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Understanding and controlling hepatobiliary function
Best Practice & Research Clinical Gastroenterology Vol. 16, No. 6, pp. 1025±1034, 2002
doi:10.1053/bega.2002.0340, available online at http://www.idealibrary.com on
13 Understanding and controlling hepatobiliary function Ronald P. J. Oude Elferink Laboratory of Experimental Hepatology, AMC Liver Center, Academic Medical Center, Amsterdam, The Netherlands
Over the past decade, enormous progress has been made in identifying the mechanisms that underlie hepatobiliary excretion. A set of transport proteins mediates the canalicular transport of most important bile constituents. With the discovery of these transporter genes, the mechanism of bile formation could be partly elucidated and genetic defects caused by mutations in these genes identi®ed. This progress is crucial not only for paediatric and adult hepatology, but also for pharmacology, because the characterization of these transport systems provides tools for the prediction of the pharmacokinetics of drugs. Indeed, there is a growing interest on the part of the pharmaceutical industry for research into transport systems in general and hepatobiliary secretion in particular. For all of these transporter genes, knockout mice have been bred that allow one to assess the in vivo function of each of these transporters with regard to their role in physiology and drug elimination. Key words: bile; cholestasis; transport; ABC transporters.
TRANSPORT PROTEINS AND THE ENTEROHEPATIC CYCLING OF DRUGS AND TOXINS Nearly all canalicular transport proteins are so-called ATP-binding cassette (ABC) transporters. These comprise a large family of transporter genes with at least 48 members in humans. The transport proteins are involved in highly diverse transport steps across the plasma membrane and the membranes of intracellular organelles.1 The transport is driven by ATP hydrolysis, which enables uphill transport. A substantial fraction of these transporters mediate the extrusion of unwanted compounds from the cell. Because of this, the cell is defended against noxious compounds but, by virtue of their expression in the apical membrane of epithelia, these transporters also take care of the whole-body elimination of the unwanted drugs and toxins. Several of these transporters are expressed both in the apical membrane of enterocytes and in the canalicular membrane of hepatocytes. They thus have a double function as gate-keepers against toxic products. First, they immediately extrude compounds that have entered gut cells back into the lumen. Second, those molecules which do enter the bloodstream are taken up by the liver and extruded by the same transporters into bile. Using animals lacking these transporter genes, it can be shown that the oral availability of drugs can be to a large extent determined by the function of these transporters. c 2002 Elsevier Science Ltd. All rights reserved. 1521±6918/02/$ - see front matter *
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MDR1 P-glycoprotein The prototype of the drug extrusion pump is MDR1 P-glycoprotein (Pgp), which excretes a very broad spectrum of amphipathic drugs out of cells, thereby conferring multidrug resistance against these substances. Indeed, this gene was discovered by its overexpression in multidrug-resistant tumour cells, but it subsequently became clear that the MDR1 gene is also expressed in normal tissues. The role of MDR1 Pgp in protecting against xenobiotics has been ®rmly established by the generation of mice lacking both the Mdr1a and Mdr1b genes (in mice, two Mdr1 genes ful®lling the function of the single MDR1 gene in humans). This knockout animal is hypersensitive to a number of toxic compounds, important sites of expression of the protein being the blood±brain barrier, the intestinal epithelium and the hepatocanalicular membrane.2,3 It can be shown, again by use of the knockout mice, that Mdr1a Pgp is involved in the biliary elimination of several amphipathic drugs, such as digoxin and vecuronium.3±5 Another important site of expression of MDR1 is the gut, where the protein is localized to the apical membrane of the enterocytes and mediates the direct elimination of compounds back into the gut lumen. As a result, MDR1 determines the oral availability of many drugs. Polymorphisms have been detected in the MDR1 gene6,7, and these may be associated with alterations in the oral bio-availability of certain drugs.8 The polymorphism C3435T has been found to be associated with reduced MDR1 Pgp expression in the duodenum, this correlating with an increased oral bio-availability of digoxin.6 It is interesting to note that Panwala et al9 detected a high incidence of colitis in Mdr1a / mice. This chronic intestinal in¯ammation could be prevented by treatment with a mix of broad-spectrum anitbiotics, suggesting that intestinal bacteria and/or their products play a role in this phenomenon. It remains to be established whether this phenomenon is the primary consequence of the absence of Mdr1a Pgp or a secondary eect. It may be hypothesized, however, that an impaired elimination of luminal toxins predisposes the animals to an in¯ammatory process. If so, reduced MDR1 expression in humans could theoretically also predispose towards intestinal in¯ammation.
MRP2 MRP2 was discovered as a liver-speci®c homologue of another multidrug resistance transporter, MRP1.10,11 The two transporters have opposite membrane localizations: MRP1 is expressed in the basolateral membrane and MRP2 in the apical membrane of epithelia. Whereas MRP1 is expressed in most cell types, MRP2 is predominantly found in hepatocytes and to a lesser extent in enterocytes and kidney proximal tubule cells.12,13 MRP1 and MRP2 have highly similar substrate speci®cities.12,13 Both transporters recognize a wide spectrum of organic anions, including unconjugated anions as well as anionic conjugates of glucuronic acid (such as conjugated bilirubin), glutathione and sulphate. It has more recently become clear that these transporters can also pump uncharged compounds. It appears that the transporters can accommodate uncharged amphipathic compounds together with a glutathione molecule in a cotransport mechanism. Experiments with plasma membrane vesicles containing MRP1 have revealed that this transporter does not to any signi®cant extent pump vincristine. The addition of GSH, however, strongly stimulated the translocation of vincristine.14 A similar involvement of glutathione was found for the MRP2-mediated transport of the uncharged carcinogen PhIP.15 This mechanism greatly enhances the versatility of these
Understanding and controlling hepatobiliary function 1027
transporters. In the intestine, MRP2 may play a role similar to that of MDR1 Pgp in the defence against toxic compounds in the gut lumen.15 Breast cancer-related protein Breast cancer-related protein (BCRP, also termed MXR or ABCP; ABCG2 gene) has more recently been identi®ed as yet another ABC transporter that is overexpressed in anthracycline-resistant cell lines.16 Upon cloning its cDNA, it turned out to be a halftransporter, of which more examples exist. These half-transporters are believed to dimerize, as either homodimers or heterodimers, into functional pumps. Transfection of ABCG2 cDNA confers resistance against mitoxantrone, topotecan, doxorubicine, daunorubicine and rhodamine 123.16 Using monoclonal antibodies against the ABCG2 gene product, Maliepaard et al17 demonstrated that this protein is present in the plasma membrane of endothelial cells and in the apical membrane of intestinal epithelial cells (both colon and small intestine) and hepatocytes. The latter ®nding strongly suggests that it is also involved in biliary secretion of amphipaths. Using a speci®c BCRP inhibitor, GF120918, Jonker et al18 showed that this transporter reduces the oral bio-availability of topotecan and other BCRP