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[CANCER RESEARCH 51. 1952-1958. April 1. 1991) Histochemical Profile of Mouse Hepatocellular Adenomas and Carcinomas Induced by a Single Dose of Diethylnitrosamine1 Hans JörgHacker,2 Hussein Mtiro,1 Peter Bannasch, and Stan D. Vesselinovitch Division of Cytopathology [H. J. //., H. M., P. B./, German Cancer Research Center, Im ,\euenheimer Feld 280, D-6900 Heidelberg, Federal Republic of Germany, and Departments of Radiology and Pathology ¡S.D. V.], University of Chicago, The Pritzker School of Medicine, Chicago, Illinois ABSTRACT In continuation of earlier studies on murine neoplastic liver lesions, we characterized by histochemical methods the phenotype of hepatocellular adenomas and carcinomas induced by single injections of diethylnitrosamine (1.25, 2.5, or 5.0 /jg/g of body weight) in 15-day-old C57BL/6 x male C3H FI mice. The hepatocellular adenomas were composed predominantly of basophilic cells but stored excessive amounts of fat and glycogen in large portions of the tumors. Irrespective of the carcinogenic dose, the adeno mas showed a consistent histochemical pattern. Glycogen synthase and phosphorylase »erehighly active in the hepatocytes that stored glycogen. In cells poor in, or free of, this polysaccharide, these enzymes were only moderately active or even inactive. In glycogen-storing parts of the adenomas, the activity of adenylate cyclase was reduced compared with normal liver parenchyma, but in fat-storing portions it was elevated. In a few adenomas, uniform increase in adenylate cyclase activity could be encountered. The levels of ATPase, acid phosphatase, and glucose-6phosphatase were either increased or decreased. Glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase showed an increased activity in all adenomas compared with preneoplastic foci, which in turn exhibited a higher glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase activity than the surrounding parenchyma or the liver of untreated controls. The hepatocellular carcinomas showed remarkable histochemical changes compared with adenomas. The levels of fat and glycogen and the activities of glycogen synthase, phosphorylase, and in most cases also that of glucose-6-phosphate dehydrogenase, were reduced significantly. In contrast, adenylate cyclase, glucose-6-phosphatase, glyceraldehyde-3phosphate dehydrogenase, and also alkaline phosphatase showed a strik ing elevation in developing carcinomas. Similar, although more pro nounced, histochemical changes were seen in the advanced hepatocellular carcinomas. These observations indicated that progression from adeno mas to hepatocellular carcinomas was associated with a change in the activity of several enzymes involved in cell membrane function, glycogen metabolism, the oxidative pentose phosphate pathway, and glycolysis. INTRODUCTION Several studies (1,2) showed that low, single doses of DEN4 their number decreased concurrently with the emergence of the hepatocellular adenomas. The hepatocellular carcinomas began to develop either within existing adenomas or within the hep atocellular parenchyma. Hepatocellular carcinomas grew rap idly, involved surrounding tissue, and metastasized to lungs (1). The temporal relationship between foci, adenomas, and carci nomas clearly pointed to time-dependent tumor progression, a phenomenon well established in several experimental systems and observed in human carcinogenesis. In a previous paper (3), the early focal lesions induced in mice by a single dose of DEN were characterized histochemically. The main feature of these lesions was a decrease in GóPase and an increase in both G6PDH and GAPDH, indi cating a shift from the gluconeogenic situation predominating in normal liver to a glucose consumption by pentose phosphate pathway and glycolysis. The focal hepatic lesions also showed increased labeling indices as well as an increase in their diam eters and volumes with time. Thus, it was concluded that the changes mentioned above acted in concert, manifesting the acquired functional and replicating potential of focal lesions. The present article reports on the histochemical characteristics of the subsequently emerging hepatocellular adenomas and carcinomas. MATERIALS AND METHODS Tumors were induced in 15-day-old male C57BL/6 x C3H F, mice by single injections of DEN (1.25, 2.5, or 5.0 ng/g of body weight) as described earlier (4). The DEN-treated and nontreated control animals were killed 44 and 68 weeks following the administration of the carcinogen. The objective was to sample primarily hepatocellular ade nomas and carcinomas for histochemical characterization. Histology. Preneoplastic