Hepatoprotective role of PXR activation and MRP3 in cholic acid-induced cholestasis

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1 British Journl of Phrmology (7) 5, & 7 Nture Publishing Group All rights reserve 7 88/7 $. RESEARCH PAPER Heptoprotetive role of PXR tivtion n MRP in holi i-inue holestsis Deprtment of Phrmeutil Sienes, Leslie Dn Fulty of Phrmy, University of Toronto, Toronto, Ontrio, Cn Bkgroun n purpose: Ativtion of the pregnne X reeptor (PXR) hs been shown to protet ginst holestti heptotoxiity. As PXR lters the expression of numerous hepti bile i trnsporters, we sought to elinete their potentil role in heptoprotetion. Experimentl pproh: Wil-type (PXR þ / þ ) n PXR-null (PXR -/- ) mie were fe % holi i () iet with or without the PXR tivtor, PCN. Liver funtion ws ssesse long with the orresponing hnges in hepti gene expression. Key results: ministrtion use signifint heptotoxiity in PXR þ / þ mie n ws ssoite with inution of severl FXR n PXR regulte genes, whih enoe for bile i trnsport n metbolizing proteins. Compre to lone, oministrtion of PCN to -fe PXR þ / þ mie signifintly erese heptotoxiity n ws ssoite with inution of MRP mrna s well s CYPA mrna n funtionl tivity. Unexpetely, PXR -/- mie, whih expresse signifintly higher bsl n -inue levels of MRP, MRP, OST, OSTb, OATP n CYPA, were rmtilly less sensitive to heptotoxiity thn PXR þ / þ mie. Conlusions: Protetion of PXR þ / þ mie ginst -inue heptotoxiity by PCN is ssoite with the inution of MRP n CYPA expression. Resistne ginst -inue heptotoxiity in PXR -/- mie my result from higher bsl n inue expression of bile i trnsporters, prtiulrly MRP. These finings emphsize the importne of trnsport by MRP n metbolism s mjor protetive pthwys ginst holestti liver injury. British Journl of Phrmology (7) 5, 67 76; oi:.8/sj.bjp.775; publishe online 6 April 7 Keywors: PXR; trnsporters; ytohrome P5; holestsis; bile is; heptotoxiity Abbrevitions: ALP, lkline phosphtse; ALT, lnine minotrnsferse; AST, sprtte minotrnsferse; BA, bile i; BSEP, bile slt export pump; 7-BQ, 7-benzyloxyquinoline;, holi i; R, onstitutive nrostne reeptor; CYP, ytohrome P5; FXR, frnesoi X reeptor; 7-HQ, 7-hyroxyquinoline; MDR, multirug resistne; MRP, multirug resistne-ssoite protein; NTCP, soium-turoholte trnsporting polypeptie; OATP, orgni nion trnsporting polypeptie; OST, orgni solute trnsporter; PCN, 5-pregnen-b-ol--one-6rbonitrile; PXR, pregnne X reeptor Introution Bile is (BAs) re enogenous holesterol-erive ompouns tht ply n importnt role in the igestion n bsorption of lipis n hyrophobi ompouns from the intestine. Despite their physiologil importne, the surftnt properties of BAs rener them toxi to membrne omponents of ells n elevte levels of BAs n result in mge to tissues suh s the liver n bile ut. Uner holestti onitions, BA levels re mrkely inrese in the liver n this is thought to ontribute to further heptoellulr injury (Bremmelgr n Alme, 98; Fisher et l., 996). Elevte intrhepti BA levels use Corresponene: Dr M Piquette-Miller, Leslie Dn Fulty of Phrmy, University of Toronto, College St., Toronto, Ontrio, Cn M5S M. E-mil: m.piquette.miller@utoronto. Reeive November 6; revise 8 Jnury 7; epte Jnury 7; publishe online 6 April 7 hnges in hepti gene expression tht le to inrese BA efflux s well s erese biosynthesis n uptke (Fikert et l., ; Zollner et l., ; Ano et l., 5). However, these intrinsi ptive hnges re not suffiient in proteting the liver ginst the high intrhepti BA onentrtions seen uring holestsis. Thus strtegies to minimize toxi BA onentrtions re of interest for the evelopment of therpies to tret holestti liver iseses. One potentil pproh tht hs reently emerge involves the use of tivtors of nuler hormone reeptors suh s the pregnne X reeptor (PXR), frnesoi X reeptor (FXR) n onstitutive nrostne reeptor (R). These lign-tivte trnsription ftors oorintely regulte genes involve in BA biosynthesis, metbolism n trnsport. In prtiulr, PXR regultes the expression of ytohrome P5 (CYP)A (Bertilsson et l., 998) n CYP7A (Stuinger et l., ), whih re respetively involve in

