No Increase of Apoptosis in Regressing Mouse Liver after Withdrawal of Growth Stimuli or Food Restriction
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《毒物学科学杂志》
Medizinische Universitt Wien, Univ. Klinik für Innere MedizinI, Abtl. Institut für Krebsforschung, Borschkegasse 8a, A-1090 Wien
ABSTRACT
In short-term in vivo experiments, liver growth and regression in mice with high (C3H/He), intermediate (B6C3F1) or low (C57BL/6J) susceptibility to hepatocarcinogenesis was compared. Liver growth was induced by dietary administration of phenobarbital (PB; 750 ppm) or nafenopin (NAF; 500 ppm). PB or NAF treatment for 7 days produced moderate increases of liver DNA (15% or 25–28%, respectively) along with pronounced hypertrophy. Liver growth was strongest in C3H/He mice. Cessation of PB or NAF treatment led to a rapid regression of liver hypertrophy. However, the enhanced hepatic DNA content persisted for at least 2 weeks in all mouse strains. Apoptosis was not increased at any time after cessation of treatment in all strains. Food restriction to 60% of the ad libitum intake did not amplify either regression of liver hyperplasia or the occurrence of apoptosis. No strain difference in the occurrence of apoptosis was detected. Mouse hepatocytes in liver regressing after mitogen withdrawal do not enter apoptosis as readily as rat hepatocytes.
Key Words: phenobarbital; nafenopin; C3H/He; B6C3F1; C57Bl/6J; liver weight; protein content; DNA content; DNA synthesis; apoptosis.
INTRODUCTION
Cancer risk assessment of industrial chemicals, pesticides, food additives, drugs, and other substances is largely based upon lifetime bioassays with mice and rats. Among the species often used for this purpose, some mouse strains (B6C3F1, C3H/He) exhibit a high, others (C57BL/6J, NMRI) a low susceptibility to spontaneous and chemically induced hepatocarcinogenesis (Diwan et al., 1990; Fausto, 1999; Gold et al., 1998; Klaunig et al., 2003; Takahashi et al., 2002). Elucidation of the underlying causes is of importance from a basic scientific as well as from a practical toxicological point of view. A great deal of attention has been paid to genes which, according to the multistage concept of carcinogenesis, may affect either initiation or promotion (Dragani, 2003; Feo and Pascale, 1998; Maronpot et al., 1995; Poole and Drinkwater, 1996; Ogawa et al., 1999).
In our studies on rat hepatocarcinogenesis, we have previously demonstrated that apoptosis constitutes a protective mechanism against cancer formation, and in 1984 we were the first to show that inhibition of apoptosis by liver tumor promoters accelerated the manifestation of frank neoplasia (Bursch et al., 1984; Schulte-Hermann et al., 1995). However, relatively little is known about the role of apoptosis in determining susceptibility to liver cancer in different mouse strains. Based upon different long-term experimental models, somewhat conflicting data on apoptosis in mouse hepatocarcinogenesis have been reported (Goldsworthy and Fransson-Steen, 2002; James and Roberts, 1996; Kamendulis et al., 2001; Sanders and Thorgeirsson, 2000; Stevensson et al., 1999).
Consequently, we addressed the question whether the high cancer susceptibility of B6C3F1 and C3H/He mice may result from low efficiency or even failure of cancer defense by apoptosis. In a first series of experiments we performed comparative short-term in vivo studies on liver growth (cell proliferation) and involution (apoptosis). C3H/He, C57Bl6/J, and B6C3F1 mice were treated with the "classical" tumor promoter phenobarbital (PB) or the peroxisome proliferator nafenopin (NAF) as model compounds. Short-term in vivo studies with mice and rats have been successfully used to characterize the liver response to drugs and industrial chemicals in view of their hepatocarcinogenic potency (for review, see Huber et al., 1996; Schulte-Hermann et al., 1995; Whysner et al., 1996). The studies addressing apoptosis regulation in normal mouse liver served to complement and to better understand the results of a long-term carcinogenesis study with these mouse strains (see Bursch et al., accompanying manuscript). The in vivo approach was chosen because the actual rates of cell replication and of apoptosis integrate survival- and death-controlling factors in the context of the organism's genetic background.
Here we report on the results of the short-term experiments (7- to 14-day PB or NAF treatment followed by withdrawal) focused on cell proliferation and apoptosis. The results revealed that PB/NAF-induced mouse liver growth largely is due to cell enlargement (hypertrophy). In all strains of mice under study, liver regression upon cessation of PB/NAF treatment was not associated with an increase in apoptoses; this observation was confirmed by biochemical analysis of liver DNA content. Furthermore, food restriction did not amplify liver regression and the occurrence of apoptosis. Thus, as to strain differences in cancer susceptibility, C3H/He mice exhibited a more pronounced DNA synthesis response to PB or NAF than C57Bl/6J, thereby revealing a positive correlation between short-term effects and cancer susceptibility. However, no difference among the mouse strains with respect to the occurrence of apoptosis was detected. Surprisingly, mouse hepatocytes do not appear to enter apoptosis as readily as rat hepatocytes (species difference).
