[CANCER RESEARCH 38, 4634-4639, December 1978]0008-5472/78/0038-0000$02.00

Effects of Dimethylnitrosamineon Induction of Cholangiocarcinoma inOpisthorchis viverrini-infected Syrian Golden Hamsters1

Witaya Thamavit, Natth Bhamarapravati,2 Somphong Sahaphong, Suvajra Vajrasthira,3 and SubhkijAngsubhakorn

Department of Pathobiology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 4, Thailand

ABSTRACT

Opisthorchiasis is an endemic parasitic disease innortheastern Thailand. The concomitant occurrence ofthe parasite and chobangiocarcinomaas well as the mcidenceof suchtumorsare higherinthat partof the countrythan in other areas. Nitrosatingagents, nitrosatablesubstances,and nitrosaminecompoundsare commonlypresent in several kinds of stable foods in that region. Tostudy the interactionsbetween the parasite and the nitrosocompounds,we dividedSyriangoldenhamstersintofour groups:GroupI , untreated;Group2, dimethylnitrosamine (DMN, 00025% or 25 ppm)-treated;Group3, 100metacercariae-treated;and Group4, DMN (0.0025%or 25ppm) plus 100 metacercariae-treated groups. The animalsthat received both DMN and parasites (Group4) developed cholangiocarcinoma(100%) and cholangiofibrosis(100%). The tumor was not observed in the group thatreceived either DMN (Group 2) or parasites alone (Group3), aithoughcholangiofibrosiswas foundin someanimalsin the DMN group(Group2). It is postulatedthat cholangiocarcinomas in these animals arose because the DMNexerted a carcinogenic effect on the altered prolIferatingepithelialcells of bile ductsthat had been stimulatedbythe parasite.These findingssuggestthat the combinationof DMN ingestionand liver fluke infestationmay play animportant robe in the carclnogenesis of the intrahepatic

ductneoplasmsin humanbeings.

INTRODUCTION

Liven fluke infection of man in northeastern Thailand andwest Malaysia is caused by Opisthorchis viverrini (5, 26),whereas in other Asian countries it is caused by 0. fe!ineusand C!onorchis sinensis (26). The association of liver flukesand cholangiocarcinoma as well as an increased incidenceof such cancer in the endemic areas have been reported (4,8, 10-12, 23, 26—28).Nitnosamine compounds formed fromnitnosating agents, particularly nitrites, nitrates, and nitnosatable secondary on tertiary amines, are well known to behighly carcinogenic (14). A kind of fermented fish product,namely Pla na, is popularly eaten by the northeastern Thaipeople. Migasena and Changbumrung (15) found that thisfood contained components necessary for nitrosamine for

I This investigation was supported in part by National Research Council

of Thailand.2 Present address: Department of Pathology, Faculty of Medicine, Rama

thibodi Hospital,MahidolUniversity,Bangkok,Thailand.3 Present address: Department of Parasitology, Faculty of Tropical Medi

cine, Mahidol University, Bangkok, Thailand.ReceivedMarch22, 1978;acceptedAugustii , 1978.

mationand thata dietcontaining20% Pbaracaused fattymetamorphosis in rat liver after feeding for 6 weeks (16). Inaddition to Pla ra, other preserved protein foodstuffs, suchas dried fish, sausage, dried shrimp, cured pork, and saltymeat, as well as the common salts and ground waterconsumed by local populations, have been reported (17) tocontain high levels of either nitrosating agents and nitrosatable substances or nitrosating agents alone. NitrosaminesinPIaraor nitrosaminesformed inthestomach (14)afterconsuming fermented fish have also been assigned a contnibutory role in the high incidence of primary liven carcinoma in Thailand (15). DMN,4 the simplest form of nitrosamine, has been reportedto induce hepatocellularcarcinoma in various animals (14) and cholangiocarcinoma,particularly in hamsters (9, 14, 25). These reasons promptedus to study the interaction between the liver fluke and thenitrosamine compound. A preliminary communication onthe occurrence of cholangiocarcinoma in liver fluke-infested hamsters treated with DMN was recently published(2).

MATERIALSAND METHODS

Preparationof Metacercariae. Preparationof metacercanal cysts of 0. viverrini obtained from the northeasternregion of Thailand was carried out as described elsewhere(3).

