Generation of high yields of Syrian hamster cholangiocellular carcinomas and hepatocellular nodules by combined nitrite and aminopyrine administration and Opisthorchis viverrini infection

Combined administration of 0.1% nitrite and 0.1% aminopyrine in the drinking water for eight to ten weeks resulted in subsequent development of both hepatocellular nodules and cholangiofibrotic lesions/cholangiocellular carcinomas in Syrian golden hamsters. Additional prior dosing with Opisthorchis viverrini metacercariae (100/animal) induced inflammatory and proliferative changes in the livers of infected hamsters and was associated with a significant increase in yields of hepatocellular and cholangiocellular preneoplastic and neoplastic lesions. Thus, environmental factors thought to be casually related to the high levels of human liver cancer observed in the Northeastern provinces of Thailand were sufficient to bring about development of equivalent tumors in experimental animals. The results indicate that parasite associated liver injury and non-specific compensatory regeneration may play an important role in generation of both hepatocellular and cholangiocellular carcinomas in man.     

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.     

Time-dependent Modulation of Liver Lesion Development in Opisthorchis-infected Syrian Hamster by an Antihelminthic Drug, Praziquantel

In the North-east of Thailand, repeated antihelminthic therapy has been introduced for control of the opisthorchiasis known to be a major risk factor for cholangiocellular carcinomas. What influence this may have on tumorigenesis, however, remains unclear. The effects of administration of praziquantel, an antihelminthic drug, at different time points subsequent to infection with Opisthorchis viverrini (OV) on 2,2'-dihydroxy-di- n -propylnitrosamine (DHPN)-initiated lesion development in the liver of female Syrian hamsters were therefore investigated. Praziquantel (250 mg/kg body weight, i.p.) was given 4, 12 or 20 weeks after infection of DHPN-treated animals (two 1000 mg/kg i.p. injections at weeks 0 and 2) with 60 OV metacercariae (at week 4). Survivors at week 38 were killed and examined. It was found that whereas praziquantel administration at the earlier two time points was effective at reducing hepatocellular nodule development, the results for cholangiocellular lesions were less pronounced, significant reduction only being evident in hamsters treated 4 weeks after parasite infestation. The findings thus indicate that enhancement of DHPN-initiated bile duct carcinogenesis by opisthorchiasis is both rapid and to a large degree irreversible. Hepatocellular lesion development in this model, on the other hand, appears to correlate more closely with the duration of parasite-associated proliferative stimulus.     

Carcinogenic effect of nitrosomorpholine administered in the drinking water to Syrian golden hamsters

The long term carcinogenic effect of nitrosomorpholine (NM) was tested in Syrian golden hamsters (Mesocricetus auratus W.). Groups of 30 females and 30 males were given 0.010%, 0.005% and 0.001% NM in their drinking water. The animals developed neoplasms in the larynx, and trachea (papillary polyps, papillomas and epidermoid carcinomas) and in the liver (hepatocellular adenomas and carcinomas). In addition to these, cholangiocellular and endothelial liver tumours were observed. The overall tumour frequency was dose dependent.     

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.     

Effects of phenobarbital and secondary bile acids on liver, gallbladder, and pancreas carcinogenesis initiated by N-nitrosobis (2-hydroxypropyl)amine in hamsters

The effects of dietary administration of phenobarbital [(PB) CAS: 50-06-6] or the secondary bile acids, deoxycholic acid [(DCA) CAS: 83-44-3] and lithocholic acid [(LCA) CAS: 434-13-9], on tumorigenesis in the liver, gallbladder, and pancreas were investigated in male Syrian golden hamsters after carcinogenic initiation by N-nitrosobis(2-hydroxypropyl)amine [(BHP) CAS: 53609-64-6]. BHP [500 mg/kg (body wt)] was injected sc once weekly for 5 weeks. The animals were then maintained on a basal diet or a diet containing either 0.05% PB, 0.1% DCA, 0.5% DCA, or 0.5% LCA for 30 weeks. DCA enhanced the development of cholangiocarcinomas without influencing that of hepatocellular lesions. PB promoted the induction of hepatocellular carcinomas but not that of cholangiocarcinomas. LCA was without effect on the induction of either hepatocellular carcinomas or cholangiocarcinomas. DCA at a dose of 0.5% enhanced the induction of polyps in the gallbladder. Both DCA, at a dose of 0.1%, and LCA significantly enhanced the induction of pancreas carcinomas. PB had no effect on the induction of polyps in the gallbladder or of pancreas carcinomas. These data document that different tumors may be differentially promoted following initiation with a common carcinogen.     

