Antigenic phenotypes common to rat oval cells, primary hepatocellular carcinomas and developing bile ducts

The shared expression of monoclonal antibody-defined antigens by oval cells and by bile ducts, neoplastic nodules and primary hepatocellular carcinomas (PHC) has provided support for the ability of oval cells to undergo differentiation along ductular or hepatocyte lineages and/or to progress to hepatocellular carcinoma. With the aim of obtaining additional insight into this process, we have combined serial section and double labeling immunofluorescence analysis to determine if phenotypes expressed in vitro by four rat oval cell lines and the H5D.61 hepatocellular carcinoma cell line and in situ by ethionine- induced primary hepatocellular carcinomas reproduce antigenic patterns occurring during normal liver development. Analysis using monoclonal antibodies specific for the oval cell antigens OV6 and OC.2 and hepatocyte markers HBD.1 and H.4 defined subpopulations in four oval cell lines and neoplastic hepatocytes in PHC and H5D.61 with OC.2-/OV6+ and OC.2+/OV6+ phenotypes. Cells with an OC2+/OV6- phenotype were rarely observed in cell lines or primary tumors. In contrast, areas composed of OV6+/H.4+ cells were frequently found in PHC. Examination of fetal and neonatal rat livers demonstrated the stage-specific appearance of three of these phenotypes during liver development. The OC.2+/OV6- phenotype appeared transiently prior to embryonic day (ED) 18 in a subpopulation of HBD.1+ hepatoblasts. OV6 expression was first detected at ED18 on developing bile ducts that were negative for OC.2. These newly formed ducts rapidly acquired OC.2, starting with ducts in the hilar region and spreading outward towards the periphery. This OC.2 expression gradient persisted in the newborn rat liver but became more skewed towards doubly positive cells, with OC.2-/OV6+ cells being found primarily in the periphery. Hepatocytes expressing both OV6 and H.4 were not observed in fetal liver but appeared in neonatal liver in close proximity to OV6+ interlobular ducts. From these findings, it was concluded that oval cells and PHC display phenotypes representing normal stages in liver development, suggesting that oval cells and cells within ethionine-induced PHC are capable of initiating but are unable to complete pathways of hepatocytic or biliary differentiation.      

Different lineages of chemically induced hepatocellular carcinoma in rats defined by monoclonal antibodies

Different lineages of hepatocellular carcinoma (HCC) were identified by the application of selected monoclonal antibodies to the study of the sequential histopathological changes which occurred during two regimens of chemical carcinogenesis in the rat. One regimen, that of Solt-Farber, caused prominent oval cell proliferation and large multiple neoplastic nodules, and the other regimen, continuous administration of diethylnitrosamine, produced minimal oval cell proliferation and a few small nodules. However, both regimens produce HCC in most exposed rats. Three monoclonal antibodies to liver cells, OV-6, H-4, and T-6, were selected on the basis of different tissue staining. OV-6 stains the cytoskeleton of bile duct cells, oval cells, and HCC but not that of hepatocytes. H-4 stains the cytoplasm of hepatocytes but of not hepatomas. T-6 stains the cytoskeleton of HCC only. In the Solt-Farber model, the monoclonal antibodies identified groups of hepatocytes within the persistent neoplastic nodules which had acquired the OV-6 epitope and had lost the H-4 epitope. HCC derived from this regimen had the same staining pattern, suggesting that the OV-6 positive H-4 negative hepatocytes were the precursors of the HCC. The presence within the nodules of oval cells, atypical duct structures, cells intermediate between duct cells and hepatocytes, and nodular hepatocytes all containing the OV-6 epitope raises the possibility that any of these cell types could serve as the precursor of the OV-6 positive hepatocytes that arose within the nodule. In the continuous diethylnitrosamine regimen a different staining pattern was seen. T-6 positive hepatocytes first appeared in periportal areas by the 5th week. These cells increased in numbers during the later weeks and with rare exceptions neither acquired the OV-6 epitope nor completely lost the H-4 epitope. Most HCC derived by the continuous diethylnitrosamine regimen were T-6 positive and OV-6 negative, suggesting a direct lineage from the periportal T-6 positive hepatocytes. These findings indicate that the lineage and phenotype of chemically induced HCC may vary with the carcinogenic regimen used and that HCC which arise in nodules may originate from cell types other than typical nodular cells.     

