Endocrine Disruption In Vertebrates Research Articles

Developmental assays using invasive cane toads, Rhinella marina, reveal safety concerns of a common formulation of the rice herbicide, butachlor.

Identifying the adverse impacts of pesticide exposure is essential to guide regulations that are protective of wildlife and human health. Within rice ecosystems, amphibians are valuable indicators because pesticide applications coincide with sensitive reproductive and developmental life stages. We conducted two experiments using wild cane toads (Rhinella marina) to test 1) whether environmentally relevant exposure to a commercial formulation of butachlor, an acetanilide herbicide used extensively in rice, affects amphibian development and 2) whether cane toad tadpoles are capable of acclimatizing to sub-lethal exposure. First, we exposed wild cane toads to 0.002, 0.02, or 0.2mg/L of butachlor (Machete EC), during distinct development stages (as eggs and hatchlings, as tadpoles, or continuously) for 12 days. Next, we exposed a subset of animals from the first experiment to a second, lethal concentration and examined survivorship. We found that cane toads exposed to butachlor developed slower and weighed less than controls, and that development of the thyroid gland was affected: exposed individuals had smaller thyroid glands and thyrocyte cells, and more individual follicles. Analyses of the transcriptome revealed that butachlor exposure resulted in downregulation of transcripts related to metabolic processes, anatomic structure development, immune system function, and response to stress. Last, we observed evidence of acclimatization, where animals exposed to butachlor early in life performed better than naïve animals during a second exposure. Our findings indicate that the commercial formulation of butachlor, Machete EC, causes thyroid endocrine disruption in vertebrates, and suggest that exposure in lowland irrigated rice fields presents a concern for wildlife and human health. Furthermore, we establish that developmental assays with cane toads can be used to screen for adverse effects of pesticides in rice fields.

Ferredoxin 1b Deficiency Leads to Testis Disorganization, Impaired Spermatogenesis, and Feminization in Zebrafish.

The roles of steroids in zebrafish sex differentiation, gonadal development, and function of the adult gonad are poorly understood. Herein, we used ferredoxin 1b (fdx1b) mutant zebrafish to explore such processes. Fdx1b is an essential electron-providing cofactor to mitochondrial steroidogenic enzymes, which are crucial for glucocorticoid and androgen production in vertebrates. Fdx1b-/- zebrafish mutants develop into viable adults in which concentrations of androgens and cortisol are significantly reduced. Adult fdx1b-/- mutant zebrafish display predominantly female secondary sex characteristics but may possess either ovaries or testes, confirming that androgen signaling is dispensable for testicular differentiation in this species, as previously demonstrated in androgen receptor mutant zebrafish. Adult male fdx1b-/- mutant zebrafish exhibit reduced characteristic breeding behaviors and impaired sperm production, resulting in infertility in standard breeding scenarios. However, eggs collected from wild-type females can be fertilized by the sperm of fdx1b-/- mutant males by in vitro fertilization. The testes of fdx1b-/- mutant males are disorganized and lack defined seminiferous tubule structure. Expression of several promale and spermatogenic genes is decreased in the testes of fdx1b-/- mutant males, including promale transcription factor sox9a and spermatogenic genes igf3 and insl3. This study establishes an androgen- and cortisol-deficient fdx1b zebrafish mutant as a model for understanding the effects of steroid deficiency on sex development and reproductive function. This model will be particularly useful for further investigation of the roles of steroids in spermatogenesis, gonadal development, and regulation of reproductive behavior, thus enabling further elucidation of the physiological consequences of endocrine disruption in vertebrates.

Effects of single exposure and binary mixtures of ultraviolet filters octocrylene and 2-ethylhexyl 4-(dimethylamino) benzoate on gene expression in the freshwater insect Chironomus riparius.

