1) receptors are functional in plasma membrane and nucleus as shown by VIP stimulation of cAMP production in both cell lines. In addition, VIP increases its own intracellular and extracellular levels, and could be involved in the regulation of VPAC(1)-receptor traffic from the plasma membrane to the nucleus. These results support new concepts on function and regulation of nuclear GPCRs which could have an impact on development of new therapeutic drugs.

The use of manure as an agricultural amendment is increasing the release of steroid hormones into the environment. Most research in this field has focused on estrogenic phenomena, with less attention paid to androgenic substances. The present study assessed androgenic activity in broiler manure using in vitro approaches based on cells stably transfected with androgen receptor. Leaching experiments were also performed to observe whether endocrine disruptors present in manure pass through a soil column and potentially reach groundwater. In parallel, an analytical chemistry method was used to determine the contribution of the most important natural androgens to androgenicity. Samplings were performed at 4 farms in 2 seasons. All but 2 samples showed androgen activity. In leakage experiments, however, no androgenic activity was detectable in leachates or in soils after leaching. According to the analytical results, androgenicity can be attributed mainly (but not completely) to androstenedione, and dihydrotestosterone. Similarly to the bioassays, chemical analysis did not reveal the presence of any androgen in leachates or soils. These results point to a rapid degradation of the substances responsible for androgenic activity in soils under the experimental conditions of the present study. However, the long-term effects associated with the constant and intensive application of manure to agricultural land require further attention. Environ Toxicol Chem 2017;36:1746-1754. © 2016 SETAC.

Previous studies have shown that vasoactive intestinal peptide (VIP) and its receptors (VPAC(1) and VPAC(2) receptors) are involved in promotion and growth of many human tumours including breast cancer. Here we investigated whether VIP regulates the expression of the main angiogenic factor, vascular endothelial cell growth factor (VEGF) in human oestrogen-dependent (T47D) and oestrogen-independent (MDA-MB-4687) breast cancer cells. Semiquantitative and quantitative real-time RT-PCRs were used at mRNA level whereas enzyme immunoanalysis was performed at protein level. Both cancer cell lines expressed VIP and VPAC(1) (but not VPAC(2)) receptors that were functional as shown by VIP stimulation of adenylate cyclase activity. VIP induced VEGF expression at both mRNA and protein levels following a time-dependent pattern. The responses were faster in T47D than in MDA-MB-468 cells. The observed VIP regulation of VEGF expression appears to be modulated at least by the cAMP/protein kinase A (PKA) and the phosphoinositide 3-kinase (PI3-K) signalling systems as shown by studies of adenylate cyclase stimulation and using specific kinase inhibitors such as H89 and wortmannin. These actions suggest a proangiogenic potential of VIP in breast cancer.

Aquatic organisms are often exposed to mixtures of low levels of pollutants whose presence and effects can pass easily unnoticed if only traditional monitoring strategies are employed. The main aim of this work was to assess the presence and effects of trace levels of pollutants in a scarcely affected area through the combination of chemical and biological approaches. Sediments were collected along a river with little anthropogenic pressure and assayed for cytochrome P450 (Cyp1a)-dependent ethoxyresorufin-O-deethylase (EROD) activity with the rainbow trout gonadal cell line RTG-2. Chemical analyses were performed in these sediments using two-dimensional gas chromatography-time-of-flight mass spectrometry. Sediment samples induced EROD activity, and chemical analyses evidenced the presence of a wide variety of contaminants in the range of nanograms per gram of dry weight. Correlation analysis between EROD induction and chemical analyses data showed an r value of 0.840 (p < 0.05). In addition, fish from a fish farm located downstream of the sampling points exhibited high hepatic EROD levels as well as an induced expression of cyp1a and cyp3a. In conclusion, only an appropriate combination of biological and chemical techniques allowed the detection of the presence of trace levels of contaminants in a theoretically nonaffected river. © Springer Science+Business Media 2015.

