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.

Comparing an in-person workshop and a postal Delphi survey for involving health service users in health care and health research prioritization.

INTRODUCTION: To involve health service users in health care and health research priority setting, different methods exist. Which method is most suitable under which circumstances is unknown. We compared a postal Delphi survey and an in-person workshop to involve health service users in priority settings for rehabilitative care and research in Germany. METHODS: One hundred and eighty-four former rehabilitants were randomly assigned to a postal Delphi survey (n = 152) or an in-person workshop (n = 32). Two hundred and seventy-six employees in rehabilitation were also invited to the Delphi Survey. The methodological comparison refers only to the sample of rehabilitants. Within each method, the participants agreed on the top 10 priorities for practice improvement and research in rehabilitative care. The priorities were compared descriptively. Participants' satisfaction was measured with the Public and Patient Engagement Evaluation Tool. The usability of both methods was compared based on the effort, time and material costs required for implementation. RESULTS: Seventy-five former rehabilitants and 41 employees in rehabilitation completed both Delphi survey rounds. Eleven former rehabilitants participated in the in-person workshop. Priorities for practice improvement showed a high degree of overlap between both methods whereas research priorities differed greatly. Participants of the in-person workshop felt significantly better prepared, more listened to and more likely to feel that different views on the topics were discussed. Participants of the Delphi survey expressed difficulties in understanding all survey questions. The Delphi survey was more elaborate in preparation and implementation but caused lower material costs. CONCLUSION: The differences in research priorities between the two methods could be due to the different samples, differences in the individual interests of participants or differences in the prioritization process. In-person workshops seem to be more appropriate for complex topics, where clarifications of questions and deeper discussions are needed. Delphi surveys seem to be more suitable for easily understandable topics, larger sample sizes and when fewer financial resources are available. PATIENT OR PUBLIC CONTRIBUTION: The different study phases were supported by employees in rehabilitation and former rehabilitants (e.g., developing study documents, and interpreting results).

[Need for Action and Research in Psychosomatic and Orthopaedic Rehabilitation from the Point of View of Rehabilitants and Individuals who work in Rehabilitative Care]. / Handlungs- und Forschungsbedarf in der Psychosomatischen und Orthopädischen Rehabilitation aus der Sicht von Rehabilitand:innen und Mitarbeitenden in der Rehabilitation.

PURPOSE: The aim of this study was to assess the need for practical action and research in psychosomatic and orthopaedic rehabilitation from the perspective of rehabilitants and individuals who work in rehabilitative care. METHODS: The project was divided into an identification and a prioritization phase. In the identification phase, 3872 former rehabilitants, 235 employees from three rehabilitation clinics and 31 employees of the German Pension Insurance Oldenburg-Bremen (DRV OL-HB) were invited to participate in a written survey. The participants were asked to name relevant needs for action and research for psychosomatic and orthopaedic rehabilitation. The answers were evaluated qualitatively using an inductively developed coding system. Practical fields of action and research questions were formulated from the categories of the coding system. In the prioritization phase, the identified needs were ranked. For this purpose, 32 rehabilitants were invited to a prioritization workshop and 152 rehabilitants, 239 clinic employees and 37 employees of the DRV OL-HB to a two-round written Delphi survey. The resulting prioritized lists from both methods were combined into a top 10 list. RESULTS: In the identification phase, 217 rehabilitants, 32 clinic employees and 13 employees of the DRV OL-HB participated in the survey, in the prioritization phase, 75 rehabilitants, 33 clinic employees and 8 employees of the DRV OL-HB in both survey rounds of the Delphi survey, and 11 rehabilitants in the prioritization workshop. A need for practical action primarily in the implementation of holistic and individual rehabilitation, in quality assurance and in the education and participation of rehabilitants was identified, as also a need for research primarily on access to rehabilitation, structures in the rehabilitation setting (e. g., inter-agency cooperation), the design of rehabilitative interventions (more individualized, more suitable for everyday life), and the motivation of rehabilitants. CONCLUSION: The identified needs for action and research include many topics that have already been identified as problems in previous research projects and by various actors in rehabilitation. In the future, there needs to be increased focus on the development of strategies for dealing with and solving the identified needs, as well as on the implementation of these strategies.

Participation preferences of health service users in health care decision-making regarding rehabilitative care in Germany-A cross-sectional study.