MATERIAL AND METHODS
Animals, husbandry and induction of liver growth.
Seven- to eight-week-old female and male C57Bl/6J, C3H/He and B6C3F1 mice were obtained from Zentralinstitut für Versuchstierzucht, Hannover, Germany, or from Institut für Labortierkunde und Genetik, Himberg, Austria. The animals were housed individually and adapted for 4–6 weeks to a reversed light-dark-rhythm (lights on 10 P.M. to 10 A.M., lights off from 10 A.M. to 10 P.M.) with food (standard maintenance diet Altromin 1321N, Altromin, Lage, Germany) and tap water ad libitum. Animals were sacrificed at the end of the lighting period, when apoptosis is expected to be at its diurnal maximum. Liver growth was induced by phenobarbital (PB) or nafenopin (NAF); both compounds were administered via the diet, PB (5-ethyl-5-phenyl-barbituric acid, Fluka Chemie AG, Buchs, Switzerland): 2 days 500 ppm, followed by 5 days 750 ppm; NAF (nafenopin, gift from Ciba-Geigy, Basel, Switzerland): 7 days 500 ppm. The effective dose per kg body weight was calculated from food consumption (Table 1). PB-doses consumed were similar in all groups except for male C3H/He, whose PB intake was somewhat higher than in female C3H/He (20%). Overall, five independent animal experiments were performed. All experiments were performed according to Austrian and EU regulations for animal care and treatment.
Food restriction.
To study the effect of food restriction on liver regression and apoptosis, mice were pretreated with PB for 7 days as described above. Along with the stop of PB-treatment, the mice
DNA synthesis.
The rate of DNA synthesis was measured based upon BrdU-incorporation into DNA. For continuous infusion of BrdU (5-bromo-2'-deoxyuridine, Sigma Chemicals, St. Louis, MO) osmotic minipumps (Alza Corporation, Palo Alto, CA) were implanted sc under isoflurane anesthesia (Abbott Laboratories, Queensborough, UK; 1.5% isoflurane [v/v] in 60% N2O and 40% O2 [v/v]); 3-day pumps (Alzet model 1003D) and 7-day pumps (Alzet model 2001) were used to deliver a BrdU solution (20 mg BrdU/ml PBS) at a rate of 1 μl/h.
Sacrifice, biochemical, and histological procedures.
Animals were anaesthetized with isoflurane, decapitated, and exsanguinated. The liver was quickly excised, and the gall bladder was carefully removed. For the biochemical determination of hepatic DNA and protein content, representative liver samples from the large median lobe were frozen, stored in liquid nitrogen, and processed as described elsewhere (Grasl-Kraupp et al., 1994). Other liver specimens of the same lobe were cut into 4- to 5-mm thick slices and fixed either with Carnoy's solution or 4% neutral buffered formalin according to Lillie (Pearse, 1980). Samples fixed in Carnoy's solution were transferred into isopropanol after 24 h and were stored in isopropanol until being processed for histology. Formalin-fixed tissue was processed after a fixation time of 24 to 48 h. Sections (3 to 5 μm) were mounted on APES-coated glass slides (3-aminopropyl-triethoxysilane, Fluka Chemie AG, Buchs, Switzerland) to ensure adhesion during processing. H&E (hematoxylin and eosin) staining was performed according to standard protocols. For the detection of BrdU-incorporation, formalin fixed liver specimens were processed as described in detail elsewhere (Chabicovsky et al., 2003) using a mouse monoclonal antibody to BrdU (Boehringer-Mannheim, Mannheim, Germany; 1:800 in PBS/0.1% BSA). Biotinylated rabbit anti-mouse IgG (DAKO A/S, Glostrup, Denmark; 1:100 in PBS/0.1% BSA) was used as second antibody, followed by peroxidase-conjugated streptavidin (DAKO A/S, Glostrup, Denmark; 1:100 in PBS/0.1% BSA) and visualized with DAB (3,3'-diaminobenzidine tetrahydrochloride). Sections were counterstained with Mayer's hematoxylin and mounted with Entellan.
Labeling index and apoptotic index.
The rate of DNA synthesis was determined histologically in BrdU-stained liver sections; a section of duodenum, a tissue with a high cell proliferation rate, was included on each slide to confirm the systemic delivery of BrdU to the animal. At least 2000 hepatocyte nuclei per animal were counted using a Nikon Microphot-FXA microscope (Tokyo, Japan). The labeling index (LI) was expressed as percentage of labeled hepatocyte nuclei in the total population of nuclei counted. For quantitative determination of apoptosis, 3000–4000 hepatocytes per animal were scored. Hepatocytes exhibiting chromatin condensation typical of apoptosis, apoptotic bodies with or without chromatin located intra- or extracellularly were recorded. The total number of apoptoses was expressed as percentage of the total number of hepatocytes counted; the low apoptotic counts as determined by scoring 3000–4000 hepatocytes per liver section were verified by screening whole liver sections. The validity of this method for quantitative determination of apoptoses with H&E-stained liver sections has been demonstrated previously (Chabicovsky et al., 2003; Schulte-Hermann et al., 1995).
