Apoptosis in Stages of Mouse Hepatocarcinogenesis: Failure to Counterbalance Cell Proliferation and to Account for Strain Differences in Tumor Susceptibility

Wilfried Bursch*,1, Monika Chabicovsky*,2, Ute Wastl*, Bettina Grasl-Kraupp*, Krystina Bukowska*, Henryk Taper{dagger} and Rolf Schulte-Hermann*

* Medizinische Universität Wien, Univ. Klinik für Innere MedizinI, Abtl. Institut für Krebsforschung, Borschkegasse 8a, A-1090 Wien, and {dagger} Unité de Biochemie Toxicologique et Cancérologique, Université Catholique Louvain, UCL 7369, B-1200 Bruxelles

1 To whom correspondence should be addressed. Fax : ++43-1-4277 9651. E-mail: wilfried.bursch{at}meduniwien.ac.at.

Received July 20, 2004; accepted February 14, 2005


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 Conclusions
 REFERENCES
 
C3H/He and B6C3F1 show much higher liver cancer susceptibility than C57BL/6J mice. We studied the hypothesis that this difference might result from failure of apoptosis. Hepatocarcinogenesis was induced by a single dose of N-nitrosodiethylamine (NDEA), followed by phenobarbital (PB) for up to 90 weeks. We observed (1) earlier appearance of putative preneoplastic foci (PPF), hepatocellular adenoma (HCA), and carcinoma (HCC) in C3H/He than in C57Bl/6J mice and (2) an increase of hepatocellular DNA synthesis in C3H/He and C57Bl/6J mice, compared to normal liver, via PPF and HCA to HCC. PB enhanced DNA synthesis and growth of PPF, in the C3H/He strain only, and of HCA and HCC of both strains. Apoptoses were rare in unaltered livers as well as in preneoplastic lesions, but tended to increase in HCA and HCC of both strains. PB lowered apoptotic activity in PPF of C3H/He mice, but enhanced it in HCA and HCC of C57Bl/6J mice at late stages. In conclusion, the strain difference in growth rates of PPF and tumors is largely determined by higher rates of cell proliferation in C3H/He mice, with and without promotion by PB. Moreover, in C57Bl/6J mice the promoting effect of PB was restricted to HCA and HCC and was not seen in PPF. Apoptosis was generally low and was not a major cause of the strain difference in tumor susceptibility. In contrast with rat liver, inhibition of apoptosis appears to be a minor determinant of tumor promotion in mice.

Key Words: B6C3F1; C3H/He; C57BL/6J; hepatocarcinogenesis; tumor susceptibility; apoptosis; cell proliferation; phenobarbital.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 Conclusions
 REFERENCES
 
Rodent species used for lifetime cancer studies on chemicals include mouse strains exhibiting high (C3H/He, B6C3F1) or low (C57Bl/6J) susceptibility to hepatocarcinogenesis induced either spontaneously or chemically (for review see Dragani, 2003Go; Fausto, 1999Go; Gold et al., 1998Go; Klaunig et al., 2003Go; Whysner et al., 1996Go). From a practical toxicological point of view, the use of mouse liver tumor data for human risk assessment has been challenged and thereby initiated an ongoing discussion on how to adapt current carcinogenicity testing protocols (Ashby, 2001Go; Maronpot and Boorman, 1996Go). Elucidation of the underlying causes for the genetically predisposed cancer susceptibility of mouse strains still awaits completion. Such knowledge should improve the scientific basis for interpretation of animal studies and may facilitate future decisions on test strategies; it also might help to understand individual susceptibility of humans to development of hepatocellular carcinoma. Strain variations in cancer susceptibility do not result from different metabolism and genotoxicity of carcinogens (for review see Whysner et al., 1996Go) but have been attributed to specific hepatocarcinogen-susceptibility (HCS) genes (Dragani, 2003Go; Poole and Drinkwater, 1996Go) and to differences in the growth rate of putative preneoplastic cell populations (Carter et al., 2003Go; Goldsworthy et al., 2002Go; Hanigan et al., 1988Go; Klaunig et al., 2003Go; Lee, 2000Go; Pereira, 1993Go; Takahashi et al., 2002Go; Whysner et al., 1996Go).

In early studies on tumor promotion in rat liver, we have demonstrated that preneoplastic foci, in spite of a very high rate of cell proliferation, grew only slowly because cell birth was efficiently counterbalanced by apoptosis (Bursch et al., 1984Go). Thus, apoptosis was shown to constitute an innate tissue defense against carcinogens by preventing survival of initiated cells. We also demonstrated that phenobarbital and other liver tumor promoters inhibited apoptosis and thereby accelerated liver tumor formation (Bursch et al., 1992Go; Grasl-Kraupp et al., 1997Go; Luebeck et al., 1995Go; Schulte-Hermann et al., 1990Go). Block of apoptosis as prevailing mechanism of liver tumor promotion has been confirmed and extended by others in many rodent studies, mostly using rat liver models (Kamendulis et al., 2001Go; Luebeck et al., 1995Go; Oliver and Roberts, 2002Go; Pitot et al., 2000Go; Schwarz et al., 2000Go; Tharappel et al., 2002Go).

