Production Bile Duct- Hyperplasia Gallstones .Production of Bile Duct-Hyperplasia and Gallstones

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  • Journal of Clinical InvestigationVol. 45, No. 8, 1966

    Production of Bile Duct- Hyperplasia and Gallstones byLithocholic Acid *

    ROBERT H. PALMER t AND ZDENEK HRUBAN(From the Departments of Medicine and Pathology, and the Argonne Cancer Research

    Hospital,4 University of Chicago, Chicago, Ill.)

    Lithocholic acid 1 is an important metabolite ofcholesterol in man and other animals. Interest inits biological properties stems from its markedtoxicity. It is the most potent of the naturally oc-curring steroids that produce intense fever and in-flammation in man (2, 3) and inflammation in anumber of other species (3, 4). It is also one ofthe most active steroid hemolysins (5). In addi-tion, its oral administration produces cirrhosis ofthe liver in rabbits (6) and ductular cell hyper-plasia in a variety of species, including rodents(7), reptiles (8), and primates (9). In view of itsphysiological occurrence, its known toxic proper-ties, and its potential relevance to human disease,we have investigated the effects of feeding largeamounts of lithocholic acid to rats. The resultingbile duct hyperplasia and choledocholithiasis aredescribed in this report.

    Methods

    The acute effects of lithocholic acid and sodium litho-cholate on Sprague-Dawley rats were investigated in

    * Submitted for publication August 23, 1965; acceptedApril 21, 1966.

    Presented in part at the Annual Meeting of the Ameri-can Society for Clinical Investigation, Atlantic City, N. J.,May 2, 1965. A preliminary report has been published(1).Supported in part by U. S. Public Health Service re-

    search grant CA-05310 from the National Cancer -In-stitute.t Address requests for reprints to Dr. Robert H.

    Palmer, Dept. of Medicine, University of Chicago, 950 E.59th St., Chicago, Ill. 60637.t Operated by the University of Chicago for the U. S.

    Atomic Energy Commission.1 Trivial names of bile acids used in this report are as

    follows: lithocholic acid, 3a-hydroxy-5p-cholanoic acid;6P-hydroxylithocholic acid, 3a,6fi-dihydroxy-5p-cholanoicacid; hyodeoxycholic acid, 3a,6a-dihydroxy-5,f-cholanoicacid; chenodeoxycholic acid, 3a,7a-dihydroxy-5p-cholanoicacid; deoxycholic acid, 3a,12a-dihydroiy-SjS-cholanoicacid; cholic acid, 3a,7a,12a-trihydroxy-5f8-cholanoic acid.

    experiments I and II and chronic effects in experimentsIII and IV. Lithocholic acid was obtained commer-cially 2; no quantitatively significant bile acid contami-nants were observed when it was analyzed by thin layerchromatography [system S15 of Eneroth (10)]. Thesodium salt was prepared by neutralization of the acidwith sodium hydroxide.

    In the first experiment, 5 male rats, average weight372 g, were force fed 300 mg sodium lithocholate per kgbody weight per day in a liquid diet (11) containing 3.6g casein hydrolysate per kg body weight per day. Therats were fed 3 times daily at 8-hour intervals and killedwith ether on the sixth day.

    In the second experiment, 9 males and 9 females, av-erage weight 171 g, were divided into 3 equal groups andforce fed the basic liquid diet as described above. Onegroup served as controls. A second group was fed litho-cholic acid, 300 mg per kg body weight per day, in thediet. The third group received sodium lithocholate, 300mg per kg body weight per day, in the diet All ratswere killed with ether on the twelfth day.

    In the third experiment, 10 male and 10 female controlrats (average weights: males, 193 g; females, 157 g) werefed ad libitum an 8% protein diet 8 containing 8% casein,78% starch, 10% vegetable oil, and 4% salt mixtureU.S.P. XIV, to which had been added 1 kg Vitamin DietFortification Mixture2 per 100 pounds diet. A similargroup of rats (average weights: males, 193 g; females,157 g) was fed the same diet containing 1% lithocholicacid. The powdered diets were mixed with sufficient wa-ter to permit molding into conveniently sized balls.Generally a 1- to 2-weeks' supply was made up and re-frigerated. A ball was placed in each cage every 2 to 4days, depending on spillage. At the end of the fourthmonth the surviving animals were lklled by decapitation.

    In the fourth experiment, 5 control and 15 treated rats(150 to 200 g) were force fed the liquid diet of the firstexperiment for 1 month and then placed on the solid dietof the third experiment ad libitum. The treated animalsreceived sodium lithocholate, 300 mg per kg per day inthe liquid diet and 1% in the solid diet. After 4 months,the surviving animals were killed.At autopsy, livers were weighed and tissue samples

    fixed in buffered formalin and Carnoy's fluid. Sectionswere stained with hematoxylin-eosin, Mallory trichrome,periodic acid Schiff, and methyl green-pyronine.

    2Nutritional Biochemicals Corp., Cleveland, Ohio.8 "Low 8% Protein," Nutritional Biochemicals Corp.

