Effects Of Phthalate Esters On Human Cytochrome P450 Enzyme And Human Hepatoma HepG2 Cells

ObjectivePhthalate esters(PAEs)was one of the plasticizers widely used in industrial products.Long-term exposure to this kind of plasticizer could bring some potential harm to human health.In this study,cocktail probe method was used to explore the effects of 20 phthalate esters plasticizers on CYP450 enzymes in human liver microsomes,and the effects of PAEs on human hepatoma Hep G2 cells were preliminatively explored through cell culture experiments.MethodsLiquid chromatography tandem mass spectrometry(LC-MS/MS)was used to detect and analyze the inhibitory potential of 20 common PAEs on 9 CYP450 isoforms that phase Ⅰ drug metabolizing enzyme in human liver microsomes by cocktail method.The inhibition types were determined by time-dependent inhibition(TDI)tests of single-point inhibition and enzyme inhibition kinetics analysis.The inhibitory effects were evaluated by in vitro-in vivo extrapolation(IVIVE).The inhibition mechanisms of PAEs towards CYP450 isoforms were investigated by molecular docking simulation.The effects of PAEs on Hep G2 cells viability were explored by CCK8.TUNEL and JC-1 were used to detect the effects of PAEs on apoptosis of Hep G2 cells,and the effects of PAEs on the expression of CYP450 isoforms protein in Hep G2 cells were investigated by Western Blot.ResultsThe results of in vitro preliminary screening inhibition experiment showed that among 20 kinds of PAEs,only DCHP and MOP showed strong no timedependent and no NADPH-dependent competitive inhibition on CYP1A2 and CYP2C9 isoforms enzymes in human liver microsomes,respectively.The values of its half-inhibitory concentration(IC50)were 34.06±3.51 μmol/L and16.51±1.66 μmol/L,respectively.The kinetic parameters(Ki)were 18.31±3.86μmol/L and 12.32±1.88 μmol/L,respectively.The IVIVE results predicted that the exposure concentration of DCHP and MOP were 4.58±0.96 μmol/L and3.08±0.47 μmol/L in vivo,respectively.And CCK8 assays showed that DCHP and MOP inhibited the proliferation of Hep G2 cells.TUNEL assays showed that DCHP and MOP could induce apoptosis of Hep G2 cells.The results of JC-1probe showed that DCHP and MOP could change the mitochondrial membrane potential of Hep G2 cells.Western Blot results showed that DCHP could reduce the expression of CYP1A2 and MOP could reduce the expression of CYP2C9 in Hep G2 cells.ConclusionsIn this study,DCHP and MOP were found to inhibit CYP1A2 and CYP2C9 isoforms of human cytochrome P450 enzyme,respectively.DCHP and MOP can inhibit the proliferation of Hep G2 cells,DCHP and MOP can reduce the expression of CYP protein.Long-term exposure to these two phthalates may affect the normal metabolic function mediated by CYP450 enzymes and bring potential harm to human health.