BACKGROUND: Involving patients and citizens in health care decision-making is considered increasingly important in Germany. Participatory structures have been implemented, especially in rehabilitative care. However, it is unknown whether and to what extent German patients and citizens want to participate in decisions that exceed their own medical treatment. OBJECTIVE: This study aimed to survey participation preferences and associated factors of health service users in decisions regarding rehabilitative care at micro, meso and macro levels. METHODS: A questionnaire was sent to 3872 former rehabilitants. We collected participation preferences using the Control Preference Scale or an adapted form. Possible influencing factors were examined using logistic regression models. RESULTS: The response rate was 5.7% (n = 217). At all decision-making levels, joint decision-making was preferred. At the macro level, preferences for actively participating were the highest. Preferences were significantly interrelated between decision-making levels. At the micro level, an orthopaedic indication significantly decreased the desire for participation compared to psychosomatic indications (odds ratio = 0.44, p = .019). DISCUSSION: Participants wanted to be equally involved in decision-making as experts. Higher preferences for active participation at the macro level might be due to dissatisfaction with the current health care organisation and lack of trust in politicians. Compared to the general public, our study sample was older (73.3% between 50 and 69 years) and more often chronically ill-factors associated with increased participation preferences in the literature. CONCLUSION: Contrary to the identified preferences, participation opportunities in the German health care system are rare. Further research on participation preferences and structures that enable meaningful involvement are needed.

ERGO: Breaking Down the Wall between Human Health and Environmental Testing of Endocrine Disrupters.

ERGO (EndocRine Guideline Optimization) is the acronym of a European Union-funded research and innovation action, that aims to break down the wall between mammalian and non-mammalian vertebrate regulatory testing of endocrine disruptors (EDs), by identifying, developing and aligning thyroid-related biomarkers and endpoints (B/E) for the linkage of effects between vertebrate classes. To achieve this, an adverse outcome pathway (AOP) network covering various modes of thyroid hormone disruption (THD) in multiple vertebrate classes will be developed. The AOP development will be based on existing and new data from in vitro and in vivo experiments with fish, amphibians and mammals, using a battery of different THDs. This will provide the scientifically plausible and evidence-based foundation for the selection of B/E and assays in lower vertebrates, predictive of human health outcomes. These assays will be prioritized for validation at OECD (Organization for Economic Cooperation and Development) level. ERGO will re-think ED testing strategies from in silico methods to in vivo testing and develop, optimize and validate existing in vivo and early life-stage OECD guidelines, as well as new in vitro protocols for THD. This strategy will reduce requirements for animal testing by preventing duplication of testing in mammals and non-mammalian vertebrates and increase the screening capacity to enable more chemicals to be tested for ED properties.

Nectin-2 in ovarian cancer: How is it expressed and what might be its functional role?

Nectin-2 is an adhesion molecule that has been reported to play a role in tumor growth, metastasis and tumor angiogenesis. Herein, we investigated Nectin-2 in ovarian cancer patients and in cell culture. Tumor as well as peritoneal biopsies of 60 ovarian cancer patients and 22 controls were dual stained for Nectin-2 and CD31 using immunohistochemistry. Gene expression of Nectin-2 was quantified by real-time PCR and differences analyzed in relation to various tumor characteristics. In the serum of patients, vascular endothelial growth factor (VEGF) was quantified by ELISA. Effect of VEGF on Nectin-2 expression as well as permeability was investigated in HUVEC. In tumor biopsies, Nectin-2 protein was mainly localized in tumor cells, whereas in peritoneal biopsies, clear colocalization was found in the vasculature. T3 patients had a significantly higher percentage of positive lymph nodes and this correlated with survival. Nectin-2 was significantly upregulated in tumor biopsies in patients with lymph node metastasis and with residual tumor >1 cm after surgery. Nectin-2 expression was significantly suppressed in the peritoneal endothelium of patients associated with significantly increased VEGF serum levels. In cell culture, VEGF stimulation led to a significant downregulation of Nectin-2 which was reversed by VEGF-inhibition. In addition, Nectin-2 knockdown in endothelial cells was associated with significantly increased endothelial permeability. Nectin-2 expression in ovarian cancer may support tumor cell adhesion, leading to growth and lymph node metastasis. In addition, VEGF-induced Nectin-2 suppression in peritoneal endothelium may support an increase in vascular permeability leading to ascites production.

