JournalofHepatology, 1992; 14: 151-156 @ 1992 Eisevier Science Publishers B.V. Ali rights resewed.

0168-8278/92/$05.00

1.51

HEPAT 01048

Gary C.

Vitale’,

Yong

Siow’,

Peter W. Bake? and Alfred Cuschiet?

‘Department of Surgery, Universityof Louisville School of Medicine, .Louisville. KY, United Statesof America, 2Department of Surgery, Universityof Birmingham, Birmingham and 3Department of Sulgery, Universityof Dundee, Dundee, United Kingdom

(Received 2 April 1990)

The etiology of hepatocellular dysfunction resulting from chronic biliary obstruction is not clearly understood. Alterations in bile acid metabolism due to changes in microsomal cytochrome P-450 enzyme activities may have a fundamental role in cholestatic liver injury. This study examines the very early changes in both biliary bile acids and hepatic microsomal cytochrome P-450 content after bile duct obstruction in the rat and the effects of the restoration of bile flow after 3 days of biliary obstruction. We found that early induction of cytochrome p-450 may be a fundamental step in the generation of cholestatic liver injury mediated by hepatotoxic bile acids. The rapid reversal of bile acid changes with reconstituted bile flow indicate that the liver is able to quickly recover when obstruction is relieved . characterization of this fundamental process may ultimately provide a means of modulation of cholestatic hepatotoxicity.

Bile duct obstruction leads to the accumulation in the liver of compounds normally excreted in bile (1). Some of these compounds of either endogenous or exogenous origin are potentially toxic. Unrelieved biliary obstruction can lead to acute and chronic liver failure in surgical patients with cholestasis. The etiology of the hepatocellular dysfunction is not clearly understood, but early interference of cellular processes by retained biliary metabolites may lead to irreparable cellular damage. Bile secretory products are metabolites that generally have hydrophobic properties. Bile and chenodeoxycholic acids, in particular, by nature of their hydrophobicity are potentially toxic (2,3). During biliary obstruction, some of the aberrant hepatocellular morphology observed has been attributed to the interactions of accumulating bile acids (43. In addition, significant alterations in bile acid metabolism has led to the formation of atypical bile acids in humans, which may also be hepatotoxic (6,7). The formation of normal physiologic bile acids from cholesterol involves a series of reactions catalyzed by the enzymes of both the endoplasmic reticulum (microsomes)

and mitochondria (8). These reactions, which lead to the shortening of the side chain of the cholesterol molecule (mitochondrial) and hydroxylation at different positions of the steroid ring (microsomal), have specific dependency on the involvement of different cytochrome P-450 isozymes. In contrast t? the atypical bile acids found in association with other gastkntestinal disorders that are the products of gut bacterial metabolism, atypical bile acids formed during cholestasis are the results of further modifications of physiologic bile acids by liver cytochrome P450 isozymes (1,9,10). Distinct changes in the acid composition of bile after biliary obstruction have been reported in the rat (llJ2). Although these compositional changes are different than those found in humans with obstructive jaundice, they are also the products of alterations in the activities of microsoma1 cytochrome P-450 enzyme or enzymes of the liver; therefore, early changes in the activities of the microsoma1 enzymes are likely to have a fundamental role in cholestatic liver injury. This study focuses on the characterization of the early changes in both biliary bile acids and

Correspondence: Dr. Gary C. Wale, University of Louisville, Department

of Surgery, Louisville, KY 40292, U.S.A.

G.C. VITALE et al.

152

hepatic microsomal cytochrome P-450 content after bile duct obstruction in the rat. The effects of restored bile flow on acid composition after biliary obstruction were also investigated.

