Aromatase in zebrafish: a potential target for endocrine disrupting chemicals

In zebrafish, two isoforms of the aromatase gene exist, namely cyp19a1 and cyp19a2, expressed predominantly in the gonads and brain, respectively. In this study, we focus on characterizing the specificity of antibodies against the aromatase isoforms, and on (xeno)estrogen-induced changes of individual cyp19a2 mRNA concentrations in the brains of adult male zebrafish. Among three polyclonal antibodies studied, the one against CYP19A2 was found to be specific in Western blots and immunohistochemistry. Real-time RT-PCR analyses revealed strong interindividual variation of cyp19a2 levels in the brains of adult male zebrafish. After a three-week-exposure to (xeno)estrogens, mean values of cyp19a2 mRNA levels tended to increase, with significant induction at 200 ng 17beta-estradiol/L, but interindividual variation of cyp19a2 expression was maintained.

Interference of endocrine disrupting chemicals with aromatase CYP19 expression or activity, and consequences for reproduction of teleost fish

Many natural and synthetic compounds present in the environment exert a number of adverse effects on the exposed organisms, leading to endocrine disruption, for which they were termed endocrine disrupting chemicals (EDCs). A decrease in reproduction success is one of the most well-documented signs of endocrine disruption in fish. Estrogens are steroid hormones involved in the control of important reproduction-related processes, including sexual differentiation, maturation and a variety of others. Careful spatial and temporal balance of estrogens in the body is crucial for proper functioning. At the final step of estrogen biosynthesis, cytochrome P450 aromatase, encoded by the cyp19 gene, converts androgens into estrogens. Modulation of aromatase CYP19 expression and function can dramatically alter the rate of estrogen production, disturbing the local and systemic levels of estrogens. In the present review, the current progress in CYP19 characterization in teleost fish is summarized and the potential of several classes of EDCs to interfere with CYP19 expression and activity is discussed. Two cyp19 genes are present in most teleosts, cyp19a and cyp19b, primarily expressed in the ovary and brain, respectively. Both aromatase CYP19 isoforms are involved in the sexual differentiation and regulation of the reproductive cycle and male reproductive behavior in diverse teleost species. Alteration of aromatase CYP19 expression and/or activity, be it upregulation or downregulation, may lead to diverse disturbances of the above mentioned processes. Prediction of multiple transcriptional regulatory elements in the promoters of teleost cyp19 genes suggests the possibility for several EDC classes to affect cyp19 expression on the transcriptional level. These sites include cAMP responsive elements, a steroidogenic factor 1/adrenal 4 binding protein site, an estrogen-responsive element (ERE), half-EREs, dioxin-responsive elements, and elements related to diverse other nuclear receptors (peroxisome proliferator activated receptor, retinoid X receptor, retinoic acid receptor). Certain compounds including phytoestrogens, xenoestrogens, fungicides and organotins may modulate aromatase CYP19 activity on the post-transcriptional level. As is shown in this review, diverse EDCs may affect the expression and/or activity of aromatase cyp19 genes through a variety of mechanisms, many of which need further characterization in order to improve the prediction of risks posed by a contaminated environment to teleost fish population.

Endocrine disrupting compounds: can they target the immune system of fish?

Endocrine disruption, in particular disruption by estrogen-active compounds, has been identified as an important ecotoxicological hazard in the aquatic environment. Research on the impact of endocrine disrupting compounds (EDCs) on wildlife has focused on disturbances of the reproductive system. However, there is increasing evidence that EDCs affect a variety of physiological systems other than the reproductive system. Here, we discuss if EDCs may be able to affect the immune system of fish, as this would have direct implications for individual fitness and population growth. Evidence suggesting an immunomodulatory role of estrogens in fish comes from the following findings: (a) estrogen receptors are expressed in piscine immune organs, (b) immune gene expression is modulated by estrogen exposure, and (c) pathogen susceptibility of fish increases under estrogen exposure.

