The morphofunctional and biochemical characteristics of ...

ISSN 2079-0597, Russian Journal of Genetics: Applied Research, 2016, Vol. 6, No. 4, pp. 454–462. © Pleiades Publishing, Ltd., 2016. Original Russian Text © G.A. Maksimova, M.Y. Pakharukova, E.V. Kashina, N.A. Zhukova, M.N. Lvova, M.V. Khvostov, D.S. Baev, A.V. Katokhin, T.G. Tolstikova, V.A. Mordvinov, 2015, published in Vavilovskii Zhurnal Genetiki i Selektsii, 2015, Vol. 19, No. 4, pp. 466–473.

The Morphofunctional and Biochemical Characteristics of Opisthorchiasis-Associated Cholangiocarcinoma in a Syrian Hamster Model G. A. Maksimovaa, M. Y. Pakharukovaa, E. V. Kashinaa, N. A. Zhukovab, M. N. Lvovaa, M. V. Khvostovb, D. S. Baevb, A. V. Katokhina, T. G. Tolstikovab, and V. A. Mordvinova, c aInstitute

of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, Novosibirsk, Russia Institute of Organic Chemistry, Siberian Branch, Russian Academy of Sciences, Novosibirsk, Russia c Institute of Molecular Biology and Biophysics, Novosibirsk, Russia e-mail: [email protected]; [email protected]

b

Received June 24, 2015; in final form, July 14, 2015

Abstract—The validity of the experimental models of pathologies is one of the key challenges in translational medicine. Cholangiocarcinoma, or bile duct cancer, ranks second among oncological diseases of the liver. There is a strong association between bile duct cancer and parasitic infestation of the liver caused by trematodes in the Opisthorchiidae family. We recently demonstrated that cholangiocarcinoma can develop in Syrian hamsters (Mesocricetus auratus) infected by Opisthorchis felineus and administered with dimethylnitrosamine. However, there is still no description of how this experimental model can be used in translational research. The aim of this work was to study the morphological, functional, and biochemical characteristics during cholangiocarcinoma development in Syrian hamsters infected by O. felineus and administered with dimethylnitrosamine. The experiment lasted 30 weeks with combined administration of dimethylnitrosamine in drinking water at a dose of 12.5 ppm and a single injection of 50 metacercariae O. felineus. It was shown that the development of cholangiocarcinoma (18 weeks) increased the total number of basophils, eosinophils, and monocytes; the relative number of granulocytes, as well as the amount of total and direct bilirubin; and the cholesterol and ALT levels; however, it reduced the relative number of lymphocytes. Based on pathological, morphometric, and biochemical analyses, our model has characteristics similar to those in patients with opisthorchiasis-associated cholangiocarcinoma. Thus, this model can be used to test anticancer drugs, to study the mechanisms of cholangiocarcinogenesis, and to search for molecular markers for early diagnosis of bile duct cancer. Keywords: cholangiocarcinoma, Syrian hamsters Mesocricetus auratus, opisthorchiasis, liver DOI: 10.1134/S2079059716040134

INTRODUCTION Cholangiocarcinoma, or bile duct cancer, ranks second among the malicious diseases of the liver and is known to be an aggressive form of cancer; it progresses rapidly and is difficult to treatment. Usually, cholangiocarcinoma is diagnosed at the late stages, and most patients die within five years. The main approach to bile duct cancer consists of the surgical removal of the tumor. It has been reported that the frequency of cholangiocarcinoma has increased worldwide. However, our knowledge of this type of cancer remains quite limited and fragmental (Khuntikeo et al., 2015). The study of cholangiocarcinoma is complicated by the lack of models of this disease appropriate for in vivo investigations. It is known that bile duct cancer is closely associated with the helminthic invasion of liver induced by trematodes Clonorchis sinensis and Opisthorchis viver-

