Comparison of lesions induced in the Syrian golden hamster by diethylnitrosamine, dimethylhydrazine, and dibutylnitrosamine: influence of subsequent butylated hydroxyanisole treatment

The target organ specificity of the carcinogens diethylnitrosamine [(DENA) CAS: 55-18-5], dimethylhydrazine [(DMH) CAS: 57-14-7], and dibutylnitrosamine [(DBN) CAS: 924-16-3] was examined in Syrian golden hamsters. Groups of male animals were given 8 weekly injections of one of these carcinogens and then were maintained on a basal diet or a diet supplemented with 1% butylated hydroxyanisole [(BHA) CAS: 25013-16-5], or they were given the respective carcinogens in the drinking water until they were sacrificed at week 34. While DENA specifically induced tracheal polyps and hepatocellular foci and nodules, DMH administration was associated with development of both hepatocellular and hemangiocellular liver lesions as well as forestomach papillomas and adenocarcinomas of the large intestine. DBN induced lesions in the urinary bladder, forestomach, and trachea, in addition to a few preneoplastic foci in the liver and lungs. In all organs studied, preneoplastic and neoplastic populations were essentially similar to those observed in other experimental animals, with colon and tracheal lesions demonstrating alteration in polysaccharide metabolism. While inhibiting the development of hepatocellular lesions, especially in the group initiated with DENA, and while itself inducing extensive papillomatous forestomach hyperplasia, BHA administration did not exert a significant modifying influence on tumorigenesis in other organs. The present results demonstrate the efficacy of Syrian golden hamster studies for investigation of comparative neoplasia. Of particular interest in this respect were differences in the degree of phenotypic instability demonstrated by glutathione S-transferase placental form-positive foci induced by the 3 carcinogens, which indicated a possible qualitative variation in "initiation."     

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.     

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.     

Detection of antibodies in sera from patients with Opisthorchiasis

The indirect immunofluorescent antibody technique (IFA) was used for detection of antibodies in sera of patients with Opisthorchiasis. Antibodies to fluke worm and egg antigens were detected in 166 of 205 (81%) patients. The test showed that only the IgG class of antibodies reacting exclusively with integumental wall of the worm (AW) were positive in 46.8% (96/205), reacting only with the wall of intact eggs in 11 out of 205 (5.4%) and antibodies to both fluke and their egg antigens were present in 28.8% (59/205). In addition, 5.4% (11/205) of patients' sera were positive for autoantibodies producing a speckled antinuclear antibodies (ANA) pattern. The sera positive for only AW contained detectable autoantibodies to other cell antigens including: anti-smooth muscle antibodies of 9.4% (9/96), antimitochondrial antibodies of 3.1% (3/96), anti-liver/kidney microsomes of 1% (1/96) and anti-parietal cell antibodies of 1% (1/96). Autoantibodies were undetectable in sera from normal subjects. Among the ANA positive sera, 55% (6/11) exhibited antibodies against an extractable nuclear antigen (ENA) by a tanned red cell hemagglutination assay. This finding may suggest that the autoantibody response was due to the cross reaction between worm antigen and self antigen or it may be the result of polyclonal activation of B lymphocytes in these patients.     

Immunohistochemically demonstrated suppressed expression of tryptophan oxygenase, a marker for liver differentiation, within putative preneoplastic rat liver lesions

The behaviour of rat liver putative preneoplastic lesions withrespect to the enzyme tryptophan oxygenase (TO), a liver-specificdifferentiation marker, and a possible growth-related marker,glucose-6-phosphate dehydrogenase (G6PD) was investigated duringand after their induction by diethylnitros-amine initiationand subsequent ‘selection pressure’. Using specificantibodies to rat liver TO and G6PD and the avidin-biotin complexmethod for immunohistochemical staining it was demonstratedthat all of the nodular lesions showing increased expressionof G6PD during the induction phase were also negative or deficientin TO enzyme protein. With the onset of ‘phenotypic instability’or loss of marker enzymes, a gradual return to normal expressionof TO activity was evident. Administration of dexamethasoneand L-tryptophan 11 weeks after cessation of carcinogen treatmentallowed differentiation between morphologically altered, apparentlypersisting lesions in which no, or little, enzyme inductionwas apparent and instable lesions showing a strong increasein levels of TO protein. Thus, persisting nodular lesions sharea common lack of response to normal homeostatic physiologicalcontrol.     

