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Biochimica et Biophysica Acta 1588 (2002) 139–148 www.bba-direct.com

Selective inhibition of CYP27A1 and of chenodeoxycholic acid synthesis in cholestatic hamster liver

Yasushi Matsuzaki a,*, Bernard Bouscarel b, Tadashi Ikegami a, Akira Honda a,c, Mikio Doy c, Susan Ceryak b, Sugano Fukushima a, Shigemasa Yoshida a, Junichi Shoda a, Naomi Tanaka a

aDepartment of Gastroenterology and Hepatology, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba City, 305-8575 Ibaraki, Japan bDepartment of Medicine, The George Washington University, Washington, DC, USA cIbaraki Prefectural Institute of Public Health, Mito, Ibaraki, Japan Received 18 April 2002; received in revised form 20 June 2002; accepted 20 June 2002

Abstract

The aim of this study was to explore the regulation of serum cholic acid (CA)/chenodeoxycholic acid (CDCA) ratio in cholestatic hamster induced by ligation of the common bile duct for 48 h. The serum concentration of total bile acids and CA/CDCA ratio were significantly elevated, and the serum proportion of unconjugated bile acids to total bile acids was reduced in the cholestatic hamster similar to that in patients with obstructive jaundice. The hepatic CA/CDCA ratio increased from 3.6 to 11.0 ( P < 0.05) along with a 2.9-fold elevation in CA concentration ( P < 0.05) while the CDCA level remained unchanged. The hepatic mRNA and protein level as well as microsomal activity of the 7a-hydroxylase, 7a-hydroxy-4-cholesten-3-one 12a-hydroxylase and 5h-cholestane-3a,7a,12a-triol 25-hydroxylase were not significantly affected in cholestatic hamsters. In contrast, the mitochondrial activity and mass of the sterol 27-hydroxylase were significantly reduced, while its mRNA levels remained normal in bile duct-ligated hamster. In conclusion, biosynthetic pathway via mitochondrial sterol 27-hydroxylase was preferentially inhibited in bile duct-ligated hamsters. The suppression of CYP27A1 is, at least in part, responsible for the relative decreased production of CDCA and increased CA/CDCA ratio in the liver, bile and serum of cholestatic hamsters. D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Bile acid synthesis; Cholestasis; Cholesterol 7a-hydroxylase (CYP7A1); 7a-Hydroxy-4-cholesten-3-one 12a-hydroxylase (CYP8B1); 5h- Cholestane-3a,7a,12a-triol 25-hydroxylase (CYP3A)

1. Introduction acid biosynthesis under cholestatic conditions remains unknown. The total serum bile acid concentration increases in Production of CA and CDCA from cholesterol is princi- patients with various hepatobiliary diseases: e.g., acute pally regulated by two key , namely microsomal and chronic hepatitis, liver cirrhosis and intra- and extra- cholesterol 7a-hydroxylase (CYP7A1) and mitochondrial hepatic cholestasis [1–3]. However, the composition of sterol 27-hydroxylase (CYP27A1) [4]. CYP7A1 is the first serum bile acids in patients with cholestasis is very different and rate-limiting enzyme in the classic pathway, while from that in patients with other hepatobiliary diseases. In CYP27A1 is the key enzyme in the alternative pathway as cholestasis, the ratio of the serum concentration of the depicted in Fig. 1. The microsomal 7a-hydroxy-4-cholesten- primary bile acid cholic acid (CA), to chenodeoxycholic 3-one 12a-hydroxylase (CYP8B1) is an additional key acid (CDCA) increases to more than one [1–3], while the enzyme involved in bile acid biosynthesis [5]. This enzyme proportion of unconjugated bile acids is reduced [2,3]. is known to catalyze 12a-hydroxylation of 7a-hydroxy-4- However, the mechanism(s) responsible for the altered bile cholesten-3-one which is an intermediate in the classic path- way. The role of CYP8B1 in the alternative pathway has not been clarified, while it is believed that the alternative pathway * Corresponding author. Tel.: +81-298-53-3138; fax: +81-298-53-3138. is mostly involved in CDCA synthesis [6]. However, recent E-mail address: [email protected] (Y. Matsuzaki). studies have suggested that 27-hydroxycholesterol could be

