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Arch Pharm Res Vol 28, No 3, 249-268, 2005 ~rt~i~ez of ~armata ]~e~eart~ http://apr.psk.or.kr Induction of Phase I II and III Drug Metabolism/Transport by Xenobiotics Changjiang Xu Christina Yong-Tao Li and Ah -Ng Tony Kong Department of Pharmaceutics, Ernest Mario School of Pharmacy, Piscataway, NJ 08854, USA Rutgers, The State University of New Jersey, Received No vem ber 18, 2004) Drug metabolizing enzym es DMEs) play central roles in the metabolism, elimination and detoxification of xenobiotics and drugs introduced into the human body. Most of the tissues and organs in our body are well equipped with diverse and various DMEs including phase I, phase II metabolizing enzymes and phase III transporters, which are present in abundance either at the basal unstimulated level, and/or are inducible at elevated level after exposure to xenobiotics. Recently, many important advances have been made in the mechanisms that reg- ulate the expression of these drug metabolism genes. Various nuclear receptors including the aryl hydrocarbon receptor AhR), orphan nuclear receptors, and nuclear factor-erythoroid 2 p45-related factor 2 Nrf2) have been shown to be the key mediators of drug-i nduced changes in phase I, phase II metabolizing enzymes as well as phase III transporters involved in efflux mechanisms. For instance, the expression of CYP1 genes can be induced by AhR , which dimerizes with the AhR nuclear translocator Arnt), in response to many polycyclic aromatic hydrocarbon PAHs). Similarly, the steroid family of orphan nuclear receptors, the constitutive androstane receptor CAR) and pregnane X receptor PXR), both heterodimerize with the ret- inoid X receptor RXR), are shown to transcriptionally activate the promoters of CYP2B and CYP3A gene expression by xenobiotics such as phenobarbital-like compounds CAR) and dexam ethasone and rifampin-type of agents PXR). The peroxisome proliferator activated receptor PPAR), which is one of the first characterized members of the nuclear hormone receptor, also dimerizes with RXR and has been shown to be activated by lipid lowering agent fibrate-type of compounds leading to transcriptional activation of the promoters on CYP 4A gene. CYP7A was recognized as the first target gene of the liver X receptor LXR), in which the elimination of cholesterol depends on CYP7A. Farnesoid X receptor FXR) was identified as a bile acid recep tor, and its activation results in the inhibition of hepatic acid biosyn thes is and increased transport of bile acids from intestinal lumen to the liver, and CYP7A is one of its target genes. The transcriptional activation by these receptors upon binding to the promoters located at the 5-flanking region of these CYP genes generally leads to the induction of their mRNA gene expression. The physiological and the pharmacological implications of common partner of RX R for CAR, PXR , PPAR , LXR and FXR receptors largely remain unknown and are under intense investigations. For the phase II DMEs, phase II gene inducers such as the phenolic compo unds butylated hydroxyanisol BHA), tert-butylhydroquinone tBHQ), green tea polyphenol GTP), -)-epigallocatechin-3-gallate EG CG ) and the isothiocyanates PE ITC, sul- foraphane) generally appear to be electrophiles. They generally po ssess electrophilic-medi- ated stress response, resulting in the activation of bZIP transcription factors Nrf2 which dimerizes with Mafs and binds to the antioxidant/electrophile response element ARE/EpRE) promoter, which is located in many phase II DMEs as well as many cellular defensive enzymes such as hem e oxygenase-1 HO-1), with the subse quen t induction of the expression of these genes. Phase III transporters, for example, P-glycoprotein P-gp), multidrug resistance-associ- ated proteins MRPs), and organic anion transporting polypeptide 2 OATP2) are expressed in many tissues such as the liver, intestine, kidney, and brain, a nd play crucial roles in drug absorption, distribution, and excretion. Th e orphan nuclear receptors PX R and CAR have been shown to be involved in the regulation of these transporters. Along with phase I and phase II enzyme induction, pretreatment with several kinds of inducers has been shown to alter the Correspondence to: A h-N g Tony Kong, Glaxo Professor of Pharmaceutics, Department of Pharmaceutics, Ernest Mario School of Phar- macy, Rutgers, The State University of N ew Jersey, 160 Frelinghuysen Road, Ro om 228, Piscataway, NJ 088 54, US A Tel: 732-445-3831 ext. 226 , Fax : 732-445-3134 E-mail: KongT @ ci.rutgers.edu 249

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    Arch P harm Res Vo l 28, No 3 , 249-268, 20 05~rt~ i~e z of

    ~armata ]~e~eart~http://apr.psk.or.kr

    I n d u c t io n o f P h a s e I II a n d III D r u g M e t a b o l is m / T r a n s p o r t b yX e n o b i o t i c sChangj iang Xu Chr is t ina Yong-Tao L i and Ah -Ng Tony KongDepartment of Pharmaceut ics, Ernest Mario School of Pharmacy,P iscataway, NJ 08854, USA Rutgers, The State Univers i ty of New Jersey,

    Received No vem ber 18, 2004)Drug metabo l iz ing enzym es DME s) p lay cen tra l ro les in the metabo lism, e l imina t ion anddetox if ica t ion o f xenob io t ics and d rugs in t roduced in to the human body . Most o f the t issuesand o rgans in our body a re we l l equ ipped wi th d iverse and var ious DMEs inc lud ing phase I ,phase I I metabo l iz ing enzy me s and phase I II t ranspor te rs , wh ich a re p resen t in abund anceeither at the basal unstimulated level, and /or are inducib le at e levated level after expos ure toxenob io tics . Recent ly, ma ny impor tan t a dvanc es have been m ade in the mecha n isms tha t reg-u la te the express ion o f these d rug m etabo lism genes . Var ious nuc lear recep to rs inc lud ing thearyl hydrocarbon receptor AhR ), orphan nuc lear receptors, and nuclear factor-erythoroid 2p45-re la ted fac to r 2 Nr f2 ) have been shown to be the key med iato rs o f d rug- induced change sin phase I , phase I I metabol iz ing enzym es as we l l as ph ase I I I t ranspor te rs invo lved in e f f luxmechan isms. For ins tance , the express ion o f CYP1 gene s can be induced by AhR , wh ichd imer izes wi th the Ah R nuc lear t rans loca to r Arn t ), in respon se to many po lycyc lic a rom at ichydrocarbon PAH s). Similar ly, the steroid family of orphan n uclear receptors, the constitut iveandros tan e recep to r CA R) and p regnane X recep to r PXR), bo th he te rod imer ize wi th the re t -ino id X recep to r RX R), a re show n to t ranscr ip t iona l ly ac t iva te the p romoters o f CYP 2B andCY P3 A g e n e e x p re s s io n b y x e n o b io t i cs s u c h a s p h e n ob a rb i ta l -l ik e c o mp o u n d s CAR) a n ddexam ethaso ne and r ifamp in -type o f agen ts PXR). The perox isom e pro l ife ra to r ac t iva tedrecepto r PPAR ), wh ich is one o f the fi rst charac te r ized me mb ers o f the nuc lear hormon erecepto r , a lso d imer izes wi th RXR and has been shown to be ac t iva ted by l ip id lower ing agentf ib ra te -type o f com poun ds lead ing to transcrip tiona l ac t iva t ion o f the p rom oters on CYP 4Agene. C YP 7A w as recogn ized as the f irs t ta rge t gene o f the l ive r X recep to r LXR), in wh ichthe e limina tion o f cho les te ro l depe nds on CY P7A . Farneso id X recep to r FXR ) was iden t if iedas a b ile acid recep tor, and its act ivat ion results in the inhib it ion of hepatic acid b iosyn thes isand inc reased t ranspor t o f b i le ac ids f rom in tes t ina l lumen to the l ive r, and CY P7 A is one o f i tsta rge t genes . The t ranscrip tiona l ac t iva t ion by these recep to rs upon b ind ing to the p rom otersloca ted a t the 5 - f lanking reg ion o f these C YP genes genera lly leads to the induc t ion o f the i rm RN A gene express ion . Th e phys io log ica l and the pharmaco log ical imp l ica t ions o f com mo npar tner o f RX R fo r CA R, PXR , PPAR , LXR and FXR recep to rs la rgely remain unknown andare under in tense inves t iga t ions . For the phase I I DM Es, ph ase I I gene inducers such as thepheno l ic compo unds bu ty la ted hydroxyan iso l BHA), te rt -bu ty lhydroqu inone tBHQ), g reen teapolyphenol GTP ), -)-epigal locatechin-3-gallate EG CG ) and the isothiocyanates PE ITC , sul-fo raphane) genera l ly appear to be e lec troph iles . The y genera l ly po ssess e lec t roph i lic -med i-ated stress response, result ing in the activat ion o f bZIP transcription factors N rf2 wh ichd ime r izes wi th Mafs and b inds to the an t iox idan t/e lec t roph i le respon se e lement AR E/E pRE )promoter , wh ich is loca ted in many phase I I DMEs as we l l as many ce l lu la r de fens ive enzymessuch as hem e oxygenase-1 HO-1) , wi th the subse quen t induc t ion o f the express ion o f thesegenes. P hase III transporters, for exam ple, P-glycoprotein P-gp), mult idrug resistance-associ-a ted pro te ins MR Ps) , and o rgan ic an ion transport ing po lypeptide 2 OATP 2) a re expre ssed inm any t issu es such a s the l ive r, in test ine, k idney, and brain, a nd p lay crucia l ro les in drugabsorp tion , d is tr ibu t ion , and excre tion . Th e o rphan nuc lear recep to rs PX R and CA R have beenshow n to be invo lved in the regu la tion o f these t ransporte rs. A long w i th ph ase I and ph ase I Ienzy me induc tion , p re t rea tment wi th severa l k inds o f inducers has been show n to a l te r the

    Correspondence to: A h-N g Ton y Kong, Glax o Professor of P harmaceutics, Departm ent of P harmaceutics, Ernest Ma rio School of Pha r-macy, Rutgers, The State University of N ew Jersey, 160 Frel inghuysen Road, Ro om 22 8, Piscataway, NJ 088 54, US ATel: 732-445-3831 ext. 226 , Fax : 732-445-3134E-mail: [email protected] ci.rutgers.edu

    249

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    250 C . Xu et a l .

    express ion of phase I I I t ransporters , and a l ter the excret ion o f xenobio t i cs , which impl ies thatphase I I I t ransporters may a lso be s im i lar l y regula ted in a coord inated fash ion, and prov idesan important mean to protect the body f rom xenobio t i cs insu l t s . I t appears that in genera l ,expo sure to phase I , pha se I I and ph ase I II gen e inducers m ay t r igge r ce llu lar s t ressresponse lead ing to the increase in the i r gene express ion, which u l t imate ly enhance the e l im i -nat ion and c learance of these xenobio t ics and /or o ther ce l lu lar s tresses inc lud ing harm fu lreact i ve in termediates such as react i ve oxygen spec ies (ROS), so that the body w i l l removethe s t ress expedi t ious ly . Consequent ly , th is homeostat ic re spo nse of the body p lays a cen-t ra l ro le in the protection o f the bod y against env i ronmenta l insu lt s such as those e l ic i ted b yexposure to xenobio t ics .Key words: Phase I metabol i z ing enzyme s, Ph ase I I metabol i z ing enzyme s, P-Glycoprote in ,Mul t idrug resistance-associated protein, Organic anion t ransport ing polypept ide 2, Aryl hydro-carbon receptor , Pregnane X receptor , Const i tu t i ve androstane receptor , Perox isome pro l i fera-tor act i vated receptor , L iver X receptor , Farnesoid X receptor , Ret ino id X receptor , Nuc learfactor-erythoroid 2 p45-related factor 2