Screening and testing for endocrine disruption in fish-biomarkers as "signposts," not "traffic lights," in risk assessment

Biomarkers are currently best used as mechanistic "signposts" rather than as "traffic lights" in the environmental risk assessment of endocrine-disrupting chemicals (EDCs). In field studies, biomarkers of exposure [e.g., vitellogenin (VTG) induction in male fish] are powerful tools for tracking single substances and mixtures of concern. Biomarkers also provide linkage between field and laboratory data, thereby playing an important role in directing the need for and design of fish chronic tests for EDCs. It is the adverse effect end points (e.g., altered development, growth, and/or reproduction) from such tests that are most valuable for calculating adverseNOEC (no observed effect concentration) or adverseEC10 (effective concentration for a 10% response) and subsequently deriving predicted no effect concentrations (PNECs). With current uncertainties, biomarkerNOEC or biomarkerEC10 data should not be used in isolation to derive PNECs. In the future, however, there may be scope to increasingly use biomarker data in environmental decision making, if plausible linkages can be made across levels of organization such that adverse outcomes might be envisaged relative to biomarker responses. For biomarkers to fulfil their potential, they should be mechanistically relevant and reproducible (as measured by interlaboratory comparisons of the same protocol). VTG is a good example of such a biomarker in that it provides an insight to the mode of action (estrogenicity) that is vital to fish reproductive health. Interlaboratory reproducibility data for VTG are also encouraging; recent comparisons (using the same immunoassay protocol) have provided coefficients of variation (CVs) of 38-55% (comparable to published CVs of 19-58% for fish survival and growth end points used in regulatory test guidelines). While concern over environmental xenoestrogens has led to the evaluation of reproductive biomarkers in fish, it must be remembered that many substances act via diverse mechanisms of action such that the environmental risk assessment for EDCs is a broad and complex issue. Also, biomarkers such as secondary sexual characteristics, gonadosomatic indices, plasma steroids, and gonadal histology have significant potential for guiding interspecies assessments of EDCs and designing fish chronic tests. To strengthen the utility of EDC biomarkers in fish, we need to establish a historical control database (also considering natural variability) to help differentiate between statistically detectable versus biologically significant responses. In conclusion, as research continues to develop a range of useful EDC biomarkers, environmental decision-making needs to move forward, and it is proposed that the "biomarkers as signposts" approach is a pragmatic way forward in the current risk assessment of EDCs.

Developmental toxicity and endocrine disrupting potency of 4-azapyrene, benzo[b]fluorene and retene in the zebrafish Danio rerio

This study examined the developmental toxicity of the polycyclic aromatic hydrocarbons (PAHs) 11H-benzo(b)fluorene (BBF) and 4-azapyrene (AP) in comparison to the known teratogen retene. Developmental toxicity assays were performed in zebrafish embryos exposed for 120 h. BBF and retene induced a similar dioxin-like phenotype, whereas AP showed distinct effects, particularly craniofacial malformations. Microarray analysis revealed that for BBF and retene, drug metabolism pathways were induced, which were confirmed by subsequent studies of cyp1a gene expression. For AP, microarray analysis revealed the regulation of genes involved in retinoid metabolism and hematological functions. Studies with a panel of CALUX((R)) bioassays to screen for endocrine disrupting activity of the compounds also revealed novel antagonistic effects of BBF and retene on androgen and progesterone receptors. Classification analysis revealed distinct gene expression profiles for both individual and combined PAH exposure. This study highlights the potential health risk of non priority PAHs.

Expression of zebra fish aromatase cyp19a and cyp19b genes in response to the ligands of estrogen receptor and aryl hydrocarbon receptor

