Xenoestrogens cause estrogen receptor-dependent R-loop formation and DNA damage

Background: The hormone 17β-estradiol (E2) plays an important role in breast cell proliferation and development but prolonged exposures and higher levels of E2 have been linked to genomic instability and breast cancer. Recent studies show E2 induced transactivations lead to DNA damage. A variety of environmental chemicals mimic the activities of estrogen, referred to as xenoestrogens. Thus, our hypothesis is to determine whether xenoestrogens stimulate transcriptional response and induce DNA damage similarly like E2. Methods: The T-47D breast cancer cell line is used as a model because it expresses both estrogen receptors (ERα u0026 ERβ) as found in normal breast tissue. Cells were exposed a physiologic range of E2 (17β-estradiol) [0.5-100nM] and the xenoestrogens, e.g., benzophenone-3 (BP-3) [0.5-50µM] and propyl paraben (PP) [0.5-10µM] with or without ER-antagonist ICI (1µM) for 24 hours. Transcriptional responses were quantified using an integrated ERE-luciferase reporter and qPCR for endogenous genes. Proliferation was monitored using the Alamar Blue assay. DNA damage was determined using γ-H2AX. R-loops were detected using the S9.6 antibody against DNA-RNA hybrids in the genome. Results: Exposure of T-47D cells to physiologic levels of E2, PP and BP3 causes significant increase in DNA double-strand breaks (DSBs) as determined by γ-H2AX staining. We demonstrate a concomitant increase in R-loop formation with increase in γH2AX intensity. Treatment with RNaseH depletes R-loop intensity, corroborating the result. E2 and PP induce transactivation as determined by Luciferase reporter assay as well as expression of ER-response genes (PGR and AREG), whereas BP3 has modest effect on transactivation of PGR. ER transactivation as well as R-loop formation by E2, PP and BP3 were abrogated with ICI, demonstrating ER-dependency of R-loop formation. Consistent with the transcriptional response, proliferation studies determined that E2 showed highest proliferation followed by PP. However, BP3 showed no increase in proliferation. Conclusions: Our results show that xenoestrogens have strikingly different activities in transactivation and proliferation. However, both xenoestrogens appear to stimulate R-loop accumulation that leads to DSB in an ER-dependent manner. Citation Format: Karen A. Dunphy, Prabin Dhangada Majhi, Aman Sharma, Amy L. Roberts, Elizabeth A. Daniele, Sallie S. Schneider, D Joseph Jerry. Xenoestrogens cause estrogen receptor-dependent R-loop formation and DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3750.