Di-2-ethylhexyl phthalate substitutes DEHA and DEHTP differentially regulate adipogenesis in 3T3-L1 cells

Abstract Objectives Phthalates are plasticizers that are ubiquitously present in consumer products. Di-2-ethylhexyl phthalate (DEHP) was widely used but deemed toxic due to the effects of its bioactive metabolite, mono-(2-ethylhexyl) phthalate (MEHP), which has been linked to many negative health outcomes including increased adipogenesis. DEHP has been increasingly replaced with alternative plasticizers such as DEHTP and di-(2-ethylhexyl) adipate (DEHA). The health effects of these substitutes in general and on adipocyte development remain less understood. The objective of this study was to assess the effects of DEHTP, DEHA, and their metabolites MEHTP and MEHA. Design The adipogenic effects of alternative plasticizers and their metabolites were evaluated using the murine 3T3-L1 preadipocyte, which can be induced to differentiate into mature adipocytes using appropriate inducers. Methods 3T3L1 cells were treated with 0.01 to 100 µm of test compounds throughout differentiation, in the presence of insulin and 3-isobutyl-1-methylxanthine. Lipid accumulation, mRNA expression, and protein expression of various adipogenic markers were evaluated. Results Our results show that MEHA and MEHTP exposures significantly increased mRNA expression of adipogenic markers in a dose-dependent manner. Lipid accumulation and protein expression of adipogenic markers were increased by MEHA with increasing concentration but not by MEHTP. Both MEHA and MEHTP moderately activated the mouse peroxisome proliferator-activated receptor γ in a dose-dependent manner, suggesting the receptor's involvement. Conclusions Overall, these results illustrate that DEHP alternatives' metabolites may pose health risks by promoting or disrupting adipocyte differentiation through distinct mechanisms, underscoring the need to evaluate their unintended metabolic effects.