Search

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.

Thyroid hormones (THs) 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 thyroid hormone 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. Many compounds belonging to a wide range of chemical classes have been identified as EDCs, notably those affecting thyroid hormone signaling. We hypothesized that embryonic exposure to a mixture of chemicals (containing phenols, phthalates, pesticides, heavy metals, perfluorinated -, polychlorinated, and polybrominated compound) commonly found in the human amniotic fluid could lead to altered brain development to assess its effect on thyroid hormone signaling and neurodevelopment in an amphibian model (Xenopus laevis), highly sensitive to thyroid disruption. Newly hatched tadpoles were exposed for eight days to either TH (thyroxine, T4 10nM) or the amniotic mixture (1x concentration) and gene expression was analyzed in the brains of exposed tadpoles using both RT-qPCR and RNA sequencing. Results indicate that whilst some overlap on TH-dependent genes exist, 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

Breast cancer (BC) remains one of the most commonly diagnosed malignancies in women. There is increasing interest in the development of non-invasive screening methods. Volatile organic compounds (VOCs) emitted through the metabolism of cancer cells are possible novel cancer biomarkers. This study aims to identify the existence of BC-specific VOCs in the sweat of BC patients. Sweat samples from the breast and hand area were collected from 21 BC participants before and after breast tumor ablation. Thermal desorption coupled with two-dimensional gas chromatography and mass spectrometry was used to analyze VOCs. A total of 761 volatiles from a homemade human odor library were screened on each chromatogram. From those 761 VOCs, a minimum of 77 VOCs were detected within the BC samples. Principal component analysis showed that VOCs differ between the pre- and post-surgery status of the BC patients. The Tree-based Pipeline Optimization Tool identified logistic regression as the best-performing machine learning model. Logistic regression modeling identified VOCs that distinguish the pre-and post-surgery state in BC patients on both the breast and hand area with sensitivities close to 1. Further, Shapley additive explanations and the probe variable method identified the most important and pertinent VOCs distinguishing pre- and post-operative status which are mostly of distinct origin for the hand and breast region. Results suggest the possibility to identify endogenous metabolites linked to BC, hence proposing this innovative pipeline as a stepstone to discovering potential BC biomarkers. Large-scale studies in a multi-centered VOC analysis setting must be carried out to validate obtained findings.

Convergent evidence associates exposure to endocrine disrupting chemicals (EDCs) with major human diseases, even at regulation-compliant concentrations. This might be because humans are exposed to EDC mixtures, whereas chemical regulation is based on a risk assessment of individual compounds. Here, we developed a mixture-centered risk assessment strategy that integrates epidemiological and experimental evidence. We identified that exposure to an EDC mixture in early pregnancy is associated with language delay in offspring. At human-relevant concentrations, this mixture disrupted hormone-regulated and disease-relevant regulatory networks in human brain organoids and in the model organisms Xenopus leavis and Danio rerio , as well as behavioral responses. Reinterrogating epidemiological data, we found that up to 54% of the children had prenatal exposures above experimentally derived levels of concern, reaching, for the upper decile compared with the lowest decile of exposure, a 3.3 times higher risk of language delay.

International audience; 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.

Background: Remote medical scent detection of cancer and infectious diseases with dogs and rats has been an increasing field of research these last 20 years. If validated, the possibility of implementing such a technique in the clinic raises many hopes. This systematic review was performed to determine the evidence and performance of such methods and assess their potential relevance in the clinic.Methods: Pubmed and Web of Science databases were independently searched based on PRISMA standards. We included studies aiming at detecting cancers and infectious diseases affecting humans with dogs or rats. We excluded studies using other animals, studies aiming to detect agricultural diseases, diseases affecting animals, and others such as diabetes and neurodegenerative diseases. Only original articles were included. Data about patients’ selection, samples, animal characteristics, animal training and testing configurations, and performances were recorded.Results: A total of 62 studies were included. Sensitivity and specificity varied a lot among studies: While some publications report low sensitivities of 17% and specificities around 29%, others achieve rates of 100% sensitivity and specificity. Only 6 studies were evaluated in a double-blind screening like situation. In general, the risk of performance bias was high in most evaluated studies, and the quality of the evidence found was low.Conclusions: Medical detection using animals’ sense of smell lacks evidence and performances so far to be applied in the clinic. What odours the animals detect is not well understood. Further research should be conducted, focusing on patient selection, samples (choice of materials, standardization), and testing conditions. Interpolations of such results to free running detection (direct contact with humans) should be taken with extreme caution.

