Mutagenesis, Vol. 14, No. 4, 403-410,
July 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
A micromethod for the in vitro micronucleus assay
1 Laboratoire de Toxicologie Génétique, Institut Pasteur de Lille, 59019 Lille Cedex and 2 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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A micromethod for the in vitro micronucleus assay was developed using L5178Y cells to enable the rapid screening of a large number of molecules. The method is quick, simple to perform and needs very small amounts of compound, i.e. <10 mg. In this methodology, three types of treatment were carried out in parallel, enabling an optimal detection of both aneugenic and clastogenic compounds: two treatments without metabolic activation with or without a recovery period after a 24 h continuous treatment and one treatment with metabolic activation by Aroclor 1254-induced rat or hamster liver S9 mix. Seventeen known genotoxins (12 clastogens and five aneugens) and seven known non-genotoxins were tested. The in vitro micronucleus micromethod using L5178Y cells exhibited good sensitivity (16 positive/17 known genotoxins tested) and specificity (7 negative/7 known non-genotoxins tested) for the 24 test compounds studied with or without metabolic activation. Furthermore, this test showed a good correlation with other in vitro micronucleus tests performed using macromethods with various mammalian cell cultures. We conclude that the in vitro micronucleus micromethod with L5178Y cells could be used in the earliest stages of development of new molecules as a preliminary short-term screening assay before starting regulatory tests.
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The increasing numbers of new molecules synthesized in the pharmaceutical, chemical and cosmetic industries requires the use of assays providing rapid results and requiring small amounts of test material. As regulatory tests cannot reasonably be performed at an early stage of chemical development due to their complex protocols (duration, cost and need for large amounts of compound), we tried to miniaturize a chromosomal mutation assay, the in vitro micronucleus (MN) test. In fact, the in vitro MN test is known to be well adapted to the pre-screening stage as it requires lower quantities of compound (500 mg are generally sufficient for the evaluation) and slide reading is easily and quickly performed. Moreover, it is able to demonstrate structural and numerical chromosomal aberrations in the same assay (Marzin, 1997
). In fact, this methodology can simultaneously detect mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction (Kirsch-Volders et al., 1997).
The first requirement was to use microplates in order to decrease the amount of test compound. Thus, cells growing in suspension, which do not need trypsinization, were chosen. It was also necessary to use cells with rapid growth, a feature of cell lines, and with good karyotypic stability. L5178Y cells, used in the mouse lymphoma assay (MLA), a test in which the effect of genotoxic compounds inducing gene mutation and chromosomal aberrations has been well validated (Sofuni et al., 1996), can also be used for the in vitro MN test because this cell line fulfils all of the above conditions. Indeed, Stopper et al. (1993a,b, 1994) demonstrated the suitability of this cell line for the measurement of in vitro chemically induced MN.
The conditions of treatment, duration of treatment and harvesting time both with and without metabolic activation were initially determined by studying the kinetics of MN induction for well-known direct and indirect acting genotoxins. Then, several other well-established potent clastogens and aneugens, acting through a variety of mechanisms, as well as several known non-mutagenic compounds, were investigated to validate the method.
Data obtained in this micromethod were compared with those reported in selected literature on the in vitro MN test.
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Cell culture
L5178Y TK+/– clone 3.7.2C mouse lymphoma cells obtained in 1991 from ECACC (Porton Down, Salisbury, UK) were cultured in suspension in FM10, i.e. Fisher medium (FM0; Gibco BRL, Paisley, UK) supplemented with 200 U/ml penicillin, 50 µg/ml streptomycin, 2.5 µg/ml amphotericin B, 200 µg/ml L-glutamine, 200 µg/ml sodium pyruvate, 500 µg/ml pluronic acid and 10% (v/v) heat-inactivated horse serum (Gibco BRL). Cell cultures were grown in a humidified atmosphere with 5% CO2 in air at 37°C. Each new batch of cells was tested to confirm the absence of Mycoplasma contamination. In addition, the strain was routinely checked for both its karyotypic stability (n = 40 ± 2) and the prevalence of polyploid cells, which was ~10/1000 cells. The doubling time was 10–12 h.