    I N T R O D U C T I O N O F P H A S E I P H A S E II D R U GM E T A B O L I Z IN G E N Z Y M E S A N D P H A S E IIID R U G T R A N S P O R T E R SD r u g m e t a b o l i z i n g e n z y m e s ( D M E s ) p l a y c e n t r a l r o l e s

    i n t he m e t abo l i sm , e l i m i na t i on and / o r de t ox i f i ca t i on o fx e n o b i o ti c s o r e x o g e n o u s c o m p o u n d s i n t r o d u c e d i n to th eb o d y ( M e y e r , 1 9 9 6 ) . I n g e n e r a l , D M E s p r o t e c t t h e b o d ya g a i n s t t h e p o t e n t ia l h a r m f u l e x p o s u r e t o x e n o b i o t ic s f r o mt h e e n v i r o n m e n t a s w e l l a s c e r t a in e n d o b i o t ic s . I n o r d e r t om i n i m i z e t h e p o t e n t ia l i n ju r y c a u s e d b y th e s e c o m p o u n d s ,m o s t o f t h e t i s s u e s a n d o r g a n s a r e w e l l e q u i p p e d w i t hd i v e r s e a n d v a r i o u s D M E s i n c l u d i n g p h a s e I , p h a s e I Im e t a b o l i z i n g e n z y m e s a s w e l l a s p h a s e I I I t r a n s p o r t e r s ,w h i c h a r e p r e s e n t i n a b u n d a n c e e i t h e r a t t h e b a s a lun i n duc ed l eve l , an d / o r i nduc i b l e a t e l eva t ed l eve l a f t e rx e n o b i o ti c s e x p o s u r e ( M e y e r , 1 9 9 6 ; R u s h m o r e a n d K o n g ,2 0 0 2 ; W a n g a n d L e C l u y s e , 2 0 0 3 ) .

    P h a s e I D M E s c o n s i s t p r im a r i ly o f th e c y t o c h r o m e P 4 5 0( C Y P ) s u p e r f a m i l y o f m i c r o s o m a l e n z y m e s , w h i c h a r ef oun d ab un dan t l y in the l i ve r , gas t ro i n t es t i na l t rac t , l ungand k i dney , cons i s t i ng o f f am i l i es and sub f am i l i es o fe n z y m e s t h a t a r e c l a s s i f i e d b a s e d o n t h e i r a m i n o a c i ds e q u e n c e i d e n t i t i e s o r s i m i l a r i t i e s ( G o n z a l e z a n d N e b e r t ,1 9 9 0 ; G u e n g e r i c h , 2 0 0 3 ; M e y e r , 1 9 9 6 ; N e b e r t e t a L 1991 ;N e l s o n e t a L 1 9 9 6 ) . M o r e t h a n t h i r t y - s i x g e n e f a m i l i e sh a v e b e e n d e s c r i b e d t o d a t e . T w e l v e f a m i l i e s e x i s t i n a l lm a m m a l s , w h i c h c o m p r i s e t w e n t y - t w o s u b f a m i l i e s . I nh u m a n , f i v e C Y P g e n e f a m i l i e s , s u c h a s C Y P 1 , C Y P 2 ,C Y P 3 , C Y P 4 a n d C Y P 7 a r e b e l i e v e d t o p l a y c r u c i a l r o l e si n h e p a t i c a s w e l l a s e x t r a - h e p a t i c m e t a b o l i s m a n de l i m i n a t i o n o f x e n o b i o t i c s a n d d r u g s ( G o n z a l e z a n dN e b e r t , 1 9 9 0 ; L e w i s , 2 0 0 3 ; N e b e r t e t a L 1 9 9 1 ; N e l s o ne t a L 1 9 9 6 ; P a s c u s s i e t a L 2 0 0 3 b ; S i m p s o n , 1 9 9 7 ;W a x m a n , 1 9 9 9 ) .

    T h e p h a s e I I m e t a b o l i z i n g o r c o n j u g a t i n g e n z y m e s ,c o n s i s t i n g o f m a n y s u p e r f a m i l y o f e n z y m e s i n c l u d i n gs u l f o t r a n s f e r a s e s ( S U L T ) ( B a n o g l u , 2 0 0 0 ; W e i n s h i l b o u m

    e t a l . 1 9 9 7 ) , a n d U D P - g l u c u r o n o s y l t r a n s f e r a s e s ( U G T )( I nnocen t i e t a L 2 0 0 2 ; K i n g e t a L 2 0 0 0 ; M a c k e n z i e e t a L1 9 9 7 ; T u k e y a n d S t r a s s b u r g , 2 0 0 0 ) , D T - d i a p h o r a s e o rN A D ( P ) H : q u i n o n e o x i d o r e d u c t a s e ( N Q O ) o r N A D ( P ) H :m e n a d i o n e r e d u c t a s e ( N M O ) ( J a i s w a l , 1 9 9 4 ; K o n g e t a L2 0 0 1 a ) , e p o x i d e h y d r o l a s e s ( E P H ) ( G u e n t h n e r e t a l .1989 ; H i nson an d Fo rke r t, 1995 ) , g l u t a t h i one S - t rans f e rase s( G S T ) ( M o s c o w a n d D i x o n , 1 9 9 3 ; S c h i l te r e t a L 1 9 9 3 ;T e w a n d R o n a i , 1 9 9 9 ) a n d N - a c e t y l t r a n s f e r a s e s ( N A T )(Va t s i s e t a L 1 9 9 5 ) . E a c h s u p e r f a m i l y o f p h a s e I I D M E sc o n s i s t s o f f a m i li e s a n d s u b f a m i l ie s o f g e n e s e n c o d i n g t h eva r i ous i so f o rm s w i t h d i f f e ren t subs t ra t e spec i f i c i t y , t i ssuea n d d e v e l o p m e n t a l e x p r e s s i o n , a s w e l l a s i n d u c i b i l i t y a n di n h i b i t o r y b y x e n o b i o t i c s ( H i n s o n a n d F o r k e r t , 1 9 9 5 ;Sch i l t e r e t a L 1993) . I n gene ra l , con j uga t i on w i t h phase I ID M E s g e n e r a l l y in c r e a s e s h y d ro p h ili cit y, a n d t h e r e b ye n h a n c e e x c r e t i o n i n t h e b i l e a n d / o r t h e u r i n e a n d c o n s e -que n t l y a de t ox i f ica t i on e f f ect . A l t hou gh und er ce r t a i ns i t u a t i o n s , c o n j u g a t i o n w i t h p h a s e I I e n z y m e s c o u l d r e s u l tin a c t iv a t e d m e t a b o l i t e s a n d i n c r e a s e t o x i c it y ( C h e n e t a L2 0 0 0 ; H i n s o n a n d F o r k e r t , 1 9 9 5 ; K o n g e t a L 2 0 0 0 ;R u s h m o r e a n d K o n g , 2 0 0 2 ; S c h i l t e r e t a L 1993) . Fo re x a m p l e , r e a c t i v e e l e c t r o p h i l e s a r e t y p i c a l l y c o n j u g a t e dw i t h g l u t a th i o n e ( G S H ) c a t a l y z e d b y v a r io u s G S T s , a n dhave been i m p l i ca t ed w i t h t he po t en t i a l o f f o rm i ng reac t i vei n t e r m e d i a t e s i n p a r t i c u l a r w h e n G S H l e v e l s i n t h e c e l l sa r e a t t e n u a t e d , c o n s e q u e n t l y r e s u l t i n g i n t o x i c o l o g i c a le f f e c t s ( B o l t o n a n d C h a n g , 2 0 0 1 ; B o l t o n e t a L 2 0 0 0 ) . O nt h e o t h e r h a n d , t h e S U L T ( B a n o g l u , 2 0 0 0 ) a n d U G T( S u g a t a n i e t a L 2 0 0 1 ; T u k e y a n d S t r a ss b u r g , 2 0 0 0 ) w h i c hc a t a l y z e s u l fa t io n a n d g l u c u r o n i d a t i o n , m a y p l a y i m p o r t a n tr o l e s i n t h e c o n j u g a t i o n a n d u l t i m a t e l y e x c r e t i o n a n de l i m i n a t i o n o f m a n y d r u g s a n d x e n o b i o t i c s c o n t a i n i n gh y d r o x y l ( O H ) fu n c t i o n a l g r o u p e i t h e r p r e s e n t in t h e p a r e n ts t r u c t u r e a n d / o r a f t e r b i o t r a n s f o r m a t i o n b y t h e p h a s e Ie n z y m e s s u c h a s t h e C Y P s ( B a n o g l u , 2 0 0 0 ; K i n g e t a L2 0 0 0 ; S c h i l te r e t a L 1 9 9 3 ; S i m p s o n , 1 9 9 7 ) .

    Phase I I I t ranspo r t e rs , i nc l ud i ng P -g l ycop ro t e i n (P -gp )

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    Regulation of Drug Metabolism and Drug Transport 251

    Brinkmann and Eichelbaum, 2001), mul t idrug resistance-associated protein MRP ) Kerb e t a L 2001), and organicanion t ransporting polypept ide 2 OAT P2) Ti rona andKim, 2002) are expressed in many t issues such as thel iver, intest ine, k idn ey, and brain , wh ere the y p rovide aformidable barr ier against drug penetrat ion, and playcrucial ro les in drug ab sorption, distribution, and excre tionBrinkmann and E ichelbaum , 2001; Kim , 2003; Mizuno e t

    aL 2003; Staudinger e t a L 2003). P -gp wa s f irs t reportedto be associated wi th mul t idrug resistance MD R) incancer chemotherapy. P-gp and MRP ut i l ize the energyfrom the hydrolys is of ATP to substrate t ransport acrossthe ce l l mem brane, and are cal led AT P binding casset teABC) t ransporters Mizuno e t a l . 200 3). ABC transporters

    belong to one of the largest superfami l ies of proteins, andei ther import or export a broad range of substrates thatinc lude amino acids, ions, sugars, l ip ids, xenobiotics, andman y therapeut ic drugs Dean e t a L 2001; Kerb e t a L2001). There are only exporters in the eukaryotes. Inhum an, 46 ABC transporters have been ident if ied Deane t a L 2001 ; Mizuno e t a L 2003). A l l ABC transporters a recomp osed of two nucleot ide binding dom ains NBD s) andtwo transmembrane domains TMDs) . The NBD is a lsocalled an ABC, is the hal lmark feature of this tranporterfami ly . The role of TMD is to recognize and med iate thepassage of substrates across the cel l mem branes Deane t a L 2001; Kerb e t a L 2001). A long wi th P-gp or MD R1;ABCB1, the MDR subfamily inc ludes MD R3 ABCB4) ,BSEP or SP-gp; ABCB 11) Br inkmann and Eichelbaum,2001) . The MRP subfami ly cons is ts o f MRP1 ABCC1) ,MRP2 ABCC2) , MRP3 ABCC3) , MR P4 ABCC4) ,MR P5 ABCC5) , MRP6 ABCC6) , MR P7 ABCC10) ,MRP8 ABCC11) , and MRP9 ABCC12) Br inkmann andEichelbaum, 2001; Dean e t a L 2001; Kerb e t a L 2001;Mizuno e t a L 2003) . M RP 1 and MR P3 are typica ll ylocated on the basolateral membrane of polar ized cel ls ,whereas MRP2 is general ly local ized to the apicalme mb rane cana licular in l iver), which im plies that in liver,MRP2-mediated t ransport leads to increased excret ioninto bi le, but MRP1 - and MR P3-me diated t ransport intoblood leads to increased excret ion into the u r ine. Organicanion transport ing po lypept ide 2 OA TP 2; SLC21 A5) is amember of the organic anion t ransport ing polypept idefami ly that mediates sodium- and ATP-independentt ransport of a var iety of s t ructural ly unrelated endogen ousand exogenous compounds, inc lud ing con jugated andunconjugated bi l i rubin, conjugated steroids, neutral com-pounds, some type I I organic cat ions, thyroid hormonesT3 and T4, and bi le salts Reichel e t a L 1999; Shi tara e taL 2002). OATP2 is local ized in the hepatic sinusoidalmem brane, wi th select ive expression in the m idzonal toper ivenous hepatocytes. P- gp , MR P and O ATP2 are al lexpressed on the brush-border m emb rane o f the intest inal

    enterocytes, and excrete thei r substrates as wel l asxenobiot ics/drugs into the lum en , resu l t ing in a potent ia ll imi tat ion of net absorpt ion of drugs Dean e t a L 2001;Kerb e t a L 2001; K im , 2003; M izuno e t a L 2003).