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
Liver Weight, Protein, and DNA Content
In female inbred C57Bl/6J, C3H/He, and B6C3F1 mice, phenobarbital (PB) administration in doses of 500 and 750 ppm via the diet for 7 days caused an increase in liver weight by 37 to 60 % (Figs. 1A–1C). Biochemical analysis revealed marked increases in liver protein content approximately parallel to the increase in liver mass (Figs.1D–1F), but only a moderate increase in liver DNA (17–32%, Figs. 1G–1I). The increases in liver weight and liver protein content were significantly higher in C3H/He than in C57Bl/6J mice. These findings were confirmed and extended by studies on male C57Bl/6J, C3H/He, and B6C3F1 mice (Fig. 2). After PB treatment for 7 days, increases in liver mass and DNA content did not differ significantly between males and females, although they tended to be less in male mice of either strain. Furthermore, continuing PB treatment until day 14 did not result in further increase either in liver weight/ protein or in liver DNA content (Fig. 2). Male C57Bl/6J, C3H/He, and B6C3F1 mice were also treated with nafenopin (NAF, 750 ppm) resulting in an increase in liver mass by 85–113% (Figs. 2D–2F) and liver DNA content by 25–28% (Figs. 2G–2I). Notably, NAF, at the dose level chosen, caused a much more pronounced liver enlargement than PB in all three strains of mice.
As summarized in Figure 3, the magnitude of liver growth induced by short-term treatment with PB was clearly more pronounced in C3H/He mice than in C57BL/6J mice, independent of dose and sex.
Liver DNA Synthesis
The rate of hepatocyte DNA synthesis was measured in males by continuous infusion of BrdU for 3 and 7 days. As shown by histological analysis, in the livers of control male C3H/He, C57BL/6J, and B6C3F1 mice about 0.4% (3 days) and 1% (7 days) of hepatocyte nuclei were labeled with BrdU without exhibiting a significant difference among the mouse strains (Fig. 4). PB treatment resulted in significant increases in the number of BrdU-labeled nuclei to about 9–12% within 7 days (Fig. 4). As observed previously, the mouse liver growth response to PB was preferentially brought about by pericentral (zone 3) hepatocytes (data not shown; Schulte-Hermann et al., 1995). Overall, the hepatocellular labeling indices in PB treated animals did not differ significantly among the three mouse strains, although its stimulation above control level tended to be stronger in C3H/He (9-fold above controls) than in C57BL/6J (6-fold above controls) and B6C3F1 (7-fold above controls). No mitoses were detected in liver sections (3000–4000 hepatocytes scored) of control C3H/He, C57BL/6J, and B6C3F1 mice. Slightly but insignificantly enhanced numbers of mitoses were found in C57Bl/6J (0.03 ± 0.04%) and C3H/He (0.05 ± 0.02%) mice after PB for 3 days. In immunohistochemically stained sections, all mitoses were positive for BrdU-incorporation (not shown). Thus, a clear strain difference in mitotic activity, with or without PB treatment, was not detected.
Liver Regression and Apoptosis after PB or NAF Withdrawal
Discontinuation of PB or NAF treatment of C3H/He, C57BL/6J, and B6C3F1 mice resulted in a decrease of liver weight and liver protein content back to control level within 1–2 weeks (Figs. 1A–1G and 2A–2F). The enhanced liver DNA content, however, persisted at least until 14 days after cessation of PB or NAF treatment (Figs. 1 and 2G–2I).
Apoptoses were extremely rare at any time point after cessation of treatment in control and withdrawal groups of all mouse strains (Table 2). The lack of increased numbers of apoptoses in involuting livers of mice is in accordance with the persistent enhancement of liver DNA.
Effects of Food Restriction after Cessation of PB Treatment
In an attempt to amplify liver regression and the occurrence of apoptoses, PB-pretreated mice were subjected to a 40% food restriction (FR) along with PB-withdrawal; FR has been previously shown to induce apoptoses in normal, hyperplastic, and preneoplastic cell populations of rat liver (Grasl-Kraupp et al., 1994). In the present study, body weight of FR animals decreased steadily within the first week of food restriction, reaching the lowest levels (approx. 70% of the initial body weight) within 7 to 9 days; body weight loss did not differ among the mouse strains (Figs. 5A and 5B). Changes in liver weight, DNA and protein content, and hepatocellular size are illustrated by data on C3H/He mice; essentially the same observations were made with C57BL/6J and B6C3F1 mice (not shown). Liver weight and liver protein decreased by approximately 55–60% within one week of FR, but no further decrease occurred during the second week of FR (Figs. 5C and 5D). The decrease of liver weight and protein content led to a significant increase in the number of hepatocytes per microscopic field in food-restricted animals, indicating cellular hypotrophy (Fig. 5F). Even food restriction for up to 14 days did not result in a significant decrease liver DNA content as compared to animals fed ad libitum (Fig. 5E). Accordingly, histological analysis revealed no significant increase in apoptotic activity at all time points investigated (2, 4, 7, and 14 days FR; Table 2).
