Opisthorchis viverrini excretory-secretory products suppress GLUT8 of cholangiocytes.

Opisthorchis viverrini infection and the subsequent bile duct cancer it induces remains a significant public health problem in Southeast Asia. Opisthorchiasis has been reported to cause reduced plasma glucose levels among infected patients. The underlying mechanism for this phenomenon is unclear. In the present study, evidence is presented to support the hypothesis that O. viverrini exploits host cholangiocyte glucose transporters (GLUTs) in a similar manner to that of rodent intestinal nematodes, to feed on unabsorbed glucose in the bile for survival. GLUT levels in a cholangiocyte H69 cell line co-cultured with excretory-secretory products of O. viverrini were examined using qPCR and immunoblotting. GLUT 8 mRNA and expressed proteins were found to be downregulated in H69 cells in the presence of O. viverrini. This suggests that O. viverrini alters glucose metabolism in cells within its vicinity by limiting transporter expression resulting in increased bile glucose that it can utilize and potentially explains the previously reported anti-insulin effect of opisthorchiasis.

Opisthorchis viverrini infection induces metabolic disturbances in hamsters fed with high fat/high fructose diets: Implications for liver and kidney pathologies.

A combination of Opisthorchis viverrini infection and high fat/high fructose diets (HFa/HFr) intake is likely to enhance fatty liver and kidney pathologies. Here we investigated the combined effects of chronic O. viverrini infection and HFa/HFr intake on liver and kidney pathologies, metabolism, and gut microbiome in hamsters. Animals were infected with O. viverrini and fed with either standard chow (OV group) or HFa/HFr diet (OH group) and non-infected hamsters were fed with either standard chow (NC) or HFa/HFr diet (HF) for 8 months. The OH group exhibited dyslipidemia and the highest severity of fatty liver. Tubular damage, inflammatory cell infiltration, and tubular fibrosis were the most prominently observed in this group, supported by increased expression of KIM-1, HMGB-1, and MCP-1. Urinary 1H NMR metabolic profiles revealed that tauro-ß-muricholic acid level was increased in the OV and OH groups, whereas metabolites involved in the TCA cycle and gut microbiota-associated metabolites (phenylacetylglycine, trimethylamine, and trimethylamine-N-oxide) were lower in OV, HF and OH groups compared to the NC group. Gut microbial profiles of the OH group were also different from other groups. In conclusion, O. viverrini infection and HFa/HFr diet-induced disturbance of metabolites and gut microbiota associated with concurrent liver and kidney pathologies in hamsters.

Plasma checkpoint protein 1 (Chk1) as a potential diagnostic biomarker for opisthorchiasis and cholangiocarcinoma.

BACKGROUND: Patients infected with a parasite often develop opisthorchiasis viverrini, which often progresses into cholangiocarcinoma (CCA) due to the asymptomatic nature of the infection. Currently, there are no effective diagnostic methods for opisthorchiasis or cholangiocarcinoma. OBJECTIVE: The aim of this study was to identify the host-responsive protein that can be developed as a diagnostic biomarker of opisthorchiasis and cholangiocarcinoma. METHODS: Plasma samples were collected from non-OVCCA, OV, and CCA subjects, and the proteomes were investigated by LC-MS/MS. Venn diagrams and protein network prediction by STITCH were used to identify the potential biomarkers. The level of candidate protein, the plasma checkpoint protein 1 (Chk1), was measured by indirect enzyme-linked immunosorbent assay (ELISA). RESULTS: Chk1 was present in the center of the protein network analysis in both the OV and CCA groups. In addition, the plasma Chk1 levels were significantly increased in both groups (P< 0.05). The sensitivity of the opisthorchiasis viverrini and cholangiocarcinoma was 59.38% and 65.62%, respectively, while the specificity of both was 85.71%. CONCLUSION: Chk1 was identified by differential plasma proteomes and was increased in O. viverrini-infected and cholangiocarcinoma-derived plasma samples. Higher levels of plasma Chk1 levels may serve as a potential diagnostic biomarker for opisthorchiasis and cholangiocarcinoma.

