Virchows Archiv B Cell Pathol (1992) 62:3-8
VirchowsArchivB CellPathology
IncludingMolecularPathology
9 Springer-Verlag 1992
Original articles
Glycogen phosphorylase hyperactive foci of altered hepatocytes in aged rats H. Enzmann ~, H. Zerban 2, E. Ltiser 1, and P. Bannasch 2 1 BayerAG, Institut fiir ToxikologieIC, Friedrich-Ebert-Strage217-333, W-5600 Wuppertal, Federal Republic of Germany 2 Abteilungfiir Cytopathologie,Deutsches Krebsforschungszentrum,Im NeuenheimerFeld 280, W-6900 Heidelberg, Federal Republicof Germany Received October 24 / Accepted December 23, 1991
Summary. In untreated 12- to 24-month-old rats, the enzyme histochemical pattern of 45 focal hepatic lesions was investigated in serial sections. In addition to previously characterized glycogen storage loci, a new type of enzymatically altered hepatic focus was found. The outstanding feature of this was an increased glycogen phosphorylase activity. The frequent appearance of glycogen phosphorylase hyperactive foci simultaneously exhibiting excessive glycogen storage suggests a close relationship to the well known glycogen storage foci representing an early stage in the sequence of cellular changes which lead to hepatic tumors. Key words: Hepatocarcinogenesis - Aged rats - Foci of altered hepatocytes - Enzyme histochemistry - Glycogen phosphorylase
Introduction Various types of foci of altered hepatocytes (FAH) have been described in detail and are regarded as preneoplastic lesions (Bannasch et al. 1989) although the relationship between the different types of foci is controversial. A number of experiments indicated a sequential development from glycogenotic (clear or acidophilic) cell foci through mixed and basophilic cell foci to benign and malignant hepatocellular neoplasms (Moore et al. 1982; Bannasch etal. 1985; Enzmann and Bannasch 1987). Other authors have suggested a stable phenotype with the independent development of different types of foci (Goldsworthy et al. 1985; Peraino et al. 1984). Although those two concepts are contradictory, they are both based on the assumption that different phenotypes of foci reflect differences in carcinogenic potential. Experimental support for this assumption has been provided by Baba and coworkers (1989) who showed that foci with a markedly increased glucose-6-phosphate dehyOffprint requests to: H. Enzmann
drogenase activity exhibit more pronounced proliferation and are probably endowed with a greater carcinogenic potential than foci showing a lesser increase in glucose-6-phosphate dehydrogenase activity. Several phenotypically different types of foci have been described (Bannasch et al. 1989), some of which are not discernible in hematoxylin and eosin stained sections (Enzmann et al. 1989). A considerable number of enzyme histochemical and immunohistochemical markers for the comprehensive detection of these lesions has been suggested during recent years (for review see Moore and Kitagawa 1986). However, it is perhaps worth remembering that modulation of the phenotype may substantially alter the results of quantitative investigations, particularly those dependent on a single marker. The majority of untreated rats aged more than 1 year exhibit FAH (Ogawa et al. 1981 ; Schulte-Hermann et al. 1983; Harada et al. 1989) and in aged male Sprague Dawley rats, glycogen phosphorylase hyperactive foci are a frequent finding. Although foci with increased glycogen phosphorylase activity are only occasionally mentioned in carcinogen-treated rats (Seelmann-Eggebert etal. 1987; Wenske 1991), they have been observed more frequently in mice (Vesselinovitch et al. 1985). This paper presents a detailed description of the histochemical pattern of this type of FAH.
