Alberta Mandich

Estrogenicity profile and estrogenic compounds determined in river sediments by chemical analysis, ELISA and yeast assays

An effects-directed strategy was applied to bed sediments of a polluted tributary in order to isolate and identify the major estrogenic chemicals it discharges into the River Po, the principal Italian watercourse. Sediment extract was concentrated by solid phase extraction and then fractioned into 10 fractions by reversed phase high performance liquid chromatography (RP-HPLC). Estrogenic activity of whole extract and fractions were determined using a recombinant yeast assay containing the human estrogen receptor (YES). The 10 fractions and whole extract were analysed for target compounds, e.g. estrone (E1), 17beta-estradiol (E2), estriol (E3), 4-nonylphenol (NP), 4-tert-octylphenol (t-OP), bisphenol A (BPA), using both liquid chromatography-tandem mass spectrometry (LC-MS/MS) and non-competitive enzyme-linked immunosorbent assays (ELISA). The YES assay determined high estrogenic activity in whole sediment (15.6 ng/g EE2 equivalents), and positive results for fractions nr 1, 2, 6, 7 and 8. E1, E3 and NP were the main estrogenic chemicals, however, other unidentified compounds contributed to sediment estrogenicity, particularly for polar fractions nr 1 and 2. A GC-MS screening performed in scan mode identified other potential contributors such as phthalates (DBP, BBP), and OP isomers. A next sampling campaign extended to other tributaries and receiving stretches of the River Po confirmed E1, E3 and NP as major estrogenic chemicals potentially threatening other sites of the main river. In general, target compound ELISAs have been shown to be suitable tools for a rapid screening of wide areas or large numbers of environmental samples for estrogenic risk. The potential for interferences suggests however to use cautiously the concentration values obtained from some of the immunoassays.

Xenobiotic-contaminated diets affect hepatic lipid metabolism: Implications for liver steatosis in Sparus aurata juveniles

The metabolic effects induced by feed contaminated with a lower or a higher concentration of -nonylpnenol (NP), 4-tert-octylphenol (t-OP) or bisphenol A (BPA), three environmental endocrine disruptors, were assessed in juvenile sea bream liver. Histological analysis demonstrated that all these three xenobiotics induced hepatic lipid accumulation and steatosis. These findings prompted analysis of the expression of the major molecules involved in lipid metabolism: peroxisome proliferator activated receptors (which is encoded by ppars), fatty acid synthase (encoded by fas), lipoprotein lipase (encoded by lpl) and hormone-sensitive lipase (encoded by hsl). The enzymes encoded by ppars and fas are in fact responsible for lipid accumulation, whereas lpl- and hsl- encoded proteins play a pivotal role in fat mobilization. The three xenobiotics modulated ppar mRNA expression: pparα mRNA expression was induced by the higher dose of each contaminant; pparβ mRNA expression was upregulated by the lower doses and in BPA2 fish ppary mRNA overexpression was induced by all pollutants. These data agreed with the lipid accumulation profiles documented by histology. Fas mRNA levels were modulated by the two NP doses and the higher BPA concentration. Lpl mRNA was significantly upregulated in all experimental groups except for BPA1 fish while hsl mRNA was significantly downregulated in all groups except for t-OP2 and BPA1 fish. The plasma concentrations of cortisol, the primary stress biomarker, were correlated with the levels of pepck mRNA level. This gene encodes phosphoenolpyruvate carboxykinase which is one of the key enzymes of gluconeogenesis. Pepck mRNA was significantly overexpressed in fish exposed to NP2 and both t-OP doses. Finally, the genes encoding cyclooxygenase 2 (cox2) and 5-lipoxygenase (5 lox), the products of which are involved in the inflammatory response, transcriptions were significantly upregulated in NP and BPA fish, whereas they were unchanged in t-OP specimens. The present findings suggest that dietary xenobiotic contamination can give rise to metabolic disorders also in fish and highlight the potential for their vertical transfer through the trophic levels and ultimately to humans.

Expression of stress proteins 70 in tilapia (Oreochromis mossambicus) during confinement and crowding stress

The knowledge of fish biology of stress response to unfavourable environmental conditions is of interest for both basic and applied purpose, providing useful information for fish farming and environmental risk assessment. In this study, tilapias (Oreochromis mossambicus), confined in nets inside the tanks for 4 or 5 h, were examined for induction of Hsp 70 in various organs by western blotting and for cortisol levels in the blood. In net‐confined fish, cortisol, primary stress indicator, was found to consistently increase with differences between 4 and 5 h of confinement. Hsp 70 expression, evaluated in striated and cardiac muscle, gill, liver and brain showed fluctuations in confined animals. Indeed, increase or decrease of Hsp 70 expression in stressed fish was modified in organ‐ and stress duration‐dependent manner and subjective fluctuations were minimal. Morphological observations (LM and EM) were carried out on liver, brain and gill from control and stressed animals. In particular, the liver underwent major modifications while the brain was only slightly modified and the gills were unmodified during confinement stress. Taken together, our data suggest that Hsp 70 expression could be an indicator of stress state in fish when measured in the significant responsive organs.