Opisthorchis viverrini antigens up-regulates the expression of CD80 and MHC class II in JAWSII mouse dendritic cells and promotes IL-10 and TGF-ß secretions.

Dendritic cells (DCs) are antigen-presenting cells (APC) involved in the initiation of immune responses. Maturation of DCs is characterized by the high expression of major histocompatibility complex (MHC) class II and co-stimulatory clusters of differentiation (CD) 40, CD80, and CD86 molecules. Matured DCs are required for T cell differentiation and proliferation. However, the response of DCs to Opisthorchis viverrini antigens has not yet been understood. Therefore, this study sought to determine the expression of surface molecules of JAWSII mouse DCs stimulated by crude somatic (CS) and excretory-secretory (ES) antigens of O. viverrini. ES antigen significantly induced only mRNA expression of CD80 and MHC class II in JAWSII mouse DCs, while CS antigen promoted up-regulation of both mRNA and protein levels of CD80 and MHC class II, indicating relative maturation of JAWII mouse DCs. Moreover, the secreted cytokines from the co-cultures of O. viverrini antigens stimulated JAWSII DC with naïve CD4+ T cells was determined. Significantly increased levels of immunosuppressive cytokines interleukin (IL)-10 and transforming growth factor beta (TGF-ß) were found. The up-regulation of these cytokines may indicate the response of regulatory T cells (Treg) to CS antigen-stimulated JAWSII DC. These findings may lead to a better understanding of the role that DCs play in O. viverrini infection.

A tumorigenic cell line derived from a hamster cholangiocarcinoma associated with Opisthorchis felineus liver fluke infection.

AIMS: The food-born trematode Opisthorchis felineus colonizes bile ducts of the liver of fish-eating mammals including humans. There is growing evidence that this liver fluke is a risk factor for cholangiocarcinoma (CCA). Cancer cell lines are necessary for drug screening and for identifying protein markers of CCA. The aim was to establish a cell line derived from cholangiocarcinoma associated with opisthorchiasis felinea. MAIN METHODS: Allotransplantation, immunohistochemistry, karyotype analysis, cell culture techniques, immunocytochemistry and real-time PCR. KEY FINDINGS: Here we repot the establishment of first CCA cell line, CCA-OF, from a primary tumor of an experimental CCA in Syrian hamsters treated with low doses of dimethyl nitrosamine and associated with O. felineus infection. The cell line was found to be allotransplantable. Expression of epithelial and mesenchymal markers (cytokeratin 7, glycosyltransferase exostosin 1, Ca2+-dependent phospholipid-binding protein annexin A1 and vimentin) was demonstrated by immunostaining of the primary tumors, CCA-OF cells, and allotransplants. CCA-OF cells were found to express presumed CCA biomarkers previously detected in both human and experimental tumors associated with the liver fluke infection. The cells were diploid-like (2n = 42-46) with complex chromosomal rearrangements and have morphological features of epithelial-like cells. The usefulness of the CCA-OF cell model for antitumor activity testing was demonstrated by an analysis of effects of resveratrol treatment. It was shown that resveratrol treatment inhibited the proliferation and the migration ability of CCA-OF cells. SIGNIFICANCE: Thus, the allotransplantable CCA-OF cell line can be used in studies on helminth-associated cholangiocarcinogenesis and for the testing of antitumor drugs.

Effect of Silicon Dioxide Nanoparticles on Syrian Hamsters Infected by Opisthorchis felineus: 1H MRS Study of the Brain.

In recent years, silicon dioxide nanoparticles have been widely used in medicine and the pharmaceutical industry, however, their effect on the brain has hardly been studied. We assessed the effects of long-term consumption of 5-nm amorphous silicon dioxide nanoparticles (SiO2-NPs) by Syrian hamsters infected with the trematodes Opisthorchis felineus on the hippocampus and frontal cortex. Spectroscopic determination of brain neurometabolites, performed using a horizontal Magnetic Resonance Imaging system at 11.7 Tesla magnetic field, has shown that the ratio of the excitatory neurotransmitters (glutamate + glutamine + aspartate) to the inhibitory ones (GABA + glycine) was higher in the animals infected with O. felineus. However, pre-consumption of the SiO2-NPs solution prevented this imbalance. In addition, the protective effect of SiO2-NPs on the level of myo-inositol and glycine was found. It is concluded that the use of SiO2-NPs can neutralize the negative effects of infectious factors on the brain.

