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Published in final edited form as: Parasitol Int. 2015 Oct 9;66(4):458–463. doi: 10.1016/j.parint.2015.10.002

Effect of Opisthorchis felineus infection and dimethylnitrosamine administration on the induction of cholangiocarcinoma in Syrian hamsters

Galina A Maksimova a, Maria Y Pakharukova a,*, Elena V Kashina a, Natalya A Zhukova b, Anna V Kovner a, Maria N Lvova a, Alexey V Katokhin a, Tatyana G Tolstikova b, Banchob Sripa c, Viatcheslav A Mordvinov a,d
PMCID: PMC4956575  NIHMSID: NIHMS800597  PMID: 26453019

Abstract

The food-borne liver trematode Opisthorchis felineus is an emerging source of biliary tract diseases on the territory of the former Soviet Union and Eastern Europe. This parasite along with trematodes Opisthorchis viverrini and Clonorchis sinensis belong to the triad of epidemiologically important liver flukes of the Opisthorchiidae family. It is known that O. viverrini and C. sinensis are the main risk factors of cholangiocarcinoma (CCA) in the endemic regions. The carcinogenic potential of O. felineus has not been well researched because of the absence of systematic pathomorphological, clinical, and epidemiological studies on O. felineus opisthorchiasis.

In the present study, we show the results of detailed histopathological analysis and comprehensive evaluation of inflammation, bile duct dysplasia, periductal fibrosis, bile duct hyperplasia, bile duct proliferation, egg granuloma, cysts, cholangiofibrosis, and CCA from 10 to 30 weeks following infection of Syrian hamsters with O. felineus accompanied by oral administration of dimethylnitrosamine (DMN). The results revealed that O. felineus contributes to bile duct cancer development in the hamster model. During the combined action of O. felineus and DMN, morphological features of the liver underwent dramatic changes at the cellular and organ levels. Already in the early stages of the experiment, we observed extensive periductal fibrosis, active inflammation, proliferation of the bile duct, bile duct dysplasia and egg granulomas. Later, against the background of all these changes, cholangiofibrosis and CCA were found.

Our work is the first step in the study of carcinogenic potential of O. felineus. Obtained data indicate the risk of CCA of patients having chronic O. felineus opisthorchiasis, and underscore the need for the development of programs for control of this helminthiasis.

Keywords: Liver fluke, Opisthorchis felineus, Cholangiocarcinoma, Hamster model, Pathology

1. Introduction

Opisthorchis felineus (Rivolta, 1884), Opisthorchis viverrini (Poirier, 1886) and Clonorchis sinensis (Loos, 1907) are three epidemiologically important species of the Opisthorchiidae family (class Trematoda). Each of these three species has discrete, though occasionally overlapping, geographical distribution: O. felineus is endemic in Europe and Russia [1,2]; C. sinensis in China, the Republic of Korea, and northern Vietnam; and O. viverrini in Southeast Asia. Together they affect more than 45 million people worldwide [3].

The area where O. felineus occurs includes vast expanses of North Eurasia as well as parts of Southern and Western Europe. Recent studies assessed the prevalence of O. felineus in Europe (reviewed in [3]), and these flukes were found on the Iberian Peninsula, the Balkan Peninsula, in Germany [5], and in Italy [6]. The Ob-Irtysh basin in Western Siberia is thought to be the world’s largest endemic centre of opisthorchiasis caused by O. felineus [1,4].

Human infection with O. felineus results from eating raw or undercooked freshwater Cyprinoid fish carrying the metacercariae of the parasite [1]. According to preliminary estimates, at least 1.6 million people in the world are infected with O. felineus [7]. The prevalence of O. felineus infection in the population of the endemic regions of Western Siberia is 10–45% according to various estimates [8–10].

The International Agency for Research on Cancer (IARC) classifies the trematodes O. viverrini and C. sinensis as group 1 carcinogens [11] and as the main factors for the development of cholangiocarcinoma (CCA) in endemic areas [12,13]. In contrast to its closest relatives, O. felineus at present is classified by IARC as a group 3 carcinogen: potentially dangerous for humans [11].

