Mutagenesis, Vol. 16, No. 3, 243-250,
May 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Apoptosis can be a confusing factor in in vitro clastogenic assays
1 Laboratoire de Toxicologie Génétique, Institut Pasteur de Lille, 1 Rue du Pr Calmette, BP 245, 59019 Lille Cedex, France, 2 INSERM U 461, Faculté de Pharmacie, Rue Jean-Baptiste Clément, 92295 Chatenay-Malabry, France and 3 Département Toxicologie-Hydrologie-Hygiène, Université de Lille II, Droit et Santé, Faculté des Sciences Pharmaceutiques et Biologiques, 59006 Lille Cedex, France
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Among the tests used to determine the mutagenic potential of chemicals, the chromosomal aberrations and micronucleus assays play an important role. These tests score either chromosomal structural aberrations at metaphase or micronuclei at interphase. One of the hallmarks of apoptosis is DNA fragmentation into 50–300 kpb leading to oligonucleosomal fragmentation that can interfere with the results of clastogenic assays. In this case, apoptosis may be a confusing factor in the evaluation of the mutagenic potential of molecules and lead to false positive results. For these reasons we have developed a cell line able to demonstrate the interference of apoptosis in two mutagenicity tests: the in vitro micronucleus test and metaphase analysis in vitro. We used a murine cytotoxic T cell line, CTLL-2 Bcl2, in which a stably transfected bcl2 gene is known to protect these cells from apoptosis induced by various stimuli. A comparison between results obtained in parental CTLL-2 cells and in CTLL-2 Bcl2 cells treated with non-genotoxic apoptosis inducers, such as dexamethasone or gliotoxin, leads us to conclude that apoptosis could give false positive results due to DNA fragmentation. Moreover, with etoposide, a clastogen that also induces apoptosis, we observed that the percentages of aberrant cells and numbers of micronuclei were significantly increased in CTLL-2 cells compared with CTLL-2 Bcl2 cells. This observation suggests that apoptosis leads to an overestimation of the genotoxic potential of chemicals. Finally, with nocodazole, an aneugen, we confirm that this model can also detect agents that have only genotoxic potential and thus allows a better estimation of the genotoxic threshold in studies with aneugens, thus avoiding overestimation of the mutagenic risk of such a compound.
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Introduction |
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The development of new chemicals requires investigation of their mutagenic potential. Several tests are now routinely performed and are required for mutagenic risk assessment before approval by various regulatory agencies. These include the Ames assay, the in vitro gene mutation assay in eukaryotic cells, the in vitro chromosome aberration assay and the in vivo micronucleus test (ICH, 1997
).
The process of repairing damaged DNA is of primary importance since DNA damage can be converted into mutations if the cell replicates before completing DNA repair (Moustacchi, 2000). Indeed, oxidants and reactive nitrogen species provoke the accumulation of mutations leading to metaplasia and dysplasia, particularly if these mutations occur in genes regulating cell division and the cell cycle (Correa and Miller, 1998). Cells respond to the burden of DNA damage by two mechanisms: DNA repair or apoptosis. The latter process removes cells with damaged DNA from the pool of replicating cells, avoiding the introduction of mutations into the genome and the associated risk of cancer (Roberts et al., 1997; Correa and Miller, 1998). Cell death may be genetically controlled and the ability of a cell to survive or die is determined by the repertoire of gene products, i.e. p53, bax, bcl2 and bcl-xL (Denis et al., 1998). Their differential expression is a critical determinant of cell and tissue sensitivity to a toxicant (Roberts et al., 1997).
Apoptosis is a form of cell death occurring under physiological conditions or in response to external stimuli, such as DNA-damaging agents, growth factor deprivation or death receptor triggering (Nagata, 1997; Ashkenazi and Dixit, 1998; Evan and Littlewood, 1998; Green and Reed, 1998). Apoptosis is characterized by biochemical features including the activation of cysteine proteases named caspases, mitochondrial permeability transition, cell membrane exposure of phosphatidylserine and DNA cleavage leading to the typical morphology of apoptotic cells, in which the nucleus appears condensed and fragmented. In most cell types DNA cleavage occurs after irreversible activation of endonucleases. An initial cleavage of DNA into 50–300 kpb induces chromatin condensation and in most cell types oligonucleosomal fragmentation follows due to double-stranded cleavage of DNA in the linker region of nucleosomes (Saraste and Pulkki, 2000).
