The Effects of Common Alternative Plasticizers on Adipogenesis in 3T3-L1

Abstract

Objective: The global rise in obesity and its associated metabolic disorders pose a major public health concern. While lifestyle factors such as reduced physical activity, high-caloric diet, and genetic predisposition are well established contributors, increasing attention has turned toward the influence of environmental chemicals. Among these Di-2-ethylhexyl phthalate (DEHP) has gained particular concern due to its widespread use and the potential endocrine-disrupting properties of its bioactive metabolite mono-(2-ethylhexyl) phthalate MEHP. Studies have linked MEHP exposure to disrupted endocrine and metabolic regulation, including promotion of adipogenesis and increased risk of obesity-related outcomes. Consequently, regulatory restrictions on DEHP have prompted the adoption of structurally related alternatives including as Di-2-ethylhexyl terephthalate (DEHTP) and Di-(2-ethylhexyl) adipate (DEHA). MEHP is considered a potential obesogen, capable of promoting adipogenic differentiation in both murine and human preadipocytes. However weather these newer DEHP alternatives and their metabolites elicit comparable adipogenic responses remains poorly characterized. This thesis aims to investigate the adipogenic effects of DEHTP, DEHA and their metabolites MEHTP and MEHA in vitro, to assess whether these compounds elicit cellular responses comparable to DEHP and MEHP, as well as assess their possible underlying mechanisms.

Design/Methods: The adipogenic effects of alternative plasticizers and their metabolites were evaluated using the murine 3T3-L1 preadipocyte, induced to differentiate into mature adipocytes using inducers such as insulin, 3-isobutyl-1-methylxanthine (IBMX), rosiglitazone (ROSI), a potent PPARγ agonist, or dexamethasone (DEX), a synthetic steroid. The adipogenic potential of test compounds was evaluated by substituting DEX and ROSI with 0.01-100 µM concentrations of each chemical during adipocyte differentiation to test for compensatory induction of adipogenesis. Anti-adipogenic effects were assessed by co-treatment of each chemical alongside DEX or ROSI to assess possible inhibitory effects. Phenotypic outcomes were quantified via lipid staining, alongside mRNA and protein expression analysis of various adipogenic markers. Activation of peroxisome proliferator-activated receptor γ (PPARγ) and retinoic X receptor (RXR) was also examined for mechanistic insights into the chemicals effects on adipogenesis.

Results: Our results show that parent compounds DEHA and DEHTP did not affect adipogenesis in this model. However, MEHA exposure significantly increased lipid accumulation and upregulated both mRNA and protein expression of adipogenic markers. MEHTP increased mRNA expression of similar markers but did not induce the corresponding increases in protein level or lipid accumulation, and in fact inhibited adipogenesis at all levels assessed in the presence of ROSI. Both MEHA and MEHTP moderately activated the PPARγ, with MEHTP also exhibiting strong activation of RXR, whereas MEHA showed only minimal RXR activation. These findings indicate that these chemicals influence adipogenesis through differential modulation of nuclear receptor activity, highlighting a complex interaction between PPARγ and RXR in regulating adipocyte development and function.

Conclusion: Overall, this thesis provides evidence that DEHP alternatives may pose health risks by promoting or disrupting adipocyte differentiation through distinct mechanisms, although these mechanisms remain to be fully elucidated. This underscores the need to evaluate the possible unintended metabolic effects of both DEHP replacement chemical as well as their metabolites.