Identification, recombinant protein production, and functional analysis of a M60-like metallopeptidase, secreted by the liver fluke Opisthorchis viverrini.

The carcinogenic liver fluke Opisthorchis viverrini (O. viverrini) is endemic in Thailand and neighboring countries including Laos PDR, Vietnam and Cambodia. Infections with O. viverrini lead to hepatobiliary abnormalities including bile duct cancer-cholangiocarcinoma (CCA). Despite decades of extensive studies, the underlying mechanisms of how this parasite survives in the bile duct and causes disease are still unclear. Therefore, this study aims to identify and characterize the most abundant protein secreted by the parasite. Proteomics and bioinformatics analysis revealed that the most abundant secretory protein is a metallopeptidase, named Ov-M60-like-1. This protein contains an N-terminal carbohydrate-binding domain and a C-terminal M60-like domain with a zinc metallopeptidase HEXXH motif. Further analysis by mass spectrometry revealed that Ov-M60-like-1 is N-glycosylated. Recombinant Ov-M60-like-1 (rOv-M60-like-1) expressed in Escherichia coli (E. coli) was able to digest bovine submaxillary mucin (BSM). The mucinase activity was inhibited by the ion chelating agent EDTA, confirming its metallopeptidase identity. The enzyme was active at temperatures ranging 25-37 °C in a broad pH range (pH 2-10). The identification of Ov-M60-like-1 mucinase as the major secretory protein of O. viverrini worms warrants further research into the role of this glycoprotein in the pathology induced by this carcinogenic worm.

Effects of Opisthorchis viverrini infection on glucose and lipid profiles in human hosts: A cross-sectional and prospective follow-up study from Thailand.

Opisthorchis viverrini (OV) infection is endemic to the Northeast Thailand where the prevalence of Type 2 Diabetes mellitus (T2DM) is higher whilst the incidence of cardiovascular diseases (CVDs) is lower than the rest of Thailand. Helminth infection has both nutritional and immunological impact on their definitive hosts. Thus, a cross-sectional study was performed to see the effects of OV infection on glucose and lipid profiles. For this purpose, 200 each of OV infected and uninfected residents were recruited and their glycated hemoglobin (HbA1c), total cholesterol, triglycerides, low- and high-density lipoproteins (LDL and HDL) levels and anthropometric measurements, including BMI were examined. Then, as the prospective follow- up study, changes of those metabolic parameters of OV positive subjects (n = 120) before and after Praziquantel (PZQ) treatment were monitored for six months. The results showed that OV infection has a protective effect against hyperglycemia (OR 0.482 and p = .04) and metabolic disease risk group (OR 0.478 and p = .03). OV positive participants had lower HbA1c (5.5% Vs. 6.01%, p = .001) but higher HDL (54.07 Vs. 49.46 mg/dL, p = .001) than OV negative participants that are statistically significant. After PZQ treatment for OV-positive subjects, their serum levels of HbA1c (p < .05) and HDL (p < .05) significantly rose during the follow up. Apparently, OV infection lowers HbA1c but increases HDL in definitive human hosts.

Inhibition of the Formation In Vitro of Putatively Carcinogenic Metabolites Derived from S. haematobium and O. viverrini by Combination of Drugs with Antioxidants.