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.     

Cholangiocellular carcinomas induced in Syrian golden hamsters administered aflatoxin B1 in large doses

The hepatocarcinogenicity of aflatoxin B1 (AFB1) was compared in male Syrian golden hamsters and inbred F344 rats. AFB1 was administered by gavage 5 days/week for 6 weeks at doses of 1 and 2 mg/kg body weight/day to rats and hamsters, respectively; rate did not survive beyond 3 weeks with doses of 2 mg/kg/day. After 6 weeks the animals either received no further treatment or were given 0.1% phenobarbital sodium in the drinking water. ATPase-deficient foci of hepatic parenchymal cells, neoplastic nodules, and hepatocellular carcinomas were observed in liver sections from AFB1-treated rats killed at 6, 14, or 23 weeks; they were not seen in sections from AFB1-treated hamsters killed at 6, 14, or 46 weeks. Each of the 50 AFB1-treated rats developed hepatocellular carcinomas by 46 weeks; many also had cholangiocarcinomas and mixed hepatocellular-cholangiocellular carcinomas. Hepatocellular carcinomas were found in only 2 of 49 AFB1-treated hamsters by 78 weeks. At this time cholangiocarcinomas were found in 15 hamsters, and microscopic cholangiomas were seen in all of the livers. Compared to the rat, the hamster was resistant to the hepatotoxic and hepatocellular carcinogenic effects of AFB1.     

Pathogenesis of dimethylnitrosamine-induced hepatocellular cancer in hamster liver and lack of enhancement by phenobarbital

The early stages of dimethylnitrosamine (CAS: 62-75-9)-induced liver cancer and the effect of administration of phenobarbital (CAS: 50-06-6) after dimethylnitrosamine were studied in Syrian golden hamsters. A single ip injection of 6 mg dimethylnitrosamine/kg (body wt) induced hepatocellular altered foci by 6 months. Most foci were composed mainly of large clear glycogen-containing cells, while smaller numbers were formed by cells with abnormally acidophilic or basophilic cytoplasms. Biliary cysts, but not biliary neoplasms, also occurred. A few neoplastic (hyperplastic) nodules and hepatocellular carcinomas developed by 12 months. gamma-Glutamyltranspeptidase activity was not present in any of the hepatocellular lesions, but foci of all types, neoplastic nodules, and carcinomas were characterized by a lack of iron accumulation. Phenobarbital at 0.05% in the diet for up to 12 months did not increase the number of lesions of any type, indicating a lack of promoting effect under these conditions.     

Dose-dependent Induction of Liver and Thyroid Neoplastic Lesions by Short-term Administration of 2-Amino-3-methylimidazo[4,5-f]quinoline Combined with Partial Hepatectomy Followed by Phenobarbital or Low Dose 3'-Methyl-4-dimethylaminoazobenzene Promotion

Dietary administration of 0.1, 0.05, or 0.025% 2-amino-3-methylimidazo[4,5- f ]quinoline (IQ) for two weeks combined with partial hepatectomy at the end of the first week and followed by long-term treatment with phenobarbital (PB) or 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) from week 3 to week 86 resulted in dose-dependent development of liver and thyroid neoplastic and preneoplastic lesions. Quantitation of glutathione S-transferase placental form (GST-P)-positive hepatocellular focal populations revealed a significant correlation of IQ concentration with lesion area, with a yield approximately equal to that generated by a similar dose of 2-acetylaminofluorene. The fact that IQ was less toxic therefore allowed greater yields of hepatocellular carcinomas to be induced. The development of thyroid tumors initiated by the IQ treatment was significantly enhanced by the administration of PB, whereas Zymbal gland tumors induced by IQ did not show any correlation with either PB or 3'-Me-DAB treatment.     

