Medium- and Long-Term Effects of Estrogenic Contaminants on the Middle River Po Fish Community as Reconstructed from a Sediment Core.

Recent studies showed that endocrine active compounds (EDs) capable to induce fish gonadal histopathologies, plasma vitellogenin and thyroid disruption, are transported by the River Lambro to the River Po, potentially affecting the fish community of the main Italian river. To assess whether fish relative abundance, composition and health were impaired by the River Lambro, a 3-year survey was undertaken in the main river. Results showed that the tributary supports in the River Po a denser fish community (+43 %), with a higher total biomass (+35 %). The survey also showed niche- and sensitivity-dependent effects, so that three benthopelagic species (bleak, topmouth gudgeon, and bitterling) were, for example, more abundant downstream from the tributary (up to 3.4×), but their sizes were significantly smaller. The present fish community was then compared with that described 30 years before in the same area of the Po River. This comparison highlighted that some fish species have disappeared and many have severely declined. To better evaluate this contrast, a sediment core of the Lambro tributary was analysed for the time trends of natural estrogens (E1, E2, E3), bisphenol A and alkylphenols. The results showed that during the last 50 years the River Lambro has been exposed to high estrogenic activities (16.1 ± 9.3 ng E2 equivalents/g), which inevitably affected also the River Po. In addition, at the time of the previous survey, six species of the main river had skewed sex ratios toward all-female populations, providing evidence that EDs and particularly (xeno)estrogens were already affecting the long-term viability of fish populations. Estrogens thus can be ascribed among the causal factors of fish qualitative and quantitative decline of the River Po, although long-term effects have been likely mitigated by nonconfinement of fish populations and nutrient enrichment.

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-17ß (E2) and testosterone (T) plasma levels and E2/T showed significantly different patterns in NP and t-OP exposed against control fish.

Aromatase mRNA expression in the brain of adult Xenopus laevis exposed to Lambro river water and endocrine disrupting compounds.

Aromatase P450 (P450 arom; Cyp19) is a key enzyme for vertebrate reproduction and brain development that catalyzes the conversion of androgens to estrogens. The aim of this study was to improve the knowledge on EDC effects by analysing their potential impact on brain P450 arom in adult Xenopus laevis exposed for 4 weeks to an environmental sample, the water of the river Lambro (LAM), the most polluted tributary of the Po river in North Italy. Other groups were exposed to individual compounds 10(-8) M tamoxifen (TAM), ethinylestradiol (EE2), flutamide (FLU) and methyldihydrotestosterone (MDHT) known for their (anti)estrogenic and (anti)androgenic modes of action. Expression of CYP19 was evaluated in brain extracts by quantitative RT-PCR, using a pair of primers located in the open reading frame (ORF) that allowed the simultaneous amplification of all transcripts (Aro-ORF) and a pair of primers specific for brain aromatase (Aro-B). Significant increase in Aro-ORF and Aro-B mRNA levels were observed in both females and males exposed to LAM. Different changes were observed for the model compounds using two pairs of primers. Aro-ORF mRNA expression was significantly increased in EE2 and MDHT exposed males and in FLU-exposed females, while it was significantly decreased in TAM exposed females. Aro-B mRNA was significantly increased in both sexes exposed to FLU and decreased in TAM exposed females. In conclusion, aromatase mRNA in the brain of X. laevis was regulated differentially in a gender specific manner by certain (anti)estrogenic and (anti)androgenic EDCs, supporting previous hypotheses that diverse compounds present in the river Lambro may induce feminization and demasculinization effects.

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.

In vivo and in vitro exposures of carp and carp tissues to graded concentrations of endocrine disrupting chemicals.

The objective of this study was to correlate morphological and biochemical parameters of reproductive activity in fish exposed to graded concentrations of endocrine disrupting chemicals. Two-year-old carps were exposed for two weeks to graded concentrations of ethynyl-estradiol, tamoxifen, and flutamide. For each chemical, morphological alterations of the gonads and liver were described according to biomarker and sex steroid levels. In parallel, isolated follicular cells (FCs) and testis fragments were incubated in the presence or absence of carp pituitary homogenate and 25-hydroxycholesterol. Media were analyzed for sex steroid level evaluations.

