Enzyme histochemical and immunohistochemical characterization of oval and parenchymal cells proliferating in livers of rats fed a choline-deficient/DL-ethionine-supplemented diet.

Male outbred Sprague-Dawley rats were fed a choline-deficient diet containing 0.10% DL-ethionine for up to 30 weeks. Liver slices from rats killed 4, 6, 10, 14, 22 and 30 weeks after starting the treatment were histochemically analyzed for the following parameters: basophilia, expression of cytokeratin 19 (which in the liver is bile duct epithelial cell-specific), glycogen content and activities of glycogen synthetase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphatase (G6PASE), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycerin-3-phosphate dehydrogenase (G3PDH), 'malic enzyme' (MDH), alkaline phosphatase (ALKPASE) and gamma-glutamyltranspeptidase (GGT). The diet induced necrosis of single parenchymal cells and a massive proliferation of oval cells within 4-6 weeks; thereafter cholangiofibroses, cystic cholangiomas and some cholangiofibromas, but no cholangiocarcinomas, were observed. Oval cells, cholangiofibroses, cystic cholangiomas and cholangiofibromas expressed cytokeratin 19, whereas parenchymal cells, foci of altered hepatocytes and hepatocellular adenomas did not; this observation does not support a precursor-product relationship between oval and parenchymal cells. SYN, PHO, G6PASE, G6PDH, GAPDH, G3PDH, MDH, ALKPASE and GGT activities were detected in oval cells; cholangiofibrotic lesions, cystic cholangiomas and cholangiofibromas stained strongly for GAPDH, G3PDH and MDH. In livers from rats fed the diet for 10 weeks, single hepatocytes storing high amounts of glycogen appeared in the parenchyma. There was no indication of a transition from the oval cell population to hepatocytes storing glycogen in excess. Foci of glycogen-storing cells were scattered all over the lobes after 14 and 22 weeks; they had increased G6PASE, G6PDH, ALKPASE and GGT activities. Mixed cell foci and hepatocellular adenomas developed within 22-30 weeks and exhibited a remarkable decrease of G6PASE activity, a strong increase of G6PDH, GAPDH, G3PDH and MDH activities as well as extremely high ALKPASE and GGT activities. The data support the concept that during hepatocarcinogenesis, a number of sequential changes in the activities of various enzymes involved in carbohydrate metabolism occur and that a correlation between morphology and enzyme pattern in the focal lesions does in fact exist. Furthermore, our results suggest that two different cell lineages are involved in the development of cholangiocellular tumors from oval cells and hepatocellular tumors from hepatocytes.     

Effect of lead nitrate on liver carbohydrate enzymes and glycogen content in the rat

Male Wistar rats were given a single i.v. injection of lead nitrate (10 mumol/100 g body wt) and were killed with matched controls 24, 48, 72 h and 20 days after the treatment. Changes of liver carbohydrate metabolism were studied histochemically testing the following parameters: glycogen content, activities of glycogen synthase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphatase (G6PASE), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In addition, gammaglutamyltransferase (GGT) activity was demonstrated. Between 24 and 48 h after lead nitrate injection there was a nearly complete loss of liver glycogen. Seventy-two hours later the polysaccharide reappeared in single hepatocytes and after 20 days the livers of the lead-treated animals not only had replenished their glycogen stores but contained even more glycogen than the matched controls. SYN and PHO activities were diminished from 24 to 72 h, but returned to control values after 20 days. G6PASE and GGT remained elevated up to 72 h before dropping to normal at 20 days after treatment. The pentose phosphate pathway enzymes G6PDH and 6PGDH showed the most remarkable changes in livers treated with lead nitrate. G6PDH was already elevated at 24 h, but only in Kupffer cells. At 48 and 72 h, when hepatocytes exhibited a highly increased mitotic rate, the levels of G6PDH, 6PGDH and GAPDH were elevated. After 20 days dehydrogenase activities were comparable to those of controls. The results of this study suggest that a single dose of lead nitrate not only stimulates proliferation of hepatocytes but also induces considerable changes in rat liver carbohydrate metabolism, especially between 24 and 72 h after administration. During that period glycogen metabolism undergoes a strong reduction, whereas gluconeogenesis and particularly the pentose phosphate pathway respond with a remarkable increase. This metabolic profile is most likely associated with lead biotransformation as well as with liver cell proliferation. It corresponds only partially to that found in preneoplastic and neoplastic liver lesions observed in chemical carcinogenesis, and is reversible, in contrast to the persistent alterations associated with neoplastic transformation.     

