Localization and function of the organic anion–transporting polypeptide Oatp2 in rat liver

GASTROENTEROLOGY 1999;117:688–695

Localization and Function of the Organic Anion–Transporting Polypeptide Oatp2 in Rat Liver CHRISTOPH REICHEL,* BO GAO,* JESSICA VAN MONTFOORT,* VALENTINO CATTORI,*,‡ CHRISTOPH RAHNER,§ BRUNO HAGENBUCH,* BRUNO STIEGER,* TOSHINORI KAMISAKO,\ and PETER J. MEIER* *Division of Clinical Pharmacology and Toxicology, Department of Internal Medicine, University Hospital, Zu¨rich, Switzerland; ‡Laboratory for Biochemistry, Swiss Federal Institute of Technology, Zu¨rich, Switzerland; §Department of Anatomy, University of Basel, Basel, Switzerland; and \Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany

Background & Aims: Multispecific organic anion–transporting polypeptides (Oatps) are involved in the transcellular movement of amphipathic compounds in many tissues including the liver, kidney, and blood–brain barrier. Recently, a high-affinity digoxin transporter (Oatp2) was cloned from rat brain and shown to be also expressed in the liver. Methods: We investigated the cellular and subcellular distribution of Oatp2 in rat liver by in situ hybridization technology and immunofluorescence microscopy and compared its substrate specificity with that of Oatp1 in complementary RNA–injected Xenopus laevis oocytes. Results: The results show a selective basolateral (sinusoidal) expression of Oatp2 in midzonal to perivenous hepatocytes, but not in periportal or the innermost layer of perivenous hepatocytes. Common substrates of both Oatp1 and Oatp2 include bile salts, steroid conjugates, thyroid hormones (T3, T4), ouabain, and the endothelin receptor antagonist BQ-123 (Michaelis constants: Oatp1, D600 ␮mol/L; Oatp2, D30 ␮mol/L). Other organic anions including sulfolithotaurocholate, bilirubin monoglucuronide, and sulfobromophthalein were transported only by Oatp1. Conclusions: These results provide definite evidence for the partially overlapping and partially selective substrate specificities of Oatp1 and Oatp2. The unique acinar distribution of Oatp2 might indicate that it represents a high-affinity ‘‘backup’’ system for complete hepatocellular removal of certain cholephilic substances from portal blood plasma.

he liver efficiently extracts a large variety of amphipathic compounds from sinusoidal blood plasma. For bile salts, the major hepatocellular uptake pathway is mediated by the Na⫹/taurocholate–cotransporting polypeptides Ntcp (rodents) and NTCP (human).1 For other amphipathic albumin-bound compounds, polyspecific Na⫹-independent organic anion–transporting polypeptides (Oatps) have been cloned from rat (Oatp1) and human (OATP) liver2–5 and from rat brain (Oatp2, Oatp3).6,7 The best characterized member of the Oatp gene family of membrane transporters is Oatp1, which is

T

localized at the basolateral membrane of hepatocytes.5,8–11 On the basis of Northern blot analysis, Oatp2 is also known to be strongly expressed in the liver,6,7 but its exact function and hepatocellular in situ localization have not yet been studied. Therefore, we investigated the cellular and subcellular distribution of Oatp2 in intact rat liver by in situ hybridization and immunofluorescence microscopy. In addition, the substrate specificity of Oatp2 vs. Oatp1 was further investigated in complementary RNA (cRNA)-injected Xenopus laevis oocytes. The studies showed a selective basolateral expression of Oatp2 in midzonal to perivenous hepatocytes of normal rat liver. Furthermore, the endothelin A (ETA)-receptor antagonist BQ-123 has been identified as a new substrate for both Oatp1 and Oatp2, whereas several other Oatp1 substrates, including bilirubin monoglucuronide (BMG), sulfobromophthalein (BSP), and leukotriene C4 (LTC4), were not transported by Oatp2.