Materials and Methods

Five- to 7-month-old male Wistar rats were obtained from Charles River (Kent, United Kingdom). They were housed individually and given free access to water and food (rat diet No. 1, Special Diet Services, Essex, United Kingdom). All animals were anesthetized with sodium pentobarbital(50 mg/kg, intraperitoneal) before abdominal exploration. Three experimental groups were studied as outlined below (Fig. 1). Additionally bile was collected from a group of anesthetized normal rats (n = 9) who had not undergone previous anesthesia or sham operation. This group was studied to compare with sham-operated rats in an effort to demonstrate any anesthesia-related bile acid compositional changes. In group I, rats in the experimental group had their common bile duct exposed and cannulated with silicone rubber tubing (0.25 mm i.d. X 0.5 mm o.d., 15 cm), while those in the control group (sham) underwent a similar operation where bile duct manipulation rather than bile duct cannulation was performed. Cannula occlusion in animals of the experimental group was carried out with double 5-O silk ties. Animals were killed within 1 or 3 days postoperatively to collect bile via cannulas from the common bile duct. Cannulas were similarly placed in the sham group animals at 1 or 3 day intervals after the initial operation for bile collection from unobstructed bile ducts. Group II rats were also explored and a cannula was placed in the bile duct, with occlusion by 5-O silk ligature at the distal end of the cannula. Three days after the operation, however, rats in this group were reoperated on, bile was collected via cannula from the obstructed biliary system, and bile flow was restored by placement of the tip of the cannula into the duodenum with purse-string suture II.

III.

Bile duct cannulation and occlusion + restoration of enterohepatic circulation

Bile duct ligation and diwsion

1. Bile duct cannulation and occlusion

I Bile collection for bile acid compositional analysis Fig. 1. Animal groups studied.

Microsomal fractions prepared

closure. These animals were killed 1 day after restoration of bile flow and following bile collection (day 4). Controls for these experiments consisted of the sham operation and manipulation of the bile duct, with bile collection taking place 3 and 4 days postoperatively via cannula placement into the unobstructed biliary system. Group III rats had bile obstruction by ligation and division, without bile duct cannulation. Control animals underwent the sham operation with bile duct dissection and manipulation but without bile duct ligation. All animals were killed 1 or 3 days after bile duct obstruction, and the livers were perfused in situ with cold saline by gravity feed via the portal vein. The portal vein was catheterized using an 8-inch (gauge 16) intravenous catheter, and perfusion started immediately after the inferior vena cava was severed. Severing the inferior vena cava allowed rapid and free drainage of perfusate, thus avoiding engorgement of the liver. Liver microsomal fractions were then prepared by differential centrifugation, as described by Remmer et al. (13). Microsomal protein concentration and cytochrome P-450 content were determined by Lowry e’i al. (14) and Gmura and Sato (15)) respectively. Bile was collected in groups I and II, initially for 10 min (0 to 10 min) then a further 20 min (11 to 30 min), from each of the experimental and control animals. Samples were placed in preweighed polystyrene containers and tightly capped. Bile samples were heated for 20 min at 60°C and stored until assayed for bile acid composition and concentration. Bile acid concentrations in the collection taken in the first 10 min were determined using 3-asteroid dehydrogenase, as described by Iwata and Yamasaki (16). Bile samples collected in the last 23 min were analyzed using high performance liquid chromatography, as previously described by Baker et al. (17).

Results

Elevated serum bilirubin concentration was used as a simple expression for the induction of cholestasis in this study. Bilirubin levels in rats after both 1 and 3 days biliary obstruction were significantly higher compared with those obtained for all sham-operated animals. Total bile acid concentrations in the lo- and 20-min bile samples, from both sham-operated rats and rats 1 day after biliary obstruction (n = 6), were determined enzymatically. The concentration of bile acids in the first lo-min bile samples was not significantly different compared to those consecutively obtained for a further 20 min. A Student’s f-test for paired data showed 36.16 f 5.24 PM (mean -+ S.D.) and 34.62 f 6.04pM. Using the relative retention of bile acid standards ob-

REVERSIBLE TABLE

BILE ACID

CHANGES

153

1

Bile acid composition: Bile acid

bile from normal rats and rats 1 and 3 days after sham operation or biliary obstruction” Normal

Group I

(9)b Tauro$-muricholic Taurocholic Tauroursodeoxycholic Taurohyodeoxycholic Taurochenodeoxycholic Taurodeoxycholic

36.6 + 1.9 30.8 + U.2 3.2 2.2 + 5.0 zk 1.5

1 day.,3)b -__ 38.1 k 2.2 26.3 + 0.2 4.9 2.4 F 1 3 0 3c@)

