Gut, 1980, 21, 860-865 Protective effects of cholestyramine on liver cirrhosis induced by carbon tetrachloride in the rat K DE HEER, H-D SAUER, B WERNER, AND G KLOEPPEL From the Department of General Surgery and Institute of Pathology, University Hospital Hamburg, Hamburg, Federal Republic of Germany SUMMARY The influence of cholestyramine and chenodeoxycholic acid on the induction of liver cirrhosis by carbon tetrachloride was investigated in the Wistar rat. The addition of 1.3 % cholesty- ramine to the diet of the experimental animals inhibited to a large extent the induction of cirrhosis. While all the animals subjected to carbon tetrachloride exposure plus basal diet and those to carbon tetrachloride intoxication plus chenodeoxycholic acid diet developed cirrhosis, the morphological manifestation of cirrhosis occurred in the livers of only two out of 18 rats under carbon tetrachloride treatment plus cholestyramine diet. The administration of cholestyramine induces reactions which correspond to the physiological protective mechanisms of the liver. These are the bile acid binding, bacteriostatic, and microsomal enzymatic stimulating properties of cholestyramine. The central role of the liver in metabolism of the bile acids includes the synthesis, conjugation, and the maintenance of the integrity of the enterohepatic circulation. The control of the size and composition of the bile acid pool and the detoxification of nox- ious intermediate products from the intestinal tract is modified and regulated by the hepatocytes. These functions are more or less impaired during cirrhosis or other diseases of the liver. Consequently the toxic bile acids, especially the secondary bile acids, lithocholic and deoxycholic acids, in the entero- hepatic circulation are not detoxified.'-3 Whether the disturbed bile acid metabolism has an influence on the impaired liver has only been investigated to a limited extent. Furthermore, the pathogenic effects of several intermediate products of bile acid metabolism have been experimentally verified.4-8 Thus Holsti induced liver cirrhosis in the rabbit by oral administration of desiccated whole bile preparation9 and by gastric instillation of lithocholic acid.10 The question that particularly interests us, there- fore, is whether the oral application of cheno- deoxycholic acid (CDCA), the main source of lithocholic acid, and cholestyramine (CT), a quater- nary ammonium anion exchange resin, which has a strong affinity for bile salts in the intestine and thus prevents their enteral resorption, has an influence on Received for publication 13 May 1980 the induction of cirrhosis by carbon tetrachloride (CCI4) in the rat. Methods ANIMALS Four week old male Wistar rats weighing between 200 and 300 g were obtained from the Deutsche Versuchstieranstalt, Hannover, West Germany. Four animals were kept in a cage in a room with a daily cycle of alternating 12 hour periods of light and darkness. DIET The animals were maintained on a special diet obtained from the firm Dr R Scholt in Hamburg, West Germany: (1) chenodeoxycholic acid diet: 300 g basal diet was supplemented with 1 g CDCA; (2) cholestyramine diet: 300 g basal diet was sup- plemented with 4 g cholestyramine; (3) control diet consisted of basal diet without supplement. ANIMAL GROUPS The following groups were formed. Group I Twenty animals fed on the basal diet were treated with CC14. Group II Twenty animals fed on the cholestyramine diet were treated with CCI4. 860 on October 6, 2021 by guest. Protected by copyright. http://gut.bmj.com/ Gut: first published as 10.1136/gut.21.10.860 on 1 October 1980. Downloaded from
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Gut, 1980, 21, 860-865
Protective effects of cholestyramine on liver cirrhosisinduced by carbon tetrachloride in the ratK DE HEER, H-D SAUER, B WERNER, AND G KLOEPPEL
From the Department of General Surgery and Institute of Pathology, University Hospital Hamburg,Hamburg, Federal Republic of Germany
SUMMARY The influence of cholestyramine and chenodeoxycholic acid on the induction of livercirrhosis by carbon tetrachloride was investigated in the Wistar rat. The addition of 1.3 % cholesty-ramine to the diet of the experimental animals inhibited to a large extent the induction of cirrhosis.While all the animals subjected to carbon tetrachloride exposure plus basal diet and those to carbontetrachloride intoxication plus chenodeoxycholic acid diet developed cirrhosis, the morphologicalmanifestation of cirrhosis occurred in the livers of only two out of 18 rats under carbon tetrachloridetreatment plus cholestyramine diet. The administration of cholestyramine induces reactions whichcorrespond to the physiological protective mechanisms of the liver. These are the bile acid binding,bacteriostatic, and microsomal enzymatic stimulating properties of cholestyramine.