Environmental agent susceptibility assessment using existing and novel biomarkers as rapid noninvasive testing methods.

This study is part of a project aimed at developing and validating novel noninvasive methods for the detection of biomarkers of endocrine disrupters (EDs) directly in the mucus of aquatic species, to identify novel functional biomarker(s) for EDs, and to verify their applicability for field studies. The multidisciplinary approach chosen aims at the development of an integrated testing strategy utilizing in vitro protocols to identify water and sediment fractions with potential endocrine-disrupting activity; the identification, characterization, and measurement of new biomarker(s) for EDs; the development and validation of a dipstick-based test method; and the development of (computer-assisted) predictive models. Some results of the first year of the project are presented here.

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

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.

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; pparß 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.

Angiotensin II specific receptors in subcommissural organ.

In the subcommissural organ of male rats, a circumventricular organ situated inside the blood-brain barrier, specific receptors for angiotensin II were demonstrated by binding studies on homogenated membranes and by autoradiography carried out on frozen sections using 125I-angiotensin II. The receptor sites were localized in the subnuclear region of cells of the subcommissural organ. A single class of binding sites was found whose capacity was modulated by changes in the sodium plasma concentration which led to variations in plasma volume.

Rites of passage: understanding participation of children with developmental coordination disorder.

Children with developmental coordination disorder (DCD) experience difficulty participating in the typical activities of childhood and are known to have a more sedentary pattern of activities than their peers. Little research has been done to investigate the impact of these deficits on the lives of children with DCD and the importance of their participation in the typical activities of childhood. This qualitative study explored the impact of the disorder and the importance of participation for children with DCD from the perspective of the parent. Twelve in-depth interviews were conducted with parents of children with DCD who attended a university clinic specializing in using the Cognitive Orientation to daily Occupational Performance (CO-OP) approach, a cognitive-based intervention. Findings revealed that incompetence in everyday activities had serious negative effects for the children. Conversely, intervention that was focused on enablement at the activity and participation level had a significant positive impact on the children's quality of life. Emerging themes highlighted the notion that performance competency played an important role in being accepted by peers and being able "to be part of the group". As well, parents reported that successful participation built confidence in their children and allowed them to try other new activities. The World Health Organization's International Classification of Functioning, Disability, and Health provides a unique framework for analyzing and understanding the impact of the physical disability on the lives of families with children with DCD. Results illustrate how intervention that focuses on enabling children to choose their own functional goals in the area of physical activity has important implications for enabling participation and building the social networks of children with DCD.

A pilot trial of a cognitive treatment for children with developmental coordination disorder.

This pilot study compared a new treatment approach, the Cognitive Orientation to daily Occupational Performance (CO-OP) to the Contemporary Treatment Approach (CTA) to treating children with Developmental Coordination Disorder (DCD). CO-OP emphasises problem-solving strategies and guided discovery of child and task specific strategies. CTA encompasses a variety of approaches, such as neuromuscular, multi-sensory, and biomechanical, focusing on motor aspects of skill acquisition. Twenty children with a mean age of 9.05 years (S.D. = 1.23) participated in the study. All children had normal intelligence, scored below the 15th percentile on a standardised test of motor ability, and demonstrated motor difficulties significant enough to warrant referral for treatment. Pre- and post-measures included the Canadian Occupational Performance Measure (COPM), the Vineland Adaptive Behavior Scales (VABS), the Bruininks-Oseretsky Test of Motor Proficiency (BOTMP), the Developmental Test of Visual-Motor Integration-Revised (VMI), the motor items of the Self-Perception Profile for Children (SPPC), and the Performance Quality Rating Scale (PQRS). In both groups, treatment goals were child-chosen. Both treatments lead to improved COPM self-ratings of performance and satisfaction; however, improvements in the CO-OP group were greater than those in the CTA group. These results were paralleled by PQRS scores, and the Motor scores on the VABS, but not on the BOTMP measures. This outcome still needs replication as no control group was involved and because of the occurrence of pre-treatment differences between the CO-OP and CTA groups on relevant measures. Follow-up data indicated that children who received CO-OP tended to experience greater long-term maintenance of their motor goals and acquired strategies; follow-up parent-report rated CO-OP treatment as more useful than CTA treatment. Self-report, observer report, standardised assessment, and follow-up all demonstrated the effectiveness of the CO-OP approach, supporting the use of CO-OP and suggesting further investigation of this new cognitive intervention.