2 68 BA hyroxyltion n biosynthesis, s well s BA trnsporters inluing the multi-rug resistne ssoite proteins MRP (ABCC) (Kst et l., ), MRP (ABCC) (Teng et l., ) n the bile slt export pump (BSEP, ABCB) (Teng n Piquette-Miller, 5). One well known PXR tivtor, rifmpiin, is use linilly for the tretment of omplitions ssoite with holestti iseses (Hoensh et l., 985) n lithoholi i-inue liver injury is ttenute by the roent PXR tivtor, 5-pregnen-b-ol--one-6rbonitrile (PCN) (Stuinger et l., ; Xie et l., ). However, the ext mehnism behin the effet of PXR tivtion ginst BA toxiity is still unler. As trnsporters ply key role in BA homeostsis, it is likely tht the mehnism involves the moultion of trnsporters resulting in erese intrhepti BA onentrtions. Therefore, the im of this stuy ws to ompre the roles of iniviul BA trnsporters in the PXR tivtor-meite protetion ginst BA-inue heptotoxiity. We ompre the involvement of numerous BA uptke n efflux trnsporters, inluing the heteroimeri orgni solute trnsporter (OST/OSTb), whih hs only reently been foun to funtion s mjor BA trnsporter (Blltori et l., 5; Dwson et l., 5) n whih hs not yet been exmine for regultion by PXR. As holi i (), mjor BA in both humns n roents, is substntilly elevte uring holestti isese (Ltikinen n Ikonen, 977; Fisher et l., 996; Combes et l., 999), we utilize n estblishe moel of holestsis (Brone et l., 996; Rost et l., ) to inue holestti heptotoxiity. Overll, our novel finings point to ltere expression of trnsporters, prtiulrly MRP, s being integrl to heptoprotetion ginst BAs. Our stuy lso emonstrtes for the first time tht the BA trnsporters MRP n OST/OSTb re not regulte by PXR. Methos Animls The niml stuies were onute in orne with the guielines of the Cnin Counil on Animl Cre. Wiltype (PXR þ / þ ) eight- to -week-ol mle C57BL/6 mie were purhse from Chrles River Cn (Montrel, PQ, Cn). A olony of PXR / mie ws originlly obtine from Dr Christopher Sinl (Dlhousie University, Hlifx, NS, Cn) with permission from Dr Steven Kliewer (University of Texs Southwestern Meil Center, Dlls, TX, USA). Eight- to -week-ol mle PXR / mie erive from the originlly-obtine olony were use in the experiments. Animls were kept in temperture-ontrolle fility with -h light/rk yles, n llowe to limtise for week before tretment. The mie were fe either powere stnr iet or % (w/w) -supplemente iet for 5 ys, n injete ily with PCN (5 mg kg intrperitonel) or orn oil vehile ontrol, with n ¼ 6 for eh group. The -supplemente iet ws prepre in-house by ing powere to the sme stnr iet fe to ontrol nimls. Totl boy weight ws mesure eh y throughout the tretment perio. Mie were ple in metboli ges for the finl h of the stuy perio for urine olletion. Mie were fste uring the finl h to ensure tht elevtions in serum BA onentrtions were not owing to their relese from the gll bler n subsequent irultion following mel. The mie were kille h fter the finl injetion. Bloo ws ollete, n the liver ws remove; setion of the liver ws put into % prformlehye for histologil nlysis n the reminer ws snp-frozen in liqui nitrogen n kept frozen t 8C until nlyse. Determintion of mrna expression Totl RNA ws isolte from mouse liver using the QuikPrep RNA extrtion kit (Amershm Biosienes In., Pistwy, NJ, USA) oring to mnufturer s protool. DNA ws synthesize from.5 mg of RNA using the First Strn DNA synthesis kit (MBI Ferments, Flmborough, ON, Cn). The mrna levels of vrious genes were etermine by reltime quntittive PCR using LightCyler tehnology with SYBR Green I fluoresene etetion (Rohe Dignostis, Mnnhei, Germny). PCR primer sequenes hve been previously publishe (Kit et l., ; Teng n Piquette- Miller, 5; Zollner et l., 6). All mrna levels were normlize to 8S rrna. Mesurement of hepti enzymes, bilirubin n bile is in serum n urine Serum lnine minotrnsferse (ALT), sprtte minotrnsferse (AST), lkline phosphtse (ALP) n bilirubin (totl n iret) levels were mesure using regent kits from Thermo Eletron (Louisville, CO, USA) oring to mnufturer s instrutions. Totl BAs in serum n urine were mesure using kit from Trinity Bioteh (St Louis, MO, USA). BAs in serum were nlyse iretly, wheres urine ws first extrte with methnol. Histopthologil nlysis Prformlehye-fixe liver smples were re-setione in prffin (thikness of mm), stine with hemtoxylineosin t the University Helth Network Clinil Reserh Progrmme Histology Lb (Toronto, ON, Cn) n ssesse by pthologist Dr Eugene Hsieh t Sunnybrook n Women s College Helth Sienes Centre (Toronto, ON, Cn). Isoltion of liver mirosomes n mesurement of CYPA tivity Liver smples were homogenize in buffer (ph 7.) ontining.5 M surose, mm Tris-HCl n protese inhibitor oktil, entrifuge t g for min n the resulting superntnt ws entrifuge t g for 6 min. The pellet ontining the mirosoml frtion ws resuspene in the sme buffer, n protein onentrtions were mesure using the Brfor metho (Bio-R, Herules,, USA). Mirosoml CYPA tivity ws mesure using 7-benzyloxyquinoline (7-BQ) s substrte. 7-BQ is emethylte by CYPA to the fluoresent metbolite 7-hyroxyquinoline.