Chemicals
Chemicals tested are listed in Table I with the corresponding abbreviations, CAS registry numbers, sources, purities and solvents used.
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Metabolic activation system
Rat and hamster liver S9 were prepared according to Ames et al. (1975). Liver enzymes were induced with Aroclor 1254 according to Maron and Ames (1983). The S9 mix contained, per ml: 0.4 ml S9, 0.2 ml 150 mM KCl, 0.2 ml 25 mg/ml NADP and 0.2 ml 180 mg/ml glucose 6-phosphate. In the assay with metabolic activation, the FM10 was supplemented with 10% of this S9 mix (containing 40% S9).
In vitro MN test
Three types of treatment were carried out.
- (i) In the first one, without metabolic activation (24 h –S9), cells were continuously treated for 24 h and were harvested immediately.
(ii) In the second trial, without metabolic activation (24 h + 20 h –S9), cells were continuously treated for 24 h. After this treatment period, the test agent was discarded and the cells were washed twice in FM10. The cells were finally resuspended in FM10 and were further incubated for 20 h.
(iii) In the third treatment, with metabolic activation (4 h +S9), cells were treated in the presence of S9 mix for 4 h. After this treatment period, the test agent was discarded and the cells were washed twice in FM10. The cells were finally resuspended in FM10 followed by 24 h expression in fresh medium.
Each treatment was performed in duplicate and was coupled to an assessment of cytotoxicity.
Treatment of the cells
Figure 1 summarizes the different stages of treatment. Briefly, a concentrated solution was prepared in FM0 (or in DMSO if the test compound was insoluble in water) followed by a predilution performed in FM10 (supplemented with 10% S9 mix in the assay with metabolic activation) then distributed in the first row of wells of a microplate with 96 V-bottomed wells (Costar 3894; Costar, Brumath, France). Each new row of wells was filled with a half dilution (in FM10 with or without 10% S9 mix) of the solution in the previous row. Exponentially growing cells were then added to each well at 6x105 cells/ml for the 24 h –S9 and 4 h +S9 trials and at 4x105 cells/ml for the 24 h + 20 h –S9 assay.
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Cytotoxicity assay
Cytotoxicity was assessed using a 3-[4,5-dimethylthiazol-2-gl]-2,5-diphenyltetrazolium bromide (MTT; Sigma, Poole, UK) colorimetric method (Borenfreund, 1988). In the absence of marked cytotoxicity, water soluble compounds were tested up to 2000 µg/ml and at 1000 µg/ml for the DMSO soluble compounds or up to the limit of solubility if these values could not be reached. In the case of toxicity, the highest concentration was fixed according to the reduction in MTT incorporation and to the quality of the slides. At least two lower concentrations were also used for the genotoxic analysis.
Genotoxicity assay
Each centrifugation step was performed at 120 g for 5 min, followed by gentle pouring off to discard the supernatant. At the end of the recovery time, the microplates were centrifuged. The cells were first washed (0.2 ml culture medium FM0 + 0.1% pluronic acid), gently resuspended and centrifuged before hypotonic treatment (4 min with 0.2 ml FM0 diluted 1:1 v/v in distilled water + 0.1% pluronic acid). After a new centrifugation, the cells were fixed by addition of 0.1 ml ethanol/acetic acid (3:1 v/v) for at least 10 min. The cells were finally resuspended by drawing and expelling with a Pasteur pipette, dropped onto wet clean glass slides and allowed to dry at room temperature. After 24 h, the air-dried slides were stained for 10 min with 2% Giemsa water solution, rinsed, coded and analyzed. MN were analyzed in at least 500 mononucleate cells/culture of two parallel cultures (1000 mononucleate cells/dose) at 500x magnification. MN were identified according to the criteria described by Miller et al. (1995). To omit nuclear fragmentation, cells with >5 MN were not considered for the evaluation of micronucleated cells (Miller et al., 1995).
