Supplementary Material for: 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 hours to environmentally relevant CPF concentrations (10-10M and 10-8M). Three endpoints were evaluated: 1) gene expression in whole embryonic brains immediately after exposure, 2) mobility and brain morphology one week after exposure and 3) brain morphology and axon diameters at the end of metamorphosis (2 months after the exposure).
Results
CPF disrupted TH signaling 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.