Skip to Content
MilliporeSigma
  • Effects of Benzophenone-3 and Propylparaben on Estrogen Receptor-Dependent R-Loops and DNA Damage in Breast Epithelial Cells and Mice.

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

Environmental health perspectives (2020-01-16)
Prabin Dhangada Majhi, Aman Sharma, Amy L Roberts, Elizabeth Daniele, Aliza R Majewski, Lynn M Chuong, Amye L Black, Laura N Vandenberg, Sallie S Schneider, Karen A Dunphy, D Joseph Jerry
ABSTRACT

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 ( ER α ). Benzophenone-3 (BP-3) and propylparaben (PP) are xenoestrogens found in the urine of > 96 % of U.S. We investigated the effect of BP-3 and PP on estrogen receptor-dependent transactivation and DNA damage at concentrations relevant to exposures in humans. In human breast epithelial cells, DNA damage following treatment with 17 β -estradiol ( E 2 ), BP-3, and PP was determined by immunostaining with antibodies against γ - H 2 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. Concentrations of 1 μ M and 5 μ M BP-3 or PP increased DNA damage similar to that of E 2 treatment in a ER α -dependent manner. However, BP-3 and PP had limited transactivation of target genes at 1 μ M and 5 μ M concentrations. BP-3 and PP exposure caused R-loop formation in a normal human breast epithelial cell line when ER α was introduced. R-loops and DNA damage were also detected in mammary epithelial cells of mice treated with BP-3 and PP. Acute exposure to xenoestrogens (PP and BP-3) in mice induce DNA damage mediated by formation of ER α -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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Phosphatase Inhibitor Cocktail 2, aqueous solution (dark coloration may develop upon storage, which does not affect the activity)
Sigma-Aldrich
Phosphatase Inhibitor Cocktail 3, DMSO solution
Millipore
Immobilon®-P PVDF Membrane, 1 roll, 27 cm x 3.75 m, 0.45 µm pore size, Hydrophobic PVDF Transfer Membrane for western blotting.
Sigma-Aldrich
5-Bromo-2′-deoxyuridine, ≥99% (HPLC)
Sigma-Aldrich
Hydrocortisone, ≥98% (HPLC)
Sigma-Aldrich
β-Estradiol, BioReagent, powder, suitable for cell culture
Sigma-Aldrich
Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution
Sigma-Aldrich
2-Hydroxy-4-methoxybenzophenone, 98%
Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium/Nutrient Mixture F-12 Ham, With 15 mM HEPES and sodium bicarbonate, without L-glutamine and phenol red, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Propyl 4-hydroxybenzoate, ≥99%
Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium/Nutrient Mixture F-12 Ham, With L-glutamine and 15 mM HEPES, without sodium bicarbonate, powder, suitable for cell culture
Sigma-Aldrich
Anti-β-Actin antibody, Mouse monoclonal, clone AC-15, purified from hybridoma cell culture
Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
Dimethyl sulfoxide, for molecular biology