During the process of apoptosis and at the stage of chromatin condensation the original nucleus splits into a number of dense micronuclei, scattered throughout the cytoplasm (Di Baldassarre et al., 2000). These micronuclei generally appear surrounded by a double membrane system, externally outlined by ribosomes. The functional role of these micronuclei is still unknown, but it is generally accepted that they contain sequestered inactive genetic material (Columbaro et al., 1998). Consequently, in the in vitro micronucleus test a possible problem is that the very early steps of chromatin condensation due to apoptosis are not easily distinguishable from micronuclei induced by chemicals using Giemsa staining. Moreover, Simkó et al. (1998) showed in human cell lines that the increase in apoptotic cells is positively correlated with the appearance of micronuclei. Poma et al. (1999) have observed that proliferating lymphocytes undergoing apoptosis after saporin treatment showed DNA damage during the first metaphase, after which the cells die and no metaphases are observable.
A number of genes have been discovered that are involved in apoptosis, such as those belonging to the bcl2 family. The family of Bcl2-related proteins constitutes one of the most biologically relevant classes of apoptosis regulatory gene products acting at the effector stage of apoptosis, with some members functioning as suppressors of apoptosis and others as promoters of cell death (Basu and Haldar, 1996; White, 1996; Kroemer, 1997; Reed, 1997). The relative ratio of these pro- and anti-apoptotic members of the Bcl2 family (Oltvai et al., 1993; Yang and Korsmeyer, 1996) has been shown to determine the ultimate sensitivity or resistance of cells to diverse apoptotic stimuli, including chemotherapeutic drugs, radiation, growth factor deprivation and hypoxia (Selvakumaran, 1994; Kitada et al., 1996).
The properties of Bcl2 were utilized in two murine lymphocyte cell lines: CTLL-2 and CTLL-2 Bcl2. In the latter the apoptosis inhibitor protein Bcl2 is overexpressed after stable transfection. Its capacity to promote cell survival is associated with its ability to interfere with apoptosis. Bcl2 overexpression creates an imbalance between inducers and inhibitors of the Bcl2/Bax family (Korsmeyer, 1999). When Bcl2 proteins are in excess, homodimers between Bcl2 proteins are in the majority and prevent the cells from becoming apoptotic.
In this work we have studied the occurrence of apoptosis as a confounding factor in the evaluation of the genotoxic potential of xenobiotics. We used the in vitro micronucleus test and in vitro metaphase analysis, which both detect early genotoxic events, in parallel with two apoptosis detection methods, YOPRO-1 labelling (Idziorek et al., 1995), which stains apoptotic cells and is fluorescent only after intercalation between base pairs, and Annexin V staining, which measures phosphatidylserine residue expression during the early stages of apoptosis (Andree et al., 1990). This approach was conducted in the two cell lines described, thus allowing us to demonstrate interference by apoptosis in mutagenicity tests.
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Materials and methods |
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Cell lines and culture conditions
CTLL-2 is a subclone of cytotoxic T lymphocytes from mouse strain C57bl/6. The cells were routinely cultured in complete RPMI 1640 medium (Gibco BRL, Paisley, UK) containing 10% foetal calf serum (FCS) (Gibco BRL) heat-inactivated for 30 min at 56°C, 20 mg/ml sodium pyruvate (Sigma-Aldrich Chemical Co., l'Isle d'Abeau Chesnes, France), 2 mM L-glutamine (Gibco BRL), 2 mM HEPES (Sigma), 100 UI/ml penicillin (Gibco BRL), 0.1 mg/ml streptomycin (Gibco BRL) and 5x10–5 M ß-mercaptoethanol (Merck, Nogent-sur-Marne, France) supplemented with 1 ng/ml interleukin-2 (IL-2) (Chiron, France).
The CTLL-2 Bcl2 cell line was produced by stable transfection of CTLL-2 with the pSFFV neo Bcl2 plasmid (a kind gift of Dr S.Korsmeyer) containing a 1.9 kb EcoRI insert encoding the human Bcl2 protein downstream of the SFFV promoter and resistance genes to ampicillin and geniticin (Di Baldassarre et al., 2000). Briefly, CTLL-2 cells were electroporated with 10 µg plasmid using a Bio-Rad gene pulser set at 250 V and 960 µF. CTLL-2 Bcl2 cells were selected in complete medium containing 800 µg/ml G418 (Gibco) for 2 weeks and cloned by limiting dilution. Expression of Blc2 (reaching 90.2%) was measured by intracellular staining using anti-human Bcl2 (Dako, France) labelled with FITC and flow cytometry. Both cell lines were cultured in a humidified incubator containing 5% CO2 at 37°C.