The central role of the liver in metabolism of the bileacids includes the synthesis, conjugation, and themaintenance of the integrity of the enterohepaticcirculation. The control of the size and compositionof the bile acid pool and the detoxification of nox-ious intermediate products from the intestinal tractis modified and regulated by the hepatocytes. Thesefunctions are more or less impaired during cirrhosisor other diseases of the liver. Consequently the toxicbile acids, especially the secondary bile acids,lithocholic and deoxycholic acids, in the entero-hepatic circulation are not detoxified.'-3 Whetherthe disturbed bile acid metabolism has an influenceon the impaired liver has only been investigated toa limited extent.
Furthermore, the pathogenic effects of severalintermediate products of bile acid metabolism havebeen experimentally verified.4-8 Thus Holsti inducedliver cirrhosis in the rabbit by oral administration ofdesiccated whole bile preparation9 and by gastricinstillation of lithocholic acid.10The question that particularly interests us, there-
fore, is whether the oral application of cheno-deoxycholic acid (CDCA), the main source oflithocholic acid, and cholestyramine (CT), a quater-nary ammonium anion exchange resin, which has astrong affinity for bile salts in the intestine and thusprevents their enteral resorption, has an influence on
Received for publication 13 May 1980
the induction of cirrhosis by carbon tetrachloride(CCI4) in the rat.
Methods
ANIMALSFour week old male Wistar rats weighing between200 and 300 g were obtained from the DeutscheVersuchstieranstalt, Hannover, West Germany.Four animals were kept in a cage in a room with adaily cycle of alternating 12 hour periods of lightand darkness.
DIETThe animals were maintained on a special dietobtained from the firm Dr R Scholt in Hamburg,West Germany: (1) chenodeoxycholic acid diet:300 g basal diet was supplemented with 1 g CDCA;(2) cholestyramine diet: 300 g basal diet was sup-plemented with 4 g cholestyramine; (3) control dietconsisted of basal diet without supplement.
ANIMAL GROUPSThe following groups were formed.Group ITwenty animals fed on the basal diet were treatedwith CC14.Group IITwenty animals fed on the cholestyramine diet weretreated with CCI4.
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Protective effects of cholestyramine on liver cirrhosis induced by carbon tetrachloride in the rat
Group IIITwenty animals fed on the cholestyramine diet weretreated with CCI4.Group IVFive animals were allowed the basal diet and tapwater but were not treated with CC14.
DRINKING WATERSodium phenobarbitone was dissolved in tap waterat a concentration of 0.5 g/l. This was the only drink-ing water available to the animals in groups I, II,and III.
INDUCTION OF CIRRHOSISThe induction of cirrhosis in the experimentalanimals was carried out according to the method ofMcLean et al.1' The animals in groups I, II, and IIIwere placed in a wooden box with a glass front for 20minutes twice a week. Oxygen was passed througha flowmeter at 21/min, bubbling through a train oftwo wash-bottles containing CCI4 maintained at20°C, into the box. CCI4 was blown in for 10 min-
utes and the rats then left in the inhalation chamberfor an additional 10 minutes. The CC14 treatmentwas curtailed after 10 weeks. During the experi-mental period the animals were weighed every twoweeks.
NECROPSY AND HISTOLOGYSixteen weeks after the first CC14 exposure the ani-mals were killed and dissected. The liver was re-moved in toto and fixed in 3.5o% formalin. For thehistological slides 5 ,u thick paraffin slices wereprepared and stained with haematoxylin and eosinand PAS alternately.
