Effects of Benzophenone-3 and Propylparaben on Estrogen Receptor-Dependent R-Loops and DNA Damage in Breast Epithelial Cells and Mice

Background: Endocrine-disrupting chemicals have been shown to have broad effects on development, but their mutagenic actions that can lead to cancer have been less clearly demonstrated. Physiological levels of estrogen have been shown to stimulate DNA damage in breast epithelial cells through mechanisms mediated by estrogen-receptor alpha ($\text{ER}\alpha$). Benzophenone-3 (BP-3) and propylparaben (PP) are xenoestrogens found in the urine of $>96\%$ of U.S.

Objectives: We investigated the effect of BP-3 and PP on estrogen receptor-dependent transactivation and DNA damage at concentrations relevant to exposures in humans.

Methods: In human breast epithelial cells, DNA damage following treatment with $17\beta \text{-estradiol}$ ($E_2$), BP-3, and PP was determined by immunostaining with antibodies against $\gamma \text{-}H2\text{AX}$ and 53BP1. Estrogenic responses were determined using luciferase reporter assays and gene expression. Formation of R-loops was determined with DNA: RNA hybrid-specific S9.6 antibody. Short-term exposure to the chemicals was also studied in ovariectomized mice. Immunostaining of mouse mammary epithelium was performed to quantify R-loops and DNA damage in vivo.

Results: Concentrations of $1\mu M$ and $5\mu M$ BP-3 or PP increased DNA damage similar to that of $E_2$ treatment in a $\text{ER}\alpha \text{-dependent}$ manner. However, BP-3 and PP had limited transactivation of target genes at $1\mu M$ and $5\mu M$ concentrations. BP-3 and PP exposure caused R-loop formation in a normal human breast epithelial cell line when $\text{ER}\alpha$ was introduced. R-loops and DNA damage were also detected in mammary epithelial cells of mice treated with BP-3 and PP.

Conclusions: Acute exposure to xenoestrogens (PP and BP-3) in mice induce DNA damage mediated by formation of $\text{ER}\alpha \text{-dependent}$ R-loops at concentrations 10-fold lower than those required for transactivation. Exposure to these xenoestrogens may cause deleterious estrogenic responses, such as DNA damage, in susceptible individuals. https://doi.org/10.1289/EHP5221.