1.1.1. Taxonomy

Opisthorchis viverrini (O. viverrini) and Clonorchis sinensis (C. sinensis) are pathologically important foodborne members of the genus Opisthorchis; family, Opisthorchiidae; order, Digenea; class, Trematoda; phylum, Platyhelminths; and kingdom, Animalia. They belong to the same genus (Opisthorchis) but to different species based on morphology; nonetheless, the genus Clonorchis is so well established in the medical literature that the term is retained here.

1.1.2. Structure

The adult of O. viverrini and C. sinensis are usually about 10–25 mm in length and 3–5 mm in width (Liu & Chen, 1998; Sripa et al., 2007).

The yellowish-brown, ovoid eggs have a distinct operculum, which opens to release the miracidum – a fully formed larva. Eggs are on average 29 μm long by 17 μm wide for C. sinensis (Liu & Chen, 1998), and 27 μm by 15 μm for O. viverrini (Sadun, 1955), and are difficult to differentiate between these two species (Kaewkes et al., 1991).

1.1.3. Structure of the genome

The genomic structures of O. viverrini and C. sinensis have not been reported.

O. viverrini is reported to have six pairs of chromosomes, i.e. 2n = 12 (Rim, 2005), to have neither CpG nor A methylations, but to contain a highly repeated DNA element that is very specific to the organism (Wongratanacheewin et al., 2003). Intra- and inter-specific variations in the gene sequences of 18S, the second internally transcribed spacer region ITS2, 28S nuclear rDNA, and of the mitochondrial cytochrome C oxidase subunit I (mtCOI) DNA are low and nearly identical (Ando et al., 2001). A comparison of the ITS2 region sequences of O. viverrini versus C. sinensis show a 95% match; the sequences differ at 28 nucleotide positions (Park, 2007).

The chromosome number of C. sinensis is 2n = 56, and the chromosomes can be divided into two groups based on their sizes, consisting of eight pairs of large and 20 pairs of small chromosomes. The mean total length of the diploid complements of liver flukes collected in the People’s Democratic Republic of China is slightly longer than that of those collected in the Republic of Korea (Park et al., 2000).

1.1.4. Host range

Three families of freshwater snails (Hydrobiidae, Bithyniidae, and Melaniidae) are first intermediate hosts (Harinasuta & Harinasuta, 1984; Liu & Chen, 1998). Of these, Parafossarulus striatulus, Alocinma longicornis (Hydrobiidae), Bithynia fuchsianus (Bithyniidae) are currently considered to be of greatest importance in China in the life cycle of C. sinensis (Lun et al., 2005).

Over 130 species of fish (belonging to 16 families) are secondary intermediate hosts (Komiya, 1966; Vichasri et al., 1982; Rim, 1986; Joo, 1988; Liu & Chen, 1998). Fish in the family Cypriniidae are the major intermediate hosts (Lun et al., 2005).

In addition to human beings, other fish-eating mammals, for example dogs, cats, pigs, minks, weasels, civets, and house rats can be definite hosts, and some may act as reservoir hosts (Wang, 1983; Lun et al., 2005; Rim, 2005). There is also evidence that rabbits, guinea-pigs, hamsters, gerbils, mice, and rats are susceptible to the parasite in a laboratory setting (Bhamarapravati et al., 1978; Wang, 1983; Boonmars et al., 2009). Cats and dogs are considered to be the most important animal hosts in the endemic regions of China (Lun et al., 2005). In contrast with many other countries, most cats and dogs are not kept as pets in rural China but roam freely in villages, and thus have easy access to the remains of raw or undercooked fish in household waste (Wang, 1983; Jiang, 2001).

1.1.5. Target organs

The adult liver flukes usually reside in the medium-sized or small intrahepatic bile ducts. In heavy infections, adult parasites may be found in the gallbladder, the extrahepatic bile duct, and the pancreatic duct (Pungpak et al., 1985; Rim, 1986, 2005; Lim, 1990; Sripa, 2003). Over 100 flukes were recovered from the gallbladder of one patient (Evans et al., 1971), and 5140 and 1348 flukes of C. sinensis were found, respectively, in the bile ducts and in pancreatic ducts of a child patient who died of clonorchiasis sinensis (Chen et al., 1963).

The pathophysiology and clinical manifestations for O. viverrini and C. sinensis and infection are very similar (Lun et al., 2005; Sripa, 2008).