rini (Platyhelminthes: Trematoda: Opisthorchiidae) (Flavell and Lucas, 1982; Sripa et al., 2007; Aunpromma et al., 2012). These helminthes induce diseases, clonorchiasis, and opisthorchiasis, which are characterized by a similar symptomatology. Another source of opisthorchiasis, which is widespread in Russia, is O. felineus, which belongs to the Opisthorchiidae family. The largest endemic centre of opisthorchiasis, induced by O. felineus, is located in Western Siberia in the Ob-Irtysh basin (Mordvinov and Furman, 2010). Common liver flukes, C. sinensis, O. viverrini, and O. felineus, parasitize in the bile ducts of the liver and gallbladder of humans and fish-eating mammals. The International Agency for Cancer Research has classified O. viverrini and С. sinensis as group 1 carcinogens (IARC, 2012) and as the main factors inducing the development of cholangiocarcinoma in endemic zones.

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Recently, we have shown that cholangiocarcinoma developed in Syrian hamsters Mesocricetus auratus on the background of experimental opisthorchiasis induced by O. felineus in the presence of dimethylnitrosamine (Maksimova et al., 2015). However, it is not yet clear whether this technology may be used to model cholangiocarcinoma in human. The present work was aimed at studying the morphological, functional, and biochemical parameters in Syrian hamsters in the development of cholangiocarcinoma in the background of the effects of dimethylnitrosamine and the invasion of O. felineus. The data of this study are necessary to perform a comparative analysis with the data of other studies dealing with the development of cholangiocarcinoma in humans, and assess the appropriateness of the chosen experimental model of cholangiocarcinogenesis for translational studies. The choice and development of an adequate experimental model provide new perspectives for screening the molecular markers of bile duct cancer and the development of early diagnostics of this disease. MATERIALS AND METHODS The objects of the study were 6–8-week-old male hamsters Mesocricetus auratus, which were obtained from SPF-vivarium (Pushchino, Russia). The animals were kept in cages, five hamsters in each, under standard conditions with free access to food and water. The experimental protocol was approved by the Commission for bioethics of the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences (protocol no. 7, December 19, 2011). Metacercaria Opisthorchis felineus was isolated from the subcutaneous muscles of the naturally infected ides Leuciscus idus, which were caught in the river Ob in Novosibirsk. The fish tissues were treated with pepsin at 37°C, and metacercaria were washed with a physiological solution and counted (Sripa and Kaewkes, 2000). The hamsters were infected per os with a dose of 50 metacercaria per animal. Animals treated with dimethylnitrosamine (DMN) were kept in a separate room. DMN was dissolved in water to a final concentration 12.5 ppm. A fresh DMN solution was prepared daily, poured into nontransparent bottles and given to hamsters to drink instead of water ad libitum. One hundred and fifty five hamsters were divided into four groups: group I⎯control animals; group II⎯ animals treated with DMN; group III⎯animals infected with O. felineus; and group IV⎯animals treated with DMN and infected with O. felineus. Animals of groups III and IV were infected at the beginning of the experiment. After one month, the infection of the hamsters was checked by modified ether-acetum coproovoscopy (Pavlukov and Berezantsev, 1991). When infection was confirmed, the water for the animals of groups II and IV was substituted for DMN. The ani-

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Fig. 1. The scheme of the experiment. Black arrow shows introduction of O. felineus; white arrow shows introduction of DMN; gray dots indicate time, when the animals were taken out from the experiment.

mals were taken out of the experiment after 10, 14, 18, 22, 26, and 30 weeks (Fig. 1). Each time 4–5 hamsters of the control group and 6 hamsters from each of the experimental group were taken. The weight of the animals and their livers was registered each time the biological material was collected. The spleen weight was measured only 26 and 30 weeks thereafter. Blood for the general analysis was taken from the orbital sinus in order to measure 18 parameters of the peripheral blood. To do this, 20 μL of blood obtained from each animal was supplemented with a 1.6 mL dilutor (Isotonic dilutor, Reamed, Russia). The obtained samples were assessed with an automatic hematological analyzer URIT-2900 (Guilin Botest Medical Electronic Co., Ltd., China). The blood serum was taken for biochemical analysis. The samples were treated in accordance with the manufacturer’s protocols to estimate the following parameters: total and conjugated bilirubin (Vector Best, Russia); total cholesterol, glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) (Olveks Diagnostikum, Russia); and total protein and lactate dehydrogenase (LDH) (Biocon, Germany). The measurements were performed with spectrophotometer Stat Fax 3300 (Awareness Technology, United States). To study the pathological modifications in the liver, it was fixed in 10% buffered formaldehyde (Biovitrum, Russia) for 16–18 h at 4°C. After the fixation, the samples were dehydrated in a series of ethanol and xylol solutions of increasing concentrations (Soyuzkhimprom, Russia) and then embedded into paraffin blocks (Biovitrum, Russia) using the histological complex Microm (Microm, United Kingdom). The paraffin blocks were used to prepare 4 μm slices, which were stained with hematoxylin and eosin, and analyzed with the light microscope Axioskop 2 Plus (Carl Zeiss, Germany). The obtained data of the animal’s weight, the relative weight of the liver and spleen, and the general and biochemical blood assays were analyzed statistically

