Modulation of Dihydroxy-di-n-propylnitrosamine-induced Liver Lesion Development in Opisthorchis-infected Syrian Hamsters by Praziquantel Treatment in Association with Butylated Hydroxyanisole or Dehydroepiandrosterone Administration

The effects of praziquantel coupled with dehydroepiandrosterone (DHEA) or butylated hydroxyanisole (BHA) administration 16 weeks subsequent to dihydroxy-di- n -propylnitrosamine (DHPN) treatment and infection with Opisthorchis viverrini (OV) on lesion development in the liver of Syrian hamsters were investigated. Animals were given 80 OV metacercariae and then two i.p. injections of DHPN (500 mg/kg body weight) 4 and 5 weeks thereafter. At week 16, groups received praziquantel (250 mg/kg, i.g.) and were placed on normal diet or diet supplemented with BHA (1%) or DHEA (0.6%) until they were killed at week 24. Histopathological assessment revealed that, whereas antihelminthic treatment alone resulted in a clear reduction in hepatocellular lesion development, effects on cholangiocellular lesions were equivocal. BHA and DHEA, in contrast, were both associated with a significant reduction in frequency of cholangiofibrosis and cholangiocellular carcinoma. The former chemical, however, increased the numbers of liver nodules while the hormone brought about a decrease as well as a shift in the phenotype of the lesions. The results thus indicate that although cholangiocellular lesion development may, unlike generation of hepatocellular nodules, be to a certain extent independent of the continued presence of parasite, it can be influenced by exogenous treatments.     

Lack of modulation effects of praziquantel on DMN-induced lesion development in the Syrian hamster liver.

The effects of repeated praziquantel administration subsequent to dimethylnitrosamine (DMN) treatment of Syrian hamsters were investigated. The antihelminthic drug was given (200 mg/kg body wt. as a suspension in corn oil, by i.g. intubation) 11 times at 2 week intervals starting at week 4 after initial 20 mg/kg DMN i.p. injections at weeks 0 and 2. Sacrifice at week 28 revealed no differences in either hepatocellular or cholangiocellular lesion development between carcinogen-initiated groups with or without antihelminthic treatment. No lesions were observed in the praziquantel alone or untreated groups. The results thus indicate no promotion potential for praziquantel on nitrosamine-induced lesions in the hamster liver.     

Repeated exposure to Opisthorchis viverrini and treatment with the antihelminthic Praziquantel lacks carcinogenic potential.

The effects of repeated Praziquantel administration, subsequent to infection and reinfection with Opisthorchis viverrini (OV), on lesion development in the Syrian hamster liver were investigated. Five applications of the antihelminthic drug were made (300 mg/kg body wt, i.g.), each time approximately 5 weeks after dosing with 60-80 OV metacercariae at weeks 0, 8, 16, 24 and 32. The animals were then maintained until week 40 when they were killed; histopathological investigation revealed no significant development of either hepatocellular of cholangiocellular preneoplastic/neoplastic lesions. The results indicate that repeated exposure to Praziquantel at levels sufficient for successful removal of parasite infestation does not itself carry carcinogenic risk.     

iNOS-dependent DNA damage via NF-kappaB expression in hamsters infected with Opisthorchis viverrini and its suppression by the antihelminthic drug praziquantel

Inflammation-mediated DNA damage triggered by Opisthorchis viverrini (OV) infection is a major risk factor of cholangiocarcinoma (CCA). We have recently reported that nitrative and oxidative DNA damage participates in CCA development caused by repeated infection with OV [Pinlaor et al., Carcinogenesis 2004; 25:1535-42]. Therefore, to clarify the preventive effect of the antihelminthic drug praziquantel against cholangiocarcinogenesis, we assessed the effect of this drug on nitrative and oxidative DNA damage, including the formation of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), and the expression of inducible nitric oxide synthase (iNOS) by immunohistochemistry in OV-infected hamsters. We also examined the expression of nuclear factor-kappaB (NF-kappaB), which functions as a tumor promoter in inflammation-associated cancer. Our results showed that although 1-week treatment with praziquantel did not kill parasites completely in hamsters on days 14 and 30, this drug dramatically reduced inflammatory cell infiltration. Double immunofluorescence staining showed that drug treatment almost completely diminished OV-induced 8-nitroguanine and 8-oxodG formation in bile duct epithelial cells. Quantitative analysis using an electrochemical detector coupled to HPLC revealed that 8-oxodG level in the liver of OV-infected hamsters was significantly decreased by drug treatment (p<0.05). Western blotting and immunohistochemistry revealed that the expression of NF-kappaB and iNOS in bile duct epithelium was reduced by drug treatment. The amount of nitrate plus nitrite in the liver and plasma was significantly decreased after drug treatment. It is concluded that praziquantel can exhibit a preventive effect against OV-induced cholangiocarcinoma by inhibiting iNOS-dependent DNA damage through not only elimination of parasites but also a potential antiinflammatory effect.     

