Enzyme histochemical and morphological phenotype ...

Carclnogenesis vol.9 no.6 pp. 1049-1054, 1988

Enzyme histochemical and morphological phenotype of amphophilic foci and amphophilic/tigroid cell adenomas in rat liver after combined treatment with dehydroepiandrosterone and iV-nitrosomorpholine Edgar Weber1, Malcolm A.Moore2 and Peter Bannasch1'3 'institute for Experimental Pathology, German Cancer Research Center, Im Neuenheimer Feld 280, D-6900 Heidelberg, FRG and 2Carcinogenesis Research Unit, School of Pathology, University of New South Wales, Kensington 2033, Australia 'To whom all correspondence should be addressed

Introduction Foci and nodules (adenomas) of altered hepatocytes preceding the development of hepatocellular carcinomas have been described in various animal species after administration of chemical carcinogens (for reviews see 1, 2). In both rats and mice the vast majority of reports have been concerned with glycogen storing, mixed cell or basophilic lesions differing from background parenchyma on the basis of morphology and enzyme phenotype (3-9). The most common histochemically demonstrated changes used for recognition of pre-neoplastic liver lesions include reduced glucose-6-phosphatase (G6Pase*) and ATPase or increased glucoses-phosphate dehydrogenase (G6PDH), y-glutamyltranspeptidase (GGT) or glutathione S-transferase placental form •Abbreviations: DHEA, dehydroepiandrosterone; NNM, W-nitrosomorpholine; GST-P, glutathione S-transferase placenta] form; CAT, catalase; G6PDH, glucose-6-phosphate dehydrogenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G6Pase, glucose-6-phosphatase; GGT, 7-glutamyltranspeptidase; SYN, glycogen synthetase; PHO, glycogen phosphorylase; SDH, succinate dehydrogenase; AP, acid phosphatase; PAS, periodic acid-Schiff. © IRL Press Limited, Oxford, England

Materials and methods One hundred and sixty male Sprague-Dawley rats (Zentralinstitut fur Versuchstierzucht, Hannover, FRG), 9 weeks of age at the commencement, were randomly distributed to cages (two per cage) and maintained under standardized conditions (12 h light—dark rhythm, 22°C room temperature). DHEA (generous gift of Schering, Berlin, FRG) was mixed with Altromin3 (Altromin, Lage) to a final concentration of 0.25%. The NNM (kindly provided by Professor Preussmann, DKFZ, Heidelberg) was dissolved in the drinking water to a concentration of 120 mg/1. The experimental groups, consisting of 160 animals, were treated with NNM for the first 7 weeks followed by pure tap water ad libitum. Half of these and half of the non-carcinogen treated rats received DHEA in their food throughout the whole experimental period, until sacrifice. Twenty control group (Altromin* and tap water), 20 DHEA alone, 20 NNM alone and 20 NNM + DHEA treated animals were killed at time points 11 and 27 weeks. Livers were excised immediately upon sacrifice under ether anesthesia.

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Male Sprague-Dawley rats were investigated after iV-nitrosomorpholine (NNM) treatment with concomitant and subsequent administration of dehydroepiandrosterone (DHEA) for development of pre-neoplastic and neoplastic liver lesions. In addition to clear, acidophilic, mixed cell and basophilic foci, a hitherto undescribed lesion type demonstrating a unique morphological and histochemical phenotype was observed in animals receiving both NNM and DHEA. The cells of the majority of these lesions for which we propose the designation amphophilic foci were characterized by increased granular acidophilia and randomly scattered cytoplasmic basophilia. Histochemically, reduced glycogen content and elevated activity of glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), acid phosphatase (AP), succinate dehydrogenase (SDH) and catalase (CAT) were evident. The lack of 7-glutamyl transpeptidase (GGT) or glutathione S-transferase placenta! form (GST-P) in foci of this type allowed clear differentiation from other NNM-induced focal lesions while suggesting certain similarities to pre-neoplastic cells induced by hypolipidemk agents. Similar enzyme histochemical patterns were characteristic for foci and later appearing nodules (adenomas) composed of amphophilic/tigroid cells the basophilic material of which was increased and frequently arranged in long striped bands. DHEA treatment, while not itself inducing any preneoplastic foci, was thus associated with altered phenotypic expression of foci and adenomas generated by NNM.

