Correlative histochemistry of some enzymes of carbohydrate metabolism in preneoplastic and neoplastic lesions in the rat liver

The livers from a total of 51 Sprague-Dawley rats treated withdifferent doses of N-nitrosomorpholine (80–120 mg/l inthe drinking water) for up to 14 weeks together with the liversof 28 control animals were histochemically investigated at thecessation of carcinogenic insult and at varying periods thereafterfor their glycogen content, basophilia and activities of variousenzymes of carbohydrate metabolism: glycogen synthetase, glycogenphosphorylase, glucose-6-phosphatase, glyceraldehyde-3-phosphatedehydrogenase and glucose-6-phosphate dehydrogenase. The enzymaticpatterns of normal tissue, preneoplastic and neoplastic lesionswere characterized and compared with reference to the morphologicallydefined stages of tumor development in the liver. The earlyappearing glycogen storing areas, localized in the peripheraland intermediate lobular regions, did not show significant changesin the histochemically demonstrable activities of the enzymestested. After cessation of the carcinogen treatment the morepronounced glycogen storage foci which developed within theaforementioned regions of the liver acinus usually showed areduction in the activities of phosphorylase and glcose-6-phosphatasewhile the activity of glucose-6-phosphate dehydrogenase, a keyenzyme for the pentose phosphate pathway, was increased. Themixed cell foci, neoplastic nodules and tumors which emergedat later stages were characterized by a progressive shift awayfrom glycogen metabolism towards glycolysis and the pentosephosphate pathway, as indicated by an increase in glyceraldehyde-3-phosphatedehydrogenase and glucose-6-phosphate dehydrogenase activities.These changes in enzyme pattern are supportive of a developmentalsequence leading from glycogen storage foci through mixed cellfoci and neoplastic nodules to hepatocellular carcinomas.     

Histochemical characterization of focal hepatic lesions induced by single diethylnitrosamine treatment in infant mice

Focal hepatocellular lesions, induced in our infant mouse system (15-day-old B6C3F1 mice) by a single carcinogenic dose of diethylnitrosamine (2.5 or 5.0 micrograms/g body weight), were characterized histochemically using toluidine blue, periodic acid-Schiff, glycogen phosphorylase, glycogen synthetase, glucose-6-phosphatase, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, ATPase, gamma-glutamyl transpeptidase, and acid phosphatase. Animals were killed 5, 12, 18, and 24 weeks following diethylnitrosamine treatment. The first focal lesions were observed in mice killed at 12 weeks. All foci showed patchy cytoplasmic basophilia and a slight decrease in the glycogen content. The early foci (12 weeks) showed no change in the levels of glycogen phosphorylase and glycogen synthetase, a strong reduction of glucose-6-phosphatase, and a high increase in glucose-6-phosphate dehydrogenase. In addition, 56% of foci in males and 86% of foci in females showed a slight rise in glyceraldehyde-3-phosphate dehydrogenase, and 12% of foci in males and 17% of foci in females had a lower acid phosphatase. The level of cytoplasmic ATPase was slightly decreased in 22% of foci. By 24 weeks, a decrease in the activity of cytoplasmic ATPase was observed in 84 and 100% of foci in males and females, respectively. The increase in the membrane ATPase was observed in 65% of foci in males and 7% of foci in females. By that time, the decrease in acid phosphatase was observed in 78% of foci in males and 37% of foci in females. The gamma-glutamyl transpeptidase failed to show any increase in its activity, indicating that this enzyme was not a "marker" of the hepatocellular lesions developing under the experimental conditions. Strong decrease in glucose-6-phosphatase in association with a manifest increase in glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase activities indicated a shift from gluconeogenesis to glycolysis. Since this metabolic shift occurred concurrently with an increase in the labeling indices and focal size, it appears that these changes act in concert, representing expression of the acquired functional and replicating potential of the focal cell population.     