substrates in the intestine by pumping these compounds back into the lumen. Thus, BCRP, as well as MDR1 and MRP2, serves as a gate-keeper in the gut by reducing the oral bio-availability of drugs and toxins. Bile salts Bile salts are pumped across the canalicular membrane via the bile salt export pump (BSEP). The BSEP gene displays a high extent of homology with MDR1 (which encodes the drug transporter described above), and on this basis the two genes fall into the same subclass of ABC transporters. Wang et al19 have reported on a mouse de®cient in the Bsep gene. Bsep / mice are cholestatic in the sense that taurocholate accumulates in the liver and little taurocholate is excreted into bile, but these mice excrete a substantial amount of tauromuricholate as well as a hitherto unde®ned tetrahydroxy bile salt, into the bile. Bsep is apparently not the only bile salt transporting system in the canalicular membrane of murine hepatocytes, another system being capable of excreting tauromuricholate and the tetrahydroxy bile salt. This escape route prevents severe and progressive cholestasis in these mice and, as a consequence, these animals have hardly any histopathological signs of liver injury. Because humans are not to any signi®cant extent capable of converting bile salts into muricholate or tetrahydroxy bile salts, this escape route is not present in them. As a consequence, mutations in the BSEP gene are expected to cause a cholestatic syndrome in humans, which is indeed the case (see below). On the basis of these observations, BSEP is believed to be the main bile salt transporter in the canalicular membrane of human hepatocytes. Biliary lipids Biliary lipids are an important component of bile. They consist mainly of phospholipid (almost exclusively phosphatidylcholine) and cholesterol. The relatively high concentration of cholesterol in human bile represents the major risk factor in gallstone formation. Upon the production of a knockout mouse lacking the Mdr2 gene, it was discovered that the encoded transporter is essential for biliary lipid secretion.20 In the absence of Mdr2 Pgp, mice secrete neither phospholipid nor cholesterol. In dierent experimental systems, it was subsequently demonstrated that the murine
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Mdr2 Pgp as well as the human orthologue MDR3 Pgp are phosphatidylcholine translocators.21±23 The MDR3 gene is homologous to MDR1 and BSEP, and all three fall into the same subclass of ABC transporters. On the basis of the data available, we have proposed a model24 in which phosphatidylcholine, after delivery to the inner lea¯et of the canalicular plasma membrane, is translocated to the outer lea¯et and subsequently resides in phosphatidylcholine-rich lipid domains. These more ¯uid, phosphatidylcholine-rich microdomains are destabilized by the canalicular bile salt molecules and, in combination with the continuous pumping of phosphatidylcholine into these domains, a vesiculation of the phosphatidylcholine-rich membrane bilayer occurs. These vesicles then pinch o to give rise to typical biliary lipid vesicles.24 The mechanism of cholesterol secretion into bile is still largely unknown. No direct evidence yet exists for the involvement of a translocator protein which would, as with Mdr2/MDR3 Pgp for phospholipids, catalyse the translocation of cholesterol across the membrane. Recent evidence suggests that the two half-transporters ABCG5 and ABCG8 are involved in the elimination of plant sterols, which have a molecular structure very similar to that of cholesterol. Like ABCG2, ABCG5 and ABCG8 are half-transporters that have to dimerize into a functional pump. The transport of sterols by the ABCG5/G8 couple has so far not been directly demonstrated, but this function can be inferred from the accumulation of plant sterols in patients with sitosterolaemia, who have a mutation in either of the two genes encoding these proteins (see below). ABCG5 and ABCG8 are expressed in the intestine and liver. DEFECTS IN CANALICULAR TRANSPORTERS All the transport proteins described and their functions have been identi®ed within the past decade. After or because of their identi®cation, inherited liver diseases caused by mutations in the respective ABC transporter genes were also recognized. Some of these have a severe phenotype and are therefore primarily paediatric disorders. The common symptoms are chronic cholestasis and cirrhosis that often progresses to liver failure. These conditions have been collectively called progressive familial intrahepatic cholestasis (PFIC), but we now know that this group consists of at least three subgroups (types 1, 2 and 3) that are caused by mutations in the FIC1, BSEP and MDR3 genes respectively. The latter two gene products have been described above. Types 1 and 2 PFIC can be distinguished from type 3 by the fact that the patients with the latter have an increased serum gamma-glutamyltransferase (GGT) activity. Progressive familial intrahepatic cholestasis type 1 and benign recurrent intrahepatic cholestasis Type 1 PFIC (formerly called Byler's disease) manifests itself primarily as a chronic intrahepatic cholestasis. The bile salt concentration is high in serum and very low in bile, suggesting that hepatic transport is impaired. In addition, patients with type 1 PFIC suer from watery diarrhoea. The jaundice in these patients is regarded as a secondary consequence of insucient bile ¯ow, the latter being dependent on bile salt secretion. A genetic screen was performed on patients from the Byler pedigree and their family members in order to identify the disease locus.25 This was located on chromosome 18q21±q22, the same chromosomal region to which a very similar disease, benign recurrent intrahepatic cholestasis (BRIC), has been localized. BRIC is
Understanding and controlling hepatobiliary function 1029
a genetic disorder in which patients suer from periods of cholestasis that are of unpredictable onset and length, and that leave no liver damage. A combined search for the two disease loci led to the identi®cation of the mutated gene FIC1 (gene code ATP8B1), and its mutations in a group of patients, including those from the original Byler pedigree.26 Many issues concerning the function of the FIC1 gene product remain puzzling. Although FIC1 is localized to the canalicular membrane of the hepatocyte27, expression in the liver is actually quite low, whereas the gene is highly expressed in intestine and pancreas.26 Expression also occurs in many other tissues. It is therefore unclear why the absence of FIC1 from many tissues leads primarily to a phenotype in the liver and intestine. FIC1 is not an ABC transporter but a P-type ATPase, the function of which is proposed to be the translocation of aminophospholipids (a ¯ippase).28 The absence of FIC1 leads to defective bile salt secretion, but how its ¯ippase activity relates to this defect is unknown. We have recently shown that mice bearing one of the mutations that have been detected in type 1 PFIC patients do not suer from cholestasis but instead display enhanced intestinal absorption of bile salts (L. Pawlikowska, in preparation). This suggests that the cholestatic phenotype in these patients may be indirect and caused by enhanced absorption of (cholestatic) bile salt (metabolites). This hypothesis would ®t with the observed relief of cholestasis in type 1 PFIC patients upon chronic bile diversion.29±32 Nevertheless, the liver must contribute to the phenotype because liver transplantation ameliorates the hepatic symptoms in these patients. Extrahepatic symptoms, such as watery diarrhoea, remain after liver transplantation. Progressive familial intrahepatic cholestasis type 2 The phenotype of this form of PFIC is very similar to that of type 1 except that type 2 patients have no extrahepatic symptoms. Strautnieks et al33 demonstrated that the disease locus of this subgroup of patients