0925-4439/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S0925-4439(02)00157-6 140 Y. Matsuzaki et al. / Biochimica et Biophysica Acta 1588 (2002) 139–148

Fig. 1. Bile acid biosynthesis from cholesterol initiated either by a series of ring modifications (classic pathway) or by side chain hydroxylation (alternative pathway). Solid arrows represent known enzymatic steps while broken arrows represent putative reactions that have yet to be confirmed. Y. Matsuzaki et al. / Biochimica et Biophysica Acta 1588 (2002) 139–148 141

2 converted into CA [7,8] while 3h-hydroxy-5-cholestenoic from MSD Isotopes; [11,11,12,12- H4]CDCA and [11,11,12, 2 acid was not [7]. Thus, CA and CDCA are synthesized by 12- H4]UDCA were supplied by the Research Laboratories both pathways, and CYP8B1 appears to be the most impor- of Nippon Kayaku Co. and Tokyo Tanabe Co. (Tokyo, tant enzyme regulating the ratio of CA to CDCA under Japan), respectively. Leupeptin, phenylmethylsulfonyl fluo- normal conditions [5,8]. ride and soybean trypsin inhibitor were purchased from In certain pathological conditions, however, CYP27A1 Sigma Chemical Co. (St. Louis, MO). The human anti- can also be important in determining CA/CDCA ratio. cholesterol 7a-hydroxylase antibody was a gift from Dr. T. Cerebrotendinous xanthomatosis (CTX) is a recessively Tanabe (National Center, Osaka, inherited disorder caused by mutations in CYP27A1 Japan). The rat anti-sterol 27-hydroxylase antibody was a located on human 2 [9–12]. In this disease, gift from Dr. D.W. Russell, University of Texas Southwestern the pool size of CDCA is markedly reduced while that of Medical Center, Dallas, TX. CA is almost normal [13]. When CYP27A1 is defective, not only 27-hydroxylation of cholesterol in the alternative 2.2. Patients pathway but also 27-hydroxylation of 5h-cholestane- 3a,7a-diol and 5h-cholestane-3a,7a,12a-triol in the classic We studied 20 human subjects. Eight had untreated pathway is blocked [14]. Therefore, in CTX, only the extrahepatic obstructive jaundice and the other 12 were microsomal 5h-cholestane-3a,7a,12a-triol 25-hydroxylase without hepatobiliary diseases. All patients provided written (CYP3A) pathway is intact for side chain hydroxylation, informed consent and this study was conducted according resulting in CA being the only bile acid synthesized [15,16] to the Declaration of Helsinki. Blood samples were (Fig. 1). obtained in the morning before breakfast after overnight In the present study, we selected the cholestatic hamster fasting, and the plasma and/or serum was stored at 20 jC with the common bile duct ligated for 48 h as model. This until analysis. model was chosen since it has both the hepatic capacity for cholesterol biosynthesis [17], as well as a bile acid compo- 2.3. Animals sition [18] similar to that found in human. In this study, we compared the bile acid concentration and composition in Adult male Golden Syrian hamsters (100–130 g body serum, bile and liver tissue, as well as CYP7A1, CYP27A1, weight) were purchased from Harlan Sprague Dowley, fed a CYP8B1 and CYP3A protein expression and activity in bile standard rodent chow diet and maintained on a 12:12 h duct-ligated and sham-operated hamsters. The results from light–dark cycle. the present study showed that in the cholestatic hamster, the increased serum CA/CDCA ratio was mainly due to the 2.4. Surgical procedure reduced hepatic CDCA production probably derived, at least in part, from the suppression of the mitochondrial After fasting for 24 h, hamsters were anesthetized with CYP27A1 protein expression and activity. sodium nembutal (50 mg/kg body weight). The ligation of the common bile duct was performed as previously described [24]. Briefly, the abdomen was opened with a midline 2. Materials and methods incision, the common bile duct was dissected from the surrounding tissue, and three ligations with silk thread were 2.1. Chemicals performed. The bile duct was cut between the second and third ligation from the gallbladder. Four hundred microliters 7a-Hydroxycholesterol was obtained from Steraloids of antibiotic solution (300,000 U/ml) containing penicillin G (Wilton, NH). 27-Hydroxycholesterol was synthesized from benzathine and penicillin G procaine (Durapen Vedco, Syr- diosgenin [19] and purified by preparative thin-layer chro- acuse, NY) were poured over the peritoneal cavity to avoid matography (TLC). 7a-Hydroxy-4-cholesten-3-one and infection. The incision of the abdominal wall was closed by 7a,12a-dihydroxy-4-cholesten-3-one were gifts from Dr. using separate sutures for the muscle layer and the skin. T. Hoshita and Dr. K. Kihira (Pharmaceutical Institute, Control hamsters underwent sham operation in which the Hiroshima University, Hiroshima, Japan). 5h-Cholestane- bile duct was not ligated. The hamsters were maintained 3a,7a,12a-triol was prepared by electrolytic coupling of under fasting conditions for 24 h after surgery. Cholestasis cholic acid with isovaleric acid according to Bergstro¨m and was suggested by the appearance of green urine and the Krabisch [20].5h-Cholestane-3a,7a,12a,25-tetrol was syn- presence of an enlarged gallbladder. After 48 h, the animals thesized from CA by the method of Dayal et al. [21]. were sacrificed at noon and blood was collected by direct 2 [25,26,26,26,27,27,27- H7]cholesterol was obtained from puncture of the heart. Livers were then immediately frozen 2 MSD Isotopes (Montreal, Canada). [ H7]7a-hydroxycholes- and stored at 70 jC until used. Serum and gallbladder bile 2 terol and [ H7]27-hydroxycholesterol were prepared by were stored at 20 jC until analysis. All animals received 2 previously described methods [22,23]. [2,2,4,4- H4]CA, humane care in compliance with the George Washington 2 2 [2,2,4,4- H4]DCA, and [2,2,4,4- H4]LCA were obtained University guidelines. 142 Y. Matsuzaki et al. / Biochimica et Biophysica Acta 1588 (2002) 139–148