Ultraviolet filters are used extensively in the production of many personal care and industrial products. These products can inadvertently pollute the environment through recreational activities. They have been associated with endocrine disruption in vertebrates but their effects in invertebrates are poorly understood. Chironomus riparius is a species of the dipteran order, with aquatic larvae that are frequently used in toxicity tests. Previously, we showed that octocrylene (OC) and 2-ethylhexyl 4-(dimethylamino) benzoate (OD-PABA) differentially affected the mRNA levels of the ecdysone receptor and Hsp70 genes. For a better understanding of their mode of action, transcriptional activity by real-time PCR was analyzed in fourth instar larvae exposed to OC, OD-PABA, or a binary mixture of both. We studied 16 genes related to the endocrine system, stress, the immune system, and biotransformation mechanisms to elucidate the putative interactions between these compounds. No response was observed for the genes involved in biotransformation, suggesting that enzymes other than cytochromes P450 and glutathione-S-transferases (GSTs) could get involved in transformation of these compounds. Similarly, no response was observed for endocrine-related genes while the stress gene HYOU1 was inhibited by OD-PABA, suggesting an effect in response to hypoxia. In addition, no significant interactions were observed following exposure to a binary mixture of these compounds. Overall, the results suggest a weak, acute response in different metabolic pathways and a lack of interaction between the compounds. Finally, new genes are identified in this organism, opening the possibility to analyze new cellular pathways as targets of toxicants.

Progestins alter photo-transduction cascade and circadian rhythm network in eyes of zebrafish (Danio rerio)

Environmental progestins are implicated in endocrine disruption in vertebrates. Additional targets that may be affected in organisms are poorly known. Here we report that progesterone (P4) and drospirenone (DRS) interfere with the photo-transduction cascade and circadian rhythm network in the eyes of zebrafish. Breeding pairs of adult zebrafish were exposed to P4 and DRS for 21 days with different measured concentrations of 7–742 ng/L and 99-13´650 ng/L, respectively. Of totally 10 key photo-transduction cascade genes analyzed, transcriptional levels of most were significantly up-regulated, or normal down-regulation was attenuated. Similarly, for some circadian rhythm genes, dose-dependent transcriptional alterations were also observed in the totally 33 genes analyzed. Significant alterations occurred even at environmental relevant levels of 7 ng/L P4. Different patterns were observed for these transcriptional alterations, of which, the nfil3 family displayed most significant changes. Furthermore, we demonstrate the importance of sampling time for the determination and interpretation of gene expression data, and put forward recommendations for sampling strategies to avoid false interpretations. Our results suggest that photo-transduction signals and circadian rhythm are potential targets for progestins. Further studies are required to assess alterations on the protein level, on physiology and behavior, as well as on implications in mammals.

Impact of three phthalate esters on the sexual reproduction of the Monogonont rotifer, Brachionus calyciflorus.

Phthalate esters are widespread contaminants that can cause endocrine disruption in vertebrates. Studies showed that molecules with hormonal activities in vertebrates and invertebrates can affect asexual and sexual reproduction in rotifers. We investigated the impact of di-hexylethyl phthalate (DEHP), di-butyl phthalate (DBP) and butylbenzyl phthalate (BBP), on the asexual and sexual reproduction of the freshwater monogonont rotifer Brachionus calyciflorus in order to determine a potential environmental risk for sexual reproduction. We observed that DEHP has no significant impact on both asexual and sexual reproduction up to 2mg/L. DBP has a positive effect on asexual reproduction at concentrations from 0.05 to 1mg/L, but depresses it at 2mg/L. Sexual reproduction is only affected at 2mg/L and the impact observed is negative. BBP displayed a negative impact on both asexual and sexual reproduction at 1 and 2mg/L. However we showed that the impacts of BBP on mixis and fertilization rates observed are due to the decrease in population growth rates at these concentrations and not to a direct impact of BBP on the mixis and the fertilization processes. Our results show that sexual reproduction in B. calyciflorus is not more sensitive than asexual reproduction to any of the substances tested which indicates the mode of action of these molecules is related to general toxicity and not to an interference with potential endocrine regulation of sexual reproduction. Comparison of effect concentrations and surface water contamination by phthalate esters suggests these compounds do not constitute a risk for primary consumers in these environments.