38.5 k 4.8 32.3 1.7 2.6 i 0.4 1.0 i 0.3

4.3 + 0.4 4.3 + 0.7

7:3’&‘2.5 5.6 + 1.0

5.3 + 1.6 11.4 + 3.2

@-murichohc Cholic

9.3 + 5.7 + N.D. 0 2W M.D. 0 1W 1. 1 +

Total (,&I)

29.0 2 5.0

Glyco-#I-muricholic Glycocholic Glycoursodeoxycholic Glycochenodeoxycholic Glycodeoxycholic

1.8 0.9

0 .4c(5)

Group II

~___ 3 days (3)b

8.2 + 4.5 5.8 + 2.8 N.D. 2 1 1.6c(2) 0’8:(‘,

2 5 3 3@) 4:2’&- 1.4 0 2 0.5f(2) 0’2’0 5~0) 1’3&1)

0 8C(t) 2.9 f 0.8

0 2cU) 0.9,0.8c(2,

38.8 + 4.0

1 day (Qb

3 days (lO)b

61.2 + 1.1 30.1 02+ zk01.4F(s) 3:9 IO:5

79.9 + 2.7 17.3 f 2.6 0.4 f 0.2’(“) 0 6 l.W(*) ] .‘8’+ - 0 .qc(*) 0 2cW

0.4,l.W 0 3cW

33.3 + 2.3

319 + 0.8 0.5 0.3@) N.D: ND. N.D. N.D. N.D.

a:3 + 0.2c(3) 0 LC(‘) I& N.D. N.D. N.D. N.D.

44.5 + 2.2

24.7 k 1.1

a Data are expressed as mean + S.E. percent distribution of total or percentage of total values for the bile acids detected only in one or two animals for each group. b Numbers in parentheses represent number of animals in each group. ’ Superscript represents the number of the bile samples (given in parentheses) analyzed in which the bile acid was found. ._ N.D. = not detected.

tamed after chromatographic separation, the major bile acids in rat bile were identified and their respective concentrations determined as a proportion of their peak size. Table 1 lists the bile acids found as a percentage of the total bile acid content in all the samples obtained from normal rats as well as from rats 1 and 3 days after either a sham procedure or cannulation and occlusion of biliaq Bow. The snperscripts indicate the number of rat bile samples obtained in each respective group where the bile acids were present. fn sham-operated and experimental rats, bile acids were predominantly present as conjugates of taurine and glycine; taurine being the more prevalent (Table 1). In both normal and sham-operated rats, tauro&muricholic and taurocholic acids, which constitute between 64-71% of total bile acid, increased to greater than 90% after I and 3 days of biliary obstruction. The only unconjugated (free) bile acids found in the rat bile were cholic and /I-muricholic acids, which were found in the bile samples from normal and sham-operated rats. These two bile acids were not present in rats after biliary obstruction. One day after ligation, the distribution percentage of tauro-@-muricholic acid was significantly greater than in the sham-operated animals. The Mann-Whitney test indicated 61.2 f 1.1% compared with 38.5 + 2.2% (Table 1, p c 0.01). At the same time, hepatic microsomal cytochrome P-450 concentration (nmol/g liver) in rats with bile duct ligation was higher compared to control, although statistical significance was not attained (Table 2). Total microsomal cytochrome P-450 content per whole

TABLE

2

Hepatic cytochrome P-450 content Sham (6Y

Group III (6)

Student’s t-test

5.5 f 0.6 56.6 + 15.2

8.5 + 3.4 99.8 +- 37.2

N.S. p 4 0.03

Three days postoperation 8.2 i 3.0 nmoYg liver 94.8 t 34.2 nmol/liver

4.8 f 1.6 58.5 i 24.0

p 4 0.03 N.S.

One day postoperation nmoYg liver nmohliver

a The number of rats in each group is shown in parentheses. Statistical analysis was by Student’s t-test. N.S. = not significant.

liver was significantly higher in rats with biliary obstruction compared to control, 99.8 + 37.2 nmol and 56.6 f 15.2 nmol0, < 0.03, mean f SD.), respectively. After 3 days, however, microsomal cytochrome P-450 content was decreased significantly in rats of the experimental group (p < 0.03), while the percentage of distribution of tauro-b-muricholic acid was progressively higher. Fig. 2 shows the effects of biliary obstruction, after both 1 and 3 day durations, on the percentage of distribution of the harmless bile acids (tauro-/3-muricholic and raurocholic) and the hepatotoxic acids (taurodeoxycholic and chenodeoxycholic). After 1 day of biliary obstruction, tauro#?-muricholic acid levels were significantly greater compared with control. This difference was even greater after 3 days of obstruction. While taurocholic acid levels 1 day

G.C. VITALE et al.