2.5. Bile acid analyses 2.6.3. CYP3A activity CYP3A activity was determined by assaying CYP3A 2.5.1. Serum and biliary bile acids activity as described previously [31]. Microsomes were Bile acid concentrations and profile in serum and incubated for 20 min at 37 jCwith5h-cholestane- gallbladder bile were determined by high-performance 3a,7a,12a-triol (25 nmol dissolved in 10 Al of acetone), liquid chromatography (HPLC) combined with a 3a- NADPH (1.2 mM), glucose-6-phosphate (3.6 mM), 2 units hydroxysteroid dehydrogenase-immobilized column ac- of glucose-6-phosphate dehydrogenase, and 100 mM potas- cording to the method of Okuyama et al. [25]. Either sium phosphate buffer (pH 7.4) containing 0.1 mM EDTA serum or bile was diluted with 100 mM potassium phos- in a total volume of 0.5 ml. The reaction was stopped by phate buffer (pH 7.2), and 5h-pregnane-3a,17a,20a-triol adding 2 ml of ethyl acetate. After the addition of 1 Agof was added as an internal standard. The mixture was 5h-cholestane-3a,7a,12a,27-tetrol as an internal recovery incubated at 64 jC for 30 min. Bile acids were then standard, tetrols were extracted twice with 2 ml of ethyl extracted with a Bond Elut C18 cartridge (Varian, Harbor acetate, purified by a Bond Elut SI cartridge, derivatized to City, CA). After evaporation, the residue was dissolved trimethylsilyl (TMS) ether and quantified by high-resolution in methanol, filtered through an Ultra-Free C3HV filter GC-MS with SIM. (0.45 Am; Millipore, Bedford, MA), and analyzed by HPLC. 2.7. Determination of CYP7A1 and CYP27A1 protein expression level 2.5.2. Hepatic bile acids Bile acid concentrations and profile in liver tissue were Proteins from microsomal or mitochondrial fractions determined by gas-chromatography mass-spectrometry (25–50 Ag) were separated by SDS-polyacrylamide gel (GC-MS) according to our previously described method electrophoresis (PAGE) according to the procedure of [26]. Briefly, the liver specimen (10 mg wet weight) was Laemmli [32]. After electrophoresis, the proteins were washed carefully with saline to remove biliary bile acids. transferred onto a polyvinylidine difluoride membrane After addition of deuterium-labeled bile acids as internal (Immobilon-P, Millipore). Transferred microsomal fraction standards and solubilization in 5% NaOH at 80 jC for 30 was probed with a rabbit anti-human CYP7A1 antibody min, bile acids were extracted by a Bond Elut C18 cartridge (1:1000) and transferred mitochondrial fraction was probed and subjected to solvolysis and enzymatic hydrolysis. The with a rabbit anti-rat CYP27A1 antibody (1:1500), respec- resulting deconjugated bile acids were converted into the tively, followed by a donkey horseradish peroxidase-labeled ethyl ester dimethylethylsilyl (DMES) ether derivatives and anti-rabbit immunoglobulin G (1:3000) (Amersham, Arling- quantified by high-resolution GC-MS with selected-ion ton Heights, IL). The immunoreactive proteins were visual- monitoring (SIM). ized by enhanced chemiluminescence using a kit from Amersham. After being exposed to the membrane, the 2.6. Assays for enzyme activities Hyperfilm (Amersham) was analyzed by densitometric scanning. 