Lithium and endocrine disruption: A concern for human health?

Lithium is a key medication for the treatment of psychiatric disorders and is also used in various industrial applications (including battery production and recycling). Here, we review published data on the endocrine-disrupting potential of lithium, with a particular focus on the thyroid hormone system. To this end, we used PubMed and Scopus databases to search for, select and review primary research addressing human and animal health endpoints during or after lithium exposure at non-teratogenic doses. Given the key role of thyroid hormones in neurodevelopmental processes, we focused at studies of the neural effects of developmental exposure to lithium in humans and animals. Our results show that lithium meets the World Health Organization's definition of a thyroid hormone system disruptor - particularly when used at therapeutic doses. When combined with knowledge of adverse outcome pathways linking molecular initiating events targeting thyroid function and neurodevelopmental outcomes, the neurodevelopmental data reported in animal experiments prompt us to suggest that lithium influences neurodevelopment. However, we cannot rule out the involvement of additional modes of action (i.e. unrelated to the thyroid hormone system) in the described neural effects. Given the increasing use of lithium salts in new technologies, attention must be paid to this emerging pollutant - particularly with regard to its potential effects at environmental doses on the thyroid hormone system and potential consequences on the developing nervous system.

Pressures of the urban environment on the endocrine system: Adverse effects and adaptation.

With an increasing collective awareness of the rapid environmental changes, questions and theories regarding the adaptability of organisms are emerging. Global warming as well as chemical and non-chemical pollution have been identified as triggers of these adaptative changes, but can we link different kinds of stressors to certain phenotypic traits? The physiological adaptation, and particularly endocrine system adaptation, of living beings to urban environments is a fascinating way of studying urban endocrinology, which has emerged as a research field in 2007. In this paper, we stress how endocrine disruption in humans and environment can be studied in the urban environment by measuring the levels of pollution, endocrine activities or adversity. We broaden the focus to include not only exposure to the chemicals that have invaded our private spheres and their effects on wild and domestic species but also non-chemical effectors such as light, noise and climate change. We argue that taking into account the various urban stress factors and their effects on the endocrine system would enable the adoption of new approaches to protect living organisms.

[Disruptors of thyroid hormones: Which consequences for human health and environment?] / Les perturbateurs des hormones thyroïdiennes : comment estimer leurs impacts sur la santé humaine et l'environnement ?

Endocrine disruptors (EDs) of chemical origin are the subject of numerous studies, some of which have led to measures aimed at limiting their use and their impact on the environment and human health. Dozens of hormones have been described and are common to all vertebrates (some chemically related messengers have also been identified in invertebrates), with variable roles that are not always known. The effects of endocrine disruptors therefore potentially concern all animal species via all endocrine axes. These effects are added to the other parameters of the exposome, leading to strong, multiple and complex adaptive pressures. The effects of EDs on reproductive and thyroid pathways have been among the most extensively studied over the last 30 years, in a large number of species. The study of the effects of EDs on thyroid pathways and brain development goes hand in hand with increasing knowledge of 1) the different roles of thyroid hormones at cellular or tissue level (particularly developing brain tissue) in many species, 2) other hormonal pathways and 3) epigenetic interactions. If we want to understand how EDs affect living organisms, we need to integrate results from complementary scientific fields within an integrated, multi-model approach (the so-called translational approach). In the present review article, we aim at reporting recent discoveries and discuss prospects for action in the fields of medicine and research. We also want to highlight the need for an integrated, multi-disciplinary approach to studying impacts and taking appropriate action.