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Relative liver weight, %

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Weeks Fig. 2. Relative weight of liver (Mean ± SD) in the experimental groups: I ( )⎯control; II ( )⎯DMN; III ( )⎯O. felineus; IV ( )⎯O. felineus + DMN. * Comparison with group I; # comparison with group II; ◊ comparison with group III. *, #,◊ p < 0.05; **, ##,◊◊ p < 0.01.

using Statistica 6.0 (Statsoft, United States) and visualized with Microsoft Office Excel 2007 and Microsoft Office Power-Point 2007 (Microsoft, United States). The confidence of the differences between the experimental groups was assessed by the Mann-Whitney U-test (p < 0.05) using Statistica 6.0 (Statsoft, United States). RESULTS It was shown that the weight of the liver of the hamsters infected with opisthorchids (groups III and IV) was twice as high as that of the control group animals beginning from the tenth week of the experiment (Fig. 2). These differences remained almost throughout the whole period of the experiment. The highest values of the relative liver weight were observed in group IV, which underwent the complex influence of O. felineus and dimethylnitrosamine (DMN). The animals in this group demonstrated an increased relative liver weight in comparison with all the other groups from the twenty-second week up to the end of the experiment (Fig. 2). In the thirtieth week of the experiment, the relative liver weight of these animals was almost triple that of the control group animals.

The increase in the relative weight of the spleen of the animals of groups II, III, and IV with respect to the control group was observed in the twenty-sixth and thirtieth weeks. The confident increase in the mass of the spleen was also revealed in the comparative analysis of group IV with groups II and III (table). The macroscopic analysis of the liver of the animals from different groups showed differences in the color of the tissue, bile, and the state of the bile duct. An important parameter was the condition of the surface liver tissue and its edges. The liver surface of the control and DMN-treated animals remained smooth, elastic, and shiny, and its edges were smooth. Their bile ducts and gallbladder were transparent and their walls were not thickened (Figs. 3a, 3b). In contrast to these groups, animals with opisthorchiasis were characterized by thickened and nontransparent bile duct walls, and beginning from the first weeks of the experiment, regardless of the presence of DMN, they lost transparency. As of the eighteenth week the liver became slightly nodular, its edges rough, and the bile acquired a dark color (Fig. 3c). Beginning from the twenty-sixth week, the liver of the animals which underwent the complex influence of helminthes and DMN was characterized by dense nodular formations of a white-yellow color with rough edges (Fig. 3d), the

Relative weight of spleen (Mean ± SD) Weeks 26 30

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IV group, % 0.16 ± 0.02*##◊◊ 0.31 ± 0.19**##◊◊

* Comparison with group I; # comparison with group II; ◊ comparison with group III. *, #,◊ p < 0.05; **, ##,◊◊ p < 0.01 RUSSIAN JOURNAL OF GENETICS: APPLIED RESEARCH

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Fig. 3. Macropreparations of liver on the thirtieth week of the experiment. a⎯group I (control); b⎯group II (DMN); c⎯group III (O. felineus); d⎯group IV (O. felineus + DMN) Scale 1 cm.

