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 In rats withdrawn from clofibrate for 16-18 weeks after 68-95 weeks of clofibrate, 0 of 14 had nodules.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.     

Consecutively occurring multiple fibroadenomas of the breast distinguished from phyllodes tumors by clonality analysis of stromal tissue

We present a woman with consecutively occurring multiple fibroadenomas of the breast distinguished from phyllodes tumor by clonality analysis of stromal tissue. Fifty-three masses developed in her right breast in an 11.5 year period since first onset. A clonality analysis, performed by polymerase chain reaction (PCR), focused on theHpa II site within exon 1 of the androgen receptor (AR) gene. Estrogen receptor (ER) α immunohistochemistry was performed with a specific polyclonal antibody against the hormone binding domain of the protein. Since all of the nodules were polyclonal in stromal tissues, they were hyperplastic rather than true neoplasms such as phyllodes tumors. The co-existence of abundant adenosis foci and numerous sub-clinical small nodules in the adjacent breast tissue also suggested proliferative stimuli throughout the breast. ER α protein was expressed only in the nuclei of epithelial cells. The possible influence of ER positive epithelial cells on stromal cell growth is discussed.     

The effect of long-term feeding of orotic acid on the incidence of foci of enzyme-altered hepatocytes and hepatic nodules in Fischer 344 rats

The present study was designed to determine the long-term effectsof orotic acid (OA), a multi-organ tumor promoter, in rats notexposed to any carcinogen. Male Fischer 344 rats (130–150g) were divided into two groups and given either a semisyntheticbasal diet (BD) or the same diet containing 1% OA. Animals fromboth groups were killed after 1 or 2 years of treatment. Fociof placental glutathione-S-transferase (GST 7–7) positivehepatocytes were observed in the livers of both BD and OA fedrats killed after 1 year. However, they were more in numberin animals receiving OA (156±21 versus 51±11/cm³).After 2 years, hepatic nodules were seen in almost all the animalsgiven OA and in 30% of the rats given BD. The nodules were oftwo main types: (i) a reddish-brown type, present in 85% ofrats exposed to OA and in 27% of rats given BD, and (ii) a greyish-whitetype, found in 50% of animals fed OA and in none of the animalsfed BD. These two types of lesions were also histologicallydifferent. Reddish-brown nodules were composed of slightly enlargedhepatocytes resembling normal surrounding tissue, while greyish-whitenodules were similar in structure and are indistinguishablefrom hepatic nodules induced by genotoxic chemical carcinogens.The results are interpreted to suggest that the foci/nodulesseen in OA-fed rats are due to a promoting effect of OA on spontaneouslyarising and/or diet-induced altered cells.     

The effect of diet on 2,6-dinitrotoluene hepatocarcinogenesis

Pectin-induced changes in microflora have been shown to elevatethe covalent binding of 2,6-dinitrotoluene (2,6-DNT)-relatedmaterials to total rat hepatic macrotnolecules. Therefore, theeffect of diets varying in pectin content on the induction offoci and hepatic tumors induced by 2,6-DNT was studied in maleF344 rats. 2,6-DNT (3.0–3.5 and 0.6–0.7 mg/kg/day)was incorporated into NIH-07 (NIH), an open formula cereal-baseddiet high in pectin content, AIN-76A (AIN), a purified pectin-freediet, or AIN-76A supplemented with 5% pectin (AP). Hepatic fociwere scored after histochemical staining for gamma-glutamyltranspeptidase (GGT), canalicular adenosine triphosphatase orglucose-6-pbosphatase following administration of test dietsfor 3, 6 and 12 months. The number of foci per cm³ of liverincreased in a dose- and time-dependent manner following incorporationof 2,6-DNT into test diets with NIH>AP>AIN. In the NIHdiet, 2,6-DNT did not alter the phenotypic distribution of foci.Animals fed control or 2,6-DNT-containing AIN and AP diets hadfew or no GGT foci throughout the study. Hepatocellular carcinomasand neoplastic nodules were observed only in rats fed NIH containing2,6-DNT. The concentrations of 2,6-DNT-related material covalentlybound to hepatic macromolecules after a single oral dose ofradiolabeled 2,6-DNT given after 12 months on the diets increasedin control rats and in rats receiving low dose 2,6-DNT in thediet with AINIt; AP< NIH. These studies show that the carcinogenicityof 2,6-DNT differs depending on whether rats are fed an NIHor AIN (± pectin) diet. The results suggest that diet-inducedalterations in the covalent binding of 2,6-DNT are not the solefactor in determining the carcinogenic response to 2,6-DNT.Furthermore, unidentified contaminants in cereal-based dietsmay influence foci and tumor production in rat liver duringcarcinogen treatment.     

