1 Rhône-Poulenc Rorer, Drug Safety Department, 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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Abbreviations: AAF, 2-acetylaminofluorene; BrdU, 5-bromodeoxyuridine; CPA, cyproterone acetate; 2,4-DAT, 2,4-diaminotoluene; 2,6-DAT, 2,6-diaminotoluene; DBC, 7H-dibenzo[c,g]carbazole; DEN, diethylnitrosamine; DMDBC, 5,9-dimethyldibenzo[c,g]carbazole; DMN, dimethylnitrosamine; HES, hematoxylin eosin saffron; LI, labelling index; MF, mutant frequency; MI, mitotic index; PAH, polycyclic aromatic hydrocarbon; p.f.u., plaque forming units.
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Introduction |
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DMDBC is metabolized into reactive species that bind covalently to liver proteins and DNA (4, 5). 32P-post-labelling studies have identified at least 15 DNA adducts induced by DMDBC in mouse liver (6). The mutagenic activity of DMDBC was established in vitro with the Ames test (Salmonella strain TA 100) and mouse liver S9 activation (7).
To further investigate the genotoxic properties of DMDBC, we tested its mutagenic potential in vivo by using the lacZ transgenic mouse (MutaTMMouse) assay (8). This model allows spontaneous and induced gene mutations to be studied in any tissue from which DNA can be extracted. Multiple shuttle vectors are integrated head-to-tail at a single site within chomosome 3 (9), and each vector contains one bacterial lacZ reporter gene as a target for mutagenesis. Each MutaTMMouse diploid cell contains 80 copies of lacZ, which encodes ß-galactosidase. The lacZ transgene is not expressed in the model mouse and does not affect the animal's physiology.
Using the MutaTMMouse positive selection system (10), the lacZ mutant frequency (MF) was determined in the liver 28 days after a single s.c. injection of DMDBC, the route of administration used in a previously reported carcinogenicity study (3). DNA damage and cell proliferation are both necessary to fix gene mutations and to initiate carcinogenesis in liver, which is a slowly proliferating tissue. Valéro et al. (3) have reported that DMDBC enhances replicative DNA synthesis in hepatocytes. To determine whether the mutagenic potential of DMDBC depends only on a genotoxic effect (DNA binding) or also on a non-genotoxic mechanism (induced cell proliferation), we examined the time-course of DNA adduct formation (32P-post-labelling assay), histological changes, cell proliferation and MF in MutaTMMice liver, 2, 4, 7, 14, 21 and 28 days after treatment.
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Materials and methods |
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Experimental design
DMDBC was synthesized as previously described (4), dissolved in corn oil and administered in a volume of 10 ml/kg by s.c. injection in the interscapular region. For the doseresponse study, mice received a single injection of the vehicle or DMDBC at 3, 10, 30, 90 or 180 mg/kg and were killed 28 days later. For the time-course study, mice received a single injection of the vehicle or DMDBC 10 or 90 mg/kg and were killed 2, 4, 7, 14, 21 and 28 days later. In both studies the liver was removed and one third of the left lateral lobe was used for MF determination. In the time-course study, a second third of this liver lobe was stored for DNA adduct analysis, and the remaining third was processed for histopathological examination. To measure 5-bromodeoxyuridine (BrdU) incorporation into nuclei, satellite groups of mice were used during the time-course study and killed 7, 14 and 28 days after treatment. Two hours before being killed, BrdU was administered to mice by i.p. injection at a dose of 100 mg/kg.