SHORT COMMUNICATION: Promotion of cholangiocarcinogenesis in the hamster liver by bile duct ligation after dimethylnitrosamine initiation

Administration of hepatocardnogenic nitrosamines before or afterinfection with the liver fluke, Opisthorchis viverrini (OV),results in marked development of cholangiocellular and hepatocellularprecancerous and cancerous lesions in the hamster liver. Thepromoting effects of OV are believed to be exerted either mechanically,chemically or immuno-logically. To test the influence of possiblemechanical effects, Syrian hamsters were initiated with a singlei.p. injection of dimethylnitrosamine (DMN) 20 mg/kg and subjected2 weeks later either to a sham operation or to complete ligationof the extrahepatic bile duct to the left lateral lobe. At theend of week 40, the animals receiving DMN-initiation and ligationhad a 60.9% incidence of cholangiofibrosis, 21.7% of mucouscystadenomas and 39.1% of cholangiocarcinomas, whereas the groupgiven DMN alone only developed cholangiofibrosis, limited to5% of the animals. In the latter case neither cystadenomas norcholangiocarcinomas were observed. The incidence of hepatocellularnodules did not differ between the two groups and no tumorouslesions developed in either the ligated or the untreated groupswithout DMN pretreatment. Complete ligation of the bile ductitself led to a series of events; obstruction of bile flow beingfollowed by dilatation, cyst formation, and necrosis of thebile duct epithelium and surrounding affected areas leadingto regenerative proliferation. The results are in line withthe conclusion that parasite-associated proliferation in targetcell populations is, at least in part, responsible for the influenceof OV on liver tumor development.     

Effect of streptozotocin diabetes on development of nitrosamine-induced pancreatic carcinoma when diabetes induction occurs after nitrosamine exposure

Diabetes mellitus has been suggested as a possible risk factorfor the development of pancreatic cancer in humans. Previousstudies in our laboratory have shown, however, that streptozotocin(STZ) diabetes inhibits the development of cancer of the exocrinepancreas in hamsters when STZ is administered prior to treatmentwith the pancreatic carcinogen N-nitrosobis(2-oxopropyl)amine(BOP). It has been reported by others that the concurrent administrationof BOP and STZ enhances pancreatic carcinogenesis in hamsters.The purpose of the present study was to determine the effectof STZ diabetes on the development of BOP-induced pancreaticcarcinoma when STZ is given following exposure to BOP. Groupsof Syrian golden hamsters were treated with either BOP only(single s.c. injection, 40 mg/kg body wt at week 0), BOP (singles.c. injection, 40 mg/kg body wt at week 0) plus STZ (50 mg/kgbody wt x3 daily i.p. doses at weeks 10, 20 or 30), STZ only(50 mg/kg body wt x3 daily i.p. doses at weeks 10, 20 or 30),or neither BOP nor STZ. The experiment was terminated at 40weeks after BOP treatment. No significant difference was seenin the incidence of pancreatic cancer between those animalsreceiving BOP only at week 0 and those receiving BOP at week0 plus STZ at weeks 10, 20 or 30 of the study. The results wouldappear to indicate that STZ diabetes, established after BOPtumor initiation, plays no apparent role in the modulation ofpancreatic carcinogenesis.     

Inhibitory effect of butylated hydroxyanisole administration on pancreatic carcinogenesis in Syrian hamsters initiated with N-nitrosobis(2-oxopropyl)amine

The modifying potential of butylated bydroxyanisole (BHA) administrationon pancreatic carcinogenesis was evaluated in 70 female Syriangolden hamsters. Groups of animals received saline, 70 mg/kgbody weight of N-nitrosobis(2-oxopropyl)-amine (BOP) or 70 mg/kgplus 20 mg/kg body weight of BOP followed by basal diet or dietcontaining 2% BHA from week 3. Although the body weights ofhamsters receiving the 2% BHA supplement decreased, caloricrestriction was not observed. All hamsters were killed at week18 and histo-pathologically examined for lesion development.The incidences of pancreatic carcinomas in hamsters receiving70 mg/kg plus 20 mg/kg body weight of carcinogen followed by2% BHA was 7.1%, significantly lower than the 64.3% evidentin hamsters given the same doses of BOP followed by basal diet.The total numbers of pancreatic lesions including carcinomas,atypical ductal hyperplasias and ductal hyperplasias and ductularproliferations in the liver were also significantly decreasedin animals receiving BOP followed by 2% BHA. The results thusindicate that both pancreatic and cholangiocellular carcinogenesisinitiated by BOP in Syrian hamsters can be inhibited by 2% BHAtreatment for a relatively short experimental period.     