Infections caused by Schistosoma haematobium and Opisthorchis viverrini are classified as carcinogenic. Although carcinogenesis might be a multifactorial process, it has been postulated that these helminth produce/excrete oxysterols and estrogen-like metabolites that might act as initiators of their infection-associated carcinogenesis. Current treatment and control of these infections rely on a single drug, praziquantel, that mainly targets the parasites and not the pathologies related to the infection including cancer. Thus, there is a need to search for novel therapeutic alternatives that might include combinations of drugs and drug repurposing. Based on these concepts, we propose a novel therapeutic strategy that combines drugs with molecule antioxidants. We evaluate the efficacy of a novel therapeutic strategy to prevent the formation of putative carcinogenic metabolites precursors and DNA adducts. Firstly, we used a methodology previously established to synthesize metabolites precursors and DNA adducts in the presence of CYP450. Then, we evaluated the inhibition of their formation induced by drugs and antioxidants alone or in combination. Drugs and resveratrol alone did not show a significant inhibitory effect while N-acetylcysteine inhibited the formation of most metabolite precursors and DNA adducts. Moreover, the combinations of classical drugs with antioxidants were more effective rather than compounds alone. This strategy might be a valuable tool to prevent the initiation of helminth infection-associated carcinogenesis.

Characteristics of liver fibrosis associated with chronic Opisthorchis felineus infection in Syrian hamsters and humans.

The food-borne liver trematode Opisthorchis felineus causes severe liver damage, including fibrosis. This study shows a comparison of the characteristics between cholangiofibrosis and periductal fibrosis in infected people and in the golden hamster as an experimental model. Comparative evaluation was carried out regarding collagen composition, the number of basic-producing cells, and extracellular-matrix degradation. The results revealed that characteristics of chronic opisthorchiasis due to O. felineus infection in humans and in Syrian hamsters are similar and include well-pronounced development of fibrotic complications in the liver parenchyma. Besides, a difference in fibrogenesis development was demonstrated between chronic O. felineus infection and noninfectious cholecystitis. In this study, we for the first time compared fibrogenesis between humans and model animals against the background of chronic O. felineus infection.

Granulin Expression in Hamsters during Opisthorchis viverrini Infection-Induced Cholangiocarcinogenesis

The secreted growth factor granulin (GRN) is upregulated during diverse epithelial cancers. GRN stimulates cell growth and development while inhibiting apoptosis. Orthologues of vertebrate granulins evolved in other animals including the liver fluke Opisthorchis viverrini. Curiously, liver fluke granulin, termed Ov-GRN-1 promotes cholangiocarcinogenesis during chronic opisthorchiasis but, by contrast, limited information is available concerning mammalian GRN during liver fluke infection-induced cholangiocarcinoma (CCA). Here we investigated the expression of mammalian granulin in the O. viverrini-associated a hamster model of opisthorchiasis and liver fluke infection-induced CCA. Male Syrian golden hamsters were assigned to one of four treatment groups, each group included 30 hamsters: 1) normal (control), 2) infected with O. viverrini (OV); 3) exposed to N-dimethylnitrosamine in drinking water (DMN); and 4) infected with O. viverrini and exposed to DMN (OVDMN). Immunohistochemistry using an anti-granulin specific probe for mammalian granulin was undertaken to monitor expression and location in hepatobiliary tissues of the hamsters. In parallel, cognate studies of transcription of mRNA and protein. Histopathological examination revealed development of proliferative lesions from the onset and eruption of CCA onwards, an outcome that was most prominent in the OVDMN hamsters. Proliferating cell nuclear antigen (PCNA) index rose continuously from initiation of infection and increased with lesion progression in OV, DMN and markedly in OVDMN hamsters. Expression of GRN in biliary was elevated in biliary epithelial cells in CCA lesions in hamsters in the DMN and OVDMN groups. Expression of GRN as assayed by western blot and RT-PCR reflected the same trend as seen with PCNA. Together the histopathogical and molecular assay based findings revealed marked expression of granulin during cholangiocarcinoma in these hamsters, and highlighted the prospect that granulin represents a potential prognostic marker for cholangiocarcinoma.

Increase in L-type amino acid transporter 1 expression during cholangiocarcinogenesis caused by liver fluke infection and its prognostic significance.