    1255

  • ROBERT H. PALMER AND ZDENEK HRUBAN

    TABLE I

    Body and liver weights of rats fed lithocholic acid for 4 months(experiment III)

    No. Inital Average weight p Average liver psurviving weight gain A SD value weight A SD value

    g I gFemale controls 10 157 70.4 12.6 .6.00 :1 0.71

  • LITHOCHOLIC ACID-INDUCED BILE DUCT PROLIFERATION AND GALLSTONES

    Results

    Morphological changes. In the acute experi-ments, the rats appeared to be normal, and nogross pathological changes were noted at autopsyexcept that one rat in the sodium lithocholategroup of experiment II had a small soft calculus inthe distal hepatic duct and some dilatation of theduct proximally. In the chronic experiments, thetreated rats lost hair and often assumed a hunchedposture. At the time of autopsy, as shown in Ta-ble I, treated female rats had not gained weight aswell as controls, but treated male rats had gainednormally. Liver weights in treated animals ofboth sexes were significantly increased over con-trols. In both chronic experiments, however, themost striking pathological feature was the presenceof large common duct calculi in all of the treatedrats (Figures 1 and 2). The stones were usuallymultiple, yellow or green, and soft and friable, butmarked variations in color, size, and shape wereapparent.

    Livers of rats fed lithocholic acid or sodiumlithocholate in both acute and chronic experimentsshowed microscopic changes that were essentiallysimilar and limited to the biliary system. The por-

    p*;. '9

    ' k

    44

    4z

    I@,~~~~~~FIG. 3. PORTAL AREA OF LIVER FROM A RAT FED LITHO-

    CHOLIC ACID FOR 4 MONTHS (EXPERIMENT III). Bile ductproliferation and pericholangitis are present. Some ductsare distended and contain amorphous material. H and Estain; x 85.

    tal areas of the liver showed marked bile duct pro-liferation and variable mononuclear cell infiltration(Figure 3). Within bile ducts, basophilic crystal-line deposits (Figure 4) with staining charac-teristics similar to those of lithocholic acid em-1)edded in agar (Figure 5) were frequently ob-served. Ductal cells, some undergoing necrosis,were seen associated with eosinophilic debris in theltimina of the bile ducts (Figure 6). A similar

    FIG. 2. RAT SHOWN IN FIGURE 1 AFTER OPENING THECOMMON DUCT. Calculi of various sizes are shown.

    1257

    ----- ------ ---- ---F 4lwa;

    FIG. 4. CRYSTALLINE BASOPHILIC MATERIAL FROM THECENTER OF A CALCULUS. H and E stain; X 1,200.

  • ROBERT H. PALMER AND ZDENEK

    FIG. 5. LITHOCHOLIC ACID IN AGAR GEL. H and Estain; X 1,200.

    eosinophilic material often surrounded the baso-philic crystalline deposits, either loosely (Figure7) or more densely (Figures 8 and 9) to formmicrocalculi. Dense infiltrates of eosinophilicleukocytes were seen around damaged bile ductsand in the wall of the extremely hypertrophiedcommon duct (Figure 10). Although focal he-patic necroses were found occasionally, the hepato-cytes were generally intact, with normal glycogen

    *'ii~owsFw wyF#--=w *fi- w~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

    V.- 5 9SRK

    FIG. 6 INTRA EPATIC BIL S.........F....A.. R Fe

    Basphiiccrysain maeil is see on.th r.:igt. the.....wall of th duc wit eqaaigcell is on th lef

    H andE Fstain >( 450 .....................

    FIGL>.6.:.INR.E I BIL DUCT FROM A RAT FED

    _pi ic crstalin maera is see on.th rigt h

    and Estin X 50

    ,Y

    FIG. 7. LIVER SECTION AS IN FIGURE 6. The large ductcontains basophilic material (B) centrally and eosinophilicmaterial (E) peripherally. H and E stain; X 185.

    preservation and the usual cytoplasmic basophilicbodies. Ultrastructural studies of rat livers fromthe first experiment showed only hyperplasia ofthe Golgi complex and an increase in the amountof smooth endoplasmic reticulum; the bile cana-liculi were not distended. Electron micrographs

    FIG. 8. CALCULUS IN A LARGE INTRAHEPATIC BILE DUCTFROM A RAT FED SODIUM LITHOCHOLATE FOR 4 MONTHS(EXPERIMENT IV). The center contains basophilic,densely clumped crystalline material; the capsule is eosin-ophilic. H and E stain; _ 465.

    HRU13AN1258

  • LITHOCHOLIC ACID-INDUCED BILE DUCT PROLIFERATION AND GALLSTONES

    FIG. 9. CALCULUS AS IN FIGURE 8. The denselypacked outer shell and the reticular inner layer are dis-cernible. H and E stain; X 1,200.

    of the calculi revealed electron dense flaky materialin a lucid matrix, similar but not identical to theappearance of calcium lithocholate preparedsimilarly.

    Composition of lithocholic acid-induced rat gall-stones. The calculi in experiment III had averagedry weights of 5.97 0.71 g for males and 4.141.75 g for females. Gallstones from 15 of the ratswere analyzed, with the results given in Table II.Eighty-five per cent of the dry weight was ac-counted for by the analyzed constituents, primarilybile acids. Small amounts of nondiazo-reactingpigments, carbohydrate, and probably protein andnucleic acids were also present, but in the presenceof large amounts of bile acids, identification wasnot