Title: Les perturbateurs des hormones thyroïdiennes : comment estimer leurs impacts sur la santé humaine et l'environnement ? Abstract: Les perturbateurs endocriniens (PE) d'origine chimique font l'objet de nombreuses études, certaines ayant permis des mesures visant à limiter leur utilisation et leurs impacts sur l'environnement et la santé humaine. Des dizaines d'hormones ont été décrites et sont communes à l'ensemble des vertébrés (certains messagers chimiquement proches ont été également répertoriés chez les invertébrés) avec des rôles variables et pas toujours connus. Les effets des PE concernent donc potentiellement toutes les espèces animales via tous les axes endocriniens ; ils s'ajoutent aux autres paramètres de l'exposome qui induisent une pression d'adaptation forte, multiple, et difficile à appréhender. Les effets des PE sur les voies de la reproduction et les voies thyroïdiennes sont parmi les plus étudiés depuis ces trente dernières années et ce, sur un grand nombre d'espèces. L'étude des effets des PE sur les voies thyroïdiennes ainsi que sur le développement cérébral va de pair avec l'augmentation des connaissances sur 1) les différents rôles des hormones thyroïdiennes au niveau cellulaire ou tissulaire (notamment le tissu cérébral en développement) chez de nombreuses espèces, 2) les autres voies hormonales et 3) les interactions épigénétiques. De façon générale, si l'on veut appréhender comment agissent les PE sur les organismes vivants, il est nécessaire d'analyser dans une approche intégrée et multi-modèles (approche dite translationnelle) les résultats issus de domaines scientifiques complémentaires. Dans cette brève revue, nous dressons un état des lieux de découvertes récentes et discutons les perspectives d'action dans les champs de la médecine et de la recherche. Nous mettons en avant la nécessité d'une approche intégrée et multidisciplinaire pour étudier les impacts des PE et prendre des mesures appropriées.

The highly and perpetually upregulated thyroglobulin gene is a hallmark of functional thyrocytes.

Abnormalities are indispensable for studying normal biological processes and mechanisms. In the present work, we draw attention to the remarkable phenomenon of a perpetually and robustly upregulated gene, the thyroglobulin gene (Tg). The gene is expressed in the thyroid gland and, as it has been recently demonstrated, forms so-called transcription loops, easily observable by light microscopy. Using this feature, we show that Tg is expressed at a high level from the moment a thyroid cell acquires its identity and both alleles remain highly active over the entire life of the cell, i.e., for months or years depending on the species. We demonstrate that this high upregulation is characteristic of thyroglobulin genes in all major vertebrate groups. We provide evidence that Tg is not influenced by the thyroid hormone status, does not oscillate round the clock and is expressed during both the exocrine and endocrine phases of thyrocyte activity. We conclude that the thyroglobulin gene represents a unique and valuable model to study the maintenance of a high transcriptional upregulation.

From Cohort to Cohort: A Similar Mixture Approach (SMACH) to Evaluate Exposures to a Mixture Leading to Thyroid-Mediated Neurodevelopmental Effects Using NHANES Data.

Prenatal exposure to a mixture (MIX N) of eight endocrine-disrupting chemicals has been associated with language delay in children in a Swedish pregnancy cohort. A novel approach was proposed linking this epidemiological association with experimental evidence, where the effect of MIX N on thyroid hormone signaling was assessed using the Xenopus eleuthero-embryonic thyroid assay (XETA OECD TG248). From this experimental data, a point of departure (PoD) was derived based on OECD guidance. Our aim in the current study was to use updated toxicokinetic models to compare exposures of women of reproductive age in the US population to MIX N using a Similar Mixture Approach (SMACH). Based on our findings, 66% of women of reproductive age in the US (roughly 38 million women) had exposures sufficiently similar to MIX N. For this subset, a Similar Mixture Risk Index (SMRIHI) was calculated comparing their exposures to the PoD. Women with SMRIHI > 1 represent 1.1 million women of reproductive age. Older women, Mexican American and other/multi race women were less likely to have high SMRIHI values compared to Non-Hispanic White women. These findings indicate that a reference mixture of chemicals identified in a Swedish cohort-and tested in an experimental model for establishment of (PoDs)-is also of health relevance in a US population.

A Mixture of Chemicals Found in Human Amniotic Fluid Disrupts Brain Gene Expression and Behavior in Xenopus laevis.

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.

Implementation of effect biomarkers in human biomonitoring studies: A systematic approach synergizing toxicological and epidemiological knowledge.

