Protective Role of Hydroxysteroid Sulfotransferase in Lithocholic Acid-induced Liver Toxicity*

Hirotaka KitadaDagger , Masaaki MiyataDagger §, Toshifumi NakamuraDagger , Aki TozawaDagger , Wataru HonmaDagger , Miki ShimadaDagger , Kiyoshi NagataDagger , Christopher J. Sinal||, Grace L. Guo, Frank J. Gonzalez, and Yasushi YamazoeDagger

From the Dagger  Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan and  Laboratory of Metabolism, NCI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, October 17, 2002, and in revised form, February 25, 2003

    ABSTRACT
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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Supplement of 1% lithocholic acid (LCA) in the diet for 5-9 days resulted in elevated levels of the marker for liver damage aspartate aminotransferase and alkaline phosphatase activities in both farnesoid X receptor (FXR)-null and wild-type female mice. The levels were clearly higher in wild-type mice than in FXR-null mice, despite the diminished expression of a bile salt export pump in the latter. Consistent with liver toxicity marker activities, serum and liver levels of bile acids, particularly LCA and taurolithocholic acid, were clearly higher in wild-type mice than in FXR-null mice after 1% LCA supplement. Marked increases in hepatic sulfating activity for LCA (5.5-fold) and hydroxysteroid sulfotransferase (St) 2a (5.8-fold) were detected in liver of FXR-null mice. A 7.4-fold higher 3alpha -sulfated bile acid concentration was observed in bile of FXR-null mice fed an LCA diet compared with that of wild-type mice. Liver St2a content was inversely correlated with levels of alkaline phosphatase. In contrast, microsomal LCA 6beta -hydroxylation was not increased and was in fact lower in FXR-null mice compared in wild-type mice. Clear decreases in mRNA encoding sodium taurocholate cotransporting polypeptide, organic anion transporting polypeptide 1, and liver-specific organic anion transporter-1 function in bile acid import were detected in LCA-fed mice. These transporter levels are higher in FXR-null mice than wild-type mice after 1% LCA supplement. No obvious changes were detected in the Mrp2, Mrp3, and Mrp4 mRNAs. These results indicate hydroxysteroid sulfotransferase-mediated LCA sulfation as a major pathway for protection against LCA-induced liver damage. Furthermore, Northern blot analysis using FXR-null, pregnane X receptor-null, and FXR-pregnane X receptor double-null mice suggests a repressive role of these nuclear receptors on basal St2a expression.

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INTRODUCTION
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Bile acids produced from cholesterol are associated with various biological functions, including absorption of fatty acids and retinoids and excretion of lipids and cholesterol. Bile acids are produced in liver and excreted into bile through canalicular bile acid transporters such as Bsep1 and Mrp2 (1). The primary bile acids excreted are deconjugated and deoxygenated by enterobacteria and are reabsorbed at intestine as secondary bile acids such as LCA and deoxycholic acid. The liver has sufficient capacity to transport bile acids into the bile duct mainly after transformation into conjugates. Thus, limited amounts of bile acids are detected in sera under normal conditions. Serum and liver bile acid levels are, however, increased dramatically after hepatic failure or bile duct obstruction. Accumulation of bile acids, particularly secondary bile acids, is believed to be a risk factor for liver toxicity.

Administration of LCA and its conjugates, which are hydrophobic secondary bile acids, causes intrahepatic cholestasis (2). Xei et al. (3) and Staudinger et al. (4) independently reported that the pregnane X receptor (PXR) is involved in protection against LCA-induced liver toxicity. Recently, Makishima et al. (5) reported that the vitamin D receptor functions as a receptor for LCA, as well as 1alpha ,25-dihydroxyvitamin D3 (5). These facts suggest the involvement of several nuclear receptors in LCA-induced toxicity.

LCA is mainly transformed to tauroconjugates and glycoconjugates in liver and pumped out to the bile duct by the Bsep. Conjugated LCA is also transformed to 3-sulfate and 3-glucuronides by sulfotransferase and UDP-glucuronosyltransferase, respectively (6, 7). LCA sulfate and LCA glucuronide were less toxic than LCA (8). These conjugations make the bile acid more hydrophilic and facilitate their elimination in the feces and urine (9-12). Bile acid sulfation is catalyzed by the hydroxysteroid sulfotransferase, St2a forms (13). In mice, two cDNAs encoding St2a4 and St2a9 were isolated (14, 15).

The farnesoid X receptor (FXR) is activated by a number of bile acids including chenodeoxycholic acid (CDCA), cholic acid, and LCA (16-18). FXR mediates a number of bile acid-dependent regulatory processes (19-25) and plays an important role in lipid homeostasis (26). FXR-null mice, in which cholesterol-bile acid regulation is disrupted, show an increase in serum total bile acid levels. Reduction of bile acid pools and fecal bile acid excretion was also shown to occur because of decreased expression of Bsep. Cholic acid feeding resulted in severe liver damage in FXR-null mice, although no apparent toxicity was detected in FXR-null mice fed control diet. Recently, CYP3A forms were reported to be elevated in FXR-null mice (27). Increased excretion of urinary bile acids was reported in FXR-null mice fed diets supplemented with 1% cholic acid, compared with wild-type mice (26). These data suggest a possible occurrence of alterations in the metabolic pathway leading to bile acids in the FXR-null mice.