1.1.6. Life cycle

The eggs produced by the mature adult worms pass down the bile duct and are excreted in the faeces. If the eggs reach a freshwater body (small ponds, streams and rivers, flooded rice fields, and reservoirs), they are ingested by snails, which act as the primary intermediate hosts. Asexual reproduction in the snail results in daily release of thousands of cercariae, 1–2 months after infection of the snail. The free-swimming cercariae penetrate the tissue of freshwater fish, which act as the secondary intermediate host, and encyst to become fully infective metacercariae under the fish’s skin or in muscle after 21 days.

Humans or other fish-eating animals are infected through the ingestion of raw or undercooked (salted, pickled, or smoked) freshwater fish that contains metacercariae. After ingestion, the metacercaria excysts in the duodenum and ascends the biliary tract through the ampulla of Vater. Maturation to adulthood takes approximately 1 month.

The life cycle of the liver flukes is shown in Fig. 1.1 (for a review, see Rim, 1986; Sripa et al., 2007).

Figure 1.1

Life cycle of Clonorchis sinensis and Opisthorchis viverrini

1.1.7. Genes and gene products

Laha et al. (2007) constructed an O. viverrini cDNA library that covers ~14% of the entire transcriptome. About 20% of contigs were assigned Gene Ontology classifications. Frequently represented protein families included those involved in physiological functions that are essential to parasitism, such as anaerobic respiration, reproduction, detoxification, surface maintenance, and feeding. An assessment of evolutionary relationships showed that O. viverrini was similar to other parasitic flukes such as C. sinensis and Schistosoma japonicum. A total of 164 O. viverrini contigs contained open reading frames (ORFs) with signal sequences, many of which were platyhelminth-specific. Moreover, ORFs representing secreted proteins with known roles in tumorigenesis were identified such as granulin, kallikrein-like serine proteases, phospholipase A2 (PLA-2), saponin-like protein, and thioredoxin peroxidase. These proteins might play a role in the pathogenesis of O. viverrini-induced cholangiocarcinoma (Laha et al., 2007). Gene expression profiling of adult O. viverrini was also constructed by the first 5′ serial analysis of gene expression (5′ SAGE) library, and vitelline B precursor protein and myoglobin were found to be the most abundant proteins (Chutiwitoonchai et al., 2008).

By using the expressed sequence tag (EST) approach, Lee et al. (2003) constructed the C. sinensis adult cDNA library. A total of 220 genes were sorted into seven functional categories including: energy metabolism (38), gene expression/RNA metabolism (21), regulatory/signalling components (14), protein metabolism/sorting (98), the structure/cytoskeleton (29), membrane transporters (10), and antigenic proteins (10). The high frequency of cysteine protease expression (30/415 randomly selected clones) suggests an important role of this protein in the metabolism and/or pathogenesis of clonorchiasis. Also identified were Cu/Zn-superoxide dismutase and glutathione-S-transferase, which are believed to play a crucial role in protecting the parasite from the host immune effector mechanisms, and are being pursued as drug targets in other parasitic infections (Lee et al., 2003). Cho et al. (2008) reported gene expression profiles in C. sinensis metacercariae compared to those of adult worms. The genes expressed more abundantly in the metacercariae were a group of structural and cytoskeletal proteins, followed by transcription and translation machinery proteins, and a group of energy metabolism proteins. In contrast, adult C. sinensis has abundant mRNA clusters encoding for regulatory and signal proteins, other metabolic proteins and enzymes, and structural and cytoskeletal proteins, in descreasing order (Cho et al., 2008). This may be explained by the fact that metacercariae in the muscles of freshwater fish are in a resting stage wherein they simply maintain a basal metabolic status, and adult C. sinensis have a high metabolic rate and produce a large numbers of eggs in mammalian hosts (Rim, 2005).

1.2.1. Prevalence, geographic distribution

Human liver fluke infection is endemic in China, Thailand, the Republic of Korea, the Democratic People's Republic of Korea, Viet Nam, Lao People's Democratic Republic, and Cambodia. Endemicity for C. sinensis is also suspected in the Russian Federation long the Amur River. Persons from Singapore and Malaysia with C. sinensis infection have been reported infrequently; many of them may be infected during travelling in other countries or through eating imported fish.

A very crude estimate of the global number of infected people is of the order of 45 million, comprising 35 million infected with C. sinensis (Korea Association of Health Promotion, 2004; Lun et al., 2005; Fang et al., 2008), and 10 million with O. viverrini (WHO, 1995; Jongsuksuntigul & Imsomboon, 2003). The geographic distribution of O. viverrini and C. sinensis is shown in Fig. 1.2.

