Fritz Klimek

Hepatocellular glycogenosis and related pattern of enzymatic changes during hepatocarcinogenesis

Systematic studies of the sequence of cellular changes during hepatocarcinogenesis induced predominantly in rats by stop experiments with N-nitrosomorpholine (NNM) led to the following main results and conclusions: The development of hepatocellular tumors is preceded by a multifocal hepatic glycogen storage disease (glycogenosis). Cytomorphological and cytochemical findings suggest a sequence of focal changes leading from clear and acidophilic glycogen storage foci through mixed cell foci and neoplastic nodules to hepatocellular carcinomas. The clear and acidophilic glycogen storage cells persisting after withdrawal of the carcinogen apparently represent a preneoplastic cell population, the neoplastic transformation of which is accompanied by a gradual reduction of glycogen and a concomitant increase in ribosomes (basophilia). The first appearance and frequency of the different liver lesions investigated was shown to depend on the dose of carcinogen administered. With increasing dose of NNM, the number of focal lesions considerably increased, and this was accompanied by an earlier development of mixed and basophilic cell populations. There was no indication of any reversibility of pronounced focal lesions under the experimental conditions chosen. On the contrary, the foci became larger and acquired phenotypic markers closer to neoplasia independent of further action of the carcinogen. Enzyme histochemically, the majority of the pronounced glycogen storage foci showed a reduction in the activities of glycogen phosphorylase and glucose-6-phosphatase while the activity of glucose-6-phosphate dehydrogenase, a key enzyme for the pentose phosphate pathway, was increased. The mixed cell foci, neoplastic nodules and carcinomas which emerged at later stages were characterized by a progressive shift away from glycogen metabolism towards glycolysis and the pentose phosphate pathway. as indicated by an increase in glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase activities. These changes in enzyme pattern are in keeping with a developmental sequence leading from glycogen storage foci through mixed cell foci and neoplastic nodules to hepatocellular carcinomas. Biochemical microanalysis of dissected glycogen storage foci and mixed cell foci revealed that the foci composed exclusively of storage cells contained on an average 100% more glycogen than the normal liver tissue. The overall glycogen content of the mixed cell foci, which were composed of both glycogenotic and glycogen-poor basophilic cells, was not distinguishable from that of normal tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Significance of Sequential Cellular Changes Inside and Outside Foci of Altered Hepatocytes During Hepatocarcinogenesis

A variety of phenotypic cellular changes emerge in the liver of different species prior to the appearance of hepatocellular adenomas and carcinomas induced by carcinogenic agents (chemicals, radiation, hepadna viruses) or develop “spontaneously.” Foci of altered hepatocytes have been studied most extensively in rats treated with chemical carcinogens; they are considered preneoplastic lesions and have been used in several laboratories as endpoints in carcinogenicity testing. The principles and problems of the morphological classification of foci of altered hepatocytes are presented. In addition to the 4 types of foci generally accepted (clear, acidophilic, basophilic and mixed cell foci), further subtypes (intermediate cell foci) or other types of foci, namely tigroid cell foci and amphophilic cell foci, have more recently been separated as distinct pathomorphological entities. Whereas the amphophilic foci might result from a modulation of clear and acidophilic cell foci, the tigroid cell foci apparently represent a stage in a separate cell lineage leading to hepatocellular adenomas. It remains open whether the tigroid cell foci may also progress to carcinomas. Extrafocal phenotypic changes of hepatocytes might also be involved in hepatocarcinogenesis. The cellular phenotypes within foci also depend strongly, among many other factors, on the dose and duration of the carcinogenic treatment. Cytomorphological, cytochemical, microbiochemical and stereological studies suggest that the predominant sequence of cellular changes during hepatocarcinogenesis leads from the clear and acidophilic cell foci storing glycogen in excess through mixed cell foci and nodules to basophilic cell populations prevailing in hepatocellular carcinomas. A multitude of metabolic aberrations is associated with the sequential cellular changes. Aberrations in carbohydrate metabolism are particularly prominent and might be causally related to the neoplastic transformation of the hepatocytes.

Biological markers of preneoplastic foci and neoplastic nodules in rodent liver

A variety of cellular lesions arise during liver carcinogenesis by chemicals in rats. The altered focus is a lesion of hepatocytes that occurs early in hepatocarcinogenesis and appears to give rise to both neoplastic nodules and carcinomas. Foci display numerous phenotypic abnormalities which together with nuclear abnormalities indicate that they are truly a new altered population. Using histochemical markers, the induction of foci can be measured as a quantitative index of hepatocarcinogenicity of genotoxic carcinogens. In addition, determination of the effect of agents on foci previously induced by genotoxic carcinogens can be used to assess the capacity of chemicals to act as liver tumor promoters.

