Metabolites of n-Butylparaben and iso-Butylparaben Exhibit Estrogenic Properties in MCF-7 and T47D Human Breast Cancer Cell Lines

Two oxidized metabolites of n-butylparaben (BuP) and iso-butylparaben (IsoBuP) discovered in human urine samples exhibit structural similarity to endogenous estrogens. We hypothesized that these metabolites bind to the human estrogen receptor (ER) and promote estrogen signaling. We tested this using models of ER-mediated cellular proliferation. The estrogenic properties of 3-hydroxy n-butyl 4-hydroxybenzoate (3OH) and 2-hydroxy iso-butyl 4-hydroxybenzoate (2OH) were determined using the ER-positive, estrogen-dependent human breast cancer cell lines MCF-7, and T47D. The 3OH metabolite induced cellular proliferation with EC50 of 8.2 mu M in MCF-7 cells. The EC50 for 3OH in T47D cells could not be reached. The 2OH metabolite induced proliferation with EC50 of 2.2 mu M and 43.0 mu M in MCF-7 and T47D cells, respectively. The EC50 for the parental IsoBuP and BuP was 0.30 and 1.2 mu M in MCF-7 cells, respectively. The expression of a pro-proliferative, estrogen-inducible gene (GREB1) was induced by these compounds and blocked by co-administration of an ER antagonist (ICI 182, 780), confirming the ER-dependence of these effects. The metabolites promoted significant ER-dependent transcriptional activity of an ERE-luciferase reporter construct at 10 and 20 mM for 2OH and 10 mu M for 3OH. Computational docking studies showed that the paraben compounds exhibited the potential for favorable ligand-binding domain interactions with human ER alpha in a manner similar to known x-ray crystal structures of 17 beta-estradiol in complex with ER alpha. We conclude that the hydroxylated metabolites of BuP and IsoBuP are weak estrogens and should be considered as additional components of potential endocrine disrupting effects upon paraben exposure.