E-screen and vitellogenin assay for the detection of the estrogenic activity of alkylphenols and trace elements

The estrogenic potential of 4-nonylphenol (4-NP), 4-octylphenol (4-OP), p-t-octylphenol (p-t-OP) and three trace elements, lead (Pb), copper (Cu) and cadmium (Cd(NO3)2 and CdCl2), were compared in two different tests, a proliferation assay with estrogen receptor-positive human MCF-7 breast cancer cells (E-screen) and the induction of vitellogenin (Vtg) in juvenile goldfish (Carassius auratus). The results showed differences in the bioassays' sensitivity and potency with the following order: E-screen > Vtg. Among alkylphenols, both in vitro and in vivo, 4-NP and 4-OP showed the highest estrogen-like activity while p-t-OP was inferior. For trace elements, Pb and Cu showed estrogenic activity in vitro and they were also active in vivo. A range of estrogenicity was observed for different species of cadmium (Cd(NO3)2 and CdCl2) which showed the highest relative proliferative effect (RPE %) in vitro, when compared with the 17β-estradiol (E2; RPE = 100%) but, Cd(NO3)2 was not estrogenic in vivo. The results suggest that an integrated approach using in vitro and in vivo assays is necessary for a correct risk assessment of the endocrine disrupting activity induced by environmental contaminants. © 2010 Elsevier Inc. All rights reserved

In vitro biomonitoring of environmental estrogens in coastal surface waters of three Italian Marine Protected Areas.

An ecotoxicogenomic study was conducted to assess the estrogenic activity in surface waters of three marine areas in the central Adriatic Sea: the Conero Park, the Torre del Cerrano Marine Protected Area and the Tremiti Islands Marine Protected Area, Italy. Potential estrogenic effect of seawater extracts from C18 cartridges was tested on Sparus aurata hepatocytes using both estrogen receptors (ERs) and vitellogenin (vtg) expression profiles as biomarkers to determine the presence of xenoestrogens. Overall, examination of extracts from 37 different stations showed estrogenic responses. The vtg gene was the most highly expressed gene in hepatocytes exposed to water samples from stations located in inner coastal waters and responded less to those from outer located stations. Vtg gene was expressed nearly 1.5-fold by surface waters from sea areas with intense maritime traffic and increased to a maximum of 3.5-fold in the waters sampled from stations located in front of river mouths. Thus, contamination of marine protected areas by biologically relevant concentrations of xenoestrogens has been observed indicating that the total exposure to anthropogenic estrogens into these environments is higher in nature due to combined presence of unknown estrogen active compounds that are not chemically monitored and with unknown impact. In addition, our study provides further evidence that S. aurata hepatocyte assay for measuring vtg mRNA levels can be used as valuable tool for identifying the presence of environmental estrogens

Combined in silico and in vitro studies of phthalates and organophosphorus compounds: effects on peroxisome proliferator activated receptors (PPARs) signalling pathways

Increasing evidence suggests that some environmental contaminants, including known endocrine disrupting chemicals (EDCs), are able to interfere with metabolic pathways by interacting with nuclear receptors. Recently, it has been demonstrated that phthalates and organometallic compounds bind to peroxisome proliferator activated receptors (PPARs) resulting in modulation of lipid metabolism at both the systemic and peripheral level. However, little is known about the metabolic impact of these pollutants on fish. In this context, we performed an in silico docking screen of an EDCs database to identify a set of ligands with conveniently high affinity for the PPARs. Kd values in the nanomolar to micromolar range, generated by the in silico model, suggest that piscine PPARs may be activated by phthalates (e.g. di-isononylphpthalate -DiNP; Di-isodecyl phthalate-DiDP) and some organophosphorus compounds (e.g. tri-m-cresyl phosphate-TmCP) at concentrations similar to those activating the homologous mammalian receptors. Because natural endogenous ligands for PPARs are involved in lipid homeostasis, we assessed the effects of compounds identified using in silico screening on Sparus aurata hepatocytes primary cultures. Generally, exposure of hepatocytes to 0.1, 1 or 10 microM of DiNP, DiDP or TmCP consistently increased both PPAR and its heterodimeric partner Retinoid X Receptor (RXR) mRNA levels at 48 h. In addition, all compounds investigated produced significant increases in the expression of the PPAR target genes, carnitine palmitoyltransferase (CPT) isoforms. In general our data show that phthalates and TmCP modulated PPAR signaling in the seabream in vitro system. The results also suggest the potential involvement of these pollutants in the modulation of mitochondrial fatty acid oxidation

