Heide Zerban

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.

Renal oncocytoma: origin from the collecting duct

SummaryThe histo- and cytogenesis of two cases of renal oncocytoma have been studied by cytomorphological and cytochemical methods. Transitions from collecting ducts into oncocytic tubules were observed at the light and electron microscopic levels. The fine structure of the oncocytes in tubules and tumors is described in detail. Cytochemically, the oncocytic tubules and oncocytomas share many characteristics with the distal nephron, especially the collecting ducts. A striking difference is the enhanced activity of succinic dehydrogenase which corresponds to the increase in the number of mitochondria in oncocytes. All the results suggest that renal oncocytoma originates from the collecting duct.

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.

Computerized Ultrasound B-Scan Texture Analysis of Experimental Fatty Liver Disease: Influence of Total Lipid Content and Fat Deposit Distribution

Statistical pattern recognition procedures allow a quantitative description of ultrasound-B-scan image texture. According to well-established animal models, different types of fatty liver disease were induced in female Wistar rats. For the correlation of the computerized ultrasound image with its underlying histology a variable tissue model based on histomorphological data, texture analysis of the histological image and biochemical measurements of total lipid, water and hydroxyprolin content was created. Whereas a regional arrangement of large fat deposits leads to a significant increase in the “mean grey level” (measure of image brightness) of the ultrasound-B-scan image, there is no difference in image brightness between normal liver tissue and liver steatosis for the tissue model with diffuse homogeneous fatty infiltration. It is demonstrated by multiple linear regression analysis that the “mean grey level” of the ultrasound-B-scan image depends not only on total lipid content but even more on the histomorphological fat deposit distribution.

Echographic tissue characterization in diffuse parenchymal liver disease: Correlation of image structure with histology

Seventy livers were examined in vitro using a computerized ultrasound B-mode data acquisition and analysis system. For tissue characterization, statistical parameters from pattern recognition algorithms describing image brightness and image structure were used. Reference classification based on histopathology as well as on chemical/morphometrical analysis led to the diagnostic classes of normal, fatty liver, fibrosis/cirrhosis and fatty fibrosis/cirrhosis. Comparing the two reference methods for ultrasound tissue characterization, re-classification based on chemical/morphometrical analysis resulted in a significant increase in diagnostic accuracy. The strong correlations between statistical ultrasound image parameters and morphometrical features reflect the relevance of our statistical approach to ultrasound tissue characterization.

Aberrant regulation of carbohydrate metabolism and metamorphosis during renal carcinogenesis

Systematic studies of the sequence of cellular changes during renal carcinogenesis induced in rats by stop experiments with N-nitrosomorpholine or streptozotocin and of human renal cell carcinomas led to the following main results and conclusions: All types of epithelial kidney tumors known from human pathology, namely clear-cell, acidophilic (granular), basophilic, chromophobic and oncocytic tumors, can be induced by the chemicals. Phenotypically altered epithelia resembling those in the tumors appear in single or multiple tubules long before unequivocal tumors develop. The progression from the preneoplastic tubular lesions to the tumors is an autogenous process which is independent of the further action of the carcinogen. At least three different types of tubular lesions can be distinguished: (a) Clear cell tubules storing glycogen in excess, (b) chromophobic or basophilic tubules frequently accumulating acid mucopolysaccharides (glycosaminoglycans, proteoglycans), and (c) oncocytic tubules accumulating atypical mitochondria. Whereas the precise site of origin of the clear cell tubules within the nephron remains unclear, the fine structural and cytochemical findings suggest that the chromophobic and basophilic tubules originate from the proximal and the oncocytic tubules from the distal nephron. Each type of tubular lesion is apparently the precursor of a cytologically specific tumor type. The well-known aberration in carbohydrate metabolism in renal tumors might occur in response to a carcinogen-induced metabolic derangement which is frequently associated with excessive storage of polysaccharides or lipids persisting for weeks and months until fast-growing tumors appear. Whereas the primary biochemical lesion leading to the persisting storage phenomena is most probably fixed at the genetic level, epigenetic changes, namely an adaptation of cellular enzymes gradually activating alternative metabolic pathways, might be responsible for the ultimate neoplastic transformation of the cell.

Foci of Altered Hepatocytes, Rat

The foci of altered hepatocytes are usually not visible with the naked eye, but they can occasionally be recognized as small, white spots on the liver surface.