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Fig. 4. Histopathological modifications of liver. Staining with hematoxylin and eosin. ×100. a⎯group I (control), thirtieth week, portal tract; b⎯group IV (O. felineus + DMN), tenth week, bile duct proliferation; c⎯group IV (O. felineus + DMN), eighteenth week, cholangiofibrosis; d⎯group IV (O. felineus + DMN), twenty-sixth week, fragment of cholangiocarcinoma; e⎯group IV (O. felineus + DMN), thirtieth week, fragment of cholangiocarcinoma.

size and number of which increased significantly by the thirtieth week of the experiment. We note that white-yellow formations were also observed in two of the six DMN-treated animals, although they appeared only on the thirtieth week. In contrast to the control animals, the histological analysis of liver of the opisthorchid-infected animals revealed a high level of bile duct epithelial and oval cell proliferation in the tenth week of the experiment, regardless of the presence of DMN. The bile duct walls of the infected animals were strongly thickened because of periductal fibrosis. The bile duct lumens of the infected animals contained polypous enlargements of different sizes, which consisted of fibroblasts surrounded with connective tissue fibers infiltrated with parasite eggs and a dark pigment.

Animals, which underwent a complex influence of helminthes and DMN were characterized by more obvious hyperplastic effects than other groups. In the eighteenth week of the experiments, we observed hepatocyte dysplasia and cholangiofibrosis formed by the bile ducts and layers of connective tissue. At this stage of the experiment, we registered the first signs of malignization, such as the irregularity of the newly formed bile duct structure and the disappearance of plasma membranes in some of the ducts (Fig. 4b). In the twenty-sixth week of the experiments, two hamsters, the liver of which carried white-yellow formations, were shown to suffer differentiated cholangiocellular carcinoma characterized by infiltrative growth (Fig. 4d). By the thirtieth week of the experiments,


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Weeks Fig. 5. General blood analysis: total number of monocytes, basophils and eosinophils (a); relative amount of lymphocytes (b) (Mean ± SD). * Comparison with group I; # comparison with group II; ◊ comparison with group III. *, #,◊ p < 0.05; **, ##,◊◊ p < 0.01.

cholangiocarcinomas were found in all animals of group IV (Fig. 4e). In contrast, animals treated only with DMN were characterized by hepatocyte dysplasia and individual microfocal necrosis of the hepatocytes. By the twentysixth week of the experiments, these animals demonstrated a small proliferation of the bile ducts, although, on the whole, the animals which did not undergo helminthic invasion did not show signs of the active proliferation of hepatocytes or fibrosis. Therefore, the first signs of malignization of the epithelium of the bile duct were observed in the liver of hamsters which underwent the complex influence of DMN and opisthorchiasis in the eighteenth week of the experiment. By the twenty-sixth week, the animals of this group developed cholangiocarcinomas. We performed a complex analysis of 18 parameters of the cellular composition of the animals’ blood. It was shown that most of the parameters of the general blood analysis, i.e., the number of lymphocytes and erythrocytes, hematocrit, the level of hemoglobin, the average level of hemoglobin in the erythrocyte, the average erythrocyte volume, the relative distribution of erythrocytes throughout the blood volume (standard deviation and variation coefficient), the number of thrombocytes, thrombocrit, and the average vol-

ume of thrombocytes, did not change, and no differences in these parameters between the groups were found (data not shown). However, several parameters of the cellular composition of the blood did change during the experiment. For example, it was shown that the total level of the mixture of basophils, monocytes, and eosinophils significantly increased in the animals which underwent the complex influence of O. felineus and DMN starting from the eighteenth week of the experiment. On the thirtieth week, in group IV, this parameter was triple that of the animals in the other groups (Fig. 5a). In group II the increase in this parameter, although insignificant, was also observed in the eighteenth and twenty-sixth weeks of the experiment. The increase in the total level of the mixture of basophils, monocytes, and eosinophils in the animals of the experimental groups is consistent with the relative increase in the number of granulocytes. The growth of this parameter was registered in the experimental animals, beginning from the eighteenth week of the experiment (data not shown). An important parameter, which, as of the eighteenth week, significantly changed in the animals which underwent the complex influence of O. felineus and DMN, was the decrease in the relative number of lymphocytes (Fig. 5b).