The site of 7-dehydrocholesterol ultraviolet photolysis in domestic fowls.

The site of the photolytic reaction leading to Vitamin D3 in fowls integument has been investigated by histochemical and biochemical analysis. The Vitamin D3 precursor, 7-dehydrocholesterol, is synthetized and stored mainly in the uropygial gland and in the same localization several amount of Vitamin D3 may be biochemically detected. 7-dehydrocholesterol and Vitamin D3 are also present although in a lesser amount in the unfeathered skin of the legs. Photolytic conversion of the Vitamin D3 precursor of uropygial origin may occur also on the dorsal feathers.

beta-Glucuronidase activity in the developing uropygial gland.

The presence of sterolic compounds and beta-glucuronidase activity have been studied in the uropygial glands of chick embryos (18th day of inc.), chickens (3 weeks after hatching) and young fowls (5 months old). Sterols are histochemically detectable only after hatching and beta-glucuronidase activity, very faint before hatching, reaches its maximum in chicken glands with a peculiar inner localization coincident with sterolic localization. It is suggested that beta-glucuronidase has in uropygial gland a double functional significance: a certain amount of activity is developed to cell proliferation whereas a more strong activity is involved in the hydrolysis of sterol glucuronides.

Occurrence of 7-dehydrocholesterol in the uropygial gland of domestic fowls.

Histochemical studies on the uropygial gland of domestic fowls have shown the presence of sterols (among which cholesterol and its esters) in the lipidic fraction of the gland secret. beta-Glucuronidase activity beside A5 3beta- and 17beta-hydroxysteroid dehydrogenase activities suggests that uropygial gland might be involved in sterols metabolism. By thin layer chromatography cholesterol and 7-dehydrocholesterol can be separated from the uropygial extracts and these compounds can be identified in gas liquid chromatography.

First observation of intersex cyprinids in the Po River (Italy).

Barbel (Barbus plebejus, Cyprinidae) were captured in the Po River, upstream and downstream from the confluence of the Lambro River, a polluted tributary of the major Italian watercourse. The gonads of the two groups of barbel have been histologically examined, and only the downstream specimens showed histo-morphological alterations that can be related to the Lambro tributary as a source to the main river of endocrine disrupting chemicals, possibly with estrogenic effects. In fact, 50% of the barbel captured (8 of 16 fish) in the downstream reach showed intersex gonads.

Histological and histochemical study of female germ cell development in the dusky grouper Epinephelus marginatus (Lowe, 1834).

The developmental stages of female germ cells were analysed in a wild population of the protogynous teleost Epinephelus marginatus (Lowe, 1834). 321 wild dusky grouper females were collected in the South Mediterranean Sea during the spawning season and their ovaries analysed using histological and histochemical techniques. Oocyte morphology, nucleus-cytoplasm ratio (N/C) range, location and movements of cytoplasmic inclusions during primary growth, vitellogenesis and final oocyte maturation were described. The distribution of proteins, lipids and carbohydrates through oocyte development was also investigated in 50 females. Lipid vesicles appeared firstly in the mid ooplasm of oocytes larger than 130 microm, at the beginning of the secondary growth phase. Immediately afterwards, small carbohydrate granules (PAS and Alcian blue positive) appeared before the occurrence of the first yolk granules. Tyrosine-enriched proteins were especially evidenced in the zona radiata interna of late vitellogenic oocytes. Specific lectin binding patterns reflected characteristic differences in the content and distribution of specific sugar moieties expressed in the oocytes during vitellogenesis and final maturation. At the end of vitellogenesis and during final maturation, follicular cells, zona radiata, and cortical alveoli were characterised by a strong increase of specific binding for WGA.

