RESUMEN
Identifying endocrine disrupting chemicals in order to limit their usage is a priority and required according to the European Regulation. There are no Organization for Economic Co-operation and Development (OECD) test guidelines based on fish available for the detection of Thyroid axis Active Chemicals (TACs). This study aimed to fill this gap by developing an assay at eleuthero-embryonic life stages in a novel medaka (Oryzias latipes) transgenic line. This transgenic line expresses green fluorescent protein (GFP) in thyrocytes, under the control of the medaka thyroglobulin gene promoter. The fluorescence expressed in the thyrocytes is inversely proportional to the thyroid axis activity. When exposed for 72 h to activators (triiodothyronine (T3) and thyroxine (T4)) or inhibitors (6-N-propylthiouracil (PTU), Tetrabromobisphenol A (TBBPA)) of the thyroid axis, the thyrocytes can change their size and express lower or higher levels of fluorescence, respectively. This reflects the regulation of thyroglobulin by the negative feedback loop of the Hypothalamic-Pituitary-Thyroid axis. T3, T4, PTU, and TBBPA induced fluorescence changes with the lowest observable effect concentrations (LOECs) of 5 µg/L, 1 µg/L, 8 mg/L, and 5 mg/L, respectively. This promising tool could be used as a rapid screening assay and also to help decipher the mechanisms by which TACs can disrupt the thyroid axis in medaka.
Asunto(s)
Oryzias , Glándula Tiroides , Animales , Glándula Tiroides/fisiología , Oryzias/fisiología , Tiroglobulina/metabolismo , Tiroglobulina/farmacología , Triyodotironina/metabolismo , Triyodotironina/farmacologíaRESUMEN
Widespread environmental antiandrogen contamination has been associated with negative impacts on biodiversity and human health. In particular, many pesticides are antiandrogenic, creating a need for robust and sensitive environmental monitoring. Our aim was to develop a sensitive and specific transgenic medaka (Oryzias latipes) model bearing an androgen responsive fluorescent reporter construct for whole organism-based environmental screening of pro- and antiandrogens. We analyzed the 5' regions of the androgen responsive three-spined stickleback (Gasterosteus aculeatus) spiggin genes in silico, revealing conserved blocks of sequence harboring androgen response elements. Identified putative promoters were cloned upstream of GFP. Germinal transgenesis with spg1-gfp led to stable medaka lines. GFP induction was exclusive to the kidney, the site of spiggin protein production in sticklebacks. Significant GFP expression was induced by three or four-day androgen treatment of newly hatched fry, but not by estrogens, mineralocorticoids, glucocorticoids or progestogens. The model responded dose-dependently to androgens, with highest sensitivity to 17MT (1.5 µg/L). In addition to flutamide, the biocides fenitrothion, vinclozolin and linuron significantly inhibited 17MT-induced GFP induction, validating the model for detection of antiandrogens. The spg1-gfp medaka model provides a sensitive, specific, and physiologically pertinent biosensor system for analyzing environmental androgen activity.
Asunto(s)
Antagonistas de Andrógenos/metabolismo , Proteínas de Peces/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Oryzias/metabolismo , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Clonación Molecular , Fluorescencia , Humanos , Datos de Secuencia Molecular , Oryzias/genética , Regiones Promotoras Genéticas/genética , Receptores Androgénicos/metabolismo , SmegmamorphaRESUMEN
Surface water receives a variety of micro-pollutants that could alter aquatic organisms' reproduction and development. It is known that a few nanograms per litre of these compounds can induce endocrine-disrupting effects in aquatic species. Many compounds are released daily in wastewater, and identifying the compounds responsible for inducing such disruption is difficult. Methods using biological analysis are therefore an alternative to chemical analysis, as the endocrine disruption potential of the stream as a whole is considered. To detect hormonal disruption of thyroid and oestrogenic functions, fluorescent Xenopus laevis tadpoles and medaka (Oryzias latipes) fish larvae bearing genetic constructs integrating hormonal responsive elements were used for physiological screens for potential endocrine disruption in streams from an urban wastewater treatment plant. The Xenopus model was used to assess thyroid disruption and the medaka model oestrogenic disruption in wastewater samples. Assays using the genetically modified organisms were conducted on 9 influent and 32 effluent samples. The thyroidal effect of wastewater was either reduced or removed by the treatment plant; no oestrogenic effect was detected in any of the wastewater samples.
