Effetti dei plasticizzanti sulla deposizione lipidica durante il differenziamento dei preadipociti 3T3-L1

Endocrine disrupting chemicals (EDCs) encompass natural or synthetic compounds present in our environment, food and everyday products that have the potential to interfere with synthesis, metabolism and action of endogenous hormones, thus resulting in perturbation of the homeostasis. Like hormones, EDCs exert their effects via binding to membrane or nuclear receptors (NRs) at nanomolar or micromolar concentrations. EDCs exposure during critical developmental phases can lead to permanent changes of the endocrine system and to an increased susceptibility to disease in adulthood. Some EDCs, called ¿obesogens¿, may cause dysfunctions of the adipose tissue: they may induce the formation of new adipocytes, alter lipid and glucose metabolism or interfere with regulation of appetite and satiety, thus contributing to the onset of obesity and related metabolic disease and disorders. Recent data report that some plasticizers used as additives to produce plastic items act as obesogens. In fact, the obesity epidemic correlates well with the increase in the use and distribution of plasticizers. In this work, we tested the effects of potentially obesogenic plasticizers on lipid deposition during differentiation of the white adipose tissue precursors 3T3-L1 cells, an in vitro model useful to study adipogenesis. The amount of fat stored during 3T3-L1 differentiation was assessed via a colorimetric assay using the Oil Red O (ORO) staining. Through an in silico screening performed on plasticizers, bisphenol A (BPA), the phthalates diisononyl phpthalate (DINP) and diisodecyl phthalate (DIDP), the benzoate diethylene glycol dibenzoate (DEGD) and the organophosphate tri-meta-cresyl phosphate (TMCP) showed higher affinities for type II NRs including PPARs, LXRs and RXRs: these receptors are known to be involved in many physiological and differentiative processes including adipogenesis. Through RT-PCR we confirmed the expression of these NRs in differentiating 3T3-L1 cells, suggesting that they may be possible targets of EDCs. During development, EDCs may interfere in the formation of the adipose tissue in all stages of adipogenesis, therefore 3T3-L1 cells were treated with each plasticizer in the early, late or both phases of their differentiation process. Treatments were made at concentrations ranging from 10 nM to 1 μM, corresponding to those actually found in the environment and in biological fluids. In all cases, plasticizers significantly enhanced lipid droplet deposition compared to the control cells. Moreover, it is known that food-contact materials (e.g. food containers, films, bottles) represent a source of human exposure to plasticizers. They are commonly made in polyvinyl chloride (PVC) and polypropylene (PP) plastics containing mixtures of plasticizers that may migrate into food concurrently. Therefore, we employed mixtures containing plasticizers (10 nM each) mimicking commercial PVC and PP plastics and assessed their effects on lipid accumulation in differentiating 3T3-L1 cells. Again, PVC and PP plastic mixtures were able to induce a significant increase in lipid accumulation. Furthermore, to better understand the molecular mechanisms underlying lipid accumulation induced by plasticizers, we performed qRT-PCR analysis. Particularly, we examined the effects of BPA, DINP and TMCP on the expression levels of NRs involved in adipocyte differentiation and in lipid metabolism including PPAR2, its target gene aP2, as well as PPARα, LXRα and RXRα. Our results indicate that the selected plasticizers may act as obesogens during differentiation of 3T3-L1 preadipocytes when used at concentrations actually found in the food.