Relatively little is known about the role of apoptosis in tumor promotion in mouse liver and on possible strain differences that might correlate with, and explain, differences in cancer susceptibility. Rather, based upon different experimental models somewhat conflicting data have been reported. For instance, growth of preneoplastic lesions, at least in early stages, has been ascribed to proliferative activity, but apoptosis was either not studied (Hanigan et al., 1988Go; Pereira, 1993Go) or did not exhibit a significant role for growth of preneoplasia (Goldsworthy and Fransson-Steen, 2002Go; James and Roberts, 1996Go; Kamendulis et al., 2001Go; Stevenson et al., 1999Go). Other studies reported apoptosis-inhibition by tumor-promoting agents (phenobarbital, peroxisome proliferators) as important determinants for tumor development in mice (James et al., 1998Go; Sanders and Thorgeirsson, 2000Go). Moreover, we have found that normal mouse hepatocytes do not undergo apoptosis as readily as rat hepatocytes in response to signals inducing regression of the liver (Bursch et al., accompanying manuscript; Chabicovsky et al., 2003Go; Parzefall et al., 2002Go).

In the present study, we investigated the role of DNA replication and apoptosis in stages of mouse liver carcinogenesis. We performed a long-term study with C3H/He, C57Bl6/J, and B6C3F1 mice using an initiation-promotion protocol with a single dose of N-nitrosodiethylamine (NDEA), with or without promotion by PB. Growth rates of phenotypically distinct preneoplastic and neoplastic lesions, their DNA synthesis, and apoptotic activity were closely analyzed. The experimental design was focused to answer the question whether the high liver cancer susceptibility in C3H/He mice specifically correlates with low efficiency or even failure of apoptotic elimination of (pre)neoplastic cells. The in vivo approach was chosen because the actual rate of cell replication and of apoptosis and, consequently, tumor development integrate survival- and death-controlling factors in the context of the organism's genetic background. The results show that PB promotes liver tumor formation in mouse strains with high as well as with low cancer susceptibility; enhanced cell proliferation was found to be the prevailing mechanism of tumor promotion. Apoptosis failed to counterbalance cell proliferation in preneoplastic and neoplastic liver tissue of both C3H/He and C57BL/6J mice, although it was somewhat enhanced in adenoma and carcinoma of C57Bl/6J mice at a late stage. Therefore, our results show that the strain difference in liver tumor susceptibility essentially results from higher rates of cell proliferation in C3H/He mice in all stages of carcinogenesis; apoptosis or its failure plays a minor role only.


    MATERIAL AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 Conclusions
 REFERENCES
 
Animals, husbandry, and treatment.
Male B6C3F1, C3H/He, and C57Bl/6J (specific-pathogen free) mice were purchased from the Institut für Labortierkunde und -genetik (Himberg, Austria). Mice were housed individually in Macrolon cages on standard softwood bedding (Altromin, Lage, Germany) under standardized environmental conditions and an inverted 12-h light-dark rhythm, with light from 10 P.M. to 10 A.M. and dark from 10 A.M. to 10 P.M. Food (Altromin, 1321N) and tap water were provided ad libitum. Body weight and food consumption were recorded weekly.

Five-week-old mice received a single intraperitoneal injection of NDEA, 90 mg/kg of body weight (b.w.; NDEA obtained from Sigma-Aldrich, Wien, Austria), freshly dissolved in sterile 0.9% saline (20 ml/kg of b.w.). After 2 weeks of recovery, mice were fed either standard diet ("NDEA->0") or a diet containing phenobarbital (5-ethyl-5-phenyl-barbituric acid, PB; Fluka Chemie, Buchs, Switzerland) for up to 90 weeks ("NDEA->PB"); other groups of animals received PB alone ("0->PB") or no treatment at all (control "0->0"). In addition, a subgroup of NDEA->PB-C3H/He mice was put off PB from week 50 onward, until sacrifice 3 weeks after PB-withdrawal. Details of the experimental design including time points of investigation and number of animals are depicted in Figure 1. The PB concentration in the diet was adjusted in the range of 0.05–0.07% to provide a constant daily intake of 90 mg PB/kg b.w.



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FIG. 1. Study design and treatment regimen. Numbers indicate the number of mice/group of each strain (C57Bl/6J, C3H/He, and B6C3F1) sacrificed at the respective time point (abscissa). Solid arrows, NDEA treatment (1 x 90 mg/kg b.w., ip); open bars, PB treatment (90 mg/kg b.w. per day). 0->0, control; 0->PB, PB only; NDEA->0, NDEA only; NDEA->PB, PB treatment 2 weeks after injection with NDEA; NDEA->PB->0, NDEA and PB as described before, followed by PB withdrawal 3 weeks before sacrifice.

 
Two weeks before sacrifice, mice were subjected to a feeding rhythm with food offered 9 h per day (10 A.M to 7 P.M.). This feeding rhythm serves to synchronize diurnal rhythms of hepatic DNA synthesis and mitosis in rats and mice (Bursch et al., 1992Go; Grasl-Kraupp et al., 1994Go). The rhythm also synchronizes apoptotic activity in the liver of rats and—according to preliminary data—of mice (Bursch et al., 1992Go, and unpublished observations). The feeding rhythm allows collection of all or most cells undergoing DNA replication and apoptosis occurring per day in a single peak. (LI and AI from tumors, in particular from HCC, are to be considered with the caveat that synchronization by the feeding rhythm may be less efficient in these lesions. In this case actual birth or death rates of tumor cells would be higher than LI/AI counts.) For cells in S-phase, mice were pulse labeled with a single ip injection of 100 mg BrdU/kg body weight (5-bromo-2'-deoxyuridine; Sigma Chemical, Germany; 20 mg BrdU per ml phosphate buffered saline, pH adjusted to 7.2 ± 0.2) at 7 P.M., i.e., 2 h before the peak of DNA synthesis. Sacrifice was performed at 9 A.M. at the peak of apoptotic activity.