    Therefore, the regulat ion of gene expression of var iousphase I , phase I I DMEs and phase I I I t ransporters haspotent ia l impact on the metabol ism, el iminat ion, pharma-cokinetics/dynamics, toxicokinetics/dynamics, drug-druginteract ions of many therapeut ic agents, as wel l as thei rabi l ity in the protect ion of the h um an body against exposureof environmental xenobiot ics Guen gerich, 200 3; Rushmo reand K ong, 2002; Wang and LeCluyse, 2003).R E C E P T O R S I N V O L V E D I N T H E R E G U L A -T I O N O F P H A S E I P H A S E II M E T A B O L I Z I N GE N Z Y M E S A N D P H A S E I II T R A N S P O R T E R S

    The human body has evolved versat i le inducible metab-ol iz ing enzym es and ef f lux t ransporters to fac il itate themetabol ism and e l iminat ion of potent ia lly ha rm ful drugs,and/or xenobiot ics that are introduced f rom the envi roment.The enzymat ic symtem inc ludes phase I enzymes, suchas CY P superfami ly Gueng erich, 2003; Lewis, 2003), aswel l as phase I I enzymes, such as GST and UGTMackenzie e t a L 1997; Tew and Ro nai , 1999). The ef f lux

    transporter system includes phase I I I ABC proteins, suchas P -gp, MRP2, and OA TP2 which remove the parentdrugs, metabol ites, and xenobiot ics f rom cel ls Dean e ta L 2001; Lew is, 2003; Me yer , 1996 ; Mizuno e t a L 2003;Wang and LeCluyse, 2003). In order to understand theregulat ion of gene expression o f phase I , phase I I metab-ol iz ing enzyme s and p hase I II ef f lux t ransporters, onewould need to address the s ignal ing mechanism involv ingthe ary l hydrocarbon receptor AhR) Hahn, 2002; Rowlandsand Gustafsson, 1997), the orphan nuclear receptorsMoore e t a L 2000; Wang and LeCluyse, 2003) , and o ther

    relevant t ranscr ipt ion factors and /or s igna l t ransduct ioncascades Kumar and Thompson, 1 99 9; Wang andLeCluyse, 2003) at the molecular level .

    The AhR and orphan nuclear receptors comprise agene superfami ly encoding the t ranscr ipt ion factors thatsense endo genous, such as smal l l ipophi l ic hormon es,and exogenous, such as drugs, xenobiot ics and t ransferinto cellu lar responses by regulat ing the expression ofthei r target genes Levine and Perdew, 2001; Wan g andLeCluyse, 2003). Regulat ion of gene expression at thetranscr ipt ional level by AhR and orpha n n uclear receptorsplays a crucia l role in the m etabol ism and c learance ofdrugs and xenobiot ics that are introduced into the bodyfor the purpose of protect ion the body f rom the envi ron-me ntal insults Li e t a L 1998; Rushmore and Kon g, 2002;Wang and LeCluyse, 2003).

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    Aryl hyd rocarbon receptorAhR is a m em ber o f the bas ic -he l ix - loop-he lix (bHLH ) -

    Per-Arnt -S im (PAS) gene super family of t ranscr ipt ionfac tor , and has been s tud ied fo r more than 30 years(Hahn, 2002; Ro w land s and Gusta fsson, 1997) . AhR isknown to recognize a range of chemical s t ructures,inc lud ing non-aromat ic a nd non-ha logen ated com pou nds(Elfer ink, 2003; H ahn, 2002; R ow land s and Gustafsson,1997) . The AhR is highly polymorphic, especial ly whencompared w i th orphan nuc lear receptors , such as PXRand C A R ( H onk ak os k i e t a L 2003; Wil lson and K l iewer ,2002) . The bHLH mot i f exsists in many t ranscr ipt ionfactors su ch as M yc and M ax tha t f unct ions as seque nce-speci f ic t ranscr iptional regulat ion. T his m ot i f p lays a role inboth DNA binding (basic region) and protein dimer izat ion(HLH) (Hahn, 2002; Huang e t a l . 2004; K ikuchi e t a L2003) .

    The Ah receptor nuclear t ranslocator (Arnt ) , is notrequired for nuc lear translocat ion pe r se, but is required togenera te an AhR-Arn t complex w i th a greater a f f in i t y f o rnuclear ext racts upon cel l d isrupt ion(Heid e t a L 2000;Kikuchi e t a L 2003) . The un l iganded Ah R is f ound a lmos texclusively in the cytoplasm of the cel l , and t reatment withl igand causes a t ime-dependent movement o f t he AhRinto the nucleus. The Arnt protein, on the other hand, isfound to be exclusively nuclear with or wi thout l igand.Thus, l igand m ay serve to in i tiate translocat ion of the AhRto the nuc leus whe re d imer iza t ion o f these tw o par tnerscan occur . In Arnt -def ic ient cel ls , the AhR can st i l lt r ans locate to t he nuc leus in t he ce l l , a p rocess there foreindependent of Arnt (Hahn, 2002; K ikuchi e t a L 2003;Levine and Perdew, 2001) .

    AhR can b ind to DNA a s a heteromer ic complex . I t wa sdem ons t r a t ed t ha t bo t h t he bH LH and P A S dom a ins a r erequired for DNA-binding, and thus presumably for d imer i-zat ion with Arnt . The basic region of Arnt is not requiredfor dimer izat ion, but both hel ix regions, and ei ther the N-terminal or C- terminal hal f of the PAS domain, areessent ia l . The AhR and Arnt proteins have a s inglet ransact ive domain (TAD) in their C- terminals, compr is ingam ino ac ids 521 - 640 i n t he A hR and am ino ac ids 582 -774 in A rn t (Huang e t a L 2004; K ikuchi e t a L 2003) .

    The un l iganded AhR ex is t s in t he cy toso l complexedwith a dimer of Hsp90, which maintains the Ah R in a ligand-binding conformat ion and prevents nuc lear t ranslocat ionand/or dimer izat ion with Arnt (Heid e t a L 2000) . Thehydrophob ic AhR l igands enter t he ce l l by d i f f us ion andare bound by the Hsp90-assoc ia ted AhR. L igand b ind ingcau ses a conformat ional cha ng e resul ting in a receptorspec ies w i th an inc reased a f f in i t y f o r DNA and a muchslow er rate of ligand dissociat ion. This event is ass ocia tedw i th nuc lear trans locat ion and an exchan ge o f Hs p90 fo rArnt (Hahn, 2002; Heid e t a L 2000) . I t has been shown

    that in a pur i f ied sys tem the AhR-Hsp90 complex is no tdissociated by the addit ion of l igand. Ult im ately, therecogn i t ion o f DRE enhancer sequences by the AhR-Arn tcom plex resul ts in the t ransact ivation of target gene s(Hahn, 2002; Heid e t a L 2000; Lev ine and Perdew,2001) .Orphan nuclear receptors

    Orpha n receptor is a subc lass o f nuc lear receptors tha tbinds to steroid-based l igands, suc h as cor t isol, est radiol ,progesterone, aldosterone, tes tostero ne and v itamin D.A l l t he known orphan receptors share two modu la torydomains , one is t he h igh ly conse ved D NA-b ind ing dom ain(DBD), the other is the l igand binding domain (LBD)(Kumar and Thompson, 1999 ; Wang and LeC luyse, 2003) .Th e D BD is character ized by two C 4- type z inc fingers,l inks the receptor to the speci f ic promoter regions of i tsta rget genes , t e rmed hormone response e lement (HRE)or xenob io t ic respon se e lement (XRE) . The DB D canrecogn ize the respo nse e lements tha t conta ins one or twoconse nsus core ha l f- s it es re la ted to the he xam er ACA AC A(s tero id receptors ) o r AGG TC A (es trogen receptors andso on) (Kumar and Thompson, 1999; Wang and LeC luyse,2003) . D i f ferent orphan nuclear receptors bind to theirrespon se e lement e i ther as hom odimers, as heterod imersw i th the R XR, or as mono me rs . The L BD is loca ted in t hecarboxy- terminal por t ion of the r e ce p t o r and not on lys e r v e s as a docking s i te for l igands, but a lso containsdimer izat ion m ot i fs : t ranscript ional act ivation doma ins,suc h as the act ivat ion funct ion 2 (AF-2) h el ix , and theseq uen ce me diat ing the nu clear local izat ion of the receptor .Ligand binding induces s igni f icant conformat ional changesin the fo ld ing the LBD, and leads to the recrui tment ofcoact ivator proteins and co-integrators, and trancact ivat iono f t he ta r ge t genes ( K um ar and Thom ps on , 1999 ; W angand LeCluyse, 2003) .Pregnane X receptor PXR )

    PXR wa s f ir s t c loned f rom mo use l iver , t hen i ts homolo-gou s counterpar ts in rat , rabbi t and hu ma n we re ident if ied(Kliewer e t a / . 1998) . Or thologous receptors f rom di f ferentspec ies were g iven unre la ted names a t f i r s t due to thelack o f a common nomenc la ture sys tem. The humanreceptor of P has also bee n referred to as steroid andxenobiot ic receptor (SXR) or pregnane act ivated receptor(PAR) ( e t a / . 2000a, 2000b, 2001) . A nomenclaturesys tem has been dev ised fo r t he nuc lear receptorsupef family recent ly . Acc ording to th is system, P XR h asbeen c lass fied a s N R l l2 . The gen e fami ly is des ignatedby an Arabic numeral , the supef family is indicated by acapita l let ter , and indiv idual gene members are ident i f iedby the second A rab ic numera l (Dussau l t and Forman,2002; K l iewer e t a / . 1998 ; K um ar and Thom ps on , 1999 ;

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    W a n g a n d L e C l u y se , 2 0 0 3 ).A l l P X R s ( h u m a n , m o u s e , r a t a n d s o o n ) a r e p r e d o m i -

    n a n t l y e x p r e s s e d i n t h e l i v e r a n d i n t e s t i n e , a n d t o a l o w e rl eve l i n t he k i dney and l ung . The t i ssue -spec i f i c d i s t r i bu -t io n p a t t er n o f P X R s e x p r e s s i o n r e s e m b l e s t h a t of C Y P 3 A( B e i g n e u x e t a L 2 0 0 2 ; C o u m o u l e t a l . 2 0 0 2 ) . T h e r e i sm o r e t h a n 9 5 % s e q u e n c e h o m o l o g y i n t h e D B D r e g i o n s ,b u t o n l y 7 5 - 8 0 % a m i n o a c i d h o m o l o g y i n t h e L B D o f P X Rb e t w e e n t h e d i f f e r e n t s p e c i e s ( D u s s a u l t a n d F o r m a n ,2 0 0 2 ; W a n g a n d L e C l u y se , 2 0 0 3 ) .Const i tut ive androstane receptor CAR)

    T h e o r p h a n n u c l e a r r e c e p t o r C A R ( N R 1 1 3) w a s id e n ti fie di n 1 9 9 4 ( B a e s e t a L 1994) . I t wa s o r i g i na l l y de f i ned ascons t i t u t i ve l y ac t i va t ed recep t o r , be ca use i t f rom s ah e t e r o d i m e r w i t h R X R w h i c h b i n d s t o r e t i n o i c a c i dr e s p o n s e e l e m e n t s a n d t r a n s a c t i v a t e s t a r g e t g e n e s i n t h ea b s e n c e o f l i g a n d s ( H o n k a k o s k i e t a L 1 9 9 8 b ) . C A R i sm a i n l y e x p r e s s e d i n li v e r , a n d l e s s a b u n d a n c e i n t h ei n te s t in e ( W a n g a n d L e C l u y s e , 2 0 0 3 ; W e i e t a L 2 0 0 2 ) . T w om e t a b o l i t e s o f a n d r o s t a n e , a n d r o s t a n o l a n d a n d r o s t e n o lw e r e f o u n d t o b e th e e n d o g e n o u s C A R l ig a n d s . B o t h o ft h e m a c t a s a n t a g o n i s t s b y d i s s o c i a t i n g C A R f r o m i t sc o a c t i v a t o r a n d i n h i b i t i n g t h e t r a n s c a c t i v a t i o n o f C A Ri n s t e a d o f a c t iv a t in g C A R ( F r a s e r e t a L 2 0 0 3 ; G o o d w i n e ta L 2 0 0 2 ; H o n k a k o s k i e t a L 2 0 0 3 ; P a s c u s s i e t a L 2 0 0 3 b ) .

