Modeling mixtures of thyroid gland function disruptors in a vertebrate alternative model, the zebrafish eleutheroembryo
Introduction
Maternal thyroxine (T4) plays an essential role in fetal brain development before the onset of fetal thyroid function, and even mild and transitory deficits in free-T4 in pregnant women can produce irreversible neurological effects in their offspring (Raldúa et al., 2012). Although iodine deficiency is, without doubt, the major cause of severe thyroid gland dysfunction throughout the world, a number of natural-occurring and synthetic chemicals also have the ability to impair the thyroid gland function even in iodine-sufficient areas (Howdeshell, 2002). An important group of these thyroid gland function disruptors (TGFD) acts directly on the thyroid follicles (direct-TGFDs) inhibiting the thyroid TH synthesis by different modes of actions (MoA) (Raldúa et al., 2012). Some of these direct-TGFDs, such as perchlorate, inhibit the iodide uptake by the sodium-iodide symporter (NIS). Other chemicals, including thionamides, inhibit iodide organification by thyroid peroxidase (TPO). Different reports indicate that, even in situations of high iodine nutritional status, exposure of women of childbearing age to some individual environmental TGFDs already leads to hypothyroxinemia. However, it is the daily exposure through the diet, drinking water, air and pharmaceuticals to “cocktail” of many different TGFDs with different MoAs that has raised the highest concern for the potential additive or synergic (i.e. greater than additive) effects on the development of mild hypothyroxinemia during early pregnancy (Brown, 2003, Zoeller and Crofton, 2000).
Currently there are limited number of studies in the peer-reviewed literature analyzing the effect of mixtures of thyroid disrupting chemicals in vivo. Although combined effect assessments should be based on the comparison of an expected effect of a mixture based on knowledge about the individual component behavior and the observed effects in a mixture experiment (Berenbaum, 1985), most of these studies have analyzed the effect of mixtures without assessing the effects of individual mixture components (Khan et al., 2005, McLanahan et al., 2007), an approach useful for case-by-case basis, but not for the assessment of combination effects in terms of additivity, synergism, or antagonism (Kortenkamp et al., 2007). Only a few in vivo studies provide the experimental design suitable for coupling an individual and a combined effect of thyroid disruptors (Crofton et al., 2005, Flippin et al., 2009). Models for assessing the risk of the exposure to multiple chemicals often use the concepts of concentration addition (CA, also referred as dose addition) and response addition (RA, also referred as independent action) to describe the effects of mixtures of chemicals affecting one specific endpoint through similar or dissimilar mode of actions, respectively. Crofton et al. (2005) investigated the hypothesis that the CA model predicted the effect of a complex mixture of polyhalogenated aromatic hydrocarbons (PHAHs) on reducing circulating levels of T4 in young female rats. While there was no deviation from additivity at the lowest doses, a greater-than-additive effect was found at the highest mixture doses. Flippin et al. (2009) tested the hypothesis that circulating T4 levels in rat exposed to a mixture of direct-TGFDs and chemicals inducing T4 clearance in the liver (PHAHs) could be best predicted by an integrated addition model. While the RA model overestimated the effect of the mixture, the CA one and the integrated addition models provided better predictions.
Zebrafish is a vertebrate model organism increasingly used for assessing drug toxicity and safety and numerous studies have confirmed that mammalian and zebrafish toxicity profiles are very similar (Raldúa et al., 2012). Although in vitro assays are widely used in toxicology and safety pharmacology, the results are frequently not predictive of in vivo effects. Thus, zebrafish are increasingly used in pharmacological and toxicological screenings as an intermediate step after cell-based evaluation, to prioritize drug candidates for conventional animal testing, thus reducing the number and cost of mammalian studies (McGrath and Li, 2008). It is important to note that according to the new EU Directive 2010/63/EU on protection of animals used for scientific purposes, zebrafish pre-hatched [usually from 0 to 72 h post fertilization (hpf)] and non-feeding post-hatched embryos (eleutheroembryos, since hatching to 120 hpf) are not defined as protected and, therefore, do not fall into the regulatory frameworks dealing with animal experimentation (Strähle et al., 2012).
The origin and growth of the thyroid gland in zebrafish have been extensively studied and described, opening the possibility to use this model for assessing the potential of chemical pollutants and drugs to disrupt thyroid gland function (reviewed in Raldúa et al., 2012). Recently, we developed a simple, rapid zebrafish eleutheroembryo bioassay, the T4 immunofluorescence quantitative disruption test (TIQDT; Raldúa and Babin, 2009), to measure impairment of the thyroid function as a decrease in the intrafollicular T4-content (IT4C), and a high concordance between TIQDT on zebrafish and mammalian published data was found (Thienpont et al., 2011). Moreover, concentration–response analysis in this model provides information about the thyroid disrupting potency and hazard of chemicals impairing the thyroid hormone synthesis. Since TIQDT assay is performed on zebrafish eleutheroembryos, is an alternative method compliant with the 3R principles (relative replacement of animal tests), a target for many international regulatory bodies.
Our present study tested the hypothesis that the CA model best predicted the decrease in the IT4C in zebrafish exposed to a mixture of seven well-characterized mammalian TPO inhibitors. The study also evaluated the hypothesis that an RA model predicted better effects on the IT4C of binary TPO–NIS mixtures of direct-TGFDs with reported dissimilar MoA.
Section snippets
Chemicals
Potassium perchlorate (KClO4), potassium thiocyanate (KSCN), methimazole (MMI), 6-propyl-2-thiouracil (PTU), phloroglucinol, sulfamethoxazole (SMX), benzophenone-2 (BP2), amitrole, and 2-imidazolidinethion (ethylenethiourea, ETU) were purchased from Sigma-Aldrich (St. Louis, MO), with a range of purity between 97% for BP2 and 100% for MMI (see Supplementary Data, Table S1, for details). MMI, PTU, BP2, phloroglucinol, SMX, amitrole, and ETU were selected as representative of TPO-inhibitors.
Single substance toxicity
The decrease in the IT4C observed for the nine studied individual chemicals followed a sigmoid curve (Fig. 1), which could be modeled by the Hill regression function of Eq. (1). IT4C exhibited an important variability inside each experimental group, as indicated by the error bars in Fig. 1. To overcome the statistical problem generated by the high intra-group variability at least 18 embryos were analyzed in each experimental group. By using this sample size, parameters of single-curve fit were
Discussion
The present study tested the hypotheses that in zebrafish embryos exposed to a mixture of direct-TGFDs with a similar MoA (TPO-inhibitors), joint effects observed in the decrease of IT4C were additive and well predicted by a CA model, whereas the RA model predicted better joint additive effects on the decrease in the intrafollicular T4-content in zebrafish embryos exposed to binary mixtures of direct-TGFDs with different MoA (TPO- vs NIS-inhibitors).
Single TGFD raw data generated in a previous
Conflict of interest statement
None of the authors has competing interests with regard to this work.
Funding information
This study was supported by the Spanish MICINN grant CTM2011-30471-C02-01. D.R. was also partially supported for the French National Research Agency grant ANR-10-BLAN-1140/CHONDRO-X.
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