DISCUSSION
The present study revealed that the liver-tumor-susceptible C3H/He mice exhibited a more pronounced (although not thoroughly significant) increase in liver mass, liver protein, and liver DNA content than the less susceptible C57Bl/6J mice. Notably B6C3F1, the F1-generation of paternal C3H/He and maternal C57Bl/6J, tended to an intermediate response. Relative increases in liver mass and liver protein exceeded those of liver DNA content, suggesting that mouse liver enlargement in response to PB and NAF largely reflects hypertrophy; the latter was confirmed by histomorphometrical analysis of hepatocellular size (not shown). In summary, these observations confirm and extend results of previous studies on PB and NAF-induced mouse liver growth (for review, see Diwan et al., 1990; Fausto, 1999; Huber et al., 1996; Kamendulis et al., 2001; Sarraf et al., 1997; Schulte-Hermann, 1995). Specifically, the present study revealed a strain-specific response pattern to short-term PB treatment with a positive correlation between the induction of liver growth and liver tumor susceptibility; a schematic presentation is given in Fig. 6.
The present study also revealed that mouse liver enlargement induced by PB is completely reversible within a few days after cessation of treatment. Similar observations on the regression of surplus liver mass upon discontinuation of PB treatment have been reported previously (for review, see Diwan et al., 1990; Schulte-Hermann et al., 1995). As to the mechanism, excess membranes of endoplasmic reticulum as well as of the peroxisomal compartment are selectively removed by autophagy (for review, see Blommaart et al., 1997). However, in contrast to hypertrophy the enhanced DNA content persisted for at least 14 days after PB withdrawal. In accordance with this observation, essentially no apoptoses were observed during liver involution after cessation of treatment. Furthermore, food restriction did not stimulate apoptosis in regressing mouse liver. We expected this to occur, as in rat liver a massive apoptotic response was observed under such conditions (Grasl-Kraupp et al., 1994; Hikita et al., 1998; Tomasi et al., 1999). However, the biochemical data of the present study (no significant loss of liver DNA) strongly support the histological findings (no increase in apoptoses). In other studies, hepatocellular apoptosis in C57Bl/6JxC3H F1 mice did not increase before 60 days of dietary restriction by 40% (James and Muskhelishvili, 1994; Kolaja et al., 1996). Taken together, these findings strongly suggest that mouse hepatocytes do not enter apoptosis as readily as rat hepatocytes; the underlying causes are not known. It should be emphasized that this conclusion apparently applies to both compounds studied, namely PB and NAF, which exert their action on hepatocytes through different receptor-signal transduction pathways (Oliver and Roberts, 2002).
Moreover, other in vivo as well as cell culture studies revealed that mouse hepatocytes are much less sensitive to the pro-apoptotic action of TGF-1 than rat hepatocytes (Chabicovsky et al., 2003; Parzefall et al., 2002). Thus, 56 μg TGF-1/kg did not induce apoptosis in mouse liver, whereas in rat liver a dose as low as 0.25 μg TGF-1/kg was found to be effective (Chabicovsky et al., 2003; Oberhammer et al., 1992; Schulte-Hermann et al., 2002a; Fig. 6). Likewise, comparative studies with primary cultures of hepatocytes from C3H and C57BL mice revealed that at any concentration between 0.1 and 100 ng/ml TGF-1 exerted only a weak proapoptotic effect (less than 1%), whereas rat hepatocytes (F344) cultured under the same conditions were readily stimulated to undergo apoptosis at concentrations of 0.3 ng/ml or higher (up to 6%, ED50 = 0.8 ng/ml; Parzefall et al., 2002; Fig. 6). Notably, mouse hepatocytes are highly sensitive to the pro-apopotic action of ligands of the TNF/NGF-receptor family (for review, see Kanzler and Galle, 2000; Schulte-Hermann et al., 2002b); it is tempting to raise the hypothesis that rats and mice might differ with respect to the relative contribution of various cytokine networks to control of hepatocellular apoptosis.
In conclusion, the present results show that the tumor-promoting efficiency of prolonged PB treatment corresponds to the short-term effects of PB in mouse liver. Thus, among the three inbred mouse strains studied, the highly cancer-susceptible C3H/He mice exhibited the strongest growth response to the tumor-promoting agent PB. Surprisingly, no strain difference with respect to the occurrence of apoptosis was found. This conclusion meets well with our observations in a long-term carcinogenesis study on C3H/He and C57Bl/6J mice, which indicates that the rate of cell proliferation largely determines susceptibility for tumorigenesis (see Bursch et al., accompanying manuscript). On the other hand, profound differences in the sensitivity to pro-apoptotic signals appear to exist between mouse and rat hepatocytes.