Presence of alpha-fetoprotein-positive cells in hepatocellular foci and microcarcinomas induced by single injections of diethylnitrosamine in infant mice

Single injections of diethylnitrosamine (5 and 50 micrograms/g body weight) in male C57BL/6J X C3HeB/FeJ F1 mice when they were 15 days old resulted in the induction of RNA-rich hepatocellular foci and nodules that contained alpha-fetoprotein (AFP)-positive hepatocytes after 20 and 28 weeks. The focal lesions were composed of 1- to 2-cell-thick plates of hepatocytes or closely packed clusters of cells, but they did not show the histological patterns that are diagnostic of trabecular hepatocellular carcinoma. AFP-positive hepatocytes were found in almost one-fourth (14 of 60) of the foci and nodules in a serially sectioned block of liver from a mouse given one injection of 50 micrograms/g body weight diethylnitrosamine and killed at 28 weeks. In general, the presence or absence of AFP-positive cells correlated with the size of the foci and nodules. All six nodules with diameters greater than 1.5 mm contained AFP-positive cells, while all 12 foci smaller than 0.24 mm in diameter were negative for AFP. However, among the 42 foci that were intermediate in size, there were 8 AFP-positive foci, the sizes of which appeared rather randomly distributed among the negative foci. Reactive changes in hepatocytes could be ruled out as a cause of the induction of AFP because the foci first appeared many weeks after the administration of diethylnitrosamine in these mice. Since bile ductules or oval cells, which occasionally appeared in these foci, were lacking entirely in AFP and since ductules are absent from the early-appearing and smallest foci, we believe that in this model the AFP-positive foci arise only from hepatocytes. The presence of AFP in the focal lesions and in tumor thrombi that extended from them into hepatic vein branches supports the hypothesis that some foci undergo progression to invasive microcarcinomas and that these in turn are precursors of late-appearing (after 1 year) metastasizing trabecular hepatocellular carcinomas.     

卵圆细胞在实验性肝癌发生过程中的演变特征

背景与目的:卵圆细胞在肝癌发生过程中的作用至今还不十分明了.本研究拟通过动态的方法观察卵圆细胞在肝癌发生过程中的演变规律,揭示卵圆细胞与肝癌发生之间的关系.方法:构建实验性肝癌的大鼠诱癌模型,运用常规HE染色、免疫组织化学和爱新蓝特殊染色等方法,动态观察卵圆细胞在肝癌发生过程中的演变规律.结果:HE和免疫组化结果显示,在诱癌的第4周即可见散在的卵圆细胞在门管区附近出现.卵圆细胞OV-6免疫染色呈阳性.在诱癌的第8周和第14周,OV-6阳性细胞逐渐增多,并向肝小叶实质内深入,将肝组织分割成假小叶状.到诱癌的第17周和第24周,多个癌灶出现,同时OV-6阳性细胞的总体数量下降,癌灶内可观察到OV-6阳性细胞.爱新蓝特殊染色显示,诱发的肿瘤属混合性肝癌,其中胆管上皮细胞癌爱新蓝染色呈阳性,肝细胞癌染色呈阴性.结论:卵圆细胞在肝癌的发生过程中可能扮演重要的角色.     