    C A R i s l o c a t e d i n t h e c y t o p l a s m o f h e p a t o c y t e s i n t h ea b s e n c e o f l ig a n d s , a n d i t is tr a n s l o c a t e d i n to t h e n u c e l u sa f t e r t r e a t m e n t w i t h p h e n o b a r b i t a l - l i k e C Y P 2 B i n d u c e r s .R e c e n t s t u d i e s i n d i c a t e d t h a t a c t i v a t i o n o f C A R i s am u l t i s t ep p rocess , t he i n i t i a l s t ep i s nuc l ea r t rans l oca t i on ,w h i c h c a n b e i n d e p e n d e n t o f l i g a n d b i n d i n g , t h e f i n a l s t e pi s C a M K - d e p e n d e n t a c t i v a t i o n o f t h i s r e c e p t o r ( B a e e t a L2 0 0 4 ; M a g l i c h e t a L 2 0 0 2 ; P a q u e t e t a l . 2 0 0 0 ; W a n g a n dL e C l u y s e , 2 0 0 3 ) .Peroxisom e prol iferator act ivated receptors PPA R)

    C u r r e n t ly , t h r e e m e m b e r s o f th i s n u c l e a r r e c e p t o r fa m i l yh a v e b e e n i d e n ti fi e d a s : P P A R c ~ , P P A R ~ a n d P P A R T( G e r v o i s e t a L 2 0 0 0 ; G i l d e e t a L 2 0 0 3 ) . P P A R ~ i s m a i n l yexp res sed i n t he l i ve r , hea r t , k idney , i n t es t i ne and b row na d i p o s e t i s s u e . P P A R [ 3 i s w i d e l y e x p r e s s e d i n m o s t a d u l tt is s u e s , a n d t h e b r a i n , k i d n e y a n d i n t e s ti n e a r e t h e h i g h e s te x p r e s s e d t is s u e s . P P A R T i s m a i n l y exs i t ed i n t he sp l ee n ,i n te s t in e a n d f a t c e ll s , a n d i t i s c o m p o s e d o f tw o s u b m e m -b e rs , n a m e d P P A R T 1 a n d P P A R ~ . P P A R s d e m o n s t r a t e dd i s t i nc t bu t ove r l app i ng phys i o l og i ca l f unc t i ons (G i l de e taL 2 0 0 3 ; I s s e m a n n a n d G r e e n , 1 9 9 0 ; R u s h m o r e a n dK o n g , 2 0 0 2 ; T u g w o o d e t a L 1 9 9 2 ; W a n g a n d L e C l u y s e ,2 0 0 3 ) .

    A t th e v e r y b e g i n n in g , P P A R ~ w a s f o u n d t o b eac t i va t ed by com pou nd s t ha t cau se p ro l if e ra t ion o f li ve rpe rox i som es , hype rp l as i a and hepa t i c ca rc i nogenes i s i n

    roden t s , however , subsequen t l y , s t ud i es sugges t ed t ha tP P A R s m a y p l a y a c r u c i a l r o l e i n t h e r e g u l a t i o n o fl ip o p r o t e in a n d f a t ty a c i d m e t a b o l i s m ( G e r v o i s e t a l . 2 0 0 0 ;G i l de e t a L 2 0 0 3 ; S c h o o n j a n s e t a L 1 9 9 6 ; Y u e t a L2 0 0 3 ) .Liver X receptor LXR )

    L i v e r X r e c e p t o r s a r e t r a n s c r i p t i o n f a c t o r s c o m m o n l yk n o w n a s c h o l e s t e r o l s e n s o r s . T h e y a r e im p o r t a n tr e g u l a t o r s o f t r a n s p o r t a n d m e t a b o l i s m o f s t e r o ls a n d f a t t ya c i d s . T h e r e a r e t w o m e m b e r s o f th i s fa m ily , L X R o ~ a n dL X R . L X R o ~ a n d L X R I3 s h a r e a h i g h d e g r e e o f a m i n oa c i d s i m i l a r i t y ( - 8 0 % ) a n d a r e c o n s i d e r e d p a r a l o g u e s .O x y s t e r o l s i n c l u d i n g 2 4 ( S ) , 2 5 - e p o x y c h o l e s t e r o l , 2 2 ( R ) -h y d r o x y c h o l e s t e r o l , a n d 2 4 ( S ) - h y d r o x y c h o l e s t e r o l , a r en a t u r a l l i g a n d s o f L X R s . S o m e L X R - m e d i a t e d g e n e si n c l u d e t h o s e a s s o c i a t e d w i t h c h o l e s t e r o l a n d b i l e a c i dm e t a b o l i s m a s w e l l a s t h o s e w i t h f a t t y a c i d s y n t h e s i s a n dregu la t ion . LXRc~ i s p redo m i na n t l y e xp res sed i n li ve r , low erl eve l i n k i dney , sp l een and i n t es t i ne . On t he con t ra ry ,L X R I 3 i s l o c a t e d i n a l m o s t e v e r y t i s s u e t e s t e d . L X R s a r em a i n l y l o c a t e d i n t h e n u c l e u s , a n d m u s t h e t e r o d i m e r i z ew i t h R X R f o r a c t i v a t i o n ( K h a n a n d V a n d e n H e u v e l , 2 0 0 3 ;L e h m a n n e t a L 1 9 9 7 ; M e n k e e t a L 2 0 0 2 ; P e e t e t a L1 9 9 8 ; V e n k a t e s w a r a n e t a L 2 0 0 0 ) .Farnesoid X receptor FXR )

    F X R w a s s h o w n t o b e a c t i v a t e d b y s u p r a p h y s i o l o g i c a lc o n c e n t r a t i o n o f f a r n e s o l i n r a t s w h e n i t w a s o r i g i n a l l yi den t if ied . S i m i l a r to o t he r o rp han nu c l ea r recep t o rs , FXRi s m a i n l y exp re ssed i n li ver and i n tes t ine , i t he t e rod i m er i zesw i th R X R a n d b i n d s t o F X R r e s p o n s e e l e m e n t ( F X R E ) i nt h e p r o m o t e r r e g i o n o f t a r g e t g e n e s . R e c e n t r e p o r t ss h o w e d t h a t F X R w a s i d e n t i f i e d a s a b i l e a c i d r e c e p t o r ,a n d w a s a c t i v a t e d b y p h y s i o l o g i c a l l i g a n d s r e s u l t e d i n t h ei nh i b i t i on o f hepa t i c b i l e ac i d b i osys t hes i s and i nc reasedt ranpor t o f b i l e ac i d f rom t he i n t es t i ne t o t he l i ve r (de lC a s t i l l o - O l i v a r e s a n d G i l , 2 0 0 0 ; M a k i s h i m a e t a / . 1999 ;W a n g e t a / . 1 9 9 9 ; W a n g a n d L e C l u y s e , 2 0 0 3 ) .Ret inoid X Rece ptor RXR )T h e r e a r e t h r e e m e m b e r s o f t h i s f am i ly , R X R c ~ , R X Ra n d R X R T . R X R ~ i s m a i n ly e x p r e s s e d i n t h e l i ve r , m u s c l e ,k i d n e y a n d l u n g , a n d t o a l o w e r l ev e l i n t h e s p l e e n , h e a r ta n d a d r e n a l g l a n d , w h e r e a s R X R ~ i s f o u n d i n a l l t i s s u e sexce p t t he l i ve r and i n t es t ine , an d RX RT is f ound i n j us t af e w t i s s u e s , s u c h a s s k e l e t a l m u s c l e , h e a r t a n d c e n t r a ln e r v o u s s y s t e m ( M a n g e l s d o r f e t a L 1 9 9 2 ; M a n g e l s d o r fa n d E v a n s , 1 9 9 5 ; W a n g a n d L e C l u y s e , 2 0 0 3 ; Z e t t e r s t r o me t a L 1 9 9 6 ) . T h e m e t a b o l i t e 9 - c i s - r e t i n o i c ac i d o f v i t am i nA w a s i n d e n t if a ie d a s a h i g h - a ff in i ty l ig a n d o f R X R s ( B a e se t a L 1 9 9 4 ) . R X R c a n f o r m h e t e r o d i m e r s w i t h o t h e ro r p h a n n u c l e a r r e c e p t o r s a s a c o m m o n p a r t n e r , a n d t h e

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    formation of a heterodimer with RXR is a critical step forfacilitating the specific binding and activation of all knownorphan nuclear receptors (Mangelsdorf and Evans, 1995;Wang and LeCluyse, 2003; Zetterstrom et aL 1996).There are two kinds of RXR heterodimers, nonpermissiveand permissive. RXR is completely silent and can only beactivated by the ligands of the partner orphan nuclearreceptor in the nonpermissive heterodimers. RXR permis-sive heterodimers can be freely activated by ligands ofboth RXR and partner nuclear receptors, such as PXR/RXR, CAR/RXR, PPAR/RXR, LXR/RXR and FXR/RXR(Mangelsdorf et aL 1992; Mangelsdorf and Evans, 1995;Wang and LeCluyse, 2003; Zetterstrom et aL 1996).Because RXRs have a broad binding ability with mostother orphan nuclear receptors, and affect the subsequentregulation of their target genens, so the RXRs are involvedin the regulation of most drug metabolizing enzymes andtransport er directly or indirectly. But the untimate role ofRXR heterodimer complexs appear to be multifacedtedand yet uncertain (Rushmore and Kong, 2002; Wang andLeCluyse, 2003).REGUL TION OF PH SE I DMEs

    It appears that in general xenobiotics exposure cantrigger certain stress respo nse to the body, and conse-quently resulting in an increase in gene expression of

    xenobiotic metabolizing enzymes or DMEs, so that thebody will be able to remove the stress insults as fast aspossible from the body (Fig. 1) (Kong et aL 2001a, 2001 b;Rushmore and Kong, 2002).