lies on chromosome 2q24. They subsequently found that these patients have mutations of their BSEP gene, which is present in this region.34 The fact that mutations of the BSEP gene lead to a virtual absence of bile salts from the bile has led to the conclusion that BSEP is the main bile salt transporter in the canalicular membrane. This is supported by studies in which rat and murine Bsep was transfected into Sf9 cells and shown to transport several bile salts.35,36 No data are yet available on the transport characteristics of human BSEP. Nonsense, missense mutations and deletions of the BSEP gene have been found in some patients with low-GGT PFIC, these individuals now being described as having type 2 PFIC.33 Very importantly, further screening by Strautnieks et al37 seems to indicate that yet another subgroup of patients exists in whom the disease locus localizes to neither the FIC1 region nor the BSEP region. This would mean that another gene is involved in low-GGT PFIC. Progressive familial intrahepatic cholestasis type 3 As mentioned above, this form of PFIC is fundamentally dierent from types 1 and 2 in that aected patients have a high serum GGT activity. The onset of this disease is somewhat later than that of the other two forms, but the histological picture is more severe, with strong bile duct proliferation and cirrhosis. The most prominent features of the disease are portal hypertension, hepatosplenomegaly, jaundice and pruritus. If untreated, the disease develops into liver failure. The genetic background of this subgroup of PFIC patients was elucidated after it was found that mice with a disruption of the Mdr2 gene, the murine orthologue of MDR3, develop a similar
1030 R. P. J. Oude Elferink
phenotype.20 This led Deleuze et al38 to investigate the possible involvement of this gene in PFIC. Subsequently, in a group of 31 patients with high-GGT PFIC, 17 were found to have a mutation in the MDR3 gene.39,40 Since the gene was not completely sequenced in all these patients, it is not clear whether the remaining 14 patients had as yet unidenti®ed mutations or whether another gene might be involved in this form of PFIC. MDR3 Pgp functions in the translocation of phosphatidylcholine, thereby facilitating the secretion of this phospholipid into the bile. The secretion of phospholipid is of crucial importance in protecting the cellular membranes of the biliary tree against the high concentrations of bile salt detergents.24 The treatment of patients with ursodeoxycholate (UDCA) appeared to be bene®cial in about half of the cases.39 Given the fact that the hepatic damage is caused by bile salts, this is a rational treatment because UDCA is a hydrophilic bile salt that has low cytotoxicity. Indeed, in Mdr2 / mice with the equivalent defect, feeding this bile salt also halted the disease process.41,42 The observation that not all patients improve on UDCA treatment may be explained by the fact that administration of this bile salt replaces the endogenous bile salt pool only insuciently. Thus, in patients who have no MDR3 Pgp activity at all, the reduction of bile salt cytotoxicity by UDCA is insucient, whereas in patients with residual phospholipid secretion, UDCA might bring the overall bile salt cytotoxicity below a critical threshold. Indeed, none of the patients with a truncated MDR3 gene improved on UDCA, whereas some of those with missense mutations did.39 The administration of higher doses of UDCA may be particularly useful in these patients. Sitosterolaemia Mutations in the ABCG5 or ABCG8 gene are the cause of sitosterolaemia. Patients with this inherited disorder accumulate a large amount of plant sterols, such as sitosterol, in their circulation. In normal individuals, the uptake of plant sterols in the gut is very low because ABCG5/ABCG8 pumps these molecules back into the gut lumen. In addition, the low amount of plant sterols that does enter the circulation is eciently excreted by ABCG5/ABCG8 in the canalicular membrane of the hepatocyte. Interestingly, these patients not only have high blood plant sterol levels, but also suer from hypercholesterolaemia, suggesting that cholesterol is also transported by this couple. Thus, in the absence of ABCG5/ABCG8, there will be an enhanced absorption of cholesterol in the gut and a decreased secretion of cholesterol in the bile. This is, however, currently speculative and remains to be demonstrated in ABCG5/ABCG8 knockout mice. Dubin±Johnson syndrome Mutations in the MRP2 gene cause Dubin±Johnson syndrome, characterized by a conjugated hyperbilirubinaemia. In contrast to its unconjugated form, glucuronidated bilirubin is not toxic, and this rare syndrome is considered to be benign. In the animal model of this syndrome, the TR rat, we were able to demonstrate that there is an enhanced oral availability of the food-derived carcinogen PhIP and probably also many other food-derived compounds. There are anecdotal reports of primary HCC, but the number of Dubin±Johnson patients is probably too small to elucidate whether this is a signi®cant side-eect of MRP2 de®ciency. It may be expected that polymorphisms in this gene leading to reduced expression or activity will be associated with an altered bioavailability of many drugs, but such polymorphisms have not yet been characterized.
Understanding and controlling hepatobiliary function 1031
REGULATION OF TRANSPORTER EXPRESSION Over the past few years, it has been shown that the expression of ABC transporters, including those responsible for bile formation, is subject to tight regulation. In particular, the family of nuclear receptors plays a prominent role in the transcriptional control of ABC transporter genes.43,44 The common theme in this family is that dimers of RXR (the retinoid receptor that binds retinoic acid) and a ligand-speci®c counterpart bind to a responsive element in the promoter region of a gene and thereby regulate transcription. In this fashion, BSEP expression is controlled by the nuclear receptor pair RXR/FXR, in which FXR is the farnesoid receptor, which has high anity for bile salts. Hence, the binding of bile salts to FXR in the dimer RXR/ FXR induces transcription of the BSEP gene, leading to enhanced protein levels and increased biliary bile salt secretion. ABCG5 and ABCG8 are located on chromosome 2p21 in a close head-to-head arrangement. These genes are co-ordinately expressed and are strongly induced by LXR (the `liver-speci®c' receptor), the latter nuclear receptor being a sensor for cholesterol as it binds oxysterols. Many genes involved in cholesterol metabolism and transport are regulated by LXR. The observation that ABCG5 and ABCG8 are regulated by LXR again suggests that this transporter couple is involved in the transport of cholesterol. A recent report suggests that MRP2 is regulated by three nuclear receptors, FXR, CAR and PXR.44 PXR (in rodents) and SXR (in humans) are receptors that accomodate as the ligand a large number of xenobiotics, for example rifampicin (SXR only), hyperforin (from St. John's wort), dexamethasone, the HIV protease inhibitor ritonavir, taxol and others.44 MDR1 expression is also regulated by SXR.45 Interestingly, the latter nuclear receptors also induce cytochrome P450 enzymes from the important CYP3A family. Hence, certain drugs induce their own metabolism as well as their secretion by the induction of CYP isoforms and drug pumps respectively. This will become an important issue in pharmacokinetics and drug interaction. THE FUTURE OF HEPATOBILIARY TRANSPORT RESEARCH Genetic disorders associated with the most important transport proteins in the canalicular membrane have now been identi®ed and characterized. These disorders are of minor importance for hepatology in general because they are extremely rare and con®ned mostly to the paediatric arena. This will, however, change in the future because the monogenic inherited disorders are only the tip of the iceberg. These disorders are the simplest situation encountered in transport defects, namely the complete absence of a single transport protein. Polymorphisms causing a reduction in transport protein level and/or activity will, however, be much more frequent because the consequences of such polymorphisms are much less severe. Such polymorphisms will nevertheless predispose individuals to cholestasis and/or other forms of liver disease under conditions of reduced liver function that are caused by other genetic or environmental conditions. A good example of this situation is MDR3 de®ciency. A complete absence of this phospholipid-translocating protein causes severe (type 3 PFIC) liver disease, as described above. More recently, it was, however, found that heterozygous females in the family of a type 3 PFIC index patient developed cholestasis of pregnancy.46 Under normal conditions, these women do not have clinical symptoms, but in the last trimester of pregnancy there is apparently a general impairment of bile formation that,
