Developmental instability is associated with estrogenic endocrine disruption in the Chilean native fish species, Trichomycterus areolatus.

Endocrine disrupting compounds (EDCs) are widespread contaminants that alter the normal functioning of the endocrine system. While they cause dysfunctions in essential biological processes, it is unclear whether EDCs also impact developmental stability. In the present study, we investigated the occurrence of estrogenic endocrine disrupting compounds in a small watershed of south-central Chile impacted by anthropogenic activities. Then, we assessed their relationship with internal levels of estrogenic active compounds and fluctuating asymmetry (FA), a proxy of developmental stability in organisms with bilateral symmetry, in a native fish species (Trichomycterus areolatus). Yeast estrogenic screen assays were performed to measure estrogenic activity in river sediments and in male fish tissues collected from 17 sites along the Chillán watershed, and geometric morphometrics used to estimate fluctuating asymmetry based on the shapes of 248 fish skulls. Estrogenic activity was detected both in sediments and male fish tissues at concentrations of up to 1005 ng and 83 ng 17ß-estradiol equivalent/kg dw, respectively. No significant correlation was found between the two. However, fish tissue estrogenicity, water temperature and dissolved oxygen explained >80% of the FA population variation. By showing a significant relationship between estrogenic activity and FA of T. areolatus, our results indicate that developmental stability can be altered by estrogenic endocrine disruption, and that FA can be a useful indicator of sub-lethal stress in T. areolatus populations.

Molecular isolation and characterization of the kisspeptin system, KISS and GPR54 genes in roach Rutilus rutilus.

The reproduction of vertebrates is regulated by endocrine and neuro-endocrine signaling molecules acting along the brain-pituitary-gonad (BPG) axis. The understanding of the neuroendocrine role played in reproductive function has been recently revolutionized since the KiSS1/GPR54 (KiSS1r) system was discovered in 2003 in human and mice. Kisspeptins, neuropeptides that are encoded by the KiSS genes, have been recognized as essential in the regulation of the gonadotropic axis. They have been shown to play key roles in puberty onset and reproduction by regulating the gonadotropin secretion in mammals while physiological roles in vertebrates are still poorly known. In order to provide new knowledge on basic reproductive physiology in fish as well as new tools to assess impacts of endocrine disrupting compounds (EDCs), the neurotransmitter system, i.e., gene/receptor, KISS/GPR54 might constitute an appropriate biomarker. This study provides new understandings on the neuroendocrine regulation of roach reproduction as well as new molecular tools to be used as biomarkers of endocrine disruption. This work completes the set of biomarkers already validated in this species.

High estradiol exposure disrupts the reproductive cycle of the clam Ruditapes decussatus in a sex-specific way.

Bivalve species may be susceptible to environmental estrogenic compounds including estradiol (E2). However, they are able to biotransform the hormone quite readily and inactivate its estrogenic action. To study the long-term effects of elevated free E2 tissue levels, we transiently exceeded the biotransformation capacity of the clam Ruditapes decussatus by exposing them with high E2 concentrations (400 ng/L) and subsequently study the consequences on gametogenesis during the following reproductive cycle. Exposure to 400 ngE2/L led to a significant increase in tissue free E2 levels, which reached 10-50 ng E2Eq/gww. No deleterious effect on gonado-somatic index (GSI), condition index (CI), or ability to respond to the stress on stress test could be detected after a month of exposure, suggesting the absence of negative effects on the clam's health. However, a marked increase in gametogenesis could be observed in both sexes during the exposure. Subsequent transplantation of the clams in the field allowed the normal development of the male clams and maturation of the gonads without any detrimental effect observed after 4 months. In contrast, in early July, all female clams formerly exposed to E2 showed lower health status, and only ovaries with atretic oocytes while all control and indigenous females were normal and mature. These results show a sex-specific effect of high E2 exposure and suggest either a direct or indirect role for E2 in R. decussatus' reproduction.

Sensitive periods for 17ß-estradiol exposure during immune system development in sea bass head kidney.