Compounds
Dexamethasone, gliotoxin, etoposide and nocodazole were purchased from Sigma (Sigma-Aldrich Chemical Co.); methane methylsulphonate (MMS) was purchased from Aldrich. The test compounds were dissolved in DMSO (final concentration not exceeding 0.2%) and stored at –20°C. The first two compounds are not carcinogens in humans whereas the others are possible carcinogens (class 2B in the IARC classification).
These compounds were studied up to concentrations inducing apoptosis and/or cytotoxicity (measured by Trypan Blue exclusion) in >50% of the cells according to OECD guidelines (OECD, 1997).
Immunoblotting assay
Expression of p53 was confirmed by immunoblotting using a mouse mAb directed against murine p53 protein (mAb-3; Calbiochem, Meudon, France). Cells were washed and lysed at 4°C for 45 min in phosphate-buffered saline (PBS) with Nonidet P40 (Boehringer Mannheim, Mannheim, Germany) and anti-protease (Complete EDTA-free; Boehringer Mannheim). Extracted proteins were separated by SDS–acrylamide electrophoresis in a 12% gel. After transfer to nitrocellulose the blots were blocked with PBS containing 0.1% Tween and 5% non-fat milk for 1 h and incubated with the specific antibody at 2.5 µg/ml for an additional 2 h at room temperature. After three washes with PBS containing 0.1% Tween, filters were incubated with the secondary antibody (rabbit anti-mouse IgG, dilution 1:1000) conjugated to horseradish peroxidase (Sigma-Aldrich Chemical Co.) for 45 min and then washed with PBS containing 0.1% Tween. Visualization was with an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Little Chalfont, UK).
Mutagenesis assays In vitro micronucleus test
CTLL-2 or CTLL-2 Bcl2 cells (75 000 cells/ml) were cultured in complete RPMI medium containing 25 pg/ml IL-2 (a concentration that avoids apoptosis due to IL-2 deprivation for the duration of the assay; Singh et al., 1994). The cells were treated for 15 h with the compounds at different concentrations. Cells were harvested after centrifugation for 5 min at 200 g and resuspended in a hypotonic solution (1 vol. RPMI 1640, 0.6 vol. water, 2% FCS) for 5 min.
The cells were centrifuged for 5 min at 200 g and fixed with 10 ml of Carnoy II fixative mixture (3 vol. ethanol, 1 vol. acetic acid) for 10 min. After another centrifugation the cells were spread on slides and stained with Giemsa dye (Sigma) diluted at 5% in water. Micronucleated cells were analysed in at least 1000 cells/culture of three parallel cultures (3000 cells/dose) at 500x magnification. The criteria for micronucleus determination are that the intensity of stained micronuclei is inferior to the principal nucleus, its diameter is inferior to the principal nucleus, it is round with a nuclear membrane, it is not connected to the principal nucleus, there is no overlap with the principal nucleus and it is within the cytoplasm.
In vitro metaphase analysis test CTLL-2 or CTLL-2 Bcl2 cells (75 000 cells/ml), cultured in complete RPMI medium containing 25 pg/ml IL-2, were treated for 15 h with the compounds at different concentrations. Two hours prior to harvesting 0.1 µg/ml colcemid (Fluka/Sigma-Aldrich Chemical Co.) was added. Cells were harvested after centrifugation for 5 min at 200 g and resuspended in a hypotonic solution (75 mM KCl at 37°C) for 5 min. Cells were centrifuged for 5 min at 200 g and fixed with 10 ml of Carnoy I fixative mixture (3 vol. methanol, 1 vol. acetic acid) for 12 min. After a further centrifugation the cells were spread on slides and stained for 10 min with Giemsa dye diluted at 4% in water. At least 100 cells/culture (100 cells/dose) were screened for aberrant cells at 1000x magnification, scoring chromosome and chromatid-type aberrations.
