There has been a 300-fold increase in the number and quantity of chemicals released into the environment in the last 50 years26. Many chemicals are now ubiquitous in the environment and in humans, including in pregnant women6. In this study, the rationale was to select chemicals found ubiquitously, test their thyroid disrupting effects individually and then, to represent intra-uterine exposure, test a mixture at concentrations found in amniotic fluid.

Focus on Thyroid disruption

We emphasised disruption of thyroid hormone signalling because (i) of the tight dependence of brain development on maternal levels of thyroid hormone4 (ii) a 3 day screening assay undergoing OECD validation is available and (iii) this hormonal axis is highly sensitive to endocrine disruption27. This sensitivity could relate to the complexity of thyroid hormone production that includes iodine uptake through specific symporters and a highly-regulated organification process28, but also to specific enzymes controlling thyroid hormone availability in peripheral targets1. A salient point is that thyroid hormones are the most complex halogenated molecules produced by vertebrates and the only one to contain iodine. Interestingly, eight of the 15 compounds tested here are halogenated, all of which showed thyroid signalling disruption, while most non-halogenated molecules (i.e. BPA, napthol and benzophenone-3) were inactive in the XETA test at relevant concentrations. Only two non-halogenated molecules were active: mercury and lead. Mercury chelates selenium29, an element required for synthesis and activity of all deiodinases30. This feature links mercury to interference with thyroid hormone activation/inactivation via deiodination, through selenocysteine deiodinases that are active in Xenopus tadpoles at this developmental stage20,31. These enzymes finely tune the bioavailability of the active form of thyroid hormone (T 3 ) in each cell.

Increased TH signalling with individual compounds and mixtures

These deiodination processes, and more generally the complexity of thyroid hormone signalling, could also contribute to explaining why the majority of individual chemicals and the mixture induced increase in thyroid hormone availability (after 72 h). It is worth pointing out that we did not observe additive effects of the individual compounds used as a mixture. Indeed, at the concentrations used in the mixture, certain compounds could be exerting negative effects whilst other exert positive effects. Given the multiple possible pathways affected, the overall readout on transcription could be muted. Similarly, the effect of one compound could override that of others. Previous epidemiological and experimental data on some individual chemicals could have led to predict decreased thyroid hormone availability. For instance, high PCB or BDE exposure depresses circulating thyroid hormone levels in humans and different species32,33. However, many of these experiments were based on long term exposure where increased clearance is subsequent to transient interactions with distributor proteins. In such cases, one could well expect a transient increase in thyroid hormone availability as observed in our experimental model34. Moreover, brain T 3 increased bioavailability is also strongly suggested by the mixture-induced increased expression of dio2 encoding for activating enzyme D2 (Fig. 2b). In this light, it should be borne in mind that our in vivo screening readout encompasses multiple levels of thyroid hormone signalling axis which could, by definition, indicate multiple levels of disruption. Indeed, this screening model offers the huge advantage of detecting chemicals interacting directly or indirectly with TH signalling whatever the level of disruption, but requires deeper investigations to identify specific mode of action. The thyroid disruption property of the mixture and the subsequent fluorescence increase, while possibly indirect for certain components, is clearly shown by the co-exposure with antagonist NH-3 that abrogated 10x mixture induced fluorescence in presence or absence of T 3 (Fig. S2a and b).

Early stages of development are considered to be amongst the most vulnerable windows for exposure as they represent ongoing organogenesis35. Studying mammalian embryos at these early stages is challenging due to their intrauterine development and limited numbers of embryos per mother. Hence, there is a need for more tractable models. The free-living amphibian X. laevis tadpole, provides large-scale screening tools and allows easy access to early developmental stages. A further advantage of the Xenopus system is the high homology of thyroid hormone signalling with mammals that is not fully shared by other free-living aquatic models such as teleosts.

After having established the thyroid disrupting effect of the mixture, rationale was then to mimic an embryonic exposure during a critical period for brain development. For that purpose mixture alone was applied to embryos before thyroid gland formation during neurogenesis. Our results show that mixture exposure affects T 3 –dependent transcription, cellular responses and behaviour. These multiple early developmental effects could well be interrelated.

Among the brain-expressed genes significantly modified were numerous actors implicated in thyroid hormone signalling, such as deiodinases, TRs, thyroid hormone transporters, and thyroid hormone targets including determinants of neural development. Expression of the activating/inactivating deiodinase, D1 was significantly decreased, whilst that of the activating deiodinase, D2, was significantly increased. These findings fit with the results from the XETA test that displayed a dose-dependent increase in thyroid hormone signalling following chemical mixture exposure, reflecting greater bioavailability of the hormone.