An increasing body of evidence suggests that sex steroids play an important role in the development and regulation of vertebrate immune defense. Therefore, compounds with estrogenic activity may influence the immune system via receptor-mediated pathways. The presence of estrogen receptors in immune cells and organs during the early stages of development may indicate that female steroid hormones are involved in the maturation of the fish immune system. This is of particular importance, as some marine fish are probably exposed to sources of exogenous estrogens while they reside in their estuarine nursery grounds. In this study, the influence of 17ß-estradiol (E2) on estrogen receptor and cytokine gene expression was assessed in juvenile sea bass (Dicentrarchus labrax) together with characterization of the head kidney leukocyte populations and corresponding phagocytic activity during organ regionalization from 98 to 239 dph. E2 exposure, beginning at 90 dph resulted in indirect and delayed modifications of interleukin 1ß and estrogen receptor α gene expression, which may affect B-lymphocyte proliferation in the sea bass head kidney. The E2 treatment of 120 dph fish led to an increase in estrogen receptor ß2 and a decrease in transforming growth factor ß1 gene expression, which coincided with decreased phagocytic activity of head kidney lymphocytes and monocytes/macrophages. Additionally, these changes were observed during developmental periods described as critical phases for B-lymphocyte development in mammals. Consequently, exogenous estrogens have the potential to modify the innate immune response in juvenile sea bass and to exert detrimental effects on head kidney development. Copyright © 2015 John Wiley & Sons, Ltd.

TP53 and FGFR3 Gene Mutation Assessment in Urine: Pilot Study for Bladder Cancer Diagnosis.

AIM: To assess, in a prospective clinical research study, a new non-invasive and reliable test to accurately detect tumor protein 53 (TP53) and fibroblast growth factor receptor-3 (FGFR3) mutations in cells in urine. MATERIALS AND METHODS: TP53 mutations were analyzed using the functional analysis of separated allele in yeast (FASAY) method, which allows functional analysis of the P53 protein, and FGFR3 mutations were assessed with the SNaPshot system, detecting the eight most frequent point-mutations of this gene. Chi-square test or Fisher's exact test were used to compare TP53 and FGFR3 mutations in the tumors according to tumor stage and grade. RESULTS: TP53 and FGFR3 mutations in bladder tumors increased and decreased respectively with increasing tumor stage and cellular grade (p<0.05 and p<0.001, respectively). A total of 103 tumor/urinary sediment couples were analyzed. TP53 or FGFR3 mutations were observed in 76 tumors. The sensitivity for the detection of this type of mutation in urine was 46%, the specificity was 81%, the positive predictive value was 94% and the negative predictive value was 37%. CONCLUSION: Our original data confirmed the feasibility of TP53 and FGFR3 mutation detection in urine sediment. These measurements, together with urine cytology, may increase tumor detection. The sensitivity of the TP53/FGFR3 phenotype test in the urine was less than 50% and was not able to replace standard cystoscopy in the diagnosis of bladder tumors.

17ß-Estradiol induces changes in cytokine levels in head kidney and blood of juvenile sea bass (Dicentrarchus labrax, L., 1758).

The cytokine network is involved in the immune system communication. As estrogens influence the cytokine expression in mammals, this study investigated the impact of exogenous estrogenic pollutants on selected cytokines in Dicentrarchus labrax. The gene expression of Interleukin 6, Tumour Necrosis Factor α, Transforming Growth Factor ß1 and Interleukin 1ß was assessed and accomplished with protein measurements in the blood for the last two. Impacts through 17ß-estradiol mainly occurred at the beginning of organ regionalisation, thus falling together with a developmentally induced increase of Interleukin 1ß and Tumour Necrosis Factor α gene expression in 102 dph fish. 17ß-estradiol depressed this modification after 35 days of exposure and the cytokine gene expression tended to be generally down-regulated independently of the 17ß-estradiol concentrations after 56 days of exposure. This impact was confirmed at the protein level, showing that 17ß-estradiol affects the fine control of the cytokine network in sea bass.