Materials and methods Male Sprague-Dawleyrats (purchased from Zentralinstitutfiir Versuchstierzucht, Hannover, FRG) were kept under constant conditions (22~ C, 12-h light-dark cycle) and received Altromin (Lage/ Lippe) and tap water ad libitum. The animals were not fasted prior to excising the livers under ether anaesthesia between 9:00 and 11:00 a.m. Liver slices (about 5 mm thickness) were frozen immediately by immersing in isopentane at -150 ~ C and stored at - 80~ C until investigated. Demonstration of glycogen phosphorylasewas performed on semipermeable membranes (Hacker et al. 1988). The final concentrations were: glucose-l-phosphate (Boehringer Mannheim) 50 mM, Coffein (Merck) 10 mM, ethylenediaminetetraaceticacid disodium salt (Merck) 10 raM, sodium fluoride (Merck) 17 raM,
dithiothreitol (Sigma) 10mM, morpholinoethanesulfonic acid (Merck) 100 mM, pH 6.5. The reaction was performed at 30~ for 2 h. The reaction products were stained with Lugol's iodine (Merck) through the membrane for 15 min. The part of the semipermeable membrane carrying the sections was washed in water for 5 min. The sections were mounted in iodoglycerol(1 part Lugol's iodine, 4 parts glycerol) and stored in light-proof boxes at 4~ C until investigations. Serial sectionswere stained with hemalumand eosin. Reactions for glucose-6-phosphatedehydrogenase(Meijer and de Vries 1974), adenosine triphosphatase and glucose-6-phosphatase (Wachstein and Meisel 1957), pyruvate kinase (Klimek et al. 1988) and mitochondrial glycerol-3-phosphate dehydrogenase (Enzmann et al. 1989) were performed as described previously. In order to obtain a representative sample of loci in aged animals the histochemical pattern of all foci identifiedby any of these markers were investigated. Results
Foci of altered hepatocytes in untreated rats aged more than 1 year exhibited an enzyme histochemical pattern which has not been described previously in chemically induced lesions. The outstanding feature was an increased activity of the glycogen phosphorylase. After incubation with 0.05 M glucose-l-phosphate, staining with Lugol's iodine revealed a red-brown reaction product in the cytoplasm of hepatocytes of rats sacrificed between 9: 00 and 11 : 00 a.m. Clear cell foci were occasionally found in sections stained with hemalum and eosin. The PAS reaction revealed an enhanced storage of glycogen in these lesions, in which the activity of glycogen phosphorylase was also frequently elevated. The glycogen phosphorylase hyperactive foci were predominantly localized in the periportal region. In some of these foci, the reaction product after staining with Lugol's iodine was slightly blue tinged rather than the usual brownish color of the polyglucane-iodine complex (Fig. 5). Washing the stained sections in water resulted in complete loss of the reddish-brown staining in practically all hepatocytes. In contrast, the staining in the foci remained practically uninfluenced by the washing process. In the decolorized sections the focal lesions stood out as striking groups of reddish-brown stained cells. The decolorization of the section in water was due to the removal of the reaction product, not to a destruction of the polyglucane-iodine complex, as repeated application of Lugol's iodine did not restore the staining. Exposure to light, and storage at room temperature resulted in complete fading of the staining after a few days. This could be at least partly reversed by a second staining with Lugol's iodine, indicating that fading is due to the instability of the glycogen-iodine complex rather than to the disappearance of the reaction product. The enzyme pattern of 45 foci was investigated in serial sections. None of the seven markers investigated detected all the foci, although the enhanced storage of glycogen was the most sensitive. A comparison of the different markers is given in Table 1. For the identification of focal lesions, only clearcut alterations permitted a sufficiently reliable distinction between a focus and the surrounding parenchymal cells. However, minimal alteration may give a clue to the metabolic activity of
Table 1. Detection of phenotypicallyaltered loci by selected histochemical markers
Enhanced storage of glycogen Increased activity of glycogen phosphorylase Increased activity of glucose-6phosphate dehydrogenase Decreased activity of glycogen phosphorylase Decreased activity of adenosine triphosphatase Decreased activity of glucose-6phosphatase Increased activity of pyruvate kinase Increased activity of glucose-6phosphatase Increased activity of mitochondrial glycerol3-phosphate dehydrogenase
Number of f o c i
Clearcut alteration
Borderline alteration
45
32 (71%)
0
43
23 (53%)
1
(2%)
45
21 (47%)
3
(7%)
43
12 (28%)
1
(2%)
42
11 (26%)
5 (12%)
45
4
(9%)
4
(9%)
38
2
(5%)
6 (16%)
45
1
(2%)
2
(4%)
45
1
(2%)
2
(4%)
an individual focus without allowing its unequivocal identification. It should be noted that increases and decreases in glycogen phosphorylase activity are given separately in Table 1. Alterations in glycogen phosphorylase activity were most frequent in foci with enhanced storage of glycogen and occurred in 90% (27/30) of such lesions. Conversely, only 61% (8/13) of foci without excessive glycogen accumulation were detected by the histochemical reaction for this enzyme. A decrease in glycogen phosphorylase activity corresponded with an increase in glucose-6phosphate dehydrogenase activity, whereas foci with high glycogen phosphorylase activity only occasionally exhibited an increased glucose-6-phosphate dehydrogenase activity (Fig. 1). Similarly, five of seven (71%) loci showing decreased glucose-6-phosphatase activity had enhanced pyruvate kinase activity whereas only seven of a total of 38 (18%) foci investigated for pyruvate kinase exhibited increased activity. Because of the complementary expression of glycogen phosphorylase and glucose-6-phosphate dehydrogenase in focal lesions in untreated rats, simultaneous staining for these enzymes detected more FAH than any other combination of two
Fig. 1. Correlation between the expressionof glycogenphosphoryl-
ase and glucose-6-phosphatedehydrogenasein focal lesions
markers. Nevertheless, the enhanced storage of glycogen proved to be the most sensitive single marker.