Figure 1.2

Distribution of Liver fluke infection in Asia

1.2.2. Transmission and risk factors for infection

The definitive host is infected by the liver fluke primarily through the ingestion of raw (dried, pickled or salted) or undercooked infected fish, which contain metacercariae – this is the infective stage in the life cycle of liver flukes (Sithithaworn & Haswell-Elkins, 2003). Many surveys show that people in Thailand (Kaewpitoon et al., 2008), Viet Nam (Dang et al., 2008), China (Fang et al., 2008; Lun et al., 2005), Laos (Hohmann et al., 2001), and the Republic of Korea (Lim et al., 2006) have these eating habits.

In southern China and among the Cantonese population in the Hong Kong Special Administrative Region, raw fish is traditionally eaten after being dipped in rice porridge. Alternatively, large fish are sliced and eaten with ginger and garlic known as “yushen.” This mode of transmission tends to increase with age. In contrast, many children in hilly areas of Guangdong and eastern China such as Jiangsu, Shandong, and Anhui provinces, often catch fish during play, and roast them incompletely before consumption. This mode of transmission tends to decline with age (Fang et al., 2008).

The population of the Republic of Korea eat raw fish soaked in vinegar, red-pepper mash or hot bean paste with rice wine at social gatherings. The fact that men do so more frequently than women has been given as a reason for the higher prevalence of infection among men; however, in heavily endemic areas, often no significant differences are seen between the genders. When fish is abundant, raw fish is eaten very regularly as opposed to being saved for special occasions (Choi, 1984; Rim, 1986). Vietnamese people eat raw fish in salads (Kiêu et al., 1990).

In Thailand and the lowland region of Laos, three types of uncooked fish preparations are noted (Sadun, 1955; Sithithaworn & Haswell-Elkins, 2003):

• koi pla, eaten soon after preparation;
• pla som, moderately fermented, and stored for a few days to weeks; and,
• pla ra and jaewbhong, extensively fermented, highly salted fish, stored for at least 2–3 months.

Koi pla is probably the most infective dish, followed by fish preserved for <7 days, then pla ra and jaewbhong, in which viable metacercariae are rare (Sithithaworn & Haswell-Elkins, 2003).

Several factors can directly or indirectly lead to the transmission of the liver flukes to humans (for reviews, see Sithithaworn & Haswell-Elkins, 2003 for O. viverrini; Lun et al., 2005 for Clonorchis sinensis): 1) poor educational level of local residents (Jongsuksuntigul & Imsomboon, 2003); 2) lack of sanitation: it is common in some endemic regions in China, particularly in the province of Guangdong and Guangxi, that “lavatories” are built adjacent to ponds, so that human excrement containing C. sinensis eggs enters the pond water (Lun et al., 2005). Also, in Laos, 95.5% of houses in some rural villages in Bolikhamxay Province do not have a latrine, and more than half of the village people use animal and/or human faeces as fertilizer (Hohmann et al., 2001); 3) habit to eat raw or undercooked freshwater fish; 4) freshwater aquaculture is developing rapidly, but adequate testing of fish products is lacking (Fang et al., 2007); 5) dinner-set contamination from infected fish (Fang et al., 2007); and 6) the absence of systematic control activities to limit transmission in many endemic areas (Fang et al., 2007).

1.2.3. Persistency and latency

It has been reported that C. sinensis may survive up to 26 years in a human host, as has been shown in a Chinese immigrant living in Australia (Attwood & Chou, 1978). The life expectancy of O. viverrini is approximately 10 years (Sithithaworn & Haswell-Elkins, 2003).

2.1.1. Opisthorchis viverrini

The Working Group of the previous IARC Monograph on liver flukes (IARC, 1994) evaluated infection with O. viverrini based on a dozen of descriptive studies (case reports, cases series, and correlation studies), and three cross-sectional or case–control studies (Kurathong et al., 1985; Parkin et al., 1991; Haswell-Elkins et al., 1994), which demonstrated a positive association between infection with O. viverrini and cholangiocarcinoma.