Materials and Methods Chemicals [Prolyl-3,4(n)-3H]BQ-123 (43.0 Ci/mmol) was obtained from Amersham Life Science (Amersham, England). Unlabeled BQ-123 was purchased from Research Biochemicals Internet (Natick, MA). [3H]LTC4 (165 Ci/mmol), [3H]dehydroepiandrosterone sulfate (DHEAS) (16 Ci/mmol), [3H]␣ketoglutarate (280.8 Ci/mmol), and [3H]p-aminohippurate (5 Ci/mmol) were purchased from DuPont–New England Nuclear (Boston, MA). [14C]Dinitrophenylglutathione (10 mCi/mmol) was synthesized as described by Ishikawa.12 Uridine triphosphate [3H]glucuronic acid (0.6 Tbq/mmol) was obtained from Biotrend (Ko¨ln, Germany). Radiolabeled BMG was synthesized as described using microsomes from UGT1A1-transAbbreviations used in this paper: BMG, bilirubin monoglucuronide; BSP, sulfobromophthalein; cRNA, complementary RNA; DHEAS, dehydroepiandrosterone sulfate; ETA, endothelin A; LTC4, leukotriene C4; OATP, organic anion–transporting polypeptide (human); Oatp, Naⴙ-independent organic anion–transporting polypeptide (rat); SDS, sodium dodecyl sulfate. r 1999 by the American Gastroenterological Association 0016-5085/99/$10.00

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fected HeLa cells.13 [35S]BSP was prepared at a specific radioactivity of 1.5 Ci/mmol by using the method of Kurisu et al.14 The following radiolabeled bile salt derivatives were kindly provided by A. F. Hofmann and C. D. Schteingart of the University of California at San Diego (La Jolla, CA): [3H]glycocholate (30 Ci/mmol), [2-3H]taurochenodeoxycholate (14 Ci/ mmol), [2-3H]tauroursodeoxycholate (14.3 Ci/mmol), and [3H]sulfotaurolithocholate (0.79 Ci/mmol). Molecular biological enzymes and reagents were obtained from Boehringer Mannheim (Mannheim, Germany), Promega (Madison, WI), and Amersham Pharmacia Biotech (Uppsala, Sweden). All other chemicals were obtained in the highest degree of purification available from Merck (Dietikon, Switzerland), Sigma (St. Louis, MO), or Fluka (Buchs, Switzerland). Hybridomas secreting monoclonal antibodies against aminopeptidase N were the generous gift of Dr. Andrea Quaroni (Cornell University, Ithaca, NY).15 Secondary antibodies labeled with Cy2 and Cy3 were obtained from Amersham Pharmacia Biotech (Amersham, England) and Jackson ImmunoResearch Laboratories (West Grove, PA).

Animals Male Sprague–Dawley rats (RCC Ltd., Fu¨llinsdorf, Switzerland) weighing 200–300 g were used. Mature X. laevis females were purchased from the African Xenopus Facility (e.c. Noordusek, Knysna, Republic of South Africa) and kept under standard conditions as described previously.16 All studies were performed in accordance with the Swiss Federal regulations concerning animal care.

In Situ Hybridization Histochemistry Full-length antisense and sense Oatp2-cRNAs were prepared as riboprobes and labeled with digoxigenin as described previously.17 After rats were decapitated, livers were removed and frozen immediately on dry ice. Cryostat sections (12 µm) were mounted onto glass slides coated with 3-aminopropyltrienthoxysilane (Sigma) and stored at ⫺80°C until use. The sections were fixed with 2% paraformaldehyde in phosphate-buffered saline for 20 minutes, acetylated in 0.1 mol/L triethanolamine hydrochloride containing 0.25% acetic anhydride, and prehybridized for 2–3 hours at room temperature with hybridization buffer containing 50% formamide, 5⫻ standard saline citrate (SSC), 5⫻ Denhardt’s solution, 250 µg/mL yeast transfer RNA, and 500 µg/mL herring sperm DNA. The sections were subsequently hybridized overnight at 53°C with the digoxigenin-labeled antisense or sense probes dissolved in the hybridization buffer at a concentration of ⬃1 µg/mL. Sections were then sequentially washed at 53°C with SSC using a final concentration of 0.1⫻ SSC/50% formamide for 20 minutes. Thereafter, the sections were processed for immunodetection with the anti–digoxigenin-alkaline phosphatase (Boehringer Mannheim) and with nitrotetrazolium blue and X-phosphate as the unlabeled substrate (Boehringer Mannheim). The specificity of in situ hybridization histochemistry was assessed by hybridization of adjacent sections with the sense probe as the negative control.