NOTES
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 Ch. Unger is gratefully acknowledged.
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ABSTRACT
In short-term in vivo experiments, liver growth and regression in mice with high (C3H/He), intermediate (B6C3F1) or low (C57BL/6J) susceptibility to hepatocarcinogenesis was compared. Liver growth was induced by dietary administration of phenobarbital (PB; 750 ppm) or nafenopin (NAF; 500 ppm). PB or NAF treatment for 7 days produced moderate increases of liver DNA (15% or 25–28%, respectively) along with pronounced hypertrophy. Liver growth was strongest in C3H/He mice. Cessation of PB or NAF treatment led to a rapid regression of liver hypertrophy. However, the enhanced hepatic DNA content persisted for at least 2 weeks in all mouse strains. Apoptosis was not increased at any time after cessation of treatment in all strains. Food restriction to 60% of the ad libitum intake did not amplify either regression of liver hyperplasia or the occurrence of apoptosis. No strain difference in the occurrence of apoptosis was detected. Mouse hepatocytes in liver regressing after mitogen withdrawal do not enter apoptosis as readily as rat hepatocytes.
Key Words: phenobarbital; nafenopin; C3H/He; B6C3F1; C57Bl/6J; liver weight; protein content; DNA content; DNA synthesis; apoptosis.
INTRODUCTION
Cancer risk assessment of industrial chemicals, pesticides, food additives, drugs, and other substances is largely based upon lifetime bioassays with mice and rats. Among the species often used for this purpose, some mouse strains (B6C3F1, C3H/He) exhibit a high, others (C57BL/6J, NMRI) a low susceptibility to spontaneous and chemically induced hepatocarcinogenesis (Diwan et al., 1990; Fausto, 1999; Gold et al., 1998; Klaunig et al., 2003; Takahashi et al., 2002). Elucidation of the underlying causes is of importance from a basic scientific as well as from a practical toxicological point of view. A great deal of attention has been paid to genes which, according to the multistage concept of carcinogenesis, may affect either initiation or promotion (Dragani, 2003; Feo and Pascale, 1998; Maronpot et al., 1995; Poole and Drinkwater, 1996; Ogawa et al., 1999).
In our studies on rat hepatocarcinogenesis, we have previously demonstrated that apoptosis constitutes a protective mechanism against cancer formation, and in 1984 we were the first to show that inhibition of apoptosis by liver tumor promoters accelerated the manifestation of frank neoplasia (Bursch et al., 1984; Schulte-Hermann et al., 1995). However, relatively little is known about the role of apoptosis in determining susceptibility to liver cancer in different mouse strains. Based upon different long-term experimental models, somewhat conflicting data on apoptosis in mouse hepatocarcinogenesis have been reported (Goldsworthy and Fransson-Steen, 2002; James and Roberts, 1996; Kamendulis et al., 2001; Sanders and Thorgeirsson, 2000; Stevensson et al., 1999).
Consequently, we addressed the question whether the high cancer susceptibility of B6C3F1 and C3H/He mice may result from low efficiency or even failure of cancer defense by apoptosis. In a first series of experiments we performed comparative short-term in vivo studies on liver growth (cell proliferation) and involution (apoptosis). C3H/He, C57Bl6/J, and B6C3F1 mice were treated with the "classical" tumor promoter phenobarbital (PB) or the peroxisome proliferator nafenopin (NAF) as model compounds. Short-term in vivo studies with mice and rats have been successfully used to characterize the liver response to drugs and industrial chemicals in view of their hepatocarcinogenic potency (for review, see Huber et al., 1996; Schulte-Hermann et al., 1995; Whysner et al., 1996). The studies addressing apoptosis regulation in normal mouse liver served to complement and to better understand the results of a long-term carcinogenesis study with these mouse strains (see Bursch et al., accompanying manuscript). The in vivo approach was chosen because the actual rates of cell replication and of apoptosis integrate survival- and death-controlling factors in the context of the organism's genetic background.
Here we report on the results of the short-term experiments (7- to 14-day PB or NAF treatment followed by withdrawal) focused on cell proliferation and apoptosis. The results revealed that PB/NAF-induced mouse liver growth largely is due to cell enlargement (hypertrophy). In all strains of mice under study, liver regression upon cessation of PB/NAF treatment was not associated with an increase in apoptoses; this observation was confirmed by biochemical analysis of liver DNA content. Furthermore, food restriction did not amplify liver regression and the occurrence of apoptosis. Thus, as to strain differences in cancer susceptibility, C3H/He mice exhibited a more pronounced DNA synthesis response to PB or NAF than C57Bl/6J, thereby revealing a positive correlation between short-term effects and cancer susceptibility. However, no difference among the mouse strains with respect to the occurrence of apoptosis was detected. Surprisingly, mouse hepatocytes do not appear to enter apoptosis as readily as rat hepatocytes (species difference).