Methylation of DNA guanine during the course of induction of liver cancer in hamsters by hydrazine or dimethylnitrosamine

Hydrazine is carcinogenic to the mouse and rat, but three earlierstudies have reported no carcinogenicity of hydrazine in thehamster. Administration of hydrazine to mice, rats and hamstersresults in rapid methylation of liver DNA guanine for whichendogenous formaldehyde appears to be the source of the methylmoiety. Hamsters were given hydrazine sulfate at 170, 340 and510 mg/I in the drinking water for 2 years [average dose of4.6, 8.3 and 10.3 mg hydrazine (free base)/kg body wt over the2-year period], during which levels of methylation of DNA guaninein liver, kidney and lung, and histopathologic examinationsof these tissues were carried out; dimethylnitrosamine, as apositive control, was administered at 10 mg/I in the drinkingwater (average dose of 1.1 mg/kg body wt over the 4-month measurementperiod). Both 7-methylguanine and 0⁶ were readily detec tableat 6 months exposure in hamsters given hydrazine or dimethylnitrosamine;in hydrazine-treated animals only trace amounts of these basescould be detected after 12 months ex-posure; these bases wereagain detected in liver DNA at ex-posure times of 18 and 24months. Hepatocellular carcinomas were observed in hamsterstreated at the highest dose of hydrazine sulfate after 78 weeksof exposure; the incidence of liver cancer was dose-relatedover the course of the ex-periinent: 32% for hamsters exposedto 510 mg hydrazine sulfate/I, 12% for 340 mg/I and none at170 mg/I. Hamsters given dimethylnitrosamine developed highlevels of 7-methyl-guanine and even higher levels of O⁶ andboth liver cholangiocellular carcinomas (73% incidence), asreported before, and hepatocellular carcinomas (27% in-cidence),a new finding. These results demonstrate for the first timethat hydrazine is a liver carcinogen in the hamster and providenew information regarding the accumulation of DNA damage duringthe entire induction period for the car cinomas.     

High Susceptibility to Hepatocellular Carcinoma Development in LEC Rats with Hereditary Hepatitis

A remarkably high incidence of hepatocellular carcinomas was observed in long-surviving LEC rats with hereditary hepatitis. Among the 60 LEC rats examined between 12 and 28 months of age from F₂₉, and F₃₀, 55 (92%) developed putative preneoplastic and neoplastic lesions such as hyperplastic foci and nodules, and hepatocellular carcinomas. Of these, hepatocellular carcinomas were observed with a high frequency (46/55; 84%). All rats of advanced age that survived more than 18 months developed hepatocellular carcinomas. These results suggest that the development of liver tumors in LEC rats is an age-associated phenomenon with serial hepatic alterations after the subsidence of acute hepatitis. The long-surviving rats had no normal tissue and showed chronic hepatitis in nontumorous tissues of the liver. Cholangiofibrosis was also found in most rats with hepatic lesions. Metastasis of hepatocellular carcinomas was found in four rats. Histologically, the hepatocellular carcinomas were of a well-differentiated type with a typical trabecular structure. Thus, LEC rats seem to be a promising animal model for studying the pathogenesis of hepatitis and hepatocellular carcinoma.     

Sex differences in the dietary modulation of pancreatic cancer in Syrian hamsters treated continuously with N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine

The effect of continuous week-long administration of N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) by s.c. implanted osmotic pumps was examined in male and female Syrian hamsters given access to three isocaloric synthetic diets containing 10 (LP), 20 (MP) and 30% (HP) casein respectively. At a total dose of 210 mg/kg, toxicity of HPOP, expressed as acute liver injury, was greater in male than in female hamsters. Such toxicity increased with protein intake in male, but not in female animals. Twenty-five weeks after initiation, female and male hamsters developed pancreatic ductal adenocarcinomas and to a lesser degree liver cholangiocellular and hepatocellular carcinomas. The highest incidence of pancreatic cancer was observed under HP diet regimens and was 75 and 67% in female and male hamsters respectively. Decrease of protein intake resulted in the reduction of the incidence of pancreatic cancer, which was more striking in males than in females. In males fed the MP and LP diets respectively, such an incidence declined to 33 and 6%. Although significant differences in the incidence of pancreatic cancer were not observed among female groups fed the above three diets, the multiplicity and the number of such tumors were significantly greater at the HP than the LP level. Differences in the incidence of pancreatic tumors between males and females were statistically significant only at the LP level. However, such differences were also significant at the MP level when comparisons were based on tumor number and multiplicity. Since the incidence, tumor multiplicity and size were generally greater in female than male hamsters, estrogenic hormones may play a role in the development of pancreatic ductal adenocarcinomas in this species.     

Is heterozygous alpha-1-antitrypsin deficiency type PIZ a risk factor for primary liver carcinoma?