Animalsand ExperimentalDesign.Syriangolden hamsters (SEATO Medical Research Laboratory Center, Bangkok,Thailand)aged 3 to4 weeks were kept3 ina cage inatemperature-controlled room at 27°.They were fed a stockdiet [Zuellig (Gold Coin Mills) PTE. Ltd., Singapore]throughout the 22 weeks of the experimental period. Theanimals were divided into 4 treatment groups: Group 1,untreated; Group 2, 0.0025% DMN (MERCK-Schuchardt,Schuchardt, 8011 Hohenbnunn bei MUnchen, Germany);Group 3, 100 metacencaniae; on Group 4, 0.0025% DMN plus100 metacercaniae. The untreated (Group 1) and the metacencania groups (Group 3) were composed of 18 animals,whereas the other 2 groups were composed of 21 animals.Metacercaniaewere administeredtotheanimalsviaintragastric tube. For the combined DMN and metacercaniaetreated group (Group 4), 0.0025% DMN in drinking waterwas administered as soon as the parasitic eggs were detected in the stool (4 weeks after infection). For the animalstreated with DMN alone (Group 2), DMN was not given untilthe animals reached the age of 7 to 8 weeks. DMN in bothgroups was withdrawn after 10 weeks of administration,and these animals were left untreated for another 8 weeks,

4 The abbreviation used is: DMN, dimethylnitrosamine.

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Probability between 2 groups: untreatedversus DMN, p 0.025 to 0.05; untreatedversusmetacercariae,p < 0.005;untreatedversusDMNplus metacercariae,p < 0.005;DMNversus

metacercaniae,p 0.2 to 0.3; DMN versus DMN metacercaniae,p < 0.005; metacercariaeversusDMNplusmetacencaniae,p < 0.005.Average

waterAverage DMN Total DMNinintake/animal/intake/animal/ take/animal % of liverweight/Experimental

group day (ml)day (mg) (mg) bodyweight1

. Untreated 8.893.32 ±0.06@2.DMN 7.300.18 12.783.63±0.153.Metacercariae 8.993.74±0.114.DMN + metacercariae 7.350.18 12.86 5.88 ±0.23a

(Mean ± SE.)

Effects of DMN on Cholangiocarcinoma

except that 5 animals in the DMN group were left to 23weeks. At the end of the experiment, the animals werestarved overnight prior to sacrifice. Before the liver andother organs were removed, a small piece of tumorous livertissue was aseptically removed from each of 6 differentsuspected tumor-bearing animals and further minced intoseveral tiny pieces. The pieces were then mixed with a fewdrops of 10% chboramphenicol solution (Kemicitine succinate; Carlo Erba, Italy). Each of the 6 preparations wasinoculatedi.m.intothe righthind leg of 6 respectivehomologous animals. The transplanted animals were kept 3in a cage for observation. After the transplanted tumorshowed growth, the tumors from 2 of these were transplanted further into 2 new recipients. The organs of theexperimental animals were fixed in 10%buffered formalin,and representative areas were embedded in paraffin. Five

@mthick sections were stained with hematoxylin and eosin,Masson trichrome stain, McManus's method for glycogen(periodic acid-Schiff), Gomori's method for reticulum, andMayer mucicarmine method for mucin (13). General laboratory cane of animals in this experiment followed theprocedure used in our previous work (24).

RESULTS

The averagedaily intake of water and DMN and the totalDMN intake, as well as the percentage of liver weight, aresummarized in Table 1.

The averageweights of the animals as a function of timeafter the treatment are shown graphically in Chart 1. Comparedto Groups 1 to3,thegrowth rateoftheanimalsinGroup 4 was decreased from the middle period of DMNadministration to the end of the second week after DMNwithdrawal.

GrossFindings.Therewerenogrosslesionsof the liversin the untreated group. In the DMN group the livers of someanimals had a few pink discolored patches of soft consistency on the surface of some lobes, and light nodularity withsmall white areas were seen in the livers of a few animals.Most of the liver of animals in the metacercaria-treatedgroup showed outstanding nodubanity with many areas offirm pink discolored patches on the surface. Gall bladderand cystic duct were considerably dilated. Compared toGroups 1 to 3, Group 4 had bangerlivers. The surface wasnodular with hemorrhagic areas and irregularly distributedlarge white hard areas. In most animals there were more

than one of these prominent white firm nodules measuring0.3 to 1 cm on the surface (Fig. 1). A few large mucinouscysts varying from 0.5 to 1.5 cm in diameter were encountered in 3 animals.