Characterization of cytoskeletal components in epithelial and mesenchymal liver tumors by electron and immunofluorescence microscopy

SummaryIntermediate-sized filaments and microfilaments were studied by electron and immunofluorescence microscopy using antibodies to their constitutive proteins in various rat liver tumors induced by nitrosamines. Hepatocellular carcinomas contained both tonofilaments and microfilaments which could be demonstrated light-microscopically by antibodies to prekeratin and actin, respectively, and were predominantly localized, in a somewhat irregular pattern, in peripheral cytoplasmic regions. The hepatocellular tumors did not show significant amounts of intermediate-sized filaments of the vimentintype. Cholangiofibromas exhibited, within the epithelial duct component, actin-positive microfilaments organized in a terminal web and prekeratinpositive tonofilaments, the latter often being present in large amounts. By contrast, the mesenchymal component of the cholangiofibromas and the cells of angiosarcomas or undifferentiated sarcomas were characterized by intermediate filaments stainable with antibodies against vimentin, but not with those to prekeratin. In addition to vimentin, the mesenchymal tumors contained small amounts of actin. In many cells of the sarcomas a pronounced accumulation of intermediate-sized filaments of the vimentin type, frequently forming whorl-like aggregates, was evident. The characteristic “vimentin storage cells”, many of which were readily identifiable in H&E sections by large fibrillar cytoplasmic inclusions, were pathognomonic for sarcomas, especially for angiosarcomas. The detection of early stages of angiosarcomas by antibodies to vimentin was also facilitated.Our results do not support the concept that presence of an excess of contractile proteins such as actin is an important prerequisite for the metastasic spread of malignant cells. The observations show that antibodies to the different classes of intermediate filaments are a powerful tool for the classification and differential diagnosis of epithelial and mesenchymal tumors of the liver and probably also of other sites. The accumulation of prekeratin in many ductular cells of the cholangiofibromas and the considerable storage of vimentin in sarcoma cells seem to indicate a severe disturbance of the cell metabolism which might be related to the neoplastic transformation.ZusammenfassungIntermediäre Filamente und Microfilamente wurden in verschiedenen Typen von Nitrosamin-induzierten Lebertumoren der Ratte elektronenmikroskopisch und mit Hilfe von Antikörpern gegen ihre konstitutiven Proteine — immunfluorescenzmikroskopisch untersucht. Hepatocelluläre Carcinome enthielten sowohl Tonofilamente als auch Microfilamente, die lichtmikroskopisch durch Antikörper gegen Präkeratin bzw. Actin besonders in peripheren Cytoplasmabereichen darzustellen waren. Ihre Anordnung war unregelmäßiger als in normalen Hepatocyten. Cholangiofibrome zeigten in ihrer epithelialen Gangkomponente Actin-positive Microfilamente, die ein terminales Gespinst bildeten, und Präkeratin-positive, gegenüber der Norm oft deutlich vermehrte Tonofilamente. Im Gegensatz dazu waren die Zellen der mesenchymalen Komponente der Cholangiofibrome ebenso wie die Tumorzellen der Angiosarkome und der undifferenzierten Sarkome durch Filamente gekennzeichnet, die mit Antikörpern gegen Vimentin, nicht aber mit solchen gegen Präkeratin positiv reagierten. Neben Vimentin enthielten die mesenchymalen Tumoren geringe Mengen von Actin. In vielen Zellen der Sarkome war eine starke Anhäufung von Filamenten des Vimentintyps festzustellen. Sie bildeten z.T. wirbelartige Aggregate. Die charakteristischen „Vimentinspeicherzellen“ waren lichtmikroskopisch häufig schon im H&E Präparat an ihren großen fibrillären Cytoplasmaeinschlüssen leicht zu erkennen. Sie waren pathognomonisch für die Sarkome, speziell die Angiosarkome. Die Erfassung von Frühstadien der Angiosarkome wurde durch die Verwendung von Antikörpern gegen Vimentin erleichtert.Unsere Ergebnisse sind nicht mit der Vorstellung zu vereinbaren, daß eine Vermehrung von kontraktilen Proteinen eine wesentliche Voraussetzung für die metastatische Ausbreitung von Tumorzellen ist. Die Beobachtungen belegen, daß Antikörper gegen verschiedene Klassen von intermediären Filamenten ein ausgezeichnetes Hilfsmittel für die Klassifizierung und Differentialdiagnose von epithelialen und mesenchymalen Tumoren in der Leber und vermutlich auch in anderen Organen sind. Die Akkumulation von Präkeratin in vielen Gangepithelien der Cholangiofibrome und die starke Speicherung von Vimentin in Sarkomzellen zeigen Störungen des Zellstoffwechsels an, die in einem engen Zusammenhang mit der neoplastischen Transformation stehen könnten.

Renal Cell Adenoma and Carcinoma, Rat

Renal cell adenomas and carcinomas are epithelial tumors which are predominantly located in the cortex. They are usually well demarcated from the surrounding tissue and have a firm consistency. Large tumors of this type, which may reach a diameter of several centimeters, are readily visible with the naked eye as grayish-white or grayish-yellow masses projecting from the cortex.Frequently, the tumors contain red spots due to hemorrhage. Cystic tumors or tumor components may appear as translucent vesicles at the surface of the kidney.

