Effect of mitogenic or regenerative cell proliferation on lacz mutant frequency in the liver of MutaTMMice treated with 5,9-dimethyldibenzo[c,g]carbazole
Françoise Tombolan1,2,
Dominique Renault1,
Dominique Brault1,
Magali Guffroy1,
François Périn3 and
Véronique Thybaud1,4
1 Rhône-Poulenc Rorer, Non-Clinical Safety Assessment, 13 quai Jules Guesde, BP14 94403 Vitry-sur-Seine Cedex,
2 CNRS UPR 42, 94801 Villejuif and
3 Institut Curie-Recherche, Centre Universitaire, 91405 Orsay Cedex, France
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Abstract
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The purpose of this work was to investigate the impact of cell proliferation on liver mutagenesis. The genotoxic hepatocarcinogen 5,9-dimethyldibenzo[c,g]carbazole (DMDBC) was administered to lacZ transgenic MutaTMMice at a non-hepatotoxic dose of 10 mg/kg, which induces only a slight increase in the liver lacZ mutant frequency (MF). To determine if cell proliferation stimuli enhanced DMDBC mutagenicity, MF was analyzed in mice first receiving DMDBC 10 mg/kg, then ~2 weeks later, either carbon tetrachloride (CCl4, a cytotoxic agent inducing regenerative cell proliferation) or phenobarbital (PB, a mitogenic agent inducing direct hyperplasia). In preliminary studies, the extent of cell proliferation induced by CCl4, PB and DMDBC was determined in non-transgenic CD2F1 mice by means of 5-bromodeoxyuridine labeling. The labeling index was significantly increased after CCl4 and PB, while no change was detected with DMDBC. MF was then determined in MutaTMMice 28 days after initial DMDBC treatment. No increase in MF was detected in mice receiving CCl4 or PB alone. A 2- to 3-fold increase in MF was detected in mice treated with 10 mg/kg DMDBC alone. In contrast, MF was markedly increased in mice receiving DMDBC followed by proliferative treatment (15-fold with CCl4 and 25-fold with PB). These results demonstrate that expression of DMDBC-induced mutations in mouse liver largely depends on the induction of cell proliferation (by a cytotoxic or mitogenic stimulus) and illustrate that MutaTMMouse is a valuable tool to investigate the early events of liver carcinogenesis.
Abbreviations: BrdU, 5-bromodeoxyuridine; CCl4, carbon tetrachloride; DMDBC, 5,9-dimethyl-dibenzo[c,g]carbazole; LI, labeling index; LW, liver weight; MF, mutant frequency; PB, phenobarbital; p.f.u., plaque-forming units.
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Introduction
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Genomic mutations are key events in carcinogenesis. More than one such genetic event is necessary for cancer to arise, and current models of carcinogenesis are based on multistep mechanisms. Cell proliferation plays a pivotal role in carcinogenesis, contributing to the creation of `initiated' cells by favouring conversion of DNA lesions into heritable mutations (1,2) and enabling clonal expansion of initiated cells (3). Induction of cell proliferation is particularly important for carcinogenesis in slowly proliferating tissues such as the liver. This organ is of particular interest, because of its high tumor susceptibility and the possibility of triggering cell proliferation by various manipulations. Two mechanisms are known to induce cell proliferation in the liver: (i) regenerative or compensatory growth, which restores hepatic parenchyma lost through necrosis or hepatectomy; and (ii) direct mitogenic growth, which results in liver enlargement with no evidence of necrosis. Cell proliferation can be induced through these two mechanisms by carbon tetrachloride (CCl4) (4,5) and phenobarbital (PB) (6,7), respectively.
While the effect of cell proliferation on tumorigenesis has been extensively studied, its impact on the expression of mutations has rarely been addressed. To further investigate the role of cell proliferation on liver mutagenesis, we examined the effect of regenerative and mitogenic cell proliferation induced by CCl4 and PB, respectively, on lacZ mutant frequency (MF) in the transgenic MutaTMMouse mutagenicity assay (8,9). Each diploid cell of this mouse model carries 80 copies of the bacterial lacZ reporter transgene for the detection of spontaneous and induced mutations. In this study, 5,9-dimethyldibenzo[c,g]carbazole (DMDBC) (10), a synthetic derivative of the environmental pollutant 7H-dibenzo[c,g]carbazole, was used to generate DNA lesions. DMDBC is a potent genotoxic carcinogen for mouse liver (11). Previous studies of MutaTMMouse liver showed that a single administration of DMDBC at 10 or 90 mg/kg induced a similar level of persistent DNA adducts (12). However, only a weak increase (2-fold) in MF and no change in cell proliferation were detected at 10 mg/kg, whereas 90 mg/kg induced a marked increase in MF (44-fold) and regenerative cell proliferation (12,13). These results strongly suggested that regenerative cell proliferation induced by a high dose of DMDBC allowed DMDBCDNA adducts to be fixed as stable mutations. To determine if secondary stimuli of cell proliferation enhanced the MF after a low dose of DMDBC, MF was analyzed in mice first receiving 10 mg/kg DMDBC and then, ~2 weeks later, either CCl4 (a cytotoxic agent inducing regenerative cell proliferation) or PB (a mitogenic agent inducing direct hyperplasia).