(GST-P). Sequential studies have revealed a switch from gluconeogenesis to increased pentose phosphate pathway and glycolysis within liver foci and nodules (5,10). Comparison of different markers has also suggested a coordinated pattern of change in larger lesions (11 — 13) although the question of heterogeneity of different enzyme alterations remains equivocal (2). Recently the finding that pre-neoplastic liver lesions induced by hypolipidemic agents markedly differ from those usually associated with hepatocarcinogens with regard to altered enzyme phenotype (14) has brought the significance of such changes for the process of hepatocarcinogenesis into question. It has been demonstrated that dehydroepiandrosterone (DHEA) and other related hormone analogs which are reported to be inhibitors of the mammalian G6PDH (15,16) may inhibit development of different tumours. For example DHEA treatment results in a decrease in spontaneous breast cancer formation (17), lung tumour induction (18) and colon tumorigenesis (19) in mice. Furthermore DHEA or 3/3-methylandrost-5-en-17-one have been demonstrated to inhibit the induction of skin papillomas and carcinomas by 7,12-dimethylbenzanthracene or their promotion by 12-O-tetradecanoylphorbol-13-acetate (20,21) while inhibiting DNA synthesis in mouse epidermis (22). The fact that G6PDH is increased in pre-neoplastic and neoplastic lesions arising in almost all these tissues (23-26) brings into question the mechanisms involved in DHEA hormonal modulation of carcinogenesis. While it has been shown that a decrease in G6PDH activity after treatment with DHEA correlates with inhibition of foci development in the Solt—Farber system (27), long-term administration of the hormone results in increased hepatic activity (28) of this key enzyme of the pentose phosphate pathway (29). Although subsequent DHEA treatment was associated with a reduction in total foci numbers after initiation by dihydroxy-din-propylnitrosamine, an increase in basophilic lesions was observed (28). Similarly, concomitant administration of DHEA and Nnitrosomorpholine (NNM) with hormone treatment being continued after withdrawal of carcinogen resulted in the appearance of large numbers of lesions which demonstrated morphological differences from adenomas and carcinomas usually associated with NNM hepatocarcinogenesis (30). The present investigation was aimed at defining the enzyme histochemical and morphological phenotype of these unusual lesions.

E.Weber, M.A.Moore and P.Bannasch

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Fig. 1. (a) Amphophilic focus induced by NNM + DHEA. H&E X200. (b) Amphophilic/tigroid cell focus with beginning compression of the surrounding parenchyma. H&E X200. (c) Detail of amphophilic focus. H&E xfSOO. (d) Detail of amphophilic/tigroid cell focus. H&E X800.

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DHEA and NNM in rat liver

Table I. Frequency of clear/mixed-cell and amphophilic/tigroid cell lesions in animals treated with NNM + DHEA Time period (weeks)1

No. of animals

No. of clear/mixedcell foci per cm2 b (% total lesions)

No. of amphophilic/tigroid cell foci per cm2 (% total lesions)

Incidence of mixedcell adenomas (%)

Incidence of amphophilic/ tigroid adenomas (%)

7 + 4 7 + 20

20 20

9.17 ± 7.07 (77) 25.01 ± 13.21 (79)

2.71 ± 2.30 (23) 6.69 ± 4.51 (21)

0 (0) 9(45)

0 (0) 11(55)

"Seven weeks treatment + time period after withdrawal of carcinogen. •"Numbers represent mean and standard deviation.

Table n. Percentage of lesions in NNM + DHEA-treated rat liver sihowing definite enzyme histochemical changes Histochemical 'marker'

Amphophilic/tigroid cell foci Intensity of reaction* No. of investigated foci 1 n.c.

1

Amphophilic/tigroid cell adenomas Intensity of reaction* No. of investigated adenomas 1 n.c.

