Effects of parabens on adipocyte differentiation

Abstract

Parabens are a group of alkyl esters of p-hydroxybenzoic acid that include methylparaben, ethylparaben, propylparaben, butylparaben, and benzylparaben. Paraben esters and their salts are widely used as preservatives in cosmetics, toiletries, food, and pharmaceuticals. Humans are exposed to parabens through the use of such products from dermal contact, ingestion, and inhalation. However, research on the effects of parabens on health is limited, and the effects of parabens on adipogenesis have not been systematically studied. Here, we report that (1) parabens promote adipogenesis (or adipocyte differentiation) in murine 3T3-L1 cells, as revealed by adipocyte morphology, lipid accumulation, and mRNA expression of adipocyte-specific markers; (2) the adipogenic potency of parabens is increased with increasing length of the linear alkyl chain in the following potency ranking order: methyl- < ethyl- < propyl- < butylparaben. The extension of the linear alkyl chain with an aromatic ring in benzylparaben further augments the adipogenic ability, whereas 4-hydroxybenzoic acid, the common metabolite of all parabens, and the structurally related benzoic acid (without the OH group) are inactive in promoting 3T3-L1 adipocyte differentiation; (3) parabens activate glucocorticoid receptor and/or peroxisome proliferator-activated receptor γ in 3T3-L1 preadipocytes; however, no direct binding to, or modulation of, the ligand binding domain of the glucocorticoid receptor by parabens was detected by glucocorticoid receptor competitor assays; and lastly, (4) parabens, butyl- and benzylparaben in particular, also promote adipose conversion of human adipose-derived multipotent stromal cells. Our results suggest that parabens may contribute to obesity epidemic, and the role of parabens in adipogenesis in vivo needs to be examined further.

FIG. 1.

Chemical structure of parabens, their common metabolite 4-hydroxybenzoic acid, and structurally related benzoic acid.

FIG. 2.

Parabens promote 3T3-L1 adipocyte differentiation. Confluent 3T3-L1 preadipocytes were induced to differentiate with butylparaben or DMSO (a) or with various selected parabens, the common metabolite 4-hydroxybenzoic acid or the structurally related benzoic acid (b), in the presence or absence of the differentiation cocktail (cortisone 5µM, MIX 0.5mM, and insulin 170nM—CMI) during the 7-day differentiation process. Oil Red O (ORO) staining of lipid accumulation (left panel) and adipocyte morphology (right panel) and quantification of ORO absorbance (c) at D7 are shown. (d) 3T3-L1 preadipocytes were differentiated in the presence or absence of increasing doses of selected parabens (1, 10, 100µM) during the differentiation process. Relative mRNA expression of adipocyte markers was analyzed. The relative mRNA expression was normalized to 36B4 and expressed as fold of that of the 0µM sample (set at 1) for each paraben. Data are mean ± SE (n = 3). One-way ANOVA was performed followed by multiple comparison tests with Student-Newman-Keuls method for structure-function relationship (c, right panel) or dose response (d) or Holm-Sidak method (c, left panel) to compare with the respective control. Different letters indicate significant difference (p < 0.05) (c, right panel). The bar indicates the dose-dependent responses (d). *, **, p < 0.05, and p < 0.01, respectively. Scale bar = 127µm.

FIG. 3.

The effect of exposure stage to paraben on 3T3-L1 adipocyte differentiation. Confluent 3T3-L1 preadipocytes were induced to differentiate with the differentiation cocktail CMI in the presence of butylparaben (100µM) or DMSO at indicated time shown in (a). Oil Red O (ORO) staining of lipid accumulation (b), quantification of ORO absorbance (c), and relative mRNA expression of adipocyte markers at D7 (d) are shown. The relative gene expression was normalized to 36B4 and expressed as fold of DMSO 0–3 samples (set at 1). Data are the mean ± SE (n = 3). One-way ANOVA was performed followed by multiple comparison tests with Student-Newman-Keuls method to compare among different treatment groups. Different letters indicate significant difference (p < 0.05).

FIG. 4.