L-type amino acid transporter 1 (LAT1) is highly expressed in various human cancers, including cholangiocarcinoma (CCA), the most common cancer in Northeast Thailand. Chronic inflammation and oxidative stress induced by liver fluke, Opisthorchis viverrini, infection has been recognized as the major cause of CCA in this area. We show here that an increased expression of LAT1 and its co-functional protein CD98 are found during carcinogenesis induced by Ov in hamster CCA tissues. We also demonstrate that oxidative stress induced by H2O2 is time-dependent and dramatically activates LAT1 and CD98 expression in immortal cholangiocytes (MMNK1). In addition, H2O2 treatment increased LAT1 and CD98 expression, as well as an activated form of AKT and mTOR in MMNK1 and CCA cell lines (KKU-M055 and KKU-M213). We also show that suppression of PI3K/AKT pathway activity with a dual PI3K/mTOR inhibitor, BEZ235, causes a reduction in LAT1 and CD98 expression in KKU-M055 and KKU-M213 in parallel with a reduction of activated AKT and mTOR. Interestingly, high expression of LAT1 in human CCA tissues is a significant prognostic factor for shorter survival. Taken together, our data show that LAT1 expression is significantly associated with CCA progression and cholangiocarcinogenesis induced by oxidative stress. Moreover, the expression of LAT1 and CD98 in CCA is possibly regulated by the PI3K/AKT signaling pathway.

Proteomic characterization of the internalization of Opisthorchis viverrini excretory/secretory products in human cells.

The association between liver fluke infection caused by Opisthorchis viverrini and cholangiocarcinoma (CCA - hepatic cancer of the bile duct epithelium) has been well established. Multiple mechanisms play a role in the development of CCA, but the excretory/secretory products released by O. viverrini (OvES) represent the major interface between the parasite and its host, and their uptake by biliary epithelial cells has been suggested to be responsible for proliferation of cholangiocytes, the cells that line the biliary epithelium. Despite recent progress in the study of the molecular basis of O. viverrini-host interactions, little is known about the effects that OvES induces upon internalization by host cells. In the present study we incubated non-cancerous human cholangiocytes (H69) and human colon cancer (CaCo-2) cells with OvES and performed a time-course quantitative proteomic analysis on the cells to determine the early changes induced by the parasite. Different KEGG pathways were altered in H69 cells compared to Caco-2 cells: glycolysis/gluconeogenesis and protein processing in the endoplasmic reticulum. In addition, the Reactome pathway analysis showed a predominance of proteins involved in cellular pathways related to apoptosis and apoptotic execution phase in H69 cells after incubation with OvES. The present study provides the first proteomic analysis to address the molecular mechanisms by which OvES products interact with host cells, and Sheds light on the cellular processes involved in O. viverrini-induced CCA.

Upregulation of endothelial nitric oxide synthase (eNOS) and its upstream regulators in Opisthorchis viverrini associated cholangiocarcinoma and its clinical significance.

Endothelial nitric oxide synthase (eNOS) is an isoform of the enzyme nitric oxide synthase (NOS) which is constitutively expressed in endothelial cells and plays important roles in vasodilation. We previously reported the importance of eNOS activation in cholangiocarcinoma (CCA) tissues and cell lines. The present study aims to investigate the relative abundance of eNOS and phosphorylated eNOS (P-eNOS) and their upstream regulators VEGFR3, VEGFC, EphA3 and ephrin-A1, in the Opisthorchis viverrini (Ov)/N-nitrosodimethylamine (NDMA)-induced hamster CCA model and in human CCA by semiquantitative immunohistochemical analysis of the relevant tissues. Results from the hamster model suggested an increase in eNOS and P-eNOS and upstream regulators during CCA genesis. In human CCA, high immunohistochemical staining intensity of all investigated proteins was associated with the presence of metastasis. A pairwise analysis of the staining data for eNOS and its upstream regulators showed that a concurrent increase in eNOS/VEGFR3, eNOS/ephrin-A1, eNOS/VEGFC and eNOS/EphA3 was significantly associated with metastasis. An increase in eNOS/VEGFR3, eNOS/ephrin-A1 was also associated with non-papillary type CCA. Additionally, an increase in eNOS and P-eNOS was significantly correlated with a high micro-vessel level (P=0.04). Our results indicate that the development of CCA involves upregulation of eNOS and P-eNOS and their regulators. This may drive angiogenesis and metastasis in CCA.

