A Novel Transgenic Model to Study Thyroid Axis Activity in Early Life Stage Medaka.

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.

Evaluation of the endocrine activity of surface water samples using aquatic eleuthero-embryos-A comparison with in vitro assays.

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.

Electrochemical degradation of 17α-ethinylestradiol: Transformation products, degradation pathways and in vivo assessment of estrogenic activity.

Conventional water treatment methods are not efficient in eliminating endocrine disrupting compounds (EDCs) in wastewater. Electrochemical Advanced Oxidation Processes (eAOPs) offer a promising alternative, as they electro-generate highly reactive species that oxidize EDCs. However, these processes produce a wide spectrum of transformation products (TPs) with unknown chemical and biological properties. Therefore, a comprehensive chemical and biological evaluation of these remediation technologies is necessary before they can be safely applied in real-life situations. In this study, 17α-ethinylestradiol (EE2), a persistent estrogen, was electrochemically degraded using a boron doped diamond anode with sodium sulfate (Na2SO4) and sodium chloride (NaCl) as supporting electrolytes. Ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry was used for the quantification of EE2 and the identification of TPs. Estrogenic activity was assessed using a transgenic medaka fish line. At optimal operating conditions, EE2 removal reached over 99.9% after 120 min and 2 min, using Na2SO4 and NaCl, respectively. The combined EE2 quantification and in vivo estrogenic assessment demonstrated the overall estrogenic activity was consistently reduced with the degradation of EE2, but not completely eradicated. The identification and time monitoring of TPs showed that the radical agents readily oxidized the phenolic A-ring of EE2, leading to the generation of hydroxylated and/or halogenated TPs and ring-opening products. eAOP revealed to be a promising technique for the removal of EE2 from water. However, caution should be exercised with respect to the generation of potentially toxic TPs.

Transgenic Medaka Identify Embryonic Periods Sensitive to Disruption of Sex Determination.

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.

Effect-based trigger values for in vitro and in vivo bioassays performed on surface water extracts supporting the environmental quality standards (EQS) of the European Water Framework Directive.

Effect-based methods including cell-based bioassays, reporter gene assays and whole-organism assays have been applied for decades in water quality monitoring and testing of enriched solid-phase extracts. There is no common EU-wide agreement on what level of bioassay response in water extracts is acceptable. At present, bioassay results are only benchmarked against each other but not against a consented measure of chemical water quality. The EU environmental quality standards (EQS) differentiate between acceptable and unacceptable surface water concentrations for individual chemicals but cannot capture the thousands of chemicals in water and their biological action as mixtures. We developed a method that reads across from existing EQS and includes additional mixture considerations with the goal that the derived effect-based trigger values (EBT) indicate acceptable risk for complex mixtures as they occur in surface water. Advantages and limitations of various approaches to read across from EQS are discussed and distilled to an algorithm that translates EQS into their corresponding bioanalytical equivalent concentrations (BEQ). The proposed EBT derivation method was applied to 48 in vitro bioassays with 32 of them having sufficient information to yield preliminary EBTs. To assess the practicability and robustness of the proposed approach, we compared the tentative EBTs with observed environmental effects. The proposed method only gives guidance on how to derive EBTs but does not propose final EBTs for implementation. The EBTs for some bioassays such as those for estrogenicity are already mature and could be implemented into regulation in the near future, while for others it will still take a few iterations until we can be confident of the power of the proposed EBTs to differentiate good from poor water quality with respect to chemical contamination.

Identification of Unknown Antiandrogenic Compounds in Surface Waters by Effect-Directed Analysis (EDA) Using a Parallel Fractionation Approach.

Among all the nuclear-receptor mediated endocrine disruptive effects, antiandrogenicity is frequently observed in aquatic environments and may pose a risk to aquatic organisms. Linking these effects to responsible chemicals is challenging and a great share of antiandrogenic activity detected in the environment has not been explained yet. To identify drivers of this effect at a hot spot of antiandrogenicity in the German river Holtemme, we applied effect-directed analysis (EDA) including a parallel fractionation approach, a downscaled luciferase reporter gene cell-based anti-AR-CALUX assay and LC-HRMS/MS nontarget screening. We identified and confirmed the highly potent antiandrogen 4-methyl-7-diethylaminocoumarin (C47) and two derivatives in the active fractions. The relative potency of C47 to the reference compound flutamide was over 5.2, whereas the derivatives were less potent. C47 was detected at a concentration of 13.7 µg/L, equal to 71.4 µg flutamide equivalents per liter (FEq/L) in the nonconcentrated water extract that was posing an antiandrogenic activity equal to 45.5 (±13.7 SD) FEq/L. Thus, C47 was quantitatively confirmed as the major cause of the measured effect in vitro. Finally, the antiandrogenic activity of C47 and one derivate was confirmed in vivo in spiggin-gfp Medaka. An endocrine disrupting effect of C47 was observed already at the concentration equal to the concentration in the nonconcentrated water extract, underlining the high risk posed by this compound to the aquatic ecosystem. This is of some concern since C47 is used in a number of consumer products indicating environmental as well as human exposure.