Expression of hepatocyte and oval cell antigens in hepatocellular carcinomas produced by oncogene-transfected liver epithelial cells

We have established an in vivo/in vitro system in which epithelial cells ("oval cells") isolated from livers of rats fed a carcinogenic diet for a very brief period are placed in culture and transfected with an oncogene. Injection s.c. into nude mice of oval cells transfected with the activated c-Ha-ras (EJ oncogene) produces tumors with morphological features of differentiated hepatocellular carcinomas. Using monoclonal antibodies that can recognize hepatocyte, oval cell, and tumor antigens, we investigated the expression of these antigens in oval cells in culture, transfected with either the EJ oncogene or the normal c-Ha-ras allele and in tumors derived from the oncogene-transfected cells. We show that EJ-transfected cells and most particularly the tumors they produce expressed hepatocyte and oval cell antigens not detectable in untransfected cells or cells transfected with the normal c-Ha-ras gene. Furthermore, we found that in cloned tumor cells, the expression of hepatocyte antigens could be induced by changes in culture conditions and was accompanied by a decrease in the expression of oval cell markers. Trabecular hepatocellular carcinomas had higher reactivity toward monoclonal antibodies recognizing hepatocyte antigens while tumors with glandular architecture reacted predominantly with monoclonal antibodies against oval cells. We conclude that, in addition to its tumorigenic effect, the EJ oncogene induced the differentiation of tumor cells toward the hepatocyte lineage. In addition, the data provide further confirmation that oval cells can serve as progenitors of differentiated hepatocellular carcinomas.     

The resistant hepatocyte model of carcinogenesis in the rat: the apparent independent development of oval cell proliferation and early nodules

The early cellular changes in the Solt-Farber resistant hepatocytemodel of carcinogenesis have been studied to clarify the relationshipof oval cell proliferation to the development of early hepatocytenodules. Cellular proliferation, intermediate filament profilesand the expression of specific cytochrome P450 enzymes wereexamined. At 24 h after partial hepatectomy (PH) many of thebile ductular cells were in S phase, but over the next few daysDNA synthesis progressively decreased in the portal bile ductsand was more common in arborizing ductules (oval cells) radiatingfrom the portal areas. These cells strongly expressed cytokeratins8 and 19 and vimentin, and from 1 week after PH they frequentlyunderwent differentiation either into hepatocytes, expressingcytochrome P450 enzymes, or into intestinal-type cells. Fivedays after PH, numerous basophilic foci were discernible, andthese expanded rapidly. The ductular cells swirled around thefoci, but their antigenic profile clearly indicated that thesecells were not involved in the development of these early nodules.In normal hepatocytes, cytokeratin 8 immunoreactivity was distinctlymembranous in location, and could only be readily detected inperiportal hepatocytes. In the basophilic hepatocyte foci, overexpressionof cytokeratin 8 was consistently associated with cells organizinginto acini, with expression reminiscent of authentic bile ducts,possibly indicating a structure-function relationship. In conclusion,early foci and nodules in this model are derived from resistanthepatocytes and not ductular oval cells, the latter being afacultative multipotential stem cell compartment.     

Induced drug resistance inhibits selection of initiated cells and cancer development

Compounds exerting a mitoinhibitory effect on normal hepatocytes are potent promoters in the resistant hepatocyte model of chemical carcinogenesis in combination with stimulation of regenerative growth by partial hepatectomy or treatment with carbon tetrachloride. 2- Acetylaminofluorene (2-AAF) almost completely inhibits liver cell regeneration after partial hepatectomy, allowing only resistant cells to participate in regenerative growth. After initiation by diethylnitrosamine and promotion with 2-AAF and partial hepatectomy (PH), focal growth of initiated cells generates liver lesions which occupy 40% of the hepatic volume three weeks after PH. In this work the mechanism for the anti promoting effects of phenobarbital and 3- methylcholantrene were investigated as well as their effects on the development of malignant hepatocellular carcinoma in the resistant hepatocyte model. Treatment with phenobarbital or, especially, 3- methylcholanthrene rendered normal rat hepatocytes resistant to the mitoinhibitory effect of 2-AAF. In combination with 2-AAF/PH, 3- methylcholanthrene shortened the regenerative growth period to less than one week. In the Solt-Farber protocol for experimental hepatocarcinogenesis, treatment with phenobarbital or 3- methylcholanthrene during promotion with 2-AAF/PH permitted hepatocytes surrounding the focal lesions to respond with regenerative growth. The foci and surrounding liver grew until the liver/body mass index reached the control value. With phenobarbital treatment the total focal volume was 20% of the liver volume three weeks after PH, whereas the corresponding value in the case of 3-methylcholanthrene was only 1%. Labelling index data supported the conclusion that growth of the liver lesions in the resistant hepatocyte model was dependent on differential inhibition of normal hepatocyte growth by the promoter and that the size of the foci obtained was related to the length of time after PH required to complete liver regeneration. 3-methylcholanthrene induced 2- AAF resistance prevented the development of large persistent nodules and hepatocellular carcinoma while phenobarbital delayed cancer development with several month. The data thus supports the idea that the degree of clonal expansion during promotion determines the size of the population at risk for malignant transformation, as well as the final frequency of carcinomas.      