Morphogenese und Mikromorphologie epithelialer Nierentumoren bei Nitrosomorpholin-vergifteten Ratten@@@Morphogenesis and micromorphology of epithelial tumors induced in the rat kidney by nitrosomorpholine: IV. Tubulre Lsionen und basophile Tumoren@@@IV. Tubular lesions and basophilic tumors

SummaryThe genesis of basophilic cell kidney tumors was investigated stepwise by light and electron microscopy in rats treated with N-nitrosomorpholine for a limited time (stop experiment). Seven weeks after the beginning of the experiment the kidney tubules sometimes showed unusually large “chromophobic” and basophilic cells. After a lag period of 22–97 weeks more than 60% of the animals had developed these atypical tubules. Parallel to the appearance of chromophobic tubules 50% of the carcinogen-treated animals developed basophilic cell kidney tumors. All intermediate stages between chromophobic or basophilic cell tubules and tumors were found. During the neoplastic transformation chromophobic epithelia appeared to change into basophilic cells. Some of the chromophobic renal tubules and most of the renal tubules which consisted of chromophobic and basophilic epithelia stored acid mucopolysaccharides as demonstrated by histochemical methods. The fine structure of the basophilic epitheliomas was relatively uniform. The basophilia observed under the light microscope correlated with abundant membrane-bound and free ribosomes as seen under the electron microscope. The frequent appearance of brush borders and microbodies indicated the origin of the basophilic cell tumors from proximal renal tubules. In some tumor cells many mitochondria were found. These cells resembled oncocytes. However, in contrast to typical oncocytes the mitochondria of these cells were poor in cristae or showed tubular formations of the inner membrane. In some mitochondria homogeneous condensations could be detected in the intracristal space and tooth-like formations were seen on the surface of the cristae. In perpendicular sections these cristae resembled saw blades. Acute tubular lesions and cellular regeneration, as described earlier by other authors in early stages of the development of kidney tumors, were not found. It is suggested that the storage of acid mucopolysaccharides observed in many tubules and in some renal tumors indicates adisturbance of the cellular metabolism which plays an important role in tumor development.ZusammenfassungDie Genese basophilzelliger Nierenepitheliome wurde in Stoppversuchen an N-Nitrosomorpholin-vergifteten Ratten stufenweise licht-und elektronenmikroskopisch verfolgt. Sieben Wochen nach Beginn der Giftzufuhr traten erstmals ungewöhnlich großzellige “chromophobe” oder basophile Tubuli auf. Sie waren zunächst nur bei einzelnen, nach einer Latenzzeit von 22–97 Wochen aber bei über 60% der Versuchstiere zu finden. Während des gleichen Zeitraumes entwickelten sich bei nahezu 50% der Carcinogen-vergifteten Ratten basophilzellige Nierenepitheliome. Morphologische Befunde, die als Übergänge von chromophoben oder basophilen Tubuli in Tumoren gedeutet werden können, waren häufig zu erheben. In der Regel wurden die chromophoben Epithelien offenbar schon früh im Verlaufe der neoplastischen Transformation in basophile Zellen umgewandelt. Ein Teil der chromophoben Nephronabschnitte sowie zahlreiche Tubuli, die aus einer Mischung von chromophoben und basophilen Epithelien zusammengesetzt waren, speicherten nach Ausweis histochemischer Reaktionen saure Mucopolysaccharide. Die Feinstruktur der meisten basophilen Epitheliome war sehr einheitlich. Elektronenmikroskopisches Äquivalent der lichtmikroskopisch beobachteten cytoplasmatischen Basophilie war ein großer Ribosomenreichtum. Bürstensaumstrukturen und Mikrokörper belegten die Abstammung der basophilen Nierentumoren vom proximalen Nephron. Gelegentlich fanden sich auffallend mitochondrienreiche Tumorzellen, die Onkozyten ähnelten. Im Gegensatz zu typischen Onkozyten zeichneten sich die Mitochondrien dieser Zellen entweder durch eine Cristaarmut oder durch eine tubuläre Transformation der Innenmembranen aus. In manchen Mitochondrien waren eigenartige Cristae zu beobachten, die durch eine homogene Verdichtung des intracristalen Spaltraumes sowie durch die Ausbildung von Zacken an der Cristaoberfläche auffielen. Das Schnittbild derartiger Cristae ähnelte einem Sägeblatt. Akute Tubulusläsionen und intratubuläre Zellregenerate, wie sie von zahlreichen Autoren als Frühstadien der Tumorbildung in der Niere beschrieben wurden, haben wir nicht beobachtet. Wir nehmen an, daß die in zahlreichen Tubuli und in einigen Tumoren nachweisbare Speicherung von sauren Mucopolysacchariden eine zelluläre Stoffwechselstörung anzeigt, die eine wichtige Rolle bei der Geschwulstbildung spielt.