Upregulation of the Insulin Receptor and Type I Insulin-Like Growth Factor Receptor Are Early Events in Hepatocarcinogenesis

The molecular mechanisms underlying the development of hepatocellular carcinoma (HCC) are not yet fully understood. Preneoplastic foci of altered hepatocytes regularly precede HCC in various species. The predominant earliest type of foci of altered hepatocytes, the glycogen storage focus (GSF), shows an excess of glycogen (glycogenosis) in the cytoplasm. During progression from GSF to HCC, the stored glycogen is gradually reduced, resulting in complete loss in basophilic HCC. We have previously shown that in N-nitrosomorpholine–induced hepatocarcinogenesis, insulin receptor substrate (IRS-1) is strongly expressed in GSF and reduced during progression to HCC, thus correlating with the glycogen content. In the present study, we observed increased levels of insulin receptor, IGF-I receptor (IGF-IR), IRS-2, and mitogen-activated kinase/extracellular regulated kinase-1 in GSF, following the same pattern of expression as IRS-1. We conclude that the abundance of IRS-1, IRS-2, and mitogen-activated kinase/extracellular regulated kinase-1 coincides with a concerted upregulation of both IR and IGF-IR induced by the hepatocarcinogen. Our data suggest that in early hepatocellular preneoplasia, the upregulation of IR elicits glycogenosis through IRS-1 and/or IRS-2, whereas the increased level of the IGF-IR may lead to the increased cell proliferation previously reported in GSF. Therefore, the concerted upregulation of both IR and IGF-IR may represent initial events in hepatocarcinogenesis.

Histochemical and microbiochemical demonstration of reduced pyruvate kinase activity in thioacetamide-induced neoplastic nodules of rat liver

SummaryA new method for the histochemical demonstration of pyruvate kinase (PK) activity was developed using a semi-permeable membrane and ATP-dependent phosphorylation of glucose coupled with tetrazolium reduction via glucose-6-phosphate dehydrogenase (G6PD) in order to investigate normal liver tissue nd neoplastic hepatic nodules induced by thioacetamide (TAA). A series of control reactions and comparison with microbiochemical analysis of microdissected lyophilised material were used to determine the specificity of the reaction. In agreement with earlier reports, an activity gradient in control liver decreasing from zone 3 to zone 1 was apparent both histochemically and after biochemical analysis. Liver neoplastic nodules induced by 25 weeks dietary thioacetamide administration and characterized by increased G6PD demonstrated a clear decrease in PK activity. In contrast, epithelial cells within areas of cholangio-fibrosis thought to be direct precursors for cholangioccllular tumour development were characterized by a strong increase. Comparison of the results with immunohistochemical and biochemical data from the literature indicate that the specific histochemical method described will be of great assistance in future assessment of disease and physiological alteration in activity of this key enzyme of glycolysis.

Collecting duct origin of rat renal clear cell tumors

SummaryThe renal tubular segment from which clear cell tumors originate was investigated in the kidneys of rats treated with N-nitrosomorpholine. This tumor type, which in the rat closely resembles that in man, is made up of clear and granular acidophilic cells and arises from tubules lined by clear cells. The tubular origin of the tumors was established in serial sections by demonstrating connections between both clear cell tumors and tubules lined by clear cells, and renal tubules of normal appearance. In 45 clear cell lesions (17 tumors and 28 tubules) one or more such connections were identified which belonged to the collecting system. In accordance with their localisation in the kidney, the clear cell lesions were connected predominantly to tubules of the cortical collecting system and occasionally to outer medullary collecting ducts. As previously reported, oncocytic tubules and microoncocytomas were observed to originate from the same portions of the collecting system. Rarely, microadenomas and tubules consisting of both oncocytes and clear or granular acidophilic cells were also observed in the kidneys studied.