Focal hepatic glycogenosis

Foci of altered hepatocytes (FAH) including clear cell foci excessively storing glycogen (focal hepatic glycogenosis) are well known as preneoplastic lesions in animal models of hepatocarcinogenesis induced by chemical, physical or viral agents. The occurrence of similar lesions has been studied in a series of 67 explanted and 2 resected human livers using histological and histochemical approaches. A high incidence of FAH was found in the liver of patients suffering from hepatocellular carcinoma(HCC, 14/14) and liver cirrhosis (21/42). FAH were also detected in one patient each with inborn hepatic glycogenosis type 1a, and cholangiocellular carcinoma. Two patients with focal nodular hyperplasia had FAH-like enzymatic changes within these lesions. No FAH were found in 5 donor livers. FAH excessively storing glycogen including clear and mixed cell foci predominated in most cases with these lesions. The focal hepatic glycogenosis was associated with a significantly increased cell proliferation compared to the extrafocal parenchyma, and with alterations in the activity of various enzymes. In the 175 FAH studied by enzyme histochemistry, two enzymes involved in glycogen breakdown, namely glycogen phosphorylase and glucose-6-phosphatase, showed the most consistent changes, being reduced in 98% and 95%, respectively. In addition, the activities of adenosine triphosphatase and gamma-glutamyltransferase were reduced in 46% and 53% of FAH, respectively. Inconsistent changes were observed in FAH concerning a number of other enzymes. The 14 HCCs investigated histochemically often contained clear cell populations rich in glycogen in well differentiated portions, but were poor in glycogen in moderately and poorly differentiated tumors or tumor components. There were some similarities in the enzyme histochemical pattern of HCC and FAH but also important differences were evident. In contrast to FAH, all HCCs (except one carcinoma of the fibrolamellar type) showed an increase in the activity of the mitochondrial glycerol-3-phosphate dehydrogenase, and 50% of the cases had increased glucose-6-phosphate dehydrogenase activity. The activities of glucose-6-phosphatase and gamma-glutamyltransferase usually showed a reactivation, or even an increase compared to the extrafocal parenchyma, in moderately and poorly differentiated HCCs. Our results indicate that the focal hepatic glycogenosis is a putative preneoplastic lesion in human beings similar to laboratory animals. The focal hepatic glycogenosis appears to be a frequent initial step in neoplastic transformation of hepatocytes, a process associated with a fundamental shift in energy metabolism.     

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.     

Enzyme histochemical characteristics of spontaneous and induced hepatocellular neoplasms in mice

Hepatocellular neoplasms are known to differ in enzyme activityfrom the surrounding non-neoplastic liver. We have comparedhistochemically the enzyme activity of spontaneous hepatocellulartumors in mice with tumors induced by diethylnitrosamine anddieldrin. Some neoplasms had increased activity, others haddecreased enzyme activity, yet others had the same activityas the surrounding liver. Alkaline phosphatase, glucose-6-phosphatase,succinic dehydrogenase and adenoane triphosphatase, as wellas glycogen levels were studied. Carcinomas differed from adenomasin having elevated enzyme activity significantly more oftenthan adenomas. However, the carcinomas showed elevated glycogenlevels less frequently than adenomas. Histochemically, pulmonarymetastases resembled the primary hepatocellular carcinomas fromwhich they were derived. Tumors of dieldrin animals were notablein having increased activity of all the enzymes which we studiedmore frequently than tumors of diethylnitrosamine animals orof controls. Differences in enzyme activity between the threemouse strains were slight.     

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.     