In our preliminary experiments, FXR-null male mice were more susceptible to cholic acid- (26) and LCA-induced liver damage than wild-type male mice, whereas FXR-null female mice were less susceptible to LCA-induced damage than wild-type female mice. These results suggested a specific mechanism for the protection against LCA-induced liver damage in FXR-null female mice. Thus, in the present study, the metabolic shift in bile acid metabolism was investigated with FXR-null female mice in relation to the liver toxicity of LCA. A clear increase in female-specific St2a form (5.8-fold) catalyzing LCA sulfation was detected in livers of FXR-null female mice as compared with wild-type mice. Furthermore, the hepatic level was inversely correlated with serum ALP activity a marker of liver cholestasis, indicating a critical role for St2a in protection against LCA-induced liver damage.

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Materials-- LCA, CDCA, taurolithocholic acid (tauroLCA), taurochenodeoxycholic acid (tauroCDCA), and 1,12-dodecanedicarboxylic acid were purchased from Sigma-Aldrich. [35S]PAPS (2000 mCi/mmol) was from PerkinElmer Life Sciences. SDS-PAGE molecular weight markers (low range) were from Bio-Rad. The HPLC column, Chemcosorb 5-ODS-H (6.0 × 150 mm), was purchased from Chemco Scientific Co. (Tokyo, Japan). L-column ODS (2.1 × 150 mm) was obtained from Kagakuhinnkennsakyoukai (Tokyo, Japan). Enzymepak 3alpha -HSD column was purchased from Jasco (Tokyo, Japan). 5beta -Cholanic acid-3alpha , 6beta -diol and 5beta -cholanic acid-3alpha , 6alpha -diol were purchased from Steraloids, Inc. (Newport, RI). The PXR-null mice were kindly provided by Dr. Steven A. Kliewer at the University of Texas Southwestern Medical Center under a Material Transfer Agreement with GlaxoSmithKline (4).

Animal Treatment and Sample Collection-- FXR-null mice (26) were back-crossed to strain C57BL/6 for at least five generations. Mice were housed under standard 12-h light/12-h dark cycle. Prior to the administration of special diets, mice were fed standard rodent chow (CE-2; Clea) and water ad libitum for acclimation. Experimental diets contained 0.5 or 1% (w/w) LCA mixed with the control diet (CE-2). Age-matched groups of 8- to 12-week-old animals were used for all experiments and were allowed to access to water ad libitum. Bile, blood, and tissue samples were taken for the biochemical assay after 9 or 5 days. Microsomal and cytosolic protein concentrations were determined by the method of Lowry et al. (28). Total RNA was prepared from livers using the ULTRASPEC II RNA isolation system (Biotecx Lab., Houston, TX). The total RNA content was determined by measuring the absorbance at 260 nm using a spectrophotometer (Beckman DU 640).

Serum AST and ALP Activities and Determination of Bile Acid-- Serum AST activity was estimated by the POP-TOOS method using a commercial kit, Transaminase CII-B-test Wako (Wako, Osaka, Japan). Serum ALP activity was estimated by the Bessey-Lowry method using Alkaliphospha B-test Wako (Wako, Osaka, Japan). Bile, liver, and serum 3alpha -hydroxy bile acid concentrations were estimated by an enzyme-colorimetric method using Total bile acid-test Wako (Wako, Osaka, Japan). 3alpha -Sulfated bile acid concentrations were determined by an enzyme-colorimetric method using UBASTEC-AUTO (Daiichi Pure Chemical, Tokyo, Japan) (29, 30). All absorptions were measured using spectrophotometer (Beckman DU 640). The liver 3alpha -hydroxy bile acid contents were measured by HPLC. A portion (100 µl) of liver homogenate was mixed with 1 ml of ethanol containing 2 nmol of androstandiol and treated at 85 °C for 1 min and then centrifuged at 1,000 × g for 5 min. After the supernatant was isolated, the precipitate was extracted twice with 1 ml of ethanol. Combined extracts were dried and re-dissolved in 200 µl of methanol. HPLC analyses were performed with a Jasco intelligent model PU-980 pump (Jasco, Tokyo, Japan), Waters M-45 pump (Waters, Milford, MA) and FP-920S fluorescence detector (Jasco). The bile acid extracts were separated at 35 °C with L-column ODS (2.1 × 150 mm) (Kagakuhinnkennsakyoukai, Tokyo, Japan). The eluates were mixed with an NAD+ solution prior to introduction to 3alpha -hydroxysteroid dehydrogenase immobilized on Enzymepak 3alpha -HSD column. NADH produced was measured by the fluorescence using an excitation wavelength of 365 nm and an emission wavelength of 470 nm. The separation was started at 0.5 ml/min with a 60-min linear gradient of solution A (10 mM phosphate buffer, pH 7.2/acetonitrile (60:40))/solution B (30 mM phosphate buffer, pH 7.2/acetonitrile (80:20)) mixture (25:75) to solution A/solution B mixture (55:45) and then continued with solution A/solution B mixture (55:45) for 25 min. The eluates were passed through a 3alpha -HSD column after mixing with solution C (10 mM phosphate buffer, pH 7.2, 1 mM EDTA, 0.05% 2-mercaptoethanol, and 0.3 mM NAD+) (1:1).