1032 R. P. J. Oude Elferink
combined with a decrease in the level of MDR3 expression, leads to hepatic damage. Indeed, other genetic forms of cholestasis may be unmasked by the general impairment of bile formation during pregnancy.47 Several individuals with gallstones have also been reported to have mutations of the MDR3 gene that did not occur in the control population.48 This suggests that mutations in the MDR3 gene may predispose to gallstone formation. Finally, an interesting recent preliminary report by Thompson et al49 suggested that mutations in the MDR3 gene may predispose to the development of primary biliary cirrhosis as they found MDR3 mutations in patients with primary biliary cirrhosis who didn't demonstrate anti-mitochondrial antibodies. These observations illustrate how mutations of transporter genes are not only relevant for rare inherited diseases, but may also play a role in more common disease phenotypes. Canalicular transport proteins will also become an important issue in identifying the targets of drug-induced liver disease. Although liver disease associated with a particular drug is quite rare, the whole range of drug-induced liver disease is substantial, each case probably occurring against the genetic background of a patient predisposed to the side-eects of the drug. It has been known for quite a long time that drug-metabolizing enzymes play a major role in drug handling by the liver and that polymorphisms in the genes encoding these enzymes determine the behaviour of a drug in a particular individual (e.g. rapid versus slow acetylators). This knowledge will also be developed for the spectrum of transporter genes because it is clear that several of the canalicular transporters, such as MDR1 Pgp, MRP2 and BCRP, play an important role in drug clearance. We will therefore in future probably identify polymorphisms in these genes that cause reduced drug clearance with enhanced liver toxicity as a consequence. Drug-metabolizing enzymes, as well as transporters, constitute a large group of potential target genes for polymorphisms; collectively, these polymorphic sequences will have to be put on microarrays for screening patient groups with drug-induced liver disease. This will be of great importance for the further delineation of the role of transporters in liver toxicity arising from certain drugs and for diagnosis in patients.
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Understanding and controlling hepatobiliary function 1033 8. Lown KS, Mayo RR, Leichtman AB et al. Role of intestinal P-glycoprotein (mdr1) in interpatient variation in the oral bioavailability of cyclosporine. Clinical Pharmacology and Therapeutics 1997; 62: 248±260. 9. Panwala CM, Jones JC & Viney JL. A novel model of in¯ammatory bowel disease: mice de®cient for the multiple drug resistance gene, mdr1a, spontaneously develop colitis. Journal of Immunology 1998; 161: 5733±5744. 10. Paulusma CC, Bosma PJ, Zaman GJ et al. Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene. Science 1996; 271: 1126±1128. 11. Buchler M, Konig J, Brom M et al. cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMrp, reveals a novel conjugate export pump de®cient in hyperbilirubinemic mutant rats. Journal of Biological Chemistry 1996; 271: 15091±15098. 12. Konig J, Nies AT, Cui YH et al. Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate speci®city, and MRP2-mediated drug resistance. Biochimica et Biophysica Acta ± Biomembranes 1999; 1461: 377±394. 13. Keppler D & Konig J. Hepatic secretion of conjugated drugs and endogenous substances. Seminars in Liver Disease 2000; 20: 265±272. 14. Loe DW, Deeley RG & Cole SP. Characterization of vincristine transport by the M(r) 190,000 multidrug resistance protein (MRP): evidence for cotransport with reduced glutathione. Cancer Research 1998; 58: 5130±5136. 15. Dietrich CG, de Waart DR, Ottenho R et al. Increased bioavailability of the food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in MRP2-de®cient rats. Molecular Pharmacology 2001; 59: 974±980. 16. Doyle LA, Yang W, Abruzzo LV et al. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proceedings of the National Academy of Sciences of the USA 1998; 95: 15665±15670. 17. Maliepaard M, Scheer GL, Faneyte IF et al. Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues. Cancer Research 2001; 61: 3458±3464. 18. Jonker JW, Smit JW, Brinkuis RF et al. Role of breast cancer resistance protein in the bioavailability and fetal penetration of topotecan. Journal of the National Cancer Institute 2000; 92: 1651±1656. 19. Wang R, Salem M, Yousef IM et al. Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis. Proceedings of the National Academy of Sciences of the USA 2001; 98: 2011±2016. 20. Smit JJM, Schinkel AH, Oude Elferink RPJ et al. Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell 1993; 75: 451±462. 21. Ruetz S & Gros P. Phosphatidylcholine translocase: a physiological role for the mdr2 gene. Cell 1994; 77: 1071±1082. 22. Smith AJ, Timmermans-Hereijgers JLPM, Roelofsen B et al. The human MDR3 P-glycoprotein promotes translocation of phosphatidylcholine through the plasma membrane of ®broblasts from transgenic mice. FEBS Letters 1994; 354: 263±266. 23. van Helvoort A, Smith AJ, Sprong H et al. MDR1 P-glycoprotein is a lipid translocase of broad speci®city, while MDR3 P-glycoprotein speci®cally translocates phosphatidylcholine. Cell 1996; 87: 507±517. 24. Oude Elferink RP, Tytgat GN & Groen AK. Hepatic canalicular membrane. 1. The role of mdr2 P-glycoprotein in hepatobiliary lipid transport. FASEB Journal 1997; 11: 19±28. 25. Carlton VEH, Knisely AS & Freimer NB. Mapping of a locus for progressive familial intrahepatic cholestasis (Byler disease) to 18q21±q22, the benign recurrent intrahepatic cholestasis region. Human Molecular Genetics 1995; 4: 1049±1053. 26. Bull LN, van Eijk MJ, Pawlikowska L et al. A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis. Nature Genetics 1998; 18: 219±224. 27. Eppens EF, van Mil SWC, de Vree JM et al. FIC1, the protein aected in two forms of hereditary cholestasis, is localized in the canalicular membrane of the hepatocyte. Journal of Hepatology 2001; 35: 436±443. 28. Ujhazy P, Ortiz D, Misra S et al. Familial intrahepatic cholestasis 1: studies of localization and function. Hepatology 2001; 34: 768±775. 29. Whitington PF & Whitington GL. Partial external diversion of bile for the treatment of intractable pruritus associated with intrahepatic cholestasis. Gastroenterology 1988; 95: 130±136. 30. Emond JC & Whitington PF. Selective surgical management of progressive familial intrahepatic cholestasis (Byler's disease). Journal of Pediatric Surgery 1995; 30: 1635±1641. 31. Ismail H, Kalicinski P, Markiewicz M et al. Treatment of progressive familial intrahepatic cholestasis: liver transplantation or partial external biliary diversion. Pediatric Transplantation 1999; 3: 219±224.
