North-Eastern Brazil saw intensive application of the insecticide pyriproxyfen (PPF) during the microcephaly outbreak caused by the Zika virus (ZIKV). ZIKV requires the neural RNA-binding protein Musashi-1 to replicate. Thyroid hormone (TH) represses MSI1. PPF is a suspected TH disruptor. We hypothesized that co-exposure to the main metabolite of PPF, 4'-OH-PPF, could exacerbate ZIKV effects through increased MSI1 expression. Exposing an in vivo reporter model, Xenopus laevis, to 4'-OH-PPF decreased TH signaling and increased msi1 mRNA and protein, confirming TH-antagonistic properties. Next, we investigated the metabolite's effects on mouse subventricular zone-derived neural stem cells (NSCs). Exposure to 4'-OH-PPF dose-dependently reduced neuroprogenitor proliferation and dysregulated genes implicated in neurogliogenesis. The highest dose induced Msi1 mRNA and protein, increasing cell apoptosis and the ratio of neurons to glial cells. Given these effects of the metabolite alone, we considered if combined infection with ZIKV worsened neurogenic events. Only at the fourth and last day of incubation did co-exposure of 4'-OH-PPF and ZIKV decrease viral replication, but viral RNA copies stayed within the same order of magnitude. Intracellular RNA content of NSCs was decreased in the combined presence of 4'-OH-PPF and ZIKV, suggesting a synergistic block of transcriptional machinery. Seven out of 12 tested key genes in TH signaling and neuroglial commitment were dysregulated by co-exposure, of which four were unaltered when exposed to 4'-OH-PPF alone. We conclude that 4'-OH-PPF is an active TH-antagonist, altering NSC processes known to underlie correct cortical development. A combination of the TH-disrupting metabolite and ZIKV could aggravate the microcephaly phenotype.

Wildlife is increasingly exposed to environmental pollution, but data illustrating to what extent this exposure can impact health and survival of endangered species is missing. In humans, hair matrix analysis is a reliable tool for assessing cumulative exposure to organic pollutants such as pesticides but has rarely been used in other primates for this purpose. LC/MS-MS and GC/MS-MS multi-residue methods were used to screen the presence of 152 organic pollutants and their metabolites belonging to 21 different chemical families in hair samples from our closest relative, the chimpanzee. Samples were collected from 20 wild chimpanzees in Sebitoli, Kibale National Park, Uganda and 9 captive chimpanzees in the Réserve Africaine de Sigean, France. In total, 90 chemicals were detected, 60 in wild chimpanzees and 79 in captive chimpanzees. The median concentrations of detected chemicals in captive individuals were significantly higher than those in wild chimpanzees. Hair from the captive individuals at RAS was sampled a second time after 6 months in an environment of reduced exposure to these pollutants (diet of organic food, decreased use of plastic food and water containers). The number of chemicals detected in captive chimpanzees reduced from 79 to 63, and their concentrations were also significantly reduced. In the present study we report for the first time the use of hair analysis to detect organic pollutants in primate hair. We conclude that both wild and captive chimpanzees are exposed to a large range of different chemicals through their diet. Our study provides surprising and alarming evidence that besides the direct threats of poaching, deforestation and diseases, wild chimpanzees might be endangered by indirect consequences of anthropic activities. As chimpanzees are our closest relatives, our results should be considered as an alert for human health as well.