3 69 A NADPH-generting system (5 mm MgCl, 7.5 mm gluose- 6-phosphte,. mm NADP þ n unit ml gluose-6- phosphte ehyrogense) ws e to smples ontining.5 mg ml of mirosoml protein n 5 mm 7-BQ. Retions were inubte t 7C for min n terminte by the ition of. ml % trihloroeti i. Smples were vortexe, entrifuge ( g for 5 min) n superntnt liquots were mesure t exittion n emission wvelengths of n 5 nm, respetively, using Spetr Mx Gemini XS miroplte fluorimeter (Moleulr Devies, Sunnyvle,, USA). Beuse 7-BQ is lso metbolize to smll extent by other CYP isoforms (Renwik et l., ), retions in the presene of the CYPA inhibitor ketoonzole ( mm) were lso performe, n vlues were subtrte from the inhibitor-free mesurements to obtin metboli tivity ue to CYPA only. Sttistil nlysis All stuies were performe using n ¼ or more. Differenes between the tretment groups were nlyse using ANOVA n signifine ws etermine by the Tukey test. In ll ses, Po.5 ws onsiere to be sttistilly signifint. Anlysis ws performe using SigmStt. (SPSS In., Chigo, IL, USA). Drugs n hemils Primers for PCR were synthesize by the DNA Synthesis Centre, Hospitl for Sik Chilren (Toronto, ON, Cn). 7-BQ ws purhse from BD Gentest (Woburn, MA, USA). Brfor regent for protein quntifition ws from Bio-R Lbortories (Herules,, USA). All other regents n hemils inluing n PCN were purhse from Sigm-Alrih Cn (Okville, ON, Cn). Results Effet of feeing on liver funtion n heptoprotetion by PXR tivtion feeing use signifint heptotoxiity in PXR þ / þ mie s inite by n inrese in serum ALT, AST n ALP levels (Tble ). Totl n iret bilirubin levels in serum were lso signifintly inrese by in PXR þ / þ mie. Coministrtion of PCN signifintly ttenute heptotoxiity in -fe PXR þ / þ mie s emonstrte by 7 78% reution in ALT, AST n ALP levels s ompre to lone. Likewise, totl n iret bilirubin levels lso erese (by 7 n 8%, respetively). Of prtiulr interest ws the fining tht PXR / mie emonstrte rmtilly lower suseptibility to -inue heptotoxiity. Although feeing i use smll but signifint egree of heptotoxiity in PXR / mie, ALT, AST n ALP levels were 7-, 5- n 6.5-fol lower, respetively, in PXR / s ompre to PXR þ / þ mie following feeing. This inites tht PXR / mie re onsierbly protete from BA-inue heptotoxiity. In ition, totl n iret bilirubin levels in serum were not inrese by in PXR / mie. -meite hnges in serum ALT n AST levels were not ltere by the ition of PCN tretment in PXR / mie, onfirming PXR-epenent mehnism of heptoprotetion by PCN. Tretment of either PXR þ / þ or PXR / mie with PCN lone i not signifintly impt bilirubin levels or the other mrkers of heptotoxiity. Totl boy weight ws lso mesure throughout the tretment perio beuse heptotoxiity is often ssoite with erese ppetite n weight loss. Consistent with the serum biohemistry finings, feeing use severe weight loss in PXR þ / þ mie n this ws prevente by nerly 5% in mie given PCN long with (Tble ). Weight loss lso ourre in -fe PXR / mie, lthough this ws signifintly less pronoune ompre to PXR þ / þ mie. Also in greement with the serum biohemistry Tble Effet of feeing with or without o-tretment with PXR tivtors on liver funtion n totl boy weight ALT (U/l) AST (U/l) ALP (U/l) Totl bilirubin (mm) Diret bilirubin (mm) Chnge in totl boy weight (%) PXR þ / þ () þ PCN () NC (6) þ PCN (5) b 878 b 67 b.7. b b PXR / () þ PCN () () þ PCN () Abbrevitions: ALP, lkline phosphtse; ALT, lnine mino trnsferse; AST, sprtte minotrnsferse;, holi i; PXR, pregnne X reeptor; PCN, 5-pregnen-b-ol--one-6-rbonitrile; NC, no hnge. PXR þ / þ n PXR / mie were fe stnr how or % -supplemente iet for 5 ys with or without injetion of the PXR tivtor PCN. Numbers in brkets inite the number of mie in eh tretment group. Bloo smples were nlyse for ALT, AST, ALP n bilirubin levels s initors of liver funtion. Mie were weighe throughout the tretment perio; the hnge in weight represents the ifferene in weight between the strt n en of tretment. Results re presente s men enzyme or bilirubin onentrtions7s.e.m. Signifint from ontrol Po.5. b Signifint from Po.5.