Results
During the CCI4 administration five animals fromgroup I, four in group II, and two in group III died.These were not considered in the final evaluation ofthe results. The weight measurement showed no sig-nificant differences between the various groups.Only the control group steadily gained weight.
Fig. 1 Rat liver after CC14 exposure: cirrhosis with complete destruction of normal architecture and theformation of regeneration nodules (arrow). PAS, x 84. (Original magnifications are given in this and the followingfigures.)
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MACROSCOPIC FINDINGSIn all the animals in group I (only CCI4 treatment)and group II (CCI4 treatment plus CDCA diet) theliver had a fine nodular surface and a firm consis-tency, while most of the animals in group III(CCI4 treatment plus CT diet) had livers with asmooth surface and normal consistency. Only twoof 18 animals in this group had livers with a finenodular surface and firm consistency.
MICROSCOPIC FINDINGSIn group I (only CC14 treatment) and group II(CC14 treatment plus CDCA diet) a completecirrhosis developed in all the animals (Table). Thecirrhosis was highly active in the majority of theanimals. Regeneration nodules of varying sizes andremnants of the original liver lobules were seenbetween broad scars and thinner connective tissuesepta which were infiltrated mostly with histio-cytes (Fig 1). In some livers the cirrhosis wasless obvious, the parenchyma being irregularly sep-arated by thin strands of connective tissue. Thesesepta were scantily infiltrated with histiocytes (Fig. 2)There was no cholestasis.
A comparison of the histological pictures of theliver in group I (only CCI4 treatment) and group II(CC14 treatment plus CDCA diet) showed that ingroup lI the condensation of fibrous septa and theinfiltration with histiocytes were more pronouncedthan in the group I.
In group III (CCI4 treatment plus CT diet) oneanimal developed a complete cirrhosis with mildactivity. In another animal only parts of the livershowed nodularity, while the other parts displayed a
Fig. 2 Rat liver after CC14 exposure and chenodeoxycholic acid administration: cirrhosis with irregular separationof the liver parenchyma by strands of connective tissue (arrows). PAS, x 196.
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Protective effects of cholestyramine on liver cirrhosis induced by carbon tetrachloride in the rat
Fig. 3 Rat liver after CC14 exposure and cholestyramine dietary supplement: liver tissue with fibrotic changesbut persisting basic architecture. Moderate fibrosis is seen in the portal field (white arrow) and the centrilobulararea (asterisk). Groups ofPAS-positive histiocytes at the margin of the centrilobular fibrosis (black arrow).PAS, x 196.
distinctive fibrosis of the portal tracts and centrilobu-lar areas (incomplete cirrhosis). In contrast, onlyfibrotic but no cirrhotic changes were found in theremaining 16 animals. The fibrosis developed in theportal and centrilobular areas (Fig. 3) and was ofvarying intensity. It was marked in four animals andslight in 12 animals (Table). Within the centrilobularscars and the adjacent parenchyma there weregroups of histiocytes laden with ceroid pigment,a consequence of single hepatocyte necrosis (Fig. 3).The hepatocytes showed distinct anisokaryosis of thenuclei. Bile duct proliferation was lacking. In theuntreated animals (group IV) no pathologicalchanges were found.