Histological Findings. The main histological findings inthe livers were classified according to the scheme of Edwards and White (7) and Stewart and Snell (21).There wereno significant changes in the untreated group. The livers ofmost animals in the DMN group developed a low degree ofbile ductule proliferation, glandular lesions, cystic lesions,mucinous cyst, and cholangiofibrosis. In some animals inthis group, in addition, bile duct hyperplasia associatedwith penicholangitis or cholangitis and peliosis hepatis (10)were seen, and megabocytosis(9) of parenchymalcells wascommon. Five animals left untreated longer than 23 weeksafter DMN withdrawal resumed a nearly normal appearanceexcept for a few megabocytesand dilated bile ducts in someareas. The characteristic hepatic alterations of animals inthe metacercania group have been fully described elsewhere (3). The early pathological changes consisted of anacute inflammatory reaction involving the bile ducts of thesecond order and the portal connective tissue, especiallythe large veins. As the flukes developed into adults, theyinduced hyperplasia of the bile duct epithelium and adenomatous formation. Granubomatous responses to the adultworms and ova were present. Resolution of the granubomas

0 I 2 3 4 5 6 7 8 9 eli 2 3 45 @I7SWEEKS

Chart 1. Growth curves of hamsters treated with DMN, metacercaria of 0.viverrini, or both. These are compared to the untreated Group 1.All figures are derived from animals in the DMN plus metacercaria group.

Table 1

Summary of water and DMN intake and percentage of liver weight per body weight ofexperimentalanimals

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led to peniductal and portal scarring. Bile ductube proliferation associated with peniportal fibrosis and linking up between portal areas by fibrous bands gave the appearance ofmultibobular cirrhosis in most hepatic lobes. In the DMNplus metacercania group, mucin-producing chobangiocarcinomas developed (Fig. 2) with or without chobangiofibrosis (Fig. 3) in all 15 animals. These lesions were cornposedof various-sizedirregular glands, frequently papillaryor stratified. Most tumors contained central necrotic areas,while at the periphery, where abundant mitotic figures wereseen, they showed an infiltrative pattern. The neoplasticcells lining the glands showed an increased nuclear cytoplasmic ratio and elongated vesicular or hyperchromaticnuclei with prominent nucleoli. Pale, foamy eosinophilicgranular material with or without fragmented neutrophils ormacrophageswas observedin the glandular lumina. Metastases to other organs were not detected. In addition to thelesions found in the metacercaniagroup, papillary formation of main intrahepatic duct, glandular and cystic lesions,mucinous cysts and cholangiofibrosis were prominent. Bileduct hyperplasia associated with either penicholangitis orcholangitis and peliosis hepatis were more frequently seenin Group 4 than in Group 2. In addition, the extensive

dilatation and periductal fibrosis of large bile ducts andclear cell foci (20)that were seen in the metacercaniagroup(Group 3) were also noted in Group 4. There were granuIomasassociatedwith either parasiteeggs or parasitesonly(Fig. 4). Three animals of Group 4 died a few weeks afterDMN administration, apparently from cannibalism. Theother 3 animals lost from Group 4 during the study died atthe 10th, 12th, and 13thweeks, with excessiveproliferationof bile ducts and parenchymal cell megabocytosis throughout all the hepatic lobes. The livers of 3 animals in Group 2sacrificed at corresponding periods exhibited only a fewsmall areas of proliferating ducts and megabocytosis.In 3animals each from the untreated (Group 1) and metacercaria groups (Group 3), the hepatic alterations were similarto those found in the animals sacrificed at the end of theexperiment. It was clear from our histopathobogicalstudythat DMN alone caused mild alteration of the bile ductulesand the second-order bile duct that immediately follow thebile ductubes,whereas the liver fluke alone caused markedalteration of the bile ductules and moderated alteration ofthe second-order bile duct, especially the portion beforethey leave the hepatic lobes where most of the parasitesreside. Combination of DMN and the parasites causedseverealteration of the intrahepatic bile ducts at all levels.The average number of parasites counted from sectionstaken from 4 different lobes were about equal in bothparasitic Groups 3 and 4, although parasites in the DMNplus metacercania group (Group 4) seemed to contain asmaller number of eggs in the bodies than in the groupreceiving parasitesalone (Group 3).