    The steroid family of the orphan receptors, PXR andCAR can heterodimerize with the RXR, and have beenshown to transcriptionally induced CYP3A (Anakk et aL2004; Coumoul et aL 2002; Lehmann et aL 1998) andCYP2B (Bae et aL 2004; Beigneux et aL 2002) gene ex-pression by xenobiotics such as dexamethasone/rifampintype of compounds and phenobarbital-like compounds(Bae et aL 2004; Coumoul et aL 2002; Honkakoski et aL2003; Willson and Kliewer, 2002). PPAR is one of the veryfirst members to be identified in this orphan nuclearreceptor superfamily, and it can also heterodimerize withRXR, that was initially found to be activated by the lipidlowering agent fibrate-type of compounds and otherchemicals. Previously it was found to increase the levelsof peroxisomes in rodents, and later it was shown toincrease the gene expression of CYP4A enzymes (Gervoiset aL 2000; Rushmore and Kong, 2002; Simpson, 1997;Zhou et aL 2002). LXR (Menke et aL 2002) and FXR(Wolters et aL 2002) receptors are involved in theregulation of CYP7A in mediating the elimination ofcholesterol and bosynthesis and excretion of hepatic bileacids. These diverse array of naturally occurring orsynthetic compounds are primarily metabolized by CYP

    Fig. 1. A schematic representationof drugs/chemicals/xenobiotics-induced tress response eading to the activation of specific receptor-mediatedgene expression of phase I drug metabolizing enzymes, the cytochrome p450s, phase II drug metabolizing enzymes, other stress enzymes, andphase III transporters,which result n the enhancementof detoxification of the xenobiotics and a potential homeostaticcell survival response.

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    Regulat ion of Drug Metabol ism and Drug Transpor t 255

    enz y m es i n t he body , and t hey r ange f rom endoge nouscom pou nds su ch as the s tero ids and cho les tero l t o d rugsas w e l l as po tent ia l carc inogen s found in t he env ironment( Lehm ann e t a L 1997 ; V enk a t es w ar an e t a L 2000; Wange t a L 1999) . The oxidized products are general ly morepolar , can be excreted direct ly and/or fur ther conjugatedby the phase I I DMEs and u l t imate ly e l im inated f rom thebody, and consequent ly detoxi f icat ion (Hinson and Forker t ,1995; Meyer , 1996) . However , in some si tuat ions, notablyprocarc inogens, they may be metabol ized to more react ivespecies, and potent ia l ly promot ing toxic i ty and carc ino-genic i ty (Banoglu, 2000; Guenger ich, 2003; Schi l ter e t a L1993) .Regula tion o f CY P by Ah R

    W hen Ah R is bound by po lycyc l ic a romat ic hydrocarbo n(PAH), such as dioxin and 3-methylchoranthrene (3-MC),AhR t rans locates f rom the cy top lasm to the nuc leus ,heterodimer izes with Arnt , and act ivates t ranscr ipt ionthrough the XR E located in the prom oters o f CYP1 fami lygenes (L i e t a L 1998; Nakaj ima e t a L 2003) . Therequ i rement o f AhR in CYP1 express ion induc t ion wasdemonst ra ted in AhR-nu l l mutant m ice (Gonza lez andFernandez-Salguero, 1998; Shimizu e t a L 2000) . The ex-press ion o f CYP1 gen es induced b y the AhR, in respo nseto PAHs or ha logenated aromat ic hydrocarbon l igandssuch as benzo[a]pyrene and 2,3,7,8- tet rachlorodibenzo-p-dioxin (TCDD ) or dioxin is wel l establ ished (Gonzalez andFernandez-Salguero, 1998; Levine and Perdew, 2001; Lie t a L 1998; Nakaj ima e t a L 2003; Shimizu e t a L 2000) .Regula t ion o f CY P 3A by P X R

    Systemat ic delet ion analys is has dem ons t rated thatPXR response elem ent (PXR RE) is located in the promoterreg ion o f CYP3A. The PXR response e lement is e i t her ad i rec t repeat o f t he ha l f - s i t e TGAACT spaced by th reebase pairs (DR3) or an ever ted or inver ted repeat of theTGAACT ha l f - s i t e spaced by s ix base pa i r s (ER6 andIR6) (Dussau l t and Forman, 2002; K l iewer e t a L 1998;W a n g e t a L 2003; Wang and LeC luyse, 2003; X ie e t a L2000b) . PXR can bind to and t ransact ivate these responseelements af ter act ivat ion by CYP3A inducers, and PXR isthe predominant regulator of the xenobiot ic- responsiveexpression o f CYP 3A gen es (Anakk e t a L 2004; Coumoule t a L 2002; K l iewer e t a L 1998; X ie e t a L 2001) .

    Th ere are impor tant species-speci f ic PX R activat ionpro f i les to suppor t t he regu la t ion o f CYP3A by PXR. Forexample , r i fampic in is a we l l - known and potent inducer o fCY P3A in rabb it and hum an l iver , bu t no t in ra t andmouse l i ver , and was found to be a potent ac t iva tor o fhuman and rabb i t PXR, bu t no t o f ra t o r mouse PXR( J ones e t a L 2000) . On the o ther hand, PCN is a po tent

    ra t and mo use C YP 3A inducer, bu t no t o f human o r rabb itCYP3A, and was found a lso to be a potent ra t PXRact ivator , having very l i t t le ef fect on hu ma n an d rabbitP X R ( J ones e t a L 2000; Staudinger e t a L 2001a, 2001b) .Treatment of PXR-nul l mice with PCN fai led to induceCYP3A express ion prov id ing def in i t i ve proof f o r PXRregu la t ion o f CY P3A express ion (S taud inger e t a l . 2001 b;X ie e t a L 2000a) . Replacem ent o f the mP XR with itshum an or tho logue resu lted in t he xenob io tic respon se inth is humanized mouse, a nd the resp onse to xenob io ticst imulat ion resembled that in human (Anakk e t a L 2004;Staudinger e t a L 2001b; X ie e t a L 2000a, 2001) .Regula t ion o f CY P 2B by CAR

    CYP2B is potent ly induced by phenobarbi ta l in mostmamm al ian spec ies . S tudy show ed tha t CAR can b ind toa 5 1-base-pa i r m in imum sequ ence located in the 5 -f lank ing reg ion o f t he CYP2B genes , and th is sequencewa s requ i red fo r phenobarb ita l induct ion , and w as na me das phenoba rbi ta l- response element module (PBR EM )(Honkakoski and Neg ish i, 1997) . PBREM is com pose d o ftwo n uc lear receptor b ind ing s i t es (NR 1 and N R2) as we l las a nucle ar factor 1 (NF1) binding s i te. Both NR1 andN R 2 a r e D R 4 m o t if s ( R am s den e t a L ~ 1999; Sueyoshi e ta L 1999 ; Wang and LeC luyse, 2003). The h igh ly conservedNR1 site is cr it ical for conferr ing phenobarbital responsive-ness, the funct ion of NF1 s i te is st i l l unclear (Honkakoskie t a L 1998b; Sueyoshi and Negishi , 2001) . Af ter t rans-fec tion w i th som e kno wn nu c lear receptors , such as RXR,CAR, o r LXR, us ing PBR EM reporter assay to s tudy the i rabi l i ty to bind and t ransact ivate the PBREM, only CARwas found to be ab le to s t imu la te PBREM repor ter geneexpression (Honkakoski e t a l . 1998b) . Subsequent ly , NR1-a f f in i t y choromatography was used to pur i f y t he pro te intha t bound to PBREM, and tha t bo th b ind ing assay andWestern b lo t assay demonst ra ted tha t CAR was theprotein that mediated the phenobarbi ta l induct ion response(Honkakoski e t a L 1998b ; K aw am ot o e t a L 1999; Paquete t a L 2000; Sueyoshi e t a L 1999; Sueyoshi and Negishi ,2001) .Regula t ion o f CY P 4A by P P AR

    PPAR is act ivated by a l igand- induced conformat ionalst ructure change, and binds to speci f ic upst ream region ofi ts target genes refer red to as peroxisome prol i feratorr es pons e e lem en ts ( P P R E s ) ( Lam be and Tu gw ood ,1996 ; Tugw ood e t a L 1996) . CY P4A cou ld be induced bya number of peroxisome prol i ferators, such as c lof ibrate,v ia the act ivat ion of PPARo~. CY P4 A plays a cent ral ro le inthe hydroxylat ion of fat ty acid der ivat ives and cholesterolmetabo l ism (Rushmore and Kong, 2002; S impson, 1997;Y u e t a L 2003 ; Zhou e t a L 2002) .

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    256 C. Xu e t aL

    R e g u l a t i o n o f C Y P 7 A b y L X R a n d F X RLXR can recognize a direct repeat of two s imi lar

    hexanucleot ide hal f -s i tes se para ted by 4 base pairs (DR4 )in the upst ream regions of their target genes, refer red toas an LX R E ( Lehm ann e t a L 1997; Wang and LeC luyse,2003) . The en dogenou s oxysterols, such as the metabol i tesof cholesterol , are select ive LXR l igands. The cholesterolis metabol ized to hydrophi l ic b i le acids by CYP7A(cholesterol 7~ -hydroxylase), and a cholesterol-r ich diet inrat can upregulate CYP7A (Jet inek e t a L 1990; Je l inekand Russe l l , 1990; Menke e t a L 2002; Peet e t a L 1998;Wang and LeC luyse, 2003) . CYP7A was recogn ized asthe f i r s t t a rget gene o f LXR (Lehmann e t a L 1997) . TheDR4-LXRE is loca ted in t he prox imal p romoter reg ion o fCYP 7A, and i t can b e spec if ica l ly boun d and ac t iva ted b yL X R ( M e n k e e t a L 2002; Peet e t a L 1998) . LXRo~knocko ut m ice are phenotyp ica l ly normal whe n fed w i thlow cholesterol d iet , but cholesterol accum ulat ion, chron ichepa tomega ly deve lopm ent an d l iver f unc tion impa i rmentoccurred as compared to their w i ld- type counterpar tswhe n the kn ock out m ice were fed w i th a h igh cho les tero ld ie t (2%) , beca use the LXRo~ knocko ut m ice cou ld no tregu la te the CYP7A gene express ion and the b i le ac idb iosynthes is (Peet e t a L 1998 ). The LX R E o f C Y P 7A i s amuch s t ronger res pons e e lem ent f o r transcrip tion ac t iva-t ion by LXRo~ han by LXRI3 . A l though LXR ~ express ion isnormal in LXRo~ knock out m ice , i ts p resen ce cou ld no tprevent the cholesterol accumulat ion and l iver funct iond isorder when these mice wer9 fed w i th h igh cho les tero ld ie t (Lehmann e t a L 1997 ; V enk a t es w ar an e t a L 2000;Wang and LeC luyse, 2003) .