Analysis of tail coiling activity of zebrafish (Danio rerio) embryos allows for the differentiation of neurotoxicants with different modes of action.

In (eco)toxicology, there is a critical need for efficient methods to evaluate the neurotoxic potential of environmental chemicals. Recent studies proposed analysis of early coiling activity in zebrafish embryos as a powerful tool for the identification of neurotoxic compounds. In order to demonstrate that the analysis of early tail movements of zebrafish embryos allows for the discrimination of neurotoxicants acting via different mechanisms, the present study investigated the effects of four different neurotoxicants on the embryogenesis (fish embryo toxicity test) and early tail coiling movements of zebrafish embryos. Cadmium predominantly increased the frequency of tail coiling at the late pharyngula stage. Dichlorvos delayed embryonic development and caused convulsive tail movements resulting in prolonged duration of tail coils. Embryos exposed to teratogenic concentrations of fluoxetine and citalopram displayed absence of spontaneous tail movements at 24 h post-fertilization. In contrast, a non-teratogenic test concentration of citalopram decreased coiling frequency at multiple time points. Results demonstrated that the analysis of tail coiling movements of zebrafish embryos has the potential to discriminate neurotoxic compounds with different primary modes of action. In addition, chemical-induced effects on coiling activity were shown to potentially overlap with effects on embryogenesis. Further studies are needed to clarify the interplay of unspecific developmental toxicity of neurotoxic chemicals and effects resulting from specific neurotoxic mechanisms.

Thyroid Hormone Disruptors Interfere with Molecular Pathways of Eye Development and Function in Zebrafish.

The effects of thyroid hormone disrupting chemicals (THDCs) on eye development of zebrafish were investigated. We expected THDC exposure to cause transcriptional changes of vision-related genes, which find their phenotypic anchoring in eye malformations and dysfunction, as observed in our previous studies. Zebrafish were exposed from 0 to 5 days post fertilization (dpf) to either propylthiouracil (PTU), a thyroid hormone synthesis inhibitor, or tetrabromobisphenol-A (TBBPA), which interacts with thyroid hormone receptors. Full genome microarray analyses of RNA isolated from eye tissue revealed that the number of affected transcripts was substantially higher in PTU- than in TBBPA-treated larvae. However, multiple components of phototransduction (e.g., phosphodiesterase, opsins) were responsive to both THDC exposures. Yet, the response pattern for the gene ontology (GO)-class "sensory perception" differed between treatments, with over 90% down-regulation in PTU-exposed fish, compared to over 80% up-regulation in TBBPA-exposed fish. Additionally, the reversibility of effects after recovery in clean water for three days was investigated. Transcriptional patterns in the eyes were still altered and partly overlapped between 5 and 8 dpf, showing that no full recovery occurred within the time period investigated. However, pathways involved in repair mechanisms were significantly upregulated, which indicates activation of regeneration processes.

[Amyotrophic Lateral Sclerosis - Challenges of Family Caregivers]. / Amyotrophe Lateralsklerose ­ Herausforderungen von pflegenden Angehörigen.

BACKGROUND: Patients who suffer from amyotrophic lateral sclerosis sometimes show pathological changes in behaviour and cognition. Some even develop frontotemporal dementia. The family caregivers of the patients are faced with additional challenges. AIM: To describe the effect of changes in behaviour and cognition on the emotional state and the perceived stress and burdens of family caregivers. METHOD: Different search terms were used for searching in several databases. Ten trials were included in the study. Further examinations of this topic on the basis of additional literature are described in the discussion section. RESULTS: Changes in behaviour and cognition can lead to symptoms of anxiety and depression in family caregivers. The findings also show a correlation between apathy, disinhibition and executive dysfunction combined with an increase in caregiver burden. CONCLUSION: Changes in behaviour and cognition in patients with ALS have a negative impact on the emotional state of family caregivers and contribute to increased feelings of being burdened. Early and detailed information concerning possible disease-induced changes is necessary.

Intrafollicular thyroid hormone staining in whole-mount zebrafish (Danio rerio) embryos for the detection of thyroid hormone synthesis disruption.