Mutant frequency determination
High-molecular-weight genomic DNA was isolated from fresh liver samples as described by Brault et al. (11). Five microlitres of DNA was mixed with a -phage packaging extract (Transpack; Stratagene, La Jolla, CA) as recommended by the manufacturer. 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 previously described (12). The phage suspension was adsorbed to 2 ml of bacterial culture (~2x109 cells) for 30 min at 37°C. Phage titration and mutant selection were performed according to the positive selection method (10) using phenyl-ß,D-galactoside in the selection plates as described by Renault et al. (12). Mutant frequency was expressed as the ratio between the number of plaque forming units (p.f.u.) in the mutant selection plates and the total number of p.f.u. plated (derived from the titration plates), as described by Dean and Myrh (13). Mutant frequencies in treated and control groups were compared by using Student's t-test after log transformation of the data as recommended by Callahan and Short (14). Plaques derived from selection were sampled from the agar plates and the mutant phenotype of the phages was confirmed 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 MutaTMMouse Instruction Manual.
32P-post-labelling analysis of DNA adducts
Liver samples were frozen in liquid nitrogen and DNA was isolated after grinding the tissue in a mortar filled with liquid nitrogen. The tissue powder was suspended in 1 mM EDTA, 1% SDS, 1 mg/ml Pronase E and incubated for at least 3 h at 37°C. Then, solvent extractions and Rnase digestion were conducted as described by Gupta (15). Using the nuclease P1 enhancement procedure of the 32P-post-labelling assay (16), DMDBCDNA adducts were labelled, isolated and quantified as described by Périn-Roussel et al. (17). Data are expressed as total DNA adducts per 108 nucleotides and correspond to all DMDBC-derived adduct spots detected in a given DNA sample.
Histopathology and evaluation of cell proliferation
Liver samples from animals in all the groups, and small-intestine specimens (a positive control for BrdU incorporation) from mice in the satellite groups 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 sectioned (5 µm). Liver sections were stained with hematoxylin, eosin and saffron (HES) for light microscopy and scoring of mitotic figures; 30003500 hepatocellular nuclei per animal were counted. The mitotic index (MI) was expressed as the number of mitotic figures per 1000 nuclei. Liver cell proliferation was assessed by BrdU immunohistochemistry in mice from the satellite groups, with an anti-BrdU mouse monoclonal antibody (Becton Dickinson Immunocytometry Systems, San José, CA). Sections of small intestine were used as positive immunohistochemical controls. Briefly, tissue sections were deparaffinized, exposed to 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity, treated with 2 N HCl for 30 min, and incubated with normal horse serum for 15 min. The sections were then incubated for 1 h with an anti-BrdU monoclonal antibody, followed by peroxidase-conjugated goat anti-mouse IgG for 30 min. Immunopositive nuclei were visualized with the chromagen diaminobenzidine, and the sections were counterstained with hematoxylin. A labelling index (LI) was calculated as the percentage of BrdU-immunopositive nuclei among the 30003500 hepatocellular nuclei scored per animal. For both MI and LI, treated and control groups were compared with Student's t-test.
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Results |
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Time-course study
In this study DNA adducts (detected by 32P-post-labelling), histological changes, cell proliferation and MF were assessed in liver 2, 4, 7, 14, 21 and 28 days after a single s.c. injection of 10 or 90 mg/kg DMDBC. The results are summarized in Figure 1.
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Total DNA adduct levels are reported in Table II. In the liver of mice treated with 10 mg/kg, DNA binding reached a level of 200600 adducts per 108 nucleotides on day 4 and remained stable until day 28. The 90 mg/kg dose induced a very high adduct level (>1500 per 108 nucleotides) in the liver of two animals (nos 38 and 39) out of five on day 2. In the other three animals the DNA adduct level was ~400 per 108 nucleotides. At later sampling times the value remained constant at 300500 adducts per 108 nucleotides.