Fumonisin B1-induced hepatocellular and cholangiocellular tumors in male Fischer 344 rats: potentiating effects of 2-acetylaminofluorene on oval cell proliferation and neoplastic development in a discontinued feeding study

Fumonisin B1 (FB1) is a naturally occurring mycotoxin produced by Fusarium verticillioides. Dietary exposure to FB1 has been linked to human cancer in certain parts of the world, and treatment with FB1 causes oval cell proliferation and liver tumors in rats. To study the potential role of oval (liver progenitor) cells in the cellular pathogenesis of FB1-induced liver tumors, we gave male F344 rats prolonged treatment with FB1 for 25 weeks, followed by return to control diet until 50 weeks ('stop study'). The time course of FB1-induced liver lesions was followed by examination of serial liver biopsies at set time intervals and post-mortem liver tissue at the end of the study. The effects of different FB1 treatment regimens (5 versus 25 weeks), as well as the modulating effect of 2-acetylaminofluorene (2-AAF), on the kinetics of oval cell proliferation and development of liver tumors were compared. Prolonged treatment with FB1 in normal diet caused persistent oval cell proliferation and generation of both hepatic adenomas and cholangiofibromas (CFs). These liver lesions occurred in the setting of chronic toxic hepatitis and liver fibrosis/cirrhosis, similar to that seen in human hepatocarcinogenesis. Some adenomas and CFs were dysplastic, and one post-mortem liver contained a hepatocellular carcinoma. OV-6+ oval cells were noted in close relation to proliferative neoplastic liver lesions, and some of these lesions expressed OV-6, suggesting that all these cell types were derived from a common progenitor cell. 2-AAF enhanced the size of FB1-induced glutathione S-transferase pi+ hepatocellular lesions and the incidence of CFs in post-mortem liver specimens, but this was not statistically significant. In conclusion, this study supports the involvement of dietary FB1 in liver carcinogenesis in male F344 rats. Oval cells may be the source of both the hepatocellular and cholangiocellular tumors induced by prolonged treatment with FB1. 2-AAF appears to have an enhancing effect on FB1-induced liver tumors, presumably due to its potent inhibitory effects on hepatocyte regeneration.     

In situ hybridization studies on expression of albumin and alpha-fetoprotein during the early stage of neoplastic transformation in rat liver

The expressions of albumin and alpha-fetoprotein (AFP) genes were studied in early preneoplastic liver lesions produced by the Solt-Farber protocol using "in situ" hybridization with single stranded RNA probes. In normal rat liver, albumin was expressed at a lower level in the centrilobular than in the periportal areas of the liver acinus, whereas the bile duct epithelium did not show any expression. Five weeks after initiation with diethylnitrosamine, islands of hepatocytes were present which showed heterogeneous expression of albumin and were surrounded by cells comprised of albumin negative hepatocytes and oval cells. gamma-Glutamyltranspeptidase positive foci of enzyme altered cells were located in albumin positive areas. Albumin expression gradually decreased in permanent nodules but increased in the hepatocytes outside the nodules during the first five months after initiation with diethylnitrosamine. Remodeling nodules, which were partly gamma-glutamyltranspeptidase and albumin positive, were also present. However, no consistent correlation was found between gamma-glutamyltranspeptidase positive and albumin negative areas during the first 5 months after initiation. Occasionally, cells showing an elevated expression of albumin were found in permanent nodules. These cells were located in the vicinity of oval type cells, which also showed a weak expression of albumin. AFP was expressed at high level in oval cells 5 weeks after the initiation. However, oval cells observed at later time points, either around the neoplastic nodules or inside the nodules showed only low expression of AFP. Hepatocytes in the enzyme-altered foci and in neoplastic nodules were always negative for AFP. The presence of strongly albumin positive cells inside the neoplastic nodules in close proximity to oval type cells suggests that these cells may be derived from primitive "stem-cell"-like oval cells.     