Apoptosis detection Annexin V–FITC method Cells were washed in culture medium and resuspended in HEPES buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 5 mM KCl, 5 mM CaCl2)/NaOH at a concentration of 106 cells/ml. Cells were stained with Annexin V–FITC (10 µg/ml final concentration) and propidium iodide (0.5 µg/ml final concentration) for 15 min in the dark at room temperature. Fluorescence of at least 10 000 cells was then assessed using bivariate flow cytometry (Epics II; Coulter, Margency, France) and the percentages of viable, apoptotic and secondary necrotic cells were measured (FITC–/IP– are viable cells; FITC+/IP– cells are early apoptotic cells but still viable; FITC+/IP+ are late apoptotic cells no longer viable). Annexin V staining was performed in parallel with mutagenicity assays. The percentages of FITC+/IP– cells, corresponding to cells in the early stage of apoptosis, are those used in the studies of each compound.
YOPRO-1 method Cells were washed in RPMI 1640 medium and YOPRO-1 (10 µM) was added (1 µl/106 cells/ml) to the cells for 10 min (20). Fluorescence of at least 10 000 cells was then assessed using bivariate flow cytometry (Epics II) and the percentage of apoptotic cells (YOPRO-1+ cells) was measured. YOPRO-1 staining was performed in parallel with mutagenicity assays.
DNA extraction and gel electrophoresis analysis
Treated cells were washed in PBS and incubated for 30 min at 4°C in lysis buffer (5 mM Tris, 20 mM EDTA, 0.5% Triton X-100; all from Sigma). The DNA from the cell lysate was precipitated with 1/10 vol. 3 M sodium acetate and 2 vol. cold absolute ethanol for 1 h at –80°C. The DNA was then incubated for 5 h at 37°C with RNase (500 µg/ml; Boehringer Mannheim) and extraction buffer (10 mM Tris, 5 mM EDTA). DNA was then incubated overnight at 65°C with proteinase K (1.25 mg/ml), 10x proteinase K buffer (100 mM Tris, 100 mM EDTA, 250 mM NaCl) and extraction buffer. The proteins were extracted with water-saturated phenol. The aqueous phase was harvested and phenol was eliminated with chloroform:isoamyl alcohol (24:1). The DNA from the aqueous phase was precipitated with 1/10 vol. 3 M sodium acetate and 2 vol. cold absolute ethanol for 1 h at –80°C. The DNA was washed in cold absolute ethanol and dissolved in extraction buffer. DNA (10 µg) was separated electrophoretically at 30 mA for 2–3 h on a 1.2% agarose gel containing 0.5 µg/ml ethidium bromide and visualized with UV light.
Statistical analysis
Statistical analysis of the micronucleus test results was performed by analysis of variance (ANOVA) and multiple comparisons were made by Dunnett analysis (Statview v.4.0) to assess where the difference in the dependent variable value arose in each cell line. The comparisons between pairs of groups for each dose in the two cell lines were made by Student's t-test. The two types of aberrations (gaps and breaks) were analysed using Student's t-test, because although the standard deviations found did not demonstrate that the distribution was normal, this test can be used because of the large number of samples (100 cells/culture/dose). In the figures the values for number of micronucleated cells differ significantly from the corresponding control value (aP < 0.05, bP < 0.01) and the number of micronucleated CTLL-2 cells differs significantly from the corresponding CTLL-2 Bcl2 value at the same dose (cP < 0.01).
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Results |
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Expression of p53 in CTLL-2 and CTLL-2 Bcl2 cells
Apoptosis can occur by a p53-dependent pathway as well as by a p53-independent pathway (Vogt Sionov and Haupt, 1999
). Therefore, it is important to verify that CTLL-2 cells express this protein.
Genotoxins are known to induce p53 expression in order to control the cell cycle (Yang and Duerksen-Hugues, 1998). CTLL-2 and CTLL-2 Bcl2 cells were treated with 10 and 100 µM MMS, an alkylating agent known to induce p53 expression (Zhan et al., 1993), and were examined by western blotting for expression of p53 and its induction by genotoxic stress.
The results showed that wild-type p53 is normally present in the two cell lines without genotoxic treatment and its expression is increased by treatment with MMS in both cell lines.