Animals were anaesthetized with isoflurane, decapitated, and exsanguinated. The liver was quickly excised and weighed upon removal of the gall bladder. Macroscopically visible hepatocellular lesions were excised, registered, and sampled individually. Representative liver samples from the large median lobe were cut into 4- to 5-mm thick slices and fixed with Carnoy's solution or 4% neutral buffered formalin and processed for histological analysis as described in detail elsewhere (Grasl-Kraupp et al., 1994Go); the remaining liver tissue was frozen for biochemical and molecular analyses to be published elsewhere. The duodenum, a tissue with a high proliferation rate, was taken at necropsy to serve as a positive control to confirm the systemic availability of BrdU injected. Duodenum samples were fixed in 4% neutral buffered formalin (Lillie) before routine processing and paraffin embedding.

The animal experiments were performed according to the Austrian and EU regulations for animal care and treatment.

Quantitative Analysis of Histological Parameters
In hematoxylin and eosin (H&E) stained sections, putative preneoplastic foci (PPF), hepatocellular adenoma (HCA), and carcinoma (HCC) were diagnosed according to published criteria (Bannasch and Gössner, 1994Go; Evans et al., 1992Go; Kraupp-Grasl et al., 1991Go; Maronpot et al., 1987Go; Turusov et al., 1979Go).

Histopathological classification of liver tumors.
According to histopathological criteria, the macroscopically visible lesions were classified as either hepatocellular adenoma (HCA) or carcinoma (HCC) according to the following criteria: (1) HCA are larger than one lobulus with signs of compression at the border (particularly in large nodules). They were diagnosed as benign noduli if less than three criteria described for hepatocellular carcinomas were applicable. Characteristic staining patterns as described for preneoplastic foci (eosinophilic/clear cell, basophilic/amphophilic, vacuolated, tigroid or mixed type, see below) were also observed in nodules. (2) HCC. Three or more of the following criteria were required for diagnosing a tumor as malignant: (a) basophilia, (b) undifferentiated trabecular structure, (c) evidence of invasive growth and occasionally of metastasis, (d) nuclei larger than in the surrounding tissue and rich in chromatin, (e) high incidence of mitosis, or appearance of atypical mitotic figures, respectively. Finally, in diagnosing hepatocellular carcinoma, three stages were distinguished, namely, Stage I: trabecular structure well identifiable; Stage II: trabecular structure poorly identifiable; Stage III: no trabecular structure, anaplastic cells (Turusov, 1979Go).

Morphometric analysis.
The area of each tissue section analyzed to determine the number of PPF per square cm was determined with an image analyzer software (Lucia, Laboratory Imaging GmbH). A mean of 30 (±10) mm2 liver section was analyzed per animal. A large number of tumors and PFF was scored by more than one independent observer, and essentially the same results were obtained.

Apoptotic index (AI).
Apoptoses were identified in normal liver (NL), PPF, HCA, and HCC using H&E stained liver sections and were quantified as described previously (Bursch et al., 1992Go; Grasl-Kraupp et al., 1994Go). Briefly, hepatocytes with chromatin condensation typical of early stages of apoptosis, as well as intra- and extracellular apoptotic bodies, with or without visible chromatin, were recorded; their total number was expressed as percentage of the total number of hepatocytes scored in the respective cell population (%AI, apoptotic index). The morphology of apoptotic cell residues as demonstrated by H&E staining provides reliable data of apoptosis in hepatic foci and tumors, including eosinophilic lesions (Bursch et al., 1984Go; Grasl-Kraupp et al., 1994Go, 1997Go; for review Bursch et al., 1992Go). Furthermore, the reliability and sensitivity of these procedures for quantitative determination of apoptoses has been verified by the TUNEL technique (Chabicovsky et al., 2003Go; Grasl-Kraupp et al., 1995Go). Labeling Index (LI): BrdU-incorporation into DNA was visualized according to standard procedures; the number of BrdU-positive nuclei per total number of hepatocyte nuclei was calculated (%LI, labeling index; Grasl-Kraupp et al., 1997Go). The histological analysis of LI and apoptosis was necessary for calculation of the growth rate of PPF (see below). PPF (1) amount, at most, to 0.7% of the total liver mass (see results section), and (2) individual foci, particularly in early stages of hepatocarcinogenesis, are very small. These features of PPF preclude a quantitative analysis of cell proliferation and apoptosis from biochemical/molecular parameters to be measured in liver homogenates or dissected PPF.

Statistics.
If not stated otherwise, means (±SD) are given; data were analyzed by ANOVA, followed by Tukey-Kramer multiple comparisons test. The significance level was set at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 Conclusions
 REFERENCES
 
Body and Liver Weights
At necropsy, the body weight of NDEA-treated C3H/He mice was slightly less than that of control animals; no such trend was observed with C57Bl/6J and B6C3F1 animals (Table 1). Relative liver weights were increased in PB-treated animals at all time points studied (Table 1); in low susceptible C57Bl/6J mice, this increase was, on average, 33–39% (0->PB vs. 0->0) and somewhat more pronounced in the highly susceptible strains (average 37–68%). Liver enlargement at late time points in NDEA->0 and NDEA->PB mice was excessive and obviously resulted from formation of large liver tumors (see below).


