Biochemical and Molecular ToxicologyENVR 442/TOXC 442/BIOC 442The Role of Transporters (Phase III)in Xenobiotic DispositionKim L.R. Brouwer, PharmD, PhDWilliam R. Kenan Distinguished Professor and Chair,Division of Pharmacotherapy & Experimental TherapeuticsUNC Eshelman School of [email protected]; 919-962-7030Organic Anion TransportersSolute Carrier Family 22Transporter Tissue distribution Transport mechanismSubstratesOAT1 kidney, brain, choroid plexusantiport (dicarboxylates)PAH, PSP, methotrexate (MTX), cidofovir, ochratoxin A (OTA), cephaloridine, indoxyl sulfate, AZT, penicillins, cAMP, cGMP, PGE2, etc.OAT2 kidney, liver ?PAH, MTX, salicylate, acetylsalicylate, PGE2OAT3 kidney, liver, bone, brain, eyeantiport (dicarboxylates)PAH, OTA, salicylate, estrone-sulfate, cAMP, PGE2, cimetidineOAT4 kidney, placenta antiport (dicarboxylates)PAH, OTA, DHEA-sulfate, estrone-sulfate, AZT, cimetidine, MTXF. RusselOrganic Anion Transporting PolypeptidesSolute Carrier Family 21/SLCOTransporter Tissue distribution Transport mechanismSubstratesOatp1 (1a1) kidney, liver antiport(GSH)BSP,ouabain, taurocholate, estradiol glucuronide (E217βG), estrone sulfate, DHEA sulfate, aldosterone, cortisol, enalapril, thyroxine, triiodo-L-thyronine (T3), leukotriene C4, PGE2, ochratoxin AOatp3 (1a5) kidney, lung, retina, liver?taurocholate, thyroxine, T3Oatp5 (1a6) kidney ??Oat-k1/k2 (1a3_v1/v2)OATP-A (1A2)OATP-H (4C1)kidneybrain, kidney, liverkidney?antiport?(GSH)?MTX, folate, DHEA sulfate, E217βG ochratoxin A, digoxin, MTX, AZTBSP, DHEA, estradiol glucuronide, estrone sulfate, thyroxine, T3, ochratoxin A, bile acids, fexofenadine, oubain, rocuronium, chlorambucildigoxin, ouabain, thyroxine, T3, cAMP, MTXF. RusselOrganic Cation TransportersSolute Carrier Family 22Transporter Tissue distribution Transport mechanismSubstratesOCT1 liver uniportMPP+, TEA, acyclovir, ganciclovirOCT2 kidney, brain, neurons uniportTEA, MPP+, NMN, monoamines, amantadineOCT3 liver, skeletal muscle, placenta, kidney, heart, brainuniportMPP+, guanidine, monoamines, cimetidine, tyramineOCTN1 kidney, skeletal muscle, placenta, prostate, heartantiport (H+)TEA, MPP+, carnitine, quinidine, verapamil, pyrilamineOCTN2 skeletal muscle, kidney, placenta, liver, intestine, heart, etc.uniport Na+-carnitine cotransportTEA, MPP+, carnitine, choline, quinidine, verapamil, pyrilamine, valproateF. RusselTransporter Tissue distribution Transport mechanismSubstratesMRP1 kidney, (liver), lung, intestine, brainpump (ATP)glutathione, glucuronide, and sulfate conjugates, anticancer agents, GSH, GSSG, PAHMRP2 kidney, liver, intestine pump (ATP) glutathione, glucuronide, and sulfate conjugates, PAH, GSH, GSSG, cisplatin, methotrexate, ochratoxin AMRP4 kidney, liver, intestine, brain, prostatepump (ATP)cidofovir, PMEA, AZTMP, MTX, PAH, cAMP, cGMP, prostaglandinsMRP6MDR1kidney, liverkidney, liver, intestine, brain, placenta, lungpump (ATP)pump (ATP)BQ123, glutathione conjugatesE217βG, calcein, rhodamine 123, digoxin, anticancer drugs, verapamil, anti-HIV drugs, steroid hormonesMultidrug Resistance TransportersATP-Binding Cassette subfamily (ABCB/ABCC)outinNH2COOHATPATPoutinNH2COOHATPATPoutinoutinNH2COOHATPATPoutinoutinNH2COOHATPATPMRP1MRP2MRP6MRP4MRP5MDR1F. RusselAyrton & Morgan, Xenobiotica 31:469, 2001Organ Distribution of Transport Proteins BSEPMdr1a P-gpMdr1b P-gpBBBGallbladderLiverStomachIntestineKidneyAdapted fromSchinkel, 1997Organ Distribution of Multidrug Resistance Mdr1 P-glycoproteinP-gp Expression in Murine Brain Capillary Endothelial CellsGraff and Pollack, 2005Ivermectin Toxicity inMdr1a(-/-) and Mdr1a(+/+) MiceSchinkel et al., Cell, 77:491, 1994Maintenance of Barrier Function:Xenobiotic Transporters in the BrainHo and Kim, Clin Pharmacol Ther 78:260, 2005P-glycoprotein stainingEndothelial cell stainingCo-localizationMaintenance of Barrier Function:Endothelial Cells Lining the Olfactory BulbGraff and Pollack, Pharm Res 22:86, 2005Maintenance of Barrier Function in Sanctuary Site TissuesPlacentaLeslie et al., Tox Appl Pharmacol 204:216, 2005Maintenance of Barrier Function:Xenobiotic Transporters in the IntestineHo and Kim, Clin Pharmacol Ther 78:260, 2005Role of Mrp1 in Intestinal Toxicity of MethotrexateIntestinal toxicity induced by methotrexate treatment in wild-type [Mrp1(+/+)] and Mrp1 gene knockout [Mrp1(−/−)] mice in vivo. Mrp1 is localized primarily in proliferative cells in crypts where it is involved in active efflux of methotrexate as a defensive mechanism to protect the small intestinal epithelial cells from toxicity.Tissue sections from ‘lower’ part of the small intestine were analyzed for morphology with H&E staining (top panel) and for S-phase cells with immunostaining using anti-BrdU antibody (bottom panel).Ho and Kim, Clin Pharmacol Ther 78:260, 2005Xenobiotic Transporters in the KidneyHo and Kim, Clin Pharmacol Ther 78:260, 2005Effect of Probenecid on Renal Content and Urinary Excretion of Cadmium (Cd) in Mice Mice were injected i.p. with saline or probenecid (1 mmol/kg), and injected i.v. 30 min later with either Cd alone (1 mg Cd/kg, 74 kBq 109Cd) or Cd with dithiocarbamate chelating agents (1:30 molar ratio); urine samples were collected for 3 h and renal Cd content was determined from radioactivity.Kamenosono et al., Comp Biochem Physiol C Toxicol Pharmacol 132:61, 2002NPT1pOatv1OAT1/3MRP6α-KGOat1/3Mrp6α-KGOAT4MRP2/4α-KGOat4Mrp2/4α-KGOCT2Oct1/2MDR1OCTN1/2H+Mdr1a/1bOctn1/2H+OATP4C1Oatp4c1OATP1A2GSHGSHOatp1a1Oatp1a3(Oat-k1/k2)Oatp1a4/a5blood bloodurinehumanratSpecies differencesF. RusselAvailable Models To ExamineRenal Transport Processes Intact kidney in vivo Isolated perfused kidney Isolated perfused or nonperfused tubules Cultured renal cells Isolated plasma membrane vesicles(basolateral or brush border)Hepatic Elimination: Phase I (P450s),Phase II (conjugation) & Phase III (transport)biletight junctionsinusoidalmembranehepatocytecanalicularmembraneMetaboliteuptakeegressblood flowreabsorptionbileblood flowsinusoidalmembraneParentegressIntracellularSequestrationprotein bindingBile and Urine as Complementary Pathways for Excretion of Foreign Compounds in Rats: Molecular Weight Threshold Hypothesis Hirom et al., Xenobiotica 6:55-64, 1976% of dose recovered inMW Bile UrineSulphathiazole 255 3.1 844.8 ---aSuccinylsulphacetamide 314 1.8 626.3 ---aGlutarylsulphathiazole 369 42 4785 ---a---b831,2,3,6-Tetrahydrophthalyl- 407 45 34sulphathiazole 81 ---a---b83Bromophenol blue 670 69
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