The statistical significance of differences between groups was determined using the 
2 test. The criteria for determining a positive result were a concentration-related increase in the number of micronucleated cells and a statistically significant increase over the spontaneous level in at least one treatment schedule.
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Results |
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The treatment schedules were determined by studying the kinetics of chemically induced MN. On the one hand, two direct genotoxins, a potent clastogen (mitomycin C, MMC) and a well-known aneugen (diethylstilbestrol, DES) were tested at different concentrations without metabolic activation by varying both the treatment period and the recovery period (Tables II–IV
). On the other hand, the kinetics of MN induction with metabolic activation by rat liver S9 mix was investigated using an indirect genotoxin (cyclophosphamide, CPA) and varying only the recovery period (Table V). In this last case, the treatment duration was limited to 4 h due to the relative cytotoxicity of the S9 mix and the short life of this enzymatic system.
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The chemically induced MN frequencies were determined and we decided to implement three treatments: 24 h –S9; 24 h + 20 h –S9; 4 h +S9. In order to validate this protocol, several direct and indirect genotoxins, acting through a variety of mechanisms, were studied. The results are summarized in Tables VI and VII
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As expected, all the aneugenic compounds studied gave clear-cut positive responses without metabolic activation (Table VI
): diazepam (DIA), griseofulvin (GRI) and paclitaxel (TAX) behaved like DES and did not require any reincubation: they clearly increased MN after both immediate (24 h –S9) and prolonged harvest (24 h + 20 h –S9). MN induction could not be quantified at high concentrations of TAX and GRI in the 24 h + 20 h procedure, due to the fragmentation of nuclei. The 24 h treatment followed by immediate harvest was, however, insufficient for all aneugens, as chloral hydrate (CHLO) required the 20 h recovery period to reveal its genotoxic potential.
Similarly, all of the clastogenic chemicals studied without S9 mix showed a clearly genotoxic activity with clear dose–effect relationships, especially with a further 20 h culture after the end of the treatment (Table VI). As a matter of fact, although all the clastogens were detected with the 24 h protocol, they were more clearly positive with the 24 h + 20 h schedule.
In the presence of metabolic activation by rat liver S9 mix, all the indirect genotoxins tested revealed significant genotoxic potential, except ß-naphtylamine (ß-NA) (Table VII). The results for ß-NA, although equivocal (5 MN at 1.79 µg/ml versus 1.5 MN for the control), were not statistically significant. A possible explanation for this finding is that rat liver S9 contains too low an activity of N-acetyltransferase (NAT), an enzyme essential for the metabolism of aromatic amines. Indeed, Prival et al. (1984) showed that hamster liver S9 was more effective than rat liver S9 in revealing the genotoxicity of aromatic amines. We therefore tested both 
-naphtylamine (-NA) and ß-NA in the microscale MN test on L5178Y cells as previously described in the presence of Aroclor 1254-induced hamster liver S9 mix. In this independant assay, these two aromatic amines gave positive results, as shown in Table VIII.
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Finally, in order to demonstrate the specificity of the test, we studied several non-genotoxic compounds. Each of them gave unequivocal negative responses both with and without metabolic activation when tested up to 2000 µg/ml or to the limit of solubility or cytotoxicity (Tables IX and X
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Choice of the cell line
L5178Y cells are widely used in mutagenesis (e.g. MLA) and are suitable for measuring MN induction by chemicals (Stopper et al., 1993a
,b, 1994). As regards their p53 status, Storer et al. (1997) demonstrated that the mouse lymphoma L5178Y Tk+/– 3.7.2 C cell line was heterozygous for a codon 170 mutation in the p53 tumor suppressor gene and therefore suggested that this cell line may be abnormally susceptible to the induction of genetic alteration. More recently, Clark et al. (1998) showed that L5178Y Tk+/– 3.7.2 C mouse lymphoma cells have two mutant Trp53 alleles which contributed to their ability to detect the major types of mutational damage. In contrast, these authors concluded that this ability to integrate across mutational events further supported the use of the assay in chemical and drug safety studies. As a matter of fact, a recent evaluation of the MLA database by the Environmental Protection Agency's Gene-Tox Mouse Lymphoma Committee gave no support to Storer's contention that the assay gives positive results for a large number of non-carcinogens (Clark et al., 1998).