Discussion
In this paper a new type of focal hepatic lesion has been investigated. Foci of altered hepatocytes induced by chemical carcinogens usually demonstrate decreased glycogen phosphorylase activity (Hacker et al. 1982). In untreated controls, however, 53% of foci exhibited an increase in glycogen phosphorylase activity and only 28% showed a reduction. The significance of foci with increased glycogen phosphorylase activity in relation to the phenotypically different types of FAH, and in particular to the glycogen storage foci generally accepted as indicators of an early carcinogenic response, needs further clarification. The development and biological significance of the glycogen phosphorylase hyperactive foci may be unrelated to other focal lesions, or they may be part of a developmental sequence involving phenotypically different foci. Experimental results from a number of earlier studies suggest that phenotypically different loci reflect various stages in the process of hepatocarcinogenesis and that transitions from early phenotypes to late phenotypes occur according to an ordered sequence, leading from glycogenotic through mixed and basophilic foci to hepatocellular tumors (Bannasch 1968; Moore et al. 1982; Enzmann and Bannasch 1987). As Figs. 2-4 illustrate, the occurrence of an increased glycogen phosphorylase activity may precede the glycogenotic clear cell phenotype (Fig. 2A, B), although small foci with enhanced glycogen storage may still exhibit the enhanced glycogen phosphorylase activity (Fig. 3 A, B). Whereas the excessive storage of glycogen persists until an increase in ribosomes and an enhanced proliferation occur, indicating
the transition of the altered hepatocytes to the mixed or basophilic cellular phenotypes (Bannasch et al. 1988), the increased glycogen phosphorylase activity gradually disappears in glycogen storage foci (Fig. 4A, B). However in these glycogenotic foci, the enzyme protein is still immunologically detectable and may even be increased. Only in more advanced lesions does the enzymatic activity and the immunologicaUy detectable amount of glycogen phosphorylase decrease (SeelmannEggebert et al. 1987). Maguire and Rabes (1989) showed that proliferating hepatocytes in the periportal areas are the preferred sites for initiation by some chemical hepatocarcinogens. Therefore, the finding that foci with increased glycogen phosphorylase activity are predominantly found in the zone I, as defined by Rappaport (Fig. 2A, B), is consistent with the concept that these lesions may represent the first step in an ordered sequence of phenotypically different foci. Baba and coworkers (1989) demonstrated, that foci with high glucose-6-phosphate dehydrogenase activity showed enhanced proliferation and are probably more likely to progress to hepatocellular tumors than foci without increased activity of this enzyme. In our investigation, glycogen storing foci with high glycogen phosphorylase activity normally failed to exhibit increased glucose-6-phosphate dehydrogenase activity, whereas glycogen storage foci with decreased glycogen phosphorylase activity frequently showed an increased glucose-6phosphate dehydrogenase activity. This further supports the hypothesis, that glycogen storing foci with high glycogen phosphorylase activity are phenotypically and developmentally less closely related to hepatocellular tumors, but may be precurser lesions of obviously preneoplastic glycogen storing foci high in glucose-6-phosphate dehydrogenase and low in glycogen phosphorylase activity. The observation that the polyglucane-iodine complex is more resistant to water in the glycogen phosphorylase hyperactive foci than in the surrounding liver may be due to a qualitatively different reaction product as well as to a different intracellular localization. The slightly acidophilic appearance of the lesions may indicate a close association of the glycogen with membranes of the smooth endoplasmic reticulum resulting in the observed water resistance of the reaction product. The different violet tinges found in some loci after staining with Lugol's iodine (Fig. 5) may reflect a particular association of glycogen with the protein. Alternatively, it may indicate that the reaction product in the glycogen phosphorylase hyperactive foci is less ramified than in the surrounding hepatocytes (Thorn 1990), suggesting a relative lack of the branching enzyme. These potentially longer unramified chains might explain why glycogen is less easily mobilized in foci than in surrounding hepatocytes after fasting. The apparent contradiction of an increased glycogen phosphorylase activity and a simultaneously enhanced storage of glycogen in the same foci is difficult to understand. In rats and mice, there is experimental evidence that a shift in carbohydrate metabolism is an integral
Figs. 2-4. Relation between foci characterized by an increased water-resistant reaction product of glycogen phosphorylase and the appearance of morphologically detectable alterations in serial sections stained with hemalum and eosin (HE)