Currently, primary liver cancer is the leading cancer in Thailand in men (annual standardized ratio [ASR], 33.4/100000 population), and the third in women (ASR, 12.3/100000) (Khuhaprema & Srivatanakul, 2007), with cholangiocarcinoma being the predominant type. In addition, the highest incidence of liver cancer (ASR of up to 113.4/100000 in men) is found in the north-eastern regions where O. viverrini is endemic, and is 20 times higher than that in the south of Thailand where O. viverrini is rare (Sripa & Pairojkul, 2008). Furthermore, the proportion of histologically verified cases of cholangiocarcinoma in men diagnosed with liver cancer in the north-eastern regions has been reported to be as high as 85.5% compared to 10% in the south (Khuhaprema & Srivatanakul, 2007). A recent correlation study (Sriamporn et al., 2004) found a significant positive association between the incidence cases of cholangiocarcinoma from the cancer registry and O. viverrini infection in Khon Kaen, a province in north-east Thailand, with the highest incidence of cholangiocarcinoma cancer in the world (see Table 2.1).

Table 2.1

Descriptive study of Opisthorchis viverrini and liver cholangiocarcinoma.

Table 2.2 presents the results from all the available cross-sectional and case–control studies, all conducted in Thailand (descriptive studies are not presented). The odds ratios ranged from 1.3–27.1. The highest relative risk, reported by Honjo et al. (2005), was adjusted for sex, age, residence, alcohol consumption, and smoking. Haswell-Elkins et al. (1994) reported adjusted prevalence odds ratios (POR) of 1.7 in the light infection group, 3.2 in the moderate infection group, and 14.1 in the heavy infection group (based on 14 exposed cases stratified by intensity of infection).

Table 2.2

Cross-sectional and case–control studies on Opisthorchis viverrini infection and cholangiocarcinoma.

2.1.2 Clonorchis sinensis

The Working Group of the previous IARC Monograph on liver flukes (IARC, 1994) evaluated infection with C. sinensis as probably carcinogenic to humans (Group 2A), based on nine case series and three case–control studies (Gibson, 1971; Kim, 1974; Chung & Lee, 1976). Since then, several studies have been published, and are summarized here.

The incidence of primary liver cancer in the Republic of Korea is the highest in the world (ASR, 44.9 in men and 12.0 in women), with a proportion of microscopically verified cases of cholangiocarcinoma of 22.3% and 36.1% in men and women, respectively (Curado et al., 2007). According to the Korean Cancer Registry, the incidences of cholangiocarcinoma vary by geographic area, with up to 4-fold differences (Shin et al., 2008). The region with the highest incidence (7.2/100000 in men) was reported to be that with the highest prevalence of C. sinensis infection in a nationwide survey conducted 20 years ago (Seo et al., 1981).

A recent correlation study from the Republic of Korea showed a high correlation between the endemicity of C. sinensis infection with the incidence as well as mortality of cholangiocarcinoma (Lim et al., 2006; Table 2.3).

Table 2.3

Descriptive study of Clonorchis sinensis infection and cholangiocarcinoma.

Since the previous IARC Monograph, two case series from China have been published, both supporting a relationship between C. sinensis and cholangiocarcinoma (Cheng et al., 2000; Wang et al., 2003; Table 2.4). Furthermore, three case–control studies have been published from the Republic of Korea (Table 2.5). All three showed significant positive associations between C. sinensis infection and cholangiocarcinoma. The study by Choi et al. (2006) reported an (unadjusted) odds ratio for any evidence of infection of 7.3 (95%CI: 3.9–13.3). Shin et al. (1996) reported an odds ratio of 2.7 (95%CI: 1.1–6.4), adjusted for alcohol consumption, smoking, hepatitis B and C, and Lee et al. (2008) found an odds ratio of 13.6 (95%CI: 6.1–30.3) after adjusting for hepatitis B, alcohol consumption, and liver cirrhosis.

Table 2.4

Case series and case reports of cholangiocarcinoma associated with Clonorchis sinensis.

Table 2.5

Cross-sectional and case-control studies on Clonorchis sinensis infection and cholangiocarcinoma.

In two of the studies (Shin et al., 1996; Choi et al., 2006), higher odds ratios were reported for evidence of past C. sinensis infection (i.e. based on positive history, serology, skin test, radiology) compared to current infection (i.e. based on positive stool microscopy or pathology).

2.2.1. Opisthorchis viverrini

A correlation analysis of the prevalence of O. viverrini infection and liver cancer incidence, conducted in five regions with different frequencies of cholangiocarcinoma and hepatocellular carcinoma (HCC), showed little geographic variation in the incidence of HCC, with a correlation of −0.37 (P = 0.54) for antibody titre ≥ 1:40, and of 0.02 (P = 0.96) for faecal egg count (Srivatanakul et al., 1991a).

