Municipal wastewater affects adipose deposition in male mice and increases 3T3-L1 cell differentiation

• Sewage watewaters (TW) sampled from a Municipal Treatment Sewage Plant are biologically active in mice and in vitro.
• In male mice TW promotes an increase in adipose mass deposition following exposure from foetal to adult life. A similar effect is produced by BPA.
• The expression of key genes of the adipose metabolism is increased in epidydimal fat both by TW and BPA.
• An extract from the TW which concentrated the contained chemicals by a factor of 1000, is able to stimulate 3T3-L1 adipocyte differentiation.
• The major chemicals contained in the TW, bisphenol A and nonylphenol, are able to promote adipocyte differentiation at the low environmentally relevant doses as are found in the TW.

Trace concentration of EDs (endocrine disrupting compounds) in water bodies caused by wastewater treatment plant effluents is a recognized problem for the health of aquatic organisms and their potential to affect human health. In this paper we show that continuous exposure of male mice from early development to the adult life (140 days) to unrestricted drinking of wastewater collected from a municipal sewage treatment plant, is associated with an increased adipose deposition and weight gain during adulthood because of altered body homeostasis. In parallel, bisphenol A (BPA) at the administration dose of 5 μg/kg/body weight, shows an increasing effect on total body weight and fat mass. In vitro, a solid phase extract (SPE) of the wastewater (eTW), caused stimulation of 3T3-L1 adipocyte differentiation at dilutions of 0.4 and 1 % in the final culture medium which contained a concentration of BPA of 40 nM and 90 nM respectively. Pure BPA also promoted adipocytes differentiation at the concentration of 50 and 80 μM. BPA effect in 3T3-L1 cells was associated to the specific activation of the estrogen receptor alpha (ERα) in undifferentiated cells and the estrogen receptor beta (ERβ) in differentiated cells. BPA also activated the Peroxisome Proliferator Activated Receptor gamma (PPARγ) upregulating a minimal 3XPPARE luciferase reporter and the PPARγ-target promoter of the aP2 gene in adipose cells, while it was not effective in preadipocytes. The pure estrogen receptor agonist diethylstilbestrol (DES) played an opposite action to that of BPA inhibiting PPARγ activity in adipocytes, preventing cell differentiation, activating ERα in preadipocytes and inhibiting ERα and ERβ regulation in adipocytes.The results of this work show that the drinking of chemically-contaminated wastewater promotes fat deposition in male mice and that EDs present in sewage are likely responsible for this effect through a nuclear receptor-mediated mechanism.