MATERIAL AND METHODS
Animals, husbandry and induction of liver growth.
Seven- to eight-week-old female and male C57Bl/6J, C3H/He and B6C3F1 mice were obtained from Zentralinstitut für Versuchstierzucht, Hannover, Germany, or from Institut für Labortierkunde und Genetik, Himberg, Austria. The animals were housed individually and adapted for 4–6 weeks to a reversed light-dark-rhythm (lights on 10 P.M. to 10 A.M., lights off from 10 A.M. to 10 P.M.) with food (standard maintenance diet Altromin 1321N, Altromin, Lage, Germany) and tap water ad libitum. Animals were sacrificed at the end of the lighting period, when apoptosis is expected to be at its diurnal maximum. Liver growth was induced by phenobarbital (PB) or nafenopin (NAF); both compounds were administered via the diet, PB (5-ethyl-5-phenyl-barbituric acid, Fluka Chemie AG, Buchs, Switzerland): 2 days 500 ppm, followed by 5 days 750 ppm; NAF (nafenopin, gift from Ciba-Geigy, Basel, Switzerland): 7 days 500 ppm. The effective dose per kg body weight was calculated from food consumption (Table 1). PB-doses consumed were similar in all groups except for male C3H/He, whose PB intake was somewhat higher than in female C3H/He (20%). Overall, five independent animal experiments were performed. All experiments were performed according to Austrian and EU regulations for animal care and treatment.
Food restriction.
To study the effect of food restriction on liver regression and apoptosis, mice were pretreated with PB for 7 days as described above. Along with the stop of PB-treatment, the mice
DNA synthesis.
The rate of DNA synthesis was measured based upon BrdU-incorporation into DNA. For continuous infusion of BrdU (5-bromo-2'-deoxyuridine, Sigma Chemicals, St. Louis, MO) osmotic minipumps (Alza Corporation, Palo Alto, CA) were implanted sc under isoflurane anesthesia (Abbott Laboratories, Queensborough, UK; 1.5% isoflurane [v/v] in 60% N2O and 40% O2 [v/v]); 3-day pumps (Alzet model 1003D) and 7-day pumps (Alzet model 2001) were used to deliver a BrdU solution (20 mg BrdU/ml PBS) at a rate of 1 μl/h.
Sacrifice, biochemical, and histological procedures.
Animals were anaesthetized with isoflurane, decapitated, and exsanguinated. The liver was quickly excised, and the gall bladder was carefully removed. For the biochemical determination of hepatic DNA and protein content, representative liver samples from the large median lobe were frozen, stored in liquid nitrogen, and processed as described elsewhere (Grasl-Kraupp et al., 1994). Other liver specimens of the same lobe were cut into 4- to 5-mm thick slices and fixed either with Carnoy's solution or 4% neutral buffered formalin according to Lillie (Pearse, 1980). Samples fixed in Carnoy's solution were transferred into isopropanol after 24 h and were stored in isopropanol until being processed for histology. Formalin-fixed tissue was processed after a fixation time of 24 to 48 h. Sections (3 to 5 μm) were mounted on APES-coated glass slides (3-aminopropyl-triethoxysilane, Fluka Chemie AG, Buchs, Switzerland) to ensure adhesion during processing. H&E (hematoxylin and eosin) staining was performed according to standard protocols. For the detection of BrdU-incorporation, formalin fixed liver specimens were processed as described in detail elsewhere (Chabicovsky et al., 2003) using a mouse monoclonal antibody to BrdU (Boehringer-Mannheim, Mannheim, Germany; 1:800 in PBS/0.1% BSA). Biotinylated rabbit anti-mouse IgG (DAKO A/S, Glostrup, Denmark; 1:100 in PBS/0.1% BSA) was used as second antibody, followed by peroxidase-conjugated streptavidin (DAKO A/S, Glostrup, Denmark; 1:100 in PBS/0.1% BSA) and visualized with DAB (3,3'-diaminobenzidine tetrahydrochloride). Sections were counterstained with Mayer's hematoxylin and mounted with Entellan.
Labeling index and apoptotic index.
The rate of DNA synthesis was determined histologically in BrdU-stained liver sections; a section of duodenum, a tissue with a high cell proliferation rate, was included on each slide to confirm the systemic delivery of BrdU to the animal. At least 2000 hepatocyte nuclei per animal were counted using a Nikon Microphot-FXA microscope (Tokyo, Japan). The labeling index (LI) was expressed as percentage of labeled hepatocyte nuclei in the total population of nuclei counted. For quantitative determination of apoptosis, 3000–4000 hepatocytes per animal were scored. Hepatocytes exhibiting chromatin condensation typical of apoptosis, apoptotic bodies with or without chromatin located intra- or extracellularly were recorded. The total number of apoptoses was expressed as percentage of the total number of hepatocytes counted; the low apoptotic counts as determined by scoring 3000–4000 hepatocytes per liver section were verified by screening whole liver sections. The validity of this method for quantitative determination of apoptoses with H&E-stained liver sections has been demonstrated previously (Chabicovsky et al., 2003; Schulte-Hermann et al., 1995).