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Materials and methods
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Animals
Male MutaTMMice (strain 40.6) developed by Gossen et al. (8) were supplied by Covance (Denver, CO). Male CD2F1/CrlBR mice were supplied by Charles River (Saint-Aubin-lès-Elbeuf, France). MutaTMMice and CD2F1 mice both result from the crossing of Balb/C and DBA/2 mouse strains, but MutaTMMice are transgenic (their genome harbours several copies of the bacterial lacZ transgene as a reporter gene for mutagenesis; the transgene is not expressed in the animals). The animals were ~8 weeks old when the experiments began. They were kept in standard conditions of temperature (20 ± 2°C) and humidity (50 ± 10%) with a 12 h lightdark cycle.
Chemicals and dosing
DMDBC was synthesized as described previously (10), dissolved in corn oil and administered at a dose of 10 mg/kg in a volume of 10 ml/kg by s.c. injection in the interscapular region. CCl4 (Prolabo, Paris, France) was dissolved in corn oil and administered by gavage at a dose of 80 mg/kg in a volume of 10 ml/kg. Sodium phenobarbital (Rhône-Poulenc Rorer, Vitry, France) was mixed at 1500 p.p.m. in the AO4C P1 diet (U.A.R., Villemoisson, France).
Experimental design of CD2F1 mice studies
Three experiments involved CD2F1 mice. In the first, mice received a single injection of 10 mg/kg DMDBC, and four mice were killed at each time point (7, 14 and 28 days later). In the second experiment, mice were treated once with 80 mg/kg CCl4, and four mice were killed at each time point (1, 2, 3, 4 and 7 days later). In the third experiment, mice were fed ad libitum for 5 days with a diet containing 1500 p.p.m. PB, and three mice were killed at each time point (1, 2, 3, 4, 5, 6, 7, 8 and 9 days after the first day of treatment). In each of the three experiments, control mice received the vehicle alone; two or three mice were killed at the first and last sampling times. In all experiments the mice received a single i.p. injection of 5-bromodeoxyuridine (BrdU) at a dose of 100 mg/kg 2 h before killing, to label cells in S phase. The liver and small intestine (positive control) were removed and processed for histopathological examination and immunohistochemistry. Liver and body weights were recorded at all sampling times.
Experimental design of MutaTMMice studies
Two experiments were done with MutaTMMice (Figure 1
). In the first (CCl4 study), mice received a single injection of 10 mg/kg DMDBC and, 14 days later, a single dose of 80 mg/kg CCl4 as a cytotoxic stimulus for regenerative cell proliferation. Four groups of five mice were used (vehicle only, DMDBC only, CCl4 only and DMDBC plus CCl4). In the second experiment (PB study), mice received a single injection of 10 mg/kg DMDBC and were fed ad libitum with a diet supplemented with 1500 p.p.m. PB for 5 days from day 11 to day 15, as a direct mitogenic stimulus. Four groups of five mice were used (vehicle only, DMDBC only, PB only and DMDBC plus PB). Sufficient time was left between DMDBC exposure and the proliferative treatments to limit the effects of CCl4 and PB on DMDBC distribution, metabolism and elimination, given the well-known induction of several drug-metabolizing enzymes by PB. The animals were weighed and killed 28 days after DMDBC treatment. The liver was removed and weighed, and a specimen was stored at 80°C for MF determination. Another specimen was processed for histopathological examination.

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Fig. 1. Experimental design of the studies with MutaTMMice. (A) Effect of regenerative cell proliferation induced by CCl4. (B) Effect of mitogenic cell proliferation induced by PB.