North-Eastern Brazil saw intensive application of the insecticide pyriproxyfen (PPF) during the microcephaly outbreak caused by Zika virus (ZIKV). ZIKV requires the neural RNA-binding protein Musashi-1 to replicate. TH represses MSI1. Being a suspected TH disruptor, we hypothesized that co-exposure to the main metabolite of PPF, 4’-OH-PPF, would exacerbate ZIKV effects through increased MSI1 expression. This was tested using in vitro mouse neurospheres and an in vivo TH signaling reporter model, Xenopus laevis. TH signaling was decreased by 4’-OH-PPF in both models. In mouse-derived neurospheres the metabolite reduced neuroprogenitor proliferation as well as markers of neuronal differentiation. The results demonstrated that 4’-OH-PPF significantly induced MSI1 at both the mRNA and protein level, as well as Fasn mRNA. Other TH target genes were also significantly modified. Importantly, several key genes implicated in neuroprogenitor fate and commitment were not dysregulated by 4’-OH-PPF alone, but were in combination with ZIKV infection. These included the neuroprogenitor markers Nestin, Egfr, Gfap, Dlx2 and Dcx. Unexpectedly, 4’-OH-PPF decreased ZIKV replication, although only at the fourth and last day of incubation, and RNA copy numbers stayed within the same order of magnitude. However, intracellular RNA content of neuroprogenitors was significantly decreased in the combined presence of the PPF metabolite and ZIKV. We conclude that 4’-OH-PPF interferes with TH action in vivo and in vitro, inhibiting neuroprogenitor proliferation. In the presence of ZIKV, TH signaling pathways crucial for cortical development are significantly impacted. This provides another example of viral effects that are exacerbated by drug or pesticide use.Significance statementIn 2015, an increase in children born with unusually small heads (microcephaly) in North-Eastern Brazil was linked to infection with the ZIKA virus. An insecticide with thyroid hormone disruptive properties was used in the same areas. We investigated whether simultaneous exposure to the insecticide could increase viral susceptibility. The main metabolite 4’-OH-PPF dysregulated thyroid hormone signaling pathways crucial for brain development in both models used. Neural stem cells proliferated less and contained more Musashi-1, a protein the virus needs to replicate. Infecting stem cells pre-exposed to the endocrine disruptor did not amplify viral replication, but aggravated expression of genes implicated in brain development. Our results suggest the insecticide is particularly deleterious to brain development in areas with ZIKA virus prevalence.

International audience; Plant Protection Products, more commonly referred to as pesticides and biocides, are used to control a wide range of yield-reducing pests including insects, fungi, nematodes, and weeds. Concern has been raised that some pesticides may act as endocrine disrupting chemicals (EDCs) with the potential to interfere with the hormone systems of non-target invertebrates and vertebrates, including humans. EDCs act at low doses and particularly vulnerable periods of exposure include pre-and perinatal development. Of critical concern is the number of pesticides with the potential to interfere with the developing nervous system and brain, notably with thyroid hormone signaling. Across vertebrates, thyroid hormone orchestrates metamorphosis, brain development, and metabolism. Pesticide action on thyroid homeostasis can involve interference with TH production and its control, displacement from distributor proteins and liver metabolism. Here we focused on thyroid endpoints for each of the different classes of pesticides reviewing epidemiological and experimental studies carried out both in in vivo and in vitro. We conclude first, that many pesticides were placed on the market with insufficient testing, other than acute or chronic toxicity, and second, that thyroid-specific endpoints for neurodevelopmental effects and mixture assessment are largely absent from regulatory directives.

