Impact of BPA, BPF and Mixture N1 on DNA-Methylation of GRIN2B and NR3C1 during human neuroprogenitor cell differentiation

Sammanfattning: Endocrine disrupting chemicals (EDCs) are ubiquitous and their adverse impact on nature, wildlife and humans is extensively researched. We are constantly exposed to EDCs, such as the widespread and extensively researched Bisphenol A, as well as its substitute Bisphenol F, which is coming into wider use, even though it is much less is researched and limited information is available about its endocrine effects. Realistically, we are exposed to mixtures rather than single substances. In the Swedish Environmental Longitudinal, Mother and Child, Asthma and allergy (SELMA) study, the co-exposure of EDCs was assessed. Based on the SELMA cohort data, a study identified a mixture of EDCs, Mixture N1, which is associated with delayed language development. In recent years, it has been hypothesized that epigenetic alterations are one of the underlying mechanisms for the effect of EDC exposures. For example, EDC induced changes in DNA Methylation of the promoter region of a gene might lead to altered gene expression, which can result in adverse health effects. Several studies already indicate an impact of the formerly introduced chemicals/mixtures on the DNA methylation on genes such as NR3C1 and GRIN2B in animals. However, limited research is available on the impact on NR3C1 and GRIN2B in the early human brain, which is of interest since both genes are crucial for the development of the brain and altered gene expression often leads to adverse effects. This study aimed to investigate the impact of BPA, BPF and Mixture N1 on NR3C1 and GRIN2B in the developing human brain as well as establish a protocol for differentiation of human stem cells into neuroprogenitor cells that express GRIN2B and NR3C1. In the end stem cells were differentiated in vitro into neural progenitor cells (NPCs) using the protocol of Hosseini et al. (2020). During the differentiation, the cells were exposed to different concentrations of the former mentioned chemicals. Afterwards, RNA and DNA were extracted, followed by a qPCR and bisulfite-pyrosequencing to investigate the changes in gene expression and DNA methylation of NR3C1 and GRIN2B. This study established the differentiation protocol but revealed no significant results regarding the chemical exposure. However, some chemical exposures showed a clear tendency towards an impact of the chemicals on the gene expression and the DNA methylation. Furthermore, a negative correlation between DNA methylation at 2 CpG sites and gene expression in NR3C1 could be observed. In conclusion, the DNA methylation at promoter region in NR3C1 is important for the gene expression.