Discussion
The most important result of this experimentalstudy is the fact that the induction of liver cirrhosisby carbon tetrachloride in the rat is strongly modifiedby simultaneous administration of 1-3 % cholestyra-
mine. Only two out of 18 animals developed cirrhosisof the liver, while the remaining 16 showed scantilydistributed single hepatocyte necrosis with mildcentrilobular cicatrisation.Under physiological conditions the toxic litho-
cholic acid is formed by 7-alpha bacterial dehydroxy-lation of chenodeocholate in the intestine (usuallyin the colon and terminal ileum). Because of poorabsorption only part of the lithocholate reaches theliver via the enterohepatic circulation. An importantaspect of the lithocholate detoxification whichoccurs mainly in the liver in humans and in the rat isesterification with sulphate.2 Furthermore, the intactliver has the ability to convert lithocholate into thenon-toxic hyodeoxycholic acid.12 This physiologicaldetoxification process is catalysed by the enzymes7-alpha and 12-alpha hydroxylase.13 With impair-ment of liver functions, clinically by cirrhosis andexperimentally by CCI4 intoxication, the detoxifica-tion of lithocholic acid is more or less retarded.Because of severe liver damage a relative deficiencyof 12-alpha hydroxylase activity develops resulting
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in an increased formation of chenodeoxycholic acidinstead of cholic acid, which is transformed bycolonic bacteria into lithocholic acid.5The liver damage in the experimental animals
caused by CC14 inhalation consists of a partialparenchymal necrosis and a consequent reduction inthe microsomal enzymatic activity. Because in therat 80% of bile acids are located in the microsomes,'4the hydroxylation reactions which take place inthese organelles are further impeded. Changes inoptical absorption spectra produced by addition ofCCI4 to microsomes suggest a close attachment ofCC14 to cytochrome P450.16 Similarly, bile salts areknown to interact with microsomal elements,specially cytochrom P450. It is, therefore, conceiv-able that the simultaneous administration of CCI4and CDCA causes an increased inhibition of cyto-chrome P450. The inhibition of cytochrome P450leads to increased formation of lithocholate directlyfrom cholesterol or indirectly via dehydroxylation ofchenodeoxycholate in the enterohepatic circulation.'6The cytotoxic effects of bile salts are related to
their amphipatic properties and involves interactionwith membranes and organelles. The incorporationwith lysosomes plays an especially important role incausing tissue injury."7 18
Cholestyramine, which has a strong affinity forbile acids and bacteriostatic effects, causes a re-duction in the bacterial population of the colon and asequestration of primary bile salts preventing it frombiotransformation into lithocholate.19 Consequently,less lithocholate is produced after cholestyramineadministration. In addition, this bile acid bindingresin stimulates the enzymes 7-alpha and 12-alphahydroxylases," a deficiency of the latter leads toincreased serum lithocholic acid concentrationswhich perpetuate liver injury.5
Studies on the effects of carbon monoxide onhydroxylation in the biosynthesis and metabolism ofbile acids have revealed that the 12-alpha hydroxyla-tion may not involve a cytochrome P450, a carbon-monoxide sensitive haemoprotein.13 This is anindication that the stimulation of 12-alpha hydroxy-lation after cholestyramine does not lead to analteration of the levels of cytochrome P450. Thus,when CC14 and cholestyramine are simultaneouslyadministered, the latter causes an increase in theactivity of microsomal hydroxylation reactions butleaves the level of microsomal cytochrome P450unchanged. The resulting condition maintainsother microsomal oxidative reactions to a consider-able extent, compared with the group treated withCC14 alone. Whether these effects of cholestyraminereduce the damage caused by CCI4 intoxication orrepair the damage afterwards merits additionalinvestigation.
It is, therefore, possible that, through the ad-ministration of cholestyramine, mechanisms aretriggered which correspond to the physiologicalprotective reactions of the liver. This could explainthe effect of cholestyramine in modifying the livercirrhosis induced by carbon tetrachloride.
References
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12Einarsson K. On the formation of hyodeoxycholic acidin the rat. J Biol Chem 1966; 241: 534-9.
1Johannson G. Effect of cholestyramine and diet onhydroxylations in the biosynthesis and metabolism ofbile acids. Eur JBiochem 1970; 17: 292-5.
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",Garner RC, McLean AEM. Increased susceptibility tocarbon tetrachloride poisoning in the rat after pre-treatment with oral phenobarbitone. Biochem Pharma-col 1969; 18: 645-50.
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'6Palmer RH. Bile acids, liver injury, and liver disease.Arch Intern Med 1972; 130: 606-17.
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