Investigation Of The Aryl Hydrocarbon Receptor Induction By The Endogenous And Xeno Aromatic Hydrocarbon Compounds

Aromatic hydrocarbon compounds are widely distributed in nature which have important influence on human health.The aryl hydrocarbon receptor(AhR)is a ligand-dependent transcription factor that can be activated by synthetic and natural chemicals,and it mediates the toxic and biological effects of AhR ligand.The activation of AhR is regulated by ligands,in order to explore AhR response to ligands,the recombinant Hepa1.6-pGl4.43 cells and HepG2-pGl4.43 cells were constructed in this research.A variety of endogenous and xeno aromatic compounds were selected to stimulate the recombinant cells,to investigate AhR response.The main contents and results are as follows:1.Construction of recombinant cell lines Hepa1.6-pGl4.43 and HepG2-pGl4.43 and screening of stable strains.The pGl4.43 [luc2P/XRE/Hygro] plasmid vector which luciferase reporter under the control of three XRE enhancers was stable transfected into human hepatoma cell line HepG2(wild type)and mice hepatoma cell line Hepa1.6(wild type).The concentration of hygromycin B was 800 g/mL and 200 g/mL,respectively.The results showed that two recombinant cells had high sensitivity,100 pM TCDD and 10 nM FICZ could induce the luciferase activity in a dose dependent manner.2.To verify the accuracy of the recombinant cells in response to ligand.In experiment,wild type HepG2 and Hepa1.6 were treated with the same concentration of TCDD and FICZ for the expression of CYP1A1,CYP1B1.Data showed the same trend of the two types of cells.In this article,we had successfully constructed cell lines which response to aromatic hydrocarbon compounds with high sensitivity.It can be used to explore AhR response to the aromatic hydrocarbon compounds.3.Exploring the response of AhR to its ligand.In this study,we screened five kinds of xeno,seven kinds of dietary and four kinds of endogenous compounds to determine their response in AhR.The results showed that exogenous ligands and endogenous ligands are agonists of AhR.Most of the lower concentrations of dietary compounds can activate AhR,which is a weak agonist of AhR.However,at high concentrations,some dietary compounds have the effect of inhibiting AhR,which is AhR antagonist.Experimental study on the co-effect of dietary compounds with FICZ/TCDD in recombinant rat hepatoma cells,dietary compounds enhance AhR response to FICZ and high concentration of 3',4'-DMF,QUE,RES,CUR inhibited AhR response to TCDD,however,API and DAI has been a synergistic trend.Experimental study on the co-effect of dietary compounds with FICZ/TCDD in recombinant human hepatoma cells.Low concentration of API,QUE,RES,CUR enhance AhR response to FICZ/TCDD,however,the high concentration showed antagonistic trend and DAI,3',4'-DMF always been the trend of synergy.4.To verify the inhibitory effect of dietary compounds.The viability of wild-type cells were evaluated by MTT colorimetric assay.The results showed that the inhibition rate of cell activity exhibited dose dependent manner.Moreover,the cytotoxicity of AhR ligands to human hepatoma cells was significantly higher than that of rat hepatoma cells.We successfully established the recombinant cells using luciferase reporter gene,which is sensitive simple and rapid for AhR ligands analysis.This will bring significant value for further investigation on physiological and biological role of AhR ligands in vivo via the AhR inductive pathway.