These mouse lymphoma cells show several other advantages, such as: growth in suspension (no need for trypsinization), rapid growth (doubling time of ~10–12 h), unsynchronized division, stable karyotype and low and reproducible incidence of spontaneous MN (Kirchner et al., 1993). One could expect that this continuous cell line would give limited intra- and inter-laboratory variation in the number of spontaneous MN. However, there is a good correlation between the mean spontaneous MN frequency in our studies and those obtained by Kirchner et al. (1993); 4.5 ± 2.4/2000 mononucleate cells (range 0–10 MN/2000 mononucleate cells) and 3.7 ± 0.6/2000 mononucleate cells, respectively. This rate is lower than the baseline level observed in cultured human T lymphocytes. For instance, Villani et al. (1995) reported a baseline frequency of MN in T lymphocytes of 48 unexposed humans averaging 10.9 ± 5.7/1000 binucleate cells with a high inter-individual variability.
As part of a screening assay and in order to save time, the slide reading was done on 1000 mononucleate L5178Y cells/dose. To refine the test, the analysis may be performed on 1000 mononucleate cells per culture, i.e. 2000 mononucleate cells/dose.
Use of cytochalasin B
Cytochalasin B is an inhibitor of actin polymerization which blocks mitotic cytokinesis. It is currently used in in vitro MN tests, particularly on human lymphocytes (Fenech and Morley, 1985), to ensure discrimination between cells which have undergone one division after exposure to the chemicals (binucleate cells) and undivided cells (mononucleate cells). It is usually used in a range of final concentrations from 3 to 6 µg/ml. However, cytochalasin B has the ability to induce nuclear extrusion (Carter, 1967). It also causes DNA fragmentation in a number of cells lines, particularly in T lymphoma cell lines, as early as 2 h after the start of incubation at a final concentration of 5 µg/ml (Kolber et al., 1990). In our preliminary experiments, treatment of L5178Y cells with 2 µg/ml cytochalasin B for 8 h indeed induced DNA fragmentation and a high frequency of pycnotic nuclei. This explains why, in order to avoid co-treatment with a bioactive chemical, we did not apply cytochalasin B treatment in our study.
Justification of the choice of the protocol
To investigate the optimum times to measure the possible genotoxic potential of any compound, we varied both the treatment time and the harvesting time.
In a first series of trials carried out without metabolic activation, we studied the chemically induced MN frequencies of a potent clastogenic agent, MMC, and of an aneugenic agent, DES.
With 0.025 µg/ml MMC, the kinetics of MN induction (Table II) showed that the maximum response and the best quality of smears was obtained when cells were reincubated for 20 h after the 24 h continuous treatment. A 48 h continuous treatment induced a slightly higher response but increased the cytoxicity and decreased the quality of the cells, particularly cytoplasm integrity (data not shown).
The rate of induced MN reached a peak when cells were harvested immediately after 24 h exposure to 10 µg/ml DES (Table IV). In parallel, we treated the cells for a shorter time (4 h) and a longer time (48 h): on the one hand, we found that 4 h, which is the treatment time used by Stopper et al. (1993a,b, 1994), was insufficient to demonstrate the genotoxic activity of DES (Table III); on the other hand, a 48 h continuous treatment appeared to be very toxic. Thus, we retained two different harvesting times without metabolic activation: the first one without recovery time (immediate harvest) and the second one with 20 h of reculture.
In the same way, we determined the best recovery time with metabolic activation by scoring the number of MN induced by CPA. We decided to limit the time of treatment to 4 h, according to OECD guidelines, because of the toxicity of the S9 mix. Table V shows that the response obtained with 10 µg/ml CPA was similar at 20, 24 and 28 h reincubation. In addition, at 5 µg/ml CPA, the 24 h harvest time corresponded to the beginning of the plateau. The 4 h + 24 h treatment schedule was then retained as it was also more convenient for the assay.