DNA fragmentation assay
We first verified that in CTLL-2 cells DNA fragmentation could be seen when apoptosis occurs in this model. DNA electrophoresis from CTLL-2 cells treated with dexamethasone showed a typical DNA ladder, indicating that oligonucleosomal fragmentation was present (Figure 1). However, no DNA fragmentation was present when CTLL-2 Bcl2 cells were treated with dexamethasone, demonstrating that overexpression of Bcl2 in CTLL-2 cells protects them from apoptosis (Figure 2).
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Evaluation with compounds inducing apoptosis in CTLL-2 cells
To assess whether apoptosis can lead to chromosome and chromatid breakage we compared the results obtained in parental CTLL-2 cells with those obtained in CTLL-2 cells stably transfected with the apoptosis inhibitor gene bcl2 and treated with either dexamethasone or gliotoxin, which are apoptotic but not genotoxic compounds. Two tests were used: the in vitro micronucleus test and metaphase analysis. Apoptosis was evaluated with Annexin V and YOPRO-1 staining, which quantify two different outcomes of apoptosis.
In the in vitro micronucleus test we observed a dose-dependent increase in the number of micronucleated cells in CTLL-2 cells treated with dexamethasone, with maxima of 38.3 and 39.6
at 75 and 150 nM, respectively. This increase was statistically significant at doses of 18.75 nM and higher (Figure 2A
). Similar results were obtained in the in vitro metaphase analysis, where dexamethasone significantly increased the percentage of aberrant cells at doses of 37.5 nM and higher (Figure 2B). The percentage of aberrant cells was reduced at the highest dose of dexamethasone used (150 nM). This effect could be the consequence of the anti-proliferative action of glucocorticoids. Chromosome and chromatid breaks, but never chromatid exchanges, were observed following dexamethasone treatment (Tables I and II). However, when CTLL-2 Bcl2 cells were treated with dexamethasone we did not observe an increase in micronucleated cells or aberrant cells at any of the doses tested (Figure 2A and B).
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Gliotoxin, another apoptosis inducer, was studied at a range of doses from 25 to 200 nM (Figure 3
). In the in vitro micronucleus test gliotoxin was positive at 100 and 200 nM in CTLL-2 cells (P < 0.01, Figure 3A) whereas in the metaphase analysis test a statistically significant difference was present at >50 nM (Figure 3B). Concentrations in the metaphase analysis assay were not tested over 50 nM in CTLL-2 cells due to the absence of mitosis. Apoptosis was present at 100 and 200 nM using either Annexin V–FITC staining or the YOPRO-1 assay (Figure 3C and D). Again, when CTLL-2 Bcl2 cells were treated with gliotoxin neither micronuclei, aberrant cells nor apoptosis were present at a significantly higher level compared with control cells at concentrations up to 200 nM (Figure 3A–D).
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These studies using compounds known to induce apoptosis but devoid of genotoxicity demonstrated: (i) micronucleated cells or aberrant cell number did not increase following treatment with either dexamethasone or gliotoxin in CTLL-2 Bcl2 cells whereas they did increase in parental CTLL-2 cells; (ii) the presence of micronucleated cells or aberrant cells was correlated with the occurrence of apoptosis.
Evaluation with compounds inducing both genotoxicity and apoptosis in CTLL-2 cells
We compared the results obtained in the two murine cell lines with compounds that are both genotoxic and apoptotic to test whether apoptosis could lead to an overestimation of the genotoxic potential of chemicals using mutagenicity tests.
Etoposide, studied in a range of concentrations from 62.5 to 500 nM, induced a dose-dependent increase of the number of micronucleated cells in both CTLL-2 and CTLL-2 Bcl2 cells (Figure 4A). This effect was also observed in the metaphase analysis test, with a dose-dependent increase in the percentage of aberrant cells in both cell lines. This increase was statistically significant from 62.5 nM in CTLL-2 cells and from 125 nM in CTLL-2 Bcl2 cells (Figure 4B and Tables I and II). Apoptosis was present in CTLL-2 cells treated with etoposide, with 29% apoptotic cells at the higher dose, but not in CTLL-2 Bcl2 cells, suggesting that etoposide provoked genotoxicity independently of apoptosis. However, the numbers of micronucleated cells and the percentages of aberrant cells were always significantly higher in parental CTLL-2 cells compared with CTLL-2 Bcl2 cells. This difference is probably the consequence of DNA fragmentation during apoptosis that occurs in parental CTLL-2 cells leading to higher numbers in both tests compared with CTLL-2 Bcl2 cells.