1034 R. P. J. Oude Elferink 32. Ng VL, Ryckman FC, Porta G et al. Long-term outcome after partial external biliary diversion for intractable pruritus in patients with intrahepatic cholestasis. Journal of Pediatric Gastroenterology and Nutrition 2000; 30: 152±156. 33. Strautnieks SS, Kagalwalla AF, Tanner MS et al. Identi®cation of a locus for progressive familial intrahepatic cholestasis PFIC2 on chromosome 2q24. American Journal of Human Genetics 1997; 61: 630±633. 34. Strautnieks SS, Bull LN, Knisely AS et al. A gene encoding a liver-speci®c abc transporter is mutated in progressive familial intrahepatic cholestasis. Nature Genetics 1998; 20: 233±238. 35. Gerlo T, Stieger B, Hagenbuch B et al. The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver. Journal of Biological Chemistry 1998; 273: 10046±10050. 36. Noe J, Hagenbuch B, Meier PJ & St-Pierre MV. Characterization of the mouse bile salt export pump overexpressed in the baculovirus system. Hepatology 2001; 33: 1223±1231. 37. Strautnieks SS, Byrne J, Soler A et al. Progressive familial intrahepatic cholestasis: mutation analysis and evidence for a third locus, 2001; 184. 38. Deleuze JF, Jacquemin E, Dubuisson C et al. Defect of multidrug-resistance 3 gene expression in a subtype of progressive familial intrahepatic cholestasis. Hepatology 1996; 23: 904±908. 39. Jacquemin E, de Vree JML, Cresteil D et al. The wide spectrum of MDR3 de®ciency in patients with progressive familial intrahepatic cholestasis type 3: from neonatal cholestasis to cirrhosis of adulthood. Gastroenterology 2001; 120: 1448±1458. 40. de Vree JM, Jacquemin E, Sturm E et al. Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis. Proceedings of the National Academy of Sciences of the USA 1998; 95: 282±287. 41. Van Nieuwkerk CM, OudeElferink RP, Groen AK et al. Eects of ursodeoxycholate and cholate feeding on liver disease in FVB mice with a disrupted mdr2 P-glycoprotein gene. Gastroenterology 1996; 111: 165±171. 42. van Nieuwkerk CM, Groen AK, Ottenho R et al. The role of bile salt composition in liver pathology of mdr2 ( / ) mice: dierences between males and females. Journal of Hepatology 1997; 26: 138±145. 43. Muller M. Transcriptional control of hepatocanalicular transporter gene expression. Seminars in Liver Disease 2000; 20: 323±337. 44. Karpen S. Nuclear receptor regulation of hepatic function. Journal of Hepatology 2002 (in press). 45. Synold TW, Dussault I & Forman BM. The orphan nuclear receptor SXR coordinately regulates drug metabolism and eux. Nature Medicine 2001; 7: 584±590. 46. Jacquemin E, Cresteil D, Manouvrier S et al. Heterozygous non-sense mutation of the MDR3 gene in familial intrahepatic cholestasis of pregnancy. Lancet 1999; 353: 210±211. 47. Lindberg MC. Hepatobiliary complications of oral contraceptives. Journal of General Internal Medicine 1992; 7: 199±209. 48. Rosmorduc O, Hermelin B & Poupon R. MDR3 gene defect in adults with symptomatic intrahepatic and gallbladder cholesterol cholelithiasis. Gastroenterology 2001; 120: 1459±1467. 49. Thompson RJSS, Gerred S, Kniseley A et al. Adult onset cholangiopathy (AMA ve PBC) due to mutations in ABCB4 2001; 184.
doi:10.1053/bega.2002.0340, available online at http://www.idealibrary.com on
13 Understanding and controlling hepatobiliary function Ronald P. J. Oude Elferink Laboratory of Experimental Hepatology, AMC Liver Center, Academic Medical Center, Amsterdam, The Netherlands
Over the past decade, enormous progress has been made in identifying the mechanisms that underlie hepatobiliary excretion. A set of transport proteins mediates the canalicular transport of most important bile constituents. With the discovery of these transporter genes, the mechanism of bile formation could be partly elucidated and genetic defects caused by mutations in these genes identi®ed. This progress is crucial not only for paediatric and adult hepatology, but also for pharmacology, because the characterization of these transport systems provides tools for the prediction of the pharmacokinetics of drugs. Indeed, there is a growing interest on the part of the pharmaceutical industry for research into transport systems in general and hepatobiliary secretion in particular. For all of these transporter genes, knockout mice have been bred that allow one to assess the in vivo function of each of these transporters with regard to their role in physiology and drug elimination. Key words: bile; cholestasis; transport; ABC transporters.