1

40

100

0

0

20

100

0

0

Glyccraldehyde-3-phosphate dehydrogenase

38

3

39

58

20

0

15

85

Glucose-6-phosphate dehydrogenase

40

5

20

75

21

0

0

100

Glucose-6-phosphatase

39

51

15

34

20

85

10

5

Acid phosphatase

39

13

44

43

21

19

38

43

7-glutamyltranspeptidase

39

0

100

0

21

0

100

0

Catalase

40

0

55

45

20

5

80

15

Succinate dehydrogenase

40

8

35

57

21

19

19

62

*l decrease; n.c. no change; t increase. For light and enzyme histochemistry, two adjacent slices of all liver lobes were cut with a razor blade. One slice was fixed in Carnoy's fluid and automatically processed for subsequent paraffin sectioning. The other portion was immediately frozen in liquid nitrogen pre-cooled isopentane at approximately - 140°C and stored at —80°C. Serial paraffin sections were cut at 2 /un and stained with hematoxylin and eosin (H&E), the periodic acid-Schiff (PAS) reaction and alcian blue. Selected serial sections were stained for binding of specific antibody to GST-P (generous gift of Professor K.Sato, Hirosald University, Japan; raised and purified as described earlier, 31) using the avtdin-biotin-peroxidase complex (ABC) method (Vectastain Kit, Vector Laboratories, Burlingame, California) (after 32). The activity of the following enzymes was demonstrated in 6 /tin serial sections of frozen liver: glyceraldchyde-3-phosphate dehydrogenase (GAPDH), G6Pase, G6PDH, acid phosphatase (AP), GGT, succinate dehydrogenase (SDH) (after 5,33) and catalase (CAT) by a modification of the method described for electron microscopy (34). In addition, a PAS/toluidine blue reaction was performed. Counts of mitooc figures within different focal subpopulations were performed at the microscope. For each lesion type (see Table III) assessment of number of mitoses per 10 000 hepatocytes was made.

Results

Foci of altered hepatocytes were only observed in NNM-treated animals. Table I shows the frequency of hepatic lesions in animals treated with both NNM and DHEA. Since the foci observed in the group treated with NNM alone were almost exclusively of glycogen storing or mixed cell character this group was not directly relevant to the present report. Four weeks after cessation of the carcinogen no adenomas and only a small number of foci were detectable. Twenty weeks after termination of the NNM treatment a total of 20 adenomas appeared while the number of foci was more than doubled. Approximately 20% of all foci were composed of large cells showing a homogeneous, granular acidophilic cytoplasm with randomly scattered basophilia (see Figure

la and c). Adenomas belonging to this type of lesion showed smaller cells with an increase in basophilia of tigroid character (3), dilated sinusoids and compression of the surrounding parenchyma (see Figure lb and d). For these types of lesion the terms amphophilic and amphophilic/tigroid cell are proposed. The results of enzyme histochemical investigation of these lesion types are summarized in Table II. A total of 40 foci and 21 adenomas were studied. The majority of foci and adenomas shared a common phenotype whereby glycogen content was decreased, CAT, SDH, AP, GAPDH and G6PDH were partly increased and G6Pase was either increased or decreased in activity (see Figure 2). Both foci and adenomas were totally negative for GGT and the immunohistochemical demonstration of GST-Prevealeda lack of staining in the vast majority of foci and in all adenomas (see Figure 3). Data for mitotic indices are summarized in Table III. Amphophilic foci demonstrated a comparable rate to that for clear cell foci, and amphophilic/tigroid cell foci to mixed cell foci, and amphophilic/tigroid cell adenomas to mixed cell adenomas. Discussion

The present investigation clearly demonstrated that long-term DHEA administration at a concentration of 0.25% in the diet exerts a marked modulating effect on a proportion of NNMinduced hepatocellular focal lesions. Thus the amphophilic foci and amphophilic/tigroid cell adenomas could be differentiated from the more usual glycogen storing foci and mixed cell adenomas associated with nitrosamine (35) and other hepatocarcinogen treatment (36-38) on both morphological and enzyme histochemical grounds.

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Fig. 2. Enzyme histochemistry of serial sections through an amphophilic focus induced in rat liver after treatment with NNM and DHEA. (a) PAS, (b) GAPDH, (c) CAT, (d) G6PDH, (e) G6Pase, (f) AP, (g) GGT, (h) SDH. X160.