All 6 specimens of the tumor transplanted to homologous

animals were first palpable as a small firm nodules at 3.5 to5 months after transplantation, and most of these tumorsgrew larger to 1 cm in diameter at 1 to 1.5 months and to 4cm at 3 to 4.5 months after being first palpated (Fig. 5). Thetumors grew and infiltrated surrounding tissues in glandubarand papillary patterns that were lined by single, tallcolumnar or pseudostratified columnar cells (Fig. 6). Mi

totic figures were numerous, and the tumor cells yielded apositive mucin stain. Large cysts were found in the transplanted tumors of animals that were observed longer.Metastasesto other organs of the transplanted tumors werenot seen. Perceptible growth of the transplanted tumor ofsecond generation was observedafter 3 weeks.

DISCUSSION

The growth curve indicated that the animals receivingboth DMN and parasites (Group 4) were less tolerant thanwere the animals treated with either agent alone (Groups 2and 3). The tumors that occurred in DMN-treated flukeinfested animals (Group 4) were cholangiocarcinoma; theyfulfilled the histological criteria discussed by Reddy et al.(18) and also were transplantable. Three animals, deadbetweenthe 10th and 13thweeks, showed excessiveproliferation of epithelial cells of the second-order ducts following from the bile ductules, but the ductal arrangement wasregular. This type of alteration extended beyond the portalareas and into the lobules. Later, there was prominentfibrosis around those ducts, and the proliferating cellsshowed hyperchromatic, enlarged, and stratified nuclei. Wepostulated that those atypical epitheliab cells were transformed into neoplastic cells either before or after fibrosisoccurred. The cause of the excessive proliferation of bileducts in Group 4 is not known. However, mechanicaltrauma, metabolites (19) from the parasites, or both, arepossible causes of alteration of the bile duct integrity,rendering it more vulnerable to the carcinogenic action ofDMN.

Replicating stages of cells during the proliferative process are known to be sensitive to the action of carcinogens.During cell proliferation methylation of DNA might be expected to be increased compared to nonreplicating cellswhere less DNA may be exposed (6). There is also a goodcorrelation of the sites of methylation with the sites ofneoplastic transformation (22). We believe that cholangiocarcinomas in these animals arose becauseDMNexerted acarcinogenic effect on the altered proliferating epithelialcells of the bile ducts. There was positive stain for mucin inseveral places of the tumor cells in all tumor-bearing animals. Nodular hyperplasia, considered by some to be aprecancerous stage of hepatocellular carcinoma (20), wasencountered at the end of the experiment in only oneanimal of Group 4. The mucin production as well as thehistological pattern of the tumor cells suggest that this is acholangiocarcinoma, eventhough someauthors mayclaimthat liver cell carcinoma may produce mucin. The presenceof a rare hyperplastic nodule in only one group of animalsalso mitigates against the formation of hepatoceblular carcinoma in these animals. The immunopathobogicalmechanisms underlying the pathogenesisof liver fluke infestationin hamsters may involve both the humoral and cell-me

diated immune system (3). The time when DMN was introduced into fluke-infested hamsters was the time whenimmunological reactions are fully active, based on thefindings of large amounts of immune complex in the gbmeruli, around the fluke and around the ova (S. Boonpucknavig and N. Bhamarapravati,personal observation cited inRef.3) and epithelioid granubomaaround the flukes and the

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Effects of DMN on Cholangiocarcinoma

ova. In the present study, even though no direct immunological data have been presented, one cannot avoid considering how these phenomena, which were triggered by aparasitic infestation, interfere with the immune surveillancesystem against neoplastic transformation. It was shown byTomatis et a!. (25) that cholangiocarcinoma may developafter an average time of 30 weeks using a high dosage ofDMN,that is, after the consumption of 19mg or nearly onethird more than the amount consumed by the animals inthis exDeniment. This may explain why tumors did notdevelop in the DMN group in this experiment. It is evidentthat the presence of the liver flukes not only shortened thelatent period for neoplastic transformation but reduced theneed for a large dose of DMN. The dosage of liver flukesand of DMNused in our experiment is still considered to behigh and would rarely be encountered in the human situation where both liver fluke infestation and nitrosamineconsumption occur over a period of severalyears. A studyby Bhamarapravati and Nimsomburana (1) indicated thatthe peak age of chobangiocarcinomain humans at the timeof death was 51 years. Thus there seems to be a distinctpossibility that the carcinogenic action of both agents inhumans is a cumulative effect operating over a period ofyears.

