A mixture of chemicals commonly detected in human amniotic fluid has been found to perturb thyroid signalling, development of neurons and glia in the brain, and behavioural outcomes in offspring, when tested in a frog model of embryogenesis. The findings show the exquisite sensitivity of the developing organism to environmental contaminants.
Environmental chemicals are implicated as factors contributing to the increased prevalence of neurodevelopmental disorders 1. However, whether the environment has a causal role in the development of autism spectrum disorders, attention-deficit/hyperactivity disorder or other neurodevelopmental disorders is unclear. One reason for the con- troversy is that the human evidence is based on correlations between tissue concentrations of chemicals and neurobehavioural outcomes, each assessed at one or few times in an individual’s life. These studies cannot account for the real-world situation: that is, all wildlife, including humans, come into contact with a large number of chemicals in various mixtures, dosages and via different routes throughout their lives. Furthermore, dynamic changes in cellular and organ physiology in early life mean that even very low-level exposure to chemicals during sensitive periods might permanently change the developmental trajectory and lead to long-lasting dysfunction. This concept is particularly important when endocrine- disrupting chemicals (EDCs), defined as exogenous chemicals that interfere with any aspect of hormone action, are in the mix2. The endocrine system has a key role in determining how an organism responds and adapts to its environment. Disruptions to hormone signalling and metabolism, and actions of hormones on their receptors, potentially underlie the pathophysiological changes caused by exposures to EDCs 3.
“Exposure to multiple chemicals is inevitable… we live in a chemical soup..” – Linda Birnbaum (NIEHS)
The recent study by Fini, Bilal and colleagues demonstrates the power of a free-living embryonic model in assessing changes to early neurodevelopment and behaviour induced by exposure to a mixture of human-relevant environmental contaminants that perturb thyroid signalling 4.
In mammals, a pregnant mother’s exposure to the external world can inadvertently introduce EDCs into the exquisitely calibrated environment that comprises an embryo’s own hormones, together with maternal and placental hormones. Owing to the key impor- tance of proper thyroid hormone signalling in neurodevelopment, Fini and colleagues tested 15 chemicals found ubiquitously in the amniotic fluid of pregnant women in the USA for thyroid-disrupting activity. By using realistic exposure levels, and testing chemicals individually and in mixtures, the investigators have considerably advanced previous work in this area.
Embryonic xenopus brains (1 week post fertilization), treated or untreated with the mixture of 15 chemicals. (Upper panel) The mixture exposure results in a reduction of neuron number and volume, and a reduction in oligodendrocyte volume. (Lower panel) The mixture exposure increases the number of proliferating cells. © Scientific Reports
Fini, Bilal and colleagues used transgenic Xenopus laevis tadpoles that contained a thyroid hormone-responsive reporter gene. In these frogs, the gene for green fluorescent protein protein was coupled to the gene for a transcription factor sensitive to thyroid hormone activity, thereby enabling direct fluorescent visualization of changes in the action of thyroid hormone in the tadpoles. In the presence of thyroid hormone, many of the chemicals alone or in a mixture exaggerated the thyroid hormone activity response in the whole embryo but were insufficient to stimulate thyroid hormone activity alone 4. This result suggests a mechanism of increased bio- availability of thyroid hormone rather than activation of thyroid hormone receptors by the mixture.
…contaminants in amniotic fluid undoubtedly affect the development of our exposed children
Consistent with its thyroid-disrupting effects, the EDC mixture also perturbed proliferation and differentiation of neurons and glia 4. Changes in gene transcription, and downstream changes in the number of proliferating cells and the ratio of differentiated neurons to oligodendrocytes in the brains of exposed tadpoles, were also observed. Of particular interest, expression of the pluripotency marker sox2 was reduced in the brains of tadpoles treated with thyroid hormone and exposed to the mixture, which suggests that increased thyroid hormone activity signals the timing of neural differentiation. A limited precedence exists for effects of EDCs on the birth, maintenance and pruning of neurons during neuro-development. Another EDC, bisphenol A, acting through a different hormonal pathway that involves androgen and oestrogen signalling, was recently shown to induce precocious neurogenesis in the hypothalamus of developing zebrafish embryos 5. In the study by Fini, Bilal and colleagues, bisphenol A was a chemical component of the amniotic fluid mixture, but it did not increase thyroid hormone activity4. Nevertheless, its inclusion in the mixture might have contributed to the morphological changes observed in the brains of exposed tadpoles, something that requires further study.