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DHEA and NNM in rat liver

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In contrast to the well-known acidophilic cell foci storing glycogen in excess (1), the amphophilic foci are poor in, or free of, glycogen and show a densely granular instead of a reticular cytoplasmic acidophilia. The appearance of an increased structural basophilia in cells of pre-neoplastic hepatic foci and the corresponding neoplastic lesions has been described by some authors earlier. Recently termed intermediate basophilic in the mouse (9) and tigroid in the rat (3), such lesions appear to be preferentially induced by single doses of carcinogen. They share, to a certain extent, the characteristics of the presently described populations, for example, with regard to the lack of GGT and decrease in glycogen (3,9). Similarly, the focal lesions induced by the peroxisome proliferator group of hepatocarcinogens do not demonstrate any increase in either GGT or GST-P (14). In addition, they present a morphological picture almost identical to amphophilic/ tigroid cell lesions (39). Whether this is also the case for methapyrilene-induced hepatocellular foci and nodules (40,41) is a question of interest. Since no evidence of lesion induction by DHEA itself has been gained from the present or previous studies (28,42) the hormone must have brought about a modulation of the phenotype of NNMinduced populations. Data published earlier clearly showed that this is a post-carcinogen treatment process and not dependent on altered carcinogen metabolism (28,42). Although the underlying mechanisms remain to be elucidated a number of possibilities do present themselves. For example, the fact that DHEA has been demonstrated to inhibit G6PDH raises the question of whether a direct reduction in the normally very high activity in foci and adenomas (5) could be involved. While other long-term studies have indicated that the hormone in fact can lead to increased liver G6PDH after long-term application, the amphophilic cells usually demonstrated moderate activity in this key enzyme of the pentose phosphate shunt. Since a relatively high rate of proliferation was observed it would appear unlikely that a lack of ribose-5-phosphate units could be responsible for the modulation of phenotype. It is also unlikely that reduction in NADPH could be involved given the reported massive increase in malic enzyme by DHEA (43). Perhaps of more direct significance to the action of hormone on foci and adenoma character is the increase in CAT and SDH, suggestive of elevated peroxisome and mitochondrial numbers or metabolism. In view of the morphological and enzyme phenotypic similarities with peroxisomal proliferator-induced lesions (14,39), the fact that a number of biochemical effects are shared

TaMe ID. Mitotic index of pre-neoplastic and neoplastic lesions in rats treated with NNM and DHEA Type of lesion

No. of investigated lesions

No. of mitoses/ 10 000 hepatocytes

Clear cell focus Mixed cell focus Amphophilic focus Amphophilic/tigroid cell focus Amphophilic/tigToid cell adenoma Mixed cell adenoma

18 13 20 10 15 8

10 30 9 28 83 59

by the hormone and this group of hepatocarcinogens, especially in alteration of lipid metabolism (44,45), is of great interest. Thus in addition to the common increase in CAT, an increased acylCoA hydrolase is worthy of note. Future investigation should be concentrated on elucidating the biochemical background of different foci types since only a comprehensive approach will allow definition of whether the heterogeneity in enzyme expression reported for focal lesions by a number of authors is determined by chance (46) or can be satisfactorily explained by separate lines of development as appears more likely from the results of the present and previous investigations performed in this laboratory (3,5,10). This question is of obvious importance given the recent interest in exploring the biochemical character of such lesions as an aid to explaining the mechanisms underlying hepatocarcinogenesis. In conclusion, the present investigation has shown that even at a dose level of 0.25% (in contrast to the 0.6% used previously by other authors, 17-21,28) the hormone DHEA exerts significant effects on hepatocarcinogenesis. Although no significant reduction in development of hepatic foci or adenomas was apparent either in terms of numbers or proliferation rate, longer application was associated with a reduction in the malignant potential of hepatocarcinomas induced (30). Further investigation of the potential beneficial effects of DHEA would appear warranted.

Acknowledgements The authors would like to express their appreciation to Erika Rlsinger, Gabriele Schmidt and Ditmar Greulich for excellent technical assistance. Many thanks are also due to Antje Groh for patient secretarial help. M.A.M. is a recipient of a guest scientist stipendium from the German Cancer Research Center.

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Fig. 3. Amphophilic focus and clear cell focus induced in rat liver after treatment with NNM and DHEA. (a) H&E, (b) GST-P. Note the lack of GST-P staining in the amphophilic lesion. X40.


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