154

ro-@-muricholic acid levels were greatly reduced compared with levels found without restoration of bile flow (p c 0.002). This reduction in the levels of tauro-p-muricholic acid occurred with a corresponding elevation of the dihydroxy bile acid content.

100

g

80

2

60

Ii? ltn zi

40

a

Discussion M

20

0

A

B

A

C

1 DAY

0

C

3 DAYS

Fig. 2. Percent distribution of bile acids after 1 and 3 day intervals of biliary obstruction. Values are mean zk S.D. The bide acids shown are: A = tauro-@-muricholic acid; B = taurocholic acid; and C = taurochenodeoxycholic and taurodeoxycholic acid. Open bars represent sham; closed bars represent bile duct ligation and division (group III). Statistical analyses was by Mann-Whitney test. “p C 0.05, “p < 0.02 and cp 4 6.X compared with respective control groups.

100

b

0 TWO-~muricholic

Taurocholic

Tourodeoxycholic & Taurochenodeoxycholic

Fig. 3. Percent distribution of bileacidsfollowingrestorationof bile flow after three days of biliary obstruction. Values are mean + S.D. Open bars represent normal; closed bars bile duct cannulation and occiusion (group I); striped bars, bide duct occlusion with restoration of enterohepatic circulation (group II). Statistical analysis was by Mann-Whitney test. *p c 0.02, bp c 0.01 compared to normal, ‘p c 0.002, and “p < 0.001 compared to without restoration of bile flow.

after biliary obstruction were similir, levels of this bile acid were significantly lower compared with control after 3 days. Taurodeoxycholic and taurochenodeoxycholic acids were lower in the bile duct ligated rat groups compared with their respective control group. The effects of the restoration of enterohepatic circulation on bile acid composition after 3 days of biliary obstruction are shown in Fig. 3. Tauro-#I-muricholicacid levels were significantly elevated 3 days after biliary obstruction, with a concomitant reduction in levels of the dihydroxy bile acids as well as taurodeoxycholic and taurochenodeoxycholic acids. One day after the restoration of bile flow, however, tau-

The synthesis of bile acids from cholesterol is catalyzed by enzymes of the endoplasmic reticulum (microsomes). The enzymatic reactions that occur in the microsomes involve the addition of hydroxyl groups to the steroid nucleus. Most, if not all, of this series of enzymatic reactions are catalyzed by the enzymes of the cytochrome P-450 system. The specific P-450 isozyme associated with bile acid biosynthetic reactions, however, have not been fully elucidated. The overall result of these enzymatic reactions is the conversion of a highly insoluble cholesterol molecule to a relatively soluble bile acid molecule that possesses unique detergent properties. Hepatic dysfunction in extrahepatic jaundice may oc. cur with increased levels of the bile acids of greater detergent properties; in particular, the dihydroxylated bile acids such as chenodeoxycholic acid. Feathery hepatocyte degeneration is seen histologically in humans after bile duct obstruction (5). After bile duct ligation in the rat, the secondary bile acids (deoxycholic and hyodeoxycholic) disappear due to the interruption of enterohepatic circulation (9). Levels of the primary dihydroxylated bile acids also decrease with a corresponding rise in the concentration of the less toxic trihydroxylated bile acids and tauro@-muricholicacid. This preferred pathway for the metabolism of chenodeoxycholic acid in the rat has been thought to be a protective mechanism in cholestasis (11,12). Since /I-muricholic acid is the product of 6/3-hydroxylation of chenodeoxycholic acid (18), we would therefore expect a transient increase in the hepatic content of the highly toxic dihydroxy bile acids. In the absence of this hydroxylation reaction, hepatic accumulation of chenodeoxycholic acid would occur as has been demonstrated in the hamster and human during biliary obstruction (19,20). Chenodeoxycholic acid, a dihydroxy bile acid, is more hydrophobic than the trihydroxy bile, cholic, and &muricholic acids, and therefore has greater affinity for binding to lipid-rich membranes of intracellular organelles (12,21). This phenomenon is believed to have a role in hepatocellular injury (20,22-24). Cholic acid is not known to exhibit similar hepatocelluiar toxicity (25). The rapid reversal of this process, with decreasing tauro-@muricholic acid and a corresponding rise in the di-