    The binding and act ivat ion of FXR by bi le acidsaccum ulat ion wa s fol lowd by the t ranscr iptional act ivationof i leal b i le acid-binding protein ( IBABP), that resul ted inthe increase of b i le acid reabsorpt ion (Makishima e t a L1999; Wang and LeC luyse, 2003) . FXR can negat ive lyregu la te CYP7A express ion by b ind ing to i t s b i le ac idl igands , bu t un t i ll now there is no ev idence show ing tha tFXR can b ind d irec tly to t he C YP7 A promoter reg ion . FXRis the main regulator in facil i tat ing bile acid reabsorpt ion( IBABP act ivat ion) and i t is an inhibi tor of CYP7A(cholesterol hyd roxylase) (del Cast i l lo-Ol ivares and G i l ,2000; Denson e t a L 2001; Wan g and LeC luyse, 2003) .C r o s s t a lk a m o n g t h e o r p h a n n u c l e a r r e c e p to r s

    The ind iv idua l respo nse e lements o f t he d if fe rent pha seI CYP genes can be ac t iva ted by more than one s ing lenuc lear receptor , and i t is com m only referred to as cross-ta lk . R ecent s tudy found tha t PX R can b ind to thePBREM located in t he 5 ' - f lank ing reg ion o f t he CYP2B(Pascussi e t a L 2003b; Sueyosh i and Neg ish i , 2001;Wang and LeC luyse, 2003) . Dexamethasone is a l igandfor mo use P XR bu t no t an ac tiva tor f o r mou se CAR, s tud y

    revealed that i t could potent ly induce CYP2B10 expres-s ion in mouse hepatocy tes (Wang and LeC luyse, 2003;Wei e t a L 2002) . PXR act ivators such as r i fampin,phenob arbi ta l , phenytoin and c lot r imazo le (an PXR act ivatorbut CAR deac t iva tor ) can e f f ic ien t ly induce CYP2B6 ex-press ion in human hepatocy tes (Honkak osk i e t a L 1998a,1998b; X ie e t a L 2000b) . A l l PXR ac t iva tors can t rans-ac t iva te CY P2B 6 hPB REM repor ter gen e express ion a fte rco t rans fec t ion o f hPXR w i th the CYP2B6 hPBREM orNR1 repor ter vectors in huaman hepatocytes. A distalxenob io tic respons ive e nha ncer modu le (XREM ) wa sfound to be located in t he prom oter o f t he C Y P 2 B 6 g e n erecently , and both P XR a nd C AR can b ind to and ac t iva teth is nove l XREM (Wang e t a l . 2003) . Transfect ion of bothPBREM and XREM was found to max imal ly ac t iva teCYP2B6 repor ter gene (Wang e t a L 2003 ; W ang andLeCluyse, 2003) . A l l these resul ts st rongly suppor t thenot ion that PXR plays an impor tant ro le in the regulat ionof C Y P 2 B gene . B o t h C Y P 2C 8 and C Y P 2C 9 ex p r es s ioncan be induced by PXR ac t iva tors such as r i f ampin , SR-12813 and pac l i t axe l in human hepatocy tes , sugges t ingt ha t a P X R r es pons e e lem en t m ay be p r es en t i n t hepromoter reg ion o f t hese genes (Pascuss i e t a L 2003b;W a n g e t a L 2003) . To date , severa l DR4 and DR5elements have been found in the upst ream 5' - f lankingregion of CYP2C9 star t s i te. The role of PXR in thetranscript ional regulat ion of C Y P 2 C gene express ion isst il l unclear , and n eed s futher invest igat ion of the upst reamregion of the C Y P 2 C gene promoters (Ferguson e t a L2002; Pascuss i e t a L 2003b; Wang and LeC luyse, 2003) .CYP7A was a lso repor ted to be regu la ted by PXR(Staudinger e t a L 2001a, 2001b; Wang and LeC luyse,2003; Waxman, 1999) .

    A l though C AR a nd PXR we re or ig ina lly identi fied as theregu la tors o f CYP2B and CYP3A, respec t ive ly , t here area lot of cross- talk between the induct ion of these targetgene s by thes e two com pound s . Th is is due in par t t o thefac t t ha t bo th CAR and PXR can recongn ize o therresponse e lements such as DR3, DR4 or ER6, andresu lt ing in the induc t ion o f CYP 3A and C YP2 B b y e i thercommon or se lec t ive l igands . Both CAR and PXR canregu la te the CYP3 A and CY P2B gen e express ion by the i rspeci f ic l igands in CV-1 cel ls a s wel l as in hepatocytes(Wang e t a L 2003; Wang and LeC luyse, 2003; X ie e t a L2000b, 2001 ).

    UGT s play an impor tant ro le in phas e I I metabol ism, andthey are mainly expressed in the l iver . Phenobarbi ta l hasbeen used for the t reatment of Cr ig ler -Naj jar syndrome forqu i te some t ime, and i t was repor ted to induce UGT1A1(Innocent i e t a L 2002; Sugatani e t a L 2001) . UG T1A 1 isthe speci f ic isozyme responsible for b i l i rubin conjugat ionand detox if ica t ion . A 29 0-base -pa i r d is ta l enhan cercontaining three p utat ive nuclear receptor mo t ifs was fou nd

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    Regulation of Drug Metabolism and Drug Transport 257

    and identified to be necessary for UGT1A1 induction byphenobarbital, and it was considered to be correlated withthe regulation by CAR (Pascussi et aL 2003a; Ritter et aL1999; Sugatani et aL 2001; Wang and LeCluyse, 2003).

    Overall, the transcriptional regulation of phase I drugmetabolizing enzymes is a multifaceted and complicatedprocess. A single orphan nuclear receptor may mediatethe induction of multiple targe t genes, and conversely, asingle gene may be coregulated by multiple orphannuclear receptors and ligands.REGUL TION OF PH SE II DMEs

    The role of phase II conjugation in the metabolism ofdrugs and xenobiotics in the human body has beenstudied for a long time however, the mechanism of phaseII gene s regulation remains unclear until recently. Manystructurally unrelated chemicals including the PAHs,barbiturates and many naturally occurring cancerchemopreventive agents including phenolic antioxidants,isothiocyanates and flavonoids were all found to inducephase II genes (Chen et aL 2000; Hu et aL 2004; Keumet aL 2003; Owuor and Kong, 2002; Schilter et aL 1993;Shen et aL 2004). Further studies of the promoters of

    some of the phase II genes revealed the existence ofseveral cis-acting regulatory elements, such as the anti-oxidant response element (ARE)/electrophile responseeiement (EpRE), xenobiotic-responsive element (XRE)/aromatic hydrocarbon responsive element (AhRE), activatorprotein-1 (AP-1), and nuclear factor -kappa B (NF-~B)binding sites in their 5 -flanking regula tory region (Hu etaL 2004; Itoh et aL 1997; Keum et aL 2003; Kong et aL2001a; Rushmore and Kong, 2002; Shen et aL 2004).Most recent findings suggest and support the key role ofthe ARE/EpR E in the regulation of expression of somephase II genes such as NQO, GST, and UGT by phenolicantioxidants and other naturally occurring cancer chemo-preventive agents (Chen et aL 2000; Hu et aL 2004;Keum et aL 2003; Kong et aL 2001a, 2001b; Owuor andKong, 2002; Rushmore and Kong, 2002; Shen et aL2004). Recently, several ARE/EpRE-binding proteins havebeen proposed and identified, including the members ofbasic leucine zipper transcription factor (bZIP) family,Nrfl, Nrf2, and small Maf proteins. A nuclear protein ARE-BP1, has also been described to bind constitutively to theARE-inducible sequence, the GC box, and to be activatedby tBHQ possibly through a post-translational mechanism(Itoh et aL 1997; Owuor and Kong, 2002), the exact

    Fig. 2. A schematic representationof drugs/chemicals/xenobiotics nduces stress response eading o the potential sulfhydryl modification of Keapl-Nrf2 and/or activation of the signaling pathways such as the non-receptor-mediated MAPK (ERK, JNK, and p38), PKC, PI3K and PERK. Theactivation of these signaling pathways eads to the activation of transcription factors such as Nrf2/Maf and increase in ARE-mediated geneexpression including he phase II DMEs (GST, NQO, UGT) as well as other cellular defensive enzymes (GCL, HO-1), which ultimately results n theincrease of detoxification of the xenobiotics and/or generated ROS, eading o a potential homeostaticcell survival response.

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    258 C. Xu e t aL

    identi ty of this protein is sti l l unclear, presumably could berelated to N rf2/Maf complex. Th e central role of Nrf2 in thetranscr ipt ional act ivation of ARE-reporter genes has beenconf i rmed recent ly in other ARE-m ediated gen es inc ludinghum an ~-glutamylcysteine l igase GC L), and mo use hem eoxygenase-1 HO-1) Chen e t a l . 2000; Kong e t a l . 2000,2001 a , 2001 b ; Ow uor and Kong, 2002; Shen e t a l . 2004).The induct ion of NQ O and GS T by the phenol ic ant iox idantBHA wa s largely el iminated in the intest ine and l iver ofNrf2-/- m ice, and the gene expression o f several detoxi-f icat ion enzymes inc luding NQO was markedly reduced inthe lung of Nrf2 - / - mice I toh e t a L 199 7) . Th is lack o fphase I I DM E induct ion in Nrf2 - / - mice strongly suggeststhat Nrf2 is the most l ikely transcriptional factor involved inthe transcription activation of ARE-mediated phase IIgenes and cel lu lar defense genes induct ion Chan andKan, 199 9; I toh e t a L 1997 ; K w ak e t a L 2001).

    Qust ions remain over the past few years as to how N rf2is transcriptionally activated by such diverse chemicalcompounds. Several models have been proposed and putforward as dep icted in Fig. 2 and the biolog ical real ityprobably inv olve the convergent of some or al l of thesemul t ip le s ignal ing pathways depending on the chemicalstructures, cel l or t issues types, the gene of interest and inconjuct ion wi th other s ignal ing events that are yet to beuncovered.

    Previously, our group has shown that the mi togen-act ivated protein k inase MAP Ks) are involved in theregulat ion of the ARE in a Nrf2-dependent manner usingtransient transfection studies as well as kinase specif icchemical inhibitors Yu e t a L 2000a). We found that theextracellu lar s ignal -regulated k inase 2 and 5 ERK2 ,ERK5 ), and c-Ju n N-terminal k inase 1 JN K1) upregulatedthe A R E K eum e t a L 2003; Shen e t a L 2004; Yu e t a L1999), whi le the p38 M AP K appears to suppress i t Yu e ta L 2000b).

    Th e phosphat idy l inosi to l 3-k inase PI3K) has beenpostulated to be a posi t ive regulator of ARE in IMR-32neuroblastoma by the use of PI3K chemical inhibi tor,wortmannin Lee e t a l . 2001). Kang e t a l . provided furtherevidence that PI3K may be involved in Nrf2 nucleart ranslocat ion in response to tBH Q-induced oxidat ive stressin conjunct ion wi th cytoplasmic act in rearrangement Kange t a L 2002). Furthermore, Huang e t a L have reported thatprotein k inase C PKC) can di rect ly phosphorylated N rf2Huang e t a L 2000) and Ser-40 appears to be a s i te of

    potent ia l phosphorylat ion Hu ang e t a l . 2002). Furthermore,Cull inan e t a L have indicated that Nrf2 is directlyphosphorylated b y PERK, a t ransmem brane t ranscr ipt ionfactor, fol lowing the accumulation of unfolded proteins inthe endoplasmic ret iculum ER) Cul l inan and Diehl , 2004;Cull inan e t a L 2003). Ta ken together, these resul ts suggestthat mult iple kinase pathways are involved in the tran-

    scriptional activation of ARE.The most compel ing regulatory mechanism of act ivat ion

    of Nrf2 other than phosphorylat ion, have been reportedrecent ly . Dinkova-Kostova e t a L have shown that phase I Iinducers, most of wh ich are strong electrophi les, can resul tin di rect c leavage of Nrf2-Keapl comp lex by modi fy ingKe ap l at cysteine residues through Michael react ionDinkova-Kostova, 2001, 2002a, 2002b). To support th is

    hypothesis, Wakabayashi e t a L recent ly show n that two ofthe 15 cysteine residues Cys273 and Cys288) in Ke ap lmay play an important ro le in releasing Nrf2 in responseto electrophiles and oxidative stress via the formation ofan intermolecular disulf ide bridge, at least in the test tubeWakabayashi e t a L 2004). Quest ions remain whether

    this wil l occur in cel ls or i n v i v o t issues. In addi t ion, thepresence o f an ARE-l ike sequence in the promo ter regionof Nrf2 have also been shown, which may be responsiblefor sustaining the durat ion of ARE act ivat ion by providingthe binding s i te of Nrf2 i tsel f , as a feedb ack controlmechanism Kwak e t a L 2002). Interestingly, strongphase II inducers such as cadm ium, tert -buty lhydroquinonetBHQ), and beta-naphthof lavone ~-NF) did not af fect