4 7 + PCN PXR +/+ PXR / Figure Histologil hnges in mouse liver following n þ PCN tretment. PXR þ / þ n PXR / mie were fe % supplemente iet for 5 ys with or without otretment with the PXR tivtor PCN (5 mg kg ). The liver ws remove n setion ws fixe n stine with hemtoxylin-eosin. Imges re shown t mgnifition. Lrge res with egenertive heptoytes re evient in the setions from -fe PXR þ / þ mie, wheres only few res with inrese mrophge infiltrtion in the sinusois re notble in those from þ PCN trete mie. Likewise in the -fe PXR / mie, some inflmmtion represente by lustere mrophges is present. results, H&E-stine liver smples from -fe PXR þ / þ mie showe tissue mge hrterize by lrge zones of ysplsti n egenertive heptoytes (Figure ). This ws bsent in the liver from PXR þ / þ mie o-trete with PCN. On the other hn, no mjor histologil bnormlities were observe in the livers of -fe PXR / mie other thn some res of mil inflmmtion. Totl bile i levels in serum n urine following feeing Totl BA levels in serum n urine were lso mesure to ssess BA istribution (Figure ). Signifint - to -fol inreses in serum BAs were seen in the -fe PXR þ / þ n PXR / mie s ompre to ontrols. Serum BA levels were similr between -fe PXR þ / þ n PXR / mie. Coministrtion of PCN impose signifint (%) reution in serum BAs in the PXR þ / þ mie. feeing lso use - n -fol elevtion of urinry totl BAs versus ontrol levels in PXR þ / þ n PXR / mie, respetively. Interestingly, in ontrst to the hnges seen in serum, urinry BAs were not further ltere by ministrtion of PCN to the -fe PXR þ / þ mie. PCN ministrtion lone h no signifint effet on BA levels in either the serum or urine of ll mie fe stnr iets. Renl lerne of BAs ws signifintly higher (Po.5) in - ( mlh ), PCN- (.7. mlh ) n þ PCN- (9.679 mlh ) trete mie ompre to ontrols (7.7 mlh ) but signifint ifferenes were not seen between n -PCN trete mie. Bsl expression of efflux trnsporters n CYPA in PXR þ / þ versus PXR / mie To investigte the reson for the reltive insensitivity of PXR / mie to toxiity, we ompre PXR / n PXR þ / þ mie for ifferenes in the bsl levels of genes involve in BA homeostsis. Figure epits expression levels of genes Totl serum bile is (µmol/l) b Totl urine bile is (µmol/l) PXR +/+ PCN PXR +/+ PCN # +PCN +PCN PXR / PCN PXR / PCN tht were signifintly ifferent between PXR þ / þ n PXR / mie. The bsl levels of MRP, MRP n CYPA were - to -fol higher in PXR / mie thn in PXR þ / þ mie. +PCN +PCN Figure Totl bile i levels in serum n urine. Serum () ws ollete from mie (n ¼ 6) t the time of kille, fter h of fsting. (b) Urine ws ollete from mie for h before kille. Totl BA levels were nlyse s esribe in Methos. Results re presente s men BA levels7s.e.m. Po.5 versus ontrol; # Po.5 versus.