Dynamic performance analysis: a framework for understanding occupational performance.

Occupational therapy is now consistently described as a profession concerned with enabling occupation. A crucial step in enabling occupation is understanding the occupational performance of our clients. Dynamic Performance Analysis (DPA) is a new approach to occupational analysis that focuses on the client's actual performance. DPA, acknowledging that optimal performance is the product of the interaction of person, environment, and occupation, and thus highly individualistic, places the client and his or her occupation, in interaction with the environment, at the center of the analysis process. Embedded in a top-down framework, DPA is a dynamic, iterative process, carried out as the client performs the occupation. The purpose of DPA is to identify where performance breaks down and test out solutions. In this article, the rationale, origins, and basic assumptions of DPA are discussed, and a detailed description of the DPA process together with two clinical examples is presented.

In vivo exposure of carp to graded concentrations of bisphenol A.

A wide range of environmental pollutants commonly termed endocrine disrupters (ED) can mimic steroid hormones causing adverse health effects. Recent studies showed that bisphenol A (BPA) together with other estrogenic chemicals, may be responsible for the disrupting effects observed in fish of the middle River Po. The present study has investigated the potential role of BPA in inducing this kind of effects and to this aim, 1-year-old (k1) common carp were exposed for 14 days to graded concentrations of BPA (1, 10, 100, and 1000microg/L). Histological alterations of gonads were described and compared to vitellogenin (VTG) and sex steroid levels. In carp males, BPA caused severe alterations of testis structure starting from 1microg/L. Several specimens lost the typical lobular structure showing spermatogenic cysts intermingled with free spermatozoa often degenerating into the lumen. Oocyte atresia was observed starting from 1micro BPA/L, concerning 57.1% of females at the highest concentration (1000microg BPA/L). A few carp from the same highest treatment also showed intersexuality (27%). Oestradiol-17beta (E2) significantly decreased both in 1 and 10microg BPA/L exposed carp reaching again values not significantly different from control in 1000microg BPA/L. At this concentration, a significant reduction of testosterone (T) was observed in both males and females. The mean plasma concentration of VTG significantly increased in 1000microg BPA/L exposed carp, even if a growing number of responsive carp was observed starting from the environmental concentrations (1 and 10microg BPA/L).

Investigating the estrogenic risk along the river Po and its intermediate section.

Different endpoints have been used to investigate the occurrence of estrogenic risk along the Po River, particularly its middle section. An in vitro assay based on recombinant yeast could not detect estrogenic activity in bed sediments of the Italian river or in bile samples of five Cyprinid species, with the only exception being one carp (Cyprinus carpio) captured downstream of the River Lambro, a polluted tributary of the middle River Po. Chemical analyses of fish bile and water samples from the same middle section showed diffuse contamination by moderately low levels of estrogenic chemicals (estrone [E1], 17beta-estradiol, estriol [E3], 17alpha-ethinylestradiol, 4-nonylphenol [NP], 4-tert-octylphenol [tOP], 4-n-octylphenol, and bisphenol A) but they were of limited help in understanding the risk present in the downstream area where intersex barbel were previously found. In contrast, the analyses of River Lambro waters showed that this tributary is a source to the middle River Po of all eight estrogens investigated. Analyses of bed sediments and macroinvertebrates from the same area consistently showed at least two levels of contamination, with the downstream stretch showing higher concentrations of natural steroids (E1 and E3) and xenoestrogens (NP and tOP). Accordingly, new histologic examinations undertaken on young barbel (Barbus sp.) showed intersex gonads only in the individuals captured in the downstream stretch, thereby confirming previous results. Present findings confirm the occurrence of disrupting conditions in the middle River Po and provide the first suggestions of cause-effect relationships.