Hyperplastic foci in precancerous rat liver: light microscopic and electron microscopic study.

4-Dimethylaminoazobenzene was fed to Wistar-derived, male, albino rats after hepatic siderosis had been induced by including ferric citrate in the diet. Iron-free foci of hepatocytes developed and this characteristic enabled them to be recognized macroscopically in the brown parenchyma. Five such lesions, each 1 mm or less in diameter, were studied by light and electron microscopy. The cells in the foci were larger than those surrounding the foci and had a granular and moderately basophilic cytoplasm. Ultrastructurally, the cells closely resembled normal hepatocytes. They possessed well-developed rough endoplasmic reticulum, numerous free ribosomes, peroxisomes, bile canaliculi, and cytoplasmic junctional complexes, but only small stores of glycogen were observed. Occasional ferritin-laden lysosomes persisted in some cells. These foci were regarded as hyperplastic. Possibly, they evolved into hyperplastic nodules either of the basophilic or vacuolated type. These foci should be clearly distinguished from hyperbasophilic foci that consisted of very poorly differentiated cells.     

Adenocarcinoma of the kidney. IV. Electron microscopic study of the development of renal adenocarcinomas induced in rats by N-(4'-fluoro-4-biphenylyl)acetamide

Ultrastructural features of various stages in the histogenesis of renal adenocarcinoma induced in F344 rats by the carcinogen N-(4'-fluoro-4-biphenylyl)acetamide (FBPA) are described. FBPA was added to the diet up to 48 weeks; animals were killed at intervals from 4 to 52 weeks. Characterized were foci of cellular alterations followed by foci of proliferation with dysplasia leading to solid and cystic lesions, carcinoma in situ, and finally, carcinoma. Other lesions included foam cell nodules. Alterations also were noted in distal tubules. By electron microscopy, subtle changes in some proximal tubule cells of the pars recta were noted by 4 weeks (foci of cellular alteration). By 12 weeks there was evidence of dedifferentiation and reduction of cell polarity, thickening of basal lamina, alterations in endoplasmic reticulum and mitochondria, and increases in residual bodies. Nuclei of these cells possessed large nucleoli. From 24 to 36 weeks foci of proliferation were seen and consisted of tumor cells arranged in solid and cystic configurations. In solid lesions (less than 3 mm), in addition to the above alterations, cells showed a microvillus brush border in unusual locations, small and irregular mitochondria, multiple Golgi complexes and basal lamina, and loss of cell polarity. From 36 to 52 weeks carcinoma in situ consisted of cells similar to tumor cells described above. In addition, intracellular canaliculi were present. Areas of tumor cell degeneration and necrosis also were seen. Cells of larger carcinomas (greater than 3 mm), also observed at this interval, were identical to those of carcinoma in situ. Calcification of necrotic debris was noted.     

Ethanol interactions with a choline-deficient,ethionine-supplemented feeding regime potentiate pre-neoplastic cellular alterations in rat liver