HEPATIC LOCALIZATION AND FUNCTION OF Oatp2 689

Antibodies and Western Blotting The antiserum against a fusion protein spanning the C-terminal 40 amino acids of Oatp1 has been characterized previously.5,18 Oatp2 antibodies were raised against a synthetic peptide consisting of the 15 carboxy-terminal amino acids of Oatp2 (amino acids 648–661)6 coupled to keyhole limpet hemocyanine at its C terminus via an additional tyrosine. Rabbits were immunized as described previously.19 The specificity of the antiserum was tested with isolated basolateral rat liver plasma membranes.20 Sodium dodecyl sulfate (SDS)– polyacrylamide gel electrophoresis and Western blotting were performed according to standard procedures.21,22

Immunofluorescence Studies Rat livers were fixed by perfusion and tissue sections (0.5–1 µm) incubated with the antisera as described previously.19 Micrographs were taken with a Zeiss Axiophot epifluorescence microscope (Zeiss, Oberkochen, Germany).

Transport Assays in X. laevis Oocytes In vitro synthesis of Oatp1- and Oatp2-cRNAs was performed from the cloned cDNAs as described previously.4 Oocytes were prepared and incubated overnight at 18°C.4,23 Healthy oocytes were microinjected with cRNAs encoding Oatp1 or Oatp2 or with the same volume of H2O as controls and subsequently cultured for 2–3 days to allow the expression of the carriers in the oocyte plasma membrane. Tracer uptake studies were performed in a medium that consisted of 100 mmol/L choline chloride, 2 mmol/L KCl, 1 mmol/L CaCl2, 1 mmol/L MgCl2, and 10 mmol/L HEPES adjusted to pH 7.5 with Tris. Ten to 15 oocytes were prewashed in the incubation medium and then incubated at 25°C in 100 µL of the same medium containing the radiolabeled substrate. Subsequently the oocytes were washed with 3 mL of ice-cold incubation buffer. Each oocyte was dissolved in 10% SDS. After addition of 5 mL of scintillation fluid (Ultima Gold; Canberra Packard, Zu¨rich, Switzerland), the oocyte-associated radioactivity was measured in a Packard Tri-Carb 2200 CA liquid scintillation analyzer (Canberra Packard).

Statistical Analysis Data are expressed as means ⫾ SD. Kinetic parameters were determined by computer-based nonlinear regression analysis of initial uptake values fitted to the Michaelis–Menten equation. To compare uptakes between oocytes injected with H2O and different cRNAs, the nonparametric Mann–Whitney U test with post hoc adjustment for multiple comparisons was used.

Results Cellular and Subcellular Distribution of Oatp2 The reactivity of the polyclonal antibodies with basolateral rat liver plasma membrane proteins is illus-

690 REICHEL ET AL.

trated in Figure 1. Whereas the Oatp1 antiserum reacted with an ⬃80-kilodalton protein, the Oatp2 antiserum reacted with a basolateral protein of ⬃92 kilodaltons, indicating that both antisera do not cross-react and specifically recognize their respective antigens. Furthermore, the molecular mass of ⬃92 kilodaltons indicates that the native liver Oatp2 is considerably more glycosylated than its analogue at the blood–brain barrier (⬃76 kilodaltons).17 More extensive N-glycosylation of the mature basolateral Oatp2 may also explain the discrepancy between our results and the molecular mass of ⬃76 kilodaltons recently reported in total rat liver microsomes.24 A study using in situ hybridization technology showed that the Oatp1-mRNA is homogeneously distributed within the liver cell acinus.25 This homogeneous parenchymal distribution of Oatp1-mRNA contrasts to a more compartmentalized expression of Oatp2 mRNA in midzonal to pericentral hepatocytes (Figure 2). On the level of the individual carrier proteins, both Oatp1 and Oatp2 are expressed at the basolateral membrane of hepatocytes, and their parenchymal distribution pattern was found to parallel the expression of their mRNAs (Figure 3). Thus, whereas Oatp1 is expressed at the basolateral membrane of all hepatocytes to approximately similar extents (Figure 3A), Oatp2 expression is concentrated around the central hepatic veins (Figure 3B). In periportal hepatocytes, Oatp2 was detected either not at all or only to a minor extent (Figure 3C). In addition, and most interestingly, the innermost 1–2 cell layers around the central veins were also virtually devoid of Oatp2 (Figure 3D). Neither Oatp1 nor Oatp2 was detected in nonparenchymal cells or in bile ductular cells to any significant extent. Functional Differences Between Oatp1 and Oatp2 As summarized in Table 1, Oatp1 and Oatp2 show partially overlapping and partially distinct substrate

Figure 1. Specificity of the Oatp1 and Oatp2 antibodies. Antibodies against the C-terminal ends of Oatp1 and Oatp2 were raised in rabbits and used for Western blotting of basolateral liver plasma membrane proteins as described in Materials and Methods. Initial SDS– polyacrylamide gel electrophoresis was performed with 100 µg of basolateral liver plasma membrane protein in each lane. The differences in the mean molecular mass of Oatp1 and Oatp2 indicate the specificity of the antisera used.

