Modeling mixtures of thyroid gland function disruptors in a vertebrate alternative model, the zebrafish eleutheroembryo.

Maternal thyroxine (T4) plays an essential role in fetal brain development, and even mild and transitory deficits in free-T4 in pregnant women can produce irreversible neurological effects in their offspring. Women of childbearing age are daily exposed to mixtures of chemicals disrupting the thyroid gland function (TGFDs) through the diet, drinking water, air and pharmaceuticals, which has raised the highest concern for the potential additive or synergic effects on the development of mild hypothyroxinemia during early pregnancy. Recently we demonstrated that zebrafish eleutheroembryos provide a suitable alternative model for screening chemicals impairing the thyroid hormone synthesis. The present study used the intrafollicular T4-content (IT4C) of zebrafish eleutheroembryos as integrative endpoint for testing the hypotheses that the effect of mixtures of TGFDs with a similar mode of action [inhibition of thyroid peroxidase (TPO)] was well predicted by a concentration addition concept (CA) model, whereas the response addition concept (RA) model predicted better the effect of dissimilarly acting binary mixtures of TGFDs [TPO-inhibitors and sodium-iodide symporter (NIS)-inhibitors]. However, CA model provided better prediction of joint effects than RA in five out of the six tested mixtures. The exception being the mixture MMI (TPO-inhibitor)-KClO4 (NIS-inhibitor) dosed at a fixed ratio of EC10 that provided similar CA and RA predictions and hence it was difficult to get any conclusive result. There results support the phenomenological similarity criterion stating that the concept of concentration addition could be extended to mixture constituents having common apical endpoints or common adverse outcomes.

Zebrafish eleutheroembryos provide a suitable vertebrate model for screening chemicals that impair thyroid hormone synthesis.

Thyroxine-immunofluorescence quantitative disruption test (TIQDT) was designed to provide a simple, rapid, alternative bioassay for assessing the potential of chemical pollutants and drugs to disrupt thyroid gland function. This study demonstrated that zebrafish eleutheroembryos provided a suitable vertebrate model, not only for screening the potential thyroid disrupting effect of molecules, but also for estimating the potential hazards associated with exposure to chemicals directly impairing thyroxine (T4) synthesis. Amitrole, potassium perchlorate, potassium thiocyanate, methimazole (MMI), phloroglucinol, 6-propyl-2-thiouracil, ethylenethiourea, benzophenone-2, resorcinol, pyrazole, sulfamethoxazole, sodium bromide, mancozeb, and genistein were classified as thyroid gland function disruptors. Concordance between TIQDT on zebrafish and mammalian published data was very high and the physiological relevance of T4-intrafollicular content was clearly higher than regulation at the transcriptional level of tg or slc5a5. Moreover, concentration-response analysis provided information about the thyroid disrupting potency and hazard of selected positive compounds. Finally, the effect of perchlorate, but not MMI, was completely rescued by low-micromolar amounts of iodide. TIQDT performed on zebrafish eleutheroembryos is an alternative whole-organism screening assay that provides relevant information for environmental and human risk assessments.

Effects of BDE-209 contaminated sediments on zebrafish development and potential implications to human health.

Polybrominated diphenyl ethers are compounds widely used as flame-retardants, which are of increasing environmental concern due to their persistence, and potential adverse effects. This study had two objectives. First, we assessed if BDE-209 in sediment was bioavailable and bioaccumulated into zebrafish embryos. Secondly, we assessed the potential impact on human and environmental health of bioavailable BDE-209 using human in vitro cell assays and zebrafish embryos. Zebrafish were exposed from 4h to 8days post-fertilization to sediments spiked with 12.5mg/kg of BDE-209. Zebrafish larvae accumulated ten fold more BDE-209 than controls in unspiked sediment after 8days. BDE-209 impacted expression of neurological pathways and altered behavior of larvae, although BDE-209 had no visible affect on thyroid function or motoneuron and neuromast development. Zebrafish data and in silico predictions suggested that BDE-209 would also interact with key human transcription factors and receptors. We therefore tested these predictions using mammalian in vitro assays. BDE-209 activated human aryl hydrocarbon receptor, peroxisome proliferator activating receptors, CF/b-cat, activator protein 1, Oct-MLP, and the estrogen receptor-related alpha (ERRα) receptor in cell-based assays. BDE-209 also inhibited human acetylcholinesterase activity. The observation that BDE-209 can be bioaccumulated from contaminated sediment highlights the need to consider this as a potential environmental exposure route. Once accumulated, our data also show that BDE-209 has the potential to cause impacts on both human and environmental health.

Zebrafish eleutheroembryos as an alternative system for screening chemicals disrupting the mammalian thyroid gland morphogenesis and function.

The importance and irreversibility of the effects of thyroid hormone deficiency on human brain development highlight the importance of identifying environmental agents that interfere with thyroid gland morphogenesis and function. Zebrafish eleutheroembryos are currently used by many pharmaceutical companies in drug discovery as a vertebrate model, not subjected to regulations for animal experiments, that provides an intermediate step between in vitro and rodent assay. The mechanisms of zebrafish thyroid development are generally comparable to those in humans, and moreover, molecular and functional studies of zebrafish thyroid follicles have demonstrated a high degree of conservation with upper vertebrates, opening up the possibility of designing alternative methods for screening individual chemicals and mixtures that impairing thyroid gland morphogenesis and/or function. Analysis of the intrafollicular thyroxine-content of zebrafish larvae exposed to potential disruptors has proved to be a reliable, physiologically relevant endpoint to estimate effects of chemicals on the mammalian thyroid gland.

Triiodothyronine-induced changes in the zebrafish transcriptome during the eleutheroembryonic stage: implications for bisphenol A developmental toxicity.

Thyroid disruption during early development is a current matter of concern due to its significant human health implications. We present here a transcriptome analysis of thyroid hormone-regulated genes in zebrafish during the eleutheroembryonic stage (days 2-5 post fertilization) to detect potential markers of thyroid disruption. Exposure to 3,5,3'-triiodo-l-thyroxine (T3, 50 nM) induced changes in a minor portion (less than 2%) of the zebrafish transcriptome, with a significant fraction of genes involved in the haematopoietic system, eye formation, and ossification/skeletal system, including the thyroid receptor thra gene. Some of the transcriptomic changes were reflected macroscopically, as an allometric decrease of eye size and an increase on thra hybridization signal in the skeletal tissue. Using this information, changes on transcription of three genes (adult alpha globin gene si:ch211-5 k11.6, embryonic globin gene hbae3, and long wavelength cone opsin gene opn1/w1) were analyzed to monitor the effect of the suspected thyroid disrupter bisphenol A (BPA) on the thyroid system during this period of development of zebrafish. BPA acted as a weak T3 agonist when tested alone, but it strongly enhanced the effect of subsaturating concentrations of T3. In thyroxine immunofluorescence quantitative disruption tests (TIQDT), BPA did not prevent the ability of thyroid follicles to synthesize thyroxine, a landmark for direct goitrogens. Our results suggest that BPA potentiates the effect of endogenous T3 in early development and demonstrate the requirement for the use of in vivo, multi-endpoint methods to evaluate thyroid disruption hazards on early developmental processes in vertebrates.