Brain cytochrome P450 aromatase activity in roach (Rutilus rutilus): seasonal variations and impact of environmental contaminants.

P450 aromatase catalyses the conversion of C19 androgens to C18 estrogens which is thought to be essential for the regulation of the reproductive function. In this study, brain aromatase activity (AA) was measured monthly over a reproductive cycle in wild roach (Rutilus rutilus) sampled in a reference site in Normandy. AA peaked during the breeding season, reaching 35 fmol mg(-1)min(-1) in both male and female fish, and was low during the rest of the year except for a significant rise in October. AA was correlated with ovary maturation (measured either as gonado-somatic index or by histological analysis of the gonads) and plasma sex-steroid levels (11-ketotestosterone in males and 17-ß-estradiol in females). Measurements of AA in polluted sites showed that activity was significantly upregulated in sites with fish showing high levels of plasma vitellogenin and large proportion of intersexuality (20-50%) thus suggesting the occurrence of estrogenic compounds and their involvement in AA modulation.

Identification of reproduction-specific genes associated with maturation and estrogen exposure in a marine bivalve Mytilus edulis.

BACKGROUND: While it is established that vertebrate-like steroids, particularly estrogens (estradiol, estrone) and androgens (testosterone), are present in various tissues of molluscs, it is still unclear what role these play in reproductive endocrinology in such organisms. This is despite the significant commercial shellfishery interest in several bivalve species and their decline. METHODOLOGY/PRINCIPAL FINDINGS: Using suppression subtraction hybridisation of mussel gonad samples at two stages (early and mature) of gametogenesis and (in parallel) following controlled laboratory estrogen exposure, we isolate several differentially regulated genes including testis-specific kinases, vitelline lysin and envelope sequences. CONCLUSIONS: The differentially expressed mRNAs isolated provide evidence that mussels may be impacted by exogenous estrogen exposure.

Reference gene selection for qPCR in mussel, Mytilus edulis, during gametogenesis and exogenous estrogen exposure.

PURPOSE: The aim of this study is to develop a normalization method for real-time PCR data by analyzing the most stably expressed control genes in mussel (Mytilus edulis) reproductive tissue. METHODS: To facilitate this, six candidate genes, including several commonly used in the literature, were investigated in mussels at different stages of gametogenesis and following experimental exposure to a model estrogen (17b-estradiol). GeNorm and NormFinder softwares were employed to assess the stability of the reference genes. RESULTS: Our results demonstrate that the most stable reference genes are not the same in mussels at different stages of gametogenesis and in experimentally E2-exposed mussels. Interestingly, HEL (helicase) and ACT (actin) mRNA expression levels were most affected by the stage of gametogenesis and yet, in molluscan studies, ACT is possibly the most frequently used reference gene. CONCLUSIONS: We demonstrate that the experimental results are highly dependent on the reference gene chosen and that statistically significant contrasting differences between sample groups are present or absent depending on the reference gene employed.

Effects of estrogen exposure in mussels, Mytilus edulis, at different stages of gametogenesis.

Mytilus edulis were exposed to 17beta-estradiol (E2) and the synthetic estrogens ethinyl estradiol (EE2) and estradiol benzoate (EB) for 10 days. Two exposures were performed to determine their effect on vitellogenin (VTG) and estrogen receptor 2 (ER2) mRNA expression at different stages of the reproductive cycle. Significant natural variation was not observed in VTG mRNA expression, though ER2 mRNA expression displayed significantly lower values during January, February and July compared with other times of the year. A significant increase in VTG and ER2 mRNA expression was observed in mussels exposed to estrogens at the early stage of gametogenesis. In contrast, mature mussels displayed no statistically significant change in the VTG or ER2 mRNA expression. The data presented suggests that the reproductive physiology of molluscs, in terms of VTG and ER2 mRNA expression, may be susceptible to damage by environmental estrogens at certain points in their gametogenesis process.