Many endocrine-disrupting chemicals act via estrogen receptor (ER) or aryl hydrocarbon receptor (AhR). To investigate the interference between ER and AhR, we studied the effects of 17beta-estradiol (E2) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the expression of zebra fish cyp19a (zfcyp19a) and cyp19b (zfcyp19b) genes, encoding aromatase P450, an important steroidogenic enzyme. In vivo (mRNA quantification in exposed zebra fish larvae) and in vitro (activity of zfcyp19-luciferase reporter genes in cell cultures in response to chemicals and zebra fish transcription factors) assays were used. None of the treatments affected zfcyp19a, excluding the slight upregulation by E2 observed in vitro. Strong upregulation of zfcyp19b by E2 in both assays was downregulated by TCDD. This effect could be rescued by the addition of an AhR antagonist. Antiestrogenic effect of TCDD on the zfcyp19b expression in the brain was also observed on the protein level, assessed by immunohistochemistry. TCDD alone did not affect zfcyp19b expression in vivo or promoter activity in the presence of zebra fish AhR2 and AhR nuclear translocator 2b (ARNT2b) in vitro. However, in the presence of zebra fish ERalpha, AhR2, and ARNT2b, TCDD led to a slight upregulation of promoter activity, which was eliminated by either an ER or AhR antagonist. Studies with mutated reporter gene constructs indicated that both mechanisms of TCDD action in vitro were independent of dioxin-responsive elements (DREs) predicted in the promoter. This study shows the usefulness of in vivo zebra fish larvae and in vitro zfcyp19b reporter gene assays for evaluation of estrogenic chemical actions, provides data on the functionality of DREs predicted in zfcyp19 promoters and shows the effects of cross talk between ER and AhR on zfcyp19b expression. The antiestrogenic effect of TCDD demonstrated raises further concerns about the neuroendocrine effects of AhR ligands.

Vitellogenin synthesis in primary cultures of fish liver cells as endpoint for in vitro screening of the (anti)estrogenic activity of chemical substances

Concern over possible adverse effects of endocrine-disrupting compounds on fish has caused the development of appropriate testing methods. In vitro screening assays may provide initial information on endocrine activities of a test compound and thereby may direct and optimize subsequent testing. Induction of vitellogenin (VTG) is used as a biomarker of exposure of fish to estrogen-active substances. Since VTG induction can be measured not only in vivo but also in fish hepatocytes in vitro, the use of VTG induction response in isolated fish liver cells has been suggested as in vitro screen for identifying estrogenic-active substances. The main advantages of the hepatocyte VTG assay are considered its ability to detect effects of estrogenic metabolites, since hepatocytes in vitro remain metabolically competent, and its ability to detect both estrogenic and anti-estrogenic effects. In this article, we critically review the current knowledge on the VTG response of cultured fish hepatocytes to (anti)estrogenic substances. In particular, we discuss the sensitivity, specificity, and variability of the VTG hepatocyte assay. In addition, we review the available data on culture factors influencing basal and induced VTG production, the response to natural and synthetic estrogens as well as to xenoestrogens, the detection of indirect estrogens, and the sources of assay variability. The VTG induction in cultured fish hepatocytes is clearly influenced by culture conditions (medium composition, temperature, etc.) and culture system (hepatocyte monolayers, aggregates, liver slices, etc.). The currently available database on estrogen-mediated VTG induction in cultured teleost hepatocytes is too small to support conclusive statements on whether there exist systematic differences of the VTG response between in vitro culture systems, VTG analytical methods or fish species. The VTG hepatocyte assay detects sensitively natural and synthetic estrogens, whereas the response to xenoestrogens appears to be more variable. The detection of weak estrogens can be critical due to the overshadow with cytotoxic concentrations. Moreover, the VTG hepatocyte assay is able to detect antiestrogens as well as indirect estrogens, i.e substances which require metabolic activation to induce an estrogenic response. Nevertheless, more chemicals need to be analysed to corroborate this statement. It will be necessary to establish standardized protocols to minimize assay variability, and to develop a set of pass-fail criteria as well as cut-offs for designating positive and negative responses.

Zebrafish (Danio rerio) as a model organism for investigating endocrine disruption