Cholangiocarcinoma in Experimental Hamsters with Long-standing Opisthorchis viverrini Infection

Liver fluke infection of Opisthorchis viverrini (O. viverrini) is closely associated with several hepatobiliarydiseases including cholangiocarcinoma (CCA), but no reports have described these diseases in chronic andlong-standing experimental opisthorchiasis in hamsters more than 10 months of age. A longer period of infectioncould induce different pathological lesions. To prove the hypothesis, we therefore sequentially investigatedhistological changes of the hepatobiliary system in 4 groups of hamster: O. viverrini infection (OV group) for upto 20 months, O. viverrini infection combined with short-term DMN (OV+DMN group) until 7 months, longtermtreatment with DMN (DMN group) to 7 months and normal control group for up to 20 months. Pathologicalchanges in hamsters of the OV group gradually increased. Induction of CCA in this study was apparent with allthree protocols. Importantly, this is the first report of CCA-induction in hamsters solely with long-termopisthorchiasis for up to 20 months. Although the histopathology of CCA in the OV group showed some differencesin appearance from the OV+DMN and DMN groups, overall, O. viverrini itself can really induce CCA. Inaddition, this study confirms the previous studies both in vitro and in vivo on of effects of parasites and theirmetabolic products inducing cell proliferation, resulting in cholangiocarcinogenesis.     

Streptozotocin prevents development of nitrosamine-induced pancreatic cancer in the Syrian hamster

Administration of the nitrosamine carcinogen N-nitroso-bis (2-oxopropyl) amine (BOP) by subcutaneous injection (5 mg/kg/week) led to the development of invasive pancreatic ductular adenocarcinoma in 100% of normal Syrian hamsters by 24 weeks. Pretreatment of a second group of hamsters with the beta-cell toxin streptozotocin in a diabetogenic dose (50 mg/kg i.p. X 3) completely prevented the development of pancreatic cancer when BOP was subsequently administered. The mechanism of blockade by streptozotocin is unknown. This study suggests the potential importance of the endocrine pancreas in exocrine pancreatic carcinogenesis.     

N-nitrosodiethylamine-induced changes in the liver of syrian hamster with superinvasive opistorchiasis

Trematoda O. felinius-induced hepatic lesions were investigated in Syrian golden hamsters with superinvasive opistorchiasis. One hundred hamsters were divided into 4 groups: (1)--control; (2) N-nitrosodiethylamine (DENA), i.p., twice a week, 3 weeks, total dose 72 mg/kg; (3) metacercariae O. felinius, with drinking water, 3 injections per day, once in 2 weeks, and (4) metacercariae O. felinius, as in group 3, followed by DENA, as in group 2. Animals were sacrificed 12 months after the beginning of the study. No changes in the liver were found in group 2. Reddish protrusions, up to 4 cm in diameter, appeared on liver surfaces in groups 3 and 4. Group 4 featured the highest relative and absolute weights of liver as well as clusters of oval cells and cholangiocellular tubules and cholangiofibrosis (in group 3, they were less visible). Electron microscopic examination identified hepatocytes with destructive changes to plasmalemma, nucleus and cytoplasmic organelles. Also, perisinusoidal cells (Ito cells) occurred. Tumor-bearing animals showed low hepatic cytochrome P-450. It is suggested that proliferative growth in the liver was stimulated by opistorchis invasion.     

Rapid production of pancreatic carcinoma by initiation with N-nitroso-bis(2-oxopropyl)amine and repeated augmentation pressure in hamsters

A rapid-production model incorporating the principle of selection by resistance to cytotoxicity demonstrated earlier for liver carcinogenesis in rats was established for pancreatic carcinoma development in Syrian hamsters. Adenocarcinomas were induced in 84% of treated animals by 10 weeks after initiation with 70 mg of N-nitroso-bis(2-oxopropyl)amine (BOP) per kg of body weight when augmentation pressure (choline-deficient diet combined with DL-ethionine and L-methionine and administration of 20 mg/kg BOP upon return to basal diet) was applied three times. A 52% yield of cholangiocellular tumors also resulted from this experimental protocol.     