A new in vitro screening bioassay for the ecotoxicological evaluation of the estrogenic responses of environmental chemicals using roach (Rutilus rutilus) liver explant culture.

There is growing evidence that many chemicals released in the environment are able to disturb the normal endocrinology of organisms affecting the structure and function of their reproductive system. This has prompted the scientific community to develop appropriate testing methods to identify active compounds and elucidate mechanisms of action. Of particular interest are in vitro screening methods that can document the effects of these endocrine disrupting compounds on fish. In this study, an in vitro bioassay was developed in the roach (Rutilus rutilus) for evaluating the estrogenicity or antiestrogenicity potency of environmental pollutants by measuring vitellogenin (VTG) induction in cultured liver explants. The cell viability was assessed by the measurement of nonspecific esterase activity using a fluorescein diacetate hydrolysis assay. Results showed that explants could be cultured for 72 h without any significant loss of activity. Dose-dependent responses have been measured with estrogenic model compounds such as 17-ß-estradiol (E2) and 17-α-ethynylestradiol (EE2) or antiestrogenic compounds such as tamoxifen. Lowest observable effective concentrations were 1 nM for E2, 1 nM for EE2, and 100 nM for tamoxifen, showing a good sensitivity of the test system. Estrogenicity of butyl 4-hydroxybenzoate, 4-nonylphenol, and bisphenol A was tested. bisphenol A (100 µM) or butylparaben induced a twofold increase in VTG production when compared with 100 nM E2, whereas this production was only 20% with 100 µM 4-nonylphenol. Overall, this study shows that the bioassay could provide valuable information on endocrine disrupting chemicals including metabolites and mixtures of compounds.

Estrogens disrupt serotonin receptor and cyclooxygenase mRNA expression in the gonads of mussels (Mytilus edulis).

Estrogenic contaminants in the aquatic environment are associated with feminisation of male fish, however their effects on some invertebrate species, such as bivalve molluscs, have yet to be characterised. Gametogenesis represents a critical step in the reproductive process and is subjected to hormonal control by serotonin (5-HT), prostaglandins (synthesised by cyclooxygenases-COX) and steroids such as 17beta-estradiol (E2). Here, we examine the responses of 5-HT receptor and COX mRNA expression in mussels, Mytilus edulis, exposed to estrogenic compounds during different stages of their reproductive cycle. In mature mussels, 5-HT receptor mRNA expression decreased following E2 exposure. The opposite trend was observed in mussels at early gametogenesis stages. COX mRNA expression levels at both stages were generally decreased by E2 exposure. Mussels at early gametogenesis stages were also exposed to ethynylestradiol (EE2) and estradiol benzoate (EB) and a significant increase in 5-HT receptor mRNA expression was observed with both xeno-estrogens. COX expression levels were increased with EB exposure but no significant effects were found with EE2 exposure. These results show that the natural estrogen, E2, as well as the synthetic estrogen, EE2, induce alterations, dependent on reproductive stage, in the mRNA expression levels of 5-HT receptor and/or COX in the marine bivalve M. edulis.

Seasonal variations and alterations of sex steroid levels during the reproductive cycle of male roach (Rutilus rutilus).

Seasonal variations of plasma steroid concentrations i.e. progesterone (P), 11-ketotestosterone (11-KT) and 17-ß-estradiol (E2) were determined immunoenzymatically during a whole reproductive cycle in male roach (Rutilus rutilus) caught in a reference site. Plasma 11-KT concentrations were significantly correlated with gonad growth, expressed as the gonado-somatic index (R² =0.60; p<0.05) and highest concentrations (757 ± 99 pg ml⁻¹ ) coincided with the final testis maturation in March. E2 and P concentrations peaked once during the reproductive cycle. E2 synthesis was significantly induced during the spawning period while P concentration peaked at the very start of the gametogenesis (June) thus suggesting specific roles of these steroids in the reproductive cycle. Comparison of reference levels were then made with plasma steroid concentrations from male roach sampled in polluted areas in the North of France. A significant decrease of E2 (50-60%) and 11-KT (76-84%) was measured, indicating that endocrine disrupting compounds may have interfered with the normal sex steroid synthesis. Contrary to the E2 and 11-KT sex steroids, plasma P concentration was not significantly affected in fish inhabiting impacted areas.