LCA 6alpha - and 6beta -Hydroxylase Activities-- LCA 6alpha - and 6beta -hydroxylations were determined by HPLC. A typical incubation mixture consisted of 0.1 M phosphate buffer, pH 7.4, 4.8 mM MgCl2, 0.32 mM NADP+, 2.4 mM glucose 6-phosphate, 0.26 units/ml glucose-6-phosphate dehydrogenase, 0.25 mM LCA, and 250 µg of microsomal protein in a final volume of 500 µl. The mixture was incubated for 20 min at 37 °C. The incubation was terminated by addition of 1 ml of ethyl acetate. After incubation, 1,12-dodecanedicarboxylic acid was added to the incubation mixture as an internal standard. The mixture was centrifuged at 700 × g for 5 min. An organic phase was evaporated to dryness under nitrogen. The residue was dissolved in 0.1 ml of acetonitrile containing 10 mM 3-bromomethyl-7-methoxy-1,4-benzoxazin-2-one and 0.1 ml of 56 mM 18-crown-6 in acetonitrile saturated with K2CO3, and the resulting solution was allowed to stand at 40 °C for 30 min. Following the addition of 50 µl of 2% acetic acid, the solution was subjected to HPLC analyses (31). HPLC analyses were performed with Jasco intelligent model PU-980 pump and FP-920S fluorescence detector. The derivatives were separated with a 5-ODS-H column (6.0 × 150 mm) and detected by using an excitation wavelength of 355 nm and an emission wavelength of 450 nm. The column was eluted with acetonitrile containing 3% THF/1% acetic acid (52:48) for 15 min and then with a 35-min linear gradient of acetonitrile containing 3% THF/1% acetic acid (52:48) to acetonitrile containing 3% THF. The final elution with acetonitrile containing 3% THF was continued for 10 min. LCA 6alpha - and 6beta -hydroxylations were also determined using a 3alpha -HSD column. The data were consistent with the data obtained with the post-labeling method described above.

LCA Sulfating Activity-- Sulfating activities were determined by monitoring the radioactivities of the metabolites obtained with [35S]PAPS as a sulfate donor after thin layer chromatography. A typical incubation mixture consisted of 50 mM Tris-HCl buffer, pH 7.4, 1 mM dithiothreitol, 20 mM MgCl2, 10 µM LCA, 5 µM [35S]PAPS, and 1 µg of cytosolic protein in a final volume of 10 µl. The reaction was initiated by addition of [35S]PAPS and terminated by addition of 5 µl of chilled acetonitrile after incubation at 37 °C for 20 min. LCA dissolved in Me2SO was added to make the final Me2SO concentration 1%. A portion (10 µl) of the reaction mixtures was applied to a thin layer plate (thin layer chromatography aluminum plate silica gel 60; Merck). Metabolites on the chromatogram were developed with a solvent system of chloroform/methanol/ammonia/water (60:35:0.5:7.5). The radioactive spots were analyzed by a FLA-3000 image analyzer (Fuji Film, Tokyo, Japan). The apparent kinetic parameters were developed from assays carried out with several concentrations of each substrate (10 nM-1 µM LCA and tauroLCA, 1-25 µM CDCA and tauroCDCA). Each reaction was linear within the concentrations examined.

Western Blot Analysis-- Cytosolic protein (3-10 µg/lane) were separated by 12% SDS-PAGE and transferred to a nitrocellulose sheet. The sheet was immunostained with a polyclonal antibody (1:2000 dilution) raised against the purified recombinant ST2A1 protein (32), horseradish peroxidase-conjugated goat anti-rabbit IgG, 3,3'-diaminobenzidine, and hydrogen peroxide. The stained sheets were scanned with an Epson GT-8700 scanner, and their intensities were measured by use of the NIH image (version 1.59) software (Bethesda, MD).