Sequential changes in glycogen content, expression of glucose transporters and enzymic patterns during development of clear/acidophilic cell tumors in rat kidney.

Renal clear cell tubules and clear/acidophilic cell tumors were induced in male Sprague-Dawley rats by 7 weeks oral administration (stop model) of N-nitrosomorpholine (NNM) at a concentration of 12 mg/100 ml in the drinking water. Twelve, 23 and 34 weeks after withdrawal of NNM serial cryostat sections of the kidneys were histochemically analyzed for the following parameters: glucose transporter proteins (GLUT1, GLUT2), glycogen content and the activities of glycogen synthase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphatase (G6Pase), glucose-6-phosphate dehydrogenase (G6PDH), hexokinase (HK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PK), succinate dehydrogenase (SDH), malate dehydrogenase (MDH), alkaline phosphatase (ALP), acid phosphatase (ACP) and gamma-glutamyltransferase (GGT). Clear cell (glycogenotic) tubules first appeared at 23 weeks, and clear/acidophilic cell tumors at 34 weeks after withdrawal of the carcinogen. G6Pase, ALP, GGT and GLUT2 were absent in clear cell tubules, clear/acidophilic cell tubules, and clear/acidophilic cell tumors indicating a sequential origin of all these types of lesions from the collecting duct system, in line with previous morphological findings. In comparison to the collecting duct epithelium, glycogenotic tubules demonstrated an increased activity of PHO and reduced activities of glycolytic and mitochondrial enzymes, which were accompanied by a strongly reduced expression of GLUT1. Moderately increased activities of glycolytic and mitochondrial enzymes were observed in the clear cells of clear/acidophilic cell tubules and tumors compared with those in glycogenotic tubules. They had slightly increased activities of the glycolytic enzymes GAPDH and PK compared with normal collecting duct epithelium, while most of them were nearly lacking in GLUT1. Our findings suggest that glycogen storage is not due to an increased uptake of glucose from the blood, but results from a disturbance in intracellular flux of metabolites. The development of clear cell tubules from the normal collecting duct epithelium is accompanied by a markedly decreased expression of GLUT1 along with a reduction in glycolytic and mitochondrial enzymes. This reduction of enzyme activities is replaced by an increase in enzyme activities in clear/acidophilic cell tumors indicating a fundamental shift in carbohydrate metabolism during progression from preneoplastic to neoplastic lesions.     

Expression of L- and M2-pyruvate kinases in proliferating oval cells and cholangiocellular lesions developing in the livers of rats fed a methyl-deficient diet

Male outbred Sprague-Dawley rats were fed a cholinedeficientdiet containing 0.1% w/w DL-ethionine (CDE) for up to 22 weeks.The expression of the pyruvate kinase isoenzymes L (L-PK) andM₂ (M₂ was immunohistochemically analyzed in liver slices fromrats killed 4, 10, 14 and 22 weeks after starting the treatment.M₂ was detected in bile duct epithelial cells of untreated ratsand in proliferating oval cells, cholangiofibroses and cholangiofibromasof CDE-fed animals. Thus, M₂ can be viewed as a positive markerof the bile duct epithellial/oval cell compartment. L-PK, aparenchymal cell-specific protein in untreated rat liver, wasnot present In proliferating oval cells, but was consistentlyobserved In cells that were part of the ductal structures inthe cholangiofibroses and cholanglofibromas. Based on theirmorphology, the L-PK-posltlve duct cells were undoubtedly partof the bile duct epithellal cell lineage and no L-PK-positivehepatocyte-like cells were observed in the ducts. Hence, thisstudy clearly shows that the mere presence of a liver parenchymalcell marker in cells of the bile duct epithelial/oval cell compartmentdoes not necessarily preclude that these cells are undergoinga differentiation into preneoplastic parenchymal cells, as haspreviously been suggested.     