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It is interesting to note that with the non-genotoxic apoptosis inducers (dexamethasone and gliotoxin) chromosome breaks but no exchanges were observed (Tables I and II
). In contrast, with etoposide chromosome breaks and exchanges were observable. On the one hand, this difference arises from the difference in primary DNA damage. On the other hand, this difference is due to the fact that in apoptotic cells no DNA repair occurred while in cells with primary DNA damage repair occurred leading to chromosome exchanges. In practice, a high level of breaks without exchanges induced by a test compound could alert one to suspect a role of apoptosis in breakage induction.
The aneugenic molecule nocodazole, studied over a range of doses from 40 to 100 nM, was also able to induce both an increase in the number of micronucleated cells (354/1000 at the higher concentration) and in the percentage of apoptotic cells (22% at the highest concentration) in parental CTLL-2 cells (Figure 5). In CTLL-2 Bcl2 cells nocodazole did not induce apoptosis but significantly increased in a dosedependent manner the number of micronucleated cells (Figure 5A), leading to a statistically significant difference in the number of aberrant cells between the two cell lines. No metaphase analyses were conducted since the OECD guidelines (OECD, 1997) note that this assay is not pertinent to the study of aneugenic compounds. Again, like etoposide, the number of micronucleated cells was higher in parental cells compared with CTLL-2 Bcl2 cells, suggesting that apoptosis participates in the production of micronucleated cells in the parental CTLL-2 line (Figure 5A).
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Discussion |
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The overestimation of chromosomal aberrations due to interference of apoptotic DNA fragmentation is a problem previously mentioned by several authors without clear discrimination between the two phenomena (clastogenesis and apoptosis): Singh et al. (1994) concluded that dexamethasone induced a positive response in metaphase analysis in vitro in human lymphocytes when a long incubation time was used.
Moreover, in an investigation of the mechanism of induction of nuclear anomalies, Guo et al. (1998) observed that 
-irradiation led to a low number of micronucleated cells correlated with a low apoptotic cell frequency. Thus they proposed that -radiation, an apoptosis inducing agent and also a true clastogen, induced both processes and indistinguishable micronuclei. In contrast, Kirsch-Volders et al. (1997) showed, in a study of the in vitro micronucleus test, that cells suffering injury by chemicals, especially genotoxins, could undergo apoptosis. In that case, depending on the degree of chromatin condensation, round shaped vesicles containing chromatin fragments could be observed in the cytoplasm as micronuclei. They stressed the fact that the frequency of micronucleated cells included apoptotic cells. In the same way, Ronen and Heddle (1984), studying nuclear damage after treatment of mice with carcinogens, noticed that among the anomalies some appeared as micronuclei or apoptotic nuclei, but sometimes micronuclei were indistinguishable from apoptotic nuclei, leading to the conclusion of both of the two sorts of anomalies and thus false results.
For these reasons it is necessary to distinguish DNA fragmentation due to apoptosis from that due to a true genotoxic effect in mutagenicity tests in order to have a better understanding of the chromosomal aberrations observed. This differentiation is difficult to perform in the case of apoptosis induction associated with a high primary DNA damage level or with aneugenic events. To address this question we developed a cell model using T lymphocytes of murine origin which overexpressed the anti-apoptotic protein Bcl2. Bcl2 was chosen since it protects cells against a large number of external apoptotic stimuli, such as UV, radiation, genotoxic agents and hormones. During this work we compared the results obtained in the CTLL-2 parental cell line with that obtained in CTLL-2 cells transfected with the bcl2 gene.
CTLL-2 cells are able to respond to genotoxic stress, as shown by p53 induction following MMS treatment. Moreover, these cells show oligonucleosomal DNA fragmentation during the process of apoptosis.
We first addressed the question whether DNA fragmentation occurring during apoptosis could give false positive results in mutagenicity assays such as the in vitro micronucleus test and in vitro metaphase analysis. These two tests permit the detection of primary DNA damage due to a clastogenic treatment. Two non-genotoxic molecules that are well-known apoptosis inducers were studied, dexamethasone and gliotoxin.
