ABSTRACT
Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases, which can cause serious complications and gradually increase the mortality rate. However, the effects of NAFLD on drug-metabolizing enzymes and transporters remain unclear, which may cause some confusion regarding patient medication. In this study, a NAFLD rat model was constructed by feeding rats with methionine and choline deficiency diets for 6 weeks, and the mRNA and protein levels of drug-metabolizing enzymes and transporter were analyzed by real-time fluorescent quantitative PCR and Western blot, respectively. The activity of drug-metabolizing enzymes was detected by cocktail methods. In the NAFLD rat model, the mRNA expression of phase I enzymes, phase II enzymes, and transporters decreased. At the protein level, only CYP1A1, CYP1B1, CYP2C11, and CYP2J3 presented a decrease. In addition, the activities of CYP1A2, CYP2B1, CYP2C11, CYP2D1, CYP3A2, UGT1A1, UGT1A3, UGT1A6, and UGT1A9 decreased. These changes may be caused by the alteration of FXR, HNF4α, LXRα, LXRß, PXR, and RXR. In conclusion, NAFLD changes the expression and activity of hepatic drug-metabolizing enzymes and transporters in rats, which may affect drug metabolism and pharmacokinetics. In clinical medication, drug monitoring should be strengthened to avoid potential risks.
Subject(s)
Choline Deficiency , Cytochrome P-450 Enzyme System , Liver , Non-alcoholic Fatty Liver Disease , Rats, Sprague-Dawley , Animals , Non-alcoholic Fatty Liver Disease/metabolism , Non-alcoholic Fatty Liver Disease/enzymology , Male , Liver/metabolism , Liver/enzymology , Cytochrome P-450 Enzyme System/metabolism , Cytochrome P-450 Enzyme System/genetics , Choline Deficiency/complications , Disease Models, Animal , RNA, Messenger/metabolism , RNA, Messenger/genetics , Methionine/metabolism , Rats , Membrane Transport Proteins/metabolism , Membrane Transport Proteins/genetics , Gene Expression Regulation, EnzymologicABSTRACT
Organic anion-transporting polypeptides 1B1 (OATP1B1) plays a crucial role in the transport of statins. However, there are too few animal models related to OATP1B1, especially humanized animal models. In this study, the human SLCO1B1 cDNA was inserted into the second exon of the rat Slco1b2 gene using CRISPR/Cas9 technology. Pharmacokinetic characteristics of statins were conducted in wild-type (WT), humanized OATP1B1 (hOATP1B1), and OATP1B2 knockout (OATP1B2 KO) rats, respectively. The results showed that human OATP1B1 was successfully expressed in rat liver and exhibited transport function. Furthermore, the pharmacokinetic results revealed that OATP1B1 exhibited varying uptake levels of pivastatin, rosuvastatin, and fluvastatin, leading to different levels of exposure within the body. These results were consistent with those obtained from in vitro experiments using overexpressed cell lines. In conclusion, we established a novel humanized SLCO1B1 transgenic rat model to assess the role of human OATP1B1 in the uptake of different statins. The different uptake mediated by OATP1B1 may be an important reason for the different efficacy of statins. The hOATP1B1 rat is a promising model for improving the prediction of human drug transport.
ABSTRACT
Sorafenib, an important cancer drug in clinical practice, has caused heart problems such as hypertension, myocardial infarction, and thrombosis. Although some mechanisms of sorafenib-induced cardiotoxicity have been proposed, there is still more research needed to reach a well-established definition of the causes of cardiotoxicity of sorafenib. In this report, we demonstrate that sorafenib is a potent inhibitor of the CYP2J enzyme. Sorafenib significantly inhibited the production of epoxyeicosatrienoic acids (EETs) in rat cardiac microsomes. The in vivo experimental results also showed that after the administration of sorafenib, the levels of 11,12-EET and 14,15-EET in rat plasma were significantly reduced, which was similar to the results of CYP2J gene knockout. Sorafenib decreased the levels of EETs, leading to abnormal expression of mitochondrial fusion and fission factors in heart tissue. In addition, the expression of mitochondrial energy metabolism factors (Pgc-1α, Pgc-1ß, Ampk, and Sirt1) and cardiac mechanism factors (Scn5a and Prkag2) was significantly reduced, increasing the risk of arrhythmia and heart failure. Meanwhile, the increase in injury markers Anp, CK, and CK-MB further confirmed the cardiotoxicity of sorafenib. This study is of great significance for understanding the cardiotoxicity of sorafenib, and is also a model for studying the cardiotoxicity of other drugs that inhibit CYP2J activity.