Human biomonitoring (HBM) studies have highlighted widespread daily exposure to environmental chemicals. Some of these are suspected to contribute to adverse health outcomes such as reproductive, neurological, and metabolic disorders, among other developmental and chronic impairments. One of the objectives of the H2020 European Human Biomonitoring Initiative (HBM4EU) was the development of informative effect biomarkers for application in a more systematic and harmonized way in large-scale European HBM studies. The inclusion of effect biomarkers would complement exposure data with mechanistically-based information on early and late adverse effects. For this purpose, a stepwise strategy was developed to identify and implement a panel of validated effect biomarkers in European HBM studies. This work offers an overview of the complete procedure followed, from comprehensive literature search strategies, selection of criteria for effect biomarkers and their classification and prioritization, based on toxicological data and adverse outcomes, to pilot studies for their analytical, physiological, and epidemiological validation. We present the example of one study that demonstrated the mediating role of the effect biomarker status of brain-derived neurotrophic factor BDNF in the longitudinal association between infant bisphenol A (BPA) exposure and behavioral function in adolescence. A panel of effect biomarkers has been implemented in the HBM4EU Aligned Studies as main outcomes, including traditional oxidative stress, reproductive, and thyroid hormone biomarkers. Novel biomarkers of effect, such as DNA methylation status of BDNF and kisspeptin (KISS) genes were also evaluated as molecular markers of neurological and reproductive health, respectively. A panel of effect biomarkers has also been applied in HBM4EU occupational studies, such as micronucleus analysis in lymphocytes and reticulocytes, whole blood comet assay, and malondialdehyde, 8-oxo-2'-deoxyguanosine and untargeted metabolomic profile in urine, to investigate, for example, biological changes in response to hexavalent chromium Cr(VI) exposure. The use of effect biomarkers in HBM4EU has demonstrated their ability to detect early biological effects of chemical exposure and to identify subgroups that are at higher risk. The roadmap developed in HBM4EU confirms the utility of effect biomarkers, and support one of the main objectives of HBM research, which is to link exposure biomarkers to mechanistically validated effect and susceptibility biomarkers in order to better understand the public health implications of human exposure to environmental chemicals.

Transient developmental exposure to low doses of bisphenol F negatively affects neurogliogenesis and olfactory behaviour in adult mice.

Neural stem cells in the murine subventricular zone (SVZ) reactivate during postnatal development to generate neurons and glia throughout adulthood. We previously demonstrated that a postnatal thyroid hormone (TH) peak orchestrates this remodelling, rendering this process vulnerable to endocrine disruption. We exposed mice to 2 or 200 µg/kg bw/day of the bisphenol A-replacement and suspected TH-disruptor bisphenol F (BPF) in the drinking water, from embryonic day 15 to postnatal day 21 (P21). In parallel, one group was exposed to the TH-synthesis blocker propylthiouracil (0.15 % PTU). In contrast to PTU, BPF exposure did not affect serum TH levels at P15, P21 or P60. RNA-seq on dissected SVZs at P15 revealed dysregulated neurodevelopmental genes in all treatments, although few overlapped amongst the conditions. We then investigated the phenotype at P60 to analyse long-term consequences of transient developmental exposure. As opposed to hypothyroid conditions, and despite dysregulated oligodendrogenesis-promoting genes in the P15 SVZ exposed to the highest dose of BPF, immunostainings for myelin and OLIG2/CC1 showed no impact on global myelin content nor oligodendrocyte maturation in the P60 corpus callosum, apart from a reduced thickness. The highest dose did reduce numbers of newly generated SVZ-neuroblasts with 22 %. Related to this were behavioural alterations. P60 mice previously exposed to the highest BPF dose memorized an odour less well than control animals did, although they performed better than PTU-exposed animals. All mice could discriminate new odours, but all exposed groups showed less interest in social odours. Our data indicate that perinatal exposure to low doses of BPF disrupts postnatal murine SVZ remodelling, and lowers the adult neuron/oligodendroglia output, even after exposure had been absent for 40 days. These anomalies warrant further investigation on the potential harm of alternative bisphenol compounds for human foetal brain development.

Limits of the regulatory evaluation of resorcinol as a thyroid disruptor: When limited experimental data challenge established effects in humans.