To examine the effect of ethanol on hepatocarcinogenesis induced by a choline-deficient, ethionine-supplemented (CDE) diet, rats were fed either an ethanol-supplemented diet or ethanol-free, isocaloric diet for 2 months, followed by a CDE diet or control diet for up to 8 months. Changes to cellular composition and pattern of gene expression in the liver were determined at 0 and 3 days, and 1, 2 and 3 weeks after commencing the CDE diet, using histological/immunochemical techniques and northern analysis. Oval cells in the liver were identified morphologically and by expression of pi-glutathione S-transferase (pi-GST), alpha-fetoprotein (AFP) and the embryonic isoform of pyruvate kinase (M2-PK). Oval cell numbers and changes in the pattern of gene expression induced by the CDE diet were accelerated by pre-treatment with ethanol. At all stages, the proportion of oval cells in the test group exceeded that in controls. After 1 week, oval cells had spread sufficiently from the periportal region to be observed pericentrally in test animals and by 3 weeks, extensive formation of ductal structures was apparent, which were absent in controls. Additionally, M2-PK and AFP mRNA were detected earlier, and in greater abundance in animals pre-treated with ethanol. After 8 months of CDE treatment, one or two small hepatic foci (<10 hepatocytes), strongly positive for pi-GST, were detected in the liver of ethanol-pre-treated animals. These foci were absent in CDE-treated animals; however, animals pre-treated with ethanol followed by chronic CDE treatment showed increased size (>40 hepatocytes) and numbers of foci, correlating with the extent of liver damage and varying from 5 to 50% of the liver section. Our data suggest that ethanol pre-treatment potentiates the short-term effects of the CDE diet by enhancing oval cell proliferation, while chronic CDE administration enhances the appearance of pre-malignant hepatic foci that are observed with ethanol pre-treatment alone.     

Inhibitory Effects of Inhibitors of Arachidonic Acid Metabolism on the Evolution of Rat Liver Preneoplastic Foci into Nodules and Hepatocellular Carcinomas with or without Phenobarbital Exposure

Effects of inhibitors of arachidonic acid (AA) metabolism on the evolution of preneoplastic foci into nodules and of nodules into hepatocellular carcinomas were examined in F344 male rat livers with or without phenobarbital (PB) exposure. p -Bromophenacyl bromide (BPB), acetylsalicylic acid (ASA), and quercetin (QU) were used as inhibitors of phospholipase A₂, cyclooxygenase and lipoxygenase, respectively. Preneoplastic liver foci were induced by initiation with N-nitrosodiethylamine (200 mg/kg, i.p.) followed by selection using the procedure of Cayama et al. For the nodule experiment, starting 1 week after completion of the selection procedure, animals bearing foci were given diets containing 0.05% PB plus 0.75, 1, or 1.5% of one of the inhibitors, 0.05% PB alone, or 0.75, 1 or 1.5% of inhibitor alone, or basal diet for 9 weeks. For the carcinoma experiment, 3 weeks after completion of the selection procedure, animals bearing nodules were given the same diets mentioned above for 29 weeks. BPB, ASA and QU either with or without PB accelerated the remodeling of preneoplastic foci, significantly decreasing the numbers of persistent nodules and hyperplastic nodules. ASA either with or without PB significantly decreased the number of hepatocellular carcinomas per rat. BPB and QU, however, significantly decreased the numbers of hepatocellular carcinomas with but not without PB. The results suggested an involvement of AA metabolism in the process of evolution of preneoplastic foci into nodules and hepatocellular carcinomas in rat liver with or without PB exposure.     

Increased resistance of peroxisome proliferator-induced hepatic lesions to iron overload in rats

Altered areas (AA), neoplastic nodules (NN) and hepatocellular carcinomas (HCC) induced by chronic administration of ciprofibrate and Wy-14,643 were examined for iron accumulation after systemic iron overload. Eighty percent of AA, 90% NN and 100% HCC showed increased resistance to iron accumulation. However, marked heterogeneity was observed in the amount of iron from area to area within the same lesion with some cells containing no iron, while others showing blue reaction to iron. These findings indicate, that putative preneoplastic and neoplastic hepatic lesions induced by peroxisome proliferators exclude iron in a fashion similar to that of hepatic nodules and carcinomas induced by DNA damaging carcinogens.     

