Rationale

The most frequently occurring phthalate, di(2‐ethylhexyl) phthalate (DEHP), causes adverse effects on glucose homeostasis and insulin sensitivity in several cell models and epidemiological studies. However, thus far, there is no information available on the molecular interaction of phthalates and one of the key regulators of the metabolism, the peroxisome proliferator‐activated receptor gamma (PPARγ). Since the endogenous ligand of PPARγ, 15‐deoxy‐delta‐12,14‐prostaglandin J₂ (15Δ‐PGJ₂), features structural similarity to DEHP and its main metabolites produced in human hepatic metabolism, mono(2‐ethylhexyl) phthalate (MEHP) and mono(2‐ethyl‐5‐oxohexyl) phthalate (MEOHP), we tested the hypothesis of direct interactions between PPARγ and DEHP or its transformation products.

Methods

Hydrogen/deuterium exchange mass spectrometry (HDX‐MS) and docking were conducted to obtain structural insights into the interactions and surface plasmon resonance (SPR) analysis to reveal information about binding levels. To confirm the activation of PPARγ upon ligand binding on the cellular level, the GeneBLAzer® bioassay was performed.

Results

HDX‐MS and SPR analyses demonstrated that the metabolites MEHP and MEOHP, but not DEHP itself, bind to the ligand binding pocket of PPARγ. This binding leads to typical activation‐associated conformational changes, as observed with its endogenous ligand 15Δ‐PGJ₂. Furthermore, the reporter gene assay confirmed productive interaction. DEHP was inactive up to a concentration of 14 μM, while the metabolites MEHP and MEOHP were active at low micromolar concentrations.

Conclusions

In summary, this study gives structural insights into the direct interaction of PPARγ with MEHP and MEOHP and shows that the DEHP transformation products may modulate the lipid metabolism through PPARγ pathways.