One cross-sectional study (Kurathong et al., 1985) and one case–control (Srivatanakul et al., 1991b) study were carried out in north-east Thailand to evaluate the association between O. viverrini infection and the risk for HCC (Table 2.6). Neither study showed a significant association.

Table 2.6

Cross-sectional and case–control studies on infection with liver flukes and hepatocellular carcinoma.

2.2.2. Clonorchis sinensis

A few studies have evaluated the association between C. sinensis infection and the risk for HCC (Table 2.6). One study was conducted in the Hong Kong Special Adminitrative Region (Gibson, 1971) and found no association.

Three studies were conducted in the Republic of Korea; one (Kim, 1974) in two separate regions, of low and high prevalence of C. sinensis infection, respectively; the other two studies were conducted in Pusan, one of the areas with the highest prevalence of C. sinensis infection (Chung & Lee, 1976; Shin et al., 1996). In the two earlier studies, no increased risks for HCC were observed [from crude odd ratios]. In the most recent study (Shin et al., 1996), neither C. sinensis eggs in stool samples (OR, 2.7; 95%CI: 0.9–7.7) nor a history of liver fluke infection (OR, 2.6; 95%CI: 0.6–11.3) were significantly associated with HCC in a conditional logistic regression analysis adjusted for socioeconomic status (Table 2.6).

4.2.1. Cell proliferation in vitro

Adult O. viverrini worms were co-cultured with mouse NIH-3T3 fibroblasts. Even though worms and fibroblasts were separated by Transwell membrane, fibroblast proliferation was stimulated more than 4-fold. Moreover, O. viverrini ES products increased cell proliferation by stimulating the expression of phosphorylated retinoblastoma (pRB) and cyclin D1, the key proteins in driving cells through the G1/S transition point of the cell cycle. This led to the induction of cells going into the S-phase of the cell cycle (Thuwajit et al., 2004). In similar experiments with C. sinensis, ES products, and the human embryonic kidney epithelial cell line HEK293, the ES products induce HEK293 cell proliferation, associated with the upregulation of cyclin E and the transcription factor E2F1 (Kim et al., 2008a). Furthermore, C. sinensis ES products upregulate the phosphorylation of pRB and N-nitrosodimethylamine (NDMA) upregulates cyclin-dependent kinases, and both synergistically drive the cells to proliferate (Kim et al., 2008b). An anti-apoptotic effect of C. sinensis ES products in human cholangiocarcinoma cells has been reported (Kim et al., 2009).

Gene microarrays were used to explore transcriptional changes induced in NIH-3T3 murine fibroblasts co-cultured with O. viverrini ES products. mRNAs encoding certain growth-promoting proteins such as transforming growth factor (TGF), PKC, EPS 8 and TGF-β 1I4, that are downstream of epidermal growth factor (EGF) or TGF-β-mediated signalling, were found to be overexpressed (Thuwajit et al., 2006). Moreover, human cholangiocarcinoma cell line (KKU-100) underwent excessive proliferation upon stimulation with O. viverrini worms (Sripa, 2003). The promotion of proliferation in vitro is consistent with the histopathological findings of hyperplasia of biliary epithelial cells in opisthorchiasis and clonorchiasis (Bhamarapravati et al., 1978; Sripa & Kaewkes, 2000; Rim, 2005).

4.2.2. Oval cell proliferation and differentiation in vivo

Oval cells are typically seen in response to certain liver injuries, and more than likely represent progenitor cells with the potential to differentiate along biliary or hepatocytic lineages, including into hepatic neoplasms (Sell & Leffert, 2008). Lee et al. (1997) reported the appearance of increased numbers of periductal oval cells in the portal and/or periportal areas of hamster liver infected with C. sinensis and administered NDMA.