5 7 MRP (% ontrol) OST α (% ontrol) PCN b MRP (% ontrol) PXR +/+ PXR / PXR +/+ PXR /, 9 +PCN 8 +PCN b PXR +/+ PXR / PXR +/+ PXR / OST β (% ontrol) 9, 8 +PCN 7 6 5, e.5.5, +PCN +PCN PXR +/+ PXR / PXR +/+ PXR / Figure Effet of n þ PCN tretment on the expression of hepti genes with higher bsl levels in PXR / mie: () MRP; (b) MRP; () OST; () OSTb; (e) OATP n (f) CYPA. PXR þ / þ n PXR / mie (n ¼ 6) were fe ontrol iet, % -supplemente iet or iet with PCN. Levels of mrna were mesure by RT-PCR s esribe in Methos. Vlues re presente s rtio versus PXR þ / þ ontrols7s.e.m. Po.5 versus PXR þ / þ ontrol; b Po. versus PXR þ / þ ontrol; Po.5 versus PXR þ / þ fe ; Po.5 versus PXR / ontrol. OATP (% ontrol) f CYPA (% ontrol) In ition, bsl OATP levels in PXR / mie were nerly ouble tht in PXR þ / þ mie, n tht OST n OSTb were nerly - n 6-fol higher, respetively. Severl genes were not ltere between PXR þ / þ n PXR / mie, n these re epite in Figure. Bsl levels of FXR n R mrna, whih lso regulte mny of the genes being exmine, i not iffer between PXR þ / þ n PXR / mie (Tble ). We lso onsiere whether ifferenes in -inue heptotoxiity between PXR þ / þ n PXR / mie might be relte to ifferenes in the reltive expression of genes following feeing. Inee, we observe tht -fe PXR / mie expresse more thn -fol higher levels of MRP thn their PXR þ / þ ounterprts (Po.5). Moreover, -inue heptotoxiity, s etermine by plsm ALT levels, ws foun to be signifintly orrelte to MRP mrna expression (r ¼.5, Po.5) in ll nimls. The bsolterl trnsporter OSTb ws nerly 5-fol higher in PXR / mie thn PXR þ / þ mie following feeing but no orreltions in its expression were etete with inies of heptotoxiity. No further signifint ifferenes were etete between -fe PXR / versus PXR þ / þ mie nor i the expression of other hepti genes signifintly orrelte to heptotoxiity (t not shown). Hepti gene expression in -fe n PCN-trete mie Aministrtion of the -supplemente iet to PXR þ / þ mie use signifint hnges in the expression of hepti CYPs n BA trnsporters, n these hnges re epite in Figures n. As ompre to stnr iet ontrols, the

6 7 MRP (% ontrol) MDR (% ontrol) e OATP (% ontrol) PCN +PCN +PCN b b PXR +/+ PXR / PXR +/+ PXR / PXR +/+ PXR / PXR +/+ PXR /.5. BD BD BD BD PXR +/+ PXR / PXR +/+ PXR / Figure Effet of n þ PCN tretment on the expression of hepti genes with omprble bsl levels between PXR þ / þ n PXR / mie: () MRP; (b) BSEP; () MDR; () NTCP; (e) OATP n (f) CYP7A. PXR þ / þ n PXR / mie (n ¼ 6) were fe ontrol iet, % -supplemente iet or iet with PCN n levels of hepti mrna were mesure by RT-PCR. Vlues re presente s rtio versus PXR þ / þ ontrols7s.e.m. Po.5 versus PXR þ / þ ontrol; b Po. versus PXR þ / þ ontrol; Po.5 versus PXR þ / þ fe ; Po.5 versus PXR / ontrol; BD, below etetion limit. CYP7A (% ontrol) NTCP (% ontrol) BSEP (% ontrol) f PCN +PCN Tble Effet of feeing with or without o-tretment with PCN on hepti nuler reeptor expression Reltive levels of PXR, FXR n R mrna PXR þ / þ PXR / þ PCN þ PCN PXR FXR R b, 757 Abbrevitions:, holi i; R, onstitutive nrostne reeptor; FXR, frnesoi X reeptor; PCN, 5-pregnen-b-ol--one-6-rbonitrile; PXR, pregnne X reeptor. PXR þ / þ n PXR / mie (n ¼ 6) were fe % -supplemente iet n trete with the PXR tivtor PCN or orn oil ontrol. Levels of hepti nuler reeptor mrna were mesure by RT-PCR. Vlues re presente s perentge of the mrna levels in PXR þ / þ ontrol mie (7s.e.m.). Po.5 vs PXR þ / þ ontrol. b Po.5 vs PXR þ / þ fe. Po.5 vs PXR / ontrol.