Subject(s)
Cardiotoxicity , Myocardial Infarction , Rats , Animals , Sorafenib , 8,11,14-Eicosatrienoic Acid/metabolism , 8,11,14-Eicosatrienoic Acid/pharmacology , Heart , Myocardial Infarction/chemically inducedABSTRACT
Multidrug resistance associated protein 2 (MRP2) is an important efflux transporter involved in clinical drug disposition and drug-drug interactions. The study of MRP2-mediated drug transport has become an integral part of drug discovery and development. In particular, screening of specific MRP2 inhibitors will help overcome the multidrug resistance in cancer. In this report, a new method for rapid and sensitive detection of Mrp2 function was established via using mouse small intestinal organoids. Firstly, small intestinal crypts isolated from mouse intestine were induced by Noggin, R-spondin1 and EGF to develop three-dimensional (3D) organoids. Secondly, the 3D organoids were characterized by the physical and physiological structure of Mrp2-mediated drug transport. Finally, Mrp2 fluorescent substrate 5(6)-carboxyl- 2', 7'-dichlorofluorescein (CDF) and its inhibitor MK-571 and probenecid were used to demonstrate Mrp2-mediated CDF transport in 3D organoids. The results showed that the small intestinal organoids have a physiological structure for Mrp2-mediated compound transport. Moreover, MK-571 and probenecid, inhibitors of MRP2, significantly decreased the accumulation of CDF in 3D organoids. In summary, a novel intestinal organoid model has been successfully established for the rapid and effective study of Mrp2-mediated drug transport.
Subject(s)
Intestine, Small/metabolism , Models, Biological , Multidrug Resistance-Associated Proteins/metabolism , Organoids/metabolism , Animals , Fluoresceins/pharmacology , Male , Mice, Inbred C57BL , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins/antagonists & inhibitors , Probenecid/pharmacology , Propionates/pharmacology , Quinolines/pharmacologyABSTRACT
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) technology is widely used as a tool for gene editing in rat genome site-specific engineering. Multidrug resistance 1 [MDR1 (also known as P-glycoprotein)] is a key efflux transporter that plays an important role not only in the transport of endogenous and exogenous substances, but also in tumor MDR. In this report, a novel MDR1 (Mdr1a/b) double-knockout (KO) rat model was generated by the CRISPR/Cas9 system without any off-target effect detected. Western blot results showed that MDR1 was completely absent in the liver, small intestine, brain, and kidney of KO rats. Further pharmacokinetic studies of digoxin, a typical substrate of MDR1, confirmed the deficiency of MDR1 in vivo. To determine the possible compensatory mechanism of Mdr1a/b (-/-) rats, the mRNA levels of the CYP3A subfamily and transporter-related genes were compared in the brain, liver, kidney, and small intestine of KO and wild-type rats. In general, a new Mdr1a/b (-/-) rat model has been successfully generated and characterized. This rat model is a useful tool for studying the function of MDR1 in drug absorption, tumor MDR, and drug target validation.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B/genetics , Digoxin/pharmacokinetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , Administration, Oral , Animals , Brain/metabolism , CRISPR-Cas Systems/genetics , Cytochrome P-450 CYP3A/analysis , Cytochrome P-450 CYP3A/genetics , Cytochrome P-450 CYP3A/metabolism , Digoxin/administration & dosage , Female , Gene Knockout Techniques/methods , Intestine, Small/metabolism , Kidney/metabolism , Liver/metabolism , Male , Models, Animal , RNA, Messenger/analysis , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Rats, TransgenicABSTRACT
Breast cancer resistance protein (BCRP), a key drug efflux transporter, significantly affects the therapeutic efficacy of many drugs. Thus, screening specific BCRP inhibitors and distinguishing between substrates and non-substrates of BCRP are valuable in drug discovery and development. This study presents a novel BCRP biosensor based on intestinal 3D organoids for rapid and sensitive detection of BCRP function. First, the crypts were isolated from mouse small intestine, and cultured in advanced DMEM/F12 medium to develop intestinal 3D organoids. Second, immunohistochemical studies demonstrated that BCRP protein in the organoids presented a similar expression and physiologic position to the small intestinal epithelium. Finally, the cultured organoids were treated in BCRP fluorogenic probe substrate Hoechst 33342 with or without BCRP inhibitor Ko143 and YHO-13177. The fluorescence intensity of Hoechst 33342 released from inner of the organoids was detected by microplate reader and the concentrations were calculated. Ko143 and YHO-13177 significantly inhibited the BCRP-mediated Hoechst 33342 transport in the 3D organoids. Consequently, a rapid and efficient biosensor has been successfully established to study BCRP, especially screening BCRP inhibitors in a high-throughput way.