Phytoestrogensβ-Sitosterol and Genistein Have Limited Effects on Reproductive Endpoints in a Female Fish,Betta splendens

Phytoestrogens are produced by plants and may cause endocrine disruption in vertebrates. The present study hypothesizes that phytoestrogen exposure of female Siamese fighting fish (Betta splendens) may disrupt endogenous steroid levels, change agonistic behavior expression, and potentially also disrupt oocyte development. However, only the pharmacologic dose of β-sitosterol had a significant effect on opercular flaring behavior, while we did not find significant effects of β-sitosterol or genistein on steroids or gonads. These findings are in direct contrast with previous studies on the effects of phytoestrogens in female fish. Results of the current study support previous work showing that the effects of phytoestrogen exposure may be less acute in mature female B. splendens than in other fish.

Early exposure of 17α‐ethynylestradiol and diethylstilbestrol induces morphological changes and alters ovarian steroidogenic pathway enzyme gene expression in catfish, Clarias gariepinus

Environmental estrogens are major cause of endocrine disruption in vertebrates, including aquatic organisms. Teleosts are valuable and popular models for studying the effects of endocrine disrupting chemicals (EDCs) in the environment. In the present study, we investigated the changes caused by exposure to the synthetic estrogens 17α-ethynylestradiol (EE2 ) and diethylstilbesterol (DES) during early stages of growth and sex differentiation of air-breathing catfish, Clarias gariepinus, at the morphological, histological, and molecular levels. Catfish hatchlings, 0 day post hatch (dph) were exposed continuously to sublethal doses of EE2 (50 ng/L) and DES (10 ng/L) until 50 dph and subsequently monitored for ovarian structural changes and alteration in the gene expression of steroidogenic enzymes till adulthood. Treated fish exhibited morphological deformities such as spinal curvature, stunted growth, and yolk-sac fluid retention. In addition to ovarian atrophy, DES-treated fish showed either rudimentary or malformed ovaries. Detailed histological studies revealed precocious oocyte development as well as follicular atresia. Further, transcript levels of various steroidogenic enzyme and transcription factor genes were altered in response to EE2 and DES. Activity of the rate-limiting enzyme of estrogen biosynthesis, aromatase, in the ovary as well as the brain of treated fish was in accordance with transcript level changes. These developmental and molecular effects imparted by EE2 and DES during early life stages of catfish could demonstrate the deleterious effects of estrogen exposure and provide reliable markers for estrogenic EDCs exposure in the environment.

Identification of reliable test procedures to detect endocrine disruptive and reproduction toxic effects in aquatic ecosystems