Because of the absence of systematic epidemiological and clinical studies on opisthorchiasis in Russia, the role of O. felineus in the development of CCA in humans is unknown. Nevertheless, there are some data from several research groups showing that in the endemic regions of Western Siberia, the prevalence of liver cancer is several times higher than in Russia on average [9,14,15]. It is noteworthy that the liver cancer in O. felineus-infected patients is largely diagnosed as CCA [15,16]. Certainly, the similarity of disease manifestations between the diseases caused by O. felineus and O. viverrini [10,17–19] as well as the results of studies on the pathogenesis of these helminthiases in experimental animal models [20,21], point to fairly high probability of the involvement of O. felineus in the development of CCA. Nonetheless, to date, the biology of O. felineus and its epidemiology and carcinogenic potential have not been studied well, even worse than those of O. viverrini and C. sinensis. The research into the role of O. felineus in the development of liver cancer in the experimental opisthorchiasis model based on hamsters is one of the first stages in assessment of the carcinogenic potential of this parasite.

The aims of this study were to examine the carcinogenic potential of O. felineus infection in Syrian hamsters during administration of DMN and to conduct a detailed histopathological analysis and comprehensive evaluation of inflammation, bile duct dysplasia, periductal fibrosis, bile duct hyperplasia, bile duct proliferation, egg granuloma, cysts, cholangiofibrosis, and CCA during 30 weeks after O. felineus infection.

2. Materials and methods

2.1. The hamsters and parasites

Syrian hamsters (Mesocricetus auratus) were purchased from the stock of the Puschino Animal Facility (Russia). All of the procedures were in compliance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for animal experiments http://ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm. The animals were kept and treated according to protocols approved by the Committee on the Ethics of Animal Experiments of the Institute of Cytology and Genetics (Permit Number: 7 of 19.12.2011). Euthanasia was performed by decapitation, and all efforts were made to minimize suffering.

O. felineus metacercariae were collected from naturally infected fish (Leuciscus idus) caught in the Ob River near the city of Novosibirsk (Western Siberia) and extracted accordingly [22,23]. Territories where sample collection (fishing) took place were neither conservation areas nor private, nor otherwise protected; hence, no fishing permits were required. The fish species collected are not considered endangered or rare, and the fishing methods were in full compliance with the Federal Law N166-F3 of 20.12.2004 (ed. 18.07.2011) “Fishing and conservation of water bio-resources”.

2.2. Experimental design

One hundred and fifty five male Syrian golden hamsters, aged 6 to 8 weeks, were used. The hamsters were distributed into four groups: (I) the untreated control, (II) 12.5 ppm DMN intake (with water), (III) infection with 50 metacercariae of O. felineus, and (IV) infection with 50 metacercariae of O. felineus and 12.5 ppm DMN intake (with water). The hamsters were housed at five per cage under conventional conditions and received a stock diet and water ad libitum.

The hamsters of groups III and IV were infected with 50 metacercariae per os. After one month, we verified the infection in the hamsters by the coproovoscopy [24]. The dose of DMN was selected according to the previously published data [25–27]. After confirmation of the infection, in groups II and IV, we replaced the drinking water with a 12.5 ppm DMN solution in distilled water (ad libitum consumption). A fresh DMN solution was prepared daily and placed in nontransparent bottles. Our experimental procedure for induction of CCA in the hamsters is presented in Fig. 1. The hamsters received DMN at 12.5 ppm until the day of euthanasia. The hamsters that received DMN were kept in a separate room. The total duration of the experiment was 30 weeks. Four or five hamsters from the control group and six hamsters from each of the other groups were euthanized and necropsied on weeks (Wk) 10, 14, 18, 22, 26, and 30 post-infection (p.i.).

Fig. 1.

Fig. 1

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The experimental procedure for induction of cholangiocarcinoma (CCA) in hamsters. The black arrow denotes infection with Opisthorchis felineus, the white arrow the start of dimethylnitrosamine (DMN) administration, and the black dot corresponds to euthanasia.

2.3. Sample collection and pathological study

After each collection of biological samples, we measured the body weight and liver weight. The spleen weight was measured only after 26 and 30 weeks p.i. The liver was carefully dissected and placed in 10% buffered formalin (Biovitrum, Russia). After fixation overnight at 4 °C, the specimens were dehydrated in a graded series of ethanol solutions and then absolute ethanol, cleared in xylene, and soaked in melted paraffin. Then we embedded the specimens in paraffin using Microm (Microm, UK). Four-μm-thick slices were prepared by means of a microtome.

For histopathological analysis, the tissue slices were stained with hematoxylin and eosin by the standard method. Pathological changes were graded semiquantitatively according to previously described methods [20,27,28]. The finished slides were examined under a light microscope (Axioskop 2 Plus; Zeiss, Germany).

2.4. Statistics

The data on body weight and on the relative weight of the liver and spleen were subjected to statistical analysis in the Statistica 6.0 software (Statsoft, USA). Significance of the differences between the groups of hamsters was evaluated by the Mann–Whitney test (cutoff P < 0.05) in Statistica 6.0.