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Figure 2. Distribution of Oatp2 mRNA in intact rat liver. In situ hybridization was performed as described in Materials and Methods by using Oatp2 (A) antisense and (B) sense riboprobes. (A) Acinar distribution of Oatp2-mRNA (c, central hepatic vein; p, periportal area). (B) No specific signal was obtained with the sense riboprobe.

specificities. Functional expression studies in X. laevis oocytes identified the bile salts glycocholate, taurochenodeoxycholate, and tauroursodeoxycholate as well as the steroid conjugate DHEAS as new common substrates of both carrier proteins (Table 1). In addition, the anionic cyclopentapeptidic ETA-receptor antagonist BQ-123 was transported in a saturable manner by both Oatp1 (Michaelis constant [Km], ⬃600 µmol/L; maximal transport velocity [Vmax], ⬃250 fmol/oocyte · min) and Oatp2 (Km, ⬃30 µmol/L; Vmax, ⬃63 fmol/oocyte · min; Figure 4). Because the Km value of Oatp2 for BQ-123 is closer to the Km value reported for sodium-independent BQ-123 uptake into rat hepatocytes (⬃12 µmol/L),26 the data indicate that Oatp2 significantly contributes to the hepatic clearance of BQ-123 in normal rat liver. In addition to the common substrates, a variety of compounds were found to be transported preferentially or even selectively by either Oatp1 or Oatp2. These include

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HEPATIC LOCALIZATION AND FUNCTION OF Oatp2 691

Figure 3. Immunolocalization of Oatp1 and Oatp2 in intact rat liver. Semithin cryosections of rat liver were probed with the antisera against Oatp1 and Oatp2. Labeling was visualized with a green fluorescent secondary antibody. Bile canaliculi are visualized with a monoclonal antibody against aminopeptidase N (red fluorescence). (A) Overview showing homogeneous basolateral expression of Oatp1 throughout the hepatic lobule. (B) Overview showing compartmentalized basolateral expression of Oatp2 in midzonal to perivenous hepatocytes. (C and D) Higher-magnification views of Oatp2 expression in the (C) periportal and (D) perivenous areas. CV, central vein. PV, portal vein.

especially the cardiac glycoside digoxin for Oatp2 and the organic anions sulfolithotaurocholate, BMG, BSP, LTC4, dinitrophenyl-glutathione, and the magnetic resonance imaging compound gadoxetate for Oatp1 (Figure 5 and Table 1). The hydrophilic organic anions ␣-ketoglutarate and p-aminohippurate were not transported by Oatp1 or Oatp2 at all (Table 1).

Discussion The present study shows a unique compartmentalized basolateral expression of Oatp2 in parenchymal liver cells of normal rats on both the mRNA (Figure 2) and protein (Figure 3B–D) levels. Recently, other investigators reported similar results on the immunohistochemical level.24 Our data suggest that Oatp2 is not expressed in the periportal proliferative compartment and requires either aging and differentiation of hepatocytes within the individual liver cell plates27 and/or is dependent on regional signaling molecules and compartmentalized transcription factors.28 Its highest expression appears to

occur in hepatocytes exposed to a lower oxygen tension and in the area of most active biotransformation.27 However, Oatp2 expression is again lost at the terminal 1–2 cell layers around the central hepatic venules (Figure 3D), which show highly specialized metabolic and transport functions and selectively express several genes including glutamine synthetase, the glucose transporter GLUT-1, the ␣-ketoglutarate transporter, and the organic cation transporter rOCT1.27,29,30 The absence of Oatp2 expression in periportal hepatocytes as well as in the innermost layer of perivenous hepatocytes represents a unique transporter distribution that, to our knowledge, has not yet been described for any other hepatic membrane transport protein.27 Because the Oatp2-expressing hepatocytes lie in the area of low oxygen tension of blood,31 it is possible that oxygen plays an important role in the transcriptional regulation of the Oatp2 gene. Among the common Oatp1 and Oatp2 substrates, bile salts and steroid conjugates showed approximately the same affinities for both transport proteins, whereas