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TABLE 1 Effect of NDEA and PB Treatment on Body and Liver Weight of C57Bl/6J, C3H/He, and B6C3F1 Mice

 
Incidence and Multiplicity of Liver Tumors
The incidence of macroscopically visible liver tumors (tumor-bearing animals per group) is summarized in Table 2. Twenty weeks after a single dose of NDEA, with or without subsequent PB-treatment, no animals of either strain developed macroscopically visible lesions. Between 40 and 92 weeks of the experiment, C57Bl/6J mice exhibited a lower incidence as well as later occurrence of liver tumors as compared to C3H/He mice; B6C3F1 showed an intermediate response (Table 2). The number of tumors per animal (multiplicity) is shown in Figure 2. At 40 weeks, a low tumor multiplicity was found in NDEA->PB-treated C57Bl/6J animals, followed by gradual increase until 74 weeks of PB (Fig. 2, upper panel). In contrast, at 40 weeks C3H/He mice of the NDEA->PB group already exhibited a maximal number of tumors (Fig. 2, middle panel). C3H/He mice treated with either NDEA or PB alone developed multiple liver tumors as well, but more slowly (PB alone not before 52 weeks) and to a lesser extent (fig. 2, middle panel). In general, at 40–76 weeks C3H/He mice exhibited a significantly higher liver tumor incidence than C57Bl/6J animals. In fact, because of the high liver tumor incidence and multiplicity, NDEA->PB-treated C3H/He mice could not be assigned to the later time points of the study (n.a. at 76, 92 weeks). The B6C3F1 strain was characterized by an intermediate tumor response (Fig. 2, lower panel).


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TABLE 2 Incidence of Macroscopical Liver Tumors in C57Bl/6J, C3H/He and B6C3F1 Mice

 


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FIG. 2. Liver tumor multiplicity in C3H/He, C57Bl/6J, and B6C3F1 mice. Mean number of tumors per animal (±SD) is given; number of animals is indicated in Figure 1. Treatment is indicated below the abscissa. Control (0->0): open columns; PB only (0->PB): cross-hatched columns; NDEA only (NDEA->0): columns hatched diagonally; NDEA plus PB (NDEA->PB): black columns. 0: no lesions found in this animal group; n.a.: no animals scheduled for respective time point. Horizontal dotted lines: guide for identification of treatment and time points of investigation in upper panels. Significant differences as indicated at the columns: (1) Strain differences: NDEA->PB, ap < 0.01, C3H/He versus C57Bl/6J and B6C3F1 at 40 and 52 weeks; bp < 0.05 B6C3F1 versus C57Bl/6J at 76 weeks NDEA->0, cC3H/He versus C57Bl/6J and B6C3F1 at 52 and 76 weeks; (2) PB-Promotion (NDEA->0 vs. NDEA->PB), dp < 005 C3H/He at 40 weeks, B6C3F1 at 52 and 76 weeks.

 
Size Distribution of Liver Tumors
The size of the tumors was recorded based upon their diameter, with size classes comprising <2, 2–5, 5–10, 10–20, and >20 mm, the results are plotted in Figure 3. In NDEA- or NDEA->PB-treated C57Bl/6J mice at 40 weeks, only few and very small lesions (<2 mm) had developed (Fig. 3, upper panel). This size class remained predominant, although larger tumors gradually occurred until 76 weeks (Fig. 3, middle and lower panels). In NDEA- or NDEA->PB-treated C3H/He mice, the predominant size class observed at 40 weeks was also <2 mm, the incidence of which, however, was much higher as compared to C57Bl/6J. Furthermore, some of the C3H/He mice treated with NDEA and PB developed tumors 10 to 20 mm in diameter (Fig. 3, upper panel). At later time points, liver lesions were found in all C3H/He groups, along with a pronounced shift to larger size classes (Fig. 3, middle and lower panels). Again, B6C3F1 mice exhibited an intermediate response (Fig. 3).



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FIG. 3. Mean number of lesions per animal within different size classes. Macroscopically visible lesions in the livers of C57Bl/6J and C3H/He mice are compared 40, 52, and 76 weeks after injection of NDEA. Size classes (largest diameter) were <2 mm, 2–5 mm, 5–10 mm, 10–20 mm, >20 mm; column pattern: see top panel. Number of animals is indicated in Figure 1; tumor incidence is given in Table 2. Horizontal dotted lines: guide for identification of treatment and time points of investigation in upper panels. 0: no lesions found in this animal group.

 
Histology of Liver Tumors
Macroscopically visible liver tumors of all size classes (<2 mm to >20 mm) were histologically classified as adenoma (HCA) or carcinoma (HCC, grades 1–3); the results are summarized in Table 3. HCC (sum of grades 1–3) in C57Bl/6J mice made up 11% of all liver tumors, but 36% in C3H/He mice. Furthermore, HCC grades 2 and 3 occurred in C3H/He mice but not in C57Bl/6J mice. B6C3F1 mice showed an intermediate response (15% HCC).


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TABLE 3 Histopathological Classification of Macroscopical Liver Tumors

 
Taken together, these observations show that the C3H/He strain, with or without PB treatment, is much more susceptible to hepatocarcinogenesis than the C57Bl/6J strain, in line with previous studies (Evans et al., 1992Go; Goldsworthy and Fransson-Steen, 2000; Pereira, 1993Go; Takahashi et al., 2002Go). Therefore, the present experiment provides a reliable basis for elucidating the role of birth and death in stages of carcinogenesis in these mouse strains.

Phenotype, Number, and Size of Putative Preneoplastic Liver Foci (PPF)
Analysis of PPF was confined to C57/Bl6J and C3H/He mice. Number, size, labeling index, as well as apoptotic index of putative preneoplastic foci (PPF) were recorded separately for eosinophilic/clear, tigroid, basophilic/amphophilic, vacuolated, and mixed cell phenotypes. In NDEA->PB-treated mice eosinophilic/clear cell lesions were found to constitute about 62% of all PPF; basophilic/amphophilic, tigroid, and mixed-type foci were much less frequent (9–13% each, at 52 weeks). No strain difference in the relative frequency of these phenotypic patterns was detected, except that vacuolated PPF were observed in C57Bl/6J animals only (about 15% of PPF, NDEA->PB at 76 weeks). The low incidence of some of the PPF subtypes precluded a separate statistical analysis of their growth pattern. Therefore, for the purpose of comparing the overall development of PPF in the different mouse strains, all PPF subtypes were summarized (Fig. 4). However, rates of cell proliferation and apoptosis are also discussed separately for each subtype (below and Tables 4 and 5).