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Histopathological observations
Liver samples from animals in all studies (CD2F1 mice and MutaTMMice), and small-intestine samples from CD2F1 mice only, were preserved by immersion in Carnoy's fixative for 1 h and then transferred to 100% ethanol. Tissue samples were routinely processed, embedded in paraffin and sliced (5 µm). Liver sections were stained with hematoxylin, eosin and saffron and examined by light microscopy.
Evaluation of cell proliferation
Cell proliferation was evaluated in liver and small-intestine sections of CD2F1 mice by determination of BrdU incorporation into nuclei with an anti-BrdU mouse monoclonal antibody (Becton-Dickinson, San José, CA), as described previously (12). Small intestine, a tissue with a high cell turnover rate, was used as positive immunohistochemical control. A labeling index (LI) was calculated as the percentage of BrdU-immunopositive nuclei among 30003500 hepatocellular nuclei scored per animal.
Mutant frequency determination
High molecular weight genomic DNA was isolated from frozen liver samples as described in the Stratagene Instruction Manual (14). Five microliters of DNA were mixed with an
-phage packaging extract (Transpack; Stratagene, La Jolla, CA) to rescue lacZ transgenes, as recommended in the Stratagene Instruction Manual (14). The reaction was stopped by adding 500 µl of SM buffer (10 mM MgSO4, 0.1% gelatin, 100 mM NaCl, 50 mM Tris, pH 8). Escherichia coli C lac recA galE Kanr (galE Ampr), developed and supplied by Ingeny (Leiden, The Netherlands), was cultured as described previously (15). The lacZ-containing phage suspension was adsorbed to 2 ml of bacterial culture (~2x109 cells) for 30 min at 37°C. Phage titration and mutant selection were done according to the positive selection method (9) using phenyl-ß,D-galactoside in the selection plates as described by Renault et al. (15). At least 200 000 plaque-forming units (p.f.u.) by animal were recorded. MF was expressed as the ratio between the number of p.f.u. in the mutant selection plates and the total number of p.f.u. (derived from the titration plates), as described by Dean and Myrh (16). Plaques derived from selection were sampled from the agar plates and the mutant phenotype of the phages was confirmed by using an E.coli C lac Tetr Ampr culture plated with X-gal (5-bromo-4-chloro-3-indolyl-ß-galactoside), as described in the Stratagene Instruction Manual (14).
Statistical analyses
Absolute and relative liver weight (LW) and BrdU LI were compared between control and treated groups by using Student's t-test.
Mutant frequencies were compared between control and treated groups by using Student's t-test after log transformation of the data, as recommended by Callahan and Short (17).
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Results
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CD2F1 mice studies
Liver weight variations, microscopic changes and cell proliferation induced by CCl4 and PB were evaluated in non-transgenic CD2F1 mice, which have a genetic background similar to that of MutaTMMice. LW and LI are reported in Table I
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After gavage of CD2F1 mice with CCl4, absolute and relative LW started to increase significantly on day 2, reached a maximum of +34 and +31% of mean control value, respectively, on day 3, and then started to decline. On day 7, absolute and relative LW remained 11% higher than control values (P < 0.01). Microscopic changes observed in the livers of CCl4-treated CD2F1 mice consisted mainly of mild to moderate centrilobular necrosis noted as early as day 1, and subsequent minimal to moderate centrilobular mineralization and mixed inflammatory cell infiltration observed from day 3 onward. Hepatocellular regeneration as evidenced by an increased number of mitotic figures was noted from day 2 to day 4. Residual changes on day 7 were mainly characterized by centrilobular accumulation of mineral-laden multinucleated giant cells admixed with macrophages and lymphocytes. Although post-necrotic hepatic mineralization is an uncommon finding, it has already been observed in our laboratory with a different test article (M.Guffroy, personal communication) and we believe this observation may be strain related. In addition, it is noteworthy that centrilobular sinusoidal congestion and red blood cell extravasation was noted in some livers from mice killed on days 2 and 3, and this observation may, at least in part, account for the increased LWs noted on these days in treated mice as compared with controls. LI, as determined by BrdU incorporation into nuclei, was unchanged on day 1, increased significantly on days 2 and 3 with a peak on day 2 (57-fold) and then returned to control level by day 7.