Enhancement of hepatocarcinogenesis in rats by dietary fructose

The effects of oral fructose on hepatocarcinogenesis were investigated with cytomorphological, cytochemical and stereological methods. Carcinogenesis was induced in male Sprague-Dawley rats by application of N-nitrosomorpholine (NNM) for 7 weeks. Afterwards, the animals received fructose in the drinking water (120 g/l) and food ad libitum (group I) or tap water and food ad libitum (group II). The incidence of hepatocellular carcinoma in rats treated with NNM plus fructose was 46% as compared to 24% in animals receiving NNM alone (P less than 0.05). There was no difference in the incidences of other malignancies between the groups (group I: 32.1%, group II: 32.0%). Morphometric evaluation of preneoplastic liver lesions indicated the enhancing effect of the fructose treatment several months before malignant tumors appeared. As early as 6 weeks after treatment the hepatic parenchyma occupied by focal lesions was increased from 6.7% in the animals which had received NNM alone to 8.5% (P less than 0.05) in animals having received NNM plus fructose. This increase was predominantly caused by an increase in glycogen storing foci (P less than 0.0005). In addition, the fructose treatment caused a histochemically detectable increase in the activity of glucose-6-phosphatase and glucose-6-phosphate dehydrogenase in both the hepatocytes of the focal lesions and the surrounding parenchyma. In the NNM plus fructose group the activity of the glucose-6-phosphatase in the foci was frequently approximately equal to the activity in the parenchyma of untreated controls. The striking increase in the activity of this enzyme in the surrounding hepatocytes, however, still sharply demarcated the lesions. The potential mechanisms by which fructose enhances hepatocarcinogenesis are discussed.     

Sequential histological and histochemical study of the rat liver during aflatoxin B1-induced carcinogenesis.

Male Wistar rats were given 50 mug of aflatoxin B1 twice a week for 4 weeks, and thereafter 75 mug twice a week for 10 weeks. Their livers were investigated histologically and histochemically for glycogen, RNA, fat, alkaline and acid phosphatases, adenosine triphosphatase, 5'-nucleotidase, glucose-6-phosphatase, glucose-6-phosphate dehydrogenase, succinic dehydrogenase, and alkaline and acid nucleases. No significant lesions occurred before 15 weeks. During this period, the liver was histochemically unchanged except for a periportal decrease of alkaline phosphatase and adenosine triphosphatase. Scattered hepatocytes with a strong glucose-6-phosphatase activity appeared. These changes represent toxic effects of aflatoxin B1 and are irrelevant to carcinogenesis. From 15 weeks onward, three types of liver cell hyperplastic foci and nodules developed. Histologically, and with respect to glycogen, fat, and RNA content, only two of these types were considered as potential precursors of hepatocarcinomas. However, all types exhibited a decrease or absence of the enzymes studied. Both histological and histochemical changes stressed the complex heterogeneity existing between and within hepatic foci and nodules. From 11 months on, hepatocarcinomas developed. The tumors disclosed similar histochemical changes. This similarity further supports the "precarcinomatous" nature of hyperplastic foci and nodules. It appears that focal changes in surface as well as in cytoplasmic and nuclear enzymes are intimately and very early linked to the carcinogenic process. Whether they are fundamental or only represent an epiphenomenon remains unclear.     

Dehydroepiandrosterone induced alterations in rat liver carbohydrate metabolism

Long-term dietary administration of the adrenal hormone dehydroepiandrosterone (DHEA) to male Sprague-Dawley rats induced significant alterations in the activities of enzymes involved in liver carbohydrate metabolism. Although glycogen synthase activity was increased and phosphorylase decreased, glycogen stores were reduced. This was presumably related to lysosomal glycogen degradation, since alpha-glucosidase was increased. All rate-limiting enzymes of glucose metabolism which were studied (glucose-6-phosphate dehydrogenase, total hexokinases, pyruvate kinase, fructose-1,6-bisphosphatase) revealed markedly reduced activity, only glucose-6-phosphatase activity was increased. These enzymatic changes point to a far-reaching metabolic shift towards energy loss via decreased glucose consumption and increased glucose output. The enzyme pattern induced by DHEA is in many respects opposite to that induced in preneoplastic and neoplastic liver lesions by chemical hepatocarcinogens.     

Development of liver tumors in transforming growth factor alpha transgenic mice.