    Nrf2 mR NA level in certain ce l l types, but a t tenuatedubiqui t inat ion and proteosomal degradat ion of Nrf2,imply ing that the act iv i ty of Nrf2 may not be determinedtransciptionally but perhaps post-translationally, at least inthese cells types Alam e t a L 2003; Nguyen e t a L 2003;Stewart e t a L 2003). F uture studies in i n v i v o animal or inhuman into the activation of these mult iple signalingpathways by xenobiot ics wi l l y ie ld bet ter ins ights intomechanisms of act ivat ion of Nrf2 leading to the induct ionof phase I I drug m etabol iz ing enzym es as wel l as cellu lardefensive enzymes and thei r b iological consequences inthe protection against environmental insults.R E G U L T I O N O F P H S E III T R N S P O R T E R S

    The major determinants of the i n v i v o systemic bioavail-abi l i ty of many drugs are due in part to the physico-chem ical properties solubi l i ty, ionization, l ipop hil ity), intes-t inal absorpt ion/permeabi l i ty and the intest inal/hepat icf irs t -pass e f fect . The P-glycoprotein P-gp) or mul t idrugresistant MD R) protein is usual ly coexpressed and co-induced wi th C YP 3A in the l iver and intest ine. I t p lays animportant role in reducing drug ab sorpt ion and enha ncingdrug el iminat ion back to the gut lum en, and i t appers thati t m ay be regulated by PXR Johnson e t a L 2003; Per lof fe t a L 2004). P-gp is expressed at the ap ical surface ofthe intest inal enterocytes, whe re i t mediates the e f f lux ofxenobiot ics into the intest inal lumen before these com-pounds can access the po rtal and subsequen t systemicci rculat ion. PXR l igands such as r i fampic in, SR-12813,51~-pregn ane-3-20-dione, clotrimazole, m ifepristone and

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    n i fed ip ine have been demonst ra ted to induce M D R 1 inhepatocy te and co lon cancer ce l l l ines (LS180 andLS174T) (Kul lak-Ubl ick and Becker , 2003; Song e t a L2004; Wang and LeCluyse, 2003) . Const i tut ively act ivatedhPXR expressed in LS180 ce l ls was ab le to induce bothP-gp and C YP 3A expression without speci f ic ligand binding.I t appe ars tha t a s im i lar P XR -depen dent mechan ism maybe also involved in M D R 1 induc t ion as compared toC Y P 3A induc t i on . E ndogenous M D R 1 gene is highlyinducible by r i fampin in human colon carc inoma cel l l ineLS174T, using DNA binding and t ransfect ions assays. ADR4 nuc lear receptor response e lement in t he ups t reamenhancer a t about 28 k i lobase pa i r s was ident i f ied as adist inct PXR binding s i te that was essent ia l for M D R 1induct ion by r i fampin (Geick e t a L 2001) .

    Most rece nt ly , ma ny s tud ies have d em onst ra ted tha tPXR act ivat ion resul ts in the induct ion of several othert ranspor ters inc luding OATP2 (Staudinger e t aL 2001a,2001b) , MRP2 or canal icular mult ispeci f ic organic aniont ranspor ter (cM OAT ) (Kast e t aL 2002) , and M RP3 (Ku llak -Ubl ick e t aL 2004; Teng e t aL 2003) . MRP2 is main lyexp ress ed in l iver, intestine, and k idne y tubules, S imi lar toPgp, MR P2 is loca lized to the ap ica l me mb ranes o f t heset issues (Chan e t a L 2004) . M RP2 is o r ig ina lly des ignatedas the cMOAT, is responsible for the bi l iary excret ion oforganic anions inc luding leukot r iene C4 (LTC4) , d ivalentbi le sal ts , and phase I I g lutathione, glucuronide, and sul-fate conjugates. Absence of th is t ranspor ter in hepato-cytes is bel ieved to be the reason for the defect in bi l iaryexcret ion of organic anions in pat ients with Dubin-Johnsonsyndrome (Kon ig e t a L 1999a, 1999b) . I t appears that thes ubs t r a t es o f M R P 2 and P gp t o s om e deg r ee a r eover lapping. The co- local izat ion of MRP2 and PgP at theapical membrane may be impor tant in drug disposi t ion,and may present a formidable barr ier to the absorpt ion ofm any d r ugs ( C han e t aL 2004) . Co-expression of MRP2wi th re levant phase I I metabo liz ing e nzym es su ch as GS Tand UG T, wh ich are found to be ex pressed notab ly a ts i tes w he r e C Y P 3A 4 , P gp and M R P a r e found i nc lud ingthe l iver , intest ine an d k idney, i t is possible that MR P2 a ndGST, UGT may play a synergist ic ro le in mediat ing drugel iminat ion (Chan e t aL 2004; Co les e t a L 2002; Turgeone t aL 2001 ). FX R , P X R and C A R appe a r t o berespons ib le fo r induc t ion o f Mrp2/MRP2 mRNA in ra t ,mouse and human hepatocy tes (Kas t e t a l . 2002) . Kaste t aL f ound tha t MRP2 mRNA leve ls were inducedfo llow ing t rea tment o f hum an or ra t hepa tocy tes w i th FX Rl igands and P X R o r C A R agon is ts . The dex am e t has one -and pregnenolone 16o~-carbonit r i le-dependent induct ionof MRP2 express ion was not ev ident in hepatocy tesder ived f rom PXR nul l mice. In cont rast , induct ion ofMRP2 by phenobarbi ta l , an act ivator of CAR, wascomparable in wi ld- type and PXR nul l mice. An unusual

    26-bp sequence was ident i f ied 440 bp ups t ream of t heMRP2 t ranscr ipt ion in i t iat ion s i te that contains an ever tedrepeat o f t he AG- I -FCA hexad se para ted by 8 nuc leo tides(ER-8) . PXR, CAR, and FXR bound with high af f in i ty tothis element as heterodimers with the RXR. Fur thermore,the iso la ted E R-8 e leme nt was capa b le o f conferr ing PXR,CAR, and FXR responsiveness with the heterologousthymidine k inase (TK) promoter . Mutat ion of the ER-8element abol ished the nuclear receptor response. Theses tud ies demonst ra te tha t MRP2 may be regu la ted bythree dist inct nuc lear receptor s ignal ing pa thw ays thatc onv e r ge on a c om m on r es pons e e lem en t i n t he 5 -f lanking region of th is gene (Kast e t a L 2002) .

    M RP 3 is a basolateral e f flux t rans por ter that t ranspo r tsbi le acids as wel l as several c l in ical ly impor tant anionicdrugs such as etoposide, methot rexate, and glucuronidecon jugates . The express ion o f MRP3 in ra t and humanl iver is low under normal condit ions but is induced dur ingcho les tasis and in the a bsen ce o f M RP2 or b i le sa l t exportpump. Up- regu la t ion o f t h is t ranspor ter appears tocompensate for the diminished abi l i ty to excrete organicanions into bile. For example, M RP 3 expresson is increasedin pa t ien ts w i th Dub in-Johnson syndrome to compensatefor a def ic iency in bi liary excret ion of organic an ions (Konige t a L 1999a, 1999b) . E levated expression of MRP3 isalso observed in the natural ly occurr ing MRP2-di f ic ienteisai hyperbil irubinemic rats (Hirohashi e t a L 1998) .MRP3 is also bel ieved to play an impor tant ro le in theenterohepat ic c irculat ion of b i les sal ts (C ha ndra andBrouwer , 2004) . PX R is also act ivated by bi le acids, whichl ikely to prevent their accumulat ion to tox ic levels(Staudinger e t a L 2001b; X ie e t a L 2001) . When thehuman hepatoma ce l l lines HuH7 and H epG 2 were t rea tedwith PX R act ivators inc luding c lot r imazo le, r i fampic in,1713-hydroxy-11J3-[4-dimethylaminophenyl]-17(z-[1 propynyl]es t ra -4 ,9-d ien-3-one (RU486) , mety rapone, n i f ed ip ine ,l i t hocho l ic ac id , and PCN, the leve ls o f MRP3 mRNAwe re induced 1 .6- t o 8-fo ld in a dose -depe nde nt manner .Cor respond ing decreases in t he mul t id rug res is tance-ass ociated pro tein-de pen den t cel lu lar retent ion of 5-carboxy f luoresce in we re a lso seen in the t rea ted H uH7ce l ls . I n v ivo s tud ies demo nst ra ted inc reased PXR mR NAand induc t ion o f MRP3 mRNA in the l i vers o f w i ld - t ypemice t reated with the PXR act ivator RU486. On the otherhand, M RP3 induc tion wa s not seen in t he RU 486- t rea tedPXR-nul l m ice. Th ese resul ts sugg est that PX R act ivat ionmay play an impor tant ro le in the regulat ion of MRP3express ion (Teng e t a L 2003) . Delet ion analys is of theMrp3 p romo ter ident if ied a basa l transcr ip t ion e lem ent a t-123/ -106, two negat ive response regions at -2723/ -1128and -530/ -443, respect ively, as w el l as two posi t ive responsereg ions a t -1063/ -943 and-302/ -157. S i te -d i rec ted muta-genes is ana lys is and ge l mob i l i t y sh i f t assays prov ided

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    260 C. Xu et aL

    evidence for Spl and Sp3 binding wi thin the -123/-105regions. These studies indicated that Spl and Sp3 maybe involved in the regulat ion of the rat Mrp3 gene (Tzengand H uang , 2002 ) . B o th MR P 3 mR N A l eve l and t hepromoter act iv i ty of MRP3 were increased about 3-fold inhum an colon cel ls by the bi le acid cheno deoxychol ic a cid(CDCA ), and that the putat ive bi le sal t- responsive elementswe re found to be located in the reg ion -229/-138, whichinclude tw o alpha-1 fetoprotein t ranscr ipt ion factor (FTF )-l ike elements. C onstruct of a spe ci fic m utat ion in the con-sensus sequence of FTF eleme nts shewed no increase inthe basa l transcr iptional act iv i ty fo l lowing CDC A treatment.In electrophoretic mobil i ty shift assay with nuclear extracts,speci f ic binding of FTF to FTF-i ike eleme nts was obse rvedwhen t reated w i th CDCA. The express ion o f FTF mRNAlevels was also markedly elevated af ter t reatment wi thCDCA, and overexpression of FTF speci f ical ly act ivatedthe M RP 3 promoter a ct iv i ty about 4-fo ld over the basalpromoter act iv i ty . These resul ts suggest that FTF mayplay an important role in the regulat ion of M RP3 expression(Inokuchi et a l . 2001).