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FIG. 4. Mean number of PPFs per cm2 unaltered tissue. Mean (±SD) per group is given, number of animals is indicated in Figure 1. All foci phenotypes were combined. n.e.: not evaluated. 0: no lesions found in this animal group. Horizontal dotted lines: guide for identification of treatment and time points of investigation in upper panels.

 

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TABLE 4 Effect of NDEA and PB Treatment on DNA Synthesis and Apoptosis in Stages of Hepatocarcinogenesis in C57Bl/6J and C3H/He Mice

 

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TABLE 5 Effect of Phenobarbital Withdrawal on DNA Synthesis and Apoptoses in NL, PPF, and HCA in the Liver of C3H/He Mice

 
In the livers of control C57Bl/6J mice (0->0), at 76 weeks no spontaneous PPF were found, while a small number was detected in NDEA->0 or NDEA->PB-treated mice (Fig. 4). PB treatment did not shift the size of eosinophilic/clear cell PPF in favor of larger classes (Fig. 5). Even at 76 weeks, large eosinophilic/clear cell PPF (>500 cells/cross section) were clearly less abundant in the NDEA->PB-treated group of C57Bl/6J mice than of C3H/He mice at 40 and 52 weeks (Fig. 5). Calculation of the volume fraction revealed that the focal tissue in the liver of C57Bl/6J mice at 76 weeks accounts for about 0.05% (without PB) and 0.08% (with PB) of the whole liver (for mathematical modeling see Grasl-Kraupp et al., 1997Go). In contrast, in the liver of C3H/He control animals (0->0), a few ("spontaneous") PPF were detected at 52 weeks; PB-treatment alone did not result in a significant increase (fig. 4). In NDEA->0 or NDEA->PB-treated C3H/He mice, however, the maximal number of PPF was reached already at 40 weeks (Fig. 4). Furthermore, PB clearly changed the size distribution of eosinophilic/clear cell PPF in favor of PPF larger than 500 cells/cross section (Fig. 5); at 52 weeks, focal tissue accounts for about 0.2% (without PB) and 0.7% (with PB) of the whole liver. The trend for decrease in PPF numbers at 52 weeks in NDEA->PB-treated mice is considered to reflect their rapid progression into advanced neoplastic lesions (cf. Fig. 2). Consequently, we did not quantitatively analyze the remaining few PPF in livers bearing a high number of neoplasms at late stages of the experiment.



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FIG. 5. Size distribution of eosinophilic/clear liver foci. The size of the eosinophilic/clear PPF was classified according to the number of cells/focus cross section; the width of the size classes is 20 cells. The number of PPF in each size class is expressed as percentage of the total number of PPF found in each group: C57Bl/6J NDEA->0 (52 weeks), 26 PPF; C57Bl/6J NDEA->PB (52 weeks), 43 PPF; C57Bl/6J NDEA->0 (76 weeks), 57 PPF; C57Bl/6J NDEA->PB (76 weeks), 55 PPF; C3H/He NDEA->0 (40 weeks), 42 PPF; C3H/He NDEA->PB (40 weeks), 25 PPF; C3H/He NDEA->0 (52 weeks), 48 PPF; C3H/He NDEA->PB (52 weeks), 41 PPF. In C3H/He 0->0 (52 weeks) and 0->PB (52 weeks) only one PPF was found in each group (134 and 128 cells, respectively) which are not included in Figure 5. Horizontal and vertical dotted line: guide for identification of treatment and time points of investigation.

 
DNA Synthesis and Apoptosis in Putative Preneoplastic Foci (PPF, HCA, and HCC)
The data on labeling indices (%LI) and apoptotic indices (%AI) in different phenotypes of PPF as well as HCA and HCC of C57Bl/6J and C3H mice are given in Table 4 and Figure 6. For easier reading lines (L) and columns (c) in Table 4 are numbered and referred to in the following description. Results obtained were:
DNA synthesis activity
(1) Different stages of carcinogenesis. In C57Bl/6J as well as C3H/He mice, DNA synthesis was lowest in phenotypically normal liver (NL, C57Bl/6J 0.03–0.6%, C3H/He 0.03–0.2%; Fig. 6; Table 4: c5), was clearly increased in eosinophilic/clear cell PPF (C57Bl/6J 0.2–0.9%, C3H/He 1.1–2.2%; Fig. 6, Table 4: c6), further enhanced in HCAs (C57Bl/6J 1.2–2.3%, C3H/He 0.8–4.5%; Fig. 6, Table 4: c7), and even more in HCCs (C57Bl/6J 1.6–3.5%; C3H/He 2.5–8.3%; Fig. 6, Table 4: c8).
(2) Effect of PB. In the liver of control animals (without NDEA), no clear effect of PB treatment on DNA synthesis could be detected (Fig.6; Table 4: c5, L6 vs. 11, L32 vs. 37). In NDEA-induced eosinophilic/clear cell foci, PB decreased DNA synthesis in C57Bl/6J mice and slighty (not significantly) induced it at 76 weeks (Fig. 6; Table 4: c6, L7 vs. 12, L15 vs. 18). In contrast, PB stimulated DNA synthesis in C3H/He mice at both time points (significant at 52 weeks; Fig. 6; Table 4: c6, L24 vs. 28, L33 vs. 39). In accordance with this observation, the size of PPF in PB-treated C57BL/6J was similar to mice without PB, but clearly increased in C3H/He (Fig. 5). HCA as well as HCC in both strains showed a clear stimulation of DNA synthesis by PB (Fig. 6; Table 4: c7 and c8, L7 vs. 12, L15 vs. 18, L24 vs. 28, L33 vs. 39).
(3) Strain difference. Overall, lesions of all stages of hepatocarcinogenesis in the C3H/He strain exhibited higher DNA synthesis activity than in C57Bl/6J mice (Figs. 6E–6H vs. 6A–6D).