Aflatoxin B1 (AFB1) and fumonisin B1 (FB1) are mycotoxins widely found as cereal contaminants and their co-occurrence in corn has been associated with a high incidence of liver cancer. Both toxins are immunotoxic, with AFB1 being a procarcinogen, and its bioactivation through specific cytochrome P450 (Cyp) enzymes, such as Cyp1A, being a requirement for hepatocarcinogenic and toxic activities. This study evaluated the effects of these mycotoxins, alone or combined, on activation and expression of Cyp1A and its transcription factor aryl hydrocarbon receptor (Ahr) in hepatoma cell line H4IIE and spleen mononuclear cells of rats. The results demonstrate that in H4IIE cells, AFB1 induced an increase in Cyp1A activity and cyp1A transcription, associated with an enhanced Ahr activity, which suggests that this toxin can act as an Ahr agonist. Moreover, FB1 caused a small rise in Cyp1A activity and cyp1A expression. Similarly in spleen cells, AFB1 and FB1 induced overexpression of cyp1A and ahr genes. This work shows that the response potency was significantly higher for the mixture, indicating the existence of an interaction between both toxins. This study proposes the Ahr pathway activation as a toxicity mechanism of AFB1 and FB1, and highlights that FB1 may increase AFB1 bioactivation. Fil: Mary, Verónica Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Valdehita, Ana. Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria; España Fil: Navas, José M.. Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria; España Fil: Rubinstein, Héctor Ramón. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Fernández-Cruz, María L.. Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria; España

Aflatoxin B1 (AFB1) and fumonisin B1 (FB1) are mycotoxins widely found as cereal contaminants and their co-occurrence in corn has been associated with a high incidence of liver cancer. Both toxins are immunotoxic, with AFB1 being a procarcinogen, and its bioactivation through specific cytochrome P450 (Cyp) enzymes, such as Cyp1A, being a requirement for hepatocarcinogenic and toxic activities. This study evaluated the effects of these mycotoxins, alone or combined, on activation and expression of Cyp1A and its transcription factor aryl hydrocarbon receptor (Ahr) in hepatoma cell line H4IIE and spleen mononuclear cells of rats. The results demonstrate that in H4IIE cells, AFB1 induced an increase in Cyp1A activity and cyp1A transcription, associated with an enhanced Ahr activity, which suggests that this toxin can act as an Ahr agonist. Moreover, FB1 caused a small rise in Cyp1A activity and cyp1A expression. Similarly in spleen cells, AFB1 and FB1 induced overexpression of cyp1A and ahr genes. This work shows that the response potency was significantly higher for the mixture, indicating the existence of an interaction between both toxins. This study proposes the Ahr pathway activation as a toxicity mechanism of AFB1 and FB1, and highlights that FB1 may increase AFB1 bioactivation.

Microparticles are produced by vesiculation of the cell plasma membrane and serve as vectors of cell-to-cell communication. Co-culture experiments have shown that hypoxia-inducible factor-α (HIF-α)-regulated-genes are up-regulated in human renal proximal tubular HK-2 cells by endothelial cell factors which might be transported inside endothelial microparticles (EMP). Here we aimed to study in HK-2 cells the effect of EMP, produced by activated endothelial cells, on HIF-α and HIF-α-regulated vascular endothelial growth factor-A (VEGF-A). EMP, at a concentration much lower than that found in plasma, increased the expression of HIF-α/VEGF-A in a COX-2/EP2 receptor dependent manner. Since the EMP/cells ratio was ∼1/1000, we hypothesized that paracrine mediators produced by HK-2 cells amplified the initial signal. This hypothesis was confirmed by two facts which also suggested that the mediators were conveyed by particles released by HK-2 cells: (i) HIF-α was up-regulated in HK-2 cells treated with the pellet obtained from the conditioned medium of the EMP-treated HK-2 cells. (ii) In transwell experiments, EMP-treated cells increased the expression of HIF-α in untreated HK-2 cells. Interestingly, we detected these cells, particles that were released by EMP-treated HK-2 cells. Depending on the pathological context, activation of HIF-α and VEGF-A signaling in renal tissue/cells may have either beneficial or harmful effects. Therefore, our results suggest that their presence in the urinary space of EMP produced by activated endothelial cells may influence the outcome of a number of renal diseases.