We studied the development of liver tumors in male transforming growth factor alpha (TGF-alpha) transgenic mice of the CD1 strain and examined the expression of the transgene by immunohistochemistry and in situ hybridization. Livers of 4-5-week-old transgenic mice contained areas of centribobular hypertrophy with low glucose-6-phosphatase activity. These areas progressively expanded, and hypertrophy and dysplasia became generalized in livers of mice at 10-12 months of age. The expression of the transgene, determined by either immunohistochemistry or in situ hybridization, was uneven in animals that were 10 weeks old or older. The positive hepatocytes formed patches with a predominant centrilobular distribution. We studied a total of 23 liver tumors (7 hepatocellular carcinomas and 16 adenomas) obtained from 11 mice at 13-15 months of age and from one 7-month-old animal which received zinc sulfate to induce the transgene. The carcinomas were well differentiated tumors, without glucose-6-phosphatase or gamma-glutamyltranspeptidase activity, that developed from the dysplastic parenchyma and occasionally within an adenoma. In all carcinomas and in 56% of the adenomas there was overexpression of the transgene in relationship to the surrounding tissue. The majority of the tumors that overexpressed TGF-alpha were alpha-fetoprotein positive, while alpha-fetoprotein staining was not detected in tumors (all adenomas) that did not show excessive transgene expression. We conclude that TGF-alpha functions as a promoter of liver carcinogenesis through its effect as an autocrine inducer of hepatocyte proliferation. Further, the data indicate that TGF-alpha overexpression may favor tumor progression.     

Influence of phenobarbital on glycogen metabolism of rat liver pretreated with N-nitrosomorpholine

Sprague-Dawley rats were treated with N-nitrosomorpholine. (NNM)alone (7 weeks, 120 mg/I in drinking water), with NNM followedby phenobarbital (PB) (750 mg/l for 6 weeks) or PB alone. Thelivers from these animals were investigated for glycogen contentand activities of glucose-6- phosphatase, glucose-6-phosphatedehydrogenase, glycogen phosphorylase and glycogen synthetase.The following parameters proved to be significantly alteredin the livers of rats treated with either NNM or PB or bothcompared with untreated controls: glycogen content was increasedand the activities of glucose-6-phosphalase and glycogen synthetasewere decreased. Although these data show some similarities inchanges of glycogen metabolism of livers treated with NNM orPB, earlier histochemical investigations revealed importantdifferences in the distribution of these alterations withinthe liver parenchyma.     

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.     

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.     

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.     

Phenotypic instability in focal and nodular lesions induced in a short term system in the rat liver

A comparative morphologic, morphometric and enzyme histochemicalinvestigation of lesions induced by short-term application ofN-nitrosomorpholine (NNM) and subsequent so-called ‘selectionpressure’ was carried out in order to assess the characteristicsof the numbers of induced putative preneoplastic populationsand to cast light on reversibility associated with this model.The glycogen storage foci, mixed cell foci and neoplastic nodulesobserved after ‘selection pressure’ were in principlesimilar to those seen after stop experiments, although alterationsin morphology and enzyme phenotype of individual cells wereusually far more pronounced after short-term induction. It wasestablished that 75% of the lesions were no longer visible 11weeks after withdrawal of induction stimuli and that a largeproportion of these remaining demonstrated heterogeneity inmorphological and histochemical markers indicative of reversionto normal phenotype. After a further 10 weeks a slight increasein number of foci associated with decrease in size and enhancedhomogeneity in phenotypic markers was established. The behaviourof foci and nodules undergoing reversion was considered withrespect to changes in basophilia and glycogen storage and activityof the enzymes glucose-6-phosphate dehydrogenase, glucose-6-phosphatase,glyceraldehyde 3-phosphate dehydrogenase, glycogen phosphorylaseand synthase, acid phosphatase and -glutamyl transpeptidaseand correlated with location of altered cellular populationswithin the liver functional acinus.     