Relevance of these results to human brain development

At first sight, given the essential role of thyroid hormones in brain development, one might think that more hormone is not problematic. However, a number of results counter this idea. First, Korevaar et al.4 in their study of mother/child pairs, showed that maternal hyperthyroidism has an equally adverse effect on children’s IQ and brain structure, as does maternal hypothyroidism. Second, many rodent studies have revealed deleterious effects of hyperthyroidism and hypothyroidism during brain development36. Finally, during neurogenesis, thyroid hormones act as differentiation signals, directly repressing the pluripotency gene Sox237. Early exposure to excess thyroid hormone could therefore induce precocious differentiation of the neural progenitor populations, with ensuing modifications of brain size and organisation. In the present study, expression of sox2 was down-regulated by exposure to mix 10x, as was the expression of a number of neural markers, including markers of neuronal (tubb2b) and oligodendrocyte (mbp) differentiation. Similarly, expression of an essential nerve growth factor, bdnf, was significantly decreased. BDNF variants have repeatedly been linked in human studies to autism spectrum disorder (ASD)38,39 as well as animal models of this neurodevelopmental disorder40,41. In our model, most of the changes in brain gene expression were only seen following exposure to mix 10x. However, it should be borne in mind that in this experimental context exposure is limited to 72 h. During this time it is probable that many of the chemicals are catabolised by the tadpoles, which are metabolically competent at this stage42. A similar situation is expected in the uterine environment, but in this case metabolites can accumulate in the amniotic fluid and prolong exposure if not removed through the umbilical cord blood and excreted by the mother. Moreover, from a legislative point of view, tolerable daily intake is calculated from no observed adverse effect level (NOAEL) in animal models with a security factor of 100 (10 for intra and 10 for interspecies differences). This also means that any result obtained in an animal model with 10 times human levels is relevant and highlight the absolute need for a better legislation.

Strikingly, in tadpoles exposed to mixture, we found that exposure to the chemical mixture at 1x concentration significantly increased proliferation in neurogenic zones (Fig. 4a,b) but also oligodendrocyte volume (Fig. 4g) whilst decreasing that of neurons (Fig. 4f). Interestingly, autopsies of brains from ASD patients have revealed changes in neuronal cell volumes43,44. Changes were also found in ratios of oligodendrocyte to neurons, with numbers of neurons being significantly reduced following short-term exposure to the chemical mixture (Fig. 4d). Again, this finding has relevance to human data. Analyses of maternal thyroid hormone levels during early pregnancy revealed that both maternal hypothyroidism and hyperthyroidism can result in changes in children’s brain structure with modifications of grey to white matter ratios4, reflecting changes in neuron to oligodendrocyte numbers.

Finally, we found that the molecular and cellular modifications resulting from mixture exposure led to marked behavioural changes as assessed by mobility tracking. Exposure to mixture significantly reduced total distance travelled by tadpoles, with mix 10x reducing distance travelled by over 50%. Maternal hypothyroidism increases the risk for many neurodevelopmental diseases characterised by behavioural problems, including ASD and Attention Deficit Hyperactivity Disorder (ADHD)45,46. We provide evidence that most of the ubiquitous compounds measured in human amniotic fluid disrupt thyroid signalling alone or if applied as a mixture. We also show that mixture exposure results in a number of T 3 –like effects in expression of key genes and neural proliferation in the brain with ensuing effects on behaviour.

Many chemicals in this mixture could also affect other endocrine pathways beside thyroid hormone. An example is phthalates that are known to affect androgen signalling47 but exposure to which has also recently been linked during pregnancy with altered maternal thyroid levels48. Importantly, epidemiological studies show that maternal exposure to many of the chemicals studied here can affect offspring IQ and/or neurodevelopmental disease risk. This is the case for PCBs that have been linked to IQ loss49 and increased ADHD risk50. As to phthalates, numerous members of this vast chemical category, have also been linked to IQ loss51 and risk for different forms of neurodevelopmental disease52,53. Further, multiple studies show that increased maternal perchlorate levels correlate negatively with offspring IQ54. Lastly, PBDEs represent yet another large chemical category where maternal exposure has repeatedly been associated with IQ loss and increased ASD risk55.