There were no appreciable compound-related histologic changes in the liver of mice dosed at 10 mg/kg, throughout the study period. In mice dosed at 90 mg/kg, microscopic liver changes were noted from day 2 to day 28 (Figure 3). On day 2, changes were observed in two of five animals examined (nos 38 and 40) and predominated in centrilobular areas, which contained scattered necrotic cells and cell debris amidst diffusely degenerate hepatocytes characterized by fairly homogenous eosinophilic cytoplasm and vesicular nuclei with marginated chromatin. There was no significant inflammatory response at this stage. Liver changes were observed in two of five animals examined on day 4 (nos 41 and 42) and consisted mainly of a loss of centrilobular hepatocytes and parenchymal collapse, accompanied by minimal to moderate infiltration by inflammatory cells. In addition, there was ongoing hepatocellular necrosis and an increased number of hepatocellular mitotic figures. Minimal karyomegaly of hepatocytes was noted in both animals. From day 7 onwards, liver changes were observed in a few to most animals examined and consisted mainly of minimal to moderate cytomegaly and karyomegaly of centrilobular and mid-zonal hepatocytes. Affected cells had an expanded cytoplasm with decreased glycogen content and enlarged, usually roundish nuclei with stippled to mottled chromatin and 1 or 2 conspicuous nucleoli.
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To detect S-phase hepatocytes, BrdU incorporation into nuclei was evaluated on days 7, 14 and 28 after treatment in satellite groups (Table III). The LI was unchanged in animals receiving 10 mg/kg, indicating no increase in replicative DNA synthesis at this dose. The LI was significantly increased on days 7 and 14 in mice treated with 90 mg/kg. Taken together, LI and MI data showed that cell proliferation was induced at 90 mg/kg but not at 10 mg/kg.
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Discussion |
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In the liver, the role of cell proliferation is often cited as a mechanism for fixing mutations but unfortunately has not been extensively experimentally addressed to date, due to the lack of appropriate models for the detection of in vivo gene mutations. The recent availability of transgenic models for the detection of in vivo gene mutations gave the opportunity to evaluate in parallel the kinetics of DNA adducts, cell proliferation and MF, in order to gain knowledge on the early events leading to carcinogenicity.
The liver MF was first analyzed 28 days after a single s.c. injection of DMDBC at doses ranging from 3 to 180 mg/kg. Groups of mice dosed at 0, 3 and 10 mg/kg demonstrated a low variability in the MF response. At 3 and 10 mg/kg, the highest individual MFs were respectively only 1.5- and 2.3-fold over the highest control value. Although groups of mice dosed at 30, 90 and 180 mg/kg demonstrated a higher variability, MFs of all animals but one in the 30 mg/kg-dosed group were increased at least 14-fold over the highest control value. At 90 and 180 mg/kg, all individual MFs were increased at least 20-fold over the highest control value. These results strongly suggest a non-linear response. Wide animal-to-animal variability has been reported in transgenic mouse mutation assays (18,20,21) and it is thus recommended to use at least five animals per group (14,22). However, the great overdispersion observed here warrants discussion. Several factors may have affected the MF response. The observed MutaTMMouse inter-animal variability may have resulted partly from differences in the genetic background, as this strain has different phenotypes. The MutaTMMouse was obtained by crossing the BALB/c and DBA/2 strains, which have markedly different metabolic capacities. BALB/c mice are Ah-responsive, while DBA/2 mice are not. The Ah receptor regulates metabolic enzymes, including cytochrome P450 1A, which is involved in polycyclic aromatic hydrocarbon (PAH) metabolism. It has been reported that the mutagenic and carcinogenic potential of PAHs for mouse strains varies with Ah responsiveness [reviewed by Nebert (23)]. Thus, the mutagenic response to DMDBC may depend in part on the metabolic status of individual mice, especially at the dose of 30 mg/kg, as additional biological events may come into play. MF variability may also be related to the use of the s.c. route, as the proportion of the dose deposited in the fatty layer could influence the rate at which DMDBC is delivered to the liver.
The increase in MF induced by DMDBC doses of 30 mg/kg and more was far larger than that obtained with reference genotoxic hepatocarcinogens such as dimethylnitrosamine (DMN), diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF). A single administration of 10 mg/kg DMN (21), 100 mg/kg DEN (24) or 100 mg/kg AAF (25) to MutaTMMice increases the liver MF ~3-fold, ~15-fold or ~2-fold, respectively. In Big Blue® transgenic mice, five administrations of 4 mg/kg DMN result in an ~10-fold increase in the liver lacI MF (26). The values observed in our study reflect the strongest mouse liver response published to date, after single or multiple dosing, and this led us to investigate the underlying mechanisms.