Fig. 2. Small focus, periportal localization, high activity of glycogen phosphorylase (A), no morphological alterations in the H.E. stain (B) Fig. 3. Focus with high activity of glycogen phosphorylase (A) and clear-acidophilic phenotype in the H.E. stain (B)
Fig. 4. Larger focus, heterogenous staining for glycogen phosphorylase (A), clear-acidophilic phenotype in the H.E. stain (B) Fig. 5. Focus with enhanced activity of glycogen phosphorylase. Note different hue of the polyglucane-iodinecomplex in the focus, indicating an atypical reaction product
part of neoplastic transformation of hepatocytes (Bannasch et al. 1984; Vesselinovitch et al. 1985). The concentration of glucose-6-phosphate within the liver is significantly elevated during the early stages of liver carcinogenesis (Enzmann et al. 1988). It is tempting to specu-
late that a carcinogen-induced elevation of glucose-6phosphate and its related metabolites may create metabolic conditions in vivo under which glycogen phosphorylase contributes to the synthesis of polyglucanes similar to the described reaction of glycogen phosphorylase
in vitro. However, under physiological conditions the enzyme has never been shown to operate in the direction o f glycogen synthesis in vivo (Stalmans 1976). Although phenotypically different foci of altered hepatocytes have not been differentiated by many authors, there is increasing evidence that foci with different phenotypes vary in their biological significance and relevance for carcinogenesis. The appearance of some of the loci with hyperactivity of glycogen phosphorylase in hematoxylin and eosin - stained serial sections suggests some connection to the "pale eosinophilic" foci described by Harada and coworkers (1989). The fact that glycogen phosphorylase hyperactive foci are the predominant phenotype in aged control rats, whereas foci in carcinogen-treated animals usually show decreased glycogen phosphorylase activity (Hacker et al. 1982; Vesselinovitch etal. 1985; Seelmann-Eggebert et al. 1987) requires further clarification. At present, the demonstration of an increased water-resistant activity of glycogen phosphorylase may be a useful additional marker for the detection of foci of altered hepatocytes and enable the clear-cut discrimination of an F A H subtype that is practically undetectable by other enzyme histochemical markers.
Acknowledgements. The authors would like to express their appreciation to Anja Skulschus and Yvonne Heiter for excellent technical assistance and to Stefanie Michels and Gabriele F611mer-Niebel for repeated retypes.
References Baba M, Yamamoto R, tishi H, Tatsuta M, Wada A (1989) Role of glucose-6-phosphate dehydrogenase on enhanced proliferation of preneoplastic and neoplastic cells in rat liver induced by N-Nitrosomorpholine. Int J Cancer 43 : 892-895 Bannasch P (1968) The cytoplasm of hepatocytes during carcinogenesis. Electron and light microscopical investigations of the nitrosomorpholine-intoxicated rat liver. Rec Res Cancer Res 19:1-100 Bannasch P, Hacker H J, Klimek F, Mayer D (1984) Hepatocellular glycogenosis and related pattern of enzymatic changes during hepatocarcinogenesis. Adv Enzyme Regul 22:97-121 Bannasch P, Zerban H, Hacker HJ (1985) Foci of altered hepatocytes, rat. In: Jones TC, Mohr U, Hunt RD (eds) Monographs on pathology of laboratory animals, digestive system. Springer, Berlin Heidelberg New York Tokyo, pp 10-30 Bannasch P, Enzmann H, Ruan Y, Weber E, Zerban H (1988) Cellular differentiation during neoplastic development in the liver. In: Roberfroid MB, Pr+at V (eds) Experimental hepatocarcinogenesis. Plenum Publishing Corp, New York, pp 89-103 Bannasch P, Enzmann H, Klimek F, Weber E, Zerban H (1989) Significance of sequential cellular changes inside and outside foci of altered hepatocytes during hepatocarcinogenesis. Toxicol Pathol 17:617-629 Enzmann H, Bannasch P (1987) Potential significance of phenotypic heterogeneity of focal lesions at different stages in hepatocarcinogenesis. Carcinogenesis 8 : 1607-1612 Enzmann H, Dettler T, Ohlhauser D, Bannasch P (1988) Elevation of glucose-6-phosphate in early stages of hepatocarcinogenesis induced in rats by N-nitrosomorpholine. Horm Metabol Res 20:128-129
Enzmann H, Ohlhauser D, Enzmann H, Dettler T, Benner U, Hacker HJ, Bannasch P (1989) Unusual histochemical pattern in preneoplastic hepatic foci characterized by hyperactivity of several enzymes. Virchows Arch [B] 57:99-108 Goldsworthy TL, Pitot HC (1985) The quantitative analysis and stability of histochemical markers of altered hepatic foci in rat liver following initiation by diethylnitrosamine administration and promotion with phenobarbital. Carcinogenesis 6:12611269 Hacker HJ, Moore MA, Mayer D, Bannasch P (1982) Correlative histochemistry of some enzymes of carbohydrate metabolism in preneoplastic and neoplastic lesions in the rat liver. Carcinogenesis 3:1265-1272 Harada T, Maronpot RR, Morris RW, Stitzel KA, Boorman GA (1989) Morphological and stereological characterization of hepatic loci of cellular alteration in control Fischer 344 rats. Toxicol Pathol 17: 579-593 Klimek F, Moore MA, Schneider E, Bannasch P (1988) Histochemical and microbiochemical demonstration of reduced pyruvate kinase activity in thioacetamide-induced neoplastic nodules of rat liver. Histochemistry 90: 37-42 Maguire S, Rabes HM (1989) Intralobular distribution of preneo9 plastic foci in rat liver after a single dose of N-methyl-N-nitrosourea (MNU) following partial hepatectomy. Carcinogenesis 10:871-874 Meijer AEFH, de Vries GP (1974) Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. IV. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (decarboxylating). Histochemistry 40:349-359 Moore MA, Kitagawa T (1986) Hepatocarcinogenesis in the rat; the effect of promoters and carcinogens in vivo and in vitro. Int Rev Cytol 101 : 125-173 Moore MA, Mayer D, Bannasch P (1982) The dose-dependence and sequential appearance of putative preneoplastic populations induced in the rat liver by stop experiments with N-nitrosomorpholine. Carcinogenesis 3:1429-1436 Ogawa K, Ono6 T, Takeuchi M (1981) Spontaneous occurrence of gamma-glutamyl transpeptidase-positive hepatocytic foci in 105-week-old Wistar and 72-week-old Fischer 344 male rats. J Natl Cancer Inst 67:407-412 Peraino C, Staffeldt EF, Carnes BA, Ludeman VA, Blomquist JA, Vesselinovitch SD (1984) Characterization of histochemically detectable altered hepatocyte foci and their relationship to hepatic tumorigenesis in rats treated once with diethylnitrosamine or benzo(a)pyrene within one day after birth. Cancer Res 44: 3340-3347 Schulte-Hermann R, Timmermann-Trosiener I, Schuppler J (1983) Promotion of spontaneous preneoplastic cells in rat liver as a possible explanation of tumor production by non-mutagenic compounds. Cancer Res 43 : 839-844 Seelmann-Eggebert G, Mayer D, Mecke D, Bannasch P (1987) Expression and regulation of glycogen phosphorylase in preneoplastic and neoplastic hepatic lesions in rats. Virchows Arch [B] 53:44-51 Stalmans W (1976) The role of the liver in the homeostasis of blood glucose. Curr Top Cell Regul 11:51-97 Thorn A (1990) a-D-oligo- and polyglucane-iodine-complexes.GIT Fachz Lab 34:1255-1263 Vesselinovitch SD, Hacker HJ, Bannasch P (1985) Histochemical characterization of focal hepatic lesions induced by single diethylnitrosamine treatment in infant mice. Cancer Res 45:27742780 Wachstein M, Meisel E (1957) Histochemistry of hepatic phosphatases at a physiological pH with special reference to the bile canaliculi. Am J Clin Pathol 27:13-23 Wenske G (1991) Induktion pr~ineoplastischer Leberl~isionen durch einmalige pr~inatale Applikation von Aflatoxin B1 an Ratten. Thesis, Fachbereich Veterin/irmedizin Universit~it Giel3en
VirchowsArchivB CellPathology
IncludingMolecularPathology
9 Springer-Verlag 1992
Original articles
Glycogen phosphorylase hyperactive foci of altered hepatocytes in aged rats H. Enzmann ~, H. Zerban 2, E. Ltiser 1, and P. Bannasch 2 1 BayerAG, Institut fiir ToxikologieIC, Friedrich-Ebert-Strage217-333, W-5600 Wuppertal, Federal Republic of Germany 2 Abteilungfiir Cytopathologie,Deutsches Krebsforschungszentrum,Im NeuenheimerFeld 280, W-6900 Heidelberg, Federal Republicof Germany Received October 24 / Accepted December 23, 1991
Summary. In untreated 12- to 24-month-old rats, the enzyme histochemical pattern of 45 focal hepatic lesions was investigated in serial sections. In addition to previously characterized glycogen storage loci, a new type of enzymatically altered hepatic focus was found. The outstanding feature of this was an increased glycogen phosphorylase activity. The frequent appearance of glycogen phosphorylase hyperactive foci simultaneously exhibiting excessive glycogen storage suggests a close relationship to the well known glycogen storage foci representing an early stage in the sequence of cellular changes which lead to hepatic tumors. Key words: Hepatocarcinogenesis - Aged rats - Foci of altered hepatocytes - Enzyme histochemistry - Glycogen phosphorylase
Introduction Various types of foci of altered hepatocytes (FAH) have been described in detail and are regarded as preneoplastic lesions (Bannasch et al. 1989) although the relationship between the different types of foci is controversial. A number of experiments indicated a sequential development from glycogenotic (clear or acidophilic) cell foci through mixed and basophilic cell foci to benign and malignant hepatocellular neoplasms (Moore et al. 1982; Bannasch etal. 1985; Enzmann and Bannasch 1987). Other authors have suggested a stable phenotype with the independent development of different types of foci (Goldsworthy et al. 1985; Peraino et al. 1984). Although those two concepts are contradictory, they are both based on the assumption that different phenotypes of foci reflect differences in carcinogenic potential. Experimental support for this assumption has been provided by Baba and coworkers (1989) who showed that foci with a markedly increased glucose-6-phosphate dehyOffprint requests to: H. Enzmann
