Effects of low-dose Bisphenol A on calcium ion influx and on genes of proliferation and differentiation in immortalized human gingival cells in vitro: The role of estrogen receptor beta.

OBJECTIVES:Relating to low-dose Bisphenol-A (BPA), there is still a lack of mechanistic studies in oral cells, representing the first targets of BPA by oral intake. The objective of this study was to investigate an assumed mechanistic interrelationship between both low-dose BPA-modulated Calcium ion (Ca2+) influx and cell behavior, and the estrogen receptor β (ERβ), in oral mucosal cells. METHODS:Indirect immunofluorescence (IIF) was conducted on estrogen receptor beta (ERβ) activity after 1, 3, and 6days in response to 39nM BPA, 15μM BPA, and 200 pM 17β-Estradiol (E2). In addition to Ca2+ concentration measurement, qPCR for proliferation and differentiation biomarkers was performed, to examine cell behavior. Fulvestrant-mediated ER inhibition was employed to seek for a mechanistic role of ERβ in regulating BPA-emanating effects. RESULTS:While both E2 and BPA yielded ERβ activation, 39nM BPA and 200 pM E2 did not change MKI67 proliferation marker expression, but reduced transcription of differentiation markers. Conversely, 15μM BPA reduced MKI67 transcription, but significantly increased differentiation gene expression and intracellular Ca2+ levels. Fulvestrant-induced ERβ inhibition yielded complete elimination of all E2- and BPA-triggered modulatory effects, suggesting a mechanistic role of activated ERβ for BPA-mediated Ca2+ influx and keratinocyte differentiation. SIGNIFICANCE:Concerning cell behavior, these findings provide significant evidence of a threshold-dependent transcription of proliferation and differentiation-related genes as well as Ca2+ influx in response to 39nM and 15μM low-dose BPA, which identify a mechanistic role of activated ERβ in oral keratinocytes.