712: The effects of exposure to BPA and BBP on the DNA methylation profile of the IGF2/H19 imprinting control region in HTR-8 cells

Abstract

Bisphenol-A (BPA) and Butyl Benzyl Phthalate (BBP) affect a range of endocrine, metabolic and developmental outcomes. While not mutational, these plasticizers may disrupt the placental and fetal epigenetic profile of imprinted genes. Disruption of two of these imprinted genes, IGF2 and H19, may lead to alterations in fetal development and programming of adult disease. Our objective was to measure the effects of exposure to BPA and BBP in first trimester extravillous HTR8 trophoblast cells by: 1.Profiling the DNA methylation of the imprinting control region (ICR) of IGF2 and H19 2.Measuring IGF2 and H19 gene expression HTR8 cells were treated with two sublethal concentrations of BPA and BBP. 5-Azacitidine (AZA), a demethylating agent, was used as a positive control. Cells were harvested on post-treatment days 1 and 4. Extracted DNA was bisulfite-treated and purified. The methylation profile of six CpG dinucleotides, part of the CTCF6 binding site of the IGF2/H19 ICR, was determined by pyrosequencing. Extracted mRNA was quantified by real-time PCR for changes in IGF2 and H19 gene expression. There were no significant differences in the methylation profiles of the groups at the various time points. The CTCF6 binding site was shown to be insensitive to treatment with AZA in HTR8 cells. The commercially-available AZA-treated “control-unmethylated DNA” from Jurkat cells was shown to be 50% methylated. Finally, no differences were found in IGF2 and H19 gene expression. This data demonstrates that the epigenetic effects of BPA and BBP are not due to changes in methylation of the CTCF6 binding site of the IGF2/H19 ICR. Additionally, BPA and BBP do not cause dysregulation of IGF2 nor H19 gene expression in HTR8 cells. Interestingly, the CTCF6 binding site appears to be stably methylated and resistant to AZA treatment in the control Jurkat cells. This suggests that a different mechanism exists to control the epigenetic profile of this ICR. This finding is now being explored in the villous 3A first trimester placental cells and freshly prepared dendritic cells.