Rat ferritin-H: cDNA cloning, differential expression and localization during hepatocarcinogenesis

Elevated serum ferritin levels, especially of the H subunit, accompany many clinical malignancies. By using the subtraction-enhanced display technique, we have recently isolated several cDNA clones which are over- expressed in rat hepatocellular carcinoma induced by diethylnitrosamine. One 830-base-pair clone was 88% similar to human ferritin-H cDNA and encoded a 182 amino acid protein which is 97% homologous to human ferritin-H chain. Hepatic mRNA levels of ferritin-H were increased markedly at the early stage of diethylnitrosamine- induced hepatocarcinogenesis in the rat (6 weeks) and appeared more than 10-fold overexpressed as the tumour progressed. In contrast, hepatic ferritin-H mRNA remained constant during liver regeneration after a 70% partial hepatectomy. In situ hybridization showed that over- expression of ferritin-H was exclusively localized to preneoplastic foci, to tumour nodules and to tumour cells invading blood vessels. These findings suggest that ferritin-H is a highly conserved protein, its over-expression during tumour development is phenotypically correlated with tumour initiation and/or progression, and it is useful as an early marker for hepatocellular carcinoma.      

Expression of the c-myc, c-fos and c-rasHa protooncogenes during sex-differentiated rat liver carcinogenesis in the resistant hepatocyte model

The expression of the protooncogenes c-myc, c-fos and c-rasHa has been studied in rats treated according to the resistant hepatocyte model. Protooncogene expression was studied in male and female rat liver during the selection phase, when the outgrowth of putative preneoplastic foci/nodules is markedly faster in males, and compared with the expression in advanced nodules and hepatocellular carcinomas in males. During the first 16 h after partial hepatectomy the expression of c-fos and c-myc showed transient, 2- to 3-fold, increases in both sexes, both in initiated and in 'control' animals, receiving the selection/promotion regiment but no diethylnitrosamine, with a maximum at 0.5 and 2-4 h respectively. c-rasHa exhibited a moderate increase (1.5-fold) at 16-24 h in all groups. A second increase in c-myc expression (2-fold) started 24 h after partial hepatectomy and lasted over the entire selection period in initiated males, while it was unchanged in females and uninitiated males. The c-fos expression also showed a short-lived increase 24 h post partial hepatectomy in initiated males. The expression of c-myc and c-fos was increased 2- to 4-fold in both preneoplastic nodules and hepatocellular carcinomas, whereas c-rasHa expression was unchanged. In conclusion, sex differences were observed in the expression of c-myc and c-fos during the early outgrowth of preneoplastic lesions, possibly reflecting a connection between the expression of these genes and the sex differentiated response to promotion in the resistant hepatocyte model. Furthermore, an overexpression also in later stages of liver carcinogenesis might indicate that expression of the protooncogenes in question is related to the entire process of multistep carcinogenesis in this model.     

Histopathology and gene expression changes in rat liver during feeding of fumonisin B1, a carcinogenic mycotoxin produced by Fusarium moniliforme.