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
Liver Weight, Protein, and DNA Content
In female inbred C57Bl/6J, C3H/He, and B6C3F1 mice, phenobarbital (PB) administration in doses of 500 and 750 ppm via the diet for 7 days caused an increase in liver weight by 37 to 60 % (Figs. 1A–1C). Biochemical analysis revealed marked increases in liver protein content approximately parallel to the increase in liver mass (Figs.1D–1F), but only a moderate increase in liver DNA (17–32%, Figs. 1G–1I). The increases in liver weight and liver protein content were significantly higher in C3H/He than in C57Bl/6J mice. These findings were confirmed and extended by studies on male C57Bl/6J, C3H/He, and B6C3F1 mice (Fig. 2). After PB treatment for 7 days, increases in liver mass and DNA content did not differ significantly between males and females, although they tended to be less in male mice of either strain. Furthermore, continuing PB treatment until day 14 did not result in further increase either in liver weight/ protein or in liver DNA content (Fig. 2). Male C57Bl/6J, C3H/He, and B6C3F1 mice were also treated with nafenopin (NAF, 750 ppm) resulting in an increase in liver mass by 85–113% (Figs. 2D–2F) and liver DNA content by 25–28% (Figs. 2G–2I). Notably, NAF, at the dose level chosen, caused a much more pronounced liver enlargement than PB in all three strains of mice.
As summarized in Figure 3, the magnitude of liver growth induced by short-term treatment with PB was clearly more pronounced in C3H/He mice than in C57BL/6J mice, independent of dose and sex.
Liver DNA Synthesis
The rate of hepatocyte DNA synthesis was measured in males by continuous infusion of BrdU for 3 and 7 days. As shown by histological analysis, in the livers of control male C3H/He, C57BL/6J, and B6C3F1 mice about 0.4% (3 days) and 1% (7 days) of hepatocyte nuclei were labeled with BrdU without exhibiting a significant difference among the mouse strains (Fig. 4). PB treatment resulted in significant increases in the number of BrdU-labeled nuclei to about 9–12% within 7 days (Fig. 4). As observed previously, the mouse liver growth response to PB was preferentially brought about by pericentral (zone 3) hepatocytes (data not shown; Schulte-Hermann et al., 1995). Overall, the hepatocellular labeling indices in PB treated animals did not differ significantly among the three mouse strains, although its stimulation above control level tended to be stronger in C3H/He (9-fold above controls) than in C57BL/6J (6-fold above controls) and B6C3F1 (7-fold above controls). No mitoses were detected in liver sections (3000–4000 hepatocytes scored) of control C3H/He, C57BL/6J, and B6C3F1 mice. Slightly but insignificantly enhanced numbers of mitoses were found in C57Bl/6J (0.03 ± 0.04%) and C3H/He (0.05 ± 0.02%) mice after PB for 3 days. In immunohistochemically stained sections, all mitoses were positive for BrdU-incorporation (not shown). Thus, a clear strain difference in mitotic activity, with or without PB treatment, was not detected.
Liver Regression and Apoptosis after PB or NAF Withdrawal
Discontinuation of PB or NAF treatment of C3H/He, C57BL/6J, and B6C3F1 mice resulted in a decrease of liver weight and liver protein content back to control level within 1–2 weeks (Figs. 1A–1G and 2A–2F). The enhanced liver DNA content, however, persisted at least until 14 days after cessation of PB or NAF treatment (Figs. 1 and 2G–2I).
Apoptoses were extremely rare at any time point after cessation of treatment in control and withdrawal groups of all mouse strains (Table 2). The lack of increased numbers of apoptoses in involuting livers of mice is in accordance with the persistent enhancement of liver DNA.
Effects of Food Restriction after Cessation of PB Treatment
In an attempt to amplify liver regression and the occurrence of apoptoses, PB-pretreated mice were subjected to a 40% food restriction (FR) along with PB-withdrawal; FR has been previously shown to induce apoptoses in normal, hyperplastic, and preneoplastic cell populations of rat liver (Grasl-Kraupp et al., 1994). In the present study, body weight of FR animals decreased steadily within the first week of food restriction, reaching the lowest levels (approx. 70% of the initial body weight) within 7 to 9 days; body weight loss did not differ among the mouse strains (Figs. 5A and 5B). Changes in liver weight, DNA and protein content, and hepatocellular size are illustrated by data on C3H/He mice; essentially the same observations were made with C57BL/6J and B6C3F1 mice (not shown). Liver weight and liver protein decreased by approximately 55–60% within one week of FR, but no further decrease occurred during the second week of FR (Figs. 5C and 5D). The decrease of liver weight and protein content led to a significant increase in the number of hepatocytes per microscopic field in food-restricted animals, indicating cellular hypotrophy (Fig. 5F). Even food restriction for up to 14 days did not result in a significant decrease liver DNA content as compared to animals fed ad libitum (Fig. 5E). Accordingly, histological analysis revealed no significant increase in apoptotic activity at all time points investigated (2, 4, 7, and 14 days FR; Table 2).