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Weeks Fig. 6. Biochemical parameters of blood: total bilirubin (a); conjugated bilirubin (b); cholesterol (c); ALT (d) (Mean ± SD). *Comparison with group I; # comparison with group II; ◊ comparison with group III. *, #,◊ p < 0.05; **, ##,◊◊ p < 0.01.

A complex analysis of nine biochemical parameters of blood, which revealed a series of significant changes in group IV, was also carried out. In particular, the control group and the groups of animals which underwent the influence of either helminths or DMN demonstrated a normal level of total and conjugated bilirubin (which is normally close to zero) (Figs. 6a, 6b). Conversely, ani-

mals which underwent the complex influence of O. felineus and DMN were characterized by an increased level of total bilirubin, beginning from the eighteenth week of the experiment. Importantly, the increase in this parameter is timely consistent with the occurrence of the first histological signs of malignization. In the twenty-sixth week the total level of biliru-


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bin was 60 umol/L, and by the end of the experiment it exceeded 200 umol/L. Changes in the level of the conjugated bilirubin was quite consistent with the dynamics of the total bilirubin concentration (Fig. 6c). Hence, the concentration of bilirubin in blood is an important parameter associated with the development of cholangiocarcinoma. Animals of group IV were characterized by a significantly increased level of cholesterol as compared with animals from other groups at the early stages of the experiment (weeks 10–18) (Fig. 6c). Moreover, in contrast to other groups, animals from group IV demonstrated an increased level of total protein in blood and a decreased level of glucose as compared with the control animals (data not shown). The amount of alanine aminotransferase (ALT) increased in all the experimental groups as compared with the control. We note that, in the early stages of the experiment, animals from group IV demonstrated the highest level of ALT (Fig. 6d). However, no significant differences in the levels of other liver enzymes (aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase) were observed between the groups. The obtained data allow us to conclude that the development of cholangiocarcinoma (group IV) is associated with the increase in the total level of basophils, monocytes, and eosinophils, as well as the relative number of granulocytes. However, the relative number of lymphocytes declined. The development of cholangiocarcinoma is also associated with an increase in the level of the total and conjugated bilirubin, cholesterol, and ALT. The majority of the parameters listed began to change in the eighteenth week of the experiment, which concurs with the occurrence of the first signs of malignization in the liver. However, in group IV the increase in the level of cholesterol and ALT was observed even before the tumor development. DISCUSSION The development of bile duct cancer in Syrian hamsters which underwent the complex influence of O. felineus amd DMN may be explained by the classical three-stage theory of carcinogenesis. According to this theory, the development of malignant tumors is induced by the synergetic influence of two agents, one of which initiates the tumor inducing event and the other facilitates the proliferation of the induced cells; i.e., it works as a tumor promotor. In the model system of cholangiocarcinoma development in Syrian hamsters, the initiator of malignant cells is DMN, which provides the genotoxic effect. It is known that DMN is contained in abundance in the environment, food, and cigar smoke. Human beings deal with this substance daily and thus, its choice as a tumor initiator is understandable.