To our knowledge this experiment showsfor the first timethe possible role of an environmental carcinogen in theneoplastic transformation of parasite-infested bibiary epitheliabcells.

ACKNOWLEDGMENTS

Professor Paul M. Newbeme reviewed the histopathology slides of thetumor. Technical assistance was provided by Chalerm Chitprasop andSurachartAemapan.Wearegratefulto Dr. PreechaUjawateefor sendingusthe fish specimensand Dr. D. V. Brownfor hiscorrectionsandsuggestions.ThemanuscriptwastypedbyBoonsongIntho.

REFERENCES

1. Bhamarapravati, N., and Nimsomburana, P. Geographical Pathology ofCirrhosis and Carcinoma of the Liver in Thailand. In: I. Arao (ed.),Proceeding of the 16th General Assembly of the Japan Medical Congress, Osaka, Japan, Vol. 3, pp. 376-382. Tokyo: Zucho Insatsu, Co.,Ltd., 1963.

2. Bhamarapravati,N., and Thamavit,W. Animal Studieson Liver FlukeInfestation, Dimethylnitrosamine, and Bile Duct Carcinoma. Lancet, 1:206-207, 1978.

3. Bhamarapravati,N., Thamavit,W., and Vajrasthlra,S. LiverChangesInHamsters Infected with a Liver Fluke of Man, Opisthorchis viverrini.American Joumal Tropical Medicine and Hygiene, 27: 787-794, 1978.

4. Bhamarapravati,N., and Viranuvatti,V. Liver Diseasesin Thailand.AnAnalysis of Liver Biopsies. Am. J. Gastroenterol., 45: 267—275,1966.

5. Bisseru,B., and Chong, L. K. Opisthorchlsviverrini (Poirier, 1886).ATrematode Parasite of Man in West Malaysia. Trop. Geograph. Med., 21:138-146, 1969.

6. Craddock, V. M. Liver Carcinoma Induced in Rats by Single Administration of Dlmethylnitrosamineafter Partial Hepatectomy.J. NatI. CancerInst.,47: 889-907,1971.

7. Edwards, J. E., and White, J. Pathologic Changes, with 5pecial Refer

enceto PigmentationandClassificationof HepaticTumorsin RatsFedp-Dimethylaminoazobenzene (Butter Yellow). J. NatI. Cancer Inst., 2:157-183, 1941.

8. Gibson,J. B. LiverParasitesandLiverCancers.In: K.Shanmugaratnam,R. Nambiar,K. K. Tan, and L. K. C. Chan (eds.), Proceedingsof theAFOCC Second Asian Cancer Conference, Liver Cancer, Cancer Problems in AsianCountries,pp. 45-48. SingaporeCancerSociety,Singapore:StamfordCollegePress,(PIE) Ltd., 1976.

9. Herroid, K.Mc. D. Histogenesisof Malignant LiverTumors Induced byDimethylnitrosamine. An Experimental Study in Syrian Hamsters. J. NatI.Cancerlnst.,39: 1099-1111,1967.

10. Hou, P. C. The Relationship between Primary Carcinoma of the Liverand Infestationwith Clonorchlssinensis.J. Pathol.Bacteriol., 72: 239-246,1956.

11. Hou, P. C. Primary Carcinoma of Bile Duct of the Liver of the Cat (Fellscatus) Infested with clonorchis sinensis. J. Pathol. Bacteriol., 87: 239-244,1964.

12. Hou, P. C. HepaticClonorchiasisand Carcinomaof the Bile Duct in aDog. J. Pathol. Bacterlol., 89: 356-367. 1965.

13. Luna, L. G. (ed.). Armed Forces Institute of Pathology, Manual ofHistologic Staining Methods.Ed. 3, Chap. 4, RoutineStaining Procedures, pp. 32-38; Chap. 6, Methods for Connective Tissue, pp. 87-88,94—95;Chap. 10, Methods for Carbohydratesand Mucoproteins, pp.158-162. New York: McGraw-Hill, Inc., 1968.

14. Magee, P. N., Montesano,R., and Preussmann,R. N-Nltroso Compounds and Related Carcinogens.In: C. E. Searle (ed), ChemicalCarcinogens, ACS Monograph 173, pp. 491-625. Washington, D.C.American Chemical Society, 1976.