lesions in DEN-treated mouse livers were classified as intermediate cell foci as defined earlier (3). Neoplastic lesions were classified as hepatocellular adenomas and frank hepato cellular carcinomas (1). Hepatocellular carcinomas that were observed as emerging within the hepatocellular adenomas were referred to as early carcinomas. No such distinction was observed in the "late" administered to infant mice resulted in a sequential emergence of preneoplastic hepatic foci, heptocellular adenomas, and hep atocellular carcinomas. The frequency of developing lesions, as well as the time of their emergence, were dose-dependent. The preneoplastic foci increased progressively until their number reached the maximal response to a given dose. Subsequently, carcinomas. Histochemistry of Enzymes and Metabolic Products. Pretreatment of tissues and histochemical procedures used were identical to those in the preceding study (3). Briefly, pieces from shock frozen liver tissue of normal (one specimen) and carcinogen-treated (3 specimens, 1 for each dose) mice were frozen onto the same tissue holder, and serial sections of all 3 pieces (0.15 cnr each) were cut simultaneously in a Cryostat (Jung, Nussloch, Federal Republic of Germany). The sections were Received 7/6/90; accepted 1/17/91. The costs of publication of this article were defrayed in part by the payment mounted onto the same slide or membrane and incubated according to of page charges. This article must therefore be hereby marked advertisement in the respective histochemical reaction. With this technique, it was pos accordance with 18 U.S.C. Section 1734 solely to indicate this fact. sible not only to produce sections of the same thickness but also to ' This work was supported in part by the National Cancer Institute Grant CAtreat them simultaneously under identical conditions for the specific 25522. H. Mtiro was a guest research scientist of the German Cancer Research Center. histochemical assays. The thickness of the sections was varied (6 to 14 : To whom requests for reprints should be addressed. urn) in accordance with the reaction intensity of the enzyme to be 3 Present address: Tanzania Tumor Center. Dar-es-Salaam, Tanzania. demonstrated. The activities of the following enzymes were demon 'The abbreviations used are: DEN. diethylnitrosamine; SYN. glycogen syn thase, EC 2.4.1.11: PHO. glycogen phosphorylase. EC 2.4.1.1; GóPase. glucosestrated: SYN, PHO, GóPase, GAPDH, ATPase, 7GT, and ACPase. 6-phosphatase, EC 3.1.3.9; G6PDH. glucose-6-phosphate dehydrogenase. EC In addition, 2 other enzymes, ADC and ALKPase, were studied in 1.1.149; GAPDH. glyceraldehyde-3-phosphate dehydrogenase. EC 1.2.1.12; preneoplastic and neoplastic lesions since they play a pivotal role in the ATPase. adenosine triphosphatase. EC 3.6.3.1: ADC. adenylate cyclase, EC regulation of cell metabolism (5) and transport processes (6, 7), respec 4.6.1.1; ALKPase, alkaline phosphatase. EC 3.1.3.1: iGT, -y-glutamyl transpeptively. Details of the histochemical assays for ADC are given by Mayer tidase, EC 2.3.2.2; ACPase, acid phosphatase, EC 3.1.3.2. 1952 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research. HISTOCHEMICAL Table 1 Number of preneoplastic fociDEN(ng/g PATTERN OF MOUSE LIVER TUMORS and neoplastic lesions studied histochemically Intermediate cell at different stages of the experiment hepatocellular hepatocellular hepatocellular carcinomasWk4400013WkcarcinomasWk440004Wk68404All carcinomasWk4400017Wk689817 wt)1.252.55.0Irrespective body 685813Late doseIrrespective of of timeAnimalsinvestigated6121230Wk4430407072Wk68022AdenomasWk4435165191Wk68202040Early et al. (8) and those for ALKPase are given by Lojda et al. (9). Furthermore, sections were stained for basophilia with toluidine blue and for the presence of neutral lipids with Fettrot B. Others were treated with either the periodic acid-Schiff reaction or Lugol's iodine to demonstrate glycogen. Evaluation of Histochemical Reactions. The histochemical reactions were evaluated under a comparison microscope (Leitz, Wetzlar, Federal Republic of Germany), which allowed exact correlation of the various histochemical parameters with specified focal lesions in the serial sections. The intensity of the histochemical reactions was estimated semiquantitatively using 5 grades (no change, increase, strong increase, decrease, strong decrease) as compared with the reaction in the concur rently processed tissue of the untreated control animals. RESULTS At 44 weeks, a large number of hepatocellular adenomas were observed in addition to the intermediate cell foci described earlier. At 68 weeks, the samples studied contained both hepa