Apoptotic activity
(4) Different stages of carcinogenesis. In phenotypically normal tissue of both mouse strains, apoptoses were undetectable or very low (C57Bl/6J 0–0.08%, C3H/He 0–0.04%; Fig. 6; Table 4: c9). PPF exhibited a slightly increased apoptotic activity (C57Bl/6J 0.05–0.1%, C3H/He 0.01–0.2%; Fig.6, Table 4, c10 vs. 9). No difference between the distinct PPF phenotypes was apparent (Table 4: c10). In HCA and HCC of both strains apoptoses were more frequent than in NL or PPF (C57Bl/6J 0.06–0.6%, C3H/He 0.02–0.3%; Fig.6, Table 4: c11 and c12) and tended to be highest in C57Bl/6J mice treated with NDEA->PB (76 weeks) (0.58%; Table 4: c12, L18; Fig. 6).
(5) PB-treatment. In C57Bl/6J mice, PB reduced apoptotic activity in PPF and HCA at 52 weeks (Fig. 6; Table 4: c10 and c11, L7–L10 vs. L12–L14), but not at 76 weeks (Fig. 6). In contrast, PB significantly increased apoptosis in HCA and HCC of C57BL/6J mice (Fig. 6; Table 4: c11 and c12, L15 vs. L18). In the C3H/He strain PB had no detectable effect on the low rate of apoptosis in eosinophilic/clear cell foci at 40 weeks, but significantly lowered apoptosis at 52 weeks (Table 4, c10, L24–L27 vs. L28–L31 and L33–L36 vs. L39–L42). No effect of PB on other PPF phenotypes was seen. In HCA and HCC, no consistent PB effect was found in the C3H/He strain.
(6) Strain difference. In phenotypically normal tissue, with or without PB treatment, the low apoptotic activity showed no strain difference (Fig. 6; Table 4: c9). However, depending on the stage of hepatocarcinogenesis, the two strains differed in their response to PB; PB was anti-apoptotic in eosinophilic/clear cell PPF in C3H/He mice and less pronounced at early stages in C57BL/6J mice. However, in HCA and HCC, PB was pro-apoptotic in C57BL/6J but not in C3H/He mice (Figs. 6M–6P vs. 6I–6L).



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FIG. 6. DNA synthesis and apoptosis in unaltered liver, eosinophilic PPF, HCA, and HCC. Means of labeling and apoptotic indices (%LI, %AI) from Table 4 are plotted. As to PPF, for the sake of clarity LI and AI of eosinophilic PPF only are shown, because they constitute the predominant phenotype. Number of animals, number of cells scored, LI and AI of other animal groups (0->0, 0->PB, further time points), tigroid, basophilic/amphophilic cell, vacuolated cell foci, mixed type PPF, and 95% confidence limits are given in Table 4. n.e. = not evaluated. Note the different scales of the ordinates for labeling index (A–H) and apoptotic index (I–P). *p < 0.05, see Table 4 for details.

 
Effect of Phenobarbital Withdrawal on DNA Synthesis and Apoptoses in the Liver of C3H/He Mice
At week 52 of the experiment, PB treatment was stopped for a subgroup of NDEA ->PB-treated C3H/He mice. As shown in Table 6, within 3 weeks of PB withdrawal the number of tumors per animal remained constant, but total mass of liver containing numerous tumors decreased by about 30%. Furthermore, PPF (eosinophilic/clear cell as well as basophilic/amphophilic) and HCA exhibited a clear decrease in DNA synthesis activity but no detectable increase of apoptotic activity.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 Conclusions
 REFERENCES
 
Preneoplastic Lesions and Tumors in C57Bl/6J and C3H/He Mice
C3H/He mice, in contrast to C57Bl/6J, are genetically predisposed to a high susceptibility to hepatocarcinogenesis. This study aimed to elucidate whether differences in the incidence of apoptosis are responsible for this strain difference. In a first step, the development of histological and macroscopical lesions, as well as their phenotypic patterns, was closely analyzed. In animals of both strains eosinophilic/clear cell foci constituted more than 60% of PPF, similar to previous observations (Evans et al., 1992Go; Kamendulis et al., 2001Go). Along with the quantitative data on DNA synthesis and apoptosis, these findings suggest that eosinophilic PPF are the preneoplastic cell population most prone to progress to HCA and HCC in our model. In support of this conclusion, Pereira (1993)Go reported that NDEA mainly induced basophilic foci and adenoma in C3H but not in C3B6F1 mice; PB treatment shifted the phenotypic pattern of lesions toward predominant eosinophilic staining. In general, the predominance of a given PPF phenotype over another in hepatocarcinogenesis is affected by the type of promoter (Bannasch et al., 2001Go).