Among the many chemicals found in avian manure, endocrine disruptors (EDs), of natural or anthropogenic origin, are of special environmental concern. Nowadays, an increasing amount of estrogens is being released into the environment via the use of manure to fertilize agricultural land. While most research in this field has focused on estrogenic phenomena, little is known about alterations related to other endocrine systems, such as the thyroidal one. Here we simultaneously assessed the potential estrogenic and thyroidal activity of poultry and broiler litter manure using in vitro approaches based on estrogen receptor (Er) and thyroid receptor (Tr) transactivation assays. In addition, leaching experiments were performed to assess whether the EDs present in the manure pass through a soil column and potentially reach the groundwater. Manure from four broiler and four poultry farms was collected in two sampling campaigns carried out in two seasons (fall and spring). Extracts from broiler and poultry manure exhibited strong thyroidal activity. Only poultry manure showed estrogenic activity, which is consistent with the low levels of estrogens expected in hatchlings. Leakage experiments were performed in columns with two kinds of arable soils sandy and loamy. No estrogenicity or thyroidal activity was detectable in soils treated with the manure or in the corresponding leachates. These results indicate that substances with estrogenic or thyroidal activity were degraded in the soil under our experimental conditions. However, the long-term effects associated with the constant and intensive application of manure to agricultural land in some regions require further research. © 2013 Elsevier B.V.

Thyroid hormones are involved in modulating the immune system in mammals. In contrast, there is no information on the role played by these hormones in the immune system of teleost fish. Here we provide initial evidence for the presence of active thyroid signaling in immune organs and cells of teleosts. We demonstrate that immune organs (head kidney and spleen) and isolated leukocytes (from head kidney and peripheral blood) of the rainbow trout ( Oncorhynchus mykiss) express both thyroid receptor α (THRA) and β (THRB). Absolute mRNA levels of THRA were significantly higher than those of THRB. THRA showed higher expression in immune organs and isolated immune cells compared to the reference organ, liver, while THRB showed the opposite. Invivo exposure of trout to triiodothryronine (T3) or the anti-thyroid agent propylthiouracil (PTU) altered THR expression in immune organs and cells. Effect of T3 and PTU over the relative expression of selected marker genes of immune cell subpopulations was also studied. Treatments changed the relative expression of markers of cytotoxic, helper and total T cells ( cd4, cd8a, trb), B lymphocytes ( mIgM) and macrophages ( csf1r). These findings suggest that the immune system of rainbow trout is responsive to thyroid hormones. © 2014 Elsevier Ltd.

Here we addressed the possible effects of trace levels of contaminants on fish by means of a combination of biomarker responses, active biomonitoring (ABM), and chemical analysis. In environmental studies, cytochromes P4501A (Cyp1A) and Cyp3A and related enzyme activities (7-ethoxyresorufin-. O-deethylase, EROD, and benzyloxy-4-[trifluoromethyl]-coumarin-. O-debenzyloxylase, BFCOD, respectively) are commonly used as biomarkers for evidencing exposure to a variety of contaminants. In a rainbow trout (Oncorhynchus mykiss) fish farm that is routinely sampled to obtain references regarding normal levels of such enzyme activities in freshwater fish, we observed a strong and punctual increase in these activities at the end of 2011. In order to shed light on the causes of this induction, we transferred some fish to a fish farm with controlled conditions and examined them using an active biomonitoring (ABM) approach. EROD activity showed a decrease of 80% from the original values after 7 days in the control farm, while BFCOD activity was also reduced after 15 days. Although not significant, a decrease in cyp1A and cyp3A mRNA levels was also observed. To determine the presence of pollutants, water and sediment samples from the river feeding the fish farm were analyzed by two-dimensional gas chromatography-time-of-flight mass spectrometry (GC. ×. GC-TOF-MS). The screening study reflected a weak inflow of pollutants in the monitored area, which is located far from any industrial activity or densely populated cities. Trace levels of polyaromatic hydrocarbons (PAHs) and personal care products (the polycyclic musk fragrance HHCB, and triclosan) were detected in sediments, at concentrations ranging from 0.01 to 38. ng/g dry weight, and in water from 4 to 441. ng/L. The approach followed in this study proved useful as a biomonitoring technique for the early detection of trace contaminants. © 2013 Elsevier B.V.