In CD2F1 mice treated with PB, absolute and relative LW started to increase significantly on day 2 of the PB-containing diet, reaching a maximum of +75 and +83% of control mean value, respectively, on day 5, the last day of treatment. From day 6 to day 9, absolute and relative LW decreased, and fell to 44 and 39%, respectively, above the control level (P < 0.001) on day 9. Microscopic changes observed in the liver of PB-treated CD2F1 mice consisted mainly of centrilobular hepatocellular hypertrophy and hyperplasia. This microscopic observation correlates with the increased liver weights noted in treated mice. Hepatocellular hypertrophy was observed from day 2 onward; it was most marked on days 4 to 6 and regressed progressively thereafter. Hepatocellular proliferation as evidenced by an increased number of mitotic figures was noted from day 3 to day 7. LI was significantly increased from day 3 to day 5 with a peak on day 4 (42-fold), then gradually declined to the control level by day 8, i.e. 3 days after PB withdrawal.
In addition, in CD2F1 mice treated with 10 mg/kg DMDBC, no changes in LW and LI were detected at any time, as compared with controls.
MutaTMMice studies
LW variations, microscopic changes and MF were determined in MutaTMMice 28 days after initial DMDBC treatment in both CCl4 and PB studies (Figure 2
; Tables II and III
).

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Fig. 2. Mutant frequency (group mean ± SD) in the livers of MutaTMMice treated with 10 mg/kg DMDBC followed by (A) CCl4 or (B) PB. Livers were sampled 28 days after DMDBC treatment. *Significantly different from control, P < 0.01; **significantly different from control, P < 0.001.
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In either study, no statistically significant differences in absolute or relative LW were found between treated and control groups on day 28.
Except for minimal residual centrilobular hepatocellular hypertrophy in occasional PB-treated mice, changes compatible with previous CCl4 (centrilobular necrosis and subsequent hepatocellular regeneration) or PB administration (centrilobular hepatocellular hypertrophy and proliferation) had subsided, and livers from CCl4 and PB-treated mice were similar to those of controls. There were no appreciable microscopic changes in the livers of mice from either study treated with DMDBC alone. Minimal hepatocellular nuclear enlargement (karyomegaly) was observed in one out of five mice from the DMDBC plus PB group and in one out of five mice from the DMDBC plus CCl4 group as compared with mice from the other study groups.
No statistically significant changes in MF were detected in mice receiving CCl4 or PB alone as compared with controls. In both CCl4 and PB studies, a statistically significant but weak MF increase (2.4- and 2.9-fold, respectively) was found in mice treated with DMDBC alone. In contrast, a statistically significant marked increase (15- and 25-fold, respectively) was measured in mice receiving DMDBC followed by CCl4 or PB.
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Discussion
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The purpose of the present study was to further investigate the impact of cell proliferation on DMDBC mutagenicity in vivo. LacZ MF was determined in MutaTMMice after a single administration of 10 mg/kg DMDBC to generate DNA adducts, followed, ~2 weeks later, by either CCl4 treatment (to induce regenerative growth) or PB treatment (to induce mitogenic growth).
Preliminary studies on non-transgenic CD2F1 mice showed that the chosen doses of CCl4 and PB resulted in a marked increase in liver cell proliferation, while treatment with 10 mg/kg DMDBC alone had no effect. This latter finding is consistent with hepatic LI and mitotic index results obtained in a previous study of MutaTMMice (12). The same CCl4, PB and DMDBC dose regimens as those used for CD2F1 mice were applied to MutaTMMice for the MF study. As the DNA adduct level induced by DMDBC reaches apparent steady state 7 days after treatment (12), CCl4 and PB were given on day 14 and from days 11 to 15, respectively. At these time points, DMDBC metabolic activation was expected to be complete. MF was analyzed 2 weeks after the proliferative treatments in order to allow the liver to return to its baseline size and cell turnover state, as illustrated by the LI and LW results.
The liver MF was not increased after treatment with CCl4 or PB alone, in agreement with previous studies using lacI or lacZ transgenic mice (1820). CCl4 and PB are negative in many genotoxicity assays (2123) but are hepatocarcinogenic after long-term oral treatment in mice (24,25). By definition, non-genotoxic carcinogens do not react directly with DNA, but they might induce mutations by increasing the rate of spontaneous genetic errors during replication, for example, or by generating DNA lesions through indirect mechanisms such as oxidative stress (26). However, all non-genotoxic carcinogens tested in lacI/lacZ transgenic animals have given negative results to date (18,19,27), even after long-term PB and heptachlor treatment at dose levels positive in 2 year carcinogenicity bioassays (28) and at chloroform dose levels with hepatocellular toxicity (29).