In order to evaluate the sensitivity of the in vitro MN micromethod on L5178Y cells, we compared our results with those previously reported in the literature on the in vitro MN test.
Results for aneugens
Similarly to the results obtained in our study, Stopper et al. (1994) observed that DES and GRI induced the formation of MN in L5178Y cells. Fritzenschaf et al. (1993) also concluded that DES was positive in Syrian hamster embryo (SHE) cells. These first findings are interesting in that Stopper et al. (1993a,b, 1994) also worked on L5178Y cells. The micromethod seems to be as sensitive as the classical method even if the times of treatment are different (24 h in our study versus 4 h in Stopper's work). Seelbach et al. (1993) also demonstrated that GRI was strongly genotoxic in V79 cells, although Migliore and Nieri (1991) found this same compound only weakly genotoxic in human lymphocytes.
Contrary to the work of these last authors, our study using the micromethod protocol revealed a statistically significant increase in the number of MN induced by DIA. Likewise, both Seelbach et al. (1993) and Lynch and Parry (1993) found DIA genotoxic in V79 and in Chinese hamster Luc2 cells, respectively.
For TAX, which we found strongly positive, no adequate result was available in the literature.
Concerning CHLO, we noted a 6-fold increase in the number of MN over the control, whereas Lynch and Parry (1993) observed a 2-fold increase in Chinese hamster Luc2 cells. The results on human lymphocytes were contradictory: Migliore and Nieri (1991) obtained a weak MN induction after both 48 and 72 h treatments (the latter gave the maximum response), whereas Van Hummelen and Kirsch-Volders (1992) as well as Vian et al. (1995) observed no dose-dependent increase in the induced MN frequency after a 48 h continuous treatment when tested up to the toxicity limit.
Thus, the in vitro MN micromethod on L5178Y cells appears to be as sensitive as other in vitro MN tests carried out on various mammalian cell lines except for the assay on human lymphocytes, which seems to be less efficient in detecting possible aneugenic activity when the reading was only performed on binucleate cells. Indeed, Elhajouji et al. (1998) found a clear increase in MN frequency in mononucleate human lymphocytes after exposure to aneugenic compounds. These authors confirmed that some mononucleate cells pass without chromatid segregation to daughter nuclei and they concluded that scoring of MN in mononucleate cells may be an additional useful end-point to distinguish clastogens from aneugens and to increase the sensitivity of the MN cytokinesis block assay.
Results for clastogens
Methylnitrosourea (MNU) is known to produce excision-repairable lesions and needs treatment with cytosine arabinoside to express its clastogenicity in the cytokinesis block method with human lymphocytes (Fenech and Neville, 1992). MNU was readily found positive in our protocol without any pretreatment at the maximum dose tested by Fenech and Neville (1992). Interestingly, a 4-fold increase in the number of micronucleated cells over the control was observed in both our study and that of Fenech and Neville (1992).
Although methyl methanesulfonate (MMS) is a potent inducer of MN in V79 cells (Lasne et al., 1984), as well as in isolated human lymphocytes (Elhajouji et al. 1997), significant differences in the levels of MN induction were noted. In our study, we found MMS strongly positive in the 24 h + 20 h treatment schedule. Zhang et al. (1995) also showed a clear genotoxic effect of MMS in the in vitro MN test on L5178Y cells.
For 1-methyl-3-nitro-nitrosoguanidine (MNNG), our data totally agreed with those reported by Matsuoka et al. (1993), using CHL cells.
Finally, our results for MMC were also comparable with those of Elhajouji et al. (1995) on human lymphocytes, Zhang et al. (1995) on L5178Y cells and Seelbach et al. (1993) on V79 cells.
Globally, whatever the mammalian cell line used, MN inductions observed were comparable for the clastogenic compounds tested.