Effect-based assessment of toxicity removal during wastewater treatment.

Wastewaters contain complex mixtures of chemicals, which can cause adverse toxic effects in the receiving environment. In the present study, the toxicity removal during wastewater treatment at seven municipal wastewater treatment plants (WWTPs) was investigated using an effect-based approach. A battery of eight bioassays was applied comprising of cytotoxicity, genotoxicity, endocrine disruption and fish embryo toxicity assays. Human cell-based CALUX assays, transgenic larval models and the fish embryo toxicity test were particularly sensitive to WWTP effluents. The results indicate that most effects were significantly reduced or completely removed during wastewater treatment (76-100%), while embryo toxicity, estrogenic activity and thyroid disruption were still detectable in the effluents suggesting that some harmful substances remain after treatment. The responsiveness of the bioassays was compared and the human cell-based CALUX assays showed highest responsiveness in the samples. Additionally, the fish embryo toxicity test and the transgenic larval models for endocrine disrupting effects showed high responsiveness at low sample concentrations in nearly all of the effluent samples. The results showed a similar effect pattern among all WWTPs investigated, indicating that the wastewater composition could be rather similar at different locations. There were no considerable differences in the toxicity removal efficiencies of the treatment plants and no correlation was observed with WWTP characteristics, such as process configuration or sludge age. This study demonstrated that a biotest battery comprising of multiple endpoints can serve as a powerful tool when assessing water quality or water treatment efficiency in a holistic manner. Rather than analyzing the concentrations of a few selected chemicals, bioassays can be used to complement traditional methods of monitoring in the future by assessing sum-parameter based effects, such as mixture effects, and tackling chemicals that are present at concentrations below chemical analytical detection limits.

Development of a bioanalytical test battery for water quality monitoring: Fingerprinting identified micropollutants and their contribution to effects in surface water.

Surface waters can contain a diverse range of organic pollutants, including pesticides, pharmaceuticals and industrial compounds. While bioassays have been used for water quality monitoring, there is limited knowledge regarding the effects of individual micropollutants and their relationship to the overall mixture effect in water samples. In this study, a battery of in vitro bioassays based on human and fish cell lines and whole organism assays using bacteria, algae, daphnids and fish embryos was assembled for use in water quality monitoring. The selection of bioassays was guided by the principles of adverse outcome pathways in order to cover relevant steps in toxicity pathways known to be triggered by environmental water samples. The effects of 34 water pollutants, which were selected based on hazard quotients, available environmental quality standards and mode of action information, were fingerprinted in the bioassay test battery. There was a relatively good agreement between the experimental results and available literature effect data. The majority of the chemicals were active in the assays indicative of apical effects, while fewer chemicals had a response in the specific reporter gene assays, but these effects were typically triggered at lower concentrations. The single chemical effect data were used to improve published mixture toxicity modeling of water samples from the Danube River. While there was a slight increase in the fraction of the bioanalytical equivalents explained for the Danube River samples, for some endpoints less than 1% of the observed effect could be explained by the studied chemicals. The new mixture models essentially confirmed previous findings from many studies monitoring water quality using both chemical analysis and bioanalytical tools. In short, our results indicate that many more chemicals contribute to the biological effect than those that are typically quantified by chemical monitoring programs or those regulated by environmental quality standards. This study not only demonstrates the utility of fingerprinting single chemicals for an improved understanding of the biological effect of pollutants, but also highlights the need to apply bioassays for water quality monitoring in order to prevent underestimation of the overall biological effect.

Using short-term bioassays to evaluate the endocrine disrupting capacity of the pesticides linuron and fenoxycarb.

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.

Effect-directed analysis supporting monitoring of aquatic environments--An in-depth overview.