DISCUSSION
The present study revealed that the liver-tumor-susceptible C3H/He mice exhibited a more pronounced (although not thoroughly significant) increase in liver mass, liver protein, and liver DNA content than the less susceptible C57Bl/6J mice. Notably B6C3F1, the F1-generation of paternal C3H/He and maternal C57Bl/6J, tended to an intermediate response. Relative increases in liver mass and liver protein exceeded those of liver DNA content, suggesting that mouse liver enlargement in response to PB and NAF largely reflects hypertrophy; the latter was confirmed by histomorphometrical analysis of hepatocellular size (not shown). In summary, these observations confirm and extend results of previous studies on PB and NAF-induced mouse liver growth (for review, see Diwan et al., 1990; Fausto, 1999; Huber et al., 1996; Kamendulis et al., 2001; Sarraf et al., 1997; Schulte-Hermann, 1995). Specifically, the present study revealed a strain-specific response pattern to short-term PB treatment with a positive correlation between the induction of liver growth and liver tumor susceptibility; a schematic presentation is given in Fig. 6.
The present study also revealed that mouse liver enlargement induced by PB is completely reversible within a few days after cessation of treatment. Similar observations on the regression of surplus liver mass upon discontinuation of PB treatment have been reported previously (for review, see Diwan et al., 1990; Schulte-Hermann et al., 1995). As to the mechanism, excess membranes of endoplasmic reticulum as well as of the peroxisomal compartment are selectively removed by autophagy (for review, see Blommaart et al., 1997). However, in contrast to hypertrophy the enhanced DNA content persisted for at least 14 days after PB withdrawal. In accordance with this observation, essentially no apoptoses were observed during liver involution after cessation of treatment. Furthermore, food restriction did not stimulate apoptosis in regressing mouse liver. We expected this to occur, as in rat liver a massive apoptotic response was observed under such conditions (Grasl-Kraupp et al., 1994; Hikita et al., 1998; Tomasi et al., 1999). However, the biochemical data of the present study (no significant loss of liver DNA) strongly support the histological findings (no increase in apoptoses). In other studies, hepatocellular apoptosis in C57Bl/6JxC3H F1 mice did not increase before 60 days of dietary restriction by 40% (James and Muskhelishvili, 1994; Kolaja et al., 1996). Taken together, these findings strongly suggest that mouse hepatocytes do not enter apoptosis as readily as rat hepatocytes; the underlying causes are not known. It should be emphasized that this conclusion apparently applies to both compounds studied, namely PB and NAF, which exert their action on hepatocytes through different receptor-signal transduction pathways (Oliver and Roberts, 2002).
Moreover, other in vivo as well as cell culture studies revealed that mouse hepatocytes are much less sensitive to the pro-apoptotic action of TGF-1 than rat hepatocytes (Chabicovsky et al., 2003; Parzefall et al., 2002). Thus, 56 μg TGF-1/kg did not induce apoptosis in mouse liver, whereas in rat liver a dose as low as 0.25 μg TGF-1/kg was found to be effective (Chabicovsky et al., 2003; Oberhammer et al., 1992; Schulte-Hermann et al., 2002a; Fig. 6). Likewise, comparative studies with primary cultures of hepatocytes from C3H and C57BL mice revealed that at any concentration between 0.1 and 100 ng/ml TGF-1 exerted only a weak proapoptotic effect (less than 1%), whereas rat hepatocytes (F344) cultured under the same conditions were readily stimulated to undergo apoptosis at concentrations of 0.3 ng/ml or higher (up to 6%, ED50 = 0.8 ng/ml; Parzefall et al., 2002; Fig. 6). Notably, mouse hepatocytes are highly sensitive to the pro-apopotic action of ligands of the TNF/NGF-receptor family (for review, see Kanzler and Galle, 2000; Schulte-Hermann et al., 2002b); it is tempting to raise the hypothesis that rats and mice might differ with respect to the relative contribution of various cytokine networks to control of hepatocellular apoptosis.
In conclusion, the present results show that the tumor-promoting efficiency of prolonged PB treatment corresponds to the short-term effects of PB in mouse liver. Thus, among the three inbred mouse strains studied, the highly cancer-susceptible C3H/He mice exhibited the strongest growth response to the tumor-promoting agent PB. Surprisingly, no strain difference with respect to the occurrence of apoptosis was found. This conclusion meets well with our observations in a long-term carcinogenesis study on C3H/He and C57Bl/6J mice, which indicates that the rate of cell proliferation largely determines susceptibility for tumorigenesis (see Bursch et al., accompanying manuscript). On the other hand, profound differences in the sensitivity to pro-apoptotic signals appear to exist between mouse and rat hepatocytes.
NOTES
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 Ch. Unger is gratefully acknowledged.
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