Andrographolide Ameliorates Beta-Naphthoflavone-Induced CYP1A Enzyme Activity and Lipid Peroxidation in Hamsters with Acute Opisthorchiasis.

BACKGROUND: Opisthorchis viverrini (OV) infection generates oxidative stress/free radicals and is considered as a primary cause ofcholangiocarcinoma since it primarily triggers sclerosing cholangitis. OBJECTIVE: In this study, the impacts of andrographolide on acute opisthorchaisis in ß-naphthoflavone (BNF)-exposed hamsters were investigated. MATERIAL AND METHOD: Ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities and Thiobarbituric acid reaction substances (TBARS) assay of andrographolide in acute opisthorchiasis in the BNF-exposed hamsters were assessed. RESULTS: The results showed that andrographolide ameliorated the hepatic CYP1A1 and CYP1A2 activities by decreases of the specific enzymatic reactions of EROD and MROD, respectively, in the BNF-exposed hamsters. Moreover, andrographolide lowered the formation of malondialdehyde in the livers and brains of the hamsters. CONCLUSION: These observations revealed the promising chemo-protective and antioxidant activities of andrographolide via suppression of the specific EROD and MROD reactions and lipid peroxidation against acute opisthorchiasis in the BNF-exposed hamsters.

Upregulation of 14-3-3 eta in chronic liver fluke infection is a potential diagnostic marker of cholangiocarcinoma.

PURPOSE: To discover protein markers in chronic/advanced opisthorchiasis for the early detection of Opisthorchis viverrini (OV)-associated cholangiocarcinoma (CCA). EXPERIMENTAL DESIGN: Liver tissues derived from normal hamsters and those with chronic/advanced opisthorchiasis (n = 5 per group) were subjected to 2DE and LC-MS/MS. Candidate protein expression was confirmed in hamster models and human CCA tissue microarray (TMA) using immunohistochemistry and Western blot. RESULT: Proteomics analysis detected 14-3-3 eta only in infected hamsters, not in uninfected controls. Immunohistochemistry and Western blot analysis confirmed low expression of 14-3-3 eta in normal hamster livers and demonstrated increased expression through time in infected livers. This protein was also observed in parasite organs, especially during the chronic phase of opisthorchiasis. Moreover, increased expression of 14-3-3 eta, relative to normal hamster livers, was observed during the early stage of CCA induced by OV infection and administration of N-nitrosodimethylamine. Immunohistochemical analysis of human TMA revealed that 14-3-3 eta was highly expressed in CCA (84.23%, 187/222 cases) but was not found in hepatocellular carcinoma or healthy liver tissues. CONCLUSIONS AND CLINICAL RELEVANCE: 14-3-3 eta protein has potential as a screening and early diagnostic marker for CCA.

Carcinogenic Parasite Secretes Growth Factor That Accelerates Wound Healing and Potentially Promotes Neoplasia.

Infection with the human liver fluke Opisthorchis viverrini induces cancer of the bile ducts, cholangiocarcinoma (CCA). Injury from feeding activities of this parasite within the human biliary tree causes extensive lesions, wounds that undergo protracted cycles of healing, and re-injury over years of chronic infection. We show that O. viverrini secreted proteins accelerated wound resolution in human cholangiocytes, an outcome that was compromised following silencing of expression of the fluke-derived gene encoding the granulin-like growth factor, Ov-GRN-1. Recombinant Ov-GRN-1 induced angiogenesis and accelerated mouse wound healing. Ov-GRN-1 was internalized by human cholangiocytes and induced gene and protein expression changes associated with wound healing and cancer pathways. Given the notable but seemingly paradoxical properties of liver fluke granulin in promoting not only wound healing but also a carcinogenic microenvironment, Ov-GRN-1 likely holds marked potential as a therapeutic wound-healing agent and as a vaccine against an infection-induced cancer of major public health significance in the developing world.

Altered Expression of Oxidative Metabolism Related Genes in Cholangiocarcinomas.