drogenase activity exhibit more pronounced proliferation and are probably endowed with a greater carcinogenic potential than foci showing a lesser increase in glucose-6-phosphate dehydrogenase activity. Several phenotypically different types of foci have been described (Bannasch et al. 1989), some of which are not discernible in hematoxylin and eosin stained sections (Enzmann et al. 1989). A considerable number of enzyme histochemical and immunohistochemical markers for the comprehensive detection of these lesions has been suggested during recent years (for review see Moore and Kitagawa 1986). However, it is perhaps worth remembering that modulation of the phenotype may substantially alter the results of quantitative investigations, particularly those dependent on a single marker. The majority of untreated rats aged more than 1 year exhibit FAH (Ogawa et al. 1981 ; Schulte-Hermann et al. 1983; Harada et al. 1989) and in aged male Sprague Dawley rats, glycogen phosphorylase hyperactive foci are a frequent finding. Although foci with increased glycogen phosphorylase activity are only occasionally mentioned in carcinogen-treated rats (Seelmann-Eggebert etal. 1987; Wenske 1991), they have been observed more frequently in mice (Vesselinovitch et al. 1985). This paper presents a detailed description of the histochemical pattern of this type of FAH.
Materials and methods Male Sprague-Dawleyrats (purchased from Zentralinstitutfiir Versuchstierzucht, Hannover, FRG) were kept under constant conditions (22~ C, 12-h light-dark cycle) and received Altromin (Lage/ Lippe) and tap water ad libitum. The animals were not fasted prior to excising the livers under ether anaesthesia between 9:00 and 11:00 a.m. Liver slices (about 5 mm thickness) were frozen immediately by immersing in isopentane at -150 ~ C and stored at - 80~ C until investigated. Demonstration of glycogen phosphorylasewas performed on semipermeable membranes (Hacker et al. 1988). The final concentrations were: glucose-l-phosphate (Boehringer Mannheim) 50 mM, Coffein (Merck) 10 mM, ethylenediaminetetraaceticacid disodium salt (Merck) 10 raM, sodium fluoride (Merck) 17 raM,
dithiothreitol (Sigma) 10mM, morpholinoethanesulfonic acid (Merck) 100 mM, pH 6.5. The reaction was performed at 30~ for 2 h. The reaction products were stained with Lugol's iodine (Merck) through the membrane for 15 min. The part of the semipermeable membrane carrying the sections was washed in water for 5 min. The sections were mounted in iodoglycerol(1 part Lugol's iodine, 4 parts glycerol) and stored in light-proof boxes at 4~ C until investigations. Serial sectionswere stained with hemalumand eosin. Reactions for glucose-6-phosphatedehydrogenase(Meijer and de Vries 1974), adenosine triphosphatase and glucose-6-phosphatase (Wachstein and Meisel 1957), pyruvate kinase (Klimek et al. 1988) and mitochondrial glycerol-3-phosphate dehydrogenase (Enzmann et al. 1989) were performed as described previously. In order to obtain a representative sample of loci in aged animals the histochemical pattern of all foci identifiedby any of these markers were investigated. Results
Foci of altered hepatocytes in untreated rats aged more than 1 year exhibited an enzyme histochemical pattern which has not been described previously in chemically induced lesions. The outstanding feature was an increased activity of the glycogen phosphorylase. After incubation with 0.05 M glucose-l-phosphate, staining with Lugol's iodine revealed a red-brown reaction product in the cytoplasm of hepatocytes of rats sacrificed between 9: 00 and 11 : 00 a.m. Clear cell foci were occasionally found in sections stained with hemalum and eosin. The PAS reaction revealed an enhanced storage of glycogen in these lesions, in which the activity of glycogen phosphorylase was also frequently elevated. The glycogen phosphorylase hyperactive foci were predominantly localized in the periportal region. In some of these foci, the reaction product after staining with Lugol's iodine was slightly blue tinged rather than the usual brownish color of the polyglucane-iodine complex (Fig. 5). Washing the stained sections in water resulted in complete loss of the reddish-brown staining in practically all hepatocytes. In contrast, the staining in the foci remained practically uninfluenced by the washing process. In the decolorized sections the focal lesions stood out as striking groups of reddish-brown stained cells. The decolorization of the section in water was due to the removal of the reaction product, not to a destruction of the polyglucane-iodine complex, as repeated application of Lugol's iodine did not restore the staining. Exposure to light, and storage at room temperature resulted in complete fading of the staining after a few days. This could be at least partly reversed by a second staining with Lugol's iodine, indicating that fading is due to the instability of the glycogen-iodine complex rather than to the disappearance of the reaction product. The enzyme pattern of 45 foci was investigated in serial sections. None of the seven markers investigated detected all the foci, although the enhanced storage of glycogen was the most sensitive. A comparison of the different markers is given in Table 1. For the identification of focal lesions, only clearcut alterations permitted a sufficiently reliable distinction between a focus and the surrounding parenchymal cells. However, minimal alteration may give a clue to the metabolic activity of