However, tumor growth requires certain conditions that would facilitate mitosis of the initiated cells. Apparently, the conditions of opisthorchiasis match these requirements. Indeed, the liver of opisthorchiasis patients increases in size; the bile duct cells actively proliferate and sometimes form cysts. Similar pathological modifications may be observed in the model experimental opisthorchiasis of Syrian hamsters. Moreover, it was shown that some proteins of opisthorchid produce mitogenic and antiapoptotic effects on the host cells, working as classical mitogens (Smout et al., 2009, Sripa et al., 2012). In our study, we observed the bile duct cell proliferation, hyperplasia and metaplasia of the epithelium of the bile duct, cholangiofibrosis, and polypuses. Therefore, O. felineus helminthes are capable stimulating cell proliferation and thus inducing precancer modifications (Lvova et al., 2012). The observed modifications of the liver induced by the complex influence of O. felineus and DMN are similar to the pathological changes induced by other common liver f lukes, such as O. viverrini and С. sinensis (Thamavit et al., 1978; Lee et al., 1993; Songserm et al., 2009). Our study revealed the association between the increase in spleen weight and the development of cholangiocarcinoma. However, the data available in literature on this point are rather contradictory. For example, it was shown that opisthorchiasis-induced cholangiocarcinoma is seldom associated with splenomegalia (Shain, 1974). Other authors have reported that the development of suppurative complications in patients with a long-term O. felineus invasion is often followed by an increase in spleen size (Brazhnikova, 1997). Interestingly, an invasion of O. viverrini did not induce an increase in spleen weight in hamsters (Flavell, D.J. and Flavell, S.U., 1986) or in humans (Upatham et al., 1982). However, the invasion of С. sinensis led to an increase in spleen weight in humans (Rohela et al., 2006). However, such an increase in spleen size was not observed in all clonorchiasis patients (Wang et al., 2004). Our study showed that the data of the general and biochemical analyses of blood in the experimental groups were mostly stable. The stability of these parameters was also observed in the studies of chronical opisthorchiasis in humans (Poddubnaya, 2009; Shchegoleva et al., 2009; Poddubnaya and Shchegoleva, 2011). At the same time, it was observed that opisthorchiasis patients with complications, such as abscess and cholangiocarcinoma, underwent changes in the cellular and biochemical parameters of blood (Brazhnikova and Tolkaeva, 2000, 2002; Tskhai et al., 2011). Our study showed that hamsters, which underwent the complex influence of O. felineus and DMN, demonstrated increased levels of granulocytes and the total number of monocytes, basophils, and eosinophils. This may point to an inflammation, which is consistent with the data on inflammation processes follow-


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ing abscesses and cholangiocarcinoma (Alperovich et al., 2010; Tskhai et al., 2011). The observed decrease in the number of lymphocytes in hamsters from group IV is also consistent with the data obtained on patients with cholangiocellular liver cancer (Brazhnikova and Tolkaeva, 2002; Alperovich et al., 2010). A similar situation was observed for the levels of bilirubin, ALT, and cholesterol in the animals from group IV. The increase in these parameters was observed in patients with chronic opisthorchiasis (Bakshtanovskaya et al., 2002; Bakshtanovskaya and Stepanova, 2005). Besides aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) more often remained at normal level (Bakshtanovskaya et al., 2002; Bakshtanovskaya and Stepanova, 2005), some patients demonstrated an increased level of AST (Poddubnaya, 2009; Soukhathammavong et al., 2015). In our study, we obtained similar data. Patients with opisthorchiasis and cholangiocarcinoma often show an increased level of alkaline phosphatase (ALP) (Brazhnikova and Tolkaeva, 2000; Bakshtanovskaya et al., 2002, Bakshtanovskaya and Stepanova, 2005). However, in the experimental animals, we did not observe significant changes in this parameter. The infection with O. viverrini also may not increase the level of ALP (Soukhathammavong et al., 2015). Summarizing the aforesaid, we may conclude that according to the data of pathomorphological, morphometric, and biochemical analyses, the model of cholangiocarcinoma induced by the complex influence of O. felineus and DMN in Syrian hamsters demonstrated parameters similar to those of human patients suffering opisthorchiasis-associated cholangiocarcinoma. Therefore, this experimental model is appropriate for testing anti-cancer drugs, investigating mechanisms of the development of cholangiocarcinoma, and screening the molecular markers for early diagnostics of bile duct cancer. ACKNOWLEDGMENTS This work was partially supported by a budget project of the Institute of Cytology and Genetics of the Russian Academy of Sciences, project no. VI.60.1.1 (State Registration no. 01201280335), grant no. 19of the partner studies of the Russian Academy of Sciences, a grant of the Ministry of Education and Science of Russian Federation, (project no. 14.621.21.0010) in the form of a subsidy dated 04.12.2014 (RFMEFI62114X0010) and subsidy agreement no. 14.619.21.0005 dated 22.08.2014 (RFMEFI61914X0005), and the Russian Foundation for Basic Research, project no. 15-0403551а.

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Translated by M. Bibov

Vol. 6

No. 4

2016