Aquatic environments are often contaminated with complex mixtures of chemicals that may pose a risk to ecosystems and human health. This contamination cannot be addressed with target analysis alone but tools are required to reduce this complexity and identify those chemicals that might cause adverse effects. Effect-directed analysis (EDA) is designed to meet this challenge and faces increasing interest in water and sediment quality monitoring. Thus, the present paper summarizes current experience with the EDA approach and the tools required, and provides practical advice on their application. The paper highlights the need for proper problem formulation and gives general advice for study design. As the EDA approach is directed by toxicity, basic principles for the selection of bioassays are given as well as a comprehensive compilation of appropriate assays, including their strengths and weaknesses. A specific focus is given to strategies for sampling, extraction and bioassay dosing since they strongly impact prioritization of toxicants in EDA. Reduction of sample complexity mainly relies on fractionation procedures, which are discussed in this paper, including quality assurance and quality control. Automated combinations of fractionation, biotesting and chemical analysis using so-called hyphenated tools can enhance the throughput and might reduce the risk of artifacts in laboratory work. The key to determining the chemical structures causing effects is analytical toxicant identification. The latest approaches, tools, software and databases for target-, suspect and non-target screening as well as unknown identification are discussed together with analytical and toxicological confirmation approaches. A better understanding of optimal use and combination of EDA tools will help to design efficient and successful toxicant identification studies in the context of quality monitoring in multiply stressed environments.

Oestrogen reporter transgenic medaka for non-invasive evaluation of aromatase activity.

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.

A comparison of high-throughput techniques for assaying circadian rhythms in plants.

Over the last two decades, the development of high-throughput techniques has enabled us to probe the plant circadian clock, a key coordinator of vital biological processes, in ways previously impossible. With the circadian clock increasingly implicated in key fitness and signalling pathways, this has opened up new avenues for understanding plant development and signalling. Our tool-kit has been constantly improving through continual development and novel techniques that increase throughput, reduce costs and allow higher resolution on the cellular and subcellular levels. With circadian assays becoming more accessible and relevant than ever to researchers, in this paper we offer a review of the techniques currently available before considering the horizons in circadian investigation at ever higher throughputs and resolutions.

Future water quality monitoring--adapting tools to deal with mixtures of pollutants in water resource management.

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.

Rapid fluorescent detection of (anti)androgens with spiggin-gfp medaka.

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.

Optimizing fluorescent protein choice for transgenic embryonic medaka models.

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.

In vivo endocrine disruption assessment of wastewater treatment plant effluents with small organisms.

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.

Expression of enzymes involved in thyroid hormone metabolism during the early development of Xenopus tropicalis.

BACKGROUND INFORMATION: There are significant indications that amphibians require TH (thyroid hormones) prior to their involvement in the regulation of metamorphosis and before the development of a functional thyroid. RESULTS: In order to investigate the potential role for TH in pre-metamorphic Xenopus tropicalis we have cloned cDNAs for, and analysed the expression of, TPO (thyroid peroxidase), 5'DII (type II iodothyronine deiodinase) and 5DIII (type III iodothyronine deiodinase), enzymes involved in TH metabolism. Zygotic expression of TPO was detected in neurula stage embryos. Expression was observed in the notochord and later in the thyroid. The notochord was also a common site of expression for 5'DII and 5DIII. Other sites of 5'DII expression are the otic vesicles, retina, liver, blood-forming region, branchial arches and brain. 5DIII is also expressed in the brain, retina, liver, developing pro-nephros, blood-forming region and branchial arches. Embryos exposed to the TPO inhibitor methimazole showed a distinctive dose-dependent phenotype of a crimped notochord and shortened axis, together with alterations in (125)I(-) uptake. CONCLUSIONS: These data suggest a novel extrathyroidal role for TH during early development, and support the proposal that embryos require thyroid signalling for normal development prior to metamorphosis.

Xenopus tropicalis peroxidasin gene is expressed within the developing neural tube and pronephric kidney.

Peroxidasin, originally identified in Drosophila, is a member of the myeloperoxidase family with a novel domain structure. It is proposed that peroxidasin is secreted and has functions associated with stabilization of the extracellular matrix. We report the identification of the Xenopus tropicalis orthologue of the peroxidasin gene. We show that the predicted protein sequence of Xenopus peroxidasin shows high sequence identity with the human orthologue and that the exon structure is highly conserved between the two species. We describe the first detailed developmental expression pattern for peroxidasin in a vertebrate species. Maternal expression of Xtpxn is localized to the animal hemisphere where it persists through early cleavage stages. Initial zygotic Xtpxn expression is detected in the developing neural tube and becomes localized to the hindbrain and midbrain. Xtpxn is expressed in the primordium of the pronephric kidney and expression persists in the pronephric tubules and duct throughout development. Potential roles for peroxidasin during early vertebrate development are discussed.