15. Migasena,P., and Changbumrung,S. The Roleof Nitrosaminesin theCausationof PrimaryCarcinoma.J. Med.Assoc.Thailand,57: 175-178,1974.

16. Mlgasena,P., Changbumrung,S., and Riganti,M. HistologicalStudiesin Rats Fed with Diet Containing 20% PIa ra (Fermented Fish). Lab.Demons., 2: 561, 1971.

17. Reansuwan, W. Determination of Some Chemical Toxic Substances inThai Preserved Protein Foods. MS. Thesis, Chulalonkom University,Bangkok, Thailand, 1977.

18. Reddy,K. P.. Buschmann,R.J., and Chomet,B. CholangiocarcinomasInducedby Feeding3'-Methyl-4-dlmethylamlnoazobenzeneto Rats.Am.J. Pathol.,87: 189-204,1977.

19. Sahal,B. N., and Srivastava,H. D. Pathologyof Liverand PancreasinOplsthorchlasisnoverca in Dogsand Pigs. Brit. Vet. J., 127:239-243,1971.

20. Squire,R. A., and Levitt, M. H. Reportof a Workshopon Classificationof Specific Hepatocellular Lesions in Rats. Cancer Res., 35: 3214—3223,1975.

21. Stewart, H. L., and Snell, K. C. The Histopathologyof ExperimentalTumors of the Uver of the Rat: A Critical Review of the Histopathogenesis. In: F. Homburger (ed.), The Physiopathology of Cancer, ed. 2, pp.85-126. New York: Paul B. Hoeber, Inc., 1959.

22. Swann,P. F., and Magee,P. N. Nitrosamine-InducedCarcinogenesis.The Alkylation of NucleicAcids of the Rat by N-Methyl-N-Nitrosourea,Dimethylnitrosamine, Dimethylsulphate and Methylmethanesulphonate.Biochem. J., 110: 39-47, 1968.

23. Tansurat,P. Oplsthorchiasis.In: R. A. Marcial-Rojas(ed.),PathologyofProtozoaandHelminthicDiseases,pp. 536-545.Baftlmore:TheWilliams&WlIklnsCo., 1971.

24. Thamavlt,W., Hiasa,Y., Ito, N., and Bhamarapravatl,N. The InhibitoryEffects of a-Benzene Hexachloride on 3'-Methyl-4-dlmethylaminoazobenzene and DL-Ethionlne Carcinogenesis In Rats. Cancer Res., 34:337-340, 1974.

25. Tomatls, L., Magee, P. N., and Shubik, P. Induction of Liver Tumors Inthe Syrian Golden Hamstersby FeedingDimethylnitrosamine.J. NatI.Cancer Inst., 33: 341-345, 1964.

26. Viranuvattl, V. Uver Fluke Infection and Infestation In Southeast Asia.Progr. Liver Dis., 4: 537-547, 1972.

27. Vlranuvatti,V., Kasemsant,D., and Bhamarapravatl,N. RetentionCystof Liver Caused by Opisthorchiasis Associated with Carcinoma. Am. J.Gastroenterol.,23:442-446,1955.

28. Viranuvatti, V., Mettiyawongse, S. Observationson Two Cases of Opisthorachiasis in Thailand. Ann. Trop. Med. Parasltol. , 47: 291-293, 1953.

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Fig. 1. Enlarged liver with 2 nodules and 2large firm white areas on the surface. One ofthe nodules was used for transplantation.Noteenlargedhilar nodeon the cystic duct.

Fig. 2. Cholanglocarcinoma from the previous figure showing various-sizedirregularglands with cellular stratification. Note homogeneous material containing fragmentedneutrophilsin the glandularlumen.H & E, x150.

Fig. 3. An area of cholangiofibrosis. H & E,x 150.

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Fig. 4. An area of granuloma trapping botha parasiteandeggs.H& E, x 150.

Fig. 5. Transplanted tumor grew and infiltrated surrounding tissue. Ruptured cystswerealsonoted.

Fig. 6. Transplanted cholangiocarcinomacomposed of irregular glands lined by single,tall columnar cells. Note striated muscle fibersin the right field. H& E, x 150.

5

DECEMBER 1978 4639

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1978;38:4634-4639. Cancer Res   Witaya Thamavit, Natth Bhamarapravati, Somphong Sahaphong, et al.   Golden Hamsters

-infected SyrianOpisthorchis viverriniCholagiocarcinoma in Effects of Dimethylnitrosamine on Induction of

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