The widely used herbicide atrazine (ATZ) has been reported to exhibit reproductive toxicity in rats, fish and amphibians, with an avian LD 50 of 5000 mg/kg. In the present work, ATZ was administered as a single oral dose of 25 or 100 mg/kg to female European quail (Coturnix coturnix coturnix) at days 0, 5 and 10 of the experiment, being the animals sampled at days 15, 30 and 45. ATZ significantly increased the expression of hepatic estrogen receptor α (ERα) at both doses at day 30. An important increase was also observed in plasma 17β-estradiol (E2) concentrations. ATZ at 100 mg/kg increased the circulating concentration of vitellogenin (Vtg), but this effect was not related with an increase in hepatic Vtg mRNA levels. ATZ had no effect on the hepatic expression of both cytochrome P450 1A4 (CYP1A4) or the related biotransformation activity ethoxyresorufin-O-deethylase (EROD). These results led to the conclusion that ATZ provokes an estrogenic effect in sexually mature females of European quail. Further studies are necessary to establish the effect on sexual development or reproduction of female and male birds in the wild. © 2012 Elsevier Inc.

9 páginas, 6 figuras, 1 tabla.-- et al., Normal functioning of the endocrine system is essential for the proper development and reproduction of animals. Substances interfering with its homeostasis are called endocrine disruptors (EDs) and may represent a risk for the health of the organism. One of the mechanisms of endocrine disruption that has attracted great attention in recent years concerns alterations in the normal functioning of the estrogen receptor (ER), but far less attention has been paid to those substances interfering with the thyroid axis, which, in fish, plays several critical roles in a variety of biological functions. In aquaculture, feedstuffs can be a source of hormones or persistent pollutants which act as potential EDs. In this study, the main purpose was to assess the possible estrogenic and thyrogenic activities of 32 commercial fish feeds. For the assessment of estrogenicity, a new estrogen receptor specific reporter gene assay using sea bass ERα (sbER α) was developed and validated. Potential thyroidal disruption was screened with a cell line permanently transfected with luciferase as reporter gene under the control of avian thyroid receptor α (THRα). The results obtained showed that 11 and 18 out of 32 assayed feeds were able to activate the sbERα or the avTHRα1, respectively. The present study is pioneer in demonstrating thyrogenic activity in fish diets commercially available and widely used in aquaculture. Given that maintaining the homeostasis in the endocrine system is critical for the proper development and reproduction of fish, any estrogenic or thyrogenic activity caused by the feedstuffs should be taken into account with regards to its potential impact on farmed fish. © 2012 Elsevier B.V., This study was funded by projects RTA 2009-00074-00-00 (MICINN) managed by the INIA, AGL 2010-22247-C03-01 and CSD 2007-00002 managed by IATS-CSIC from the Spanish Ministry of Science and Innovation.

Two rainbow trout Oncorhynchus mykiss fish farms were repeatedly sampled in order to observe the variability of ethoxyresorufin-O-deethylase (EROD) activity and of related genes in the liver. Fish coming from fish farm A exhibited EROD levels that could be considered as basal according to the scientific literature, however, EROD activity in fish coming from fish farm B was significantly increased. This was accompanied by augmented aryl hydrocarbon receptor (ahr) and cytochrome P4501A (cyp1A) messenger RNA expression and reduced oestrogen receptor (er) and vitellogenin (vtg) transcription. Only sediment extracts from the entry channel of fish farm B induced EROD activity in O. mykiss cultured cells, however, this induction could not be explained by the levels of polyaromatic hydrocarbons (PAH) and polychlorinated biphenyls (PCB) measured in the sediments. The results of this study point out that O. mykiss cultured in fish farms could be used as sentinels for indication of pollution. In this particular work, however, no conclusive evidence has been found for a relationship between the presence of PAHs and PCBs and the observed EROD induction. © 2012 The Authors. Journal of Fish Biology © 2012 The Fisheries Society of the British Isles.

