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).

Reversibility of Thyroid Hormone System-Disrupting Effects on Eye and Thyroid Follicle Development in Zebrafish (Danio rerio) Embryos.

Early vertebrate development is partially regulated by thyroid hormones (THs). Environmental pollutants that interact with the TH system (TH system-disrupting chemicals [THSDCs]) can have massively disrupting effects on this essential phase. Eye development of fish is directly regulated by THs and can, therefore, be used as a thyroid-related endpoint in endocrine disruptor testing. To evaluate the effects of THSDC-induced eye malformations during early development, zebrafish (Danio rerio) embryos were exposed for 5 days postfertilization (dpf) to either propylthiouracil, a TH synthesis inhibitor, or tetrabromobisphenol A, which interacts with TH receptors. Subsequently, one half of the embryos were exposed further to the THSDCs until 8 dpf, while the other half of the embryos were raised in clean water for 3 days to check for reversibility of effects. Continued THSDC exposure altered eye size and pigmentation and induced changes in the cellular structure of the retina. This correlated with morphological alterations of thyroid follicles as revealed by use of a transgenic zebrafish line. Interestingly, effects were partly reversible after a recovery period as short as 3 days. Results are consistent with changes in TH levels measured in different tissues of the embryos, for example, in the eyes. The results show that eye development in zebrafish embryos is very sensitive to THSDC treatment but able to recover quickly from early exposure by effective repair mechanisms. Environ Toxicol Chem 2023;42:1276-1292. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

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.

AOP Report: Thyroperoxidase Inhibition Leading to Altered Visual Function in Fish Via Altered Retinal Layer Structure.

Thyroid hormones (THs) are involved in the regulation of many important physiological and developmental processes, including vertebrate eye development. Thyroid hormone system-disrupting chemicals (THSDCs) may have severe consequences, because proper functioning of the visual system is a key factor for survival in wildlife. However, the sequence of events leading from TH system disruption (THSD) to altered eye development in fish has not yet been fully described. The development of this adverse outcome pathway (AOP) was based on an intensive literature review of studies that focused on THSD and impacts on eye development, mainly in fish. In total, approximately 120 studies (up to the end of 2021) were used in the development of this AOP linking inhibition of the key enzyme for TH synthesis, thyroperoxidase (TPO), to effects on retinal layer structure and visual function in fish (AOP-Wiki, AOP 363). In a weight-of-evidence evaluation, the confidence levels were overall moderate, with ample studies showing the link between reduced TH levels and altered retinal layer structure. However, some uncertainties about the underlying mechanism(s) remain. Although the current weight-of-evidence evaluation is based on fish, the AOP is plausibly applicable to other vertebrate classes. Through the re-use of several building blocks, this AOP is connected to the AOPs leading from TPO and deiodinase inhibition to impaired swim bladder inflation in fish (AOPs 155-159), together forming an AOP network describing THSD in fish. This AOP network addresses the lack of thyroid-related endpoints in existing fish test guidelines for the evaluation of THSDCs. Environ Toxicol Chem 2022;41:2632-2648. © 2022 SETAC.