3. Results

3.1. General findings

After 10 weeks of the experiment, the relative weight of the liver in the hamsters infected with O. felineus (groups III and IV) was almost twice than that in the control hamsters (Fig. 2). This difference persisted during almost the whole experiment. The greatest relative weight of the liver was observed in group IV, which received both O. felineus and DMN. After 22 weeks until the end of the experiment, the relative weight of the liver in this group was significantly greater than that in any other group (Fig. 2) and, after 30 weeks, was approximately threefold greater than that in the control hamsters.

Fig. 2.

Fig. 2

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The liver-to-body weight ratio (in percentage points, mean ± standard deviation). Treatment groups: I(○), control; II(▲), dimethylnitrosamine (DMN); III(◇), Opisthorchis felineus; IV(■), O. felineus + DMN. *Compared to group I, #compared to group II, ◇compared to group III; *, #, ◇ correspond to P < 0.05; **, ##, ◇◇ correspond to P < 0.01.

After 26 and 30 weeks of the experiment, we observed a significant increase in the relative weight of the spleen in hamsters of groups II, III, and IV in comparison with the control group (Suppl. 1). There was a significant increase in spleen weight in group IV compared to groups II and III (Table 1).

Table 1.

The spleen-to-body weight ratio 26 and 30 weeks post-infection (p.i.).

Weeks p.i. Group I, % Group II, % Group III, % Group IV, %
26 0.08 ± 0.01 0.12 ± 0.01* 0.11 ± 0.02* 0.16 ± 0.02*##⋄⋄
30 0.08 ± 0.01 0.12 ± 0.01* 0.11 ± 0.02* 0.31 ± 0.19**##⋄⋄
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The data are shown as mean ± standard deviation.

*

compared to group I,

#

compared to group II,

⋄

compared to group III;

*,#, ⋄

correspond to P < 0.05;

**, ##, ⋄⋄

correspond to P < 0.01.

We did not detect significant differences in body weight of the hamsters between the experimental groups (II, III, and IV) and the control group (I) at any time point of the experiment (data not shown).

Macroscopic examination of the livers revealed pronounced differences in tissue color, the state of bile ducts, and bile color among the groups (Fig. 3). A prominent indicator was the state of the liver surface and of its edges. In the control hamsters and in the DMN-treated hamsters (group II), the liver surface remained firm, smooth, and shiny, and the edges of the liver were even, whereas the walls of the extrahepatic bile ducts and of the gallbladder were transparent and not thickened; the bile was transparent and had a light yellow color (Fig. 3A, B) during almost the whole experiment. Only toward the end of the experiment, after 26 and 30 weeks, in the DMN-treated hamsters (group II), did the edges of the liver become slightly uneven.

Fig. 3.

Fig. 3

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The gross appearance of the liver, gallbladder, and extrahepatic bile ducts at 30 weeks post-infection (p.i.): A, group I (control); B, group II (dimethylnitrosamine [DMN]); C, group III (infection with Opisthorchis felineus); and D, group IV (O. felineus + DMN). The scale bar is 1 cm. The arrow indicates small whitish yellow neoplasms on the liver surface.

In contrast to groups I and II, the bile ducts in the hamsters with opisthorchiasis, regardless of DMN administration, were significantly thickened, and after the first weeks of the experiment, became opaque. The liver surface was gradually becoming nodular (small bumps; Fig. 3C, D), the edges of the organ became uneven, and the bile had a dark green color. In the group that received both the helminths and DMN, we observed after 26 weeks dense nodular whitish yellow nodules with uneven contours, and the number of these structures increased substantially during the experiment (Fig. 3D). It should be noted that in two of six hamsters receiving DMN (group II), we also observed small whitish yellow nodules on the liver surface but only after 30 weeks.

3.2. Histological findings

The results of the histopathological analysis, including comprehensive evaluation of inflammation, bile duct dysplasia, periductal fibrosis, bile duct hyperplasia, bile duct proliferation, egg granuloma, cysts, cholangiofibrosis, and CCA during the 30 weeks are shown in Fig. 4.

Fig. 4.

Fig. 4

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Histopathological findings in the experimental groups depending on the duration of the experiment. Dimethylnitrosamine (DMN): group II, OF: group III (Opisthorchis felineus), and OF&DMN: group IV (O. felineus + DMN).