PFF (eosinophilic/clear cell phenotype) of "spontaneous" origin occurred in untreated C3H/He mice at about 1 year of age, but even until the end of the experiment (92 weeks), none could be detected in the C57Bl/6J liver sections scored. After injection of NDEA the highest number of PPFs per C3H/He mouse was recorded at 40 weeks, whereas in C57Bl/6J mice significant numbers of PPF were not detected before week 76. Even at this time point the incidence of PPF tended to be lower than in C3H/He mice at 40 weeks. The present study also shows that PB enhances the number of neoplastic lesions in NDEA-treated C57Bl/6J, C3H/He, and B6C3F1 mice. This is in agreement with the known tumor-promoting effect of PB, which occurs in all three mouse strains as suggested previously (Kamendulis et al., 2001Go; Klaunig et al., 2003Go; Lee, 2000Go; Pereira, 1993Go; Takakashi et al., 2002Go; Whysner et al., 1996Go). Furthermore, the present study revealed that progression from HCA to HCC and to larger tumors occurs more readily in C3H/He (36% HCC; sum of grades 1–3) than in C57Bl/6J mice (11% HCC, grade 1 tumors only).

Taken together, the results of our study confirm and extend previous observations on susceptibility to hepatocarcinogenesis in C3H/He, C57Bl/6J, and B6C3F1 mice (Dragani, 2003Go; Fausto, 1999Go; Gold et al., 1998Go; Goldsworthy et al., 2002Go; Kamendulis et al., 2001Go; Poole and Drinkwater, 1996Go; Whysner et al., 1990). Specifically, our findings add support to the view that susceptibility to spontaneous and chemical hepatocarcinogenesis reflects quantitative differences in growth rates of PPF and tumors in C57Bl/6J, C3H/He, and B6C3F1 mice. This notion is important, because C57Bl/6J mice are often considered "resistant" to liver cancer induction. Likewise, Takahashi et al. (2002)Go recently showed that, of five mouse strains under study, all developed liver neoplasms; strains primarily differed in the latency period.

The main focus of the present study was to analyze, in quantitative terms, the role of cell birth and cell death as determinants of the growth rate of PPF and tumors and, hence, of tumor susceptibility of C3H/He and C57Bl/6J mice. Several important implications of our findings are discussed below. A schematic presentation of the major conclusions is given in Figure 7.



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FIG. 7. Divergent contribution of cell proliferation and of inhibition of apoptosis to the growth of (pre)neoplasia in the liver of C57Bl/6J and C3H/He mouse strains and of rats. See discussion for explanation. The thickness of the arrows indicates the relative contribution of a process to hepatocarcinogenesis. : no response; ?: no data on PB; {uparrow}: stimulation of cell proliferation; {perp}: inhibition of apoptosis; white: normal cell; light grey: initiated cell; dark grey: tumor cell (HCA, HCC).

 
DNA Synthesis and Apoptosis in Different Stages of Hepatocarcinogenesis in Mouse Strains and in Rats
In both strains, but clearly more pronounced in C3H/He, hepatocellular DNA synthesis activity increased from normal liver tissue via PPF and HCA to HCC. Furthermore, in all stages of carcinogenesis the rate of DNA synthesis, on average, was two to three times higher in the C3H/He than in the C57BL/6J strain. Likewise, Hanigan and colleagues (1988)Go found that the growth rate of G6Pase-deficient hepatic foci (induced by N-ethyl-N-nitrosourea) of male C3H/HeJ was about two times faster than of male C57BL/6J mice; accordingly, the thymidine labeling index of G6Pase-deficient hepatocytes was higher in C3H/HeJ than in C57BL/6J mice. In summary, the present study clearly suggests that the rate of cell birth in (pre)neoplastic lesions positively correlates with tumor susceptibility of the mouse strains studied. As to apoptosis, in untreated controls as well as in phenotypically unaltered liver of NDEA-treated C3H/He and C57Bl/6J mice, apoptotic cell death was extremely rare. At variance, Klaunig and coworkers observed a markedly enhanced apoptotic incidence in unaltered hepatocytes of B6C3F1 mice (for review see Kamendulis et al., 2001Go). This difference most likely can be explained by different experimental protocols such as the repeated injections of NDEA for a considerable period of time (2 x 35 mg/kg NDEA per week/8 weeks). In the present experiment, (pre)neoplastic lesions in the liver of C3H/He mice revealed no or, at most, a trend of an increase of apoptoses (not consistently in all groups, with PB in HCC only). Obviously, the increase in DNA synthesis activity in PPF was not associated with a concomitant increase in apoptoses. Furthermore, the tumors in NDEA->0 C3H/He mice (40 and 76 weeks) have been previously analyzed for Ha-ras mutations; neither Ha-ras wild-type nor Ha-ras mutated tumors exhibited a positive correlation between DNA synthesis and apoptotic activity (Frey et al., 2000Go).

These findings, unexpectedly, are in marked contrast to previous observations by ourselves and others in rat as well as in human liver, which clearly showed increases of both cell birth and cell death, in early and later stages of hepatocarcinogenesis (Bursch et al., 1984Go; Grasl-Kraupp et al., 1997Go). Thus, the present data suggest that preneoplastic hepatocytes of mice do not enter apoptosis as readily as preneoplastic hepatocytes of rats. Likewise, signals inducing organ regression of normal liver did not enhance apoptotic activity in C3H/He, B6C3F1, and C57Bl/6J mice, in contrast to rat liver (see Bursch et al., accompanying manuscript). In support of this species difference, TGF-ß1 had a much weaker pro-apoptotic action on mouse than on rat hepatocytes (Chabicovsky et al., 2003Go; Parzefall et al., 2002Go).