Development and implementation of a medical record system for the Istituti Clinici di Perfezionamento public network of outpatient specialty clinics in Milano, Italy

The Istituti Clinici di Perfezionamento of Milano (ICP) Hospital, Italy took the charge of managing the network of 22 outpatient specialty clinics of Milano in 2008. Within these clinics 650.000 visits are performed yearly by 255 specialists of 26 different specialties. Until 2008 these clinics have been managed by several different Public Hospitals, there was no unique standard for paper medical records and only a minority of patients encounters were registered electronically. ICP is one Public Hospital of the Lombardia Region, which had developed a Health Information System called SISS. SISS allows all the entitled public health professionals to gain access to individual patient records which contain demographic, clinical and service utilisation data registered with SISS standards. Furthermore, standards for both paper and electronic hospital records have been clearly defined by the Regione Lombardia. In this context ICP started a program for the development and implementation of an outpatient specialty clinics medical record system that took off at the end of 2009. A project team was settled, composed of specialists\u2019 representatives, quality management and organization experts from within the ICP. In six months the group defined: \u2022 The outpatient medical records general structure and format standards \u2022 The structure and content of the medical documentation for 10 main specialties that cover more than 90% the ambulatory visits and more than 75% of the specialists \u2022 The functions needed to integrate patient clinical and administrative management \u2022 The implementation plan of the medical record system, comprehensive of the evaluation of logistic, hardware, medical education and quality control needs within each of the 22 clinics \u2022 The overall medical record system architecture in order to integrate future development with the opportunities offered by the SIS

Endocrine disruptors and nuclear receptors in the control of neural progenitors proliferation

Endocrine-disrupting chemicals (EDCs), in particular plasticizers present in food wrapping materials, are very relevant to human health. Several studies have shown that EDCs may pose the greatest risk during prenatal and early postnatal development, especially when binding to members of the nuclear receptor (NR) superfamily. EDCs can mimic/suppress estrogen actions in the developing brain by binding to estrogen receptors (ERs), and also interfere with neural progenitors (NPs) homeostasis through activation of peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors (RXRs). We used the ST14A immortalized neural progenitor cell line to simulate the effects of prenatal NPs exposure to EDCs. We found by RT-PCR that the following NRs were expressed: ERα, ERβ, PPARα, PPARβ, PPARγ, RXR. Plasticizers were chosen based on their computational affinity for these receptors. NPs were exposed for 24-48 hours to endogenous and synthetic estrogens (17-β-estradiol and ethinyl estradiol) and to the following plasticizers: Bisphenol A, Diisononyl phthalate, Diisodecyl phthalate, Diethileneglicol benzoate. Both estrogens were able to increase cell proliferation by about 30% at 24hrs. Bisphenol-A (10nM) had an estrogen-like behavior in enhancing cell number, but its action was still significant at 48hrs. At 200nM concentration, all plasticizers determined an early increase in proliferation, however only DiDP and DGB were able to sustain this positive modulation for 48hrs. Ongoing studies in our lab will determine the cellular pathways activated by these EDCs in NPs, in order to earn greater insight into how these molecules may influence and perturb the neurogenic process

Endocrine disruptors present in food packages do act as lipid metabolism modulators.