Dexamethasone, a synthetic glucocorticoid, is a well-known apoptosis inducer in lymphoid cell lines (Perrin-Wolff et al., 1995; McColl et al., 1998). CTLL-2 cells respond to glucocorticoid treatment and become apoptotic (Perrin-Wolff et al., 1996). Gliotoxin, a fungal toxin, has suppressive effects, inhibiting NF
B, and induces apoptosis through elevation of the cAMP level. Then protein kinase A activation (Sutton et al., 1995) leads to hyperphosphorylation of histone H3 on serine residues to make them more sensitive to nucleases (Waring et al., 1997) and apoptosis takes place. The induction of apoptosis by dexamethasone or gliotoxin treatment was responsible for misinterpretation of the results, since it led us to conclude a genotoxic effect for the compounds, which constituted false positive responses.
We then addressed the question whether apoptosis can lead to an overestimation of the genotoxic potential of a molecule assessed by mutagenicity assays. Cells were treated with etoposide, a clastogenic agent that is also an apoptosis inducer. Etoposide leads to apoptosis by a p53-dependent pathway and recent studies suggest that activation of tyrosine kinases plays an important role in the apoptotic process induced by etoposide (Usami et al., 1998) as well as by topoisomerase II inhibition (Ferguson, 1998). Moreover, Kamesaki et al. (1993) showed that Bcl2 inhibits apoptosis induced by etoposide. Etoposide induced apoptosis, micronucleus formation and structural aberrations in CTLL-2 cells, whereas in the transfected cell line these anomalies were fewer, but still present. Moreover, metaphase analyses and micronucleus tests demonstrated that apoptosis could also give excessive positive results with genotoxic agents and apoptosis inducers. In the case of indirect mechanisms of mutagenesis it was proposed that we can determine a threshold of activity (Henderson et al., 2000; Müller and Kasper, 2000). As etoposide acts by topoisomerase inhibition it should therefore have a threshold for mutagenic activity. In the results of the clastogenicity assay apoptosis leads to an overestimation of the genotoxic effect and lowers the threshold. Under these conditions the results can lead to a false overestimation of genotoxic risk.
The induction of aneuploidy by spindle inhibitors produces a characteristic dose–response curve including a threshold (Singh et al., 1994; Kirsh-Volders et al., 2000). We noted this phenomenon in the studies of nocodazole: a statistically significant difference from the control was observed in CTLL-2 cells with 40 nM compound, whereas the threshold was shifted to 55 nM compound in the transfected cell line. Therefore, the lowest effective dose was different in the apoptotic versus non-apoptotic lines because apoptosis lowers this value. This indicates that suppression of the apoptotic effects permitted a better evaluation of the threshold value to take account of biologically relevant events and thus not overestimate the risk for the compound tested.
Since transfection with bcl2 diminished and even abolished the apoptotic component, the model developed can be envisaged as a preliminary short-term screening assay to detect true genotoxic compounds at an early stage of their development. The micronucleus test in these cells has good specificity: indeed, the two apoptosis inducers (dexamethasone and gliotoxin) gave negative results in terms of genotoxicity in CTLL-2 Bcl2 cells. It also has good sensitivity, since the two genotoxic compounds (etoposide and nocodazole) gave positive results in the two cell lines.
In order to validate the model, other aneugenic or clastogenic compounds will need to be studied. This will allow us to confirm the ability of the transfected cells to minimize the apoptotic factor observed in determination of the genotoxic potential, even if not all the apoptotic compounds induce a pathway controlled by Bcl2.
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Acknowledgments |
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We thank Chiron Laboratories for providing us with human recombinant interleukin-2 and Dr Stanley Korsmeyer for giving us the bcl2 plasmid. We also extend our appreciation to Brigitte Quatannens and Jean-Pierre Kusnierz for help with the cytofluometer. Finally, we thank CIRD Galderma laboratory for their support. Use of Bcl2-transfected cells is covered by patent no. 99-14608 (14/11/1999).
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Notes |
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4 To whom correspondance should be addressed at: Laboratoire de Toxicologie Génétique, Institut Pasteur de Lille, 1 Rue du Pr Calmette, BP 245, 59019 Lille Cedex, France. Tel: +33 3 20 87 79 75; Fax: +33 3 20 87 73 10; Email: daniel.marzin{at}pasteur-lille.fr
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Received on September 25, 2000; accepted on December 22, 2000.
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