Fumonisin B1 (FB1) is a carcinogenic mycotoxin produced by the fungus Fusarium moniliforme in corn. Feeding of FB1 to rats causes acute liver injury, chronic liver injury progressing to cirrhosis, and sometimes terminates in hepatocellular carcinoma or cholangiocarcinoma. This study describes the histolopathology and changes in gene expression in the rat liver during short-term feeding of FB1. Male Fischer rats were fed either FB1 250 mg/kg or control diet, and were killed weekly for 5 weeks. FB1 caused a predominantly zone 3 'toxic' liver injury, with hepatocyte death due to necrosis and apoptosis. Hepatocyte injury and death were mirrored by hepatic stellate cell proliferation and marked fibrosis, with progressive disturbance of architecture and formation of regenerative nodules. Despite ongoing hepatocyte mitotic activity, oval cell proliferation was noted from week 2, glutathione S-transferase pi-positive hepatic foci and nodules developed and, at later time points, oval cells were noted inside some of the 'atypical' nodules. Northern blot (mRNA) analysis of liver specimens from weeks 3 to 5 showed a progressive increase in gene expression for alpha-fetoprotein, hepatocyte growth factor, transforming growth factor alpha (TGF-alpha) and especially TGF-beta1 and c-myc. Immunostaining with LC(1-30) antibody demonstrated a progressive increase in expression of mature TGF-beta1 protein by hepatocytes over the 5 week feeding period. The overexpression of TGF-beta1 may be causally related to the prominent apoptosis and fibrosis seen with FB1-induced liver injury. Increased expression of c-myc may be involved in the cancer promoting effects of FB1.     

Hepatic foci of cellular and enzymatic alteration and nodules in rats treated with clofibrate or diethylnitrosamine followed by phenobarbital: their rate of onset and their reversibility

The histologic appearance and cytochemical characteristics of foci of hepatic cellular alteration, hepatic nodules, and hepatocellular carcinomas occurring in male Sprague-Dawley rats treated with the hypolipidemic agent clofibrate (CAS: 637-07-0), with phenobarbital (CAS: 50-06-6), or with diethylnitrosamine [(DENA) CAS: 55-18-5] followed by phenobarbital were studied after treatment periods from 1 month to 2 years. Rats treated with clofibrate revealed foci of cellular alteration that were more often basophilic and occurred slightly sooner (wk 42) than those in untreated controls (wk 60). Of 36 rats that had received 68 or more weeks of continuous clofibrate, 19 had hepatic nodules. Of the 11 nodules examined cytochemically, none was gamma-glutamyltransferase (gamma-GT) positive and 2 were positive to glucose-6-phosphate dehydrogenase (G-6-PD) under oxygen. In rats withdrawn from clofibrate for 16-18 weeks after 68-95 weeks of clofibrate, 0 of 14 had nodules. In several of these rats zones of hepatic scarring were observed, suggesting the reversibility of the nodules. Phenobarbital alone had little effect on the incidence of foci of cellular alteration, although the number of gamma-GT-positive foci was increased. DENA followed by phenobarbital led to the early appearance of foci of cellular alteration (from wk 4), of nodules (from wk 13), and of hepatocellular carcinomas (from wk 26). gamma-GT activity was raised in most of these nodules and carcinomas, while G-6-PD activity was raised in only 3 of 9 nodules but in all 9 carcinomas examined. DENA-phenobarbital given for 13 or 26 weeks followed by withdrawal of phenobarbital for 28 and 26 weeks, respectively, produced an essentially similar pattern of lesions. In view of the growing recognition of the nonspecificity gamma-GT as a marker of carcinogen-initiated foci, the value of G-6-PD (under oxygen) as a marker merits further investigation.     