Of note, the timing of exposure of the tadpoles is analogous to a particularly vulnerable window of brain development in humans during which the fetal thyroid gland has not yet developed and the only source of thyroid hormone should be from the mother’s circulation. Indeed, children’s IQ and cognitive performance are correlated with levels of maternal thyroid hormone during pregnancy 6. The finding by Fini, Bilal and colleagues that their chemical mixture can impair the ability of thyroid hormone target tissues to titrate the amount of bioactive molecules to which they are exposed might explain the changes that they observed in the brain.
In addition to effects on cell numbers,the EDC mixture also changed the volume of cells, with neurons decreased and oligodendrocytes increased in size 4. In humans, post-mortem examinations of the brains of individuals with autism spectrum disorders have shown changes in the volume of neurons during different life stages; however, the functional implications remain unclear, especially as teenagers and adults with autism and neuronal volumes similar to non-autistic controls do not recover cognitive function 7.Maternal levels of thyroid hormone have also been correlated with changes in the ratio of neurons to glia in children, and thyroid hormone is necessary for the maturation of commissural axons in the rat brain 6,8.
The importance of assessing effects of human-relevant mixtures of possible EDCs cannot be overstated. Exposure to single chemicals is almost exclusively attributable to occupational contact or acute accidental spills. With notable exceptions 9, research on mixtures has been impeded, especially in whole-animal studies, largely owing to logistical difficulties in assessing individual chemicals thoroughly in dose–response curves, and then determining whether and how to undertake the permutations and combinations of these exhaustive and exhausting studies. The advantages of conducting mixture work in a tractable transgenic Xenopus model are clear, but many questions remain to be answered. Although Fini and colleagues observed dose-dependent effects of many chemicals in their mixture, these components did not have an additive effect at the human-relevant mixture concentration. The individual constituent chemicals probably act at different levels of the thyroid hormone pathway or have opposing effects that obscure the phenotype. Furthermore, these chemicals are known to act on other hormonal systems beyond the thyroid, and hormones influence each other’s actions. The high conservation of endocrine systems and the importance of their interaction in normal physiology suggest that contaminants in amniotic fluid undoubtedly affect the development of our exposed children.
Paper: Fini J.B., Mughal B.B., Le Mével S., Leemans M., Lettmann M., Spirhanzlova P., Affaticati P., Jenett A., Demeneix B.A. Human amniotic fluid contaminants alter thyroid hormone signalling and early brain development in Xenopus embryos. Scientific Reports. 7, 43786 ; doi: 10.1038/srep43786 (2017).
1. Lyall, K. et al. The changing epidemiology of autism spectrum disorders. Annu. Rev. Public Health http://dx.doi.org/10.1146/annurev- publhealth-031816-044318 (2016).
2. Zoeller, R. T. et al. Endocrine-disrupting chemicals and public health protection: a statement of principles from The Endocrine Society. Endocrinology 9, 4097–4110 (2012).
3. Gore, A. C. et al. EDC-2: The Endocrine Society’s second scientific statement on endocrine-disrupting chemicals. Endocr. Rev. 6, E1–E150 (2015).
4. Fini, J.-B. et al. Human amniotic fluid contaminants alter thyroid hormone signalling and early brain development in Xenopus embryos. Sci. Rep. 7, 47386 (2017).
5. Kinch, C. D. et al. Low-dose exposure to bisphenol A and replacement bisphenol S induces precocious hypothalamic neurogenesis in embryonic zebrafish. Proc. Natl Acad. Sci. USA 5, 1475–1480 (2015).
6. Korevaar, T. I. et al. Association of maternal thyroid function during early pregnancy with offspring IQ and brain morphology in childhood: a population-based prospective cohort study. Lancet Diabetes Endocrinol. 1, 35–43 (2016).
7. Wegiel, J. et al. Neuronal nucleus and cytoplasm volume deficit in children with autism and volume increase in adolescents and adults. Acta Neuropathol. Commun. 3, 2 (2015).
8. Berbel, P. et al. Role of thyroid hormones in the maturation of interhemispheric connections in rats. Behav. Brain Res. 64, 9–14 (1994).
9. Crofton, K. M. et al. Thyroid-hormone-disrupting chemicals: evidence for dose-dependent additivity or synergism. Environ. Health Perspect. 11, 1549–1554 (2005).
Edit: A few video interviews prior to this publication that might help better understand and answer any questions.
English: Endocrine Disruptors: “European Commission is delaying, harming human health and the environment”
French: Perturbateurs endocriniens : La commission européenne échoue à trouver une définition