Endocrine-disrupting compounds (EDCs) are widespread in the aquatic environment and can cause alterations in development, physiological homeostasis and health of vertebrates. Zebrafish, Danio rerio, has been suggested as a model species to identify targets as well as modes of EDC action. In fact, zebrafish has been found useful in EDC screening, in EDC effects assessment and in studying targets and mechanisms of EDC action. Since many of the environmental EDCs interfere with the sex steroid system of vertebrates, most EDC studies with zebrafish addressed disruption of sexual differentiation and reproduction. However, other targets of EDCs action must not be overlooked. For using a species as a toxicological model, a good knowledge of the biological traits of this species is a pre-requisite for the rational design of test protocols and endpoints as well as for the interpretation and extrapolation of the toxicological findings. Due to the genomic resources available for zebrafish and the long experience with zebrafish in toxicity testing, it is easily possible to establish molecular endpoints for EDC effects assessment. Additionally, the zebrafish model offers a number of technical advantages including ease and cost of maintenance, rapid development, high fecundity, optical transparency of embryos supporting phenotypic screening, existence of many mutant strains, or amenability for both forward and reverse genetics. To date, the zebrafish has been mainly used to identify molecular targets of EDC action and to determine effect thresholds, while the potential of this model species to study immediate and delayed physiological consequences of molecular interactions has been instrumentalized only partly. One factor that may limit the exploitation of this potential is the still rather fragmentary knowledge of basic biological and endocrine traits of zebrafish. Information on species-specific features in endocrine processes and biological properties, however, need to be considered in establishing EDC test protocols using zebrafish, in extrapolating findings from zebrafish to other vertebrate species, and in understanding how EDC-induced gene expression changes translate into disease.

Concentration- and time-dependent effects of the synthetic estrogen, 17alpha-ethinylestradiol, on reproductive capabilities of the zebrafish, Danio rerio

Partial or full life-cycle tests are needed to assess the potential of endocrine-disrupting compounds (EDCs) to adversely affect development and reproduction of fish. Small fish species such as zebrafish, Danio rerio, are under consideration as model organisms for appropriate test protocols. The present study examines how reproductive effects resulting from exposure of zebrafish to the synthetic estrogen 17alpha-ethinylestradiol (EE2) vary with concentration (0.05 to 10 ng EE2 L(-1), nominal), and with timing/duration of exposure (partial life-cycle, full life-cycle, and two-generation exposure). Partial life-cycle exposure of the parental (F1) generation until completion of gonad differentiation (0-75 d postfertilization, dpf) impaired juvenile growth, time to sexual maturity, adult fecundity (egg production/female/day), and adult fertilization success at 1.1 ng EE2 L(-1) and higher. Lifelong exposure of the F1 generation until 177 dpf resulted in lowest observed effect concentrations (LOECs) for time to sexual maturity, fecundity, and fertilization success identical to those of the developmental test (0-75 dpf), but the slope of the concentration-response curve was steeper. Reproduction of zebrafish was completely inhibited at 9.3 ng EE2 L(-1), and this was essentially irreversible as a 3-mo depuration restored fertilization success to only a very low rate. Accordingly, elevated endogenous vitellogenin (VTG) synthesis and degenerative changes in gonad morphology persisted in depurated zebrafish. Full life-cycle exposure of the filial (F2) generation until 162 dpf impaired growth, delayed onset of spawning and reduced fecundity and fertilization success at 2.0 ng EE2 L(-1). In conclusion, results show that the impact of estrogenic agents on zebrafish sexual development and reproductive functions as well as the reversibility of effects, varies with exposure concentration (reversibility at < or = 1.1 ng EE2 L(-1) and irreversibility at 9.3 ng EE2 L(-1)), and between partial and full life-cycle exposure (exposure to 10 ng EE2 L(-1) during critical period exerted no permanent effect on sexual differentiation, but life-cycle exposure did).

Effect of volume loading with 1 liter intravenous infusions of 0.9% saline, 4% succinylated gelatine (Gelofusine) and 6% hydroxyethyl starch (Voluven) on blood volume and endocrine responses: a randomized, three-way crossover study in healthy volunteers

To study the changes in blood volume and hormones controlling sodium and water homeostasis after infusions of 0.9% saline, Gelofusine (4% succinylated gelatin in 0.7% saline, weight-average molecular weight 30 kD), and Voluven (6% hydroxyethyl starch in 0.9% saline, weight-average molecular weight 130 kD) in healthy volunteers.