BACKGROUND: It is well known that homozygotes with alpha-1-antitrypsin deficiency type PiZ are associated with an increased risk of chronic liver disease and liver carcinoma. The aim of this study was to determine whether heterozygous PiZ status is a risk factor for liver carcinoma development. METHODS: Three hundred seventeen consecutive primary liver carcinomas and the tumor-bearing liver tissue (tumor series) from adult patients were screened immunohistochemically for hepatocellular PiZ deposits. Liver specimens from 1663 consecutive adult patients (biopsy series) and liver tissue from 1030 consecutive adult autopsies (autopsy series) served as controls. The zygosity status of alpha-1-antitrypsin was verified by analysis of single strand conformational polymorphism and by sequencing DNA extracted from paraffin embedded tissue. RESULTS: The PiZ frequency in the tumor series (5.99%) was significantly higher than in the biopsy series (3.43%) or the autopsy series (1.84%). Cholangiocarcinomas and/or combined hepatocholangiocarcinomas were seen significantly more frequently in PiZ-associated liver carcinomas (57.9%) than in non-PiZ-associated carcinomas (27.2%). Cirrhosis was found in only 3 of the 19 PiZ-associated carcinomas. The remaining 16 livers showed varying stages of fibrosis or normal tissue. All nine cases with PiZ-associated liver carcinoma suitable for genetic analysis showed heterozygous PiZ mutations. CONCLUSIONS: Heterozygotes of type PiZ are associated with an increased risk of primary liver carcinoma. PiZ-associated carcinoma may develop in noncirrhotic liver tissue and without concurrent liver disease, and is frequently characterized by cholangiocellular differentiation. The site specific antibody ATZ11 is a reliable morphologic tool for detecting PiZ individuals.     

Incidence of primary cholangiocellular carcinoma of the liver in japanese patients with hepatitis C virus-related cirrhosis

BACKGROUND: Hepatitis C virus (HCV) infection is a major risk factor for the development of hepatocellular carcinoma. However, the risk factors for primary cholangiocellular carcinoma of the liver (PCC-L) have not been fully investigated. The authors determined the incidence of PCC-L in patients with HCV-related cirrhosis. METHODS: Between 1980 and 1997, the authors prospectively studied 600 consecutive patients for the appearance of PCC-L; these patients were positive for HCV and later developed cirrhosis. The follow-up period ranged from 0 to 18.5 years (median, 7.2 years). RESULTS: During the observation period, PCC-L developed in 14 patients (2.3%). Among these, 11 (1.8%) had cholangiocellular carcinomas and the other 3 (0.5%) had a combined type of hepatocellular and cholangiocellular carcinoma. Within the same period, hepatocellular carcinoma (HCC) developed in 206 patients (34.3%). The cumulative rates of newly diagnosed PCC-L were 1.6% at 5 years and 3.5% at 10 years, which was about 1000 times higher than the estimated incidence of PCC-L in the general population of Japan. PCC-L was treated by surgical resection in 3 patients who survived for > 3 years. However, the other 11 patients received palliative therapy or chemotherapy. The survival rates among PCC-L patients were 39.3%, 23. 6%, and 16.5% at the end of 1, 3, and 5 years, respectively, and were significantly lower than those of HCC (P = 0.0001). CONCLUSIONS: The results of this study show a relatively high incidence of PCC-L in patients with HCV-related cirrhosis, and also show that this type of liver cancer is associated with a relatively poor prognosis. These results indicate that HCV-related cirrhosis is a major risk factor for PCC-L in Japanese patients.     

High susceptibility to hepatocellular carcinoma development in LEC rats with hereditary hepatitis

A remarkably high incidence of hepatocellular carcinomas was observed in long-surviving LEC rats with hereditary hepatitis. Among the 60 LEC rats examined between 12 and 28 months of age from F29 and F30, 55 (92%) developed putative preneoplastic and neoplastic lesions such as hyperplastic foci and nodules, and hepatocellular carcinomas. Of these, hepatocellular carcinomas were observed with a high frequency (46/55; 84%). All rats of advanced age that survived more than 18 months developed hepatocellular carcinomas. These results suggest that the development of liver tumors in LEC rats is an age-associated phenomenon with serial hepatic alterations after the subsidence of acute hepatitis. The long-surviving rats had no normal tissue and showed chronic hepatitis in nontumorous tissues of the liver. Cholangiofibrosis was also found in most rats with hepatic lesions. Metastasis of hepatocellular carcinomas was found in four rats. Histologically, the hepatocellular carcinomas were of a well-differentiated type with a typical trabecular structure. Thus, LEC rats seem to be a promising animal model for studying the pathogenesis of hepatitis and hepatocellular carcinoma.     