4.2.3. DNA damage and adduct formation in vivo

Diffuse nitrosative and oxidative DNA damage (8-nitroguanine and 8-oxo-7, 8-dihydro-2′-deoxyguanosine [8-oxodG]) has been reported in the biliary epithelium of hamsters infected with O. viverrini (Pinlaor et al., 2003). These DNA lesions still persisted for at least 180 days post-infection. Moreover, repeated infections with liver flukes result in enhanced biliary DNA damage (Pinlaor et al., 2004a, b). This may be explained by the fact that repeated infection increased inducible nitric oxide synthase (iNOS) expression in the bile duct epithelium. The DNA damage in infected biliary cells is probably a result of the inflammatory response caused by O. viverrini because 8-nitroguanine and 8-oxodG disappear after praziquantel treatment (Pinlaor et al., 2006). However, in promoting parasite antigen dispersal, treatment with praziquantel may transiently increase inflammation, in association with increased NF-κB and iNOS expression in the bile duct epithelium and inflammatory cells, and elevated levels of plasma nitrate, of end-products of nitric oxide, and of malondialdehyde in the treated hamsters (Pinlaor et al., 2008).

Individuals infected with O. viverrini also show evidence of oxidative DNA damage. Urinary 8-oxodG levels were significantly higher in O. viverrini-infected patients (4.45 ± 0.25 µg/g creatinine) than in healthy subjects (3.03 ± 0.24 µg/g creatinine; P < 0.01). This level decreases significantly 2 months after praziquantel treatment, and is comparable with levels in healthy subjects 1 year after treatment. Urinary 8-oxodG levels were significantly correlated with leukocyte 8-oxodG levels (Thanan et al., 2008).

The excretion of lipid peroxidation-derived etheno DNA adducts – 1,N⁶-etheno-2'-deoxyadenosine (εdA) and 3,N⁴-etheno-2'-deoxycytidine (εdC) – was measured in urine samples collected from healthy volunteers and O. viverrini-infected Thai subjects. Mean excreted εdA and εdC levels were 3–4 times higher in the urine of O. viverrini-infected patients and correlated with an increased level of urinary malondialdehyde, urinary nitrate/nitrite, and plasma alkaline phosphatase. After fluke elimination by treatment with praziquantel, the level of the two etheno adducts, malondialdehyde, nitrate/nitrite, and alkaline phosphatase was decreased. The promutagenic DNA etheno adducts that are thought to derive from bile duct epithelial cells may increase the risk of developing cholangiocarcinoma in O. viverrini-infected patients. This study highlights the biomarker potential of urinary εdA and εdC levels as non-invasive risk markers for developing opisthorchiasis-related cholangiocarcinoma (Dechakhamphu et al., 2008).

[The Working Group noted that all the studies described above relate to O. viverrini; studies regarding DNA damage in response to C. sinensis infection were not available to the Working Group.]

4.3.1 O. viverrini-endemic areas

Differences in Ki-RAS mutational status have been described when comparing cholangiocarcinoma from Japanese patients (where fluke infections are very rare) with those from cholangiocarcinoma arising in patients living in areas of Thailand endemic for O. viverrini (the incidence of Ki-RAS mutation was higher in Thai patients) (Kiba et al., 1993). Hypermethylation of the promoter of the DNA mismatch repair enzyme hMLH1 has also been shown in another group of Thai patients (Limpaiboon et al., 2005). However, these studies did not specifically document liver fluke infection status in the two groups of patients.

Gene microarray transcriptional profiling of cholangiocarcinoma from Japanese versus Thai patients (again without certain knowledge of liver fluke status) led Jinawath et al. (2006) to propose a signature of O. viverrini-associated cholangiocarcinoma with an elevated expression of genes involved in xenobiotic metabolism (UGT2B11, UGT1A10, CHST4, SULT1C1) in cases from Thailand, but a lower expression of genes related to growth-factor signalling (TGFBI, PGF, IGFBP1, IGFBP3).

4.3.2. Studies in experimental animals

Few mutations of the Ki-RAS gene were observed in O. viverrini–NDMA-induced cholangiocarcinomas in hamsters (Tangkawattana et al., 2008), but TP53 overexpression was reported in nearly all O. viverrini-induced hamster cholangiocarcinomas (Tesana et al., 2000).

Loilome et al. (2006) investigated the molecular mechanism of O. viverrini–NDMA-induced cholangiocarcinogenesis in hamsters by using fluorescence differential display-PCR, and found 23 upregulated and one downregulated transcripts among 149 differentially amplified bands. Among the upregulated genes in the liver was the signal transduction protein kinase A regulatory subunit Iα (Prkar1α), which was significantly higher in cholangiocarcinoma and its precursor lesion when compared with normal liver and normal gallbladder epithelia (P < 0.05). Prkar1 expression tended to increase along with the progression of biliary transformation from hyperplasia and precancerous lesions to carcinoma.