A Study Of Regulation Of Drug Metabolism Genes And Drug Interaction By Triclosan Mediated By Nuclear Receptors

Drug metabolizing enzymes and transporter mediated drug-drug interaction(DDI)could reduce the efficacy and safety of clinical drugs,even lead to drug related toxicity.Induction of drug metabolizing enzymes and transporter is the main cause of DDI,which usually mediated by nuclear receptors.Research based on nuclear receptors is more prospective.Nuclear receptors are sensors to xenobiotics.PXR and CAR regulates the expression of numerous drug-metabolizing enzymes and transporters.Upregulation of these enzymes or transporters by PXR and CAR may cause severe DDIs leading to therapeutic efficacy or toxicity.Because of its complex and diverse biological functions,CAR has become a hot topic in the field of drug metabolism research.At the same time,because of the large difference between species,the indirect activation mode of ligands,it is a big difficult to unveil the interaction between CAR and drug-metabolizing enzymes.Triclosan is widely used in daily chemical products and considered to be safe for biosome.In recent years,a number of studies have shown that triclosan has adverse effects on endocrine disruption,muscle function and oncogenesis.In 2016,FDA banned the addition of triclosan in hand lotion and cosmetics.Triclosan can activate CAR receptor in mice,but due to the difference of species,the effect of triclosan on other species of CAR receptor is still unclear.In this study,we used triclosan as a tool activator to.On the one hand,we confirmed its induction effect on drug metabolizing enzymes and transporters on different kinds of animal,and then preliminarily unveiled whether the induction effect is closely related to CAR.On the other hand,the interaction between triclosan and aconitine or sorafenib was evaluated and the mechanism was preliminarily discussed.The experiments and results of the study are summarized as follows:1.qPCR was applied to investigate the effect of triclosan on the mRNA expression of CYPs and transporters in liver of different animals.In rat model,Cyp2b1 and Cyp3a1 were induced to 1.85-and 5.96-folds of control group,respectively after 90mg/kg administration of TCS.In mice model,Cyp2b10,Cyp3a11 and Abcb1 b were induced to6.24-,2.30-and 1.71-folds of control group,respectively after 30 mg/kg administration of TCS.After treatment of TCS in 20μM,expression of CYP3A4 and CYP2B6 was induced to 4.7 folds and 1.6 folds,respectively.In CAR knocked out mice,only Cyp3a11 was induced to 2.5 folds of control after treatment of 30mg/kg TCS.These results showed that the induction of liver metabolic enzymes and transporters by triclosan in rodents was mainly on Cyp2 b,Cyp3a and Mdr1,but there were differencesbetween species,showing that the mainly induced subtype in rat was Cyp3 a and in mice was Cyp2 b,Abcb1b in mice was induced while the relevant gene(MDR1)in rats was not induced.The induction of Cyp2b10 and Abcb1 b in CAR knockout mice disappeared while that of Cyp3a11 not,which indicate that the induction of triclosan to Cyp2b10 and Abcb1b is responsible to CAR while that of Cyp3a11 is not.2.Triclosan induced CYP3A1 activity of rat hepatocytes to 2.1-,3.2-and 4.6 folds of control group in concentration range of 2.5 μM-10 μM.The 1-hydroxymidazolam transformation activity of micrsome from rat administrated with 10,30,90 mg/kg of TCS were enhanced to 1.9-,2.3-and 3.4-fold of control group,respectively.These results showed that the CYP3A1 activity could be induced in both in vivo and in vitro models.qPCR results showed that 10,30 and 90 mg/kg of TCS induced the expression of CYP3A1 in the rat liver to 1.6-,2.5-,and 4.0-folds of the control group.The results of Western blot were consistent with qPCR.All the results together showed that both the mRNA expression and activity of CYP3A1 in rat liver could be induced by TCS.3.Compared to the vehicle control group,TCS at 10,30 and 90 mg/kg significantly reduced AUC of midazolam by 48.3,56.5 and 71.5%,respectively,with Cmax of midazolam reduced by 74.1,73.1 and 81.2%,respectively,and CL increased to 1.5-,2.3-and 3.8-fold.4.Molecular docking score of aconitine molecules to CYP3A4 enzyme crystals results in a total score of 8.307.Arg212,Glu374 and Ala370 were recognized to bond to AC and form hydrogen bonds,Phe215/108 was recognized to form hydrophobic.The results show that AC can be directly combined with CYP3A4 and may be substrate of CYP3A4.5.Treatment with concentration of 30nmol/L to 1000nmol/L of triclosan could increase the expression of some CYPs(cytochrome P450s)or ABCs(ATP binding cassettes).300 nmol/L of triclosan was the maximal effective concentration.Triclosan did not affect the expression of non-drug metabolism related genes,such as ALDH1A1,Apo E,ADRA2 A,DPYD,COMT,TPMT,HTRA or MTHFR.6.MTT assay showed that treatment with concentration of 10nmol/L to1000nmol/L of triclosan induced MHCC97-H proliferation;When coupled with TCS,the IC50 of sorafenib to MHCC97-H elevated from 1.03μmol/L to 9.64μmol/L.These results suggested that TCS could promote proliferation of HCC cell line and sorafenib coupled with TCS may reduce the inhibitory effect of sorafenib on HCC cell line.7.LC-MS/MS results showed that when sorafenib coupled with TCS,the half-life of sorafenib in MHCC97-H cells decreased from 9.70 hours to 5.43 hours,the half-life of sorafenib in HCC tumor decreased from 20.56 hours to 12.39.The results show that triclosan could promote the clearance of sorafenib in tumor cells and tumor tissues.8.PET-CT scan and quantitative detection of tumor phase by radioactivity were carried out.In both subcutaneous MHCC97-H cell tumor model and intrahepaticMHCC97-H cell tumor model,TCS acted as a tumor promoter,by elevating the volume and weight of tumor to a greater degree compared to control group.Sorafenib treatment significantly attenuated the intrahepatic growth of HCC cells in mice’s liver,and the nodules formed by HCC cells were shrinking comparing to those in control group.Triclosan attenuated the antitumor-effect of sorafenib on intrahepatic growth of HCC cells.In this paper,the nuclear receptor CAR was set as a starting point to investigate the DDI caused by an environmental chemical,called triclosan.The subtypes of CYP enzymes and transporters inducible in different species of animal were recognized and the relevance of these induction to CAR was evaluated preliminarily.The DDI between TCS and Aconitine or sorafenib was confirmed and the mechanisms of DDI were identified.DDI between TCS and Aconitine was mainly based on CYP induction,while the mechanisms of DDI between TCS and sorafenib was more complicated,including induction of drug metabolism related genes,acceleration of sorafenib clearance in HCC cell or tumors,enhancement of sorafenib resistance in HCC.