Table 1. Detection of phenotypicallyaltered loci by selected histochemical markers
Enhanced storage of glycogen Increased activity of glycogen phosphorylase Increased activity of glucose-6phosphate dehydrogenase Decreased activity of glycogen phosphorylase Decreased activity of adenosine triphosphatase Decreased activity of glucose-6phosphatase Increased activity of pyruvate kinase Increased activity of glucose-6phosphatase Increased activity of mitochondrial glycerol3-phosphate dehydrogenase
Number of f o c i
Clearcut alteration
Borderline alteration
45
32 (71%)
0
43
23 (53%)
1
(2%)
45
21 (47%)
3
(7%)
43
12 (28%)
1
(2%)
42
11 (26%)
5 (12%)
45
4
(9%)
4
(9%)
38
2
(5%)
6 (16%)
45
1
(2%)
2
(4%)
45
1
(2%)
2
(4%)
an individual focus without allowing its unequivocal identification. It should be noted that increases and decreases in glycogen phosphorylase activity are given separately in Table 1. Alterations in glycogen phosphorylase activity were most frequent in foci with enhanced storage of glycogen and occurred in 90% (27/30) of such lesions. Conversely, only 61% (8/13) of foci without excessive glycogen accumulation were detected by the histochemical reaction for this enzyme. A decrease in glycogen phosphorylase activity corresponded with an increase in glucose-6phosphate dehydrogenase activity, whereas foci with high glycogen phosphorylase activity only occasionally exhibited an increased glucose-6-phosphate dehydrogenase activity (Fig. 1). Similarly, five of seven (71%) loci showing decreased glucose-6-phosphatase activity had enhanced pyruvate kinase activity whereas only seven of a total of 38 (18%) foci investigated for pyruvate kinase exhibited increased activity. Because of the complementary expression of glycogen phosphorylase and glucose-6-phosphate dehydrogenase in focal lesions in untreated rats, simultaneous staining for these enzymes detected more FAH than any other combination of two
Fig. 1. Correlation between the expressionof glycogenphosphoryl-
ase and glucose-6-phosphatedehydrogenasein focal lesions
markers. Nevertheless, the enhanced storage of glycogen proved to be the most sensitive single marker.
Discussion
In this paper a new type of focal hepatic lesion has been investigated. Foci of altered hepatocytes induced by chemical carcinogens usually demonstrate decreased glycogen phosphorylase activity (Hacker et al. 1982). In untreated controls, however, 53% of foci exhibited an increase in glycogen phosphorylase activity and only 28% showed a reduction. The significance of foci with increased glycogen phosphorylase activity in relation to the phenotypically different types of FAH, and in particular to the glycogen storage foci generally accepted as indicators of an early carcinogenic response, needs further clarification. The development and biological significance of the glycogen phosphorylase hyperactive foci may be unrelated to other focal lesions, or they may be part of a developmental sequence involving phenotypically different foci. Experimental results from a number of earlier studies suggest that phenotypically different loci reflect various stages in the process of hepatocarcinogenesis and that transitions from early phenotypes to late phenotypes occur according to an ordered sequence, leading from glycogenotic through mixed and basophilic foci to hepatocellular tumors (Bannasch 1968; Moore et al. 1982; Enzmann and Bannasch 1987). As Figs. 2-4 illustrate, the occurrence of an increased glycogen phosphorylase activity may precede the glycogenotic clear cell phenotype (Fig. 2A, B), although small foci with enhanced glycogen storage may still exhibit the enhanced glycogen phosphorylase activity (Fig. 3 A, B). Whereas the excessive storage of glycogen persists until an increase in ribosomes and an enhanced proliferation occur, indicating
the transition of the altered hepatocytes to the mixed or basophilic cellular phenotypes (Bannasch et al. 1988), the increased glycogen phosphorylase activity gradually disappears in glycogen storage foci (Fig. 4A, B). However in these glycogenotic foci, the enzyme protein is still immunologically detectable and may even be increased. Only in more advanced lesions does the enzymatic activity and the immunologicaUy detectable amount of glycogen phosphorylase decrease (SeelmannEggebert et al. 1987). Maguire and Rabes (1989) showed that proliferating hepatocytes in the periportal areas are the preferred sites for initiation by some chemical hepatocarcinogens. Therefore, the finding that foci with increased glycogen phosphorylase activity are predominantly found in the zone I, as defined by Rappaport (Fig. 2A, B), is consistent with the concept that these lesions may represent the first step in an ordered sequence of phenotypically different foci. Baba