Northern Blot Analysis-- 10 µg of total RNA was subjected to electrophoresis in the 0.22 M formaldehyde-containing 1% agarose gel and transferred to GeneScreen Plus membrane (PerkinElmer Life Sciences). Full-length St2a9 cDNA was used as a probe. The cDNA was random-primer labeled with [alpha -32P]CTP (Amersham Biosciences). After prehybridization overnight with 15 mM sodium chloride solution containing 1.5 mM sodium citrate and 0.5% SDS, the membrane was hybridized with 32P-labeled probe (1 × 106 cpm/ml) in 0.5 M sodium phosphate buffer, pH 7.2, containing 7% SDS, 1% bovine serum albumin, and 1 mM EDTA overnight. After washing with 20 mM sodium phosphate buffer, pH 7.2, containing 1% SDS and 1 mM EDTA, the membrane was exposed to an imaging plate for 20 min. The radioactive spots were analyzed by a BAS1000 image analyzer (Fuji Film, Tokyo, Japan).

Reverse Transcription-Polymerase Chain Reaction-- Messenger RNA levels of differentially expressed genes were analyzed using reverse transcription (RT)-PCR. Single strand cDNAs were constructed using an oligo(dT) primer with the Ready-to-Go You-Prime First-strand Beads kit (Amersham Biosciences). These cDNAs provided templates for PCRs using specific primers at a denaturation temperature of 94 °C for 30 s, at an annealing temperature of 58 °C for 30 s, and at an elongation temperature of 72 °C for 30 s in the presence of dNTPs and Taq polymerase. The PCR cycle numbers were titrated for each primer pair to assure amplification in linear range. The reaction was completed by a 7-min incubation at 72 °C. PCR products were analyzed in 2% agarose gel (w/v) containing ethidium bromide for visualization. Either a set of primers specific for the nucleotide sequence of each metabolizing enzyme and transporter were followed: Bsep (bp 2094-2517 in AF133903) sense, 5'-ACAGCATTACAGCTCATTCAGAG-3' and antisense, 5'-TCCATGCTCAAAGCCAATGATCA-3'; Ntcp (bp 71-671 in U95131) sense, 5'-ACACTGCGCTCAGCGTCATTC-3' and antisense, 5'-GCCAGTAAGTGTGGTGTCATG-3'; Mrp2 (bp 386-1022 in NM 013806) sense, 5'-GGTTCCTGTCCATGTTCTGGATT-3' and antisense, 5'-GCAGCTGAGGATTCAGAAACAAA-3'; Mrp3 (bp 42-342 in AI391398) sense, 5'-CGCTCTCAGCTCACCATCAT-3' and antisense, 5'-GGTCATCCGTCTCCAAGTCA-3'; Mrp4 (bp 161-382 in W54702) sense, 5'-GGTTGGAATTGTGGGCAGAA-3' and antisense, 5'-TCGTCCGTGTGCTCATTGAA-3'; Oatp1 (bp 711-1036 in AF148218) sense, 5'-TGATACACGCTGGGTCGGTG-3' and antisense, 5'-GCTGCTCCAGGTATTTGGGC-3'; Oatp2 (bp 22-779 in AB031814) sense, 5'-GTTGCAACCCATGGGGTCAGATG-3' and antisense, 5'-GCTGGTCAGGATATTCACTCCTG-3'; Lst-1 (bp 1086-1410 in AB031959) sense, 5'-TGGTCAGACAGCATCGCAGG-3' and antisense, 5'-CCCACAGACAGGTTCCCATTG-3'; glyceraldehyde-3-phosphate dehydrogenase (bp 487-1018 in NM_008084) sense, 5'-TGCATCCTGCACCACCAACTG-3' and antisense, 5'-GTCCACCACCCTGTTGCTGTAG-3'; FXR (bp 1073-1656 in NM_009108) sense, 5'-CGGACATGCAGACCTGTTGGAAG-3' and antisense, 5'-CCAGTGGGGTTTCCTGAAGCC-3'; PXR (bp 243-710 in XM_148459) sense, 5'-GTTCCTGATTCTTCAAGGTGG-3' and antisense, 5'-TCTTCCTCTTGATCAAGGCC-3'; CYP7A1 (bp 119-701 in NM_007824) sense, 5'-CATACCTGGGCTGTGCTCTGA-3' and antisense, 5'-GCTTTATGTGCGGTCTTGAGC-3'; CYP3A11 (bp 1126-1531 in NM_007818) sense, 5'-TGATGGAGATGGAATACCTGG-3' and antisense, 5'-GGTTGAAGAAGTCCTTGTCTGC-3'.