Cell Proliferation and regulation of negative growth factors in mouse liver foci

Foci of altered hepatocytes are preneoplastic lesions capableof progressing to hepatocellular carcinomas. To Characterizethe growth of preneoplastic hepatic lesions, size of hepaticfoci was analyzed with regard to growth factor regulation andhepatocyte proliferation in focal and non-focal hepatocytes.Twelve-day-old female B6C3F1 mice were initiated with a singledose of the potent mutagen N-nitrosodiethylamine (DEN) (5 mg/kgbody weight). Beginning at 6 weeks of age, mice were exposedfor 16 weeks to 2038 p.p.m. unleaded gasoline (UG) vapor or1 p.p.m. ethinyl estradiol (EE) in the diet. Analysis of hepaticfoci demonstrated that UG significantly increased, but EE significantlydecreased the size of DEN-initiated foci. Hepatic labeling index(LI), as measured by the incorporation of 5-bromo-2'-deoxyuridine,was similar in non-focal hepatocytes at 16 weeks in all groups(0.4–0.8%) and greatly increased in hepatic foci. HepatocyteLI was significantly increased in DEN/UG foci (29%, n = 41)and significantly decreased in DEN/EE foci (6% n=23) relativeto DEN/control focal hepatocytes(18% n=25). The mean LI of focicorrelated with the focal size differences observed in the treatmentgroups. Immunohistochemical analysis with antibodies directedto the negative growth regulator transforming growth factorbetal (TGF-ß1) demonstrated a consistent decreaseof TGF-ß1 in DEN/Ct and DEN/UG hepatic foci relativeto non-lesion hepatocytes. Similar results were seen with mannose6-phosphate/insulin-like growth factor-11 receptor (M6P/IGF-IIR), which facilitates activation of latent TGF-ß1.In contrast, only 50% of DEN/EE foci had decreased levels ofTGF-ß1 and M6P/IGF-II R relative to non-focal hepatocytes.These data suggest that proliferative responses observed inhepatic foci may be correlated with foci size. In contrast,chemically induced proliferative responses in non-focal hepatocytesafter subchronic exposure cannot necessarily be used to predictproliferative effects in preneoplastic cell populations. Furthermore,these studies suggest that hepatic foci may occur by M6P/IGF-IIR enhancing activation of latent TGF-ß1 in non-focalhepatocytes but not in the focal hepatocytes, thereby affordingfocal hepatocytes a selective growth advantage.     

Inhibition by d-limonene of experimental hepatocarcinogenesis in Sprague-Dawley rats does not involve p21(ras) plasma membrane association.

The effects of d-limonene on hepatocarcinogenesis induced by N-nitrosomorpholine (NNM) and on membrane-associated p21(ras) and labeling and apoptotic indices of the liver were investigated in male Sprague-Dawley rats. Rats were given drinking water containing NNM for 8 weeks, and from the beginning of experimental week 9, they received chow pellets containing 1% or 2% limonene. The preneoplastic and neoplastic liver lesions (cellular alteration foci, neoplastic nodules and hepatocellular carcinomas), and hepatic foci staining positive for glutathione-S-transferase, placental type (GST-P) were examined microscopically and histochemically. At week 16, quantitative histologic analysis showed that oral administration of 1% or 2% limonene resulted in significant reductions in the number and mean area of GST-P-positive hepatic foci and the number of cellular alteration foci, neoplastic nodules and hepatocellular carcinomas. Limonene, at both doses, also caused significant decreases in the labeling indices and significant increases in the apoptotic indices of cellular alteration foci, neoplastic nodules, hepatocellular carcinomas and adjacent liver. However, limonene, at both doses, had no significant influence on the production of membrane-associated p21(ras) in the visible liver white nodules. These findings indicate that limonene inhibits hepatocarcinogenesis and suggest that this effect may be clearly related to its effect in inhibiting cell proliferation and in enhancing apoptosis, but not through ras oncoprotein plasma membrane association.     

Evidence of two separate mechanisms for the decrease in aryl sulfotransferase activity in rat liver during early stages of 2-acetylaminofluorene–induced hepatocarcinogenesis

