The Protein Profiles Of Drug-metabolizing Enzymes In Wild-type And Efflux Transporter Knockout FVB Mice

ObjectiveFVB mice are becoming more common laboratory models.The advantages of defined inbred background,large litters,and prominent pronuclei make the FVB mice be extensively used in transgenic,such as Bcrpl(-/-),Mrpl(-/-),Mrp2(-/-),and Mdrla(-/-)mice.It is well-accepted that substrates are metabolized through drug-metabolizing enzyme(DMEs)systems within the cells,and more water-soluble metabolites can be readily excreted by efflux transporters out of the cells.The interplay between efflux transporters and DMEs reduces substrates exposure in vivo and therefore has important effects on the oral bioavailability of drugs.To date,efflux transporter knockout mice are extensively appied for the pharmacokinetic studies.Nevertheless,few systematic studies have been conducted to determine if efflux transporters knockout had an impact on the protein amounts of DMEs.Additionaly,clinical researchs showed that the sex differences of DMEs and transporters could affect pharmacokinetic and pharmacodynamics.Traditionally,the reverse-transcriptase polymerase chain reaction(RT-PCR),enzyme-linked immunosorbent assay(ELISA),and western bloting(WB)are commonly used to detect the protein and gene expressions of drug-metabolizing enzymes(DMEs).Although these approaches are incresealy used,the application of traditional methods is unreliable and low throughput.Hence,the herein work is to develop a sensitive,rapid,stable,and high throughput isotope label-free method to profile the protein expressions of 27 DMEs in various tissues isolated from both gender five mouse genotypes including wild-type FVB,efflux transporter knockout of Bcrpl(-/-),Mrpl(-/-),Mrp2(-/-),and Mdrla(-/-)mice.This study could provide useful reference for drug design and clinical studies.MethodsIn this study,S9 fractions of hepatic and extrahepatic tissues were processed using standard differential centrifugation procedures.To construct a method for simultaneously quantifying tissue distribution and gender-specific of DMEs in wild-type(WT)FVB,Bcrp(-/-),Mrpl(-/-),Mrp2(-/-),and Mdrla(-/-)mice by using an isotope label-free UHPLC-MS/MS method.Quality samples and tissue samples were underwent tyspitic digestion and SPE.Results1.To construct a rapid,sensitive,stable,and high throughput method.The data of method validation revealed the proposed UHPLC-MS/MS method exhibited good specifity,good range(1.56-200 nM).The accuracies of QC samples met the requirement were 78.6%,75.0%,and 96.4%,respectively,at low,middle,and high concentration.The values of precision,matrix effects as well as recovery values were gained the requirements.Meanwhile,the values of stability were acceptable within three days.After the SPE,the values of recovery were 70%-120%.Considering all the aspects,a rapid,stable,and high throughput approach was developed successfully.2.The protein expressions of DMEs in various tissues of 5 mouse genotypes.The results showed that DMEs were widely expressed in the metabolic organs,and the liver was the most abundant tissue.The isoforms(i.e.,Cypla2,Cyp1b1,Cyp2c29,Cyp2c39,Cyp2d22,Cyp2el,Cyp3a11,Cyp3a25,Cyp7al,Cyp27a1;Ugtlal,Ugt1a2,Ugt1a5,Ugt1a6a,Ugtla9,Ugt2a3,Ugt2b1,Ugt2b5,Ugt2b34,Ugt2b35,Ugt2b36;Sultlal,Sult1d1,and Sult3al)were observed in the liver.The protein amounts of Cyp2c29,Ugt2b5,and Sultlal were the highest among Cyps,Ugts,and Suits.In the small intestine,we detected the protein expressions of Cyp1b1,Cyp2c29,Cyp3all,Cyp3a25,Cyp7al;Ugtlal,Ugt1a2,Ugt1a5,Ugtla6a,Ugt2a3,Ugt2b5,Ugt2b34,Ugt2b35;Sultlbl,Sultldl,and Sult2bl.Meanwhile,the protein expression levels of Cyp3all,Ugtlal,and Sultldl were the most abundant.The isoforms of Cyp1b1,Cyp2e1,Cyp7a1;Ugt1a2,Ugt2a3,Ugt2b5,Ugt2b35;Sultldl,and Sult4al were distributed in the kidney.And Cyp2el,Sultldl and Ugt2b5,Ugtla2 were the highest expressions in the males and females,respectively.The stomach covered the isoforms of Cyp1b1,Cyp7a1,Ugt1a2,Ugt1a5,Ugtla6a,Ugt2a3,Ugt2b5,Ugt2b34,and Sult1b1.The protein expressions of Ugt2b5 were at relative higher,others were at weak levels.Other organs(e.g lung,brain,spleen,and heart)had the less isoforms.3.Gender difference of important DMEs in mice.The results demonstrated that the gender differences of several isoforms were inconsistent within the five mouse genotypes,but partial isoforms exhibited the same gender differences.For instance,Cyp2c29,Cypla2,Cyp27al,Ugt2b1,Ugt2b5,and Ugt2b36 were male dominant,while Cyp2c39,Cyp2d22,Cyp7al,Ugtlal,Ugtla5,Sultlal,Sult3a1,and Sultldl were female dominant in the liver.Cyp2c29and Cyp3all displayed a male dominant patter in the small intestine.Cyp2el,Ugt1a2,and Sultld were the female-specific pattern.4.Comparisons the protein levels of DMEs in efflux transporter knockout mice and WTFVB.The results illustrated that almost isoforms were the similar protein expression levels in the different tissues of five mouse genotypes.However,partial isoforms had the significant differences.For example,in the liver,when comparing the WT FVB mice,the protein expressions of Cyp2c29,and Ugtla9 increased to about 2 times in four male knockout mice.The protein expressions of Cyp3al 1 increased to about 1.5 folds in male Mrpl(-/-)and Mrp2(-/-).Similarly,the intestinal protein amounts of Cyp2c29 in both gender Mrpl(-/-)and Mrp2(-/-)increased to about 2 folds.The protein amounts of Ugtla2 increased to 2 times in the spleen of both gender knockout mice.Conversely,the protein amounts of Ugt2b5 and Sultlbl decreased to the about 0.4 times in the kidney and stomach of male knockout mice,respectively.ConclusionIn this study,we successfully developed accurate and high-throughput method and applied for acomprehensively and systematically quantifying DMEs in WT FVB,Bcrpl(-/-),Mrpl(-/-),Mrp2(-/-),and Mdrla(-/-)mice.The present study revealed the tissue distribution and gender-specific of phase I and phase II enzymes in mice and will provide theoretical basis to further study the metabolic enzymes.