Severe hypothyroidism has been reported in humans during resorcinol therapeutic use. However, available data highlight differences in the severity of resorcinol-induced thyroid effects between humans and rodents, leading to a debate on the relevance of human data for its classification as a thyroid disruptor. The aim of this review is to illustrate some of the limitations of the evaluation framework for thyroid disrupters using resorcinol as a case study of a chemical with clear thyroid-disrupting properties in humans that could not have been identified solely from regulatory studies on animals. The reliability of human data has been called into question due to the specific exposure patterns in humans and the paucity of robust toxicokinetic data. In humans, therapeutic use of resorcinol induces severe hypothyroidism, but in rodents, thyroid disruption is limited to decreased thyroxine concentrations and histological changes in the thyroid. The adverse effects of thyroid disruption, such as impaired neurodevelopment, have not been sufficiently investigated, and experimental neurobehavioral data for resorcinol remain scarce and inconclusive. Although regulatory toxicological evaluations have not included in-depth investigations of thyroid regulation and related adverse effects, they have been used to challenge the relevance of human data. Resorcinol is an emblematic example of how the framework for regulatory evaluations of thyroid disruptors relies almost exclusively on animal studies which may not be suitable for assessing thyroid disruption. This review highlights the need to revise regulatory guidelines and to adopt strategies based on up-to-date, scientifically sound approaches to identify thyroid disruptors. The limits of the current regulatory framework for identifying thyroid disruptors can lead to opposing positions between regulatory bodies. The French Agency for Food, Environmental and Occupational Health & Safety (ANSES)'proposal to identify resorcinol as a "substance of very high concern" due to its ED properties has not been adopted by the European instances.

Short- and Long-Term Effects of Chlorpyrifos on Thyroid Hormone Axis and Brain Development in Xenopus laevis.

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.

Pyrethroids and developmental neurotoxicity - A critical review of epidemiological studies and supporting mechanistic evidence.

BACKGROUND: Pyrethroid metabolites are widely detectable in urine from the general population, including pregnant women and children. Pyrethroids are neurotoxic and suggested endocrine disruptors. Exposure during vulnerable developmental time windows may have long-term impacts on neurodevelopment. OBJECTIVE: To evaluate the epidemiological evidence for neurodevelopmental effects related to prenatal and childhood pyrethroid exposure in a systematic review and to assess biological plausibility by evaluating mechanistic evidence. METHODS: We searched PubMed and Web of Science up to September 1, 2021 and included original studies published in English in which pyrethroid exposure was measured or estimated during pregnancy or childhood and associations with neurodevelopmental outcomes in the children were investigated. The Navigation Guide Systematic Review Methodology was used to evaluate the epidemiological evidence. For mechanistic evidence, we focused on relevant key events (KEs) suggested in Adverse Outcome Pathways (AOPs) using the OECD-supported AOP-wiki platform. A systematic search combining the KEs with pyrethroids, including 26 individual compounds, was performed in the ToxCast database. RESULTS: Twenty-five epidemiological studies met the inclusion criteria, 17 presented findings on prenatal exposure, 10 on childhood exposure and two on both exposure windows. The overall body of evidence was rated as "moderate quality" with "sufficient evidence" for an association between prenatal pyrethroid exposure and adverse neurodevelopment. For childhood exposure, the overall rating was "low quality" with "limited evidence" because of cross-sectional study design. Regarding mechanistic evidence, we found that pyrethroids are able to interfere with neurodevelopmental KEs included in established AOPs for adverse neurodevelopmental. The evidence was strongest for interference with thyroid hormone (TH) function. CONCLUSION: Pyrethroids are probably human developmental neurotoxicants and adverse impacts of pyrethroid exposure on neurodevelopment are likely at exposure levels occurring in the general population. Preventive measures to reduce exposure among pregnant women and children are warranted.

Pollutants: a candidate as a new risk factor for osteoarthritis-results from a systematic literature review.

BACKGROUND: Considering non-classical environmental risk factors for osteoarthritis (OA), a systematic literature review (SLR) was performed to summarise existing knowledge on associations between OA and pollutants. METHODS: PubMed was used to identify studies reporting data on OA and pollutants in humans (examples of MeSH terms: "Pesticides" or "Polychlorinated Biphenyls" or 'Lead'). Reports included epidemiological clinical studies, pollutant assessments in ex vivo OA joint, and in vitro effects of pollutants on chondrocytes. RESULTS: Among the 193 potentially relevant articles, 14 were selected and combined with 9 articles obtained by manual search. Among these 23 articles there were: (1) 11 epidemiological studies on the relationship between OA and pollutants exposure, (2) 8 on pollutant concentrations in ex vivo OA joint, (3) 4 on the in vitro effects of pollutants on human chondrocytes. Epidemiological studies investigating mainly chlorinated and fluorinated pollutants suggested a possible link with OA. In cross-sectional studies, radiographic knee OA prevalence increased with higher serum lead levels. There was also a relationship between serum lead levels and serum/urine joint biomarkers. A high concentration of heavy metals in the cartilage tidemark was found in ex vivo joints. In vitro, the viability of chondrocytes was reduced in presence of some pollutants. However, the level of knowledge currently remains low, justifying the need for new methodologically sound studies. CONCLUSIONS: This SLR supports the hypothesis of a possible involvement of pollutants in OA disease risk. Large-scale epidemiological and biological studies and ideally big-data analysis are needed to confirm that pollutants could be risk factors for OA.

