Adipogenic effects of a combination of the endocrine-disrupting compounds bisphenol A, diethylhexylphthalate, and tributyltin

Abstract

Objective: The food contaminants bisphenol A (BPA), diethylhexylphthalate (DEHP), and tributyltin (TBT) are potent endocrine-disrupting compounds (EDC) known to interfere with adipogenesis. EDC usually act in mixtures and not as single compounds. The aim of this study was to investigate the effects of a simultaneous exposure of BPA, DEHP, and TBT on mesenchymal stem cell differentiation into adipocytes.

Methods: Multipotent murine mesenchymal stem cells (C3H10T1/2) were exposed to EDC mixtures in high concentrations, i.e. MIX-high (10 µmol/l BPA, 100 µmol/l DEHP, 100 nmol/l TBT), and in environmentally relevant concentrations, i.e. MIX-low (10 nmol/l BPA, 100 nmol/l DEHP, 1 nmol/l TBT). The exposure was performed either for the entire culture time (0-12 days) or at distinct stages of adipogenic differentiation. At day 12 of cell culture, the amount of adipocytes, triglyceride content (TG), and adipogenic marker gene expression were analyzed.

Results: MIX-high increased the development of adipocytes and the expression of adipogenic marker genes independently of the exposure window. The total TG amount was not increased. The low-concentrated EDC mixture had no obvious impact on adipogenesis.

Conclusion: In EDC mixtures, the adipogenic effect of TBT and DEHP predominates single effects of BPA. Mixture effects of EDC are not deducible from single compound experiments.

Fig. 1

a Schematic time schedule for adipogenic differentiation and EDC exposure of C3H10T1/2-MSC. Cells were cultured for 6 days until reaching 2 days post-confluence (proliferation). Adipogenic differentiation was induced by insulin 10 (µg/ml), dexamethasone (1 µmol/l), and IBMX (500 µmol/l) for 48 h (induction) and cells were terminally differentiated for 4 days including supplementation of insulin for 48 h (differentiation). Cells were exposed during the whole adipogenic differentiation period (long-term exposure) or stage-specifically during proliferation, induction, or differentiation to MIX-low (10 nmol/l BPA, 100 nmol/l DEHP, and 1 nmol/l TBT), MIX-high (10 µmol./l BPA, 100 µmol/l DEHP, and 100 nmol/l TBT), or 0.05% DMSO (vehicle control). b Verification of FABP4 as adipogenic marker gene. FABP4 mRNA amounts were quantified at day 6, day 8, and day 12 and presented as absolute transcript numbers related to the housekeeping gene 18S. All values are given as mean ± SEM; n = 3 replicates; *p < 0.05.

Fig. 2

MIX-high exposure during the entire differentiation protocol increased the development of adipocytes but not the TG content. a Adipocytes were visualized at day 12 by oil-red-o staining, scale bar = 200 µm and b quantified by flow cytometry. c Total TG content. d MIX-high increased the mRNA amounts of FABP4 and adiponectin, but not PPARγ2 and LPL. No influence was observed in MIX-low treated cells. Data are presented as relative to the corresponding vehicle control, which is set 1 and indicated by a straight line. All values are given as mean ± SEM; n = 3 replicates; *p < 0.05.

Fig. 3

Independently of the exposure interval, MIX-high increased the development of adipocytes but not the TG content. a Adipocytes were quantified by flow cytometry. b Total TG content. c MIX-high increased the mRNA amounts of FABP4 in all investigated stages. Adiponectin, PPARγ2, and LPL mRNA amounts were only increased when cells were exposed during induction. No influence was observed in MIX-low treated cells. Data are presented as relative to the corresponding vehicle control, which is set 1 and indicated by a straight line. All values are given as mean ± SEM; n = 3 replicates; *p < 0.05.