Endocrine-disrupting chemicals are known to impact multiple hormonal axes of vertebrates, among which the thyroid system is crucial for multiple developmental and physiological processes. Thus, the present study focused on the semi-quantitative visualization of intrafollicular triiodothyronine (T3) and thyroxin (T4) in zebrafish embryos as a potential test system for the detection of disrupted thyroid hormone synthesis. To this end, an antibody-based fluorescence double-staining protocol for whole-mount zebrafish embryos and larvae was adapted to simultaneously detect intrafollicular T3 and T4. During normal development until 10 days post-fertilization (dpf), the number of thyroid follicles increased along the ventral aorta. Concentrations of T4 and T3, measured by fluorescence intensity, increased until 6 dpf, but decreased thereafter. Exposure of zebrafish embryos to propylthiouracil (PTU), a known inhibitor of TH synthesis, resulted in a significant decrease in the number of follicles that stained for T3, whereas a trend for increase in follicles that stained for T4 was observed. In contrast, fluorescence intensity for both thyroid hormones decreased significantly after exposure to PTU. Overall, the zebrafish embryo appears to be suitable for the simultaneous visualization and detection of changing intrafollicular TH contents during normal development and after PTU treatment.

20 Years of fish immunotoxicology - what we know and where we are.

Despite frequent field observations of impaired immune response and increased disease incidence in contaminant-exposed wildlife populations, immunotoxic effects are rarely considered in ecotoxicological risk assessment. The aim of this study was to review the literature on immunotoxic effects of chemicals in fish to quantitatively evaluate (i) which experimental approaches were used to assess immunotoxic effects, (ii) whether immune markers exist to screen for potential immunotoxic activities of chemicals, and (iii) how predictive those parameters are for adverse alterations of fish immunocompetence and disease resistance. A total of 241 publications on fish immunotoxicity were quantitatively analyzed. The main conclusions included: (i) To date, fish immunotoxicology focused mainly on innate immune responses and immunosuppressive effects. (ii) In numerous studies, the experimental conditions are poorly documented, as for instance age or sex of the fish or the rationale for the selected exposure conditions is often missing. (iii) Although a broad variety of parameters were used to assess immunotoxicity, the rationale for the choice of measured parameters was often not given, remaining unclear how they link to the suspected immunotoxic mode of action of the chemicals. (iv) At the current state of knowledge, it is impossible to identify a set of immune parameters that could reliably screen for immunotoxic potentials of chemicals. (v) Similarly, in fish immunotoxicology there is insufficient understanding of how and when chemical-induced modulations of molecular/cellular immune changes relate to adverse alterations of fish immunocompetence, although this would be crucial to include immunotoxicity in ecotoxicological risk assessment.

Ultrastructural Alterations in Thyrocytes of Zebrafish ( Danio rerio) after Exposure to Propylthiouracil and Perchlorate.

Histopathology is a widely used approach to evaluate effects of endocrine-active chemicals in the thyroid. However, effects at an ultrastructural level have hardly been examined in fish thyroids. In the present study, zebrafish was exposed to sublethal concentrations of propylthiouracil (PTU; 0-50 mg/L) and perchlorate (PER; 0-5,000 µg/L) for 5 weeks in a modified early life-stage test. None of the treatments caused significant mortality (no observed effect concentrations for survival ≥50 mg/L [PTU] and ≥5,000 µg/L [PER]). PTU induced dose-dependent alterations in the rough endoplasmic reticulum (rER) in all exposure groups, whereas only the 2 highest PER exposure groups (500 and 5,000 µg/L) resulted in alterations of the rER. Both substances caused an increase in the numbers of lysosomes and mitochondria, with mitochondria displaying distorted cristae. Increased mitochondrial diameters were only observed in the PTU treatment. PER-exposed samples displayed an increase in apical microvilli. The highest PTU concentration (50 mg/L) showed first signs of cellular degeneration. Ultrastructural changes in zebrafish thyrocytes thus appear specific for different chemicals, most likely depending on their specific modes of action. Additional knowledge of subcellular changes in thyrocytes can help to better understand and interpret existing histological data in the future.

Reversibility of endocrine disruption in zebrafish (Danio rerio) after discontinued exposure to the estrogen 17α-ethinylestradiol.