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Influence of Bile Acid Feeding on Liver Biochemical Parameters-- 3alpha -Hydroxy bile acid levels determined colorimetrically are shown in Table I. Serum 3alpha -hydroxy bile acid levels were low in both wild-type and FXR-null female mice fed control diets, although 2-fold difference was observed between two groups. A liver 3alpha -hydroxy bile acid concentrations exhibited a 2.8-fold difference in the two groups, and the level was increased by an 1% LCA diet for 9 days. The increase in serum was more evident in wild-type mice (82-fold) than in FXR-null mice (5.3-fold), although no significant difference in food consumption was observed between FXR-null and wild-type mice. A 7.7-fold higher serum 3alpha -hydroxy bile acid level was observed in wild-type mice compared with FXR-null mice after LCA treatment. Liver 3alpha -hydroxy bile acid levels were also increased in mice treated with 1% LCA and were higher in wild-type mice than in FXR-null mice. Typical diagnostic marker activities for liver damage, serum AST activities, were slightly higher in control FXR-null mice than in wild-type mice, and ALP activities were similar between the two strains (Table I). Higher levels were detected after LCA feeding in both strains. Consistent with the bile acid levels, AST and ALP activities were higher in wild-type than in FXR-null mice after feeding LCA. In male mice fed 1% LCA diet for 9 days, AST activities, ALP activities, and liver 3alpha -hydroxy bile acid concentrations of FXR-null mice were 290 ± 280 IU/liter, 131 ± 87 IU/liter, and 715 ± 444 nmol/g liver, respectively. These of wild-type mice were 35 ± 18 IU/liter, 13 ± 2 IU/liter, and 211 ± 44 nmol/g liver, respectively.


                              
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Table I
Changes in 3alpha -hydroxy bile acid levels and liver damage diagnostic markers
The data are shown as the mean ± S.D. from three to seven mice. Serum and liver 3alpha -hydroxy bile acid levels and serum AST and ALP activities were determined as described under "Experimental Procedures." Control or 1% lithocholic acid-supplemented diets were given for 9 days to wild-type and FXR-null female mice. Basal levels of both liver bile acid and serum AST were significantly different between wild-type and FXR-null mice (p < 0.01). In LCA-treated animals, all parameter levels were significantly different between wild-type and FXR-null mice (p < 0.05). LCA, AST, and ALP indicated lithocholic acid, aspartate aminotransferase and alkaline phosphatase, respectively. 3alpha -OH bile acid indicated 3alpha -hydroxy bile acid.

LCA Metabolism in Vitro-- LCA undergoes the 6alpha - and 6beta -hydroxylations followed by sulfation prior to excretion. Thus, microsomal LCA 6alpha - and 6beta -hydroxylation activity was determined (Fig. 1). The rate of liver LCA 6beta -hydroxylation was roughly 2-fold higher in wild-type than in FXR-null mice, despite the 1.5-fold higher activity for testosterone 6beta -hydroxylation in FXR-null mice (data not shown). The rate of liver 6alpha -hydroxylation was 2-fold higher in FXR-null mice than in wild-type mice but was low compared with the 6beta -hydroxylation. Cytosolic 3-O-sulfation of LCA was low in wild-type animals but was 5.5-fold higher in FXR-null mice. The sulfating activity tended to be slightly increased after feeding 0.5% LCA.


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Fig. 1.   LCA 6alpha and 6beta -hydroxylations and sulfation in liver. A, microsomal 6alpha - and 6beta -hydroxylations of LCA. B, cytosolic sulfation of LCA. Details for microsomal 6alpha - and 6beta -hydroxylations of LCA and cytosolic sulfation of LCA are described under "Experimental Procedures." Liver samples obtained from mice treated with control, 0.5, or 1% LCA diets were used for both assays. Data are shown as the mean ± S.D. from four to six different mice. Significant differences (p < 0.01) were observed between wild-type and FXR-null mice on both the 6alpha - and 6beta -hydroxylations and sulfation.

Enzyme of LCA Sulfation-- Among cytosolic sulfotransferases, hydroxysteroid sulfotransferase termed ST2A (SULT2A) is known to catalyze sulfation of bile acids. Using anti-rat ST2A1 antibodies, cytosolic levels of mouse St2a were measured (Fig. 2A). A band of about 31 kDa detected in the liver cytosol of female mice was 5.8-fold higher with FXR-null mice than wild-type mice (Fig. 2B). Liver content of St2a was increased by the treatment of both wild-type and null mice with 0.5% LCA. A clear correlation (r2 = 0.888) was observed between individual cytosolic sulfation of LCA and St2a content as assessed by Western blotting (Fig. 3). In addition, an inverse correlation (r2 = 0.645) was observed between ALP activity and St2a content in liver of wild-type mice (Fig. 4). Upon 1% LCA treatment, no clear relationship was obtained between ALP activity and St2a content, probably because of the extensive damage to the liver.


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Fig. 2.   Relative levels of hepatic St2a and the influence of LCA supplement. A, Western blot analysis of St2a in mouse liver cytosol. Cytosolic proteins (10 µg) were subjected to SDS-PAGE on 12% gel and electrically transferred to a nitrocellulose membranes for the immunostaining with anti-rat ST2A1 antibody. B, St2a levels were quantitated as described under "Experimental Procedures." Control or 1% LCA-supplemented diets were given to wild-type and FXR-null female mice for 9 days. Data are shown as the mean ± S.D. (n = 4-6). In LCA-treated groups, significant differences (p < 0.01) were observed between wild-type and FXR-null mice.