Enzymatic and immunohistochemical experiments were conducted to evaluate the mechanistic basis for the downregulation of the important detoxication/bioactivation enzyme aryl sulfotransferase IV (AST IV) during 2- acetylaminofluorene (2AAF)–induced hepatocarcinogenesis. To distinguish between possible genotoxic and cytotoxic actions of 2AAF, three different dietary protocols were used in these experiments: group 1 received 2AAF for 12 wk, group 2 received 2AAF for 3 or 6 wk and then a control diet lacking xenobiotics for 3 or 6 wk, and group 3 received 2AAF for 3 or 6 wk and then phenobarbital for 3 or 6 wk. When hepatic AST IV activity was assessed, N-hydroxy-2AAF sulfotransferase activity was found to decrease 80–90% in response to 2AAF feeding, but activity recovered to essentially normal levels in the livers of rats subsequently placed on either control diets or diets with phenobarbital, suggesting a reversible cytotoxic mechanism for loss of AST IV activity. However, when liver sections from the rats were evaluated immunohistochemically, two distinct patterns were detected for the downregulation of AST IV expression was observed throughout the liver and among most but not all newly developed nodules. In tissue sections from rats initially fed 2AAF and then placed on a control diet (group 2) or a diet with phenobarbital (group 3), the nodules continued to show low levels of AST IV expression, while expression in the areas surrounding nodules returned to the normal, high levels. In addition, among those rats fed 2AAF for just 3 wk and then control diet or diet containing phenobarbital for 6 wk, only rats fed phenobarbital developed altered foci that stained weakly for AST IV expression. These results show that there were two kinds of 2AAF-mediated decrease in hepatic AST IV activity: a general overall loss of AST IV expression dependent on administration of 2AAF and reversible upon removal of 2AAF from the diet and a loss of AST IV expression among newly developed liver foci and nodules that persisted in the absence of 2AAF administration and appeared to be a property of 2AAF-induced subpopulations of cells. These patterns may correspond, respectively, to cytotoxic and genotoxic mechanisms of 2AAF action. © 1994 Wiley-Liss, Inc.     

Stable phenotypic expression of glutathione S-transferase placental type and unstable phenotypic expression of gamma-glutamyltransferase in rat liver preneoplastic and neoplastic lesions

Using immunohistochemical demonstration of glutathione S-transferase placental type (GST-P) and histochemical demonstration of gamma-glutamyltransferase (gamma-GT), the long-term development of preneoplastic and neoplastic lesions was followed in rats over a 50-week period. Rats were given a single i.p. injection of 200 mg/kg body weight of diethylnitrosamine (DEN), and then 2 weeks later were administered 0.02% 2-acetylaminofluorene (2-AAF) (group 1), 0.05% phenobarbital (PB) (group 2), 2.0% butylated hydroxyanisole (BHA) (group 3) or no supplement (group 4) in their diet for 6 weeks, all rats being subjected to partial hepatectomy at week 3. Hepatocellular proliferated lesions were classified as foci, nodules and hepatocellular carcinomas. Development of foci, nodules and hepatocellular carcinomas was enhanced strongly by 2-AAF and weakly by PB, and inhibited by BHA. Almost all foci and nodules were GST-P positive, although 5-10% of the GST-P-positive foci were gamma-GT negative. The areas of GST-P-positive foci and nodules increased with time in all groups. In contrast, while the areas of gamma-GT-positive lesions also increased with time in groups 2-4, they decreased from week 12 in group 1. As the percentage gamma-GT-positive area in GST-P-positive foci significantly decreased with time in all groups, the rate of phenotypic reversion of gamma-GT in foci in group 1 was revealed to be larger than the focus growing rate, whereas that in groups 2-4 was smaller. Gamma-GT-negative and GST-P-positive micro-nodules of altered morphology appeared within gamma-GT- and GST-P-positive nodules in later stages. All hepatocellular carcinomas found in this experiment consisted of GST-P-positive cells. In contrast, 37% (13/35) of the hepatocellular carcinomas were negative for gamma-GT. The results indicate GST-P to be the most accurate marker enzyme for detection of initiated cells during liver carcinogenesis and gamma-GT to be more appropriate for indicating changes of phenotypic expression in each lesion type.     