7 7 -supplemente iet inue mrna levels of MRP, MRP, MRP, BSEP, multi-rug resistne protein (MDR), OST, OSTb, OATP n CYPA, wheres CYP7A, OATP n NTCP were ownregulte. The nuler reeptors PXR n FXR, but not R, were lso inue (Tble ). To etermine potentil mehnisms of PCN-meite heptoprotetion, we exmine the pttern of hepti gene expression in -fe PXR þ / þ mie o-ministere PCN. We foun tht the mrna expression of MRP n CYPA ws signifintly higher in mie trete with þ PCN s ompre to mie fe lone (Figure b n f). Interestingly, the expression of severl FXR regulte genes inluing MDR, OST n OSTb showe pronoune ownregultory tren in þ PCN trete PXR þ / þ mie s ompre to feeing lone, suggesting possible negtive regultory feebk mehnisms. Inee, mrna levels of FXR were pproximtely 5% lower in PCN trete mie, lthough this i not reh sttistil signifine. In orer to elinete the ontribution of PXR to these hnges, we lso exmine the effet of n þ PCN tretment on gene expression in PXR / mie. Aministrtion of the -supplemente iet to PXR / mie use signifint hnges in the expression of severl FXRregulte genes inluing MDR, MRP, MRP, NTCP, OST, OSTb n CYP7A (Figures n ). MRP n MRP inution still ourre, but to signifintly lesser extent (Po.5) thn tht seen in PXR þ / þ mie (Figure n b). This suggests both PXR-epenent n inepenent pthwys of inution by. Inution of CYPA n OATP s observe in -fe PXR þ / þ mie ws not seen in -fe PXR / mie (Figure e n f), initing tht inution by ourre through PXR. Clerly, hnges in gene expression in response to BA feeing involve multiple regultory mehnisms. On the other hn, s ompre to -feeing lone, o-ministrtion of PCN to PXR / mie i not impose ny signifint hnges in gene expression. As the effet of PXR tivtion on the expression of MRP n OST/OSTb is unknown, we lso exmine the impt of PCN tretment lone in PXR þ / þ versus PXR / mie. PCN ministrtion i not signifintly lter the expression of MRP (.57. fol), OST (.7. fol) or OSTb (.7. fol) in PXR þ / þ mie. Likewise PCN tretment i not signifintly ffet these genes in PXR / mie. The metboli tivity of CYPA in isolte mirosomes lso onfirme signifint inrese in CYPA-meite metbolism of 7-BQ in -fe PXR þ / þ mie s ompre to ontrols. Metbolism ws further inue in mie ministere þ PCN thn in mie fe only (Figure 5). On the other hn, neither feeing nor þ PCN tretment h signifint impt on CYPA tivity in PXR / mie. Disussion Trnsporters in the liver ply n integrl role in BA homeostsis (Figure 6). To unerstn the reltionship between trnsporter expression n BA-inue liver injury n lso the impt of PXR tivtor-meite moultion of trnsporter expression on liver funtion, we trete PXR þ / þ CYPA tivity (nmol/mg/min) 6 8 # PXR +/+ PXR / n PXR / mie with -supplemente iet tht results in heptotoxiity long with plsm totl BA levels tht re similr to those seen in ptients with holestti isese (Bremmelgr n Alme, 98) n other niml moels of holestsis (Bossr et l., 99; Wng et l., ). Consistent with reent reports, the inrese expression of mny FXR-regulte BA trnsporters in -fe PXR þ / þ n PXR / mie suggest -meite tivtion of FXR. We lso observe lesser or bolishe -meite inution of PXR trget genes (OATP, CYPA, MRP, MRP) in PXR / mie, whih inites tht n lso tivte PXR. As n its onjugte turoholte re reltively wek PXR +PCN Figure 5 Effet of feeing with or without otretment with PCN on hepti CYPA tivity in mirosomes. Mirosomes were isolte from the liver of PXR þ / þ n PXR / mie (n ¼ 6) fe % -supplemente iet n trete with the PXR tivtor PCN or orn oil ontrol. CYPA metboli tivity ws etermine by mesuring the fluoresene generte by the emethyltion of 7-BQ to fluoresent metbolite t nm n 5 nm exittion n emission wvelengths, respetively. Results re presente s men tivity7s.e.m. Po.5 versus ontrol; # Po.5 versus. BILE DUCT MRP BSEP MDR Cholesterol BA CYP7A PXR FXR BA BA-OH CYPA MRP OST α/β MRP OATP OATP NTCP BA-OH BLOOD URINE Figure 6 Metbolism n trnsport of BA, n the propose mehnism for PCN-meite protetion ginst -inue heptotoxiity. feeing results in the fee-forwr inution of bile i efflux trnsporters n hyroxyltion by CYPA. Hepti uptke of BA by NTCP n OATP n synthesis by CYP7A re ownregulte. CYPA n MRP re further inue by the oministrtion of PCN. Thus BAs re lere from the liver by PXR tivtion-epenent hyroxyltion (CYPA) n bsolterl efflux (MRP), leing to renl elimintion. Genes in boxes with grey shing were inue by feeing, wheres those in boxes with rk shing were inue further by o-ministrtion of PCN. Genes in boxes without shing were ownregulte by feeing.