REVERSIBLE BILE ACID CHANGES

hydroxylated bile acids after restoration of bile flow, implies that there ts a sensitive biofeedback mechanism involving the 6p-hydroxylation of chenodeoxycholic acid. Although our data does not demonstrate a rise in levels of dihydroxylated bile acids early after bihary obstruction, these bile acids are observed to rise after restoration of btie flow compared with sham animals. During biliary obstruction, the increase in the rate of 6/Lhydroxyiation may obscure the detection of increased dihydroxylated bile acid levels; whereas after restoration of bile flow, the greater synthesis of these toxic bile acids can be seen. Our data showed an increase in total cytochrome P-450 content. This increase may account for the observed greater 6fi-hydroxylation of chenodeoxycholic acid to /3muricholic acid. After 3 days of bile duct ligation, however, hepatic content of cytochrome P-450 was reduced, as has also been reported by other workers (26,2?). This decrease may be due to reduced synthesis of the apoprotein, which has a half-life of about 1 day (28,29). While this may be the case, biliary obstruction may also induce qualitative changes in the isozymes, leading to continued prefferences Reichen J, Simon FR. Cholestasis. In: Arias IM, Jakoby WB, Popper H, Schachtes D, Shafritx DA, eds. The Liver: Biology and Pathobiology. New York: Raven Press, 1988; 1105-24. Leuschner U. Liver tissue injury due to chenodeoxycholic acid: metabolic pathways and toxicity. In: Paumgartner G, Stiehl A, Gerok W, eds. Biologicai Effects of Bile Acids. Lancaster: MTP Press, 1979; 191-203. PaImer RI-I. Bile acids, Liverinjury and liver disease. Arch Intern Med 1972; 130: 606-17. Popper H, Schaffner F. Pathophysiology of cholestasis. Hum Patholl970; 1: l-24. Greim H, TruIxsch D, Cxygan P, Hutterer F, Schaffner F, Pop per H. Changes of hepatic bile acid pattern in extrahepatic cholestasis in rat and man. In: Back P, Gerok W, eds. Bile Acids in Human Disease. Stuttgart: Schaffauer Verlag, 1972; 131-4. Murphy GM, Jansen FH, Billing BH. Unsaturated monohydroxy bile acids in cholestatic liver disease. Biochem J 1972; 129: 491-4. Fouin-Fortunet H, LeQuernec L, Erhnger S, Loubours F, Cohn R. Hepatic alterations during total parenteral nutrition in patients with intlammatory bowel disease: a possible consequence of lithocholic acid toxicity. Gastroenterolgy 1982; 82: 932-7. 8 Danielsson H, SjovaII J. Bile acid metabotism. Annu Kev BIOthem 1975; 44: 233-53. 9 Carey MC, CahaIane MJ. Enterohepatic circulation. In: Arias IM, Jakoby WB. Popper H, Schachter D, Shafritx DA, eds. The Liver: Biology and Pathobiology. New York: Raven Press, 1988; 573-616. 10 Hofmann AF. Bile acids. In: Arias IM, Jakoby WB, Popper H, Schachter D, Shafritx DA, eds. The Liver: Biology and Pathophysiology. New York: Raven Press, 1988; 553-72. 11 Greim H, TruIxsch D, Roboz J, et al. Mechanisms of cholestasis. V. Bile acids in normal rat livers and in those after bile duct Iigation. Gastroenterology 1972; 63: 837-45. 12 Kinugasa T, Uchida K, Kadawaki M, Takase H. Nomura Y, Saito Y. Effect of biIe acid duct ligation on bile acid metabolism in rats. J Lipid Res 1981; 22: 201-7.