After 10 weeks of the experiment, the histological analysis of the liver revealed rapid proliferation of epithelial cells of the bile ducts in the hamsters infected with the O. felineus, regardless of the DMN administration, in comparison with the control animals (Figs. 4 and 5). The lumens of the bile ducts were strongly increased, and the walls of the bile ducts were greatly thickened because of periductal fibrosis. Among the actively proliferating prismatic epithelial cells in the bile ducts, mucin secreting cells were visible well. From the beginning of the experiment, there was a pronounced inflammatory cell infiltration. In lumens of the bile ducts of the infected hamsters, we detected polyps of varying size (consisting of connective tissue), which were infiltrated by inflammatory cells, the parasite’s eggs, and a dark pigment (egg granuloma). Some of the polyps were covered with the prismatic epithelium. In the course of the experiment, we observed enhancement of bile duct proliferation and thickening of the periductal fibrosis in the hyperplastic bile ducts.

Fig. 5.

Fig. 5

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Hepatobiliary histopathological features of the hamster liver. Hematoxylin and eosin (H&E) staining, ×100 magnification. A, E, I, and M: group I (control) 18, 22, 26, and 30 wks p.i., respectively; B, F, J, and N: group II (dimethylnitrosamine [DMN]) 18, 22, 26, and 30 wks p.i., respectively; C, G, K, and O: group III (Opisthorchis felineus) 18, 22, 26, and 30 wks p.i., respectively; D, H, L, and P: group IV (O. felineus + DMN) 18, 22, 26, and 30 wks p.i., respectively.

In group IV, which received both O. felineus and DMN, the inflammation was more pronounced than that in the other groups. The extent of dysplasia of hepatocytes was found to be increased, as was the inflammatory infiltration, during the whole experiment. After 18 weeks, we discovered cysts lined with a flat epithelium. In addition, we detected cholangiofibrosis in five of six hamsters (group IV). At this time point, we observed first signs of malignancy of the small neoplasms: irregularity of the structure of the newly formed bile ductules as well as disappearance of the basal membrane in some ducts; besides, we found a CCA (small foci of cancer) in one of six hamsters (group IV; Fig. 4). After 22 weeks, in one of the six hamsters, we found an early-stage CCA. After 26 weeks, in three of the six hamsters (group IV), we found a differentiated mass-forming cholangiocellular carcinoma (Figs. 4 and 5). The tumors showed infiltrative growth (Fig. 5). After 30 weeks, in all hamsters of group IV, we detected cholangiocellular tumors of varying degrees of differentiation: from weakly differentiated to highly differentiated (Fig. 4). At this time point, the experiment had to be terminated because after 31 weeks, the hamsters in this group (IV) started to die of abdominal ascites.

In contrast, among the hamsters that received only DMN (group II), we noticed dysplasia of hepatocytes and mild inflammation; intensity of these phenomena increased in the course of the experiment (Figs. 4 and 5). After 22 weeks, we found slight bile duct proliferation, but overall, in the hamsters without the fluke infection, there was no active bile duct proliferation or periductal fibrosis, granulomas, or bile duct hyperplasia. At the end of the experiment, after 30 weeks, we noticed formation of CCA in three of six hamsters, and in the same animals, mild cholangiofibrosis, bile duct dysplasia and cysts (Fig. 4).

4. Discussion

This study shows for the first time the role of O. felineus in bile duct carcinogenesis in a hamster model during DMN administration. We showed that histopathological features of CCA appear in the liver of the hamsters already after 18 weeks p.i. At the same time point, we found cholangiofibrosis in two of six hamsters (group IV) and detected a CCA (a small focus of cancer) in one of six hamsters (group IV). After 26 weeks, in three of six hamsters (group IV), we found a mass-forming CCA, and after 30 weeks p.i., CCA was detected in all animals (group IV). By itself, the infection by an O. felineus does not lead to development of CCA during the 30 weeks of the infection, according to our findings, in line with the results of another study at the same time point of infection with O. viverrini [27]. In the present study, the pathological changes in the liver that were caused by O. felineus, or by the combined action of the O. felineus and DMN, resemble the pathological changes that were reported elsewhere regarding infection with other liver flukes: O. viverrini and C. sinensis [27,29,30,31]. After 30 weeks p.i., one half of the hamsters had CCA in the group of hamsters that received only DMN (group II). This finding is in agreement with the study [27] showing that prolonged administration of DMN to hamsters also causes liver tumors after 32 weeks. It is possible that a lower dose of DMN can increase the period of development of CCA in Group II but may not affect the period of tumor development in Group IV. This hypothesis needs empirical testing. Nevertheless, O. felineus infection substantially accelerates the development of CCA: down to 18 weeks, according to our present results. In a similar study on O. viverrini, tumors were detected after 14 weeks [27].