Enhanced O6-methylguanine-DNA alkyltransferase activity in rat liver nodules

The progression of chemical carcinogen induced hepatic lesions in the rat from altered enzyme foci to hepatocyte nodules and ultimately to hepatocellular carcinoma appears to be related to the evolution of new cell populations within these hepatic lesions. Initiator-promoter-initiator experiments in rat liver models using alkylating agents as the second genotoxic compound suggest accelerated progression toward malignancy could be the result of mutations caused by O6-alkylguanine formation in the DNA of preneoplastic liver cells. Since mutation frequency is believed to be related not only to the extent of O6-alkylguanine formation in DNA, but also to the rate of O6-alkylguanine repair, we measured the activity of the enzyme which repairs O6-alkylguanine, O6-alkylguanine-DNA alkyltransferase, in rat liver nodules. The activity of O6-methylguanine-DNA alkyltransferase in extracts of rat liver nodules 15 and 25 weeks post-initiation was approximately 1.4- and 1.8-fold greater, respectively, than comparable extracts from untreated-control and promoter only-treated rat liver tissues. Thus, the enhanced progression toward malignancy caused by treatment of rats bearing carcinogen-induced preneoplastic hepatic lesions with alkylating agents cannot be explained by a generalized deficiency of O6-alkylguanine-DNA alkyltransferase in hepatic preneoplastic lesions as a whole. These studies show that the distinctive xenobiotic resistant phenotype of rat hepatocyte nodules includes, in addition to the previously documented alterations in xenobiotic activation and detoxification enzyme activities, enhanced activity of a specific DNA repair system.     

Cell proliferation induced by triiodothyronine in rat liver is associated with nodule regression and reduction of hepatocellular carcinomas

Previous studies have demonstrated that short-term treatment with peroxisome proliferators decreased the size and number of gamma-glutamyl transpeptidase or placental glutathione S-transferase (GSTP)-positive hepatic hyperplastic lesions. In this study, we have examined the effect of the hormone triiodothyronine (T3), which, similarly to peroxisome proliferators, is a strong liver mitogen and a ligand of nuclear receptors, on the growth of GSTP-positive nodules generated by the resistant hepatocyte model and on the development of hepatocellular carcinoma. Hepatic hyperplastic nodules were induced in male Fischer rats by a single dose (150 mg/kg) of diethylnitrosamine, followed by a 2-week exposure of the animals to 2-acetylaminofluorene and partial hepatectomy. Nine weeks after diethylnitrosamine administration, rats were switched to a diet containing 4 mg/kg T3 for 1 week (experiment 1) and sacrificed during T3 feeding or were exposed to seven cycles of T3-supplemented diet (1 week/month per 7 months), and sacrificed 6 months after the last cycle (experiment 2). Results showed that T3 treatment for 1 week caused a 70% reduction in the number of GSTP-positive nodules (14/cm2 in T3-fed rats versus 44/cm2 of control animals), as well as GSTP-positive area (12% versus 43% of controls). Reduction in the number of GSTP-positive nodules observed 1 week after T3 feeding was associated with a strong increase in the labeling index of enzyme-altered nodules compared with that of controls (labeling index was 64 and 31%, respectively). No significant differences in the apoptotic index were observed between the two groups. Results from experiment 2 did reveal that although rats treated with diethylnitrosamine + 2-acetylaminofluorene developed 100% hepatocellular carcinoma and 33% of them showed lung metastasis, only 50% of rats exposed to repeated cycles of triiodothyronine developed hepatocellular carcinoma with no lung metastasis. This study indicates that cell proliferation per se might not necessarily represent a promoting condition for putative preneoplastic lesions and demonstrates an anticarcinogenic effect of T3.     

Early exposure to restraint stress enhances chemical carcinogenesis in rat liver

This study examines the effect of a stress-associated condition on chemical hepatocarcinogenesis in the rat. Rats were given diethylnitrosamine (200 mg/kg. b.w., i.p.), followed, 1 week later, by three cycles of immobilization at room temperature. Two weeks after the last cycle they were treated according to the resistant hepatocyte protocol. At 4 weeks after selection, mean size of glutathione-S-transferase 7-7 positive foci/nodules was increased in the immobilized group (0.82+/-0.22 vs. 0.25+/-0.04 mm(2) in controls). Furthermore, at the end of 1 year 10/13 animals (77%) developed hepatocellular carcinoma in the former group, while only 6/14 (43%) incidence of cancer was found in controls. These results indicate that exposure to restraint stress early during carcinogenesis enhances the development of chemically-induced hepatocellular carcinoma in the rat.     

Histopathology of neoplastic and nonneoplastic hepatic lesions in mice fed diets containing tetrachlorvinphos.

