* Medizinische Universität Wien, Univ. Klinik für Innere MedizinI, Abtl. Institut für Krebsforschung, Borschkegasse 8a, A-1090 Wien, and 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
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ABSTRACT |
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Key Words: B6C3F1; C3H/He; C57BL/6J; hepatocarcinogenesis; tumor susceptibility; apoptosis; cell proliferation; phenobarbital.
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INTRODUCTION |
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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., 1984). 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., 1992
; Grasl-Kraupp et al., 1997
; Luebeck et al., 1995
; Schulte-Hermann et al., 1990
). 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., 2001
; Luebeck et al., 1995
; Oliver and Roberts, 2002
; Pitot et al., 2000
; Schwarz et al., 2000
; Tharappel et al., 2002
).
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., 1988; Pereira, 1993
) or did not exhibit a significant role for growth of preneoplasia (Goldsworthy and Fransson-Steen, 2002
; James and Roberts, 1996
; Kamendulis et al., 2001
; Stevenson et al., 1999
). Other studies reported apoptosis-inhibition by tumor-promoting agents (phenobarbital, peroxisome proliferators) as important determinants for tumor development in mice (James et al., 1998
; Sanders and Thorgeirsson, 2000
). 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., 2003
; Parzefall et al., 2002
).
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.
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MATERIAL AND METHODS |
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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 ("NDEA0") 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 0.050.07% to provide a constant daily intake of 90 mg PB/kg b.w.
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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., 1994); 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, 1994; Evans et al., 1992
; Kraupp-Grasl et al., 1991
; Maronpot et al., 1987
; Turusov et al., 1979
).
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, 1979).
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., 1992; Grasl-Kraupp et al., 1994
). 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., 1984
; Grasl-Kraupp et al., 1994
, 1997
; for review Bursch et al., 1992
). Furthermore, the reliability and sensitivity of these procedures for quantitative determination of apoptoses has been verified by the TUNEL technique (Chabicovsky et al., 2003
; Grasl-Kraupp et al., 1995
). 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., 1997
). 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.
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RESULTS |
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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 NDEAPB-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 (913% 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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DISCUSSION |
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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., 2001; Klaunig et al., 2003
; Lee, 2000
; Pereira, 1993
; Takakashi et al., 2002
; Whysner et al., 1996
). 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 13) 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, 2003; Fausto, 1999
; Gold et al., 1998
; Goldsworthy et al., 2002
; Kamendulis et al., 2001
; Poole and Drinkwater, 1996
; 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)
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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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., 1984; Grasl-Kraupp et al., 1997
). 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., 2003
; Parzefall et al., 2002
).
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 (170220 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) 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)
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 strainsbut more pronounced in C3H/He micePB 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., 1992). 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., 1999).
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Conclusions |
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NOTES |
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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 28October 1, 2003.
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ACKNOWLEDGMENTS |
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