Enhancement of NNM-induced carcinogenesis in the rat liver by phenobarbital: a combined morphological and enzyme histochemical approach

The influence of sodium phenobarbital (PB) treatment on thesequence of N-nitrosomorpholine (NNM) induced focal preneoplasticlesions in the rat liver was investigated using a combined morphologicaland enzyme histochemical approach. Quantitative assessment ofthe different types of foci of altered hepatocytes visible inH&E sections after carcinogen application, namely the clearand acidophilic cell glycogen storage foci and mixed cell focicomprising glycogen storing cells and also more basophilic hepatocytesshowing reduction in glycogen reserves, revealed a shift towardsmixed cell character and greater size in PB-treated livers incomparison to those receiving NNM alone. Within the three doselevels of PB investigated (0.75, 0.075 or 0.0075 g/l drinkingwater) a clear dose dependence in appearance of mixed cell fociwas apparent. Assessment of alterations in the activities ofmarker enzymes observed within preneoplastic foci was carriedout by comparison of PAS preparations with sections reactedfor glucose-6-phosphate dehydrogenase (G6PDH), -glutamyl transpeptidase,glucose-6-phosphatase and adenosine triphosphatase. G6PDH provedthe most consistent enzyme marker for small glycogen storagefoci whereas larger foci of that type and mixed cell foci wereassociated with change in activity of all enzymes studied. Theresults are discussed in relation to the sequence of eventsoccurring during hepatocarcinogenesis and the influence of PBon altered cellular populations. The applicability of enzymemarkers is further considered in view of the question of heterogeneitywithin populations of preneoplastic foci.     

Enzyme histochemical and morphological phenotype of amphophilic foci and amphophilic/tigroid cell adenomas in rat liver after combined treatment with dehydroepiandrosterone and N-nitrosomorpholine

Male Sprague-Dawley rats were investigated after N-nitroso-morpholine(NNM) treatment with concomitant and subsequent administrationof dehydroepiandrosterone (DHEA) for development of pre-neoplasticand neoplastic liver lesions. In addition to clear, acidophilic,mixed cell and basophilic foci, a hitherto undescribed lesiontype demonstrating a unique morphological and histochemicalphenotype was observed in animals receiving both NNM and DHEA.The cells of the majority of these lesions for which we proposethe designation amphophilic foci were characterized by increasedgranular acidophilia and randomly scattered cytoplasmic hasophilia.Histochemically, reduced glycogen content and elevated activityof glucose-6-phosphate dehydrogenase (G6PDH) glyceraldehyde-3-phosphatedehydrogenase (GAPDH), acid phosphatase (AP), succinate dehydrogenase(SDH) and catalase (CAT) were evident. The lack of -glutamyltranspeptidase (GGT) or glutathione S-transferase placentalform (GST-P) in foci of this type allowed clear differentiationfrom other NNM-induced focal lesions while suggesting certainsimilarities to pre-neoplastic cells induced by hypolipidemicagents. Similar enzyme histochemical patterns were characteristicfor foci and later appearing nodules (adenomas) com posed ofamphophilic/tigroid cells the basophilic material of which wasIncreased and frequently arranged in long striped bands. DHEAtreatment, while not itself inducing any preneoplastic foci,was thus associated with altered phenotypic expression of fociand adenomas generated by NNM.     

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.     