tocellular adenomas and carcinomas (Table 1). The histological appearance and the histochemical pattern of the intermediate cell foci that were observed at 44 weeks did not differ from foci detected at earlier time points (3). The foci were active in SYN and PHO. In comparison with normal liver parenchyma, ADC was reduced, and ALKPase andiGT did not show a change. GoPase and ACPase activities were decreased. However, G6PDH and GAPDH were increased in comparison with the surrounding liver tissue and the parenchyma of the untreated animals. The histochemical patterns of the adenomas, early carcino mas, and the late carcinomas (fully developed carcinomas) are summarized in Table 2. For comparison, the histochemical patterns of intermediate cell foci are presented in Table 2, Table 2 Increase, decrease, or no change in the level of investigated histochemical markers in later stages of mouse hepatocarcinogenesis lesionHistochemical markersFat Type of carcinoma* carcinoma'i Column 2. Adenomas were predominantly composed of basophilic cells in addition to cells that stored excessive amounts of glycogen (Fig. 2a) and fat (Fig. Ib), respectively. The basophilic cells often formed a ring along the periphery of the adenomas. This cell population frequently showed an increase in the num ber of mitotic figures. SYN and PHO were highly active in the cells storing glycogen in excess but were only moderately active or even inactive in the basophilic cells poor in, or free of, this polysaccharide (Fig. le). The ADC was generally reduced in glycogen storage portions of adenomas (Fig. \d) but regularly elevated in fat-storing areas. In some adenomas, ADC was uniformly elevated despite a conspicuous glycogen deposition. GoPase (Fig. le), ATPase, and ACPase were increased in some but were decreased in other areas of adenomas. ALKPase was increased around the capillaries but showed a normal or de creased activity in the adenoma cells (Fig. I/). The 7GT could not be detected in any of the hepatocellular adenomas. The activity of G6PDH and GAPDH was strongly increased in all adenomas (Fig. l, g and h), exceeding that observed in the intermediate cell foci. With the exception of decreased SYN and PHO, no significant differences in the histochemical pat terns were found between the ring of basophilic cells and the rest of adenomas. The appearance of hepatocellular carcinomas was accom panied by additional histochemical changes (Fig. 2, a-h); fat, glycogen (Fig. 2a), as well as the activities of SYN, PHO (Fig. 2e), ATPase, and in many cases also that of G6PDH (Fig. 2/), were strongly reduced in comparison with adenomas. In con trast, ADC (Fig. 2c), ACPase (Fig. 2e\ and GAPDH (Fig. 2h) showed an increase in their activities. GoPase (Fig. 2c) and ALKPase (Fig. 2g) were more elevated in the early carcinomas than in the surrounding adenomas. Similarly to the preceding lesions, -yGT was lacking. The fully developed hepatocellular carcinomas showed essentially the same histochemical pattern as the early carcinomas, although most of the changes were more manifest (Fig. 3, a-h). Late DISCUSSION Glycogen Tori i SYN NCNC tori li PHO Toriî-'ftorjÃŽÃŽÃŽÃŽtori ilÃŽ ADCG iiTTNCNC]Adenoma"î" 1ÃŽÃŽ1îli PaseG6PDHGAPDHATPase 6 JÃŽÃŽ ALKPaseTGTACPaseFociNCiNCNCToriEarly1lî}NC NCî î " Compared with intermediate foci. * Focal carcinomas observed within or outside of adenomas. c Fully developed hepatocellular carcinomas. d}. increase; J, decrease; NC. no change; |j, strong increase; ({. strong decrease. ' In fat-storing portions and cells around veins, uniformly in new adenomas. ^In glycogen storage portions. Sequential histochemical changes occurring during hepato carcinogenesis from preneoplastic lesions to the hepatocellular adenomas were studied extensively to date in rats (10, 11). In mice, however, histochemical studies were of more limited scope. The present study was directed to fill this void by investigating a large number of enzymes, particularly those of alternative pathways of carbohydrate metabolism. The histochemical pattern of preneoplastic hepatic lesions induced in the infant mouse model by DEN has been described previously (3). In the present investigation, the spectrum of enzymes studied in the early lesions was complemented by demonstration of the activities of ADC and ALKPase, which are involved in signal transduction and transport processes of 1953 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research. MIMIK III MIC M I'M I I KN NI MOI SI I l\ I K II \1NKS f m • Fig. 1. Serial sections through .1hepiitocellular adenoma. Remnant liver parenclnma is visible at the left margin of the micrographs. Bar. 100 firn. a. glycogen: reduced deposition in large areas of the adenoma; A, neutral lipids: pronounced storage in some portions of the adenoma (niroir.v). Insci, higher magnification. Note intensely stained lipid droplets in contrast to nuclei of the tumor cells, c. glycogen synthase: decreased activity in large areas of the adenoma. ¡I.adenylate cyclase: increased en/yme activitv in the majority of adenoma cells, e. glucose 6-phosphatase: activity is decreased in some hut slightly increased in other parts of the adenoma. /. alkaline phosphatase: weak activity in adenoma cells but strong in capillaries (arrows). Inset, higher magnification. Note, enzyme activity is confined to the cell membrane of adenoma cells, g. glucose-6-phospliate dehydrogenase: strongly increased in adenoma. A, glyceraldehyde-3-phosphate dehydrogenase: strongly increased in adenoma. 1954 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research. HISTOCHEMICAL PATTERN OF MOUSE LIVER TUMORS Fig. 2. Serial sections through an area showing transition from adenoma (upper, lower, and left margin) to carcinoma. Bar. 100 i/m. a, glycogen: gradient leading from marked storage in the adenoma to almost complete reduction in the center of the carcinoma, h. glycogen phosphorylase: decrease activity in subpopulations of the adenoma and in all cells of the carcinoma, r. adenylate cyclase: strongly increased enzyme activity in carcinoma in contrast to the surrounding adenoma tissue, d, glucose-6-phosphatase: variable activity in adenoma but strong increase in the majority of carcinoma cells. £'. acid phosphatase: activity of the enzyme is increased at the transition /one between adenoma and carcinoma and in the central part of the carcinoma (righi upper corner), f. glucose-6-phosphate dehydrogenase: variable activity in adenoma but decreased in carcinoma, g. alkaline phosphatase: low activity in adenoma but strong increase in carcinoma, h. glyceraldehyde-3-phosphate dehydrogenase: increased activity in adenoma but even higher in carcinoma. 1955 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research. HISTOCHEMICAL ff ,•• <».•/ PATTERN OF MOl'SE LIVER Tl'MORS a *•* -. - - *r M . —¿^C^Sf :. -. .S-¿?i.&. j*%v¡ "> '£j&\:ÃŽ ^¿.V-. *•*•;:• r^. H •••jPSWfiT ^? îsln ' ^? Fig. ÃŒ. Serial sections through a frank hepatoccllular carcinoma. Kur. 100 firn, a, glycogen: s(rongl\ reduced in majority of tumor cells, h. glycogcn phosphorylase: decrease of enzyme activity throughout the tumor, r. adenylate cyclase: activity is found in plasma membrane as well as in cytoplasm of carcinoma cells, d. glucose-6phosphatase: all tumor cells show a strong increase in activity, e. acid phosphatase: strongly increased en/yme activity in all tumor cells. / glucose-6-phosphate dehydrogenase: strongly increased activity in some tumor areas but decrease in most of the carcinomas cells. #. alkaline phosphatase: strong increase in activity throughout the carcinoma, h. glyceraldehyde-3-phosphate dehydrogenase: en/yme activity is strongly increased. 1956 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research. HISTOCHEMICAl. PATTERN OF MOUSE LIVER TUMORS the plasma membrane. Whereas ADC activity was reduced, that of ALKPase remained unchanged in preneoplastic lesions. The adenomas shared a number of features with the preneo plastic foci. Thus, in addition to the pronounced basophilia, which has been known for some time, the adenomas were particularly characterized by strongly increased activities of G6PDH as detected in a number of tumor types, including hepatocellular tumors of the rat, by biochemical (12) and histochemical methods (13). In contrast to early lesions, the mu rine adenomas usually stored fat and especially glycogen in excess in large tumor areas with SYN and PHO being active at these sites. GoPase-activity, which was regularly reduced in preneoplastic foci, was frequently increased in adenomas. It is difficult to understand the significance of this histochemical pattern since in addition to the anabolic and catabolic enzymes of glycogen metabolism, key enzymes of the oxidative pentose phosphate pathway (G6PDH) and glycolysis (GAPDH) were strongly increased in their activities at the same time. In line with earlier interpretations of findings in rat liver (10, 13), it may be speculated that an increased intracellular level of the central metabolite G6P is responsible for the simultaneous activation of these alternative pathways of carbohydrate metabolism. Additional metabolic changes accompanied the transition from adenomas to carcinomas. Thus, glycogen, SYN, and PHO were reduced. Surprisingly, there was also a decrease in G6PDH activity in most carcinomas. This is at