Suppression of N-nitrosobis(2-oxopropyl)amine-induced pancreatic carcinogenesis in hamsters by pioglitazone, a ligand of peroxisome proliferator-activated receptor gamma

Fat intake and obesity are positively correlated with pancreatic cancer in humans. N-nitrosobis(2-oxopropyl)amine (BOP) induces pancreatic ductal adenocarcinomas limited to Syrian golden hamsters, other rodents not being susceptible. In the present study, we found markedly high levels of serum triglycerides (TGs) and total cholesterol (TC) in Syrian golden hamsters, but not C57BL/6 mice, ICR mice, F344 rats and Wistar rats. Consistent with this, lipoprotein lipase (LPL) activities in the liver were lower in hamsters compared with mice and rats. To examine effects of pioglitazone, a peroxisome proliferator-activated receptor gamma (PPARgamma) ligand, on LPL expression, serum lipid levels and pancreatic cancer development, 6-week-old female Syrian golden hamsters were subcutaneously injected with BOP (10 mg/kg body wt) four times in a week and thereafter fed a diet containing 800 p.p.m. pioglitazone for 22 weeks. The treatment elevated LPL mRNA expression in the liver and significantly improved hyperlipidemia with serum levels of TG and TC being decreased to 62 and 71%, respectively, of the control values. Concurrently, the incidence and multiplicity of pancreatic ductal adenocarcinomas were significantly decreased by pioglitazone in comparison with the controls (38 versus 80%, P < 0.01 and 0.55 +/- 0.15 versus 1.37 +/- 0.22, P < 0.01, respectively). The suppression rates were greater in invasive adenocarcinomas than non-invasive ones. The incidence of cholangiocellular carcinomas was also reduced. Thus, suppression of pancreatic adenocarcinoma development by pioglitazone is possibly associated with improvement in the serum lipid profile, and hyperlipidemia could be an enhancing factor for development of pancreatic cancer in hamsters.     

Carcinogenic effects of diethylstilbestrol in male Syrian golden hamsters and European hamsters.

The tumorigenic effects of sc-implanted diethylstilbestrol (DES) on male Syrian golden hamsters and European hamsters were compared. The adenohypophyses, kidneys, and testes of both species showed neoplastic responses to DES treatment. European hamsters were more sensitive than were Syrian hamsters, inasmuch as the European hamsters had a higher tumor incidence. The testicular tumors were all Leydig cell adenomas and seemed to depend on the coincident occurrence of hypophyseal neoplasms (all composed of gonadotropic cells). Of the European hamsters tested, 29% also developed liver tumors (hepatocellular adenomas, carcinomas, and cholangiocarcinomas.     

Hepatic neoplasms in the mummichog Fundulus heteroclitus from a creosote-contaminated site

High prevalences of idiopathic hepatic lesions were found in mummichog, Fundulus heteroclitus, from a site in the southern branch of the Elizabeth River, VA, contaminated with polycyclic aromatic hydrocarbons. Grossly visible hepatic lesions occurred in a total of 93% of the individuals from this site and 33% of these fish had hepatocellular carcinomas. Hepatic lesions were not detected in fish from two less contaminated sites. Lesions included foci of cellular alteration, hepatocellular adenoma, early and advanced hepatocellular carcinomas, and cholangiocellular proliferative lesions. Advanced carcinomas exhibited several distinct cellular patterns and some livers contained multiple neoplasms occupying up to 80% of the hepatic parenchyma. Sediments from the contaminated site contained extremely high concentrations (2200 mg/kg dry sediment) of polycyclic aromatic hydrocarbons, which are believed to originate from an adjacent wood treatment facility that has used creosote. Concentrations were 730- and 35-fold higher than those at the two other sites. These findings indicate a strong positive association between exposure to creosote-contaminated sediments and the high prevalence of hepatic neoplasms in a feral population of mummichog and support the putative role of polycyclic aromatic hydrocarbons in fish hepatocarcinogenesis. Additionally, they suggest that the mummichog may be a useful indicator of exposure to carcinogens in aquatic environments.