Epigenetic Changes in the HTR8 and 3A-sub E placental Cell Lines Exposed to Bisphenol A and Benzyl Butyl Phthalate

Abstract

Objective: Bisphenol A and phthalate are known endocrine disruptors and capable of inducing epigenetic changes in the human population. However, their impact on the placenta is less well studied. Our objective was to measure the effect of exposure to bisphenol A and benzyl butyl phthalate in first-trimester HTR8-SVneo and third-trimester 3A-sub E trophoblast cells by profiling the DNA methylation pattern of the imprinting control region of the IGF2 (insulin-like growth factor) and H19 genes. Methods: Human placental HTR8-SVneo and 3A-sub E cell lines were treated with two sub-lethal concentrations of bisphenol A and benzyl butyl phthalate. Demethylating agent, 5-azacytidine, was used as a positive control. Cells were harvested on post-treatment days 1 and 4. The methylation profile of six CpG dinucleotide sites, part of the CTCF 6 binding site of the IGF2/H19 imprinting control region, was determined by pyrosequencing. Results: In the first-trimester HTR8-SVneo cell line, we observed a significant increased methylation of the CpG sites 3, 4 when treated with a high concentration of bisphenol A or benzyl butyl phthalate while increased methylation at site 6 for both high and low dose treatment on day 4. Demethylation of the CpG sites 1, 4, and 6 was observed when treated with 5-azacytidine on day 4. In the third-trimester 3A-sub E cell line, no significant changes in the methylation profile were observed under any treatment conditions. Conclusions: The results of this study demonstrate the capability of epigenetic changes in human placenta cells induced by bisphenol A and benzyl butyl phthalate. The observed methylation changes only in the first-trimester HTR8-SVneo cells phthalate may reflect a window of epigenetic susceptibility related to these environmental toxicants.