A developmental hepatotoxicity study of dietary bisphenol A in Sparus aurata juveniles

Previous studies in rats have indicated that a diet enriched with Bisphenol A adversely effects metabolism and reproductive success. In rats exposed to BPA by maternal gavage, alteration in the developmental programming, higher obesity rates and reproductive anomalies were induced. Starting with this evidence, the aim of this study was to provide important insights on the effects induced by a BPA enriched diet, on the reproductive physiology and metabolism of juvenile fish, simulating the scenario occurring when wild fish fed on prey contaminated with environmental BPA. Seabream was chosen as model, as it is one of the primary commercial species valued by consumers and these results could provide important findings on adverse effects that could be passed on to humans by eating contaminated fish. A novel method for measuring BPA in the food and water by affinity chromatography was developed. Analysis of signals involved in reproduction uncovered altered levels of vtg and Zp, clearly indicating the estrogenic effect of BPA. Similarly, BPA up-regulated catd and era gene expression. A noteworthy outcome from this study was the full length cloning of two vtg encoding proteins, namely vtgA and vtgB, which are differently modulated by BPA. Cyp1a1 and EROD activity were significantly downregulated, confirming the ability of estrogenic compounds to inhibit the detoxification process. GST activity was unaffected by BPA contamination, while CAT activity was down regulated. These results collectively confirm the estrogenic effect of BPA and provide additional characterization of novel vtg genes in Sparus aurata.

Dietary administration of EDC mixtures: A focus on fish lipid metabolism

Many man-made chemical compounds are recognized as endocrine disruptors and once released into the environment are likely to spread and bioaccumulate in wild species. Due to their lipophilic nature, these substances pass through the cell membrane or bind to specific receptors activating physiological responses that in the long run can cause reproductive impairment, physiological disorders, including the occurrence of metabolic syndromes. One significant source of contamination is represented by the consumption of polluted food. As a consequence, different environmental pollutants, with similar or different modes of action, can accumulate in organisms and biomagnify along the food web, finally targeting humans. The aim of this study was to analyze, under controlled conditions, the effects induced by the consumption of contaminated diets, focusing on the effects exerted at hepatic level. Juvenile seabream were fed for 21days a diet enriched with different combinations of pollutants, nonylphenol (NP), tert-octylphenol (t-OP) and bisphenol A (BPA). The different diets containing 5mg/kg bw of each contaminant, were formulated as follows: NP+tOP, BPA+NP, BPA+tOP and NP+BPA+tOP (NBO). EDCs, at the doses administered, showed low biomagnification factor (BMF), suggesting that these pollutants hardly accumulate in muscles. The results obtained at hepatic level pinpointed the steatotic effect of all the administered diets, associated to a modulation of the expression of genes involved in lipid metabolism (ppars, fas, lpl, and hsl). Results were compared to those obtained in previous studies in which fish were fed single pollutants evidencing that the administration of mixture of contaminants exerts a milder lipogenic effect, highlighting the contrasting/antagonistic interaction establishing among chemicals. Noteworthy was the setup of a new chromatographic method to detect the presence of the selected chemical in fish muscle and the application of Fourier Transform Infrared (FT-IR) analysis to evaluate pollutant-induced changes in the liver macromolecular building.

Alkylphenolic contaminants in the diet: Sparus aurata juveniles hepatic response

A wide range of endocrine disrupter chemicals can mimic steroid hormones causing adverse health effects. Nonylphenol (NP) and t-octhylphenol (t-OP) are man-made alkylphenolic environmental contaminants possessing controversial endocrine disruption properties. This study has investigated the effects of NP and t-OP enriched diets on hepatic tissue and biotransformation activities in the liver. To this aim, sea bream juveniles were fed with commercial diet enriched with three different doses of NP (NP1: 5mg/kg bw, NP2: 50mg/kg bw and NP3: 100mg/kg bw) or t-OP (t-OP1: 5mg/kg bw, t-OP2: 50mg/kg bw and t-OP3: 100mg/kg bw) for 21 days. A significant increase of the hepatosomatic index was observed in NP1 and t-OP1. Alteration of liver morphology was observed in both NP and t-OP exposed juveniles although the most altered endpoints were observed in t-OP2 with 100% of tissue degeneration. Ethoxyresorufin-O-deethylase activity was significantly inhibited by NP and t-OP (p<0.05), while catalase activity was significantly induced, at both doses. A different pattern of protein expression of different isoforms of both vitellogenin and zona radiata protein was evidenced within the treatments. In addition, a significant increase in the abundance of the stress induced heat shock protein 70 gene in the liver of t-OP2 fish and a significant increase in the abundance of the estrogen induced cathepsin D gene in the liver of NP1 and t-OP2 fish, were observed. Finally, estradiol-17β (E2) and testosterone (T) plasma levels and E2/T showed significantly different patterns in NP and t-OP exposed against control fish.