Persistence of the cholangiocellular and hepatocellular lesions observed in 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 (CDE) for 4, 6, 10, 14 or 22 weeks followed by a standard diet for up to 59 weeks. Liver sections were histochemically analyzed for the following parameters: basophilia, glycogen content and the activities of glycogen synthase (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 stop experiments revealed that many of the oval cells proliferating during the first 4-6 weeks may undergo necrotic changes and disappear with time, whereas cholangiofibroses appearing in animals fed CDE for at least 10 weeks are persistent lesions. The sequence of lesions seen in this study, leading from persistent oval cells through cholangiofibroses to cholangiofibromas, strongly suggests that the oval cells are the precursor cells of cholangiocellular tumors. The proliferating oval cells and the hepatic foci consisting of clear and acidophilic or mixed cell populations were always spatially separated and no transitions between oval and parenchymal cells were observed. These results argue against a precursor-product relationship between oval and parenchymal cells. Both proliferating and persistent oval cells, cholangiofibroses and cholangiofibromas showed a strong staining for G6PDH, GAPDH, G3PDH, MDH, ALKPASE and GGT; low PHO, SYN and G6PASE activities were also detected in these lesions. Persistent glycogen-storage foci, which developed in all rats fed CDE for 4-14 weeks followed by a normal lab chow for over a year, had increased PHO, G6PDH, MDH, ALKPASE and GGT activities, while SYN, GAPDH and G3PDH activities remained unaltered and G6PASE activity decreased. Mixed cell foci appearing in animals fed CDE for 22 weeks followed by a normal lab chow for 59 weeks had strongly increased G6PDH, GAPDH, G3PDH, MDH, ALKPASE and GGT activities as well as decreased G6PASE activity. These results indicate that the characteristic metabolic pattern of preneoplastic hepatic foci is independent of the further administration of the carcinogenic diet. The shift from glycogen metabolism to glycolysis and the pentose phosphate pathway occurring during the later stages of CDE-induced hepatocarcinogenesis is an autogenous process apparently directing the disturbed carbohydrate metabolism towards alternative metabolic pathways. A similar metabolic shift also seems to take place during cholangiocarcinogenesis.     

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.     

Loss of adenylate cyclase activity in preneoplastic and neoplastic lesions induced in rat liver by N-nitrosomorpholine

Adenylate cyclase (AC) activity was demonstrated histochemicallyusing adenylate-(ß, -methylene)diphosphate as substratein cryostat sections of livers from 45 rats treated for 7–10weeks with N-nitrosomorpholine (NNM) (120 mg/1 drinking water)and from nine untreated control rats. The enzyme patterns ofnormal tissue, preneoplastic and neoplastic lesions were characterizedand correlated with the morphologically defined stages of tumourdevelopment in the liver. Light microscopically, the enzymeactivity of normal tissue was restricted to the plasma membrane,and was most pronounced along the bile canaliculi of the hepatocytes.In glycogen storage foci and mixed cell foci induced by NNMno, or only very weak, AC activity was visible. In the cellsof neoplastic nodules and hepatocellular carcinomas AC activitywas also clearly reduced. However, in small parts of the plasmamembrane which lined lumina resembling normal bile canaliculiand in cytoplasmic vesicles closely associated with these structures,some AC activity was occasionally detected by light and electronmicroscopy. Whereas the tissue of normal appearance surroundingthe lesions showed a marked increase in AC activity in the presenceof glucagon, forskolin and cholera toxin. AC activity in thepreneoplastic and neoplastic liver lesions could not, or couldonly weakly, be stimulated by this treatment. As demonstratedin serial sections of the foci, the reduction in AC activitycorresponded to changes in the activity of other enzymes studiedearlier in the same model. Thus the reduction in AC activitywas accompanied by a decrease in the activity of glucose-6-phosphataseand glycogen phosphorylase, and by an increase in the activityof glucose-6-phosphate dehydrogenase. The results support theconcept that the focal changes in the activity of many enzymes(including those of carbohydratemetabolism) during hepatocarcinogenesisare the consequence of aberrations in superordinate regulatorymechanisms of cell metabolism.