Results with metabolic activation
Safrole (SAF) was extensively studied in the 1980s. It was rarely found positive in in vitro genotoxicity tests, particularly in the Ames test. Sina et al. (1983) obtained equivocal results in this gene mutation test, while Gocke et al. (1981), Booth et al. (1983) and Dyrby and Ingvardsen (1983) found it negative with both Salmonella typhimurium and Escherichia coli strains. Regarding the results of the in vitro MN test, SAF was found negative in human lymphocytes by Vian et al. (1993) and in SHE cells by Fritzenschaf et al. (1993). SAF was, however, positive in the transformation test on SHE cells (Fritzenschaf et al., 1993). In our protocol, SAF gave a dose-related and statistically significant increase in the number of MN over the negative control (Table VII).
In the current study, the number of MN induced by CPA was higher than that counted by Vian et al. (1993) in human lymphocytes (the same observation was made for 7,12-dimethylbenz[a]anthracene), but close to the values observed by both Matsuoka et al. (1993), on CHL cells, and Krishna et al. (1989), on V79 cells.
Benzo[a]pyrene (B[a]P) also gave similar results in various cell lines such as CHL cells (Matsuoka et al., 1993) and human lymphocytes (Vian et al., 1993).
Only ß-NA yielded a false negative result in our initial protocol in the presence of rat liver S9 mix (Table VII). In contrast, SHE cells, which retain a broad spectrum of metabolic competence for the bioactivation of xenobiotics (particularly a high activity of NAT), revealed its genotoxicity (Fritzenschaf et al., 1993). Indeed, when we used hamster liver S9 mix, both -NA and ß-NA were found positive in the in vitro MN micromethod on L5178Y cells (Table VIII). The in vitro MN micromethod did not permit discrimination between these two isomers, which agrees with data reported by Natarajan and van Kesteren-van Leeuwen (1981) in the in vitro chromosome aberration test on CHO cells.
In conclusion, we have demonstrated that in addition to the main advantages of the in vitro MN test (objectivity in the identification of MN and detection of structural and numerical chromosomal aberrations), the in vitro MN micromethod on L5178Y cells is rapid and easy to perform (six chemicals can be simultaneously tested in duplicate on one microplate per treatment) and requires a very small quantity of compound (<10 mg). It can thus readily be implemented in the early stages of the development of new chemical entities. This micromethod is also inexpensive and does not require the use of cytochalasin B. Moreover, the in vitro MN micromethod in L5178Y cells appears to be very sensitive: all the potent genotoxins tested without metabolic activation were found clearly positive, especially in the protocol with harvest immediately after the 24 h continuous treatment for the aneugens and with a 20 h recovery time before harvest for the clastogenic compounds. For all the compounds studied, its sensitivity was at least equal to other in vitro MN tests, especially those using human lymphocytes. With metabolic activation, results are also conclusive, particularly for SAF, which we found positive; only ß-NA gave an equivocal response and revealed the present limit of the test in the presence of S9 mix. To settle this current problem, hamster liver S9 (which should be more appropriate to detect the genotoxic potential of an aromatic amine) could be used in a second study in the presence of metabolic activation, in the case of the presence of a such structure alert. Another potential drawback of the proposed assay is the testing of volatile compounds or compounds with volatile metabolites. In such a case, the problem could be dealt with by sealing the microplates, each of them containing only the volatile compound or its volatile metabolites. Furthermore, the in vitro MN micromethod on L5178Y cells seems to have good specificity: indeed, treatment with known non-genotoxic chemicals did not increase the incidence of induced MN, neither with nor without metabolic activation. For these reasons, the in vitro MN micromethod on L5178Y cells could be proposed as a very early stage in the development of a new molecule as a preliminary short-term screening assay.
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Acknowledgments |
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Dr Frank Le Curieux is acknowledged for a critical review of the manuscript.
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3 To whom correspondence should be adressed at: Laboratoire de Toxicologie Génétique, Institut Pasteur de Lille, 1 rue du Professeur Calmette, 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 November 30, 1998; accepted on March 23, 1999.
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