Quantitative stereological evaluation of four histochemical markers of altered foci in multistage hepatocarcinogenesis in the rat

Female F344/N rats dosed with diethylnitrosamine (DEN) 24 hafter partial hepatectomy were treated with the promoting agents,phenobarbital (PB) or 3,4,7,8-tetrachlorodibenzo-p-dioxin (TCDD),or the peroxisome proliferating agent, WY 14,643, for 6 months.Another group was subjected to the Solt-Farber protocol. Alteredhepatic foci (AHF) were analyzed by quantitative stereologyfrom frozen serial sections stained for gamma-gtutamyl transferase(GGT), canalicular adenosine triphosphatase (ATPase), glucose-6-phosphatase(G6Pase) and the placental isozyme of glutathione S-transferase(PGST). PGST scored more foci in all groups than GGT and ATPase.PGST marked greater focal volume than GGT or ATPase, and PGSTmarked focal volume equal to or greater than G6Pase in ratstreated with PB, TCDD or the Solt-Farber protocol. However,after treatment with WY 14,643, GGT and PGST marked much lessfocal volume than ATPase or G6Pase, and PGST scored fewer focithan G6Pase. Numerical estimations of foci scored by those markerson the basis of area of the entire tissue section (per cm²)were relatively different from those values determined by quantitativestereology. While these results confirm earlier studies, theydemonstrate the importance of quantitative stereologic analysisof AHF during multistage hepatocarcinogenesis.     

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.     

Biochemical microanalysis of glycogen content and glucose-6-phosphate dehydrogenase activity in focal lesions of the rat liver induced by N-nitrosomorpholine

Persisting focal lesions, namely glycogen storage (glycogenotic)foci and mixed cell foci, were induced in liver by treatmentof rats with the hepatocarcinogen N-nitrosomorpholine in a concentrationof 200 mg/l drinking water for 7 weeks. Four and seven weeksafter withdrawal of the carcinogen, the persisting foci weredissected from freeze-dried cryostat sections and their glycogencontent and glucose-6-phosphate dehydrogenase activity wereanalyzed with highly sensitive luminometrical tests. The focicomposed exclusively of storage cells contained on an average100% more glycogen than the surrounding tissue of normal appearanceor the liver parenchyma of untreated control animals. The overallglycogen content of the mixed cell foci, which were composedof both glycogenotic and glycogen-poor basophilic cells, wasnot distinguishable from that of the normal liver tissue. Theactivity of the G6PDH showed a clear tendency to higher valuesin the majority of the small glycogen storage foci (up to 100ng dissected material). However, in larger glycogenotic fociand in particular in the mixed cell foci the activity of thisenzyme was significantly higher (by a factor of 3 and 6, respectively)than in the surrounding tissue of normal appearance and in theliver parenchyma of untreated controls. The data support theconcept that hepatocarcinogens induce a focal hepatic glycogenstorage disease of the liver which appears to elicit adaptiveenzymatic changes gradually redirecting the disturbed carbohydratemetabolism towards other metabolic pathways, such as the pentosephosphate pathway.     

Gamma-glutamyl transpeptidase and glutathione biosynthesis in non-tumorigenic and tumorigenic rat liver oval cell lines.

Glutathione synthesis and growth properties were studied in the gamma-glutamyl transpeptidase(GGT)-negative, non-tumorigenic rat liver oval cell line OC/CDE22, and in its GGT-positive, tumorigenic counterpart line M22. gamma-Glutamylcysteine synthetase (GGCS) activities were comparable. Growth rates of M22 cells exceeded those of OC/CDE22 cells at non-limiting and limiting exogenous cysteine concentrations. A monoclonal antibody (Ab 5F10) that inhibits the transpeptidatic but not the hydrolytic activity of GGT did not affect the growth rates of OC/CDE22, and decreased those of M22 to the OC/CDE22 level. In GSH-depleted M22, but not in OC/CDE22 cells, the rate and extent of GSH repletion with exogenous cysteine and glutamine exceeded those obtained with exogenous cysteine and glutamate. With Ab 5F10, repletion with cysteine/glutamine was similar to that obtained with cysteine/glutamate. Repletion with exogenous GSH occurred only in M22 cells, and was abolished by the GGT inhibitor acivicin. Repletion with gamma-glutamylcysteine (GGC) in OC/CDE22 was resistant to acivicin whereas that in M22 was inhibited by acivicin. Repletion with exogenous GSH or cysteinylglycine (CG) required aminopeptidase activity and was lower than that obtained with cysteine. Unless reduced, CG disulfide did not support GSH repletion. The findings are compatible with the notions that (i) GGT-catalyzed transpeptidation was largely responsible for the growth advantage of M22 cells at limiting cysteine concentration, and for their high GSH content via the formation of GGC from a gamma-glutamyl donor (glutamine) and cyst(e)ine, and (ii) aminopeptidase/dipeptidase activity is rate-limiting in GSH repletion when GSH or CG serve as cysteine sources.     