The non-linear response suggests that DMDBC not only has a direct genotoxic effect (DNA binding) but also has other effects contributing to its mutagenic potential. There is strong evidence that cell proliferation plays a key role in liver mutagenesis and carcinogenesis (2629). Accordingly, we studied DNA adducts, histologic changes and cell proliferation in parallel to MF over a 28 day period after treatment with a single s.c. injection of DMDBC at 10 mg/kg (inducing only a doubling of MF) and 90 mg/kg (inducing a >40-fold increase in MF).
Interestingly, the results obtained in the time-course study (Table II) at 10 mg/kg showed a low variability in DNA adducts, cell proliferation and MF at all sampling times while a higher variability was observed at 90 mg/kg on day 2 for DNA adducts, on days 4, 7, 14 and 21 for cell proliferation and on days 4, 7 and 14 for MF (LI and/or MI data). On day 28 for the dose 90 mg/kg, the variability was limited for every end-point. This suggests that animals dosed at 90 mg/kg responded asynchronously and that a 28 day expression time was required to achieve a stable response. Finally, the global evaluation of the data suggests that the higher variability observed at 90 mg/kg most probably reflects the complexity of the biological effects elicited by DMDBC at a high dose.
At both 10 and 90 mg/kg, DMDBC induced the same 32P-post-labelled adduct spot pattern. Two animals out of five treated with 90 mg/kg and sampled on day 2 showed very high levels (1890 and 4550 adducts per 108 nucleotides). As discussed above, this variability could be attributed to differences in metabolism and toxicokinetics. Except at this early time-point, DNA adduct levels remained constant from day 4 to day 28 and averaged 200600 adducts per 108 nucleotides. It has been reported that bulky adducts formed by a number of genotoxic compounds undergo very slow removal. For instance, Hughes and Pilczyk (30) observed metabolite binding to liver DNA for at least 12 weeks after a single injection of 2-naphthylamine to mice. In another study, tamoxifen, a non-steroidal antiestrogen, formed persistent adducts with DNA in rat liver, with a half-life of 3 months (31). Even in proliferative tissues such as skin, DNA adducts induced by 7,12-dimethylbenzanthracene persist for up to 42 weeks (32). We found that DNA binding induced by DMDBC at 10 mg/kg reached a plateau 4 days after treatment. This is in good agreement with the results obtained by Szafarz et al. with a similar dose of DMDBC (5). On day 2, we found that adduct levels were ~20-fold higher in the liver of mice treated with 90 mg/kg than with 10 mg/kg. The time-course of adduct formation after 90 mg/kg, with a peak at early sampling times, was comparable to that reported by Szafarz et al. (5) after treatment with ~2 mg/kg 3-methylDBC, a derivative of DBC which is much more hepatotoxic than DMDBC. Thus, high-dose DMDBC seemed to act in the same way as 3-methylDBC.
This was confirmed by microscopic examination of the liver: DMDBC at 90 mg/kg induced early hepatocellular degeneration/necrosis, while no cytotoxicity was noted at 10 mg/kg. Lesions were observed mostly in centrilobular areas, where functionalizing enzymes such as certain cytochromes P450 are most abundant. Recently, Périn-Roussel et al. (17) found that DMDBCDNA binding in liver occurred almost exclusively in hepatocytes, probably because non-parenchymal cells lack the appropriate activation system. Thus, DNA binding and cytotoxicity induced by DMDBC are dependent on hepatocellular metabolizing capacities. It is likely that, when DMDBC metabolism generates a high level of reactive species in hepatocytes, these cells degenerate. As the decrease in the initial adduct peak induced at 90 mg/kg ran parallel to hepatocellular necrosis, and as the adduct level plateaued from day 4 until the end of the observation period, removal of DMDBC adducts might occur mainly through the elimination of badly damaged cells rather than through repair processes.