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ouabain and the ETA-receptor antagonist BQ-123, an anionic cyclopentapeptide, were transported with higher affinities by Oatp2 (Table 1 and Figure 4). Low- and high-affinity transport of BQ-123 by Oatp1 and Oatp2, respectively, could explain the previously reported diffiTable 1. Comparison of Substrate Specificities of Oatp1 and Oatp2 Oatp1 Oatp2 Km (␮mol/L) Km (␮mol/L) Bile salts Cholate

54

46

Taurocholate

19–50

35

Glycocholate

54

40

7

12

13

17

Taurochenodeoxycholate Tauroursodeoxycholate Sulfolithotaurocholate Other organic anions BMG BSP

DHEAS Estrone-3-sulfate

Estradiol-17␤-glucuronide

LT C4 Dinitrophenylglutathione ␣-Ketoglutarate

p-Aminohippurate Hormones/peptides Triiodothyronine (T3) Thyroxin (T4) BQ-123 Other compounds Digoxin Ouabain

Gadoxetate

6

NT

⫹ 1.5–3.3

NT NT

5

17

4.5–12

11

3–13

3

0.27

NT

408

NT

NT

NT

NT

NT

⫹

5.9

⫹

6.5

600 NT 1700–3000

3390

30 0.24 470

NT

Study

culties to saturate BQ-123 elimination in vivo.26 These findings indicate a potentially important role of the polyspecific Oatps for hepatic uptake of small peptides and support the concept that Oatp2 might function as a high-affinity ‘‘backup’’ system for hepatic clearance of certain cholephilic substances. For example, the distinct acinar expression of Oatp2 could account for the higher sodium-independent taurocholate uptake at increased concentrations by perivenous hepatocytes.32 Furthermore, preferential high-affinity Oatp2-mediated uptake

Meier et al.,2 Eckhardt et al.,5 Noe´ et al.6 Meier et al.,2 Eckhardt et al.,5 Noe´ et al.,6 Meier et al.9 Eckhardt et al.,5 Meier et al.,9 present study Eckhardt et al.,5 present study Eckhardt et al.,5 present study Present study

Present study Meier et al.,2 Eckhardt et al.,5 Meier et al.,9 present study Eckhardt et al.,5 present study Meier et al.,2 Eckhardt et al.,5 Noe´ et al.,6 Meier et al.9 Meier et al.,2 Eckhardt et al.,5 Noe´ et al.,6 Meier et al.,9 Kanai et al.,40 Bossuyt et al.41 Meier et al.,9 Li et al.,42 present study Meier et al.,9 Li et al.,42 present study Meier et al.,2 KullakUblick et al.4 Meier et al.,2 KullakUblick et al.4 Abe et al.,7 Friesema et al.34 Abe et al.,7 Friesema et al.34 Present study Noe´ et al.6 Meier et al.,2 Eckhardt et al.,5 Noe´ et al.6 Meier et al.,9 van Montfoort et al.36

⫹, Transported but Km value not known; NT, not transported.

Figure 4. Kinetics of (A) Oatp1- and (B) Oatp2-mediated BQ-123 uptake in cRNA-injected X. laevis oocytes. Oocytes were injected with 5 ng of Oatp1 and Oatp2 cRNAs or with an equal volume of water. They were cultured for 2.5 days at 18°C and then incubated with increasing concentrations of labeled [3H]BQ-123 and unlabeled BQ-123 in a sodium-free choline chloride medium (see Materials and Methods). Uptake measurements were performed at 30 minutes because separate experiments showed linear BQ-123 uptake up to 45 minutes. Unspecific uptake into water-injected oocytes (⬍10% of total uptake in cRNA-injected oocytes) was substracted from all uptake measurements. Results are given as means ⫾ SD of 10–15 uptake measurements. Kinetic analysis was performed by using a computerbased nonlinear regression analysis of the initial uptake values.