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Fig. 3.   Correlation between LCA sulfating activity and St2a content. Cytosolic LCA sulfating activity and St2a levels were determined as described for Fig. 2.


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Fig. 4.   Correlation between serum ALP activity and St2a content. Experimental details for the determination of ALP activity and St2a content were described under "Experimental Procedures." Liver samples obtained from wild-type mice treated with control and 0.5% LCA diets were used.

Detection of Bile Acid-- Profiles of bile acids were determined by HPLC with liver of mice fed LCA. Several bile acids with free 3-hydroxy group were detected in liver of female mice fed LCA. In the LCA supplement groups, tauroLCA was detected as a major peak in liver of wild-type and FXR-null mice. The peak was 12.8-fold higher with wild-type than with FXR-null mice (Fig. 5A). In addition, free LCA was detected as a minor but clear peak in the treated wild-type mice. Furthermore, tauroCDCA was detected as a major peak in liver of wild-type and FXR-null mice. The content was 3.2-fold higher with wild-type than with FXR-null mice (data not shown). A clear correlation was observed between ALP activity and hepatic tauroLCA content (Fig. 5B). No LCA and tauroconjugate was detected in livers of untreated female mice (less than 5 nmol/g liver), although taurocholic acid and taurodeoxycholic acid were observed. Bile and liver obtained from female mice fed 1% LCA diet for 5 days were used to determine 3alpha -sulfated bile acid level. Sulfated bile acid levels are shown in Table II. A 7.4-fold higher sulfated bile acid level was observed in bile of FXR-null mice fed an LCA diet compared with that of the wild-type mice, although no significant difference in the level of 3alpha -hydroxy bile acids was found. Hepatic 3alpha -hydroxy bile acid level was 2.1-fold higher in wild-type mice than in FXR-null mice, whereas hepatic sulfated bile acid was not detectable (<0.06 µmol/g liver) in both mice.


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Fig. 5.   Hepatic bile acid contents. The contents of bile acids were determined with HPLC. Experimental details for the assay of bile acid are described under "Experimental Procedures." Liver samples obtained from mice treated with 1% LCA diet were used for the assay. A, hepatic bile acid concentrations were significantly different between wild-type and FXR-null mice (p < 0.01). B, the correlation between hepatic tauroLCA content and ALP activity.


                              
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Table II
Changes in bile acid 3alpha -sulfate levels in liver and bile
The data are shown as the mean ± S.D. from three to four mice. 3alpha -Hydroxy bile acid and 3alpha -sulfated bile acid concentration were determined as described under "Experimental Procedures." One percent lithocholic acid-supplemented diets were given for 5 days to wild-type and FXR-null mice. 3alpha -OH and 3alpha -sulfate indicated 3alpha -hydroxy bile acid and 3alpha -sulfated bile acid, respectively.

Measurement of Kinetic Parameters for Sulfation of LCA, CDCA, and Their Tauro Conjugates-- Kinetic parameters for sulfation of major bile acid compounds, LCA, tauroLCA, CDCA, and tauroCDCA, were determined in liver of untreated FXR-null female mice. LCA and tauroLCA had more lower Km values and higher Vmax values than did CDCA and tauroCDCA (Table III). The Km value of LCA was 36.4-fold lower than that of CDCA. Tauro conjugates were found to have a higher Km value than the free compounds.


                              
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Table III
Apparent kinetic parameters for sulfation of LCA, CDCA, and their tauro conjugates by mouse cytosols
Each parameter is derived from analysis of Lineweaver-Burk plots. Assays were performed at pH 7.4 with various concentration of substrates (LCA and tauroLCA, 10 nM-1 µM; and CDCA and tauroCDCA, 1-25 µM).

Influence of PXR on the Expression of St2a-- Relative levels of St2a expression were measured by Northern blotting. Consistent with St2a protein levels, St2a mRNA level was elevated in FXR-null mice (Fig. 6). Similar increase was also detected in liver of PXR-null mice. The level of St2a mRNA was further elevated in PXR-FXR double-null mice.


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Fig. 6.   Northern blot analysis of mouse St2a. Wild-type (lanes 1 and 2), PXR-null (lanes 3 and 4), FXR-null (lanes 5 and 6), and PXR-FXR double-null (lanes 7 and 8) mice were fed a control diet. Total hepatic RNA was isolated, and 10 µg were separated on 1.1% agarose gel, transferred to a nylon membrane, and hybridized with the indicated 32P-labeled cDNA probes.