Hintergrund und Ziel Aus den Ergebnissen umfangreicher Forschungsprogramme, wie z. B. dem Schweizer Forschungsprojekt NFP50 „Hormonaktive Stoffe“ und deren Konsensplattformen zeigt sich ein zunehmender Handlungsbedarf hormonaktive Wirkungen in aquatischen Systemen nachweisen, bewerten und reduzieren zu konnen. Die EU-Strategie fur endokrine Disruptoren beinhaltet als mittelfristige Masnahmen die Beteiligung der „Organisation for Economic Co-operation and Development“ (OECD) durch die „Endocrine Disruptor Testing and Assessment Task Force“ (EDTA) und weitere Forschungsaktivitaten. Die Testverfahrensentwicklungen der OECD konnen erhebliche Beitrage zum Verstandnis des Ausmases der endokrinen Disruption leisten, sofern diese auch auf Umweltproben angewendet und fur Risikominderungsstrategien (z. B. Abwasserbehandlungen) eingesetzt werden. Ziel dieses Reviews ist es, eine Zusammenfassung und Bewertung von verfugbaren und in der Validierung befindlichen biologischen Testsystemen zu erstellen, mit denen Effekte hormonaktiver und reproduktionstoxischer Substanzen in Gewassern nachgewiesen werden konnen. Auf dieser Grundlage erfolgt eine Empfehlung einer okotoxikologischen Testpalette, die es erlaubt, die unterschiedlichen Mechanismen der hormonaktiven Wirkungen und reproduktionsrelevanten Wirkungen zuverlassig in Umweltproben zu erfassen. Der Schwerpunkt dieser Arbeit liegt auf den Testverfahren fur geschlechtshormonaktive Substanzen. Material und Methoden Ausgehend von einer Literaturrecherche und aktuellen internationalen Validierungsaktivitaten fur Nachweismoglichkeiten hormonaktiver und reproduktionstoxischer Substanzen und Wirkungen wurden 15 biologische Testverfahren (5 In-vivo- und 10 In-vitro-Testverfahren) ausgewahlt. Es wurden 8 Verfahren der OECD-EDTA erfasst und 3 von 5 In-vitro-Verfahren aus dem „Global Water Research Coalition“ (GWRC) Report: „Tools to detect estrogenic activity in environmental waters“ (Leusch 2008) ausgewahlt. Fur diese Testverfahren erfolgte eine Abfrage umfassender Kriterienprofile bei versierten Anwendern und Entwicklern, die vergleichend dargestellt und ausgewertet wurden. Ergebnisse Wichtige Kriterien bei der Auswahl der Testverfahren waren Normung/Validierung oder Verbreitung und Standardisierbarkeit der Tests sowie eine nachgewiesene Sensitivitat in der Bewertung von Umweltproben. Bei den In-vitro-Tests erfullen dies unserer Meinung nach bestimmte YES/YAS-Systeme (Yeast Estrogen/Androgen Screen Assay) und die ER/AR-Calux-Verfahren (Estrogen/Androgen Receptor). Sollte deren Anwendung durch starke zytotoxische Effekte in Umweltproben behindert werden, kann z. B. ein molekularer Rezeptorbindungsassay, wie der ELRA, Rezeptorbindungspotenziale nachweisen. Weitere molekulare Rezeptorbindungsassays sind derzeitig in der Validierung der OECD. Ebenfalls von der OECD fur Einzelsubstanzen validiert, kann der H295R Steroidgenesis Assay Modulationen der Steroidgenese, und eventuell sogar aromatasemodulierende Effekte nachweisen. Seine Anwendbarkeit auf Umweltproben wird derzeitig getestet. Fur die In-vivo-Stufe der modularen Testplattform wurden verschiedene Fischtests (z. B. der Fish Screening Assay und der Fischeitest) bewertet. Um Einflusse auf das thyroidale Hormonsystem nachzuweisen ist der Amphibian Metamorphosis Assay (in der Abfrage unter XEMA, Xenopus Metamorphosis