Identification and potential use of a soluble tumor antigen for the detection of liver-fluke-associated cholangiocarcinoma induced in a hamster model

A liver-fluke-associated cholangiocarcinoma (CCA), comparable to that occurring in humans, was induced by exposing Opisthorchis viverrini-infected hamsters to dimethylnitrosamine (DMN). Tumor masses were removed and histopathologically identified, then one portion was extracted for antigens used in the production of monoclonal antibodies (MAbs). The remaining portions were used to establish CCA cell lines. The antigens produced and secreted by these cell lines, as well as those originally present in the tissue extracts, possessed a 200-kDa glycoprotein that appeared to be immunologically distinct from other tumor markers. A specific MAb called 6E5 was used to set up a sandwich ELISA for the quantification of this antigen in the serum and bile of tumor-bearing animals. The assay system was sensitive enough to detect the antigen at concentrations below 10 ng/ml. The serum and biliary levels of this antigen were markedly elevated in animals with progressive tumors when compared with untreated controls. The serum taken serially from each animal that subsequently developed CCA showed a gradual but significant elevation of the antigen as carcinogenesis progressed. A few isolated animals exhibited a slight elevation of the antigen at a time as early as the end of DMN treatment, when the CCA should not yet have developed, judging from microscopic examination. The data from this animal model suggested that the CCA-associated soluble antigen defined by MAb 6E5 was a useful marker for the detection of tumors at an early stage of development.     

Influence of dehydroepiandrosterone, diaminopropane and butylated hydroxyanisole treatment during the induction phase of rat liver nodular lesions in a short-term system

The effects of concomitant treatment with dehydroepiandro-sterone,an inhibitor of glucose-6-phosphate dehydrogenase (G6PD), diaminopropane(DAP), an inhibitor of onuithine decarboxylase or the microsomaldrug detoxifying enzyme in-ducer butylated hydroxyanisole (BHA)during the induction phase of rat liver nodular lesion developmentwere investigated. Clear reductions in both number and sizeof foci and nodules as assayed quantitatively with the aid ofmarker enzymes G6PD, glutathione S-transferase P form or gamma-glutamyltranspeptidase were established for treatment with either DHEAor BHA. DAP in contrast did not exert influence on the numberof lesions, but brought about a significant reduction in size.The quantitative data taken together with the finding that increasedlabelling of tritiated thymidine occurred in extrafocal hepatocytepopulations in BHA-treated animals, give direct support to theview that alteration in enzyme phenotype within putative pre-neoplasticlesions plays a central role in their generation with this short-termmodel. In particular, a physiological adaptive significanceof G6PD elevation is suggested.     

Modification by sodium L-ascorbate, butylated hydroxytoluene, phenobarbital and pepleomycin of lesion development in a wide-spectrum initiation rat model

Rats were treated for 1 week each with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), 0.2% N-bis(2-hydroxypropyl)-nitrosamine (DHPN) and 0.2% N-ethyl-N-hydroxyethylnitrosamine (EHEN) in the drinking water, and then administered diet containing 5% sodium L-ascorbate (Na-AsA), 1% butylated hydroxytoluene (BHT) or 0.05% phenobarbital (PB), or weekly intraperitoneal injections of 2 mg of pepleomycin per kg body weight until week 36. Histopathological examination revealed that all exerted significant modulation effects on tumor development in the various target organs. Na-AsA was found to inhibit liver but promote renal pelvis and bladder carcinogenesis. BHT similarly decreased liver and enhanced bladder lesion development. PB, in contrast promoted hepatocarcinogenesis. However both PB and BHT were associated with increased incidences of adenomas and adenocarcinomas of the thyroid. Thus the wide-spectrum initiation model allowed confirmation of site-specific modification potential and in addition demonstrated potentiation of kidney and bladder carcinogenesis promotion by pepleomycin.     

Promoting effect of 2,4-diaminoanisole sulfate on rat thyroid carcinogenesis

Two experiments were conducted to examine the effects of 2,4-diaminoanisole sulfate (2,4-DAAS) on thyroid function and carcinogenesis in male Wistar rats. In experiment 1, feeding with 2,4-DAAS resulted in significantly elevated levels of thyroid stimulating hormone (TSH), reduced thyroxine (T4) and triiodothyronine (T3) in the serum, although the latter had almost recovered to normal levels by week 6. However, skin application did not affect serum levels. In experiment 2, administration of 0.5% 2,4-DAAS in the diet for 19 weeks, a week after a single 210 mg/100 g body weight i.p. injection of N-bis(2-hydroxypropyl)nitrosamine (DHPN), significantly increased the incidence and numbers of preneoplastic lesions (focal hyperplasia), adenomas and carcinomas developing in the thyroid gland. Histologically, brown pigment was usually observed within follicular epithelial cells in non-tumorous regions, but not in the tumors themselves.