Changes in Hepatic Expression of Bile Acid-related Genes-- Various transporters are known to participate in hepatic influx and efflux of bile acids. Hepatic levels of their specific mRNAs were compared between wild-type and FXR-null mice using RT-PCR. Bsep mRNA levels were lower in FXR-null mice than the wild-type controls (Fig. 7A). No clear change was observed in mice treated with LCA. Ntcp transporting bile acids to hepatocytes did not differ between control groups, but the mRNA level was reduced after LCA treatment. The reduction was more profound with wild-type than FXR-null animals. A similar phenomenon was observed with the anion transporter Oatp1. The levels in both wild-type and FXR-null mice were abolished after LCA treatment, although higher levels of the mRNA were observed with control FXR-null mice than control wild-type mice. Lst-1 also showed a profile similar to that of Oatp1. No obvious change was observed with Mrp2, Mrp3, and Mrp4 mRNAs. On the other hand, CYP3A11 and CYP7A1 mRNA levels were higher in FXR-null mice than wild-type mice (Fig. 7B). Furthermore, marked decrease in CYP7A1 mRNA level was observed in the wild-type mice fed LCA. In contrast, no significant change in CYP3A11 mRNA level was found in wild-type mice after LCA feeding.


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Fig. 7.   Liver expression of bile acid-related genes. Hepatic mRNA levels of bile acid-related genes were measured by RT-PCR. Hepatic mRNAs were prepared from wild-type and FXR-null female mice fed a control diet or the diet supplemented with 1% LCA for 9 days. Specific primers described under "Experimental Procedures" were used to determine levels of expression of each mRNA encoding hepatic bile acid transporters (A) and nuclear receptors and metabolizing enzymes (B).


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

In the present study, female FXR-null mice were found to be resistant to the toxic effects of LCA as compared with wild-type mice despite the decreased expression of Bsep. In LCA-fed mice, liver tauroLCA concentration correlated with a marker for liver damage, ALP activity. Furthermore, female FXR-null mice had higher LCA sulfating activity than wild-type mice. Hepatic level of St2a catalyzing LCA sulfation inversely correlated with the ALP activity, indicating that St2a plays a role in the protection against LCA-induced liver damage.

Recent studies (3, 4) suggested a protective role of activation of PXR against severe liver damage induced by LCA. Cyp3a11 mRNA detected by RT-PCR and Cyp3a proteins by Western blotting (data not shown) were higher in the liver of FXR-null mice than wild-type mice in this study consistent with what was found in an earlier report (27) showing that Cyp3a11 levels were higher in FXR-null mice. Although the catalytic activities of mouse Cyp3a forms remain unknown, human CYP3A4 is reported to catalyze LCA 6alpha - and 6beta -hydroxylations in vitro (3, 33). These data suggested the possible involvement of mouse Cyp3a in microsomal 6alpha - and 6beta -hydroxylations of LCA as a detoxification pathway of LCA-induced toxicity. However, microsomal LCA 6beta -hydroxylation activity was higher for wild-type mice than for FXR-null mice (Fig. 1). Microsomal LCA 6alpha -hydroxylation activity was very low in both mouse livers. These data, as well as in vivo profiles, indicate that CYP3A metabolism is not rate-limiting in LCA detoxification. In contrast, cytosolic sulfation of LCA and the amounts of St2a were markedly elevated in FXR-null mice. Indeed, an inverse relationship was observed between liver content of St2a and ALP activity in mice treated with LCA. These data suggest a protective role for St2a in catalyzing LCA sulfation rather than CYP3A catalyzing LCA hydroxylation in the LCA-induced liver damage, at least in female mice.

It was reported that in human, intravenously injected radiolabeled LCA were excreted rapidly and predominantly in bile, and 60% of excreted radioactivity were derived from sulfated forms (34). An increase in LCA sulfate that is poorly reabsorbed from intestine results in rapid fecal excretion of LCA (35). In bile of female FXR-null mice, sulfated bile acid concentration was 7.4-fold higher compared with that of wild-type mice, whereas 3alpha -sulfated bile acid in livers of both mice was not detectable (<0.06 µmol/g liver) (Table II). These data also support the idea that hydroxysteroid sulfotransferase-mediated LCA sulfate is rapidly excreted from liver resulting in the decrease of LCA level in liver of female FXR-null mice. Because of the efficient LCA-sulfating activity in female mice, as well as humans, female mice rather than male mice lacking the activity might represent a feasible model for human LCA-induced toxicity. Despite lacking bile acid sulfating activity, male wild-type mice are resistant to LCA-induced toxicity, indicating the presence of the protective mechanisms that are different from bile acid sulfation.

In contrast to 1% LCA feeding, no significant increase in AST and ALP activities was found in wild-type mice fed a diet supplemented with 0.25% LCA for 5 days (data not shown). Under these conditions, a rise in liver 3alpha -hydroxy bile acid levels is likely to be prevented through enhanced excretions of tauro conjugate of bile acids by Bsep and of sulfated bile acids by Mrp2, Mrp3, and Mrp4 (36-40). Further the increase in LCA intake might overwhelm the LCA excretion capacity resulting in an accumulation of hepatic bile acids in wild-type mice having lower LCA sulfating activity. Bsep expression level was not increased in mice fed with a high LCA-containing diet. Furthermore, Bsep-mediated bile acid transports were inhibited by LCA sulfate (41). The excretion pathway of LCA sulfate thus might become relatively important in the protection system of LCA-induced damage observed with mice fed high LCA diet. Because the apparent Km value of LCA sulfating activity in female mouse liver cytosol was very low (0.14 µM), hepatic LCA should be efficiently sulfated. These results support the contention that LCA sulfation is a critical determinant for regulation of hepatic LCA concentrations.