Assay) am weitesten validiert und wird bald als offizielle OECD-Richtlinie erscheinen. Da es sich auch hier um einen Vertebratentest handelt und internationale Bestrebungen bestehen Vertebratentests zu vermeiden, erfolgte nur eine optionale Empfehlung. Reproduktionstoxische Wirkungen konnen z. B. sehr sensitiv im Schneckenreproduktionstest mit Potamopyrgus antipodarum nachgewiesen werden. Dieser reagiert auch auf viele Substanzen, die in Vertebraten eine endokrine Disruption verursachen. Eine Validierung erfolgt derzeitig durch die OECD. Eine Eignung der in der modularen Testpalette vorgeschlagenen Verfahren fur Umweltprobenbewertungen wurde angegeben und kann durch mehrere Literaturquellen belegt werden. Diskussion Es wurde eine anwendungsorientierte modulare Testpalette mit verschiedenen Testverfahren vorgeschlagen, die moglichst effektiv und vielseitig die Mechanismen der endokrinen Disruption und die Reproduktionstoxizitat nachzuweisen vermogen und sich dabei in ihren Starken und unterschiedlichen Arbeitsbereichen erganzen. Es konnen fortpflanzungsrelevante Wirkungen an Invertebraten, Amphibien und Fischen und die wirkmechanistischen Ansatze der ostrogenen und androgenen Rezeptorbindung, der Steroidgenese, eventuell sogar der Aromatasemodulation erfasst werden. Neuere Forschungsergebnisse, wie z. B. der Forschungsbericht zur Gewasserrelevanz endokriner Stoffe und Arzneimittel (Moltmann et al. 2007), konnten anhand aquatisch relevanter Schadstoffe mit In-vivo-Tests nachweisen, dass endokrine Endpunkte (bei 31 von 71 untersuchten Stoffen) empfindlicher sein konnen als allgemeine okotoxikologische Endpunkte (z. B. Mortalitat, Wachstum usw.). Daher wird empfohlen endokrine Effekte nicht isoliert zu betrachten, sondern parallel mit allgemeintoxischen und fortpflanzungsrelevanten Wirkungen in okotoxikologischen Biotestpaletten integrativ zu erfassen. Schlussfolgerungen Durch die Kombination einer Literaturrecherche und der gezielten Abfrage von Informationen zu den Testverfahren konnte eine umfassende, aber auch detaillierte Ubersicht zum derzeitigen Stand der Wissenschaft und Technik gegeben werden. Dabei wurden okotoxikologische und regulative Aspekte der Testverfahren gleichermasen berucksichtigt und deren Praktikabilitat fur ein Umweltmonitoring in aquatischen Systemen bewertet. Bei der Vielfaltigkeit der endokrinen Wirkmechanismen bedarf es einer modularen Kombination von In-vivo- und In-vitro-Verfahren in einer Testpalette, um auch die fliesenden Ubergange von hormonaktiven Wirkungen bis hin zu einer manifestierten endokrinen Disruption erfassen und differenzieren zu konnen. Empfehlungen und Perspektiven Die vorgestellte Testpalette erlaubt einen umfassenden Schutz vor hormonaktiven und reproduktionsrelevanten Effekten, wobei eine Anpassung an die Interessen und Zielsetzungen der Anwender erfolgen kann. Es werden sowohl die regulativen und okotoxikologischen Anforderungen an Testsysteme als auch die aktuellen Entwicklungen in der EU und der OECD mit berucksichtigt. Eine weiterfuhrende Normung dieser Verfahren fur den regulativen Einsatz in der Umweltuberwachung und fur Risikominderungsstrategien ist zu empfehlen. Das modulare System erlaubt es, einen anwendungsorientierten Austausch der Testmodule nach dem sich kontinuierlich entwickelnden Stand der Technik ohne Neuentwicklungen auszuschliesen.