2.6.1. CYP7A1 and CYP27A1 activities Hepatic microsomes and mitochondria were prepared by 2.8. Determination of steady-state mRNA levels of CYP7A1 differential ultracentrifugation [27], and the protein deter- and CYP27A1 mined according to the method of Lowry et al. [28]. The activities of microsomal 7a-hydroxylation of cholesterol Total RNA was obtained from the stored frozen liver and mitochondrial 27-hydroxylation of cholesterol were tissues by the method of Chomczynski and Sacchi [33] with measured by high-resolution GC-MS with SIM using minor modifications using RNA-Zol B reagent (Tel-Test 2 2 [ H7]7a-hydroxycholesterol and [ H7]27-hydroxycholes- Inc., TX). Complementary DNAs (cDNAs) were synthe- terol as internal recovery standards [29]. sized from total RNA with M-MLV reverse transcriptase by the random primer method. PCR was performed using 50 2.6.2. CYP8B1 activity pmol of each primer, 200 ng of cDNA template, 1.25 units Microsomal 12a-hydroxylase activity was assayed as of Taq DNA polymerase, 200 nmol/l deoxynucleoside described by Noshiro et al. [30] with minor modifications. triphosphate, in the reaction buffer (10 mmol/l Tris–HCl, Microsomes were incubated for 10 min at 37 jC with 7a- pH 8.3, 50 mmol/l KCl, 1.5 mmol/l MgCl2, and 0.01% hydroxy-4-cholesten-3-one (6 Ag dissolved in 5 Alof gelatin) using a DNA thermal cycler (Gene-Amp PCR isopropanol), NADPH (1 mM), and 100 mM potassium system 2400; Perkin Elmer, Norwalk, CT). PCR was sub- phosphate buffer (pH 7.4) containing 0.1 mM EDTA in a jected to the respective cycles (CYP7A1, 30; CYP27A1, 35; total volume of 0.3 ml. The incubation was stopped by the and glyceraldehyde-3-phosphate dehydrogenase (G3PDH), addition of 5 ml of benzene. The formation of 7a,12a- 26) at 94, 55, and 72 jC for 1 min each, respectively. PCR dihydroxy-4-cholesten-3-one was quantified by HPLC primers were designated by referring to cDNA sequences [30]. for rat G3PDH, human, rat and rabbit CYP7A1, and human Y. Matsuzaki et al. / Biochimica et Biophysica Acta 1588 (2002) 139–148 143 and rat CYP27A1. Primer designs were as follows: hamster Table 1 CYP7A1, sense 5V-CAC/TCT/CCA/TCT/TGA/GGA/CGG- Serum and biliary bile acid profile in sham-operated and bile duct-ligated hamsters 3V, antisense 5V-GTG/GCA/AAA/TTC/CCA/AGC/CTG-3V; hamster CYP27A1, sense 5V-ACC/TTC/CTC/CCC/AAG/ Sham (n = 5) BDL (n =5) TGG/AC-3V, antisense 5V-CAG/AAC/ACA/AAC/TGG/ Serum (%) Bile (%) Serum (%) Bile (%) GTG/TTC-3V; G3PDH, sense 5V-GAA/CGG/GAA/GCT/ CA CAC/TGG/CAT/GGC-3V,antisense5V-CAG/CAA/CAG/ F33F 17 0.3 F 0.2 0.2 F 0.0 0.8 F 0.5 F F F F GGT/GGT/GGA/CCT/CA-3V. Aliquots of the reaction mix- G168338401383 T11F 937F 945F 241F 8 ture were electrophoresed on a 2% agarose gel. PCR products were stained by SYBR-GREEN I (Molecular CDCA Probe Inc., Eugene, OR) according to the manufacturer’s F17F 10 0.1 F 0.1 < 0.1 < 0.1 instructions and semi-quantified using a multi-bioimager G5F 58F 44F 18F 3 F F F F (STORM, Molecular Dynamics Inc., Sunnyvale, CA). The T831359181 data were expressed relative to the amounts of G3PDH DCA mRNA present in each specimen. F9F 9 < 0.1 < 0.1 < 0.1 G2F 23F 2 0.1 F 0.0 2 F 1 2.9. Histological examination T < 0.1 4 F 3 < 0.1 2 F 1