Effects of Phenobarbital on DNA Synthesis and Apoptosis and Their Role in Tumor Promotion in C3H/He and C57Bl/6J Mice
In unaltered liver, no unequivocal effect of PB (approx. 90 mg/kg/d) on DNA synthesis could be observed. This is in accordance with the results of short-term studies where even higher PB doses (170–220 mg/kg/d) induced only a moderate DNA synthesis response in the liver of all three strains (see Bursch et al., accompanying manuscript). Liver enlargement, however, was clearly more pronounced in C3H/He than in C57Bl6/6J mice (as in the short-term studies; see Bursch et al., accompanying manuscript).

In eosinophilic/clear cell PPF PB stimulated DNA synthesis in C3H/He mice only. In C57BL/6J mice no enhancement of DNA synthesis by PB was seen at the two time points studied. In accordance with this divergent PB-effect on DNA synthesis, PB increased the size of eosinophilic/clear cell PPF in the C3H/He but not in the C57Bl/6L strain. Likewise, Pereira (1993)Go found that PB clearly increased the bromodeoxyuridine labeling index of eosinophilic foci in C3H mice but only marginally so in B6C3F1 mice. Goldsworthy and Fransson-Steen (2002)Go did not observe such a promoting action of PB on foci growth in C3H/He mice and even a suppression of foci volume fraction in B6C3F1 and C57Bl/6J mice. Most likely, these seemingly conflicting observations can be explained by differences in the experimental protocols employed (NDEA-administration to newborn mice [15 days] in the study by Goldsworthy vs. young adult mice [5 weeks] in our study). Furthermore, in both strains—but more pronounced in C3H/He mice—PB induced a clear increase of DNA synthesis in HCA and in HCC. This stimulatory effect of PB on DNA synthesis in tumor cells is supported by the PB withdrawal study, which revealed a marked tumor regression within 3 weeks, associated with a low DNA synthesis rate.

The effects of PB on apoptosis were divergent, depending on strain and stage of hepatocarcinogenesis. Thus in C3H/He mice, PPF at early stages (40 weeks) showed a very low apoptotic activity, and consequently, a possible anti-apoptotic action of PB would not be detectable. However, a survival effect of PB became manifest at late stages, namely in eosinophilic PPF at 52 weeks and after progression to HCA at 40 weeks. Likewise, the rapid decline of liver tumor mass after PB withdrawal at 52 weeks would be consistent with a survival effect of PB treatment. However, apoptotic counts were low at 3 weeks. Possibly, at this time the rapid regression process of the tumors had already reached its end. Direct evidence of a survival effect of PB in C3H/He liver tumors therefore requires apoptosis counts at an earlier time (Bursch et al., 1992Go). In C57Bl/6J mice PB had a slight anti-apoptotic activity in PPF and HCA at 52 weeks but not at the later stage (76 weeks). Rather, at 76 weeks PB significantly increased apoptosis in HCA and HCC of C57BL/6J mice. These observations suggest that PB may exert opposite effects on hepatocellular apoptosis during hepatocarcinogenesis in the two mouse strains, being anti-apoptotic in eosinophilic PPF of C3H/He and at early stages in C57BL/6J mice, but pro-apoptotic in later stages in C57BL/6J mice.

In conclusion, in view of the overall low apoptotic activity in eosinophilic PPF of both mouse strains, apoptosis and its inhibition by PB appear to play only a minor role as a growth determinant of preneoplasia in both strains (Figs. 6 and 7). However, in HCA the anti-apoptotic effect of PB in C3H/He may contribute somewhat to the rapid tumor growth in this strain, while the pro-apoptotic effect on HCA and HCC in C57Bl/6J mice may partially explain the slow growth of tumors in C57Bl/6J. This hypothesis should be confirmed by studies on a larger series of tumors. Similar to the present findings, suppression of apoptosis in liver foci has been considered unlikely to contribute to mouse liver tumor promotion by dieldrin (Stevenson et al., 1999Go).


    Conclusions
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 Conclusions
 REFERENCES
 
The present observations along with literature data strongly suggest the following conclusions:

  1. (1) The rate of cell proliferation rather than apoptotic activity constitutes the major determinant for liver tumor development in the two mouse strains studied.
  2. (2) The high tumor susceptibility of C3H/He mice obviously results from the higher cell proliferation at all stages of carcinogenesis in this strain as compared to C57Bl/6J mice (Figs. 6 and 7). Strain differences in apoptosis in PPF do not appear to affect cancer susceptibility, while they may contribute to the different growth rates of HCA and HCC.
  3. (3) Tumor promotion by PB was more pronounced in C3H/He than in C57Bl/6J mice. This strain difference resulted from two effects: (1) enhanced replication and some inhibition of apoptosis in PPF of C3H/He mice, while PPF in C57Bl/6J mice showed little if any response, (2) stronger increase of proliferation in HCA and HCC of C3H/He than of C57Bl/6J mice (Figs. 6 and 7).
  4. (4) Normal as well as preneoplastic hepatocytes of mouse and rat exhibit profound differences in their sensitivity to pro-apoptotic signals. As a consequence, the potential of phenobarbital to act as either mitogenic or survival factor contributes to tumor promotion in rats and mice at different extents. In rats, PB stimulates DNA synthesis of preneoplastic lesions only transiently, and tumor promotion is largely effected by inhibition of apoptosis (Kamendulis et al., 2001Go; Schulte-Hermann et al., 1990Go). The reverse is true in mice.


    NOTES
 
2 Current address: Igeneon Immunotherapy of Cancer AG, Brunner Strasse 69, A-1230 Vienna. Back

Parts of this study have been presented at the 41st Congress of the European Societies of Toxicology, EUROTOX 2003 "Science for Safety," Florence, Italy, September 28–October 1, 2003.


    ACKNOWLEDGMENTS
 
The excellent technical assistance of P. Breit, B. Bublava. and C. Unger is gratefully acknowledged.


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