International audience; Thyroid hormones (THs) play critical roles in profound changes in many vertebrates, notably in mammalian neurodevelopment, although the precise molecular mechanisms of these fundamental biological processes are still being unravelled. Environmental and health concerns prompted the development of chemical safety testing and, in the context of endocrine disruption, identification of thyroid hormone axis disrupting chemicals (THADCs) remains particularly challenging. As various molecules are known to interfere with different levels of TH signalling, screening tests for THADCs may not rely solely on in vitro ligand/receptor binding to TH receptors. Therefore, alternatives to mammalian in vivo assays featuring TH-related endpoints that are more sensitive than circulatory THs and more rapid than thyroid histopathology are needed to fulfil the ambition of higher throughput screening of the myriad of environmental chemicals. After a detailed introduction of the context, we have listed current assays and parameters to assess thyroid disruption following a literature search of recent publications referring to non-mammalian models. Potential THADCs were mostly investigated in zebrafish and the frog Xenopus laevis, an amphibian model extensively used to study TH signalling.

International audience; 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. MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes. Reagents Agilent RNA 6000 Pico Kit Aquatic system water, chlorine-free Filter through an aquatic water system (e.g., Veolia custom system).

Epidemiological and experimental studies have raised questions as to whether the insecticide pyriproxyfen (PPF) could be implicated in the increased incidence of microcephaly associated with ZIKA infection during pregnancy. This pesticide is documented as a thyroid hormone (TH) disrupting chemical. We investigated whether environmentally relevant amounts of its main metabolite, 4'-OH-pyriproxyfen (4'-OH-PPF), modified TH signaling and early neuronal development. First, an in silico study revealed strong affinity of 4-OH-PPF to fit the ligand binding pocket of TH receptors (TRs). Further, in vitro assays on human cell lines showed 4'OH-PPF (> 3 mg/L ) to act as a TRα antagonist. Next, using a transgenic Xenopus TH-sensitive reporter system, Tg(thibz:GFP) tadpoles showed that 4'OH-PPF (> 10-7 mg/L) displayed TH-disruptive activity and reduced tadpole mobility (> 10-1 mg/L). Exposure to 4'OH-PPF significantly reduced Xenopus head size at levels equivalent to the maximum recommended daily intake of PPF (3x 10-1 mg/L). Most strikingly, in both the Xenopus system in vivo and in mouse neurosphere cultures, environmentally relevant concentrations of 4'OH-PPF increased expression of the gene that enables ZIKA replication: Musashi-1 (msi1) in neurogenic brain areas. We conclude that first, the PPF metabolite, 4'OH-PPF, disrupts thyroid signaling, neuronal development and behavior in Xenopus embryos, and second, that it increases Musashi-1 levels in neurogenic zones of both mouse and Xenopus, creating the potential to enhance viral replication. As PPF is used in areas with high microcephaly incidence and is readily broken down to 4'OH-PPF, these findings provide a plausible mechanism whereby PPF could, through induced changes in expression of Musashi-1, exacerbate the effects of ZIKA virus infection.

Reference genes are essential for gene expression analysis when using real-time quantitative PCR (RT-qPCR). Xenopus laevis is a popular amphibian model for studying vertebrate embryogenesis and development. Further, X. laevis is ideal for studying thyroid signaling due to its thyroid dependent metamorphosis, a stage comparable to birth in humans. When using PCR based studies, a primary concern is the choice of reference genes. Commonly used references are eef1a1, odc1, rpl8, and actnB, although there is a lack of ad hoc reference genes for X. laevis. Here, we used previously published RNA-seq data on different X. laevis stages and identified the top 14 candidate genes with respect to their expression levels as a function of developmental stage and degree of variation. We further evaluated the stability of these and other candidate genes using RT-qPCR on various stages including the unfertilised eggs, whole embryos during early development and brains during late development. We used four different statistical software packages: deltaCT, geNorm, NormFinder and BestKeeper. We report optimized reference gene pair combinations for studying development (early whole embryos), brains at later stages (metamorphosis and adult), and thyroid signalling. These reference gene pairs are suitable for studying different aspects of X. laevis development and organogenesis.