gamma-Glutamyl transpeptidase and glutathione biosynthesis in non-tumorigenic and tumorigenic rat liver oval cell lines

Glutathione synthesis and growth properties were studied in the gamma-glutamyl transpeptidase(GGT)-negative, non-tumorigenic rat liver oval cell line OC/CDE22, and in its GGT-positive, tumorigenic counterpart line M22. gamma-Glutamylcysteine synthetase (GGCS) activities were comparable. Growth rates of M22 cells exceeded those of OC/CDE22 cells at non-limiting and limiting exogenous cysteine concentrations. A monoclonal antibody (Ab 5F10) that inhibits the transpeptidatic but not the hydrolytic activity of GGT did not affect the growth rates of OC/CDE22, and decreased those of M22 to the OC/CDE22 level. In GSH-depleted M22, but not in OC/CDE22 cells, the rate and extent of GSH repletion with exogenous cysteine and glutamine exceeded those obtained with exogenous cysteine and glutamate. With Ab 5F10, repletion with cysteine/glutamine was similar to that obtained with cysteine/glutamate. Repletion with exogenous GSH occurred only in M22 cells, and was abolished by the GGT inhibitor acivicin. Repletion with gamma-glutamylcysteine (GGC) in OC/CDE22 was resistant to acivicin whereas that in M22 was inhibited by acivicin. Repletion with exogenous GSH or cysteinylglycine (CG) required aminopeptidase activity and was lower than that obtained with cysteine. Unless reduced, CG disulfide did not support GSH repletion. The findings are compatible with the notions that (i) GGT-catalyzed transpeptidation was largely responsible for the growth advantage of M22 cells at limiting cysteine concentration, and for their high GSH content via the formation of GGC from a gamma-glutamyl donor (glutamine) and cyst(e)ine, and (ii) aminopeptidase/dipeptidase activity is rate-limiting in GSH repletion when GSH or CG serve as cysteine sources.     

Sequential histochemical and morphometric studies on preneoplastic and neoplastic lesions induced in rat colon by 1 ,2-dimethylhydrazine

The sequential histochemical changes during colon carcinogenesiswere studied in male Sprague-Dawley rats given 16 weekly subcutaneousinjections of 15 mg 1,2-diinethyl- hydrazine per kg body wtand serially killed at regular in tervals. Cryostat sectionswere used to study the mucus content of the colonic mucosa withthe periodic acid Schiff's reaction, and enzyme histocheinicalmethods were applied to investigate the activity of some keyenzymes of carbohydrate metabolism at different stages of carcinogenesis.Enlarged mucus-rich crypts with a marked hypercellularlty (149%of control as determined morpbometrically) appearing very earlyduring carcinogenic treatment revealed almost normal activitiesof glucose-6-phosphatase (G6Pase), glucose-6-phosphate dehydrogenase(G6PDH) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH).Hyperbasophllic crypts lacking mucus production were observedlater and showed a loss of G6Pase, but marked increase of G6PDHand GAPDH activity. Mucus-rich signet ring cell carcinomas showedthe same enzymatic pattern as the mucus-rich crypts, whereasmucus-free adenocarcinomas and undifferentiated carcinomas revealeda loss of G6Pase and highly increased G6PDH and GAPDH activities.The results showed that focal changes in polysaccharide contentand in the activity of some enzymes of carbohydrate metabolism,as observed in various organs, also accompany the carcinogenicprocess in the colon. This supports the concept that aberrationsin carbohydrate metabolism play an important role during theprocess of carcinogenesis.     