The aim of the present study was to investigate the persistence of the feminizing effects of discontinued 17α-ethinylestradiol (EE2) exposure on zebrafish (Danio rerio). An exposure scenario covering the sensitive phase of sexual differentiation, as well as final gonad maturation was chosen to examine the estrogenic effects on sexual development of zebrafish. Two exposure scenarios were compared: continuous exposure to environmentally relevant concentrations (0.1-10 ng/L EE2) up to 100 days post-hatch (dph) and developmental exposure up to 60 dph, followed by 40 days of depuration in clean water. The persistence of effects was investigated at different biological organization levels from mRNA to population-relevant endpoints to cover a broad range of important parameters. EE2 had a strong feminizing and inhibiting effect on the sexual development of zebrafish. Brain aromatase (cyp19b) mRNA expression showed no clear response, but vitellogenin levels were significantly elevated, gonad maturation and body growth were inhibited in both genders, and sex ratios were skewed towards females and undifferentiated individuals. To a large extent, all of these effects were reversed after 40 days of recovery, leading to the conclusion that exposure to the estrogen EE2 results in very strong, but reversible underdevelopment and feminization of zebrafish. The present study is the first to show this reversibility at different levels of organization, which gives better insight into the mechanistic basis of estrogenic effects in zebrafish.

The benefits of validation of methods for toxicity testing outweigh its costs

The 4th Annual Forum on Endocrine Disrupters organized by the European Commission brought together authors of this article around the topic: "From bench to validated test guidelines: (pre)validation of test methods". Validation activities are meant to demonstrate the relevance and reliability of methods and approaches used in regulatory safety testing. These activities are essential to facilitate regulatory use, still they are largely underfunded and unattractive to the scientific community. In the last decade, there has been large amounts of funding invested in European research towards the development of approaches that can be used in regulatory decision-making, including for the identification of endocrine disrupters. There is a vast pool of candidate test methods for potential regulatory applications, but most of them will not be used due to the absence of consideration of their relevance and reliability outside the method developer's laboratory. The article explains the reasons why such a gap exists between the outputs of research projects and the uptake in a regulatory context. In parallel, there are also increasing expectations from the regulatory science community that validation becomes more efficient with respect to time and resources. This article shares some of the lessons learned and proposes paths forward for validation of new methods that are not intended as one-to-one replacements of animal studies. This includes submitting only mature methods for validation that were developed following good practices and good documentation, proposing a greater emphasis on well-documented transferability studies, and adopting a cost-sharing model between those who benefit from validated methods.

Validation activities for methods intended to be used to assess chemical safety have a cost but also bring substantial benefits when the validated methods are established as OECD Test Guidelines which results in mutual acceptance of data generated by the methods. The article discusses some of the challenges faced when method validation is underfunded and unattractive for researchers. Proposals are made to improve the current situation, gain efficiency and make validation a shared responsibility.

The generation gap in endocrine disruption: Can the integrated fish endocrine disruptor test (iFEDT) bridge the gap by assessing intergenerational effects of thyroid hormone system disruption?

Thyroid hormones (THs) act early in ontogenesis, even prior to the differentiation of thyrocytes. Maternal transfer of THs is therefore known to play an essential role in early development. Current OECD test guidelines for the assessment of TH system disruption (THSD) do not address inter- or transgenerational effects. The integrated fish endocrine disruptor test (iFEDT), a test combining parental and developmental exposure of filial fish, may fill this gap. We tested the ability of the iFEDT to detect intergenerational effects in zebrafish (Danio rerio): Parental fish were exposed to propylthiouracil (PTU), an inhibitor of TH synthesis, or not exposed. The offspring was submitted to a crossed experimental design to obtain four exposure scenarios: (1) no exposure at all, (2) parental exposure only, (3) embryonic exposure only, and (4) combined parental and embryonic exposure. Swim bladder inflation, visual motor response (VMR) and gene expression of the progeny were analysed. Parental, but not embryonic PTU exposure reduced the size of the swim bladder of 5 d old embryos, indicating the existence of intergenerational effects. The VMR test produced opposite responses in 4.5 d old embryos exposed to PTU vs. embryos derived from exposed parents. Embryonic exposure, but not parental exposure increased gene expression of thyroperoxidase, the target of PTU, most likely due to a compensatory mechanism. The gene expression of pde-6h (phosphodiesterase) was reduced by embryonic, but not parental exposure, suggesting downregulation of phototransduction pathways. Hence, adverse effects on swim bladder inflation appear more sensitive to parental than embryonic exposure and the iFEDT represents an improvement in the testing strategy for THSD.