Pheochromocytoma in rats with multiple endocrine neoplasia (MENX) shares gene expression patterns with human pheochromocytoma

Pheochromocytomas are rare neoplasias of neural crest origin arising from chromaffin cells of the adrenal medulla and sympathetic ganglia (extra-adrenal pheochromocytoma). Pheochromocytoma that develop in rats homozygous for a loss-of-function mutation in p27Kip1 (MENX syndrome) show a clear progression from hyperplasia to tumor, offering the possibility to gain insight into tumor pathobiology. We compared the gene-expression signatures of both adrenomedullary hyperplasia and pheochromocytoma with normal rat adrenal medulla. Hyperplasia and tumor show very similar transcriptome profiles, indicating early determination of the tumorigenic signature. Overrepresentation of developmentally regulated neural genes was a feature of the rat lesions. Quantitative RT-PCR validated the up-regulation of 11 genes, including some involved in neural development: Cdkn2a, Cdkn2c, Neurod1, Gal, Bmp7, and Phox2a. Overexpression of these genes precedes histological changes in affected adrenal glands. Their presence at early stages of tumorigenesis indicates they are not acquired during progression and may be a result of the lack of functional p27Kip1. Adrenal and extra-adrenal pheochromocytoma development clearly follows diverged molecular pathways in MENX rats. To correlate these findings to human pheochromocytoma, we studied nine genes overexpressed in the rat lesions in 46 sporadic and familial human pheochromocytomas. The expression of GAL, DGKH, BMP7, PHOX2A, L1CAM, TCTE1, EBF3, SOX4, and HASH1 was up-regulated, although with different frequencies. Immunohistochemical staining detected high L1CAM expression selectively in 27 human pheochromocytomas but not in 140 nonchromaffin neuroendocrine tumors. These studies reveal clues to the molecular pathways involved in rat and human pheochromocytoma and identify previously unexplored biomarkers for clinical use.

Intrapancreatic Accessory Spleen Misdiagnosed as a Nonsecreting Endocrine Tumor: Case Report and Review of the Literature

In a large series of nonselected autopsy investigations an accessory spleen was found in 10-30%. The second most common site is the pancreatic tail (17%). We report a case of intrapancreatic accessory spleen misdiagnosed as a nonsecreting neuroendocrine tumor of the pancreas. Nuclear scintigraphy may provide the definitive diagnosis of an intrapancreatic spleen and therefore prevent patients from unnecessary major surgery.

T-cell recognition of chemicals, protein allergens and drugs: towards the development of in vitro assays

Chemicals can elicit T-cell-mediated diseases such as allergic contact dermatitis and adverse drug reactions. Therefore, testing of chemicals, drugs and protein allergens for hazard identification and risk assessment is essential in regulatory toxicology. The seventh amendment of the EU Cosmetics Directive now prohibits the testing of cosmetic ingredients in mice, guinea pigs and other animal species to assess their sensitizing potential. In addition, the EU Chemicals Directive REACh requires the retesting of more than 30,000 chemicals for different toxicological endpoints, including sensitization, requiring vast numbers of animals. Therefore, alternative methods are urgently needed to eventually replace animal testing. Here, we summarize the outcome of an expert meeting in Rome on 7 November 2009 on the development of T-cell-based in vitro assays as tools in immunotoxicology to identify hazardous chemicals and drugs. In addition, we provide an overview of the development of the field over the last two decades.

Risk profiles and penetrance estimations in multiple endocrine neoplasia type 2A caused by germline RET mutations located in exon 10

Multiple endocrine neoplasia type 2 is characterized by germline mutations in RET. For exon 10, comprehensive molecular and corresponding phenotypic data are scarce. The International RET Exon 10 Consortium, comprising 27 centers from 15 countries, analyzed patients with RET exon 10 mutations for clinical-risk profiles. Presentation, age-dependent penetrance, and stage at presentation of medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism were studied. A total of 340 subjects from 103 families, age 4-86, were registered. There were 21 distinct single nucleotide germline mutations located in codons 609 (45 subjects), 611 (50), 618 (94), and 620 (151). MTC was present in 263 registrants, pheochromocytoma in 54, and hyperparathyroidism in 8 subjects. Of the patients with MTC, 53% were detected when asymptomatic, and among those with pheochromocytoma, 54%. Penetrance for MTC was 4% by age 10, 25% by 25, and 80% by 50. Codon-associated penetrance by age 50 ranged from 60% (codon 611) to 86% (620). More advanced stage and increasing risk of metastases correlated with mutation in codon position (609?620) near the juxtamembrane domain. Our data provide rigorous bases for timing of premorbid diagnosis and personalized treatment/prophylactic procedure decisions depending on specific RET exon 10 codons affected.