Estrogenic activity of cadmium, copper and zinc in the yeast estrogen screen.

Heavy metals are increasingly studied due to their apparent ability to disrupt signaling pathways of living organisms including humans. Among various mechanisms of action, metals are suspected of exerting estrogenic activity in human and wildlife. In this study, a wide range of concentration of cadmium, copper, lead, mercury and zinc (from 95.4 pM to 1 mM) alone or in combination with the natural estrogen, 17-beta estradiol, has been tested using the yeast estrogen screen, an estrogen receptor dependent transcriptional expression assay. No direct trans-activation of the estrogen-responsive element could be measured with any of the concentration of the metals tested. Nevertheless, cadmium, copper and zinc were able to potentiate the estradiol-induced response in a dose-dependent manner. Significant stimulation was obtained from 10 nM cadmium, 100 nM copper and 2 nM zinc. Maximum response led to decrease of the estradiol EC50 by a factor 10. This study indicates that cadmium, copper and zinc can act as potential endocrine disrupters by modulating the estrogenic activity of endogenous hormones.

Multixenobiotic resistance, acetyl-choline esterase activity and total oxyradical scavenging capacity of the Arctic spider crab, Hyasaraneus, following exposure to bisphenol A, tetra bromo diphenyl ether and diallyl phthalate.

The Arctic has become a sink for persistent organic pollutants (POPs) originating from lower latitudes, and relatively high levels have been found in different biota. Recent studies have identified detrimental effects on wildlife including endocrine disruption, impairment of enzyme activity, and reduced immune function. The Arctic spider crab, Hyas araneus, shown interesting potential for its use as sentinel organism in polar ecosystems. This study investigated the effect of 2,2',4,4'-tetra bromo diphenyl ether (BPDE), bisphenol A (BPA), and diallyl phthalte (DPA) on H. araneus in a three weeks exposure study. Expression of multixenibiotic resistance (MXR) proteins has been studied using the C219 monoclonal antibody which allows identifying an immunoreactive protein of 40 kDa in the digestive gland while no such protein could be observed in the gills. Expression of this protein was increased by exposure to DPA (+75%; p<0.05, n=10). All compounds significantly affected muscle acetylcholine esterase (AChE) activity (p<0.05, n=10) with 50 microg/L DPA having the strongest effect by lowering the value to 37% of control. The total oxyradical scavenging capacity measured in the digestive gland toward peroxyl, hydroxyl and peroxynitrite was also significantly reduced indicating a decreased resistance to oxidative stress generated by DPA (p<0.05, n=5). These results thus suggest the potential detrimental effects of DPA even at concentration as low as 50 microg/L on H. araneus.

In vitro study of the effects of cadmium on the activation of the estrogen response element using the YES screen.

Estrogenic potential of environmental samples is frequently assessed using receptor-based functional assays. Using the yeast estrogen screen (YES) developed by Routledge and Sumpter, we assessed the ability of cadmium to activate the estrogen receptor-mediated response. No induced transcriptional activity was observed with a range of CdCl2 concentrations (1 nm-1mM). But, when combining cadmium with the model compound 17beta-estradiol, cadmium was able to significantly potentiate the induced estrogenic response for concentrations ranging from 15 nM to 1 microM. A maximal effect was observed at 0.5 microM with a ten fold reduction of the 17beta-estradiol EC50.

Identification of the steroid fatty acid ester conjugates formed in vivo in Mytilus edulis as a result of exposure to estrogens.