Introduction: The extensive use of the insecticide chlorpyrifos (CPF) throughout the world has brought increased scrutiny on its environmental and health impact. CPF is a cholinergic neurotoxicant; however, exposure to low noncholinergic doses is associated with numerous neurodevelopmental effects in animal models. In this study, we aimed to assess CPF for its potential to disrupt thyroid hormone signalling and investigate the short- and long-term effects on neurodevelopment by using Xenopus laevis. Methods: The thyroid hormone (TH) disrupting potential of CPF was assessed using TH-sensitive transgenic Tg(thibz:eGFP) tadpoles. The consequences of early embryonic exposure were examined by exposing fertilized eggs for 72 h to environmentally relevant CPF concentrations (10−10 M and 10−8 M). Three endpoints were evaluated: (1) gene expression in whole embryonic brains immediately after exposure, (2) mobility and brain morphology 1 week after exposure, and (3) brain morphology and axon diameters at the end of metamorphosis (2 months after the exposure). Results: CPF disrupted TH signalling in Tg(thibz:eGFP) tadpoles. The expression of genes klf9, cntn4, oatp1c1, and tubb2b was downregulated in response to CPF. Tadpoles exposed to CPF exhibited increased mobility and altered brain morphology compared to control tadpoles. Early embryonic exposure of CPF affected myelinated axon diameter, with exposed animals exhibiting shifted frequency distributions of myelinated axons diameters towards smaller diameters in the hindbrain of froglets. Discussion/Conclusion: This study provides more evidence of the endocrine and neurodevelopment disrupting activity of CPF. Further experimental and epidemiological studies are warranted to determine the long-term consequences of early CPF exposure on brain development.

Thyroid hormones (TH) are essential for normal brain development, influencing neural cell differentiation, migration, and synaptogenesis. Multiple endocrine-disrupting chemicals (EDCs) are found in the environment, raising concern for their potential effects on TH signaling and the consequences on neurodevelopment and behavior. While most research on EDCs investigates the effects of individual chemicals, human health may be adversely affected by a mixture of chemicals. The potential consequences of EDC exposure on human health are far-reaching and include problems with immune function, reproductive health, and neurological development. We hypothesized that embryonic exposure to a mixture of chemicals (containing phenols, phthalates, pesticides, heavy metals, and perfluorinated, polychlorinated, and polybrominated compounds) identified as commonly found in the human amniotic fluid could lead to altered brain development. We assessed its effect on TH signaling and neurodevelopment in an amphibian model (Xenopus laevis) highly sensitive to thyroid disruption. Fertilized eggs were exposed for eight days to either TH (thyroxine, T₄ 10 nM) or the amniotic mixture (at the actual concentration) until reaching stage NF47, where we analyzed gene expression in the brains of exposed tadpoles using both RT-qPCR and RNA sequencing. The results indicate that whilst some overlap on TH-dependent genes exists, T₄ and the mixture have different gene signatures. Immunohistochemistry showed increased proliferation in the brains of T₄-treated animals, whereas no difference was observed for the amniotic mixture. Further, we demonstrated diminished tadpoles’ motility in response to T₄ and mixture exposure. As the individual chemicals composing the mixture are considered safe, these results highlight the importance of examining the effects of mixtures to improve risk assessment.

Endocrine-disrupting chemicals (EDCs), found in all categories of chemicals, are suspected to be a cause of declining well-being and human health, both as single molecules and as mixtures. It is therefore necessary to develop high throughput methods to assess the endocrine-disrupting potential of multiple chemicals currently on the market that are as yet untested. An advantage of in vivo chemical screening is that it provides a full spectrum of physiological impacts exerted by a given chemical. Xenopus laevis is an ideal model organism to test thyroid axis disruption in vivo as thyroid hormones (THs) are highly conserved across vertebrates and orchestrate tadpole metamorphosis. In particular, NF stage 45 Xenopus laevis are most apt for in vivo screening as at this stage the tadpoles possess all the main elements of thyroid hormone signaling (thyroid receptors, deiodinases transporters) and are metabolically competent, while fitting into multiple well plates, allowing the use of small amounts of test chemicals. One way to assess the endocrine-disrupting potential of chemicals or mixtures thereof is to analyze gene expression in organisms after a short time exposure to the chemical(s). Here we describe a protocol using Xenopus laevis embryos to detect endocrine disruption of the thyroid axis by analysis of gene expression and an alternative protocol for fluorescence read-out using a transgenic GFP-expressing Xenopus laevis line. Taken together, these methods allow detection of subtle changes in TH signaling by EDCs that either activate or inhibit TH signaling in vivo.