Asunto(s)
Disruptores Endocrinos/toxicidad , Aguas Residuales/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Larva/efectos de los fármacos , Oryzias , Glándula Tiroides/efectos de los fármacos , Xenopus laevisRESUMEN
Over the previous decade, numerous new approach methodologies (NAMs) have been developed and validated for the detection of endocrine activity of individual chemicals or environmental samples. These NAMs can be largely separated into three categories, in silico tools, in vitro assays, and in vivo assays using organisms or life stages not considered as laboratory animals, each with their own advantages and disadvantages. While in vitro assays provide more mechanistic information, the use of whole organisms such as fish or amphibian embryos provides a more holistic view of the net effects of an environmental sample on hormonal activity. A panel of bioassays was used to test the endocrine activity of several samples from the Danube River at Novi Sad, Serbia. The results of the in vitro assays have been published previously. Here, we present the results of the in vivo assays that were performed at the same time on the same samples. These whole organism assays are based on the use of transgenic fish and amphibian eleuthero-embryos and included the Xenopus Eleuthero-embryo Thyroid Assay (XETA), the Rapid Estrogen ACTivity In Vivo assay (REACTIV), and the Rapid Androgen Disruption Activity Reporter (RADAR) assay. Discrepancies between the different in vitro assays have previously been reported. The results of the in vivo studies also indicate discrepancies between the in vivo and in vitro data with an underestimation of the endocrine activity by the in vitro tests. Therefore, a battery of tests is advised with the initial diagnostic performed with in vivo tests to cover a wider range of modes of action and to allow the appropriate in vitro assay(s) to be selected to confirm the mode of action. PRACTITIONER POINTS: Endocrine activity was quantified in surface water using in vitro and in vivo models. The in vivo results fit with previously reported in vitro results. Higher activity was observed in water samples with in vivo models, which cover a wider range of modes of action. Endocrine activity of surface water samples may be underestimated when measured with in vitro models.
Asunto(s)
Eleutherococcus , Disruptores Endocrinos , Contaminantes Químicos del Agua , Animales , Contaminantes Químicos del Agua/análisis , Disruptores Endocrinos/toxicidad , Disruptores Endocrinos/análisis , Peces , Agua , BioensayoRESUMEN
Gonadal development in medaka (Oryzias latipes) is dependent on the synergy between estrogens and androgens. Disruption of steroid hormone levels can lead to ovo-testis. To determine the sensitive windows for hormonally induced sex reversal in medaka, we developed a novel 42sp50-GFP_ChgH-GFP transgenic medaka line, allowing the identification of female gonadal tissue by fluorescence present in developing oocytes. Germinal transgenesis resulted in a stable line exhibiting a strong green fluorescent protein signal constitutively in the ovaries and in the liver in response to estrogens. The sensitivity of this line to disruption of sex determination following 16-d chronic exposures was in the nanograms per liter range. To identify the developmental period sensitive to exogenous agents, fry were exposed to 24-h pulses of high concentrations of 17ß-estradiol (E2) or 5α-dihydrotestosterone (DHT) at various time points between days postfertilization (dpf) 0 and 12. Evaluation of phenotype followed by genotyping at 16 dpf revealed sensitivity to E2 between 1 and 8 dpf as well as 2 periods of susceptibility to DHT between 0 and 1 dpf and 4 and 8 dpf. No phenotypic sex reversal was detected after exposure to DHT or E2 on 11 or 12 dpf. The observed effects persisted to at least 24 dpf. The identified sensitive embryonic time periods for disruption of sex determination will aid future research on sex determination and the development of screening assays using early embryonic life stages. Environ Toxicol Chem 2020;39:842-851. © 2020 SETAC.