LCA Liver specimens from both sham-operated and 2-day bile F < 0.1 0.1 F 0.0 < 0.1 < 0.1 duct-ligated hamsters were fixed in 2% paraformaldehyde G < 0.1 0.4 F 0.2 < 0.1 < 0.1 and mounted. Specimens were evaluated both by light and T < 0.1 0.7 F 0.5 < 0.1 < 0.1 electron microscopy. UDCA F < 0.1 < 0.1 0.9 F 0.4 0.4 F 0.4 2.10. Statistical analysis G < 0.1 < 0.1 < 0.1 < 0.1 T < 0.1 < 0.1 0.7 F 0.4 < 0.1 Data are reported as the mean F SD. The statistical significance between the results in the different groups Total 100 100 100 100 was evaluated by the Student’s two-tailed t-test. For all Serum and bile were collected from hamsters 2 days after sham operation comparisons, significance was accepted at the level of (Sham) or bile duct ligation (BDL). F, Unconjugated bile acid; G, glycine- P < 0.05. conjugated bile acid; T, taurine-conjugated bile acid. Data are expressed as mean F SD.

3. Results centration was elevated 34-fold ( P < 0.01) in patients with obstructive jaundice while the elevation was 116-fold Fig. 2 summarizes the data comparing the effects of ( P < 0.01) in hamsters 48 h after bile duct ligation. CA/ cholestasis on the serum bile acid concentration and com- CDCA ratio was increased from 0.30 to 1.54 ( P < 0.01) in position in human vs. hamster. Serum total bile acid con- patients and from 2.0 to 6.8 ( P < 0.01) in hamsters. The pool

Fig. 2. Effects of cholestasis on total bile acid concentration (A), ratio of cholic to chenodeoxycholic acid concentration (B) and proportion of unconjugated to total bile acid concentration (C) in human and hamster sera. Solid bars represent patients with extrahepatic obstructive jaundice (n = 8) or hamster 2 days after bile duct ligation (n = 5), while open bars represent control humans (n = 12) or sham-operated hamsters (n = 5). 144 Y. Matsuzaki et al. / Biochimica et Biophysica Acta 1588 (2002) 139–148