Differential expression of alpha-fetoprotein and gamma-glutamyltranspeptidase in chemical and spontaneous hepatocarcinogenesis.

The expression of two markers of fetal liver, alpha-fetoprotein (AFP) and gamma-glutamyltranspeptidase (GGT), was studied in chemical and spontaneous hepatocarcinogenesis in mice. Serum AFP concentration increased within 3 weeks 3 weeks from the commencement of feeding of o-aminoazotuluene. This early elevation subsided about 3 months after the beginning of the administration of the carcinogen. A new, sustained elevation of the serum AFP level followed at 5 to 6 months accompanied by the appearance of liver tumors. In immunofluorescence, some small oval cells and scattered adult-type hepatocytes contained AFP during the early stage of chemical carcinogenesis. During the later phase, AFP was detected in a few of the nodular areas, in solitary hepatocytes, and in groups of carcinoma cells. GGT activity in the liver increased within 1 week after the carcinogen regimen was started, preceding the early increase of AFP production. At the final stage, the chemically induced hepatomas contained about 80 times more GGT than did normal liver. In histochemical staining, proliferating oval cells and small areas of hepatocytes stained for GGT during the early weeks, and later most nodules, small areas of nonnodular parenchyma, and carcinomas contained GGT. During spontaneous carcinogenesis in male C3HeB/FeJ mice, premalignant lesions, accompanied by a slight increase of serum AFP, precede the appearance of liver tumors. No cells staining for AFP were detected during this early stage. Once overt liver cancers had developed, AFP was readily detectable in the tumors and was localized to some but not all carcinoma cells. The corresponding serum AFP levels were highly elevated. In contrast to the high levels of GGT found during chemical carcinogenesis, no elevation of GGT was found in livers at various stages of spontaneous carcinogenesis, including cancers in eight individual mice. These results indicate that the production of AFP and GGT is not turned on as a single "genetic package," and that these two markers differ in their behaviour in liver carcinogenesis.     

Effects of low doses of N-nitrosomorpholine on the development of early stages of hepatocarcinogenesis

Male Sprague-Dawley rats received the hepatocarcinogen N-nitrosomorpholine(NNM) in the drinking water at low dose levels ranging from6 mg/1 to 60 mg/1 for 6 and 12 weeks, respectively. Foci ofaltered hepatocytes (FAH) were demonstrated histochemicallyusing changes in the activities of glucose-6-phosphate dehydrogenaseand glycogen phos-phorylase, and in the glycogen content asmarkers. Proliferating cells were detected by the immunohistochemicalreaction for proliferating cell nuclear antigen (PCNA). Thenumber and size of foci of altered hepatocytes increased ina time and dose-related manner. The dose-effect curves werenon-linear with a slight positive slope at the low doses anda markedly increased slope at higher doses. The number of PCNApositive hepatocytes showed a dose-dependent increase. In additionto the granular distribution of PCNA in the nuclei, hepatocytenuclei with homogeneously distributed PCNA occurred in animalsexposed to 60 mg/1 NNM. It is proposed that these cells arerelated to the occurrence of hepatocytes with higher ploidyinduced by NNM and may be regarded as cells in the G2 phaseof the cell cycle. The non-linear shape of the dose-response-curveof the FAH suggests that some mechanisms contribute to carcinogenesisover the whole dose range, whereas other mechanisms enhancecarcinogenesis only at higher doses. The relevance of the non-lineardose-effect curve for the risk assessment of carcinogens isdiscussed.     