Liver St2a mRNA level is strikingly elevated in FXR-null, PXR-null, and FXR-PXR double-null mice. The expression of FXR and PXR is thus likely to be directly or indirectly involved in the repression of basal St2a expression. Liver total bile acid concentration was 2.8-fold higher for FXR-null mice than for wild-type mice. Furthermore, hepatic St2a level in FXR-null and wild-type mice increased after LCA (0.5%) feeding. These results suggest the possibility that the intracellular accumulation of hepatic bile acids results in the hepatic St2a induction through nuclear receptors such as PXR and vitamin D receptor. Makishima et al. (5) have shown recently that vitamin D receptor also functions as a receptor for the secondary bile acid LCA. Runge-Morris et al. (42) have proposed, using an in vitro reporter assay, that the expression of rat hydroxysteroid sulfotransferase is stimulated by a dual mechanism in which both PXR and the glucocorticoid receptor are involved. A reporter construct carrying an FXR binding element from the rat hydroxysteroid sulfotransferase promoter is stimulated in Caco-2 cells upon CDCA treatment (43). Recently, the element was shown to mediate PXR-dependent transcriptional activation of the rat hydroxysteroid sulfotransferase (44). Our in vivo results with mice lacking PXR and/or FXR showing elevated mRNA encoding St2a were contradictory with these reports. Although we can not explain the conflicting results, our in vivo results suggest a complicated mechanism for regulating St2a expression.

The level of Bsep mRNA in control FXR-null mice was less than that of wild-type mice. Increased expression of Bsep mRNA was not observed in LCA-fed mice. This result is consistent with the recent report that LCA decreases expression of Bsep through FXR antagonist activity (45). Mrp2, Mrp3, and Mrp4, which mediate export of anionic chemicals, showed no clear changes at their mRNA levels. Relative mRNAs of transporters working for entry of anionic chemicals in liver such as Ntcp, Oatp1, and Lst-1 were all decreased under conditions of LCA supplement, which may suggest an adaptive response to high levels of LCA. The results of RT-PCR indicate that liver transporters function to lower liver bile acid levels. Bile acid sulfates are known to undergo excretion by canalicular and basolateral transporters such as Mrp2, Mrp3, and Mrp4 (37-40). No clear changes in levels of transporters working for LCA disposition were observed in the present experiments. Thus, sulfating activity of LCA, not transporting activity of LCA sulfate, might be a rate-limiting step for excreting LCA. In fact, 3alpha -sulfated bile acid level in bile correlated with hepatic St2a level and LCA sulfating activity.

There are reports showing high excretion of sulfated bile acids in liver disorders (46, 47). Urinary excretion of sulfated bile acid was shown to be high in bile-duct ligation in experimental animals and in liver failure in humans. These data also suggest the possibility that liver St2a induction is a general adaptive response to extranormal levels of bile acids, particularly of LCA in liver of mammals. The present result also implies hepatic levels of St2a sulfotransferase to be a diagnostic marker for LCA-associated liver disorder.

    ACKNOWLEDGEMENTS

The technical assistance of Hijiri Otsuka is greatly appreciated. We thank Dr. Steven A. Kliewer (University of Texas Southwestern Medical Center) for providing PXR-null mice.

    FOOTNOTES

* This work was supported by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan and by the Human Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki, Aoba-ku, Sendai 980-8578, Japan. Tel.: 81-22-217-6829; Fax: 81-22-217-6826; E-mail: miyata@mail.pharm.tohoku.ac.jp.

|| Present address: Dept. of Pharmacology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada.

Published, JBC Papers in Press, March 7, 2003, DOI 10.1074/jbc.M210634200

    ABBREVIATIONS

The abbreviations used are: Bsep, bile salt export pump; FXR, farnesoid X receptor; LCA, lithocholic acid; CDCA, chenodeoxycholic acid; St, sulfotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; ODS, octadeyl-silica; HSD, hydroxysteroid dehydrogenase; THF, tetrahydrofuran; Mrp, multidrug resistance protein; Ntcp, sodium taurocholate cotransporting polypeptide; Oatp, organic anion transporting polypeptide; Lst-1, liver-specific organic anion transporter-1; PXR, pregnane X receptor; tauroLCA, taurolithocholic acid; tauroCDCA, taurochenodeoxycholic acid; PAPS, adenosine 3'-phosphate,5'phosphosulfate; HPLC, high pressure liquid chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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