Vertebrate-type sex steroids have been detected in a number of mollusk species and may play a role in the reproductive physiology of the animal. Mollusks are also exposed to exogenous estrogenic steroids that are present in sewage effluents, and these may add to the estrogenic burden of exposed animals. We investigated the uptake of estrogens in the blue mussel, Mytlius edulis and report for the first time the identity of estrogen fatty acid ester metabolites formed in vivo in an invertebrate. We exposed mussels to waterborne radiolabeled [(14)C]-17beta-estradiol (E2) or estrone (E1) and determined the nature of their metabolites using radio-HPLC and mass spectrometry (MS). After 13 days of exposure to 10ng/L E2, concentrations of radiolabeled residues were 2428-fold higher in M. edulis soft tissues compared with the ambient water concentration of E2. All the E2 residues in the mussel were present as a lipophilic ester which, in depuration studies, had a half-life of 8.3 days. Exposure of mussels to [(14)C]-E1 (70ng/L) resulted in formation of a similar lipophilic metabolite that after hydrolysis released [(14)C]-E2. Tandem MSMS analyses of the purified steroid ester fraction isolated from mussels exposed to either E2 or E1 revealed that they had the same composition and comprised C16:0, C16:1 and C16:2 esters of E2. This work reveals that in vivo E1 is rapidly metabolized to E2 in mussels prior to conjugation to C16 fatty acid esters, proving that C17-ketoreductase and C16 fatty acid acyl-CoA:E2 acyltransferase are important enzymes for the metabolism of estrogens in M. edulis.

Evidence of endocrine alteration in the red mullet, Mullus barbatus from the NW Mediterranean.

Red mullet (Mullus barbatus) were collected from different sampling sites (NW Mediterranean) in spring and autumn, with the aim of assessing potential alterations of the endocrine system. Alkylphenols were measured in fish bile as an indicator of estrogenic exposure. Key enzymatic activities involved in both synthesis (ovarian 17beta-hydroxysteroid dehydrogenases and P450 aromatase) and metabolism of steroids were assessed together with histological alterations of the gonads. During the spring sampling, delayed gamete maturation, intersexuality, fibrosis, and depressed ovarian P450 aromatase activity were observed in organisms from the most polluted sites. During the autumn sampling, those effects were less evident, indicating that fish might be more susceptible to endocrine disrupting chemicals during the reproductive period. Nonetheless, enhanced glucuronidation of testosterone and estradiol was observed. Overall, this work provides first evidences of significant alterations in the endocrine system of red mullet from highly impacted areas in the NW Mediterranean.

Impact of endocrine toxicants on survival, development, and reproduction of the estuarine copepod Eurytemora affinis (Poppe).

Given recent reports suggesting that certain contaminants may be present in sewage effluents at levels, which may exert a deleterious impact on fish, it seems pertinent to extend ecological hazard evaluation for such substances to aquatic invertebrates. For this reason, we sought to determine whether 17beta-estradiol (E2), benzo(a)pyrene (BaP), 4-nonylphenol (NP), di(ethyl-hexyl)-phthalate (DEHP), and atrazine (A), individually or in binary mixture, can inhibit the survival, development, or reproductive output of the estuarine copepod Eurytemora affinis. In the first experiment nauplii were exposed to graded concentrations of individual contaminants to determine the 96-h LC50, 10-day no observed effect concentration (NOEC) and 10-day lowest observed effect concentration of each compound. In the second experiment newly released (<24-h-old) nauplii were exposed either to an individual contaminant at the NOEC or to binary mixtures, where each compound was used at half NOEC. The effects were monitored daily for development and sex ratio. After 10 days of exposure, adult males and females were paired and exposures continued to investigate effects on reproductive output (maximum 28 day total exposure). Based on these life cycle parameters the lowest 10-day NOECs were 6+/-4 microg L(-1) for E2, 7+/-3 microg L(-1) for NP, 12+/-3 microg L(-1) for BaP, 25+/-3 microg L(-1) for A, and 109+/-29 microg L(-1) for DEHP. BaP, NP, and DEHP inhibited naupliar development, but in binary mixture with E2 these compounds did not inhibit larval development. The results suggest that endocrine disruption could occur in copepods following exposure to estrogenic compounds, especially if they are exposed starting from embryonic development.