Decreased sensitivity to phalloidin of normal-looking rat hepatocytes after short-term 2-acetylaminofluorene feeding

Male F344/DuCrj rats were fed a diet containing 0.02% 2-acetylaminofluorene (2-AAF) for 1 or 3 weeks, and then fed a basal diet for 2 days, 2 weeks, 8 weeks, 22 weeks or 36 weeks. Hepatocytes were isolated from the liver by collagenase perfusion, and their sensitivity to phalloidin, in terms of the formation of multiple cytoplasmic blebs, was examined. The sensitivity of gamma-glutamyltransferase (GGT)-negative hepatocytes decreased on the 22nd and 36th weeks after withdrawal of 2-AAF feeding, and that of GGT-positive cells decreased on the 36th week. Induction of a small number of foci positive for the placental form of glutathione S-transferase (GSTP) was observed in the liver of all rats on the 8th, 22nd and 36th weeks after the withdrawal of the carcinogen. However, the total area of the foci was estimated to account for less than 0.2% of liver tissues even on the 36th week. Therefore, the decrease in phalloidin sensitivity of hepatocytes, particularly of GGT-negative hepatocytes, on the 22nd and 36th weeks after 2-AAF withdrawal is suggested to be a result of a decrease in the sensitivity of otherwise normal-looking hepatocytes, which may be precursors of the cells forming the preneoplastic foci.     

Decreased Sensitivity to Phalloidin of Normal-looking Rat Hepatocytes after Short-term 2-Acetylaminofluorene Feeding

Male F344/DuCrj rats were fed a diet containing 0.02% 2-acetylaminofluorene (2-AAF) for 1 or 3 weeks, and then fed a basal diet for 2 days, 2 weeks, 8 weeks, 22 weeks or 36 weeks. Hepatocytes were isolated from the liver by collagenase perfusion, and their sensitivity to phalloidin, in terms of the formation of multiple cytoplasmic blebs, was examined. The sensitivity of gamma-glutamyltransferase (GGT)-negative hepatocytes decreased on the 22nd and 36th weeks after withdrawal of 2-AAF feeding, and that of GGT-positive cells decreased on the 36th week. Induction of a small number of foci positive for the placental form of glutathione S-transferase (GSTP) was observed in the liver of all rats on the 8th, 22nd and 36th weeks after the withdrawal of the carcinogen. However, the total area of the foci was estimated to account for less than 0.2% of liver tissues even on the 36th week. Therefore, the decrease in phalloidin sensitivity of hepatocytes, particularly of GGT-negative hepatocytes, on the 22nd and 36th weeks after 2-AAF withdrawal is suggested to be a result of a decrease in the sensitivity of otherwise normal-looking hepatocytes, which may be precursors of the cells forming the preneoplastic foci.     

The effects of alternating dietary restriction and ad libitum feeding of mice on the development of diethylnitrosamine-induced liver tumours and its correlation to insulinaemia.

The effects of alternating ad libitum feeding and 30% restriction of the dietary intake on the development of diethylnitrosamine (DEN)-induced hepatic neoplasia were investigated. Dietary restriction retarded the growth of glucose-6-phosphatase-deficient (G6Pd) preneoplastic foci and subsequently that of hepatocellular adenomas and adenocarcinomas. The number of foci in standardized liver sections increased from 4.44 foci/cm2 at 12 weeks to 9.65 foci/cm2 at 24 weeks in ad libitum fed animals but only from 2.35 foci/cm2 to 3.29 foci/cm2 in restricted animals. In animals fed first ad libitum for 12 weeks and then for 12 weeks on a restricted diet, the number of G6Pd foci dropped from 4.44 at 12 weeks to 3.54 at 24 weeks. This reduction appeared to be the result of a regression of the small sized G6Pd foci. Dietary restriction was most efficient in inhibiting the development of G6Pd foci when started early in life. Conversely, the growth of foci was stimulated when the mice first had restricted access to food and thereafter were fed ad libitum. The plasma insulin concentrations after a glucose challenge increased with age. Insulinaemia was much higher in ad libitum fed compared to the restricted mice. It was correlated to the number of G6Pd foci in the liver. This study suggests that insulin, which is a known mitogen for hepatocytes in vitro, may contribute to the promotion of DEN-induced liver tumours in mice.