Parabens activate GR reporter and target gene without directly binding to, or modulating, the ligand binding of the receptor. (a) 3T3-L1 preadipocytes stably transfected with MMTV-Luc were seeded and treated with various parabens (100µM) for 18h. (b) COS-7 cells were seeded and transfected with the empty vector or the full length of GR, with MMTV-Luc reporter and β-gal control plasmid for 24h before the cells were treated with butylparaben (100µM) or DMSO in the presence or absence of Dex (1µM) for 18h. The reporter gene assays were performed. Luciferase activities were normalized with the β-gal activities. (c) Confluent 3T3-L1 preadipocytes were treated with butylparaben (100µM) or DMSO in the presence or absence of Dex (1µM) for 4h. (d) 3T3-L1 cells were transfected with siRNA targeting GR (siGR) or nontargeting control (siCON) for 24h. The cells were then treated with butylparaben or DMSO in the presence of Dex for 4h. The relative gene expression was normalized to 36B4 and expressed as fold of the respective control (set at 1). (e, f) Human GR/fluormone complex was incubated with various parabens (100µM), Dex (1µM), or cortisone (5µM) (e) or with butylparaben (10, 100µM) in combination with Dex (0.1 and 0.5µM) (f). Polarization was measured. Data are mean ± SE (n = 3). One-way ANOVA was performed followed by multiple comparison tests with Student-Newman-Keuls method for structure-function relationship (a) or to compare among different groups (b) or Holm-Sidak method (c–f) to compare with the respective control. *, **, p < 0.05, and p < 0.01, respectively; N.S., not significant.

FIG. 5.

Parabens activate PPARγ reporter and target genes. (a) 3T3-L1 preadipocytes were transiently transfected with mPPARγ-Gal4, 4xUAS-TK-luc, and β-gal for 24h and treated with various parabens (100µM) for 18h. The reporter gene assays were performed. Luciferase activities were normalized with β-gal activities. (b) Confluent 3T3-L1 cells were treated with butylparaben (100µM) or DMSO in the presence or absence of the differentiation cocktail CMI for 24h. (c) 3T3-L1 cells were transfected with siRNA targeting PPRARγ (siPPARγ) or nontargeting control (siCON) for 24h. The cells were treated with butylparaben or DMSO in the presence of CMI for further 24h. The relative gene expression was normalized to 36B4 and expressed as fold of the respective control (set at 1). Data are mean ± SE (n = 3). One-way ANOVA was performed followed by multiple comparison tests with Student-Newman-Keuls method for structure-function relationship (a) or Holm-Sidak method (b, c) to compare with the respective control. *, **, p < 0.05 and p < 0.01, respectively.

FIG. 6.

The antagonists of GR and PPARγ attenuate paraben-induced 3T3-L1 differentiation. Confluent 3T3-L1 preadipocytes were induced to differentiate with butylparaben (100µM) or DMSO in the presence of the differentiation cocktail (CMI), with or without the GR antagonist RU-486 (10µM), the PPARγ antagonist GW9662 (20µM), or BADGE (50µM) during the process. Quantification of Oil Red O absorbance (a) and relative mRNA expression of adipocyte markers (b) are shown. The relative gene expression was normalized to 36B4 and expressed as fold of the DMSO-treated samples (set at 1). Data are mean ± SE (n = 3). One-way ANOVA was performed followed by multiple comparison tests with Holm-Sidak method to compare the antagonist with the respective control. *, **, p < 0.05 and p < 0.01, respectively.

FIG. 7.

Paraben acts as a glucocorticoid-like compound to promote 3T3-L1 adipocyte differentiation. Confluent 3T3-L1 preadipocytes were induced to differentiate with butylparaben or DMSO in the presence of the medium only, MIX + Ins (without Dex), or the standard differentiation cocktail (Dex + MIX + Ins). Oil Red O (ORO) staining of lipid accumulation (a), quantification of ORO absorbance (b), and relative mRNA expression of adipocyte markers (c) are shown. The relative gene expression was normalized to 36B4 and expressed as fold of the respective control (set at 1). Data are mean ± SE (n = 3). One-way ANOVA was performed followed by multiple comparison tests with Student-Newman-Keuls method. Different letters indicate significant difference (p < 0.05) among the butylparaben-treated groups. **, p < 0.01 versus the respective control.

FIG. 8.

Parabens promote adipocyte conversion of hADSC. hADSC cells were differentiated with various parabens (50µM) (a, b) or increasing doses of butylparaben (1, 10, 50µM) (a, c) in the presence the differentiation media. Human adipocyte morphologies (a) and mRNA expression of adipocyte-specific markers (b and c) are shown. The relative gene expression was normalized to 18S and expressed as fold of the DMSO-treated samples (set at 1). Data are mean ± SE (n = 3). One-way ANOVA was performed followed by the tests with Holm-Sidak method to compare various parabens or doses with the DMSO group. *, **, p < 0.05, and p < 0.01 versus the DMSO control, respectively. Scale bar = 127µm.