Development of a Zebrafish Embryo-Based Test System for Thyroid Hormone System Disruption: 3Rs in Ecotoxicological Research.

There is increasing concern regarding pollutants disrupting the vertebrate thyroid hormone (TH) system, which is crucial for development. Thus, identification of TH system-disrupting chemicals (THSDCs) is an important requirement in the Organisation for Economic Co-operation and Development (OECD) testing framework. The current OECD approach uses different model organisms for different endocrine modalities, leading to a high number of animal tests. Alternative models compatible with the 3Rs (replacement, reduction, refinement) principle are required. Zebrafish embryos, not protected by current European Union animal welfare legislation, represent a promising model. Studies show that zebrafish swim bladder inflation and eye development are affected by THSDCs, and the respective adverse outcome pathways (AOPs) have been established. The present study compared effects of four THSDCs with distinct molecular modes of action: Propylthiouracil (PTU), potassium perchlorate, iopanoic acid, and the TH triiodothyronine (T3) were tested with a protocol based on the OECD fish embryo toxicity test (FET). Effects were analyzed according to the AOP concept from molecular over morphological to behavioral levels: Analysis of thyroid- and eye-related gene expression revealed significant effects after PTU and T3 exposure. All substances caused changes in thyroid follicle morphology of a transgenic zebrafish line expressing fluorescence in thyrocytes. Impaired eye development and swimming activity were observed in all treatments, supporting the hypothesis that THSDCs cause adverse population-relevant changes. Findings thus confirm that the FET can be amended by TH system-related endpoints into an integrated protocol comprising molecular, morphological, and behavioral endpoints for environmental risk assessment of potential endocrine disruptors, which is compatible with the 3Rs principle. Environ Toxicol Chem 2024;00:1-18. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Development of the integrated fish endocrine disruptor test (iFEDT)-Part A: Merging of existing fish test guidelines.

There has been increasing interest in endocrine-disrupting chemicals (EDCs) among scientists and public authorities over the last 30 years, notably because of their wide use and the increasing evidence of detrimental effects on humans and the environment. However, test systems for the detection of potential EDCs as well as testing strategies still require optimization. Thus, the aim of the present project was the development of an integrated test protocol that merges the existing OECD test guidelines (TGs) 229 (fish short-term reproduction assay) and 234 (fish sexual development test) and implements thyroid-related endpoints for fish. The integrated fish endocrine disruptor test (iFEDT) represents a comprehensive approach for fish testing, which covers reproduction, early development, and sexual differentiation, and will thus allow the identification of multiple endocrine-disruptive effects in fish. Using zebrafish (Danio rerio) as a model organism, two exposure tests were performed with well-studied EDCs: 6-propyl-2-thiouracil (PTU), an inhibitor of thyroid hormone synthesis, and 17α-ethinylestradiol (EE2), an estrogen receptor agonist. In part A of this article, the effects of PTU and EE2 on established endpoints of the two existing TGs are reported, whereas part B focuses on the novel thyroid-related endpoints. Results of part A document that, as expected, both PTU and EE2 had strong effects on various endocrine-related endpoints in zebrafish and their offspring. Merging of TGs 229 and 234 proved feasible, and all established biomarkers and endpoints were responsive as expected, including reproductive and morphometric changes (PTU and EE2), vitellogenin levels, sex ratio, gonad maturation, and histopathology (only for EE2) of different life stages. A validation of the iFEDT with other well-known EDCs will allow verification of the sensitivity and usability and confirm its capacity to improve the existing testing strategy for EDCs in fish. Integr Environ Assess Manag 2024;20:817-829. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Development of the integrated fish endocrine disruptor test-Part B: Implementation of thyroid-related endpoints.