and coworkers (1989) demonstrated, that foci with high glucose-6-phosphate dehydrogenase activity showed enhanced proliferation and are probably more likely to progress to hepatocellular tumors than foci without increased activity of this enzyme. In our investigation, glycogen storing foci with high glycogen phosphorylase activity normally failed to exhibit increased glucose-6-phosphate dehydrogenase activity, whereas glycogen storage foci with decreased glycogen phosphorylase activity frequently showed an increased glucose-6phosphate dehydrogenase activity. This further supports the hypothesis, that glycogen storing foci with high glycogen phosphorylase activity are phenotypically and developmentally less closely related to hepatocellular tumors, but may be precurser lesions of obviously preneoplastic glycogen storing foci high in glucose-6-phosphate dehydrogenase and low in glycogen phosphorylase activity. The observation that the polyglucane-iodine complex is more resistant to water in the glycogen phosphorylase hyperactive foci than in the surrounding liver may be due to a qualitatively different reaction product as well as to a different intracellular localization. The slightly acidophilic appearance of the lesions may indicate a close association of the glycogen with membranes of the smooth endoplasmic reticulum resulting in the observed water resistance of the reaction product. The different violet tinges found in some loci after staining with Lugol's iodine (Fig. 5) may reflect a particular association of glycogen with the protein. Alternatively, it may indicate that the reaction product in the glycogen phosphorylase hyperactive foci is less ramified than in the surrounding hepatocytes (Thorn 1990), suggesting a relative lack of the branching enzyme. These potentially longer unramified chains might explain why glycogen is less easily mobilized in foci than in surrounding hepatocytes after fasting. The apparent contradiction of an increased glycogen phosphorylase activity and a simultaneously enhanced storage of glycogen in the same foci is difficult to understand. In rats and mice, there is experimental evidence that a shift in carbohydrate metabolism is an integral
Figs. 2-4. Relation between foci characterized by an increased water-resistant reaction product of glycogen phosphorylase and the appearance of morphologically detectable alterations in serial sections stained with hemalum and eosin (HE)
Fig. 2. Small focus, periportal localization, high activity of glycogen phosphorylase (A), no morphological alterations in the H.E. stain (B) Fig. 3. Focus with high activity of glycogen phosphorylase (A) and clear-acidophilic phenotype in the H.E. stain (B)
Fig. 4. Larger focus, heterogenous staining for glycogen phosphorylase (A), clear-acidophilic phenotype in the H.E. stain (B) Fig. 5. Focus with enhanced activity of glycogen phosphorylase. Note different hue of the polyglucane-iodinecomplex in the focus, indicating an atypical reaction product
part of neoplastic transformation of hepatocytes (Bannasch et al. 1984; Vesselinovitch et al. 1985). The concentration of glucose-6-phosphate within the liver is significantly elevated during the early stages of liver carcinogenesis (Enzmann et al. 1988). It is tempting to specu-
late that a carcinogen-induced elevation of glucose-6phosphate and its related metabolites may create metabolic conditions in vivo under which glycogen phosphorylase contributes to the synthesis of polyglucanes similar to the described reaction of glycogen phosphorylase
in vitro. However, under physiological conditions the enzyme has never been shown to operate in the direction o f glycogen synthesis in vivo (Stalmans 1976). Although phenotypically different foci of altered hepatocytes have not been differentiated by many authors, there is increasing evidence that foci with different phenotypes vary in their biological significance and relevance for carcinogenesis. The appearance of some of the loci with hyperactivity of glycogen phosphorylase in hematoxylin and eosin - stained serial sections suggests some connection to the "pale eosinophilic" foci described by Harada and coworkers (1989). The fact that glycogen phosphorylase hyperactive foci are the predominant phenotype in aged control rats, whereas foci in carcinogen-treated animals usually show decreased glycogen phosphorylase activity (Hacker et al. 1982; Vesselinovitch etal. 1985; Seelmann-Eggebert et al. 1987) requires further clarification. At present, the demonstration of an increased water-resistant activity of glycogen phosphorylase may be a useful additional marker for the detection of foci of altered hepatocytes and enable the clear-cut discrimination of an F A H subtype that is practically undetectable by other enzyme histochemical markers.
Acknowledgements. The authors would like to express their appreciation to Anja Skulschus and Yvonne Heiter for excellent technical assistance and to Stefanie Michels and Gabriele F611mer-Niebel for repeated retypes.
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