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HEPATIC LOCALIZATION AND FUNCTION OF Oatp2 693

Figure 5. Comparison of various substrate transport values in Oatp1 and Oatp2 cRNA–injected X. laevis oocytes. Oocytes were injected with 1 ng of Oatp1 cRNA, 5 ng of Oatp2 cRNA, or the same volume of cRNA-free water. Uptakes of sulfo-[3H]lithotaurocholate (20 µmol/L) and [35S]BSP (4 µmol/L) were determined at 15 minutes and of [3H]BMG (0.1 µmol/L) at 30 minutes in a sodium-free choline chloride medium (see Materials and Methods). All uptake values are given as means ⫾ SD of 10–15 uptake measurements.

could be important for complete hepatic removal of highly albumin-bound compounds (low free serum concentrations)29 and/or of xenobiotic substances that cannot be taken up by Oatp1 into periportal hepatocytes because of the prevailing high bile salt gradient.33 Whether similar considerations can be extrapolated to the thyroid hormones T3 (triiodothyronine) and T4 (thyroxine) is unclear presently, because their Km values for Oatp1 have not yet been determined.34 Nevertheless, the failure of 10 µmol/L thyroxine to inhibit Oatp1-mediated BSP uptake35 suggests that Oatp1 also exhibits a lower affinity for thyroid hormones compared with Oatp2.7 Besides functional similarities, this study also shows additional differences in the substrate specificities between Oatp1 and Oatp2 (Table 1). Hence, although the organic anions sulfolithotaurocholate, BSP, and BMG are well transported by Oatp1, they are virtually not transported by Oatp2 (Figure 5). Together with the previous finding that the anionic magnetic resonance imaging agent gadoxetate is a preferred or even selective substrate of Oatp1,36 the present results further support the concept that Oatp1 preferentially transports organic anions, whereas Oatp2 is better at transporting neutral steroids such as digoxin and ouabain and possibly also thyroid hormones and cyclic peptides (Table 1). Furthermore, Oatp2 has been shown to exhibit a higher transport activity than Oatp1 for certain type II organic cations such as rocuronium, although the cloned human OATP exhibits the highest organic cation transport activities of all members of the Oatp gene family of membrane transporters.37 Thus, the spectrum of transport substrates of Oatp2 is somewhat intermediate to, but nevertheless overlapping with, the substrate specificities of Oatp1 and OATP. Although the exact structural characteristics of Oatp substrates are not yet known, it can be speculated so

far that size and hydrophobicity are two important features for qualification as an Oatp substrate. This assumption is supported by the findings that small and water-soluble organic anions such as ␣-ketoglutarate and p-aminohippurate (Table 1) as well as organic cations such as choline and tetraethylammonium37 are not transported by members of the Oatp gene family. Their transmembrane transport is rather mediated by members of the OAT (organic anion transporter) and OCT (organic cation transporter) gene families of membrane transporters, which are also highly expressed in the kidneys.38,39 In conclusion, this study shows a unique lobular expression of the polyspecific amphipathic substrate transporter Oatp2 in rat liver. Furthermore, new substrates for and new substrate specificity differences between the major hepatic organic anion–transporting polypeptides Oatp1 and Oatp2 have been identified. Its strategic localization within the liver cell plate indicates that Oatp2 plays an important role in the regulation of the concentration of amphipathic solutes in the hepatic venules and thus in the ultimate delivery of certain amphipathic substances into the systemic circulation.

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September 1999

hepatic sinusoidal organic solute transporter. J Biol Chem 1998; 273:16184–16191. Received April 1, 1999. Accepted May 27, 1999. Address requests for reprints to: Peter J. Meier-Abt, M.D., Division of Clinical Pharmacology and Toxicology, Department of Internal Medicine, University Hospital, CH-8091 Zurich, Switzerland. e-mail: [email protected]; fax: (41) 1-255-4411. Supported by the Swiss National Science Foundation (grants 31-045536.95 to P.J.M. and 31-045677.95 to B.H.); the Olga

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Mayenfisch Foundation (to B.G.), Zurich, Switzerland; the Cloetta Foundation (to B.H.), Zurich, Switzerland; the Deutsche Forschungsgemeinschaft (SFB 601/A2), Heidelberg, Germany; and the Doerenkamp Foundation (to C.R.), Germany. Dr. van Montfoort was supported by an Ubbo Emmius scholarship from the University of Groningen, The Netherlands. Dr. Kamisako was supported by the Alexander von Humboldt Foundation, Bonn, Germany. The authors thank Dr. D. Keppler, Deutsches Krebsforschungszentrum, Abteilung Tumorbiochemie, Heidelberg, Germany, for making the radiolabeled bilirubin monoglucuronide available.