Tetrachlorvinphos was fed at 8,000 or 16,000 ppm in diets to male and female (C57BL/6N X C3H/HeN)F1 mice for 80 weeks. Surviving mice were killed at 92 weeks, and all mice were completely necropsied. A high incidence of unusual nonneoplastic hepatic lesions in treated mice was present and characterized by pericellular fibrosis, hepatocyte nuclear pleomorphism, and intrasinusoidal foci of macrophages with intracytoplasmic crystalline structures. From 84 to 94% of the treated male mice and from 21 to 23% of the treated females had hepatocellular neoplasms. Only 17% of the control males and 7% of the control females had liver tumors. The induced tumors were frequently multiple in the liver, whereas the tumors in the controls were usually singular. The morphology of 241 liver tumors in 110 treated mice was different from that of tumors in controls. Liver tumors in control mice were generally composed of small basophillic hepatocytes. In treated mice, tumors were hepatocellular carcinomas composed of solid sheets of large basophilic or eosinophilic hepatocytes. Foci of prominent trabecular formation were seen in 51 tumors. Fifteen tumors were composed of small basophilic hepatocytes with oval cells interposed among them. Foci of capillary formation were noted in 3 of these tumors. In addition, 7 more typical hemangiosarcomas forming sinusoids and with thrombosis were observed.     

Effects of neonatal and adult castration and of testosterone substitution in male rats on growth of enzyme-altered hepatic foci in the resistant hepatocyte model

Marked sex differences in the growth of enzyme-altered hepatic foci have been observed in rats treated according to the "resistant hepatocyte model." The present study was performed to investigate the effect of neonatal and adult castration of male rats, with or without testosterone substitution, on the growth rate of foci during selection of initiated cells with 2-acetylaminofluorene and partial hepatectomy. Neonatal castration of male rats decreased focal growth to the same level as in female rats. Castration of adult male rats 2 wk before initiation with diethylnitrosamine also decreased the growth rate of foci, but less markedly than in neonatally castrated rats. Testosterone substitution of male rats castrated as neonates or as adults, from 10 days after initiation with diethylnitrosamine, restored focal growth to that of sham-castrated controls. Previous investigations concerning the role of gonadal hormones in sex differentiation of various liver functions indicated a role of the hypothalamo-pituitary-liver axis in mediating the effects of androgens. It is therefore also suggested that the effects of androgens on early steps of hepatocarcinogenesis observed in the present study are mediated by similar mechanisms, possibly through an influence on the metabolism of 2-acetylaminofluorene.     

Expression of N-acetylglucosaminyltransferase III in hepatic nodules generated by different models of rat liver carcinogenesis

Our earlier studies revealed that N-acetylglucosaminyltransferase III (GnTase III), which catalyzes the insertion of a bisecting N-acetylglucosamine (bi-Gn) in the complex-type N-linked glycans of cellular glycoproteins, is present in hepatic nodules promoted by the orotic acid model. Neither the bi-Gn residues nor the activity of GnTase III is detectable in normal livers and in the surrounding non-nodular liver of the rat. The present study was designed to find out whether an expression of activity of GnTase III is a phenotypic property characteristic of hepatic nodules or simply unique to nodules produced by the orotic acid model. Fischer male 344 rats were initiated with two different carcinogens namely 1,2-dimethyl-hydrazine or diethylnitrosamine and promoted by other models such as the resistant hepatocyte model and the choline deficient diet model, in addition to the orotic acid model. The hepatic nodules generated by these three different models and hepatocellular carcinomas exhibited significant levels of activity of GnTase III while non-nodular surrounding liver, regenerating liver after 2/3 partial hepatectomy or livers of age and sex matched control rats, had no detectable activity. The detection of the activity of GnTase III in nodules and in cancer is in agreement with the presence of bi-Gn residues reported in gamma-glutamyltranspeptidase of cancer tissues of rats. These results are consistent with the conclusion that the expression of GnTase III is activated during hepatocarcinogenesis and is not related to any particular initiator or promoter.