Selected endocrine disrupting compounds (Vinclozolin, Flutamide, Ketoconazole and Dicofol): Effects on survival, occurrence of males, growth, molting and reproduction of Daphnia magna

Pollution-induced endocrine disruption in vertebrates and invertebrates is a worldwide environmental problem, but relatively little is known about effects of endocrine disrupting compounds (EDCs) in planktonic crustaceans (including Daphnia magna). Aims of the present study were to investigate acute 48 h toxicity and sub-chronic (4-6 days) and chronic (21 days) effects of selected EDCs in D. magna. We have investigated both traditional endpoints as well as other parameters such as sex determination, maturation, molting or embryogenesis in order to evaluate the sensitivity and possible use of these endpoints in ecological risk assessment. We have studied effects of four model EDCs (vinclozolin, flutamide, ketoconazole and dicofol) on D. magna using (i) an acute 48 h immobilization assay, (ii) a sub-chronic, 4-6 day assay evaluating development and the sex ratio of neonates, and (iii) a chronic, 21 day assay studying number of neonates, sex of neonates, molting frequency, day of maturation and the growth of maternal organisms. Acute EC50 values in the 48 h immobilization test were as follows (mg/L): dicofol 0.2, ketoconazole 1.5, flutamide 2.7, vinclozolin >3. Short-term, 4-6 day assays with sublethal concentrations showed that the sex ratio in Daphnia was modulated by vinclozolin (decreased number of neonate males at 1 mg/L) and dicofol (increase in males at 0.1 mg/L). Flutamide (up to 1 mg/L) had no effect on the sex of neonates, but inhibited embryonic development at certain stages during chronic assay, resulting in abortions. Ketoconazole had no significant effects on the studied processes up to 1 mg/L. Sex ratio modulations by some chemicals (vinclozolin and dicofol) corresponded to the known action of these compounds in vertebrates (i.e. anti-androgenicity and anti-oestrogenicity, respectively). Our study revealed that some chemicals known to affect steroid-regulated processes in vertebrates can also affect sublethal endpoints (e.g. embryonic sex determination and/or reproduction) in invertebrates such as D. magna. A series of model vertebrate endocrine disrupters affected various sub-chronic and chronic parameters in D. magna including several endpoints that have not been previously studied in detail (such as sex determination in neonates, embryogenesis, molting and maturation). Evaluations of traditional reproduction parameters (obtained from the 21 day chronic assay). as well as the results from a rapid, 4-6 day, sub-chronic assay provide complementary information on non-lethal effects of suspected organic endocrine disrupters. It seems that there are analogies between vertebrates and invertebrates in toxicity mechanisms and in vivo effects of endocrine disruptors. However, general physiological status of organisms may also indirectly affect endpoints that are traditionally considered 'hormone regulated' (especially at higher effective concentrations as observed in this study) and these factors should be carefully considered. Further research of D. magna physiology and comparative studies with various EDCs will help to understand mechanisms of action as well as ecological risks of EDCs in the environment.

Application of Ecotoxicogenomics for Studying Endocrine Disruption in Vertebrates and Invertebrates

Chemicals released into the environment potentially disrupt the endocrine system in wild animals and humans. Developing organisms are particularly sensitive to estrogenic chemicals. Exposure to estrogens or estrogenic chemicals during critical periods of development induces persistent changes in both reproductive and nonreproductive organs, including persistent molecular alterations. Estrogen-responsive genes and critical developmental windows of various animal species, therefore, need to be identified for investigators to understand the molecular basis of estrogenic activity during embryonic development. For investigators to understand molecular mechanisms of toxicity in various species, toxicogenomics/ecotoxicogenomics, defined as the integration of genomics (transcriptomics, proteomics, metabolomics) into toxicology and ecotoxicology, need to be established as powerful tools for research. As the initial step toward using genomics to examine endocrine-disrupting chemicals, estrogen receptors and other steroid hormone receptors have been cloned in various species, including reptiles, amphibians, and fish, and alterations in the expression of these genes in response to chemicals were investigated. We are identifying estrogen-responsive genes in mouse reproductive tracts using cDNA microarrays and trying to establish microarray systems in the American alligator, roach, medaka, and water fleas (Daphnia magna). It is too early to define common estrogen-responsive genes in various animal species; however, toxicogenomics and ectotoxicogenomics provide powerful tools to help us understand the molecular mechanism of chemical toxicities in various animal species.

Survival and precopulatory behaviour of Gammarus pulex (L.) exposed to two xenoestrogens

The effects of the xenoestrogens ethinylestradiol (EE) and bisphenol A (BPA) on the survival and reproductive behaviour of the amphipod Gammarus pulex were determined in a series of bioassays. Acute toxicity of the test chemicals was determined under static conditions, revealing 10 d median lethal concentrations (LC50s) of 0.84 and 1.49 mg/L for EE and BPA, respectively. Several aspects of the reproductive behaviour including the ability of males and females to detect each other, form precopulatory guarding pairs and to continue the guarding behaviour, were examined during a 24 h exposure period over a wide range of concentrations. However, reproductive behaviour was only disrupted at relatively high concentrations where it would be unrealistic to attribute the effects to an endocrine mediated process. Consequently, changes in precopulatory guarding resulting from acute exposure, do not seem to be suitable for detecting the presence in water of xenoestrogens known to cause endocrine disruption in vertebrates.