Interference of Paraben Compounds with Estrogen Metabolism by Inhibition of 17β-Hydroxysteroid Dehydrogenases

Abstract

Parabens are effective preservatives widely used in cosmetic products and processed food, with high human exposure. Recent evidence suggests that parabens exert estrogenic effects. This work investigated the potential interference of parabens with the estrogen-activating enzyme 17β-hydroxysteroid dehydrogenase (17β-HSD) 1 and the estrogen-inactivating 17β-HSD2. A ligand-based 17β-HSD2 pharmacophore model was applied to screen a cosmetic chemicals database, followed by in vitro testing of selected paraben compounds for inhibition of 17β-HSD1 and 17β-HSD2 activities. All tested parabens and paraben-like compounds, except their common metabolite p-hydroxybenzoic acid, inhibited 17β-HSD2. Ethylparaben and ethyl vanillate inhibited 17β-HSD2 with IC₅₀ values of 4.6 ± 0.8 and 1.3 ± 0.3 µM, respectively. Additionally, parabens size-dependently inhibited 17β-HSD1, whereby hexyl- and heptylparaben were most active with IC₅₀ values of 2.6 ± 0.6 and 1.8 ± 0.3 µM. Low micromolar concentrations of hexyl- and heptylparaben decreased 17β-HSD1 activity, and ethylparaben and ethyl vanillate decreased 17β-HSD2 activity. However, regarding the very rapid metabolism of these compounds to the inactive p-hydroxybenzoic acid by esterases, it needs to be determined under which conditions low micromolar concentrations of these parabens or their mixtures can occur in target cells to effectively disturb estrogen effects in vivo.

Keywords: 17β-hydroxysteroid dehydrogenase; endocrine disrupting chemical; estrogen; in silico; in vitro; xenobiotic.

Figure A1

Predicted protein-ligand interactions of the tested parabens with the 17β-HSD1 binding site (S-docking).

Figure 1

Interconversion of potent (estradiol) and weakly active estrogens (estrone) by 17β-hydroxysteroid dehydrogenase (17β-HSD) enzymes.

Figure 2

(A) Virtual screening strategy using pharmacophore models for 17β-HSD2 inhibitors described earlier [18]. Virtual hits are required to map all features with the exception of the optional hydrogen bond acceptor. (B) Full models with exclusion volumes serving as steric restrictions for the size of hitting compounds. (C) The virtual hits phenylparaben and isopropylparaben fit to model 1 and ethylparaben, phenylparaben, propylparaben, butylparaben, isopropylparaben, and isobutylparaben fit into model 2. For reasons of clarity, exclusion volumes are not shown.

Figure 3

Chemical structures of the tested parabens, and vanillic and gallic acid derivatives.

Figure 4

(A) Inhibition of estrone formation by parabens in human embryonic kidney (HEK-293) cell lysates expressing human 17β-HSD2. The conversion of estradiol to estrone was measured in lysates of HEK-293 cells transiently transfected with 17β-HSD2 in the presence of 200 nM radiolabeled estradiol and parabens or paraben-like compounds at a final concentration of 20 µM. 17β-HSD2 inhibitor BM13 served as positive control (compound 22 in [18]). Statistical analyses were performed using one-way analysis of variance (ANOVA) test followed by a Dunnett post-hoc test. All tested compounds significantly inhibited 17β-HSD2 activity (p ≤ 0.001) expect for p-hydroxybenzoic acid, which was not significant. (B) Concentration-dependent inhibition of 17β-HSD2 by ethylparaben and ethyl vanillate. Results represent the mean ± standard deviation (SD) of three independent measurements.

Figure 5

Additive inhibitory effects of two parabens on 17β-HSD2 activity. The effect of 6 μM ethyl- (2-P) or 12 μM hexylparaben (6-P) or a mixture of both compounds on 17β-HSD2 activity was assessed in lysates of transiently-transfected HEK-293 cells. Dimethylsulfoxide (DMSO) vehicle served as a negative control and nordihydroguaiaretic acid (NDGA) as a positive control [19]. Experiments were performed three times, independently. Statistical analyses were performed using one-way ANOVA followed by a Dunnett post-hoc test. Both tested parabens and the combination significantly inhibited 17β-HSD2 (p ≤ 0.01).

Figure 6

(A) Inhibition of 17β-HSD1 activity by parabens. 17β-HSD1 activity was measured in lysates of transiently-transfected HEK-293 cells, using DMSO as a negative, and apigenin as a positive, control [20]. Statistical analyses were performed using one-way ANOVA followed by a Dunnett post-hoc test. P-hydroxybenzoic acid, methylparaben, and ethylparaben did not significantly inhibit the activity of 17β-HSD1. All other tested compounds significantly inhibited 17β-HSD1 (p ≤ 0.001). (B) Concentration-dependent inhibition of 17β-HSD1 by hexyl- and heptylparaben. The conversion of estrone to estradiol was measured in lysates of HEK-293 cells transfected with 17β-HSD1 and (C) in intact human COV434 granulosa cells endogenously expressing 17β-HSD1. Results represent the mean ± SD of three independent experiments.

Figure 7

(A,B) The most active 17β-HSD1 inhibitor heptylparaben docked into the enzymes active site. (C,D) Amino acids of the binding site are shown in ball-and-stick style (white—carbon, red—oxygen, blue—nitrogen, yellow—sulfur, green—phosphor). The cofactor NADP (NAP) is colored in salmon pink. The docked parabens are shown in stick style. Inhibitors with a smaller substituent, like benzylparaben, can also adopt a flipped position. Protein-ligand interactions are color-coded: red arrow: hydrogen bond acceptors; yellow sphere: hydrophobic. The ligand binding site surface is colored by aggregated hydrophilicity (blue)/hydrophobicity (yellow).