Table 2 Serum, hepatic and biliary bile acid concentrations in sham-operated and bile duct-ligated Hamsters Sham BDL Serum (n =5)[AM] Liver (n = 4) [nmol/g] Bile (n = 5) [mM] Serum (n =5)[AM] Liver (n = 4) [nmol/g] Bile (n = 5) [mM] CA 2.4 F 1.0 231 F 236 94 F 24 386 F 12y 680 F 227z 6.3 F 1.0y CDCA 1.2 F 1.0 63 F 57 29 F 12 57 F 10y 68 F 26 1.3 F 0.2y DCA 0.4 F 0.3 56 F 50 10 F 5 0.3 F 0.2 82 F 54 0.3 F 0.2§ LCA < 0.1a 7.8 F 8.8 1.6 F 0.7 < 0.1a 16 F 13 < 0.01a,y UDCA < 0.1a 29 F 27 0.04 F 0.02 7.6 F 3.6§ 18 F 13 0.03 F 0.02 Total 3.9 F 2.5 387 F 112 134 F 14 452 F 38y 864 F 105y 7.9 F 1.0y CA/CDCA 2.0 F 1.9 3.6 F 1.4 2.8 F 1.0 6.8 F 1.4§ 11.0 F 5.8z 5.0 F 1.2z Serum, liver and bile were collected from hamsters 2 days after sham operation (Sham) or bile duct ligation (BDL). Data are expressed as mean F SD. y P < 0.001 vs. sham-operated hamsters. z P < 0.05 vs. sham-operated hamsters. § P < 0.01 vs. sham-operated hamsters. a Limit of detection. of unconjugated bile acids was reduced from 29% to 5.2% elevated in bile duct-ligated hamsters. The highest CA/ ( P < 0.01) in patients and from 59% to 1.1% ( P < 0.01) in CDCA ratio was observed in bile duct-ligated hamster liver hamsters. tissue. In this model, the hepatic CA concentration was The respective serum and biliary bile acid compositions increased 2.9-fold ( P < 0.05) while the other bile acid levels in bile duct-ligated and sham-operated hamsters are shown were not significantly different from those in control liver. in Table 1. In sham-operated hamsters, the proportion of Table 3 shows the activity of hepatic key enzymes serum unconjugated bile acids (59%) was significantly involved in bile acid biosynthesis. Microsomal CYP7A1, greater than that in bile (0.5%). In contrast, these propor- CYP8B1 and CYP3A activities were similar in both bile tions were rather similar (1.1% vs. 1.2%) in bile duct-ligated hamsters. The ratios of glycine-conjugated to taurine-con- jugated bile acids was not significantly different between bile duct-ligated and sham-operated hamsters. Table 2 reports the respective serum, hepatic and biliary bile acid concentrations, as well as the CA/CDCA ratios in the bile duct-ligated and sham-operated hamsters. After bile duct ligation, in contrast to a markedly increased serum CA and CDCA concentration, these bile acid concentrations were significantly decreased in gallbladder bile. However, the serum and bile CA/CDCA ratios were both significantly

Table 3 Hepatic activities of mitochondrial CYP27A1, and microsomal CYP7A1, CYP8B1 and CYP3A in sham-operated, bile duct-ligated and cholestyr- amine-treated hamsters Enzymes Sham (n = 5) BDL (n = 5) CHY (n =4) pmol/min/mg protein Mitochondrial CYP27A1 1.7 F 0.3 0.8 F 0.3 * 1.7 F 0.8 Microsomal CYP7A1 1.6 F 1.0 1.4 F 0.6 4.7 F 2.3y CYP8B1 10 F 611F 3ND CYP3A 38 F 16 25 F 12 ND Livers were obtained from hamsters 2 days after either sham operation (Sham), bile duct ligation (BDL) or fed a 4% cholestyramine containing diet for 1 week (CHY). Specific activities of CYP27A1, CYP7A1, CYP8B1 and CYP3A were determined by measuring 27- and 7a-hydroxylations of cholesterol, 12a-hydroxylation of 7a-hydroxy-4-cholesten-3-one and 25- hydroxylation of 5h-cholestane-3a,7a,12a-triol, respectively. Data are Fig. 3. Western blot of hamster liver microsomal CYP7A1 and mito- expressed as mean F SD. chondrial CYP27A1. S, sham-operated (n = 4); B, bile duct-ligated (n = 4); ND, not determined. C, cholestyramine-treated (n = 4). *, Percentage of sham operation. * P < 0.005 vs. sham-operated hamsters. yP < 0.001, compared to bile duct ligation, zP < 0.05, compared to sham y P < 0.05 vs. sham-operated hamsters. operation. Y. Matsuzaki et al. / Biochimica et Biophysica Acta 1588 (2002) 139–148 145 duct-ligated and sham-operated hamsters. In contrast, the CYP7A1 protein expression level did not change signifi- mitochondrial activity of CYP27A1 was decreased by 53% cantly while that of CYP27A1 was significantly decreased ( P < 0.005) in the bile duct-ligated hamsters. In the present in the bile duct-ligated hamsters. This is in agreement with study, we also used hamsters fed a diet containing 4% the above reported results on these enzymes’ specific cholestyramine known to activate hamster microsomal activities. We also determined the steady-state mRNA CYP7A1 [34,35] as control (Table 3). The protein expres- expression levels for CYP7A1 and CYP27A1 by semi- sion levels of CYP7A1 and CYP27A1 were determined by quantitative RT-PCR (Fig. 4). However, bile duct ligation Western blotting method using specific antibodies against for 2 days did not change the mRNA expression levels of the human CYP7A1 and rat CYP27A1. As shown in Fig. 3, either of these two enzymes. Histological examination suggested dilatation of the hepatic ducts as well as increased biliary ductule prolifer- ation in the bile duct-ligated hamster liver. This was accompanied by infiltration of neutrophils and mononuclear cells. However, neither hepatic necrosis nor periportal or portal fibrosis was found. The electron microscopic findings of liver sections 48 h after bile duct ligation were charac- terized by swollen hepatocytes and a marked decreased or complete disappearance of glycogen rosettes. The endoplas- mic reticulum was dilated while the mitochondria showed no significant pathological change in either form or number. With respect to the canal of Hering, the epithelium showed irregular nuclei in shape and size.

