Efflux Transporters Drive Glucuronidation Disposition In Subcellular Organelles

ObjectivesPhase Ⅱ metabolizing enzymes mainly include UDP-glucuronosyltransferases(UGTs)and sulfotransferases(SULTs),which mediate drug metabolism and elimination that generally governs drug efficacy and toxicity.Human efflux transporters,e.g.,P-glycoprotein(P-gp),breast cancer resistance protein(BCRP),and multidrug resistance-associated proteins(MRPs),are involved in membrane active transport of drugs,xenobiotics,and endogenous substances.The coupling of phase Ⅱ metabolizing enzymes and efflux transporters play an important role in modulating drug elimination.The transporter function shows great potential in altering phase Ⅱ metabolism.Our published research has demonstrated that kaempferol mainly undergoes glucuronidation metabolism to kaempferol-3-glucuronide(K-3-G)and kaempferol-7-glucuronide(K-7-G)in vivo,of which K-3-G is the main metabolite.This metabolic pathway is mainly mediated by UGT1A9.BCRP,MRP1 and MRP2 are responsible for the excretion of K-3-G and K-7-G.Numerous publications have demonstrated that efflux transporters control intracellular glucuronidation metabolism.However,whether and how efflux transporters control glucuronidation in subcellular organelles is unclear.In this study,kaempferol was used as a model compound to elucidate the importance of efflux transporters on glucuronidation in subcellular organelles because of its autofluorescence.Methods1.Using UGT1A9,human intestinal microsomes(HIMs)and human liver microsomes(HLMs)to elucidate the characteristics of the glucuronidation metabolism of kaempferol.2.Developed and validated a sensitive and reliable UHPLC-MS/MS method to determine the intracellular/subcellular levels of kaempferol and its phase Ⅱ metabolites.Then,quantitively delineated the effect of UGT1A9 and transporter inhibitors on subcellular amounts of kaempferol and its phase Ⅱ metabolites.3.Visualized the effect of UGT1A9 and efflux transporter inhibitors on the subcellular accumulation of kaempferol by laser scanning confocal microscope(LSCM).4.Study the effects of knockout of efflux transporters on the subcellular accumulation of kaempferol in liver and intestine visualized by LSCM.5.Investigated whether UGT1A9 and efflux transporters affect the pharmacological effects of kaempferol,such as the scavenging efficiency of reactive oxygen species(ROS)and nuclear translocation of nuclear factor-E2-related factor 2(Nrf2).Results1.Glucuronidation characteristics of kaempferolUGT1A9,HIMs and HLMs mediated the glucuronidation of 3’-OH and 7’-OH of kaempferol following classic Michaelis-Menten kinetics.The clearance(CL)values of K-3-G in UGT1A9 were 3.93-fold and 10.75-fold more than those in HIMs and HLMs.respectively.The CL values of K-7-G in UGT1A9 were 3.35-fold and 1.64-fold more than those in HIMs and HLMs,respectively.2.UGT1A9 and efflux transporter inhibitors caused a rise of kaempferol and reduction of its glucuronides in subcellular organellesKaempferol was quickly absorbed and metabolized by UGT enzymes in the cytosol.mitochondria,endoplasmic reticulum,lysosome and nuclei.The maximum subcellular concentrations of kaempferol in Caco-2/TC7 cells were achieved as early as 30 min.Notably,the amounts of kaempferol in isolated mitochondria(1.50 nmol/mg)were 3-fold higher than those in nuclei/lysosomes(0.57 nmol/mg)and 2-fold higher than those in endoplasmic reticulum(0.76 nmol/mg)after incubation for 30 min.The UGT1A9 inhibitor carvacrol caused a significant rise in subcellular kaempferol,and reduction of subcellular glucuronides in a time-dependent manner.Similarly,the inhibition of transporters,e.g.,P-gp,BCRP and MRPs,resulted in marked increases in subcellular kaempferol and significant decreases in subcellular glucuronides.When the three inhibitors were used in combination,the concentrations of kaempferol were highest.3.UGT1A9 and efflux transporter inhibitors significantly increased the fluorescence densities of kaempferol in subcellular organellesConfocal images showed that the green fluorescence could co-localize with nuclei,mitochondria,endoplasmic reticulum and lysosome,confirming that kaempferol was entrapped in these subcellular organelles.The maximum green fluorescent densities(kaempferol)that accumulated in all subcellular organelles were achieved as early as 30 min.The green fluorescent densities gradually decreased after 30 minutes in the absence or presence of chemical inhibitors,indicating that the kaempferol was metabolized by UGTs and led to the disappearance of green fluorescence.UGT1A9 and efflux transporter inhibitor(carvacrol,Ko143,verapamil and MK571)alone or in combination markedly increased the green fluorescence densities and decreased the rate of fluorescence elimination in subcellular organelles.The fluorescence signal of kaempferol was highest when the three inhibitors(Ko143,verapamil and MK571)were used in combination.We also performed a correlation analysis of kaempferol content measured by UHPLC-MS/MS and LSCM.High correlations between fluorescence densities and absolute amounts were observed with correlation coefficient values of more than 0.76.4.Knockout of efflux transporters markedly increased the subcellular accumulation of kaempferol in liver and intestineAfter oral administration of kaempferol,we found that kaempferol was mainly distributed in the intestine,followed by the liver in both wild-type and efflux transporter knockout FVB mice.The increased green fluorescence was mainly overlapped with mitochondria.Kaempferol fluorescence in the liver and intestine of Mdr1a-/-(～1.56-fold),Bcrp-/-(～2.10-fold)and Mrp2-/-(～2.77-fold)mice was markedly(P<0.01)enhanced in comparison to wild-type mice.5.Kaempferol suppressed intracellular ROS formation and induced nuclear translocation of Nrf2 in a dose-dependent mannerKaempferol treatment decreased the fluorescence of intracellular ROS in a dose-dependent manner.Moreover,the ROS scavenging efficiency of kaempferol was comparable to N-acetyl-L-cysteine(NAC).Kaempferol co-treated with UGT1A9 and efflux transporter inhibitors markedly(P<0.001)decreased the levels of ROS in response to an increase in kaempferol concentrations.The chemical inhibitor group had no effect on ROS generation.Kaempferol treatment increased the fluorescence of Nrf2 in cytosol and nuclei in a dose-dependent manner.Following treatment with UGT1A9 and efflux transporter inhibitors,the expression and nuclear translocation of Nrf2 were apparently increased(P<0.01)in response to an increase in kaempferol concentrations.The chemical inhibitor group had no effect on Nrf2 expression and translocation.Conclusions1.We confirmed for the first time that efflux transporters(i.e.,P-gp,BCRP and MRPs)drive glucuronidation disposition in subcellular organelles by employing both chemical inhibition and gene knockout methods.2.UGT1A9 and Efflux transporters govern the subcellular distribution of kaempferol and corresponding pharmacological effect.3.This is the first study to demonstrate that kaempferol predominantly accumulates in mitochondria.As a useful antioxidant agent,kaempferol may play a potential role in against oxidative stress-related inflammatory diseases and aging.