Cholangiocarcinoma (CCA) is a rare but highly fatal cancer for which the molecular mechanisms and diagnostic markers are obscure. We therefore investigated the kinetic expression of isocitrate dehydrogenase-1 (IDH1), isocitrate dehydrogenase-2 (IDH2) and homogentisate 1,2-dioxygenase (HGD) during the tumorigenesis of O. viverrini infection-associated CCA in an animal model, and confirmed down-regulation of expression in human cases of opisthorchiasis-associated CCA through real time PCR. Kinetic expression of HGD, IDH1 and IDH2 in the animal model of O. viverrini infection-induced CCA was correlated with human CCA cases. In the animal model, expression of HGD was decreased at all time points (p<0.01) and expression of both IDH1 and IDH2 was decreased in the CCA group. In human cases, expression of HGD, IDH1 and IDH2 was decreased more than 2 fold in 55 cases (70.5%), 25 cases (32.1%) and 24 cases (30.8%) respectively. The present study suggests that reduction of HGD, IDH1 and IDH2 may be involve in cholangiocarcinoma genesis and may be useful for molecular diagnosis.

[Role of opisthorchis felineus on induction of bile duct cancer].

Opisthorchis felineus (Trematoda) is widespread in the Russian Federation, especially in Siberia, and other countries of Europe. Infestation of endemic area population with O. felineus reaches 80%. On animal models of the infection of closely related Opisthorchis viverrini combined with the nitrosamines' intake it has been shown that the parasite induces cholangiocarcinoma. However carcinogenic potential of O. felineus is still poorly studied. The present study is aimed to investigate the role of O. felineus in cholangiocarcinoma carcinogenesis in hamster treated additionally by dimethylnitrosamine (DMN). Golden hamsters were divided into 4 groups (15 specimens in the control group and 20 for other groups): (I) untreated control, (II) 12.5 ppm DMN solution intake, (III) infected with 50 metacercariae of O. felineus and (IV) infected with 50 metacercariae of O. felineus and 12.5 ppm DMN solution intake. According to the histological data, in the. O. felineus-infested group significant hyperplastic and dysplastic biliary changes were found considered as a precancerogenic state. Such pathological changes of bile ducts were more severe in group treated with both factors, with cholangiocarcinoma being found out at 18th week in all the animals of this group. These results demonstrate that O. felineus could play promoting role in two-step model in cholangiocarcinoma carcinogenesis and may be used to define the O.felineus group in the International Agency for Research on Cancer classification of agents, mixtures and exposures (IARC categories).

Apoptosis of cholangiocytes modulated by thioredoxin of carcinogenic liver fluke.

Chronic infection with the food-borne liver fluke, Opisthorchis viverrini, frequently induces cancer of the bile ducts, cholangiocarcinoma. Opisthorchiasis is endemic in Thailand, Lao PDR, Cambodia and Vietnam, where eating undercooked freshwater fish carrying the juvenile stage of this pathogen leads to human infection. Because inhibition of apoptosis facilitates carcinogenesis, this study investigated modulation by thioredoxin from O. viverrini of apoptosis of bile duct epithelial cells, cholangiocytes. Cells of a cholangiocyte line were incubated with the parasite enzyme after which they were exposed hydrogen peroxide. Oxidative stress-induced apoptosis was monitored using flow cytometry, growth in real time and imaging of living cells using laser confocal microscopy. Immunolocalization revealed liver fluke thioredoxin within cholangiocytes. Cells exposed to thioredoxin downregulated apoptotic genes in the mitogen activated protein kinases pathway and upregulated anti-apoptosis-related genes including apoptosis signaling kinase 1, caspase 9, caspase 8, caspase 3, survivin and others. Western blots of immunoprecipitates of cell lysates revealed binding of thioredoxin to apoptosis signaling kinase 1. Together the findings indicated that thioredoxin from O. viverrini inhibited oxidative stress-induced apoptosis of bile duct epithelial cells, which supports a role for this liver fluke oxidoreductase in opisthorchiasis-induced cholangiocarcinogenesis.