Asunto(s)
Animales Modificados Genéticamente/embriología , Disruptores Endocrinos/toxicidad , Organogénesis/efectos de los fármacos , Oryzias/embriología , Ovario/embriología , Procesos de Determinación del Sexo/efectos de los fármacos , Animales , Dihidrotestosterona/toxicidad , Estradiol/toxicidad , Femenino , Proteínas Fluorescentes Verdes/genética , Masculino , Oryzias/metabolismo , Ovario/efectos de los fármacos , Ovario/metabolismoRESUMEN
Thyroid hormones (THs) are fundamental in regulation of growth and development, particularly of the brain. THs are required for full proliferative activity of neural stem cells in the subventricular zone (SVZ) of adult mouse brains, and also affect the normal fate of progenitor cells: apoptosis. Transthyretin (TTR) is a TH distributor protein in the blood and cerebrospinal fluid. TTR secretion by the choroid plexus is involved in transport of THs from blood into cerebrospinal fluid. We investigated the regulation of neural stem cell cycle in the SVZ of adult TTR null mice. Markers for neural stem cell mitosis that are reduced during hypothyroidism, did not differ between genotypes. However, in TTR null mice the level of apoptosis, the fate of most progenitor cells, was as low as that in brains of hypothyroid wildtype mice. Thus, lack of TTR results in reduced availability of TH to progenitor cells in the SVZ. We show that proliferation and apoptosis in the SVZ neural stem cell niche are differentially affected by the lack of TTR synthesis.
Asunto(s)
Apoptosis/genética , Diferenciación Celular/genética , División Celular/genética , Neuronas/fisiología , Prealbúmina/deficiencia , Células Madre/fisiología , Animales , Apoptosis/fisiología , Diferenciación Celular/fisiología , Ventrículos Cerebrales/citología , Ratones , Ratones Noqueados , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Several short-term whole-organism bioassays based on transgenic aquatic models are now under validation by the OECD (Organization for Economic Co-operation and Development) to become standardized test guidelines for the evaluation of the endocrine activity of substances. Evaluation of the endocrine disrupting capacity of pesticides will be a domain of applicability of these future reference tests. The herbicide linuron and the insecticide fenoxycarb are two chemicals commonly used in agricultural practices. While numerous studies indicate that linuron is likely to be an endocrine disruptor, there is little information available on the effect of fenoxycarb on vertebrate endocrine systems. Using whole-organism bioassays based on transgenic Xenopus laevis tadpoles and medaka fry we assessed the potential of fenoxycarb and linuron to disrupt thyroid, androgen and estrogen signaling. In addition we used in silico approach to simulate the affinity of these two pesticides to human hormone receptors. Linuron elicited thyroid hormone-like activity in tadpoles at all concentrations tested and, showed an anti-estrogenic activity in medaka at concentrations 2.5mg/L and higher. Our experiments suggest that, in addition to its previously established anti-androgenic action, linuron exhibits thyroid hormone-like responses, as well as acting at the estrogen receptor level to inhibit estrogen signaling. Fenoxycarb on the other hand, did not cause any changes in thyroid, androgen or estrogen signaling at the concentrations tested.
Asunto(s)
Disruptores Endocrinos/farmacología , Linurona/farmacología , Plaguicidas/farmacología , Fenilcarbamatos/farmacología , Animales , Animales Modificados Genéticamente , Bioensayo , Relación Dosis-Respuesta a Droga , Larva/efectos de los fármacos , Estructura Molecular , Oryzias , Fenilcarbamatos/química , Glándula Tiroides/efectos de los fármacos , Xenopus laevisRESUMEN
Vertebrate reproduction involves complex steroid hormone interplay and inter-conversion. A critical element in maintaining sex steroid levels is the enzyme aromatase (cytochrome P450 19A1) which converts androgens to oestrogens. In turn oestrogen signalling is targeted by numerous chemicals, from pharmaceuticals to agricultural chemicals, both frequent sources of contamination in waste waters and consequently rivers. Although many models are now available to address disruption of oestrogen signalling, there are currently no published protocols allowing discrimination between alterations in testosterone metabolism and in oestrogenic signalling. It was with this limitation in mind that we optimised this protocol. We show using a 48h protocol that pre-feeding fry of the choriogenin h-gfp (chgh-gfp) medaka line are sensitive to 0.05nM EE2 (15ng/L), within the range of the lowest published observable physiological effect concentrations for medaka. In addition, co-treatment with testosterone can reveal potential effects of test substances on aromatase enzymatic activity. As the measurements are visualised in real-time without affecting embryo viability, repeated measures are possible. We demonstrate the ability of this model to detect oestrogen receptor agonists, aromatisable androgens, P450 aromatase activity modulators and selective oestrogen response modulators. Importantly, the range of this assay is physiologically relevant.
