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

Abstract

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 µM 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 µM and 43.0 µM in MCF-7 and T47D cells, respectively. The EC50 for the parental IsoBuP and BuP was 0.30 and 1.2 µM 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 µM for 2OH and 10 µM for 3OH. Computational docking studies showed that the paraben compounds exhibited the potential for favorable ligand-binding domain interactions with human ERα in a manner similar to known x-ray crystal structures of 17ß-estradiol in complex with ERα. 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.

Figure 1.

Chemical structures of 17ß-estradiol, 3OH, and 2OH paraben metabolites. * indicates chiral center.

Figure 2.

3OH and 2OH paraben metabolites induce breast cancer cell proliferation. MCF-7 and T47D cells were grown in E2-free conditions as described in Materials and Methods section. PrestoBlue cell viability assay was used as a surrogate to determine relative cell number. MCF-7 cells were treated with either 3OH (A) or 2OH (B) and T47D cells were treated with 3OH (C) or 2OH (D) at the indicated concentrations. Growth curves for A–D represent percentage of cell growth compared to DMSO (vehicle) control (0%). Points on dose response curve represent 6-day growth versus vehicle treated control ± SE (n = 6 technical replicates). Dotted line indicates growth induced by 100 pM E2.

Figure 3.

Pure anti-estrogen blocks 3OH, 2OH, respective parent compound induced breast cancer cell proliferation. MCF-7 cells were grown in E2-free conditions as described in Materials and Methods section. PrestoBlue cell viability assay was used as a surrogate to determine relative cell number. Growth induction by a fixed dose of either (A) (▪) 0.3 µM IsoBuP (solid line) or (□) 2.15 µM 2OH (dashed line) or (B) by (▪) 1.2 µM Butylparaben or (□) 8.22 µM 3OH was antagonized by the pure anti-estrogen, ICI 182, 780 (ICI). ICI was added to final concentrations ranging from 1 pM to 1 µM at log intervals. Growth curves represents percentage of cell growth compared to 2OH or IsoBuP at the fixed concentrations indicated above. Data are normalized from maximum growth (100%) to minimum growth (0%) for each treatment. Points on dose response curve represent Points represent 6-day growth versus proliferation with EC₅₀ of indicated paraben without ICI ± SE (n = 6 technical replicates).

Figure 4.

Time course induction of GREB1 expression in MCF-7 cells by the 3OH and 2OH paraben metabolites. MCF-7 cells were assayed in E2-free conditions. Cells were treated with a vehicle control (0.001% ethanol or 0.1% DMSO), or 100 pM E2, for 2, 4, and 6 h. Neither ethanol or DMSO significantly affected expression levels compared to media (CCS) alone (data not shown). Cells were also treated at 10 µM with butylparaben (BuP), IsoBuP (IsoBuP), 3OH, and 2OH. Bars represent GREB1 expression versus vehicle-treated control using the ΔΔC_T method. Bars represent the mean from 3 technical replicates ± SE. Statistical significance within each treatment group was determined by one-way ANOVA followed by Bonferroni post-hoc analysis. # = not significant; a, b, c, d, e = p ≤ .005.

Figure 5.

Induction of GREB1 expression in MCF-7 cells by the 3OH and 2OH paraben metabolites is blocked in the presence of ICI 182, 780. MCF-7 cells were assayed in E2-free conditions. Cells were treated with a vehicle control (0.001% ethanol or 0.1% DMSO), 100 pM E2, or 100 nM ICI for 6 h. Cells were also treated at 10 µM with BuP, IsoBuP (IsoBuP), 3OH, and 2OH alone or in combination with 100 nM ICI. Bars represent GREB1 expression versus vehicle-treated control using the ΔΔC_T method. Bars represent the mean from 3 technical replicates ± SE. Statistical significance between each lettered pair was determined by t-test and significant at p < .001.

Figure 6.

3OH and 2OH paraben metabolites promote significant ERE-luciferase transcriptional activity. The 2OH and 3OH paraben metabolites significantly induce ERE-luciferase activity. MCF-7 cells were co-transfected with the ERE-luciferase construct and renilla reporter plasmid in estrogen-free conditions and treated at the indicated concentrations for each compound. Relative firefly luciferase activity was plotted over renilla luciferase activity induced by treatment from each specified compound versus ethanol vehicle control for E2 and DMSO vehicle control for each paraben compound. Statistical significance between each indicated pair was determined by t-test. Bars represent the mean from 3 technical replicates ± SE. ns = not significant; *p ≤ .05; **p ≤ .001.

Figure 7.

3OH and 2OH paraben metabolites dock to ERα. Human ERα ligand-binding domain docked with (A) 2OH isomer (B) 3OH R isomer or (C) 3OH S isomer. 2OH and 3OH are colored orange and magenta, respectively. 17ß-estradiol has been overlaid with each ligand in grey for comparison. Hydrogen bonds are represented as yellow dashes. Oxygen and nitrogen atoms are colored in red and blue, respectively. The key structural features and hydrogen bonding residues are displayed and labeled.