Ultrasonography as a tool for monitoring the development and progression of cholangiocarcinoma in Opisthorchis viverrini/ dimethylnitrosamine-induced hamsters

Cholangiocarcinoma (CCA) is the most common cancer in northeastern Thailand. At present, effective diagnosis of CCA either in humans or animals is not available. Monitoring the development and progression of CCA in animal models is essential for research and development of new promising chemotherapeutics. Ultrasonography has been widely used for screening of bile duct obstruction in CCA patients. In this study, we preliminarily investigated the applicability of ultrasonography to monitor the development and progression of CCA in Syrian golden hamsters (n=8) induced by Opisthorchis viverrini (OV)/dimethylnitrosamine (DMN) administration. Ultrasonography and histopathological examination of hamsters was performed at week 0, 20, 24 and 28 of OV infection or at the start of water/Tween-80 administration to controls. The ultrasonographic images of liver parenchyma and gallbladders of OV/DMN-induced CCA hamsters showed sediments in gallbladder, thickening of gallbladder wall, and hypoechogenicity of liver parenchyma cells. The ultrasonographic images of liver tissues were found to correlate well with histopathological examination. Although ultrasonography does not directly detect the occurrence of CCA, it reflects the thickening of bile ducts and abnormality of liver tissues. It may be applied as a reliable tool for monitoring the development and progression of CCA in animal models in research and development of new promising chemotherapeutics for CCA.     

Acetaminophen Overdose Enhances Early Cholangiocarcinoma in Opisthorchiasis Hamsters

Opisthorchiasis which exerted by infection of Opisthorchis viverrini is strongly related to the incident of cholangiocarcinoma (CCA) in many Southeast Asian countries northeastern of Thailand. The O. viverrini infection is primarily caused by raw fish consumption, and repeated exposure to liver fluke. Meanwhile, acetaminophen is usually medicated to relieve pain in particularly people in northeast Thailand. Objective: This study therefore aimed at investigating effects of acetaminophen on pathogenesis in hamsters for opisthorchiasis. Methods: There were 4 groups of hamsters: i) uninfected hamster (N); ii) sole acetaminophen administration (N-Ac); iii) sole O. viverrini infection (OV); and iv) combination of O. viverrini infection and acetaminophen (OV-Ac) on pathology of hamsters for 1 month post infection. For analysis of histopathological changes through hematoxylin and eosin, Sirius red and immunohistostaining for Cytokeratin 19 (CK-19), Proliferating cell nuclear antigen (PCNA) and CA 19-9, serum’s hamsters were used detected for liver function tests and tumor-related genes expression. Results: After 1 month under these treatments, the OV-Ac showed significantly higher CCA risk, including inflammatory cells were aggregations around bile duct, new bile duct and fibrosis in subcapsular hepatic tissues, than other treatments. These pathological parameters were positively correlated with immunohistochemical staining derived from CK-19, PCNA and CA 19-9. In addition, OV-Ac had significantly higher liver function tests (ALT). Conclusion: Combined intake of liver fluke-contaminated raw fishes and acetaminophen rendered more severity of CCA than sole consumption of the contaminated raw fishes.     

DNA single-strand breaks in kidneys of Syrian hamsters treated with steroidal estrogens: hormone-induced free radical damage preceding renal malignancy

The chronic administration of estradiol by subcutaneous (s.c.)implantation into male Syrian hamsters induces kidney tumors.Free radicals generated by redox cycling between catecholestrogensand their quinones have been proposed to damage DNA and to thusmediate renal hormone-induced carcinogenesis. As part of anexamination of this postulate, we assayed by a filter elutiontechnique DNA single-strand breaks in livers and kidneys ofmale hamsters treated with estrogen by single intraperitoneal(i.p.) injection, by s.c. implant or by continuous infusionand compared values to those in untreated controls. The DNAsof hamster liver and kidney were not affected by one i.p. injectionof 5, 15 or 150 mg/kg estradiol. However, treatment of hamsterswith one 25 mg estradiol lmplantlanimal for 2 weeks elevatedby 10% the levels of DNA single-strand breaks in kidney, butonly to a minor extent in liver, which is not a target of estrogen-induced carcinogenesis. An infusion of 250 µg/day/animalof estradiol or 4-hydroxyestradiol for one week by osmotic pumpsinto hamsters resulted in a comparable increase of single-strandbreaks in kidney DNA, whereas 2-hydroxyestra- diol under theseconditions had a negligible effect. It is concluded that theinduction of DNA single-strand breakage by either estradlolor 4-hydroxyestradiol in hamster kidney supports a mechanismof estrogen-Induced carcinogenesis by free radical generationvia redox cycling between 4-hydroxy- estradiol and Its correspondingquinone.     