Asunto(s)
Aromatasa/metabolismo , Estrógenos/farmacología , Animales , Animales Modificados Genéticamente , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Oryzias/genética , Contaminantes Químicos del Agua/farmacologíaRESUMEN
Environmental quality monitoring of water resources is challenged with providing the basis for safeguarding the environment against adverse biological effects of anthropogenic chemical contamination from diffuse and point sources. While current regulatory efforts focus on monitoring and assessing a few legacy chemicals, many more anthropogenic chemicals can be detected simultaneously in our aquatic resources. However, exposure to chemical mixtures does not necessarily translate into adverse biological effects nor clearly shows whether mitigation measures are needed. Thus, the question which mixtures are present and which have associated combined effects becomes central for defining adequate monitoring and assessment strategies. Here we describe the vision of the international, EU-funded project SOLUTIONS, where three routes are explored to link the occurrence of chemical mixtures at specific sites to the assessment of adverse biological combination effects. First of all, multi-residue target and non-target screening techniques covering a broader range of anticipated chemicals co-occurring in the environment are being developed. By improving sensitivity and detection limits for known bioactive compounds of concern, new analytical chemistry data for multiple components can be obtained and used to characterise priority mixtures. This information on chemical occurrence will be used to predict mixture toxicity and to derive combined effect estimates suitable for advancing environmental quality standards. Secondly, bioanalytical tools will be explored to provide aggregate bioactivity measures integrating all components that produce common (adverse) outcomes even for mixtures of varying compositions. The ambition is to provide comprehensive arrays of effect-based tools and trait-based field observations that link multiple chemical exposures to various environmental protection goals more directly and to provide improved in situ observations for impact assessment of mixtures. Thirdly, effect-directed analysis (EDA) will be applied to identify major drivers of mixture toxicity. Refinements of EDA include the use of statistical approaches with monitoring information for guidance of experimental EDA studies. These three approaches will be explored using case studies at the Danube and Rhine river basins as well as rivers of the Iberian Peninsula. The synthesis of findings will be organised to provide guidance for future solution-oriented environmental monitoring and explore more systematic ways to assess mixture exposures and combination effects in future water quality monitoring.
Asunto(s)
Conservación de los Recursos Naturales/métodos , Monitoreo del Ambiente/métodos , Contaminantes Químicos del Agua/análisis , Recursos Hídricos/estadística & datos numéricos , Calidad del Agua/normasRESUMEN
Early-life-stage transgenic medaka are recognized as a pertinent model by the Organisation for Economic Co-operation and Development and are noncompliant with the European definition of a laboratory animal. However, autofluorescence confounds readout of fluorescent biomarkers. The authors determined the fluorescence emission spectrum of different embryonic stages of medaka submitted to a range of excitation wavelengths. This allows selection of high signal-to-noise ratio fluorescent proteins and combining multiple biomarkers within a single embryo.
Asunto(s)
Proteínas Fluorescentes Verdes/metabolismo , Oryzias/embriología , Oryzias/metabolismo , Animales , Animales Modificados Genéticamente , Biomarcadores/metabolismo , Proteínas del Huevo/genética , Fluorescencia , Proteínas Fluorescentes Verdes/genética , Azul de Metileno , Oryzias/genética , Precursores de Proteínas/genética , Relación Señal-RuidoRESUMEN
While numerous detection methods exist for environmental heavy metal monitoring, easy-to-use technologies combining rapidity with in vivo measurements are lacking. Multiwell systems exploiting transgenic tadpoles are ideal but require time-consuming placement of individuals in wells. We developed a real-time flow-through system, based on Fountain Flow cytometry, which measures in situ contaminant-induced fluorescence in transgenic amphibian larvae immersed in water samples. The system maintains the advantages of transgenic amphibians, but requires minimal human intervention. Portable and self-contained, it allows on-site measurements. Optimization exploited a transgenic Xenopus laevis bearing a chimeric gene with metal responsive elements fused to eGFP. The transgene was selectively induced by 1 microM Zn(2+). Using this tadpole we show the continuous flow method to be as rapid and sensitive as image analysis. Flow-through readings thus accelerate the overall process of data acquisition and render fluorescent monitoring of tadpoles suitable for on-site tracking of heavy metal pollution.