Given the vital role of thyroid hormones (THs) in vertebrate development, it is essential to identify chemicals that interfere with the TH system. Whereas, among nonmammalian laboratory animals, fish are the most frequently utilized test species in endocrine disruptor research, for example, in guidelines for the detection of effects on the sex hormone system, there is no test guideline (TG) using fish as models for thyroid-related effects; rather, amphibians are used. Therefore, the objective of the present project was to integrate thyroid-related endpoints for fish into a test protocol combining OECD TGs 229 (Fish Short-Term Reproduction Assay) and 234 (Fish Sexual Development Test). The resulting integrated Fish Endocrine Disruption Test (iFEDT) was designed as a comprehensive approach to covering sexual differentiation, early development, and reproduction and to identifying disruption not only of the sexual and/or reproductive system but also the TH system. Two 85-day exposure tests were performed using different well-studied endocrine disruptors: 6-propyl-2-thiouracil (PTU) and 17α-ethinylestradiol (EE2). Whereas the companion Part A of this study presents the findings on effects by PTU and EE2 on endpoints established in existing TGs, the present Part B discusses effects on novel thyroid-related endpoints such as TH levels, thyroid follicle histopathology, and eye development. 6-Propyl-2-thiouracil induced a massive proliferation of thyroid follicles in any life stage, and histopathological changes in the eyes proved to be highly sensitive for TH system disruption especially in younger life stages. For measurement of THs, further methodological development is required. 17-α-Ethinylestradiol demonstrated not only the well-known disruption of the hypothalamic-pituitary-gonadal axis, but also induced effects on thyroid follicles in adult zebrafish (Danio rerio) exposed to higher EE2 concentrations, suggesting crosstalk between endocrine axes. The novel iFEDT has thus proven capable of simultaneously capturing endocrine disruption of both the steroid and thyroid endocrine systems. Integr Environ Assess Manag 2024;20:830-845. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Enhanced identification of endocrine disruptors through integration of science-based regulatory practices and innovative methodologies: The MERLON Project.

The prevalence of hormone-related health issues caused by exposure to endocrine disrupting chemicals (EDCs) is a significant, and increasing, societal challenge. Declining fertility rates together with rising incidence rates of reproductive disorders and other endocrine-related diseases underscores the urgency in taking more action. Addressing the growing threat of EDCs in our environment demands robust and reliable test methods to assess a broad variety of endpoints relevant for endocrine disruption. EDCs also require effective regulatory frameworks, especially as the current move towards greater reliance on non-animal methods in chemical testing puts to test the current paradigm for EDC identification, which requires that an adverse effect is observed in an intact organism. Although great advances have been made in the field of predictive toxicology, disruption to the endocrine system and subsequent adverse health effects may prove particularly difficult to predict without traditional animal models. The MERLON project seeks to expedite progress by integrating multispecies molecular research, new approach methodologies (NAMs), human clinical epidemiology, and systems biology to furnish mechanistic insights and explore ways forward for NAM-based identification of EDCs. The focus is on sexual development and function, from foetal sex differentiation of the reproductive system through mini-puberty and puberty to sexual maturity. The project aims are geared towards closing existing knowledge gaps in understanding the effects of EDCs on human health to ultimately support effective regulation of EDCs in the European Union and beyond.

Developmental exposure to triclosan and benzophenone-2 causes morphological alterations in zebrafish (Danio rerio) thyroid follicles and eyes.

Thyroid hormones (THs) regulate a multitude of developmental and metabolic processes, which are responsible for vertebrate development, growth, and maintenance of homeostasis. THs also play a key role in neurogenesis of vertebrates and thus affect eye development, which is vital for foraging efficiency and for effective escape from predation. Currently, there are no validated test guidelines for the assessment of TH system-disrupting chemicals (THSDCs) in fish. Consequently, the present study was designed to demonstrate the suitability of novel thyroid-related endpoints in early life-stages of fish. Embryos of a transgenic zebrafish (Danio rerio) line expressing the reporter gene tg:mCherry in their thyrocytes were used to investigate the effects of the environmental THSDCs triclosan (TCS, antibacterial agent) and benzophenone-2 (BP-2, UV filter) on thyroid follicle and eye development. Both BP-2 and TCS caused thyroid follicle hyperplasia in transgenic zebrafish, thus confirming their role as THSDCs. The effect intensity on follicle size and fluorescence was comparable with a 1.7-fold increase for BP-2 and 1.6-fold for TCS. Alterations of the cellular structures of the retina indicate an impact of both substances on eye development, with a stronger impact of TCS. With respect to guideline development, results provide further evidence for the suitability of morphological changes in thyroid follicles and the eyes as novel endpoints for the sensitive assessment of THSD-related effects in fish.