Endocrine-disrupting chemicals (EDCs) may cause several noxious problems to both humans and wildlife and their modulations on energy balance and lipid metabolism are nowadays of great concern. Among environmental EDCs, plasticizers employed in food wrapping materials are very relevant due to their widespread distribution and persistence in the food chain. In the present work we first identify by in silico screening compounds, among the permitted plasticizers for food packaging (EU Regulation, 10/2011), with high predicted affinity for nuclear receptors known to mediate obesogenic effects, such as PPARs, LXR and RXR. The following molecules were therefore chosen for the subsequent studies: the well-known, bisphenol-A, three phthalates (di-isononylphpthalate, DiNP; Di-isodecyl phthalate, DiDP; diethylene glycol dibenzoate, DiGD) and one organophosphorus (tri-m-cresyl phosphate,TmCP).The predicted affinity for the nuclear receptors was then verified in vitro with a luciferase assay: DiNP, DiDP and DiGD were shown to have activity on PPAR α/γ at 10-5-10-7M and to induce the expression of downstream target genes (FABP4, PDK4, FGF21, CPT2). The potential obesogenic effects of the selected compounds were then tested on 3T3-L1 murine preadipocytes cells, a widely used model for adipogenesis in vitro. All the molecules, when used in a range of 10-8-10-6M, were able to significantly enhance lipid droplets deposition, both when administered during the two-days differentiation induction, as well as when maintained for all the eight-days post-differentiation period. Food plastic packages are mostly composed by a mixture of plasticizers so that many molecules at the same time may migrate and contaminate food. We consequently treated 3T3-L1 cells with mixtures of the selected plasticizers (10-8M each), coherently with some commercial plastics (pvc, polypropylene, polyethylene teraphthalate), and we observed in all the cases a significant positive modulation on lipid deposition. The obesogenic effects of the compounds were also investigated by performing qRT-PCR on target genes such as PPARs, LXR, RXR and their downstream effectors. Taken together, our data enforce the emerging awareness on energy balance and lipid metabolism modifications following environmental exposure that could be of concern in vulnerable periods, such as early postnatal life

Effects of dietary nucleotides on acute stress response and cannabinoid receptor 1 mRNAs in sole, Solea solea

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

Dietary Aloe vera components’ effects on cholesterol lowering and estrogenic responses in juvenile goldfish, Carassius auratus

Aloes are now considered a very interesting source of bioactive compounds among which phytosterols should play a major role. The present study is an attempt to investigate the hypocholesterolemic activity of Aloe vera associated with its impact on the reproductive status of juvenile goldfish. Therefore, the short- and long-term effects of feeding supplementary diet containing aloe components (20 mg aloe/g diet; 2 %) on plasma lipids, plasma vitellogenin, and hepatic estrogen receptor a/b1 mRNA levels in goldfish were examined. Results of GC–MS for phytosterols show high abundance of b-sitosterol in freeze-dried powder of Aloe vera whole leaves. Moreover, a 2 % aloe powder dietary supplement was not found estrogenic in juvenile goldfish after either 7- or 30-day treatment, but was consistent in plasma hypocholesterolemic effects following longterm exposure. The present data further support that plasma cholesterol modulation induced by phytosterols may not be related to estrogen-like activity

Plasticizers present in food packaging significantly affect lipid metabolism

Recent data suggest that endocrine-disrupting chemicals (EDCs) may alter lipid metabolism and energy balance. Among environmental EDCs, plasticizers employed in food packaging are of great concern due to their widespread distribution and high persistency in the food chain. We first selected by in silico screening several compounds from a list of plasticizers approved for food packaging (EU Regulation 10/2011) showing high affinity for lipogenic nuclear receptors (PPARs, LXR and RXR). The following plasticizers were chosen: the well-known bisphenol-A, three phthalates (diisononylphpthalate, DiNP; Di-isodecyl phthalate, DiDP; diethylene glycol dibenzoate, DiGD) and one organophosphorus compound (tri-m-cresylphosphate,TmCP).Their binding capacity to nuclear receptors was verified on HEPG2 cells transfected with receptor-luciferase reporter constructs; all phtalates (10-5-10-7 M) were shown to activate PPAR and to induce the expression of downstream target genes (FABP4, PDK4, FGF21, CPT2). The potential obesogenic effects of these compounds were then tested on differentiating 3T3-L1 preadipocyte cells. All molecules (10-8-10-6 M) were able to significantly enhance lipid droplet deposition when administered both in the two-days differentiation induction, or in the eight-days post differentiation period. Food plastic packaging is mostly composed by mixtures of plasticizers, so multiple molecules may migrate simultaneously into food. We therefore treated 3T3-L1 cells with mixtures of plasticizers (10-8 M each), mimicking commercial plastics (pvc, polypropylene, polyethylene teraphthalate); in all cases we observed a significant positive modulation of lipid deposition. The obesogenic effects of all compounds were also investigated at the molecular level by performing qRT-PCR on target genes such as PPARs, LXR, RXR and their downstream effectors. Taken together, our data enforce the emerging awareness on alteration of lipid metabolism following environmental exposure to plasticizers. This effect could be of special concern in vulnerable periods such as pregnancy and early postnatal life