TRANSPORT PROTEINS AND THE ENTEROHEPATIC CYCLING OF DRUGS AND TOXINS Nearly all canalicular transport proteins are so-called ATP-binding cassette (ABC) transporters. These comprise a large family of transporter genes with at least 48 members in humans. The transport proteins are involved in highly diverse transport steps across the plasma membrane and the membranes of intracellular organelles.1 The transport is driven by ATP hydrolysis, which enables uphill transport. A substantial fraction of these transporters mediate the extrusion of unwanted compounds from the cell. Because of this, the cell is defended against noxious compounds but, by virtue of their expression in the apical membrane of epithelia, these transporters also take care of the whole-body elimination of the unwanted drugs and toxins. Several of these transporters are expressed both in the apical membrane of enterocytes and in the canalicular membrane of hepatocytes. They thus have a double function as gate-keepers against toxic products. First, they immediately extrude compounds that have entered gut cells back into the lumen. Second, those molecules which do enter the bloodstream are taken up by the liver and extruded by the same transporters into bile. Using animals lacking these transporter genes, it can be shown that the oral availability of drugs can be to a large extent determined by the function of these transporters. c 2002 Elsevier Science Ltd. All rights reserved. 1521±6918/02/$ - see front matter *
1026 R. P. J. Oude Elferink
MDR1 P-glycoprotein The prototype of the drug extrusion pump is MDR1 P-glycoprotein (Pgp), which excretes a very broad spectrum of amphipathic drugs out of cells, thereby conferring multidrug resistance against these substances. Indeed, this gene was discovered by its overexpression in multidrug-resistant tumour cells, but it subsequently became clear that the MDR1 gene is also expressed in normal tissues. The role of MDR1 Pgp in protecting against xenobiotics has been ®rmly established by the generation of mice lacking both the Mdr1a and Mdr1b genes (in mice, two Mdr1 genes ful®lling the function of the single MDR1 gene in humans). This knockout animal is hypersensitive to a number of toxic compounds, important sites of expression of the protein being the blood±brain barrier, the intestinal epithelium and the hepatocanalicular membrane.2,3 It can be shown, again by use of the knockout mice, that Mdr1a Pgp is involved in the biliary elimination of several amphipathic drugs, such as digoxin and vecuronium.3±5 Another important site of expression of MDR1 is the gut, where the protein is localized to the apical membrane of the enterocytes and mediates the direct elimination of compounds back into the gut lumen. As a result, MDR1 determines the oral availability of many drugs. Polymorphisms have been detected in the MDR1 gene6,7, and these may be associated with alterations in the oral bio-availability of certain drugs.8 The polymorphism C3435T has been found to be associated with reduced MDR1 Pgp expression in the duodenum, this correlating with an increased oral bio-availability of digoxin.6 It is interesting to note that Panwala et al9 detected a high incidence of colitis in Mdr1a / mice. This chronic intestinal in¯ammation could be prevented by treatment with a mix of broad-spectrum anitbiotics, suggesting that intestinal bacteria and/or their products play a role in this phenomenon. It remains to be established whether this phenomenon is the primary consequence of the absence of Mdr1a Pgp or a secondary eect. It may be hypothesized, however, that an impaired elimination of luminal toxins predisposes the animals to an in¯ammatory process. If so, reduced MDR1 expression in humans could theoretically also predispose towards intestinal in¯ammation.
MRP2 MRP2 was discovered as a liver-speci®c homologue of another multidrug resistance transporter, MRP1.10,11 The two transporters have opposite membrane localizations: MRP1 is expressed in the basolateral membrane and MRP2 in the apical membrane of epithelia. Whereas MRP1 is expressed in most cell types, MRP2 is predominantly found in hepatocytes and to a lesser extent in enterocytes and kidney proximal tubule cells.12,13 MRP1 and MRP2 have highly similar substrate speci®cities.12,13 Both transporters recognize a wide spectrum of organic anions, including unconjugated anions as well as anionic conjugates of glucuronic acid (such as conjugated bilirubin), glutathione and sulphate. It has more recently become clear that these transporters can also pump uncharged compounds. It appears that the transporters can accommodate uncharged amphipathic compounds together with a glutathione molecule in a cotransport mechanism. Experiments with plasma membrane vesicles containing MRP1 have revealed that this transporter does not to any signi®cant extent pump vincristine. The addition of GSH, however, strongly stimulated the translocation of vincristine.14 A similar involvement of glutathione was found for the MRP2-mediated transport of the uncharged carcinogen PhIP.15 This mechanism greatly enhances the versatility of these
Understanding and controlling hepatobiliary function 1027
transporters. In the intestine, MRP2 may play a role similar to that of MDR1 Pgp in the defence against toxic compounds in the gut lumen.15 Breast cancer-related protein Breast cancer-related protein (BCRP, also termed MXR or ABCP; ABCG2 gene) has more recently been identi®ed as yet another ABC transporter that is overexpressed in anthracycline-resistant cell lines.16 Upon cloning its cDNA, it turned out to be a halftransporter, of which more examples exist. These half-transporters are believed to dimerize, as either homodimers or heterodimers, into functional pumps. Transfection of ABCG2 cDNA confers resistance against mitoxantrone, topotecan, doxorubicine, daunorubicine and rhodamine 123.16 Using monoclonal antibodies against the ABCG2 gene product, Maliepaard et al17 demonstrated that this protein is present in the plasma membrane of endothelial cells and in the apical membrane of intestinal epithelial cells (both colon and small intestine) and hepatocytes. The latter ®nding strongly suggests that it is also involved in biliary secretion of amphipaths. Using a speci®c BCRP inhibitor, GF120918, Jonker et al18 showed that this transporter reduces the oral bio-availability of topotecan and other BCRP substrates in the intestine by pumping these compounds back into the lumen. Thus, BCRP, as well as MDR1 and MRP2, serves as a gate-keeper in the gut by reducing the oral bio-availability of drugs and toxins. Bile salts Bile salts are pumped across the canalicular membrane via the bile salt export pump (BSEP). The BSEP gene displays a high extent of homology with MDR1 (which encodes the drug transporter described above), and on this basis the two genes fall into the same subclass of ABC transporters. Wang et al19 have reported on a mouse de®cient in the Bsep gene. Bsep / mice are cholestatic in the sense that taurocholate accumulates in the liver and little taurocholate is excreted into bile, but these mice excrete a substantial amount of tauromuricholate as well as a hitherto unde®ned tetrahydroxy bile salt, into the bile. Bsep is apparently not the only bile salt transporting system in the canalicular membrane of murine hepatocytes, another system being capable of excreting tauromuricholate and the tetrahydroxy bile salt. This escape route prevents severe and progressive cholestasis in these mice and, as a consequence, these animals have hardly any histopathological signs of liver injury. Because humans are not to any signi®cant extent capable of converting bile salts into muricholate or tetrahydroxy bile salts, this escape route is not present in them. As a consequence, mutations in the BSEP gene are expected to cause a cholestatic syndrome in humans, which is indeed the case (see below). On the basis of these observations, BSEP is believed to be the main bile salt transporter in the canalicular membrane of human hepatocytes. Biliary lipids Biliary lipids are an important component of bile. They consist mainly of phospholipid (almost exclusively phosphatidylcholine) and cholesterol. The relatively high concentration of cholesterol in human bile represents the major risk factor in gallstone formation. Upon the production of a knockout mouse lacking the Mdr2 gene, it was discovered that the encoded transporter is essential for biliary lipid secretion.20 In the absence of Mdr2 Pgp, mice secrete neither phospholipid nor cholesterol. In dierent experimental systems, it was subsequently demonstrated that the murine
1028 R. P. J. Oude Elferink