4. Discussion

Our cholestatic hamster model showed a significantly increased serum CA/CDCA ratio and a markedly decreased ratio of unconjugated to total bile acids, which are character- istic features of patients with cholestatic liver diseases. These results are different from those reported in the bile duct-ligated rats. In the cholestatic rat model, the synthesis of CA is either not changed [36] or decreased [37], whereas the h-muricholic acid production is markedly enhanced [36,38,39],andtheCA/h-muricholic acid ratio reduced [36,39]. Since h-muricholic acid is formed from CDCA [39], CA/h-muricholic acid ratio in the rat relates to the CA/ CDCA ratio in human and hamster. The effect of bile duct ligation on the total bile acid synthesis appears to be different between rats and hamsters. In bile duct-ligated rats, CYP7A1 activity was significantly up-regulated [37,40,41], whereas CYP7A1 activity was not changed in our hamster model. Similar results were also reported in rhesus monkeys [42] and humans [43] where total bile acid synthesis was suppressed during biliary obstruction. Thus, there are considerable species-specific differences in bile acid , and the hamster may be a better model than the rat for studying bile acid metabolism relevant to patients with obstructive jaundice. Abnormal flow from hepatocyte canaliculi to sinusoids is one of the important reasons for the increased level of serum Fig. 4. Relative amount of steady-state mRNA for CYP7A1, CYP27A1 and total bile acids. Actually, in bile duct-ligated hamsters, the G3PDH in the livers of sham-operated, bile duct-ligated and cholestyr- serum bile acid composition, including CA/CDCA ratio and amine-treated hamsters. S, sham-operated (n = 4); B, bile duct-ligated (n = 4); C, cholestyramine-treated (n = 4). *, Ratio of mRNA expression glycine- and taurine-conjugation pattern, were very similar level between respective enzymes and G3PDH, percentage of sham to that found in bile (Table 1). However, the elevated serum operation. yP < 0.001, compared to sham operation. CA/CDCA ratio in bile duct-ligated hamsters cannot be 146 Y. Matsuzaki et al. / Biochimica et Biophysica Acta 1588 (2002) 139–148 solely explained by this abnormal back flow from bile or ulates CYP27A1 at the transcriptional level, while CA hepatocytes to serum. The reason is that not only in serum inhibits CYP27A1 post-transcriptionally by affecting the but also in the liver and bile, the CA/CDCA ratio was stability of its mRNA [52]. It is important to know that significantly increased in the bile duct-ligated hamsters. there may be species-specific differences in the control of Therefore, altered bile acid synthesis in the cholestatic liver, CYP27A1 because in rat hepatocytes CA i.e. enhanced CA synthesis and/or decreased CDCA pro- repressed the transcription of CYP27A1 gene [53]. duction is strongly suggested. There are at least two hypotheses to explain the mech- When we measured the activity and protein expression anism(s) of decreased CYP27A1 activity in bile duct-ligated level of CYP7A1 and CYP27A1, two key enzymes in the hamster. First, as shown in human [52] and rat [53], classic and alternative bile acid biosynthetic pathways, CYP27A1 might be inhibited by an increased hepatic CA respectively, CYP7A1 was not affected but CYP27A1 was concentration in cholestasis. Hamster hepatic CYP27A1 significantly down-regulated following bile duct ligation. showed both reduced specific activity and protein expres- These results lend support to our hypothesis that in contrast sion level without decreased steady-state mRNA level, to the bile duct-ligated rats, the total bile acid synthesis was suggesting CYP27A1 to be post-transcriptionally down- not increased in the bile duct-ligated hamsters. A recent regulated. In the rat extrahepatic cholestatic model, the report by Pandak et al. [8] demonstrated that CYP8B1 was activity, protein and mRNA levels of the sodium-dependent capable of 12a-hydroxylating bile acid intermediates from taurocholate cotransporting polypeptide (ntcp) were mark- both the classic and alternative pathways. In addition, these edly reduced [54]. In contrast, 