Asunto(s)
Monitoreo del Ambiente/métodos , Metales Pesados/análisis , Contaminantes Químicos del Agua/análisis , Contaminación del Agua/análisis , Xenopus laevis/genética , Animales , Animales Modificados Genéticamente , Fluorescencia , Proteínas Fluorescentes Verdes/metabolismo , Larva/citología , Larva/efectos de los fármacos , Metalotioneína/metabolismo , Reproducibilidad de los Resultados , Elementos de Respuesta/genética , Hormonas Tiroideas/farmacología , Zinc/análisisRESUMEN
BACKGROUND: Efficient in vivo vectors are needed to exploit the enormous potential of RNA interference (RNAi). Such methods require optimisation for specific delivery routes, tissues and usages. We tested the capacity of different non-viral vectors and formulation methods for inhibition of exogenous (luciferase) gene expression when used to introduce small interfering RNA (siRNA) into the mouse brain in vivo. METHODS: Polyethylenimine (PEI)-based polyplexes and JetSI (a mixture of cationic lipids)-based lipoplexes were used to vectorise plasmid DNA encoding the firefly Photinus pyralis luciferase gene and picomolar amounts of siRNA directed against this gene. Two controls were used, DNA encoding an unrelated luciferase from Renilla reniformis and a mutated siRNA sequence. RESULTS: First, we found that linear PEI, although efficient for delivering nucleic acids to cells, did not permit development of siRNA activity within the dose range tested (<0.5 pmol). Second, various combinations of cationic lipids were tried and the best formulation was found to be a combination of JetSI with the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE). Efficient inhibition of target, firefly luciferase was obtained with exceedingly low amounts of siRNA: 78 +/- 6% inhibition at 24 h post-transfection with 0.2 pmol siRNA. This inhibition was dose-dependent and specific. No effect was seen on the control gene, co-transfected Renilla luciferase, and the control mutated siRNA sequence had no effect on the targeted firefly luciferase. CONCLUSIONS: We have optimised an efficient cationic lipoplex method for delivery of siRNA into the newborn mouse brain. Specific inhibition of exogenous target gene expression is obtained with picomolar amounts of siRNA.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Vectores Genéticos/metabolismo , Luciferasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Análisis de Varianza , Animales , Animales Recién Nacidos , Secuencia de Bases , Encéfalo/metabolismo , Femenino , Luciérnagas/enzimología , Técnicas de Transferencia de Gen , Metabolismo de los Lípidos , Masculino , Ratones , Tamaño de la Partícula , Fosfatidiletanolaminas/metabolismo , Polietileneimina/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Renilla/enzimologíaRESUMEN
The subventricular zone of the adult mammalian brain harbors the neural stem cell population with potential neural regeneration and repair capacity. We describe a nonviral technique to preferentially transfect in vivo the adult neural stem cell population and its immediate progeny based on intraventricular injection of PEI/DNA complexes. The transfected population was identified by cellular and ultra-structural evidence showing their proliferating status and expression of the specific markers GFAP and nestin. Stable activation of the lacZ reporter by cre-recombinase transfection in R26R mice demonstrated survival and migration of stem cell derivatives three months after injection. Apoptosis is thought to be the most common fate of the stem cell progeny. Overexpression of Bcl-X(L) increased number and survival time of transduced progenitors and decreased the frequency of cells immunopositive for activated Caspase-3. This method thus provides selective targeting of the stem cell population and should allow an in-depth understanding of their biology.