Mdr2 Pgp as well as the human orthologue MDR3 Pgp are phosphatidylcholine translocators.21±23 The MDR3 gene is homologous to MDR1 and BSEP, and all three fall into the same subclass of ABC transporters. On the basis of the data available, we have proposed a model24 in which phosphatidylcholine, after delivery to the inner lea¯et of the canalicular plasma membrane, is translocated to the outer lea¯et and subsequently resides in phosphatidylcholine-rich lipid domains. These more ¯uid, phosphatidylcholine-rich microdomains are destabilized by the canalicular bile salt molecules and, in combination with the continuous pumping of phosphatidylcholine into these domains, a vesiculation of the phosphatidylcholine-rich membrane bilayer occurs. These vesicles then pinch o to give rise to typical biliary lipid vesicles.24 The mechanism of cholesterol secretion into bile is still largely unknown. No direct evidence yet exists for the involvement of a translocator protein which would, as with Mdr2/MDR3 Pgp for phospholipids, catalyse the translocation of cholesterol across the membrane. Recent evidence suggests that the two half-transporters ABCG5 and ABCG8 are involved in the elimination of plant sterols, which have a molecular structure very similar to that of cholesterol. Like ABCG2, ABCG5 and ABCG8 are half-transporters that have to dimerize into a functional pump. The transport of sterols by the ABCG5/G8 couple has so far not been directly demonstrated, but this function can be inferred from the accumulation of plant sterols in patients with sitosterolaemia, who have a mutation in either of the two genes encoding these proteins (see below). ABCG5 and ABCG8 are expressed in the intestine and liver. DEFECTS IN CANALICULAR TRANSPORTERS All the transport proteins described and their functions have been identi®ed within the past decade. After or because of their identi®cation, inherited liver diseases caused by mutations in the respective ABC transporter genes were also recognized. Some of these have a severe phenotype and are therefore primarily paediatric disorders. The common symptoms are chronic cholestasis and cirrhosis that often progresses to liver failure. These conditions have been collectively called progressive familial intrahepatic cholestasis (PFIC), but we now know that this group consists of at least three subgroups (types 1, 2 and 3) that are caused by mutations in the FIC1, BSEP and MDR3 genes respectively. The latter two gene products have been described above. Types 1 and 2 PFIC can be distinguished from type 3 by the fact that the patients with the latter have an increased serum gamma-glutamyltransferase (GGT) activity. Progressive familial intrahepatic cholestasis type 1 and benign recurrent intrahepatic cholestasis Type 1 PFIC (formerly called Byler's disease) manifests itself primarily as a chronic intrahepatic cholestasis. The bile salt concentration is high in serum and very low in bile, suggesting that hepatic transport is impaired. In addition, patients with type 1 PFIC suer from watery diarrhoea. The jaundice in these patients is regarded as a secondary consequence of insucient bile ¯ow, the latter being dependent on bile salt secretion. A genetic screen was performed on patients from the Byler pedigree and their family members in order to identify the disease locus.25 This was located on chromosome 18q21±q22, the same chromosomal region to which a very similar disease, benign recurrent intrahepatic cholestasis (BRIC), has been localized. BRIC is
Understanding and controlling hepatobiliary function 1029
a genetic disorder in which patients suer from periods of cholestasis that are of unpredictable onset and length, and that leave no liver damage. A combined search for the two disease loci led to the identi®cation of the mutated gene FIC1 (gene code ATP8B1), and its mutations in a group of patients, including those from the original Byler pedigree.26 Many issues concerning the function of the FIC1 gene product remain puzzling. Although FIC1 is localized to the canalicular membrane of the hepatocyte27, expression in the liver is actually quite low, whereas the gene is highly expressed in intestine and pancreas.26 Expression also occurs in many other tissues. It is therefore unclear why the absence of FIC1 from many tissues leads primarily to a phenotype in the liver and intestine. FIC1 is not an ABC transporter but a P-type ATPase, the function of which is proposed to be the translocation of aminophospholipids (a ¯ippase).28 The absence of FIC1 leads to defective bile salt secretion, but how its ¯ippase activity relates to this defect is unknown. We have recently shown that mice bearing one of the mutations that have been detected in type 1 PFIC patients do not suer from cholestasis but instead display enhanced intestinal absorption of bile salts (L. Pawlikowska, in preparation). This suggests that the cholestatic phenotype in these patients may be indirect and caused by enhanced absorption of (cholestatic) bile salt (metabolites). This hypothesis would ®t with the observed relief of cholestasis in type 1 PFIC patients upon chronic bile diversion.29±32 Nevertheless, the liver must contribute to the phenotype because liver transplantation ameliorates the hepatic symptoms in these patients. Extrahepatic symptoms, such as watery diarrhoea, remain after liver transplantation. Progressive familial intrahepatic cholestasis type 2 The phenotype of this form of PFIC is very similar to that of type 1 except that type 2 patients have no extrahepatic symptoms. Strautnieks et al33 demonstrated that the disease locus of this subgroup of patients lies on chromosome 2q24. They subsequently found that these patients have mutations of their BSEP gene, which is present in this region.34 The fact that mutations of the BSEP gene lead to a virtual absence of bile salts from the bile has led to the conclusion that BSEP is the main bile salt transporter in the canalicular membrane. This is supported by studies in which rat and murine Bsep was transfected into Sf9 cells and shown to transport several bile salts.35,36 No data are yet available on the transport characteristics of human BSEP. Nonsense, missense mutations and deletions of the BSEP gene have been found in some patients with low-GGT PFIC, these individuals now being described as having type 2 PFIC.33 Very importantly, further screening by Strautnieks et al37 seems to indicate that yet another subgroup of patients exists in whom the disease locus localizes to neither the FIC1 region nor the BSEP region. This would mean that another gene is involved in low-GGT PFIC. Progressive familial intrahepatic cholestasis type 3 As mentioned above, this form of PFIC is fundamentally dierent from types 1 and 2 in that aected patients have a high serum GGT activity. The onset of this disease is somewhat later than that of the other two forms, but the histological picture is more severe, with strong bile duct proliferation and cirrhosis. The most prominent features of the disease are portal hypertension, hepatosplenomegaly, jaundice and pruritus. If untreated, the disease develops into liver failure. The genetic background of this subgroup of PFIC patients was elucidated after it was found that mice with a disruption of the Mdr2 gene, the murine orthologue of MDR3, develop a similar
1030 R. P. J. Oude Elferink
phenotype.20 This led Deleuze et al38 to investigate the possible involvement of this gene in PFIC. Subsequently, in a group of 31 patients with high-GGT PFIC, 17 were found to have a mutation in the MDR3 gene.39,40 Since the gene was not completely sequenced in all these patients, it is not clear whether the remaining 14 patients had as yet unidenti®ed mutations or whether another gene might be involved in this form of PFIC. MDR3 Pgp functions in the translocation of phosphatidylcholine, thereby facilitating the secretion of this phospholipid into the bile. The secretion of phospholipid is of crucial importance in protecting the cellular membranes of the biliary tree against the high concentrations of bile salt detergents.24 The treatment of patients with ursodeoxycholate (UDCA) appeared to be bene®cial in about half of the cases.39 Given the fact that the hepatic damage is caused by bile salts, this is a rational treatment because UDCA is a hydrophilic bile salt that has low cytotoxicity. Indeed, in Mdr2 / mice with the equivalent defect, feeding this bile salt also halted the disease process.41,42 The observation that not all patients improve on UDCA treatment may be explained by the fact that administration of this bile salt replaces the endogenous bile salt pool only insuciently. Thus, in patients who have no MDR3 Pgp activity at all, the reduction of bile salt cytotoxicity by UDCA is insucient, whereas in patients with residual phospholipid secretion, UDCA might bring the overall bile salt cytotoxicity below a critical threshold. Indeed, none of the patients with a truncated MDR3 gene improved on UDCA, whereas some of those with missense mutations did.39 The administration of higher doses of UDCA may be particularly useful in these patients. Sitosterolaemia Mutations in the ABCG5 or ABCG8 gene are the cause of sitosterolaemia. Patients with this inherited disorder accumulate a large amount of plant sterols, such as sitosterol, in their circulation. In normal individuals, the uptake of plant sterols in the gut is very low because ABCG5/ABCG8 pumps these molecules back into the gut lumen. In addition, the low amount of plant sterols that does enter the circulation is eciently excreted by ABCG5/ABCG8 in the canalicular membrane of the hepatocyte. Interestingly, these patients not only have high blood plant sterol levels, but also suer from hypercholesterolaemia, suggesting that cholesterol is also transported by this couple. Thus, in the absence of ABCG5/ABCG8, there will be an enhanced absorption of cholesterol in the gut and a decreased secretion of cholesterol in the bile. This is, however, currently speculative and remains to be demonstrated in ABCG5/ABCG8 knockout mice. Dubin±Johnson syndrome Mutations in the MRP2 gene cause Dubin±Johnson syndrome, characterized by a conjugated hyperbilirubinaemia. In contrast to its unconjugated form, glucuronidated bilirubin is not toxic, and this rare syndrome is considered to be benign. In the animal model of this syndrome, the TR rat, we were able to demonstrate that there is an enhanced oral availability of the food-derived carcinogen PhIP and probably also many other food-derived compounds. There are anecdotal reports of primary HCC, but the number of Dubin±Johnson patients is probably too small to elucidate whether this is a signi®cant side-eect of MRP2 de®ciency. It may be expected that polymorphisms in this gene leading to reduced expression or activity will be associated with an altered bioavailability of many drugs, but such polymorphisms have not yet been characterized.