    Select ive act ivat ion of PXR or CAR induced OATP2and MRP3 expression in wi ld- type mice but not in PXRknock out (PXR-KO) mice (Staudinger et a l . 2003).Treatment of wi ld- type mice wi th the PXR-seiect iveact ivator PCN resul ted in robust increases in Oatp2,Mrp3, and CYP3A gene expression levels. Treatment ofwi ld- type mice wi th the CAR act ivator phenobarbi talinduced only s l ight increases in Oatp2, Mrp3, and CYP3Agene expression levels. In contrast to t reatment wi th ph e-nobarbi ta l , t reatmen t of wi ld-type mice wi th the CAR -selec-t ive activator 1,4-bis[2-(3,5-dichlorophyridyloxy)]benzene(TCPOBOP) potent ly induced increases in Oatp2, Mrp3,and CY P3A gene expression leve ls . There we re nochanges in Oatp2, Mrp3, and CYP3A gene expressionwhe n PXR -KO mice were treated w i th P CN , however ,phenobarbi tal t reatment of PXR-KO mice producedrelat ively obvious increases in Oatp2, Mrp3, and CYP3Agene expression when compared wi th the phenobarbi tal -t reated w i ld-type mice (Staudinger e t a L 200 3), suggest ingthe importance of CAR and PXR in the co-regulat ion ofthese t ransporters. In the same study , MR P2 e xpressionwas s igni f icant ly induced by phenobarbi tal and this wassimi lar ly reported in rat l iver that M RP2 (cMOA T) wasfound to be induced by phenobarbi tal us ing microarraygene chip study (Rushm ore and K ong , 2002).

    OAT P2 is localized to the h epat ic s inusoidal m emb rane,wi th select ive expression in the m idzonal to per ivenoushepatocytes (Reichel et a l . 1999 ) . Expression o f O ATP2has also been detected in the brain and ret ina (Gao e t a L2002). Treatment of ra ts wi th PX R act ivator P CN , s ignif i-cant ly enhances the rat o a t p 2 gene expression (Guo e ta L 200 2a, 200 2b). Four potent ia l PXR respo nse elements

    (PXREs) we re ident i fied in the 5 - f lanking region of the ratoatp2 gene. One element (DR 3-1) is located approximately-5000 bp w i th th ree more (DR3-2, DR3-3, and DR3-4)c lustered at about -8000 bp. Resul ts f rom electrophoret icmobi l i ty shi f t assays sh ow ed that the PXR -RXR heterodi -mer binds to the DR3-2 wi th the highest af f in i ty , to theDR3-4 and DR3-1 wi th a lower af f in i ty , and weakly or notat al l to the D R 3-3 . Furthermore, a ser ies of part ia ldeletions o f the 5 -f lanking reg ion i l lustrated that both theproximal and d istal c lusters of P XREs are requ ired formaximal induct ion of rat OATP2 by PCN (Guo et a l .2002a, 2002b).C O O R D I N A T E D R E G U L A T I O N O F P H A S E IP H A S E II D M E S A N D P H A S E III T R A N S P O R T -E R S

    M any phase I and phase I I enzyme inducers sharecommon mechanisms of t ranscr ipt ional act ivat ion andshare a s imi lar bat tery of genes that are coordinatelyregulated. Many phase I I metabol i tes we re found to betransported o ut of the cel ls by P-g p, MR Ps, and O ATP2.Along wi th the phase I and phase I I enzyme induct ion,pretreatmen ts wi th seve ral types of inducers have b eenshown to al ter the excret ion of xenobiot ics, which impl iesthat phase I I I t ransport processes may also be s imi lar lyregulated. Wh ether these pha se I and pha se I I enzymeinducers coordinately regulate the so-cal led phase IIIt ransporter genes requires further studies, and suchinformat ion wou ld add to o ur knowledge of the disposi t ionand el imination of xenobiotics.

    3-methylcholanthrene (3MC) can induce the expressionof C Y P 1 A 1 C Y P I A 2 and C Y P 1 B 1 by act ivat ing the AhRregulat ing t ranscr ipt ion of the CYP1 genes. C Y P I A 1 isundetectable in the l iver of control rats but was found tobe highly induced in the l iver of 3MC treated rats.Induction of C Y P I A 2 and C Y P I B 1 were also observed inthe l i vers f rom ra ts t reated w i th 3M C (Rushmore andKon g, 2002). Ind uct io n of several phase I I enzym es,U G T 1 A 6 G S T A 1 G S T A 2 and G S T M was a lso observedin the l iver recovered f rom rats t reated w i th 3M C. 3M C isknown to induce expression of the UDP-glucuronosylt ransferase gene U G T 1 A 6 (Bock et a l . 1 9 9 8 ) a n dglutathione-S-transferase gene GSTA1 (Rushmore e t a L1990) by an AhR-dependent mechanism (Rushmore andKong, 2002) . The G S T A 2 and G S T M I genes are bothinduced by the C Y P 1 A 1 epoxide and hydroxylatedmetabol i te(s) of 3MC. B oth genes contain an ARE do mainin thei r prom oter re gion s. This c is -a c t in g element hasbeen show n to be responsive to the d iol metabol i tes of3MC that can redox cycle and produce a pro-oxidat iveenv ironment (Rushmore and Ko ng, 2002) , p resumablyanalogous to the electrophil ic actions of phenolic anti-

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    Regulat ion of Drug Metabol ism and Drug Transpor t 261

    oxidants BH A or tBH Q as de scr ibed before. The glutathione-S- t ransferases G S T A 2 and G S T M 1 were previouslyobs erve d to be 3MC - inducible in cul tured rat hepatocytes( M a h e o et aL 1997) . H igh leve ls o f mdr mRNAs wereobserved by Nor thern blot t ing in two independent rat l iverepi thel ia l (RLE) cel l l ines af ter t reatment with 3MC. 3MC-mediated m d r m R N A induc t i on w as dem ons t r a t ed t o bedose-dependent , i t occurred through enhanced expressionof the mdr 1 gene, and paral le led the induct ion of the P-gp protein expression (Fardel et aL 1996) .

    Phenobarbi ta l is a t ranscr ipt ional inducer of the ratC Y P 2 B 1 C Y P 2 B 2 a n d C Y P 3 A 1 genes . CAR is a nuc learreceptor t ha t in te rac ts w i th RXR to fo rm CAR-RXRheterod imers, wh ich b ind to the PBR E in respon se tophenobarbi ta l t reatment . C Y P 2 C 7 is a lso repor ted to beinduced by phenobarbi ta l , but pr imar i ly in female ratherthan in male rats (Honkakoski and Negishi , 1997;Rush mo re and Kong, 2002; Waxman, 1999) . Induc tion o fsevera l phase I I enzymes was observed a f te r t rea tmentwith phenobarbi ta l . A s igni f icant increase in the speci f icmR NA for m ic rosomal epox ide hydro lase (EHm ) , U G T 2 B 1G S T A 1 G S T A 2 G S T A 3 and G S T M 1 wa s a lso observedin the l ivers recovered f rom rats t reated with phenobar-bi ta l . Phenobarbi ta l is known to induce the expression ofC Y P 2B gene by t he C A R - dependen t m echan is m . N oPBRE sequence has been ident i f ied in t he promoters fo rany o f t he phase I I enzymes to da te . I n add i t ion to t hephas e I and ph ase I I enzym e induc tion , M RP2 expressionwa s s igni f icant ly induced by Phenobarbi ta l (Staudinger e taL 2003) . M RP3 w as a lso repor ted to be induced by 1 ,4-bis[2-(3,5-dichloropyr idy loxy)]benzene (TCPO BOP ), a CA Rand CYP2B inducer . However , t he data sugges ted tha tCAR may not p lay a key role in phenobarbi ta l- inducedM R P 3 ( X iong et a l . 2002) .

    E ighteen di f ferent microsomal enzyme inducers inc lud-ing TCD D, indole-3-carbinol , phenoba rbi ta l , d ia l ly l sul f ide,s p i r ono lac t one , dex am e t has one , d ie t hy lhex y lph t ha la t e ,ethoxyquin, o l tipraz, and acety lsal icy lic acid were se lectedbased upon s ix major p roposed mechan isms o f d rug-metabo l iz ing enzyme induc t ion (AhR l igands , CARact ivators, PXR l igands, PPAR l igands, EpRE act ivators,and CYP2E1 inducers) , and they did not markedlyinc rease the express ion o f MRP1 or MRP2 (Cher r ing tone t a L 2002) . How ever , MR P3 expression wa s s igni ficant lyincreased by each of the CAR act ivators (phenobarbi ta l ,390 ; 2,2 ' ,4,4 ' ,5-pentachlorobiphenyl (PCB99) , 580 ;and dial ly l sul f ide, 540 over cont rol) , and an EpR Eact ivator o lt ipraz (6 70 ove r cont rol) in the l ivers of therats. MRP3 was not s imi lar ly induced in k idney and largeintest ine, demonst rat ing that the coordinate inducibi l i ty ofMRP3 may be speci f ic to the l iver . Addit ional evidencesugg es ts tha t M RP3, wh ich is unde r the t ranscr ip t iona lregulat ion of CAR, may have l iver -speci f ic induct ion of

    M R P 3 by C A R ac t i v a t o r s , bec aus e C A R i s ex p r es s edalmost exclusively in l iver . The authors conclude that rathepat ic MRP3 is induced by CAR ac t iva tors , t husenhanc ing the vec tora l excre t ion o f some phase I Imetabol i tes f rom the l iver to the blood (Cherr ington et aL2002) . CAR has bee n descr ibed as a ce l lu la r senso r t ha tis capable of responding to chemical tox ic i ty andmed iat ing C YP 2B family induct ion (Honkako ski et a l .1998b ; K aw am ot o e t a L 1999; Sueyoshi e t a L 1999) .Act ivat ion of th is cel lu lar sensor leads to an increaseex p r es s ion o f M R P 3 , w h i c h m ay l ead t o an enhanc edabi l i ty of the l iver to el iminate organic anions into thesinusoidal b lood, thereb y reducing h epat ic tox ic i ty .

    O A TP 2 l eve ls w e r e dec r eas ed 56 t o 72 by t he A hRl igands (TCDD, indole-3-carbinol , and b-naphthof lavone) ,increased 84 to 132 by the CA R ligands (phenoba rbi ta l ,d ia l ly l sul f ide, and PC B 99) , increased 230 to 36 0 byPXR l igands (PCN, spironolactone, and dexamethasone) ,and no changes on OATP2 leve ls by PPAR l igands andAR E/EpR E ac tiva tors were observed (Guo e t a L 2002a,2002b) . There was no cor re la t ion between Oatp2 mRNAlevels with the al tered OATP2 protein levels, for example,among the PXR l igands , on ly PCN inc reased oatp2mR NA leve ls , bu t sp i rono lactone and dexa me thasone d idnot . Fur thermore, o nly PCN, bu t not spironolactone anddexam ethasone, increased the t ranscript ion of the oatp2gene as s how n by the i nc reas e am oun t o f m R N A . Thes eauthors conc luded tha t OATP2 may be coord ina tedregu la ted by the PXR-CYP3A inducers , and tha t t heregu la t ion o f OATP2 by these inducers occu rs a t bo th thet ranscr ipt ional a nd post - translat ional levels (Guo et a l .2002a, 2002b) .

    In summary, the impor tance of the coordinatedregulat ion of the p has e I II t ransp or ter wi th the ph ase I andph ase I I drug m etabol iz ing enz ym es nee ds fur ther invest i -gat ion. The addit ion of phase I l l t ranspor ters, such as P-gp, MRPs and OATPs, regu la t ion o f gene express ion byreceptors such as PXR and CAR, w i l l lend bet te r under -standing to the biological funct ions of PXR and CAR as achem ical /xenobiot ics sensor , however , mos t impor tant ly ,as a mean o f manag ing the pro tec t ion o f t he drug orxenobiot ics exposure f rom the environment . Future studieswi l l shed l ight on the roles of other receptors, t ranscr iptionfactors and s ignal ing cascades in the coordinated regu-lat ion of ph as e I, I I an d I II dru g metabo lism/transport byendoge nous c om pound s as w el l as by ex ogenous agen t sincluding environmental and nut r it ional, in the tr igger ing ofd isease s and or tox ici ties induced by these chemica ls .CKNOWLEDGEMENTSuppor ted in par t by grant R01-CA-094828 f rom

    National Inst itutes of Health.

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