Liver necrosis observed in mice treated with 90 mg/kg was followed by hepatocellular regeneration. Valéro et al. (3) observed liver cell proliferation after six s.c. injections of DMDBC ~30 mg/kg administered every 14 days. In our study, scoring of mitotic and/or S-phase hepatocytes showed that cell proliferation was significantly increased on days 7, 14, and 28. Concomitantly, the MF increased continuously from day 7 onward, probably because DNA synthesis enabled the persistent adducts to be fixed into mutations. Enhanced cell proliferation has been shown to play a key role in the induction of mutations in the liver of transgenic mice. Mirsalis et al. (26) reported that while both methylmethane sulfonate (MMS) and DMN formed methylated DNA adducts, MMS failed to induce hepatic cell proliferation and did not increase the MF, whereas DMN elevated the MF at doses that induced liver cell proliferation. Hayward et al. (33) compared two structural isomers, 2,4-diaminotoluene (2,4-DAT) and 2,6-diaminotoluene (2,6-DAT). Both are mutagenic in the in vitro Ames test, but only 2,4-DAT induces hepatocellular proliferation and is hepatocarcinogenic. In Big Blue® mouse liver, only 2,4-DAT was found to be mutagenic. The authors concluded that the cytotoxicity and compensatory cell proliferation induced by 2,4-DAT treatment led to mutation fixation during cell proliferation. In another study reported by Shane et al. (34), cell proliferation induced by partial hepatectomy largely enhanced mutant frequency in the liver of Big Blue® mice treated with N-ethyl-N-nitrosourea. Recently, Krebs et al. (35) found that cyproterone acetate (CPA) induced a dosedependent increase in mutation frequency in Big Blue® rat liver in the range 75200 mg/kg, whereas doses of 25 and 50 mg/kg were ineffective. As DNA adducts induced by CPA increased up to 75 mg/kg but reached a plateau from 75 to 200 mg/kg, the authors suggested that an additional stimulus operating at high doses of CPA (possibly proliferative activity) was required for the fixation of mutations.
We observed karyomegaly in mice treated with DMDBC at 90 mg/kg, as reported by others (3). Nuclear enlargement is a change commonly associated with hepatocarcinogen treatment (3638). It is generally accepted that nuclear enlargement indicates polyploidy (39,40), which results from rounds of replicative DNA synthesis with altered subsequent mitosis (41). A number of drugs can induce polyploidy in mammalian cells by blocking the cell cycle during G2 or M (4146). Mutations arising in genes controlling mitotic events, such as the p34cdc2 kinase gene, can also induce polyploidy (47,48). The mechanism by which DMDBC produces karyomegaly is not known. Here, nuclear enlargement correlated strongly with MF. As DMDBC-induced cytotoxicity and karyomegaly both occurred mainly in centrilobular to mid-zonal areas, damaged hepatocytes may have been favoured to undergo polyploidy and fix mutations compared with non-lesioned cells.
In conclusion, DMDBC formed DNA adducts at 10 and 90 mg/kg but induced a marked increase in MF at 90 mg/kg only. At 90 mg/kg this increase in MF was associated with hepatotoxicity and cell proliferation. These results suggest that DMDBC can act as a complete carcinogen, by the following mechanism: (i) the large amount of reactive species generated in liver by DMDBC administered at high doses (cf. high initial level of DNA adducts at 90 mg/kg in some animals) induces hepatocyte necrosis; (ii) compensatory cell proliferation fixes DNA adducts into stable mutations and amplifies the number of mutated cells; and (iii) mutations and/or chemical interference could alter cell cycle functions and induce nuclear enlargement.
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Notes |
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Acknowledgments |
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References |
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