Plasma aldosterone levels in the female tortoise Testudo hermanni Gmelin in different experimental conditions.

Abstract Plasma aldosterone concentration has been measured by radioimmunoassay in a tortoise species Testudo hermanni Gmelin. Control levels were very low, 8.26 ± 0.2 ng/100 ml, if compared with the determination carried out in other reptilian species with the same RIA method. A sodium-loaded diet for a prolonged period (30 days) depressed aldosterone levels but administration of a diuretic produced a significant elevation. Plasma aldosterone, sodium, and potassium concentrations were significantly correlated. Administration of a mammalian inhibitor of aldosterone (spironolactone) was not followed by a significant plasma aldosterone or electrolyte changes. These data are consistent in supporting the hypothesis that aldosterone is involved in hydromineral regulation, at least in terrestrial chelonians, with a direct action on the renal proximal tubule.

Exposing native cyprinid (Barbus plebejus) juveniles to river sediments leads to gonadal alterations, genotoxic effects and thyroid disruption

Juveniles (50 days post hatch) of a native cyprinid fish (Barbus plebejus) were exposed for 7 months to sediments from the River Lambro, a polluted tributary impairing the quality of the River Po for tens of kilometers from their confluence. Sediments were collected upstream of the city of Milan and downstream at the closure of the drainage basin of the River Lambro. Chemical analyses revealed the presence of a complex mixture of bioavailable endocrine-active chemicals, with higher exposure levels in the downstream section of the tributary. Mainly characterized by brominated flame retardants, alkylphenols, polychlorinated biphenyls, and minor co-occurring personal care products and natural hormones, the sediment contamination induced reproductive disorders, as well as other forms of endocrine disruption and toxicity. In particular, exposed male barbel exhibited higher biliary PAH-like metabolites, overexpression of the cyp1a gene, vitellogenin production in all specimens, the presence of oocytes (up to 22% intersex), degenerative alterations in their testis, liver fat vacuolization, a marked depression of total thyroxine (T4) and triiodothyronine (T3) plasma levels, and genotoxic damages determined as hepatic DNA adducts. These results clearly demonstrate that Lambro sediments alone are responsible for recognizable changes in the structure and function of the reproductive and, in general, the endocrine system of a native fish species. In the real environment, exposure to waterborne and food-web sources of chemicals are responsible for additional toxic loads, and the present findings thus provide evidence for a causal role of this tributary in the severe decline observed in barbel in recent decades and raise concern that the fish community of the River Po is exposed to endocrine-mediated health effects along tens of kilometres of its course.

Fish community characterization in two stretches upstream and downstream of the Lambro River confluence with the Po River.

Abstract: This study aimed to characterize the fish community in two stretches of the Po River, upstream and downstream of the Lambro River confluence, to verify the environmental effects of the presence of endocrine disrupters (EDs). More than 5000 specimens were captured, identified at the species level, and recorded for biometric parameters. Plasma, liver, and gonad fragments were sampled for histological and biochemical analyses. Few specimens belonging to carp and barbel showed gonads with the concomitant presence of male and female tissues and altered plasma levels of vitellogenin and sex steroids; thus, these species will be considered for successive environmental studies.

Atrial natriuretic peptide in trout ovarian follicles

An immunocytochemical study on atrial natriuretic peptide (ANP) was carried out in trout ovaries using an antiserum raised against human atrial natriuretic peptide (hANP). Weakly immunoreactive material was observed both in the ooplasm of the previtellogenic and the early vitellogenic oocytes and in the follicular cells of the late vitellogenic oocytes. Binding sites for hANP have been located using 125I-ANP and light-microscopic autoradiography. These studies provide evidence of specific binding of ANP-like material in both early and advanced vitellogenic oocytes. Autoradiograms indicated that the ANP-like binding sites were localized to a subpopulation of follicles. The presence of ANP-like immunoreactive material and its specific binding sites in the ovary suggest a role for atrial natriuretic peptide as a regulator of the fish ovarian function.