Specificity of cytochemical demonstration of adenylate cyclase in liver using adenylate-(β,γ-methylene) diphosphate as substrate

SummaryAdenylate cyclase activity was demonstrated cytochemically in rat liver for the first time under the light microscope using cryostat sections mounted on glass cover slips and fixed with 1% glutaraldehyde for 1 min. Adenylate-(β, γ-methylene)diphosphate (AMP-P(CH2)P) was introduced as a new substrate for adenylate cyclase. It was found that adenylate cyclase was distributed heterogenously within the liver lobule. The enzyme activity was stronger in the area surrounding the central vein. A more specific localization at the plasma membrane and less unspecific background was obtained with AMP-P(CH2)P as compared to adenylylimidodiphosphate (AMP-P(NH)P). The specificity of the enzyme reaction using AMP-P(CH2)P was proved by increased formation of reaction product in the presence of 0.05 mg/ml glucagon and 0.125 mg/ml cholera toxin, as well as by inhibition of the reaction with 0.05 mg/ml alloxan. These effects were also observed at the electron microscopic level.On the other hand, no increase in reaction was observed in the presence of glucagon with AMP-P(NH)P as a substrate for adenylate cyclase, and only a weak activation was observed after adding cholera toxin; alloxan-inhibition was not complete. These effects may be due to the presence of enzymes which hydrolyze AMP-P(NH)P nonspecifically, superimposing on the product of adenylate cyclase activity. We therefore suggest the use of AMP-P(CH2)P as substrate for histochemical adenylate cyclase demonstration in the liver.

Insulin receptor substrate-1 is over-expressed in glycogenotic but not in amphophilic preneoplastic hepatic foci induced in rats by N-nitrosomorpholine and dehydroepiandrosterone

Insulin receptor substrate-1 (IRS-1) is over-expressed in preneoplastic glycogenotic hepatic foci (GSF) and is gradually down-regulated during progression of these lesions, via mixed cell foci (MCF), to the basophilic neoplastic phenotype. The aim of the present study was to investigate the effect of dehydroepiandrosterone (DHEA), a weak hepatocarcinogen and tumour enhancer, on IRS-1 expression. Hepatocellular lesions were induced by N-nitrosomorpholine followed by DHEA. Under these conditions, many glycogen-poor amphophilic (APF) and intermediate cell foci (ICF) appear, in addition to GSF and MCF. IRS-1 was over-expressed in 215 out of 295 GSF, in 50 out of 53 MCF and in a glycogen-rich mixed cell adenoma. IRS-1 expression was not shown in 147 APF, 51 ICF and 5 amphophilic hepatocellular adenomas, and 3 out of 5 hepatocellular carcinomas showed a weak IRS-1 expression. The results suggest a close association of IRS-1 over-expression with the glycogenotic hepatocellular phenotype. The modulation and enhancement of tumour progression by DHEA is associated with a shift from glycogenosis to amphophilia and basophilia, and a down-regulation of IRS-1 expression.

CYTOCHEMICAL AND BIOCHEMICAL STUDIES ON ADENYLATE CYCLASE ACTIVITY IN PRENEOPLASTIC AND NEOPLASTIC LIVER TISSUE AND CULTURED LIVER CELLS

The ATP‐analogue adenylyl(β,γ‐methylene)diphosphonate was chosen as substrate for the cytochemical localization of adenylate cyclase (AC) activity. The tissues investigated covered normal rat liver and liver from carcinogen‐treated animals with preneoplastic lesions and hepatocellular neoplasms, as well as cultured liver cells. The AC reaction product methylene diphosphonate was precipitated with Pb2+ immediately at the place of production. This approach permitted a precise localization of AC activity by light and electron microscopy. The specificity of the AC reaction was demonstrated by control reactions, including inhibition of AC with 2′5′‐dideoxyadenosine and activation with forskolin, glucagon, and cholera toxin. Endogenous phosphatases were inhibited with tetramisole and NAD.

Histochemical Analysis of Hepatocarcinogenesis

The hybrid discipline of histochemistry, a borderline field between histology and analytical chemistry or biochemistry is concerned with the identification, localization and quantification of specific substances, reactive groups and sites of enzymatic activities in tissues, cells and cell organelles. Principally metabolic products or enzymes can also be assessed by biochemical analysis nowadays much more efficiently because of the development of new sensitive microchemical techniques. However, the data provided by this approach represent average values and cannot give any information about the true distribution of certain compounds in individual cells and organells. That individual cells differ markedly in their metabolic compartimentation is best illustrated by the kidney tubular system1 and even the liver which looks quite homogeneous uncovers at closer inspection the well known metabolic zonation2. Biochemical analysis becomes really difficult or cannot be performed at all when patholologically altered tissue has to be investigated in which cellular structure as well as metabolic activities may have changed. Because histochemistry offers a wide spectrum of effective methods to surmount such serious problems it turned out to be an indispensable tool for scientists in many fields from botany to histopathology which is frequently applied in toxicology and cancer research.