Treatment with 10 mg/kg DMDBC alone induced only a 2- to 3-fold increase in MF, in keeping with earlier results (12,13). In contrast, when DMDBC exposure was followed by CCl4 or PB treatment, MF increased 15- and 25-fold, respectively. As the lacZ transgene is not expressed in MutaTMMice, it is unlikely that the large MF increase after CCl4 or PB treatment was due to preferential proliferation of the limited number of lacZ-mutated hepatocytes induced by 10 mg/kg DMDBC. Rather, the increase in MF induced by the proliferative agents CCl4 and PB was probably due mainly to the conversion of DMDBC-induced persistent DNA adducts into mutations during hepatocyte replication.
CCl4 is hepatotoxic and induces regenerative cell proliferation while PB is a direct liver mitogen. In order to compare the effects of these two different proliferative mechanisms on liver MF, CCl4 and PB dose regimens were selected to result in a similar increase in cell proliferation, as shown by LI results in CD2F1 mice. No clear difference was seen in MF increases between MutaTMMice receiving CCl4 or PB after DMDBC administration, suggesting that MF response depends directly on the degree of cell proliferation. However, it is likely that the two proliferative agents induce further effects which could contribute to the MF response. CCl4 first causes hepatocyte loss before regenerative growth occurs. Fixation of mutations is thought to occur during the second, i.e. regenerative, phase. In contrast, PB is a direct mitogen that causes hyperplastic liver growth; when PB is withdrawn the liver returns to its initial state by eliminating the excess of cells through apoptosis (30,31). In this case, fixation of mutations is thought to occur during the first, i.e. mitogenic, phase. As the MF in the PB experiment was measured when the liver hypertrophy had already regressed, apoptosis may have modified the MF response. Moreover, it was found recently that PB alters cell-cycle G1 checkpoint functions in mouse hepatocytes (32). The authors suggest that this may prevent the detection and repair of DNA damage and, as a result, increase the frequency of mutagenic events. Thus, other factors besides cell proliferation, including apoptosis and cell-cycle checkpoint alterations were probably involved in the large MF increases obtained when 10 mg/kg DMDBC was followed by CCl4 or PB, although it is not known to what extent.
Similar large increases in MF have been reported with high doses of DMDBC (12,13). In particular, DMDBC induced regenerative cell proliferation and strongly increased the liver MF at a cytotoxic dose of 90 mg/kg (12). Our results thus demonstrate that the mutagenic potential of DMDBC in liver is highly dependent on the induction of cell proliferation, whatever its origin (DMDBC itself or a further stimulus).
Other authors have suggested that genotoxic hepatocarcinogens could increase the MF in the liver of transgenic rodents through their own proliferative properties (3337). However, few studies have addressed the role of secondary proliferative stimuli induced by agents other than the genotoxic compound itself. The results showed that when diethylnitrosamine treatment of transgenic mice was followed either by partial hepatectomy (38) or by PB feeding (20), MF increased by up to 4-fold relative to diethylnitrosamine treatment alone. The results of these studies are consistent with our findings, in that both regenerative (partial hepatectomy) and mitogenic (PB) stimuli enhanced the expression of chemical-induced mutations in the liver.
In conclusion, cell proliferation induced by two different mechanisms was found to be critical for the expression of mutations in the mouse liver after DMDBC exposure. Thus, while DMDBC is weakly mutagenic at a low dose, its mutagenicity is markedly potentiated by proliferative stimuli. This work also demonstrates the potential of in vivo mutagenicity assays. With such transgenic models, it is possible to study the impact of genotoxic and non-genotoxic effects on mutagenesis by monitoring the corresponding endpoints, and thereby determine their respective roles in early events leading to cancer. Finally, our results raise questions on risk assessment in humans exposed to low doses of a variety of chemicals, some of which are mutagenic. It is tempting to speculate that such chemicals may induce persistent DNA adducts in the liver (39), which may remain silent or be repaired unless stimulation of hepatic cell proliferation, observed, for instance, during hepatitis (40,41), cirrhosis (42), partial hepatectomy (43) or liver transplantation (44), leads to their fixation into stable mutations.
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Acknowledgments
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We are grateful to Dr Alain Sarasin (C.N.R.S.) for reviewing this manuscript and to Ms Marie-France Girault and Ms Florence Monge (Rhône-Poulenc Rorer) for their excellent technical assistance. This work received financial support from the European Union Environmental Program.
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Notes
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4 To whom correspondence should be addressed Email: veronique.thybaud{at}rp-rorer.fr 
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Received February 3, 1999;
revised March 24, 1999;
accepted March 25, 1999.