Promotion of Rat Hepatocarcinogenesis by Praziquantel

Praziquantel, the widely used anti-helminthic agent, was investigated for hepatocarcinogenesis-promoting potential using a medium-term liver bioassay system for carcinogens. F344 male rats were given a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg) and then starting 2 weeks later, received praziquantel in the diet at concentrations of 1.5 or 0.5%, or intragastrically at a dose of 1,500 mg/kg once a week for 6 weeks. Control groups received DEN or praziquantel alone. All rats were subjected to two-thirds partial hepatectomy at week 3 and killed at week 8. Development of glutathione 5-transferase placental form-positive foci in the liver was significantly increased in terms of both number and area with the 1.5% dose, while only area was affected by the 0.5% dose. The results thus indicate that praziquantel at high dose has promoting potential in rat hepatocytic tumorigenesis.     

Hepatocyte Growth Factor Enhancement of Preneoplastic Hepatic Foci Development in Rats Treated with Diethylnitrosamine and N-Ethyl-N-hydroxyethylnitrosamine

Effects of hepatocyte growth factor were investigated in a two-stage rat liver carcinogenesis protocol. Male F344 rats were first treated with diethylnitrosamine (200 mg/kg, i.p.) and then, starting two weeks later, with N-ethyl-N-hydroxyethylnitrosamine (EHEN) for 6 weeks at a dose of 0.01% in drinking water. Hepatocyte growth factor, which was injected i.v. at a dose of 200 μg/kg body weight one (at week 3) or two times (at weeks 3 and 4) during EHEN administration, significantly increased the development of preneoplastic glutathione S-transferase placental form-positive foci. Although the observed effects of hepatocyte growth factor were weaker than that of the two-thirds partial hepatectomy (PH) performed at week 3, the present results suggest that the enhancing effects of PH performed during the promotion stage may be largely mediated through induction of hepatocyte growth factor.     

Promotion of rat hepatocarcinogenesis by praziquantel.

Praziquantel, the widely used anti-helminthic agent, was investigated for hepatocarcinogenesis-promoting potential using a medium-term liver bioassay system for carcinogens. F344 male rats were given a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg) and then starting 2 weeks later, received praziquantel in the diet at concentrations of 1.5 or 0.5%, or intragastrically at a dose of 1,500 mg/kg once a week for 6 weeks. Control groups received DEN or praziquantel alone. All rats were subjected to two-thirds partial hepatectomy at week 3 and killed at week 8. Development of glutathione S-transferase placental form-positive foci in the liver was significantly increased in terms of both number and area with the 1.5% dose, while only area was affected by the 0.5% dose. The results thus indicate that praziquantel at high dose has promoting potential in rat hepatocytic tumorigenesis.     

Plasma hydroxyproline, MMP-7 and collagen I as novel predictive risk markers of hepatobiliary disease-associated cholangiocarcinoma

Chronic opisthorchiasis caused by Opisthorchis viverrini infection is characterized by advanced periductal fibrosis leading to hepatobiliary diseases (HBD), including cholangiocarcinoma (CCA). We aimed to determine fibrotic markers to differentiate HBD status including opisthorchiasis, benign biliary disease (BBD) and CCA. Candidate fibrotic markers in plasma of healthy individuals (n = 14) and patients with opisthorchiasis (n = 32, pre- and post-treatment with praziquantel), BBD (n = 31), CCA (n = 37) and other types of tumors (n = 14) were measured by ELISA and zymography. Plasma levels of hydroxyproline (HYP), collagen I, MMP-7 and TIMP2 in opisthorchiasis patients were significantly higher than those in healthy individuals, and MMP9/TIMP2 balance may be associated with tissue resorption after praziquantel treatment. HYP and TIMP-2 levels were significantly correlated with periductal fibrosis status evaluated by ultrasonography. Plasma HYP level of CCA patients was the highest among HBD patients (p < 0.05). ROC curves revealed HYP, MMP-7 and collagen I levels significantly distinguished opisthorchiasis, BBD and CCA (p < 0.001). Odd ratio (OR) analysis demonstrated these markers in opisthorchiasis were predictable for BBD risk (p < 0.05; OR = 28.50, 10.12 and 4.63 for collagen I, MMP-7 and HYP, respectively), and the risk was reduced by praziquantel treatment. Interestingly, only plasma HYP level in BBD was predictable for CCA risk (OR = 3.69; p = 0.020). In conclusion, plasma HYP, collagen I and MMP-7 may be useful as novel predictive markers of opisthorchiasis-related BBD, and HYP may be used as a diagnostic marker for CCA.