variance with the pre vious findings in rat liver (10) and does not support the concept that the increase in G6PDH activity in preneoplastic lesions merely reflects the proliferative activity of the respective tu mors. Our studies were focused in the oxidative part of the pentose phosphate pathway. There is, however, also the nonoxidative pathway, which provides precursors for ribonucleotide synthesis and glycolysis without production of NADPH. Whether both parts are acting or either of them is preferentially stimulated under normal conditions is dependent on the relative requirements for NADPH and ribose-5-phosphate (14). Thus, in the fast-growing hepatocellular carcinomas with low G6PDH activity, most of the ribonucleotide precursors might be sup plied by an elevated nonoxidative pentose phosphate pathway and glycolysis as indicated by the increased GAPDH activity observed in the present investigation. Another feature of devel oping carcinoma cells was the striking increase in ALKPase, an enzyme known to catalyze the hydrolysis of various phos phate esters, in liver, especially proteins phosphorylated at tyrosine residues ( 15). The elevation of ALKPase was correlated with an increased ADC. The ability of cyclic AMP to induce alkaline phosphatase has been described by Firestone and Heath in mouse L-cells(lo). However, an isoenzyme shift of ALKPase leading to a protein of higher catalytic activity cannot be pre cluded (6, 7, 17). The sequential cellular changes observed in the experimental model used have been shown to be consistent with earlier studies in the same infant mouse model (3), which is highly sensitive to the hepatocarcinogen DEN (4). Following treatment of the adult mice with the same carcinogen (DEN), even after its repeated applications, the frequency of preneoplastic and neoplastic lesions was significantly lower (2). Tinctorial foci similar to those observed in the infant mouse model were also induced in rat livers by a single dose of aflatoxin B, (18) and were referred to as "tigroid cell foci." However, although both types of foci share similar morphology and lack of 7GT, they are different with respect to other parameters. Thus, GoPase is regularly reduced in mouse foci but frequently unchanged in rat tigroid cell foci. On the other hand, 7GT-positive hepatocellular foci were encountered after feeding mice safrole (19). Glycogen storage foci being precursor lesions of hepatocellular carcino mas in the rat can also be found in mouse liver after adminis tration of ethylnitrosourea (20), DEN, and dieldrin (21). There fore, several factors such as species, strain, age at treatment, sex, experimental protocol, and the type of carcinogen may influence the kinetics of emerging lesions and some aspects of their phenotypic expression (22). Common characteristics, however, predominate across the lesions. The present article characterized for the first time the se quential change in the histochemical pattern taking place from small preneoplastic hepatocellular foci to adenomas and carci nomas triggered by single carcinogenic treatment of infant mice. In many respects, the observed histochemical changes during hepatocarcinogenesis in the infant mouse model are comparable with those seen during hepatocarcinogenesis in the rat. Despite some conspicuous differences in the histochemical pattern that are not fully understood as yet, a shift from glycogen metabo lism towards the pentose phosphate pathway and particularly to glycolysis seems to be a common denominator of hepatocar cinogenesis. These metabolic changes are apparently closely related to the neoplastic conversion of hepatocytes (10, 13). ACKNOWLEDGMENTS We thank Erika Filsinger for excellent histochemical work, Gabriele Becker and Erwin Zang for printing the photomicrographs. Dr. D. Mayer for critically reading the manuscript, and Brigitte Pétillonand Myrtle Tuzar for typing the manuscript. REFERENCES 1. Vesselinovitch, S. D. Mihailovich. N., and Rao. K. V. N. Morphology and metastatic nature of induced hepatic nodular lesions in C57BL x C3H F, mice. 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Mouse Liver Neoplasia, pp. 1-26. Washington-New York-London: Hemisphere Publishing Corporation, 1984. 1958 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research. Histochemical Profile of Mouse Hepatocellular Adenomas and Carcinomas Induced by a Single Dose of Diethylnitrosamine Hans Jörg Hacker, Hussein Mtiro, Peter Bannasch, et al. Cancer Res 1991;51:1952-1958. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/51/7/1952 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. 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