Endocrine-Disrupting Effects Of Phthalate Esters Through Interference With Glucocorticoid Receptor In Vitro

Phthalate esters（PAEs）are a class of endocrine-disrupting chemicals and used as plasticizers in industrial production.PAEs are also detected in food,leading to human exposure by contamination of food.PAEs can alter hormonal action by interfering with the pleiotropic response mediated by nuclear steroid receptors.Glucocorticoids（GCs）secreted by the zona fasciculata of the adrenal gland are steroid hormones regulating important physiological processes.GCs transduce their major actions via binding to glucocorticoid receptor（GR）.However,the glucocorticoid effect of PAEs is still rarely reported.In this paper,the potential endocrine-disrupting effects of PAEs through interference with GR were studied,which provided insight into the GR-mediated toxic effects induced by PAEs.The main research contents and results are as follows:（1）The recombinant plasmid pc DNA3.1（+）-h GR was constructed and a reporter-gene system for high throughput screening was established to identify the glucocorticoid effects of 21 PAEs and 4 phthalate metabolites.Among which,dicyclohexyl phthalate（DCHP）exerted antagonistic activity dose-dependently for GR.Di（2-ethylhexyl）phthalate（DEHP）,diisononyl phthalate（DINP）and monobutyl phthalate（MBP）in presence of dexamethasone could synergistically activate GR in a dose-dependent manner.The 20%relative inhibitory concentration(RIC₂₀)of DCHP was 4.518×10^-6M.The 20%relative effective concentrations(REC₂₀)of DEHP,DINP and MBP were 4.957×10^-7M,2.356×10^-8M and 6.737×10^-8M.The rest 21chemicals failed to exhibit agonistic or antagonistic effects on GR.（2）The effects of PAEs and their metabolites involved the nuclear translocation of GR were also investigated.RU486 and DCHP in presence of dexamethasone down-regulated the nuclear-protein expression levels of GR and restrained GR cytoplasmic-to-nuclear translocation.By contrast,DEHP,DINP and MBP at high doses enhanced GR nuclear translocation combined with dexamethasone,which may be a plausible explanation for the disruption of GR transcriptional response by PAEs and their metabolites.（3）The influence of PAEs and their metabolites on glucocorticoid signaling was evaluated by estimating the expression of five GC-sensitive genes by m RNA analysis.The stability of 2 reference genes HPRT and GAPDH was evaluated by Best Keeper.HPRT and GAPDH were relatively stable in Hep G2 cells and A549 cells,respectively.DCHP suppressed GC-responsive gene expression（G6Pase,PEPCK,FAS,GILZ and MKP-1）at m RNA levels.Synergistic induction of GC-responsive gene expression by DEHP,DINP and MBP co-treated with dexamethasone was also observed.Heat map cluster analysis and Circos diagram showed that the m RNA expression levels of five GC-responsive genes varied greatly.PEPCK and FAS shared similar gene expression pattern while G6Pase exhibited a different expression pattern from others.Principal component analysis（PCA）showed that the expression levels of five GC-responsive genes treated by DCHP and MBP were clearly separated and showed dose-dependent effects.（4）The protein expression levels of two key gluconeogenic enzymes were also checked by western blot.DCHP repressed protein expression levels of two key gluconeogenic enzymes G6Pase and PEPCK.DEHP,DINP and MBP co-treated with dexamethasone exhibited a synergistic induction in protein expression levels of PEPCK.However,DEHP,DINP and MBP co-treated with dexamethasone had no effects on protein expression levels of G6Pase compared with the positive control.The Pearson comparison showed that PEPCK gene expression correlated with nuclear-protein expression levels of GR（P<0.05）.（5）The binding conformation and binding stability of PAEs and their metabolites with GR were explored by molecular docking and molecular dynamics simulation.The results of molecular docking showed that DCHP,DEHP,DINP and MBP can fit into the ligand binding cavity of GR ligand binding domain（GR-LBD）.DCHP can fasten to the active site in a similar orientation to that of RU486.DEHP,DINP and MBP can bind to the active site of GR-LBD in a similar manner to the crystal pose of dexamethasone,respectively.Hydrogen-bonding and hydrophobic effects played important roles in the binding of PAEs with GR-LBD.The results of molecular dynamics simulation showed that PAE-GR-LBD complex basically achieve stability in the GR binding pocket after 10 ns.