From cohorts to molecules: Adverse impacts of endocrine disrupting mixtures.

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.

Exposure to a mixture of benzo[a]pyrene and triclosan induces multi-and transgenerational metabolic disorders associated with decreased female investment in reproduction in Silurana (Xenopus) tropicalis.

Animals must partition limited resources between their own growth and subsequent reproduction. Endocrine disruptors (ED) may cause maternal metabolic disorders that decrease successful reproduction and might be responsible for multi- and transgenerational effects in amphibians. We found that the frog Silurana (Xenopus) tropicalis, exposed to environmentally relevant concentrations of benzo[a]pyrene and triclosan throughout its life cycle, produced F1 females with delayed sexual maturity and decreased size and weight. These F1 females displayed a marked metabolic syndrome associated with decreased fasting plasma cholesterol and triglyceride concentrations and decreased gonadal development. F1 females from F0 exposed animals also had decreased reproductive investment highlighted by a decrease of oocyte lipid reserves associated with significant F2-tadpole mortality. F2 females from F0 exposed animals also displayed a marked metabolic syndrome but were able to correctly direct liver lipid metabolism to the constitution of fat bodies and oocyte yolk stores. In addition, the F2 females produced progeny that had normal mortality levels at 5 days post hatching compared to the controls suggesting a good reproductive investment. Our data confirmed that these ED, at concentrations often found in natural ponds, can induce multi- and transgenerational metabolic disorders in the progeny of amphibians that are not directly exposed. We present a hypothesis to explain the transmission of the metabolic syndrome across generations through modification of egg reserves. However, when high mortality occurred at the tadpole stage, surviving females were able to cope with metabolic costs and produce viable progeny through sufficient investment in the contents of oocyte reserves.

[What about the effects of endocrine disruptors on neurodevelopment?] / Que sait-on de l'action des perturbateurs endocriniens sur le neurodéveloppement?

WHAT ABOUT THE EFFECTS OF ENDOCRINE DISRUPTORS ON NEURODEVELOPMENT? Development of the central nervous system (CNS) relies on many endogenous factors. Thyroid hormones and sex steroid hormones are among the most studied endocrine systems for their regulation of neural functions, since early embryonic stages of development. Environmental exposure to low doses of chemicals is largely documented. The hypothesis that exposure to some of these molecules with endocrine disrupting activity, likely interfering with the production of these hormones and/or their underlying neural mechanisms is therefore plausible and supported by numerous studies. After having recalled the formation of the hypothalamo-hypophyseal axes, and the importance of thyroid and sexual hormones in neurodevelopment, we will present two examples of substances (BPA, PCBs) and their effects on brain development.

QUE SAIT-ON DE L'ACTION DES PERTURBATEURS ENDOCRINIENS SUR LE NEURODÉVELOPPEMENT ? Le développement du système nerveux central (SNC) dépend de nombreux facteurs endogènes. Les hormones thyroïdiennes et les hormones stéroïdes sexuelles figurent parmi les systèmes endocriniens les plus étudiés pour leurs effets neuraux, et ce, dès les stades embryonnaires de développement. L'exposition environnementale à de faibles doses de substances chimiques est largement documentée. L'hypothèse que certaines de ces substances, appelées perturbateurs endocriniens, puissent interférer avec la production de ces hormones, et/ou de leurs mécanismes au niveau central est donc plausible et soutenue par une multitude de travaux. Après avoir rappelé la formation des axes hypothalamo- hypophysaires et l'importance des hormones thyroïdiennes et sexuelles dans le neurodéveloppement, nous présentons deux exemples de substances (bisphénol A, polychlorobiphényles) et leurs effets sur le développement cérébral.