The natural history and dose-response characteristics of enzyme-altered foci in rat liver following phenobarbital and diethylnitrosamine administration

Enzyme-altered foci (EAF) were induced in the Liver of femalerats by 70? partial hepatectomy (PH), followed by a single intragastricadministration of diethylnitrosamine (DEN) at a dose of 10 mg/kg.The stability and response of these foci to various doses ofthe hepatic promoting agent, phenobarbital (PB), were studied.The number of yglutamyltranspeptidasepositive (GGT⁺) EAF resultingfrom PH/DEN followed by PB (0.05%) administration for 1 week,2 weeks, 1 month, 2 months, 3 months or 4 months did not significantlychange when the administration of the promoting agent was followedby a 6-month period of a diet containing no PB. These data demonstratethe stability of the fwi induced by the PH/DEN/PB regimen andindicate that the increased number of foci resulting from PBpromotion in the absence of overt hepatic necrosis are not reversibleon removal of the promoting stimulus. Chronic administrationof dose levels of PB below 0.001% in the diet failed to demonstratean increase in the number of EAF over the number in the controlanimals not promoted with PB. A linear increase in the numberof EAF was observed when rats were chronically fed doses ofPB ranging between 0.001% and 0.05% in the diet, whereas dietconcentrations of PB > 0.05% did not result in any furtherincrease in the number of EM. The number of EAF resulting fromPH/DEN followed by 0.05% PB in the diet increased during thefirst 3–4 months of promotion. Thereafter, the numberof foci did not change despite the continued administrationof PB for as long as 8 months. These data suggest the presenceof an apparent threshold (no effect level) for promotion byPB and demonstrate the presence of a maximal response of EAFto this promoting agent after initiation by a single dose ofDEN.     

Enhancement of low-dose N-2-fluorenylacetamide-induced hyperplastic liver nodules by pre-existing cirrhosis

The potentiating effect of pre-existing cirrhosis on the fonnationof hyperplastic liver nodules and/or foci was investigated byfeeding a low dose (0.008%) of 2-N-fluor-enylacetamide (FAA)in a choline-deficient (CD) diet for 32 weeks to cirrhotic andnon-cirrhotic rats. Liver cirrhosis was induced by feeding therats a CD diet for the preceding 36 weeks. The number and areaof -glutamyltranspeptidase-positive hyperplastic liver nodulesand/or foci were significantly larger in cirrhotic rats exposedto low-dose FAA than in non-cirrhotic rats similarly treated.Hyperplastic liver nodules and/or foci were not observed inrats continuously fed a CD diet alone for 68 weeks (controlcirrhotic rats). The results suggest that cirrhotic liver mightalter the metabolic response to FAA, even at low doses, andlead to enhanced induction of hyperplastic liver nodules and/orfoci.     

Effect of vitamin E on the induction and evolution of enzyme-altered foci in the liver of rats treated with diethylnitrosamine

The effects of dietary vitamin E (VE) on the steps of hepatocarcinogenesis,the induction and growth of -glutamyltranspeptidase (GGT)-positivefoci and their evolution into persistent nodules, were analyzedin the liver of rats treated with diethylnitrosamine (DEN).The induction of GGT-positive foci was inhibited by a diet containing0.36–1.5% VE given after initiation with 200 mg/kg bodyweight (b.w.) DEN for 6 weeks with partial hepatectomy (PH)on week 3. The numbers and areas of GGT-positive foci were enhancedby diets containing 036 and 0.72% VE, given for 1 week afterinitiation with 10 mg/kg b.w. DEN and PH, followed by selectionby 0.02/ 2-acetylaminofluorene (AAF) and carbon tetrachloride(CCl⁴), but these were not enhanced by a diet containing 1.5%VE. Remodeling of hyperplastic nodules was not affected by thediet containing 0.72% VE given after initiation with DEN andselection for 12 weeks. The staining characteristics of GGTwere different between remodeling and persistent nodules, exceptfor those of the glutathione-S-transferase placental form (GST-P).The results obtained suggest that VE could prevent the veryearly events during hepatocarcinogenesis, the induction of phenotypicallyaltered foci, but could no longer affect the later stages, theevolution of foci into persistent nodules.     