8 7 tivtors (Stuinger et l., ; Moore et l., ), this my our through seonry pthwy. Reently, Jung et l. (6) emonstrte tht -meite inution of PXR ours through the tivtion of FXR; hene FXR is believe to ply the primry role in gene regultion in response to. However, espite these intrinsi ptive hnges in gene expression, heptotoxiity use by feeing persiste. Aministrtion of the potent PXR tivtor, PCN, to PXR þ / þ mie substntilly ttenute inue heptotoxiity s inite by erese serum liver enzymes, bilirubin n BA levels, histologil bnormlities n hnges in totl boy weight. Thus it is highly plusible tht tivtion of PXR by PCN meites the observe heptoprotetive effets. Of prtiulr signifine ws the fining tht PXR / mie were quite tolernt to the toxi effets of in tht -inue heptotoxiity ws rmtilly ttenute in PXR / mie. In greement with previous finings (Teng n Piquette-Miller, 5), we etete signifint ifferenes in the bsl expression of severl genes involve in BA homeostsis in the PXR / mie; thus it is oneivble tht reue sensitivity in PXR / mie stems from these ifferenes. However, we lso nnot exlue the potentil for other yet-to-be-ientifie BA etoxifition n/or trnsport pthwys whih oul hve elevte tivity in PXR / mie. Our results suggest tht expression of the bsolterl nion trnsporter, MRP, is n importnt eterminnt of toxiity. Bsl n -inue MRP expression were signifintly higher in PXR / mie. Furthermore, we observe signifint orreltion between MRP expression n serum ALT mong ll n þ PCN-trete PXR þ / þ n PXR / mie. PCN-meite heptoprotetion in PXR þ / þ mie ws lso ssoite with signifint inution of MRP mrna expression, further suggesting tht sinusoil efflux is mjor route by whih PXR tivtion meites BA homeostsis. As MRP is involve in the efflux of n its min onjugte, turoholi i (Hirohshi et l., ; Akit et l., ), it is likely tht efflux by this trnsporter is essentil uring BA overlo. Elevte MRP expression is often observe in ptients with holestsis or Dubin Johnson synrome, n hs been linke to protetion ginst heptoellulr nerosis uring obstrutive holestsis in mie (Bohn et l., ). Our finings provie eviene tht upregultion of MRP is inee heptoprotetive event uring holestsis, s prt of both the inherent feeforwr response to elevte BA levels s well s, reporte here for the first time, through the exogenous PXR tivtor-meite mehnism. Heptoprotetion by PCN ws lso ssoite with signifint inution of CYPA mrna n tivity. CYPA tlyzes the hyroxyltion of BAs inluing, renering them more hyrophili n therefore less toxi (Stemn et l., ; Zollner et l., 6). Inee, CYPA inution in -fe mie is ssoite with higher proportions of hyroxylte metbolites in serum (Zollner et l., 6) n inrese proportions of hyroxylte urinry BAs hve been reporte in PCN-trete bile ut ligte mie (Wgner et l., 5). It is importnt to note tht CYPA levels in -fe PXR / mie were similr to levels in -fe PXR þ / þ mie espite the lesser toxiity in PXR / mie. This suggests tht the role of CYPA in the overll feeforwr response to feeing in PXR þ / þ mie n in the inherent resistne of PXR / mie ginst is moest. Rther, the ontribution of CYPA in heptoprotetion ppers to be ssoite exlusively with PXR tivtion by PCN in PXR þ / þ mie. On the other hn, our results o not prelue the possibility tht other metboli pthwys suh s sulphtion oul ontribute to BA etoxifition. However, sulphtion hs little relevne in mie, where it hs only been emonstrte to our with lithoholi i (Kit et l., ). It is generlly hypothesize tht the hyroxyltion of enbles the hyrophili metbolites to be more reily serete into urine. Inee we ollete high levels of BAs in the urine of -fe PXR þ / þ n PXR / mie. Surprisingly, though, while we expete the PCN-trete mie to hve further inrese hyroxylte metbolites owing to CYPA inution, there ws no ifferene in urinry totl BA levels between the -fe n þ PCN-trete PXR þ / þ mie. These finings oul reflet sturtion of renl BA trnsporters. Inee, the renl lerne of BAs ws signifintly higher in -trete mie s ompre to ontrols, however ition of PCN to tretments i not further inrese renl lerne. Of note, þ PCN tretment resulte in pronoune erese in the expression of MRP n MRP in liver. If these genes re similrly ownregulte by PCN in the kiney, this oul le to reution in renl tubulr seretion of BAs. However, regultion of these trnsporters in the kiney by PXR tivtion is still unknown. Nevertheless, espite the similr totl BA levels, we nnot isr the possibility tht the proportion of hyroxylte BAs oul hve inrese in the PCN-trete mie. Unexpetely, PCN ministrtion erese expression of the pil trnsporters MRP n BSEP to ner bsl levels in fe PXR þ / þ mie. This ws surprising beuse heptobiliry exretion is mjor route of BA elimintion. Consistent with other reports, expression of these trnsporters ws inue by the -supplemente iet, suggesting role for MRP n