Conserved role of the urotensin II receptor 4 signalling pathway to control body straightness in a tetrapod.

Urp1 and Urp2 are two neuropeptides of the urotensin II family identified in teleost fish and mainly expressed in cerebrospinal fluid (CSF)-contacting neurons. It has been recently proposed that Urp1 and Urp2 are required for correct axis formation and maintenance. Their action is thought to be mediated by the receptor Uts2r3, which is specifically expressed in dorsal somites. In support of this view, it has been demonstrated that the loss of uts2r3 results in severe scoliosis in adult zebrafish. In the present study, we report for the first time the occurrence of urp2, but not of urp1, in two tetrapod species of the Xenopus genus. In X. laevis, we show that urp2 mRNA-containing cells are CSF-contacting neurons. Furthermore, we identified utr4, the X. laevis counterparts of zebrafish uts2r3, and we demonstrate that, as in zebrafish, it is expressed in the dorsal somatic musculature. Finally, we reveal that, in X. laevis, the disruption of utr4 results in an abnormal curvature of the antero-posterior axis of the tadpoles. Taken together, our results suggest that the role of the Utr4 signalling pathway in the control of body straightness is an ancestral feature of bony vertebrates and not just a peculiarity of ray-finned fishes.

The pyriproxyfen metabolite, 4'-OH-PPF, disrupts thyroid hormone signaling in neural stem cells, modifying neurodevelopmental genes affected by ZIKA virus infection.

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.

Assessment of chemical mixtures using biomarkers of combined biological activity: A screening study in human placentas.

Humans are simultaneously exposed to complex mixtures of chemicals with limited knowledge on potential health effects, therefore improved tools for assessing these mixtures are needed. As part of the Human Biomonitoring for Europe (HBM4EU) Project, we aimed to examine the combined biological activity of chemical mixtures extracted from human placentas using one in vivo and four in vitro bioassays, also known as biomarkers of combined effect. Relevant endocrine activities (proliferative and/or reporter gene assays) and four endpoints were tested: the estrogen receptor (ER), androgen receptor (AR), and aryl hydrocarbon receptor (AhR) activities, as well as thyroid hormone (TH) signaling. Correlations among bioassays and their functional shapes were evaluated. Results showed that all placental extracts agonized or antagonized at least three of the abovementioned endpoints. Most placentas induced ER-mediated transactivation and ER-dependent cell proliferation, together with a strong inhibition of TH signaling and the AR transactivity; while the induction of the AhR was found in only one placental extract. The effects in the two estrogenic bioassays were positively and significantly correlated and the AR-antagonism activity showed a positive borderline-significant correlation with both estrogenic bioassay activities. However, the in vivo anti-thyroid activities of placental extracts were not correlated with any of the tested in vitro assays. Findings highlight the importance of comprehensively mapping the biological effects of "real-world" chemical mixtures present in human samples, through a battery of in vitro and in vivo bioassays. This approach should be a complementary tool for epidemiological studies to further elucidate the combined biological fingerprint triggered by chemical mixtures.

Transgenerational metabolic disorders and reproduction defects induced by benzo[a]pyrene in Xenopus tropicalis.

Metabolic disorders induced by endocrine disruptors (ED) may contribute to amphibian population declines but no transgenerational studies have evaluated this hypothesis. Here we show that Xenopus tropicalis, exposed from the tadpole stage, to the ED benzo[a]pyrene (BaP, 50 ng.L-1) produced F2 progeny with delayed metamorphosis and sexual maturity. At the adult stage, F2-BaP females displayed fatty liver with inflammation, tissue disorganization and metabolomic and transcriptomic signatures typical of nonalcoholic steato-hepatitis (NASH). This phenotype, similar to that observed in F0 and F1 females, was accompanied by a pancreatic insulin secretory defect. Metabolic disrupted F2-BaP females laid eggs with metabolite contents significantly different from the control and these eggs did not produce viable progeny. This study demonstrated that an ED can induce transgenerational disruption of metabolism and population collapse in amphibians under laboratory conditions. These results show that ED benzo[a]pyrene can impact metabolism over multiple generations and support epidemiological studies implicating environmental EDs in metabolic diseases in humans.