1% erential synthesis of specific bile acids. For example, t preferential induct f specific cytochrome p-&Oj isozymes with higher y alcohol has been reported (30). The production of chohc and chenodeoxycholic acids is regulated by a cytochrome P-450 dependent enzyme, 7ahydroxycholest-4-ene-3-one 12a-monooxygenase, acting on a common precursor (31,32). The induction of the specific cyctochrome P-450 isozyme with high K,,, for this precursor may, in part, be responsible for the early increased production of cholic acid during biliary obstruction. Reversion of this isozyme to the form with low K,,, for the same precursor initiated by the lower tissue bile acid concentration, however, may explain the accumulation of dihydroxylated bile acids after restoration of bile flow. The sensitivity of these enzymatic activities to revert to the favorable production of less toxic bile acids may be crucial to the development of hepatocellular injury. Modulation of this enzyme would then, potentially, allow amelioration of the hepatotoxic effect of bile duct obstruction. Characterization of this fundamental process may provide a means of modulation of cholestatic hepatotoxicity.

13 Remmer H, Greim H, Schenkman JB, Estabrook RW. Methods for the elevation of hepatic microsome mixed function oxidase levels and cytochrome P-450. Methods Enxymol 1967; 10: 703-8. 14 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265-75. 15 Omura T, Sato R. The carbon monoxide-binding pigment of Iiver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem 1964; 239: 2370-B. 16 Iwata T, Yamasaki K. Enzymatic determination and thin-layer chromatography of bile acids in blood. J Biochem 1964; 56: 424-31. 17 Baker PR, VitaIe GC, Siow YF. Medroxyprogesteione acetateand ethinylestradiol-induced changes in biliary bile acids of the rat studied by high-performance liquid chromatoghraphy. J Chromatogr 1987; 423: 63-73. 18 Voight W, Thomas PJ, Hsia SL. Enzymic studies of bile acid metabolism. I. 6 beta-hydroxylation of chenodeoxycholic acid and taurochenodeoxycholic acids by microsomal preparations of rat liver. J Biol Chem 1968; 243: 3493-9. 19 GaIeaxxi R, Javitt NB. Bile acid excretion: the alternate pathway in the hamster. J CIin Invest 1977; 60: 693-701. 20 Greim H, TruIzsch D, Cxygan P, et al. Mechanisms of cholestasis. VI. Bile acids in human livers with or without biiary obstruction. Gastroenterology 1972; 63: 846-50. 21 Carey MC. Physical-chemical properties of bile acids and their salts. In: Danielsson H, SjovaIl J, eds. Sterols and Bile Acids. New York: Elsevier, 1985; 345-403. 22 Schaffner F, Popper H. Cholestasis is the result of hypoactive hypertrophic smooth endoplasmic reticulum in the hepatocyte. Lancet 1969; ii: 355-9. 23 Miyai K, Price VM, Fischer MM. Bile acid metabolism in mammals: uhrastructural studies on the intrahepatic cholestasis induced by Iithocholic acid and ch.:nodeoxycholic acid in the rat. Lab Invest 1971; 24: 292-302. 24 Palmer RH, Bile salts and the liver. Prog Liver Dis 1982; 7: 721-42. 25 Kakis G, Yousef IM. Mechanism of cholic acid protection in li-

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G.C. VJTALE et al. fluence of phenobarbital in the turnover of hepatic microsomal cytochrome bs and cytochrome P-450 hemes in the rat. Biochim Biophys Acta 1970; 201: 20-S. 30 Thomas PE, Bandiera S, Maines SL, Ryan DE, Levin W. Regulation of cytochrome P-45Oj, a high affinity N-nitrosodiiethylamine demethylase in rat hepatic microsomes. Biochemistry 1987; 26: 2280-g. 31 Greim H. Bile acids in hepatobiliaty diseases. In: Padmanabhan NP, ed. The Bile Acids: Chemistry, Physiology and Metabolism. New York: Plenum Press, 1976; 53-80. 32 Murakami K, Okada Y, Okuda K. Purification and characterixation of 7alpha-hydroxy-4-cholesten-3-one 12alpha-monooxygenase. J Biol Chem 1982; 257: 8030-5.

Reversible bile acid changes in bile duct obstruction and its potential for hepatocellular injury.

The etiology of hepatocellular dysfunction resulting from chronic biliary obstruction is not clearly understood. Alterations in bile acid metabolism d...
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