BSEP in BA homeostsis (Fikert et l., ). The bsene of further inution by PCN my be beuse of negtive feebk whereby high biliry onentrtions of toxi BAs my inhibit further inution of pil trnsporters suh s MRP n BSEP in orer to prevent mge to the bile ut. Inee, it is thought tht elevte expression of BSEP in bile ut ligte mie is etrimentl owing to over-exposure of the lrey vulnerble bile ut to toxi BAs (Stemn et l., 6). It is lso oneivble tht heptoprotetion ginst oul be ttribute to hnges in the expression of other bsolterl BA efflux trnsporters suh s MRP or OST/ OSTb. FXR n R regulte these novel BA trnsporters (Assem et l., ; Lnrier et l., 6; Zollner et l., 6) n our results in -fe mie imply tht MRP n OST/ OSTb re involve in innte BA feebk mehnisms. However, we foun tht espite - n -fol inution of OST n OSTb, respetively, by feeing in PXR þ / þ mie, extensive toxiity still ensue. Furthermore, PCN tretment in -fe PXR þ / þ mie ws ssoite with erese rther thn inrese expression of MRP, OST

9 75 n OSTb, n hnges in their expression mong -fe PXR þ / þ or PXR / mie i not signifintly orrelte with ny inies of heptotoxiity. On the other hn, OSTb levels in PXR / mie following feeing were seven-fol higher ompre to their PXR þ / þ ounterprts, possibly initing tht the expression of this gene must reh extremely high levels in orer to ontribute to heptoprotetion. Thus hnges in the expression of MRP, OST n OSTb re not entirely onsistent with our toxiity t but, nevertheless inite tht unlike MRP, these bsolterl trnsporters o not pper to ply role in the PCNmeite heptoprotetion. Our results showing tht the impt of, PCN or þ PCN tretments on gene expression is the sme between PXR þ / þ n PXR / mie emonstrte for the first time tht MRP, OST n OSTb re not regulte by PXR. Our fining of signifint ifferenes in bsl gene expression between PXR þ / þ n PXR / mie ws not entirely unexpete. Similr trens hve been reporte in other nuler reeptor knokout moels (Guo et l., ; Sini et l., 5; Mrshll et l., 6). For exmple, hepti CYPA, MRP n MRP expression re elevte in FXR knokout mie, n this hs been linke to protetion ginst holestti liver injury through inrese BA metbolism n exretion (Shuetz et l., ; Wgner et l., ; Mrshll et l., 6; Zollner et l., 6). Sini et l. (5) lso etete higher bilirubin lerne in PXR / thn PXR þ / þ mie, whih they ssoite with elevte bsl levels of number of genes inluing MRP. Although the phenomenon of inrese gene expression in knokout mie is often reporte, the mehnism behin this effet remins unler. The ifferenes oul be interprete s being initive of PXR s negtive regultor of trnsporter expression n tht the presene of PXR promotes inue heptotoxiity. It is possible tht in n unligne stte, PXR my suppress gene expression; this might explin the higher bsl gene expression n lesser heptotoxiity in PXR / mie. On the other hn, lign-tivte PXR ts s positive regultor of gene expression. Alterntively, the ifferenes in trnsporter expression n sensitivity to between PXR þ / þ n PXR / mie oul stem not from the presene or bsene of PXR itself, but beuse of ompenstory inution of other pthwys tht regulte trnsporter genes in PXR / mie. This is n re tht is ertinly worthy of further investigtion. In onlusion, our results re suggestive of n importnt role for efflux trnsporters, prtiulrly MRP, in proteting the liver from holestti injury. We hve eluite, t lest in prt, the mehnism of PCN-meite protetion ginst -inue heptotoxiity; this involves the inution of MRP s well s CYPA. Our finings lso emonstrte tht unlike MRP, other bsolterl efflux pthwys inluing MRP n OST/OSTb re not regulte by PXR n o not ontribute to this PCN-meite effet. Our stuy signifintly s to the urrent knowlege of hnges in gene expression tht our uring holestsis n the mehnisms by whih the liver ttempts to minimize injury. Our stuy brings to the forefront the heptoprotetive role of MRP in BA trnsport uner holestti onitions n our finings provie further eviene for the potentil of BA trnsporters n PXR tivtors s foi for the evelopment of novel therpies to tret holestsis. Aknowlegements We woul like to thnk Dr Eugene Hsieh of Sunnybrook n Women s College Helth Sienes Centre (Toronto, ON, Cn) for evluting the histopthology speimens. Funing for this stuy ws provie by grnt from the Cnin Institutes of Helth Reserh (CIHR). Conflit of interest The uthors stte no onflit of interest. Referenes Akit H, Suzuki H, Hirohshi T, Tkikw H, Sugiym Y (). Trnsport tivity of humn MRP expresse in Sf9 ells: omprtive stuies with rt MRP. Phrm Res 9:. Ano H, Tsuruok S, Ymmoto H, Tkmur T, Kneko S, Fujimur A (5). Regultion of holesterol 7lph-hyroxylse mrna expression in C57BL/6 mie fe n therogeni iet. Atheroslerosis 78: Assem M, Shuetz EG, Leggs M, Sun D, Ysu K, Rei G et l. (). 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