We used an in vivo model of human experimental prostate cancer in order to shed a new light on the effects of vasoactive intestinal peptide (VIP) on tumor growth as well as its pro-metastatic potential in this disease. We used nude mice subcutaneously injected with prostate cancer androgen-independent PC3 cells for 30 days. The regulatory role of VIP on cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) expression as well as on matrix metalloproteinase-2 and 9 (MMP-2 and 9) activities was examined. A selective COX-2 inhibitor, NS-398, and curcumin were used to block VIP effects. Xenografts of VIP-treated PC3 prostate cancer cells in nude mice gave tumors that grew significantly faster than those in the untreated group. It is conceivably a result of both the trophic effect of VIP on prostate cancer cells and the proangiogenic action of the neuropeptide in the growing tumor. We show the overexpression at mRNA and/or protein levels of VIP, its main receptor VPAC 1 , the major angiogenic factor VEGF, and the pro-inflammatory enzyme COX-2 as well as the increased activity of MMP-2 and 9 in tumors derived from VIP-treated PC3 cells as compared with control group. The overexpression of the above biomarkers was suppressed in tumors derived from VIP-treated PC3 cells that had been previously incubated with curcumin or NS-398. Thus, the potential therapeutic role of curcumin and selective COX-2 inhibitors in combination with available VIP antagonists should be considered in prostate cancer therapy as supported by their inhibitory activities on tumor cell growth.

We analyzed the cross-talk between receptors for vasoactive intestinal peptide (VIP) and the human epidermal growth factor family of tyrosine kinase receptors (HER) in oestrogen-dependent (T47D) and oestrogen-independent (MDA-MB-468) human breast cancer cells. VIP treatment slowly increased the expression levels of EGFR but it rapidly augmented phosphorylation of EGFR and HER2 in both cell lines. This pattern of HERs transactivation was blocked by the specific VIP antagonist JV-1-53, supporting the direct involvement of VIP receptors in formation of P-EGFR and P-HER2. VIP-induced transactivation was also abolished by H89 (protein kinase A inhibitor), PP2 (Src inhibitor) or TAPI-1 (inhibitor of matrix metalloproteases), following a differential pattern. These results shed a new light on the specific signalling pathways involved in EGFR/HER2 transactivation by VPAC receptors and suggest the potential usefulness of VIP receptor antagonists together with current antibodies against EGFR/HER2 and/or tyrosine kinase inhibitors for breast cancer therapy.

In order to study the variability of detoxification activities associated with the cytochromes P450 1A (CYP1A) and 3A (CYP3A), several continental fish farms of Oncorhynchus mykiss were routinely sampled. In fish, CYP1A plays a key role in the biotransformation of awide variety of pollutants. This cytochrome is induced after ligand-activation of the aryl hydrocarbon receptor. CYP3A is also implicated in themetabolismof a wide variety of xenobiotics, including pharmaceuticals however its activity is mediated by the pregnane-X-receptor. CYP1A and CYP3A inductions have been widely used as biomarkers of environmental pollution in aquatic systems, in particular when measured at the enzymatic level through ethoxyresorufin-O-deethylase (EROD) and benzyloxy-4-[trifluoromethyl]-coumarin-O-debenzyloxylase (BFCOD) activities, respectively. In one of the monitored fish farms, EROD activity, which initially showed basal levels, exhibited a sharp increase difficult to explain by seasonal variability. In order to confirm the increased induction of the cytochrome activities, 32 animals were transferred to a control farm and maintained for one month. Periodic samplings of eight animals at days 0, 7, 15 and 30 were performed and both EROD and BFCOD activities in the livers measured. Under these controlled conditions, enzymatic activities associated with detoxification processes gradually decreased to basal levels indicating thepresence of some factor in the fish farm inducing the cytochromes. In order to investigate possible causes of such induction both water and sediment samples, along the river feeding the fish farm, were taken and analysed byaGCxGC-TOFMS system. This systemprovedveryuseful and allowed us to identify non-target compounds in some samples, among them personal care, pharmaceutical products, and industrial chemicals (i.e. PAHs, phthalates, alkylphenolic compounds) that couldbe related to the observed induction.