6h-hydroxylations of CDCA authors described CYP8B1 to be the sole regulator of the and LCA were significantly up-regulated and these hydro- CA/CDCA ratio. However, as seen in CTX patients, phobic bile acids were metabolized to less cytotoxic h- CYP27A1 is also an important enzyme in determining CA/ muricholic acid [37,40]. Thus, in the rat model, a signal CDCA ratio under pathological conditions. In fact, decreased seems to be generated under cholestatic conditions to CYP27A1 activity without change of CYP8B1 activity was regulate bile acid uptake and synthesis, and to diminish observed in bile duct-ligated hamsters and was associated the accumulation of potentially hepatotoxic bile acids in with an increased CA/CDCA ratio. Recent reports show that rats. Humans and hamsters do not synthesize h-muricholic induction of CYP3A family of is one of the important acid, but rather reduce the formation of CDCA by down- mechanisms for detoxification of hydrophobic bile acids in regulating CYP27A1 as a protective mechanism, which cholestasis [44,45]. The retained activity of CYP3A that is parallels the formation of h-muricholic acid in the rat. also a key enzyme in the alternative side chain cleavage Second, hydrophobic bile acids including CDCA are pathway in CA biosynthesis [46] may explain why the known to cause cellular ATP depletion due to impairment production of CA was not reduced as much as that of CDCA of mitochondrial function in rat hepatocytes [55]. Although in the bile duct-ligated hamster model. the hepatic concentration of bile acids other than CA were An interesting observation concerns the regulation of not elevated in the cholestatic hamster model, that of CDCA CYP7A1 in cholestasis. In spite of a significantly increased has been suggested to be transiently increased in the earlier hepatic total bile acid concentration, CYP7A1 was not period after bile duct ligation. CDCA has also been shown down-regulated in the cholestatic liver. CDCA is a physio- by us [24,56], as well as by others [57] to activate various logical ligand for the farnesoid X receptor (FXR), an orphan signal transduction pathways. It is therefore a possibility nuclear receptor [47–49]. The binding of CDCA to the that this bile acid can be responsible for the decreased receptor leads to the repression of CYP7A1 gene tran- CYP27A1 protein expression and/or activity through its scription. Our results showed that the hepatic CA concen- regulation of signaling mechanisms. The role for a regu- tration was increased while that of CDCA remained latory mechanism is supported by the fact that while the unchanged in the bile duct-ligated hamster, which is similar glycogen rosettes were markedly decreased, there was no to what is observed in the liver of patients with jaundice of significant pathological change in either mitochondrial short duration [50]. This may be one of the reasons for the shape or number in the cholestatic hamster liver. lack of suppression of CYP7A1 activity and expression in In summary, the hamster hepatic mitochondrial bile duct-ligated hamster, although another orphan nuclear CYP27A1 was post-transcriptionally down-regulated while receptor, LXRa [51] may also play an important role in the the microsomal CYP7A1 and CYP8B1 were not affected by regulation of CYP7A1 in cholestasis. bile duct ligation. This could explain, at least in part, the In contrast to microsomal CYP7A1, CYP8B1 and increased serum CA/CDCA ratio and the decreased CDCA CYP3A enzymes, mitochondrial CYP27A1 activity and production observed in cholestatic hamsters. protein level were significantly reduced in the liver of cholestatic hamsters. Although the regulation of CYP27A1 has not yet been clarified, Segev et al. [52] recently reported Acknowledgements that the human CYP27A1 gene had a TATA-less promoter and its transcription was initiated at a cluster of sites. This work was supported by a grant from the Ministry of Dexamethasone up-regulates and cyclosporin A down-reg- Education, Science and Culture of Japan #12670457 Y. Matsuzaki et al. / Biochimica et Biophysica Acta 1588 (2002) 139–148 147

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