Progression of carcinogen-induced foci of y-glutamyltranspeptidase-positive hepatocytes to hepatomas in rats fed a choline-deficient diet

Following previous findings that feeding a choline-deficient (CD) diet to rats strongly promotes the evolution of liver cells, initiated by a chemical carcinogen, to foci of y-glutamyltranspeptidase (yGT)-positive hepatocytes, we investigated whether a CD diet could also promote the evolution of yGT-positive foci to hepatomas. yGT-positive foci were induced in male Sprague-Dawley rats by administration of a single dose of diethyInitrosamine followed by 2 weeks' feeding on a CD diet containing 0.02% acetylaminofluorene. Immediately thereafter, one group of rats was fed a plain CD diet and the other a choline-supplemented (CS) diet, and subgroups of animals were killed periodically for analysis of the number and size of yGT-positive foci and development of hepatomas. During the first 7 weeks after switching the diets, the number and size of the foci increased in both groups. At 12 and 16 weeks, the number and size of foci began to decline in rats fed the CS diet However, in the CD group, there was a progressive increase in the size with coalescence of the foci, as well as development of neoplastic nodules. These lesions were followed at 20 and 28 weeks by development of hepatomas at a high incidence rate. In rats fed the CS diet, a few scattered yGT-positive foci, and small neoplastic nodules remained in the liver at 20 and 28 weeks, but no hepatomas developed. These results show that the CD diet is a potent promoter of the evolution of foci of altered hepatocytes to hepatomas, and that most of the yGT-positive foci are reversible lesions.     

Alteration of hepatocytes by subcarcinogenic exposure to n-2-fluorenylacetamide.

These experiments examined the effects of a single, subcarcinogenic dose of dimethylnitrosamine or N-hydroxyfluorenylacetamide when administered after a subcarcinogenic dietary regimen of N-2-fluorenylacetamide. Control rats that received either carcinogen diet alone or a single dose of carcinogen demonstrated neither hepatic nodules nor hepatocellular carcinomas. Those animals that received dimethylnitrosamine subsequent to carcinogen diet demonstrated many persistent hepatic nodules and 100% hepato-cellular carcinomas. These data support the concept that the nodules produced by subcarcinogenic ingestion of N-2-fluorenylacetamide are not composed simply of normal hepatocytes undergoing compensatory regeneration but consist of cells that have been altered by the carcinogen. One manifestation of this alteration is an increased susceptibility to further carcinogenic evolution.     

Perturbations of endogenous levels of orotic acid and carcinogenesis: effect of an arginine-deficient diet and carbamyl aspartate on hepatocarcinogenesis in the rat and the mouse

Feeding excess orotic acid (OA) In the diet promotes the carcinogenicprocess in different organs Including the liver. A number ofmetabolic and genetic disorders are associated with Increasedsynthesis of endogenous OA and some of these disorders appearto pose an Increased risk of liver cancer development. Thisstudy therefore examines whether excess OA of endogenous originalso exerts a promoting effect on hepatocarcinogenesis in themouse and the rat. Increased endogenous synthesis of OA wasachieved by (i) feeding a diet deficient in arginine (AD) and(ii) feedIng excess dietary carbamylaspartate (CA), a precursorfor the synthesis of OA. A single dose of diethylnitrosamine(DENA) was given i.p. to male Fischer 344 rats (200 mg/kg) orto male DBA/2 mice (90 mg/kg). One week later they were placedon either AD diet or the same diet supplemented with 1.3% arginine(AS) for a total of weeks. Two-thirds partial hepatectomy (PH)was performed at the end of the second week. All animals werethen transferred to a control semisynthetic basal diet for atotal of 20 weeks before they were killed. The results indicatedthat AD diet increased the incidence of hepatic nodules in bothrats (percentage area occupied by nodules was 4.7 ± 0.4in the AD group compared to a control value of 0.7 ±0.5) and mice (4/10 mice had nodules >5 mm diameter in theAD group while none in the AS group had such large nodules).In another experiment male Fischer 344 rats similarly initiatedwIth DENA were exposed to either basal diet or basal diet containing2% CA for 4 weeks coupled with PH performed at the end of thesecond week. This regimen was followed by 20 weeks of feedingbasal diet to both groups. Rats given CA developed larger hepatlcfoci and nodules (0.84 ± 0.56 mm³) compared to the controlgroup, which was fed basal diet throughout the experiment (0.07± 0.03 mm³) Further, both AD diet and dietary CA, likedietary OA, induced an increase In hepatic uridine nucleotides.Taken together, these results suggest that Increased levelsof endogenously synthesized OA, like exo genously supplied excessOA, can induce an Imbalance in hepatic nucleotide pools andcan exert a promoting effect on hepatocarcinogenesis.