The mechanisms of bile duct carcinogenesis in hamsters as a result of the combined action of O. felineus and DMN conform to the classic three-stage theory of carcinogenesis [30,32]. According to this theory, malignant tumors develop as a result of a synergistic action of two agents. One of them is the initiator of the tumor-inducing event, whereas the other promotes proliferation of the initiated cells, i.e., has properties of a tumor promoter. In the golden hamster model of CCA, the tumor initiator is DMN, which is a genotoxic compound. It is known that DMN is present in the environment, in food, and in cigarette smoke [33]. Therefore, people encounter this compound everyday, and our selection of DMN as a tumor initiator is fully justified. During opisthorchiasis, special conditions develop that facilitate proliferation of the initiated cells.

The mechanism of CCA development under the influence of liver flukes is not well understood. It is believed that liver flukes can cause damage to the bile duct epithelium in several ways: (i) via mechanical damage; (ii) as a result of action of reactive oxygen species that are produced by the host cells at the site of inflammation; and (iii) because of direct action of fluke-secreted proteins that induce cell proliferation and inhibit apoptosis [34–36]. All these processes lead to lesions in the genetic material and mutations, which get fixed after DNA replication. Accumulation of the mutations may cause malignant transformation of cholangiocytes and development of CCA [13,34,37]. Furthermore, it is known that trematodes can produce specific genotoxic compounds, in particular, oxysterols and catecholestrogens, which are specific to some carcinogenic species of the trematodes: O. viverrini and Schistosoma haematobium [37,38]. The xenobiotic metabolizing enzymes of the parasites may participate in the synthesis of such compounds [39]. Additionally, biliary microbiota may also affect biliary diseases and CCA during infection with liver flukes [40,41].

Overall, the histopathological changes in liver tissues after infection by O. felineus in our experimental model are similar to the results of analogous studies on O. viverrini and C. sinensis; the same is true for the development of CCA as a result of the combined action of O. felineus and DMN. Nevertheless, there are some differences between the previous and current results; these differences were possibly caused by peculiarities of the experimental models. Our study revealed a substantial increase in the relative weight of the liver as a result of the combined action of O. felineus and DMN, in line with similar studies on O. viverrini and C. sinensis [29,30,31]. In contrast to such studies [29, 30,42], we did not see the effects of O. felineus and/or DMN on the body weight of the hamsters. Besides, we uncovered an association between the increase in spleen weight and CCA development. In CCA during O. felineus opisthorchiasis, splenomegaly is also known to occur in people [43]. On the other hand, after infection by O. viverrini, spleen weight does not change either in hamsters [44] or in humans [45]. In humans infected with C. sinensis, the spleen enlarges [46].

5. Conclusions

O. felineus belongs to the triad of epidemiologically important trematode species of the Opisthorchiidae family, but its carcinogenic potential has been studied much less than that of O. viverrini and C. sinensis. Although the clinical manifestations during O. felineus infection are similar to the opisthorchiasis caused by O. viverrini or C. sinensis, to date, no one has conducted comprehensive evaluation of the pathological changes in the liver during O. felineus opisthorchiasis. Such results would allow for comparison of the carcinogenic potential among different species within Opisthorchiidae.

The geographic area affected by O. felineus includes large territories of Europe and Asia, and outbreaks of opisthorchiasis that is caused by this fluke can be expected to happen in many countries. The increasing migration of the population should also be taken into account as should be the tourism among various countries. Consequently, patients with O. felineus infection can be found far beyond the endemic regions. Thus, opisthorchiasis that is caused by O. felineus is becoming a global problem transcending biomedical problems of limited regions. In the present work, the carcinogenic potential of O. felineus was demonstrated for the first time in terms of development of bile duct cancer in a hamster model. This result is the first stage of the research into O. felineus carcinogenic effects on humans and raises the question of the need for epidemiological studies and programs for control of the incidence of O. felineus opisthorchiasis in the endemic regions.

Supplementary Material

Supplement 1
Supplement 2

Acknowledgments

We acknowledge support and collaboration of members of the TOPIC (Tomsk OPIsthorchiasis Consortium) [47]. This work was supported in part by the Russian Foundation for Basic Research [grant numbers 13-04-00662a, 15-04-03551a, and 15-54-45132 Ind_a] and the State Project of the Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences [grant number VI.60.1.1]. The study was also supported in part by the travel grant from Pfizer’s Moscow office (GAM grant number 1 from 24.06.2014). BS is a TRF Senior Research Scholar (RTA 568006) and is supported by the Tropical Medicine Research Center Program, National Institute of Allergy and Infectious Diseases, and NIH (grant number P50AI098639).

Footnotes

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