Xenopus is an excellent model for studying thyroid hormone signaling as it undergoes thyroid hormone–dependent metamorphosis. Despite the fact that receptors and deiodinases have been described in Xenopus, membrane transporters for these hormones are yet to be characterized. We cloned Xenopus monocarboxylate transporter 8 (mct8) and organic anion-transporting polypeptide 1C1 (oatpc1c1), focusing on these two transporters given their importance for vertebrate brain development. Protein alignment and bootstrap analysis showed that Xenopus mct8 and oatp1c1 are closer to their mammalian orthologs than their teleost counterparts. We functionally characterized the two transporters using a radiolabeled hormones in vitro uptake assay in COS-1 cells. Xenopus mct8 was found to actively transport both T3 and T4 bidirectionally. As to the thyroid precursor molecules, diiodotyrosine (DIT) and monoiodotyrosine (MIT), both human and Xenopus mct8, showed active efflux, but no influx. Again similar to humans, Xenopus oatp1c1 transported T4 but not T3, MIT, or DIT. We used reverse transcription quantitative polymerase chain reaction and in situ hybridization to characterize the temporal and spatial expression of mct8 and oatp1c1 in Xenopus. Specific expression of the transporter was observed in the brain, with increasingly strong expression as development progressed. In conclusion, these results show that Xenopus thyroid hormone transporters are functional and display marked spatiotemporal expression patterns. These features make them interesting targets to elucidate their roles in determining thyroid hormone availability during embryonic development.

Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are a group of neurodegenerative conditions that are transmissible, progressive and uniformly fatal. They occur in both humans and animals. Prion disease came to prominence after the development of bovine spongiform encephalopathy (BSE) in the 1980s and, consequently, variant Creutzfeldt–Jakob disease (vCJD) in the 1990s. BSE and vCJD have had far reaching implications for the UK economy but especially for the farming and healthcare sectors. With the advent of BSE, prices for beef originating in the UK fell dramatically and this proved crippling to much of the UK farming community. Although the peak of the vCJD epidemic appears to have passed, prevalence studies suggest that around 1 in 3000 UK adults may be infected with vCJD prions (http://www.hpa. org.uk/hpr/archives/2011/news3611.htm#cjd). As there is currently no treatment for vCJD, considerable effort has been put into disease prevention. Difficulties in the diagnosis of vCJD, lack of treatment, and inability of conventional sterilization to completely remove prion protein from surgical instruments have also resulted in significant changes in healthcare delivery. Although the implications of vCJD for the delivery of anaesthesia are limited, it is important that anaesthetists understand how to identify patients at high risk of vCJD and are aware of the relevant precautionary measures that must be taken.

In response to the 2013 ISH-CSF task force review on comorbidities in NPH we assessed 73 patients who were diagnosed with NPH and underwent shunt surgery at our tertiary neurosurgical unit between August 2008 and August 2012.

International audience; Thyroid hormones are essential for normal brain development in vertebrates. In humans, abnormal maternal thyroid hormone levels during early pregnancy are associated with decreased offspring IQ and modified brain structure. As numerous environmental chemicals disrupt thyroid hormone signalling, we questioned whether exposure to ubiquitous chemicals affects thyroid hormone responses during early neurogenesis. We established a mixture of 15 common chemicals at concentrations reported in human amniotic fluid. An in vivo larval reporter (GFP) assay served to determine integrated thyroid hormone transcriptional responses. Dose-dependent effects of short-term (72 h) exposure to single chemicals and the mixture were found. qPCR on dissected brains showed significant changes in thyroid hormone-related genes including receptors, deiodinases and neural differentiation markers. Further, exposure to mixture also modified neural proliferation as well as neuron and oligodendrocyte size. Finally, exposed tadpoles showed behavioural responses with dose-dependent reductions in mobility. In conclusion, exposure to a mixture of ubiquitous chemicals at concentrations found in human amniotic fluid affect thyroid hormone-dependent transcription, gene expression, brain development and behaviour in early embryogenesis. As thyroid hormone signalling is strongly conserved across vertebrates the results suggest that ubiquitous chemical mixtures could be exerting adverse effects on foetal human brain development.