Understanding and controlling hepatobiliary function 1031
REGULATION OF TRANSPORTER EXPRESSION Over the past few years, it has been shown that the expression of ABC transporters, including those responsible for bile formation, is subject to tight regulation. In particular, the family of nuclear receptors plays a prominent role in the transcriptional control of ABC transporter genes.43,44 The common theme in this family is that dimers of RXR (the retinoid receptor that binds retinoic acid) and a ligand-speci®c counterpart bind to a responsive element in the promoter region of a gene and thereby regulate transcription. In this fashion, BSEP expression is controlled by the nuclear receptor pair RXR/FXR, in which FXR is the farnesoid receptor, which has high anity for bile salts. Hence, the binding of bile salts to FXR in the dimer RXR/ FXR induces transcription of the BSEP gene, leading to enhanced protein levels and increased biliary bile salt secretion. ABCG5 and ABCG8 are located on chromosome 2p21 in a close head-to-head arrangement. These genes are co-ordinately expressed and are strongly induced by LXR (the `liver-speci®c' receptor), the latter nuclear receptor being a sensor for cholesterol as it binds oxysterols. Many genes involved in cholesterol metabolism and transport are regulated by LXR. The observation that ABCG5 and ABCG8 are regulated by LXR again suggests that this transporter couple is involved in the transport of cholesterol. A recent report suggests that MRP2 is regulated by three nuclear receptors, FXR, CAR and PXR.44 PXR (in rodents) and SXR (in humans) are receptors that accomodate as the ligand a large number of xenobiotics, for example rifampicin (SXR only), hyperforin (from St. John's wort), dexamethasone, the HIV protease inhibitor ritonavir, taxol and others.44 MDR1 expression is also regulated by SXR.45 Interestingly, the latter nuclear receptors also induce cytochrome P450 enzymes from the important CYP3A family. Hence, certain drugs induce their own metabolism as well as their secretion by the induction of CYP isoforms and drug pumps respectively. This will become an important issue in pharmacokinetics and drug interaction. THE FUTURE OF HEPATOBILIARY TRANSPORT RESEARCH Genetic disorders associated with the most important transport proteins in the canalicular membrane have now been identi®ed and characterized. These disorders are of minor importance for hepatology in general because they are extremely rare and con®ned mostly to the paediatric arena. This will, however, change in the future because the monogenic inherited disorders are only the tip of the iceberg. These disorders are the simplest situation encountered in transport defects, namely the complete absence of a single transport protein. Polymorphisms causing a reduction in transport protein level and/or activity will, however, be much more frequent because the consequences of such polymorphisms are much less severe. Such polymorphisms will nevertheless predispose individuals to cholestasis and/or other forms of liver disease under conditions of reduced liver function that are caused by other genetic or environmental conditions. A good example of this situation is MDR3 de®ciency. A complete absence of this phospholipid-translocating protein causes severe (type 3 PFIC) liver disease, as described above. More recently, it was, however, found that heterozygous females in the family of a type 3 PFIC index patient developed cholestasis of pregnancy.46 Under normal conditions, these women do not have clinical symptoms, but in the last trimester of pregnancy there is apparently a general impairment of bile formation that,
1032 R. P. J. Oude Elferink
combined with a decrease in the level of MDR3 expression, leads to hepatic damage. Indeed, other genetic forms of cholestasis may be unmasked by the general impairment of bile formation during pregnancy.47 Several individuals with gallstones have also been reported to have mutations of the MDR3 gene that did not occur in the control population.48 This suggests that mutations in the MDR3 gene may predispose to gallstone formation. Finally, an interesting recent preliminary report by Thompson et al49 suggested that mutations in the MDR3 gene may predispose to the development of primary biliary cirrhosis as they found MDR3 mutations in patients with primary biliary cirrhosis who didn't demonstrate anti-mitochondrial antibodies. These observations illustrate how mutations of transporter genes are not only relevant for rare inherited diseases, but may also play a role in more common disease phenotypes. Canalicular transport proteins will also become an important issue in identifying the targets of drug-induced liver disease. Although liver disease associated with a particular drug is quite rare, the whole range of drug-induced liver disease is substantial, each case probably occurring against the genetic background of a patient predisposed to the side-eects of the drug. It has been known for quite a long time that drug-metabolizing enzymes play a major role in drug handling by the liver and that polymorphisms in the genes encoding these enzymes determine the behaviour of a drug in a particular individual (e.g. rapid versus slow acetylators). This knowledge will also be developed for the spectrum of transporter genes because it is clear that several of the canalicular transporters, such as MDR1 Pgp, MRP2 and BCRP, play an important role in drug clearance. We will therefore in future probably identify polymorphisms in these genes that cause reduced drug clearance with enhanced liver toxicity as a consequence. Drug-metabolizing enzymes, as well as transporters, constitute a large group of potential target genes for polymorphisms; collectively, these polymorphic sequences will have to be put on microarrays for screening patient groups with drug-induced liver disease. This will be of great importance for the further delineation of the role of transporters in liver toxicity arising from certain drugs and for diagnosis in patients.
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