Mutagenesis, Vol. 17, No. 2, 163-170,
March 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
REVIEW |
A comparison of the in vitro Comet assay with the in vitro chromosome aberration assay using whole human blood or Chinese hamster lung cells: validation study using a range of novel pharmaceuticals
Genetic Toxicology Laboratory and 1 Biostatistic and Scientific Data Management, GlaxoSmithKline SpA, Via Alessandro Fleming 4, 37135 Verona, Italy
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The in vitro Comet assay, a sensitive, quick and relatively cheap test, could become a valid alternative to the commonly used in vitro chromosomal aberration test, in the preliminary evaluation of new chemical entities early in the development of new pharmaceuticals. A validation of the Comet assay procedure using whole human blood or CHL cells was carried out in comparison with a cytogenetic test utilizing the same target cells with the following compounds which demonstrated positive results in standard chromosomal aberration tests: two well-documented clastogens, methyl methanesulphonate and cyclophosphamide, and eight novel drugs in early development. A 3 h exposure time, in both the absence and presence of metabolic activation, was used for the in vitro Comet assay. Agreement between the results of the Comet assay and the chromosomal aberration tests was found to be satisfactory on a qualitative basis, although positive results in the Comet assay were always at higher doses than in the cytogenetic test. This indicates a reduction in sensitivity using the former genotoxicity end-point. In order to try to explain this observation, a range of exposure times (0.25, 0.5, 1, 2 and 3 h) were investigated in two further experiments to determine the optimal time for detecting Comet induction in this assay procedure. Maximum levels of DNA damage (in terms of Comet induction) were recorded at earlier sampling times (0.25–1 h) in whole human blood using the same positive doses observed in HPLT. Further studies need to be performed to confirm these findings. It is possible that strand breaks are too short lived to allow detection after a 3 h treatment period (due to preferential repair), indicating the need for shorter exposure times in some cases to optimize their detection.
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Introduction |
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The Fourth International Conference on Harmonization (ICH) Guidelines describes a standard battery of genotoxicity tests for pharmaceuticals. They can be defined as in vitro and in vivo tests designed to detect compounds that induce genetic damage. With regard to the in vitro tests for cytogenetic evaluation, the Guidelines consider human peripheral lymphocytes (HPLT), Chinese hamster ovary (CHO) and Chinese hamster lung (CHL) cells as equally acceptable. These tests are relatively expensive and quite time consuming. In contrast, the single cell gel electrophoresis (Comet) assay is rapid to perform, sensitive and inexpensive. Consequently, a validation programme for the Comet assay in comparison with chromosomal aberration tests in HPLT and CHL cells was designed (Slamenovà et al., 1997
). The aim of the study was to verify whether the Comet assay could become a valid alternative to other common cytogenetic tests in the preliminary evaluation of new chemicals early in the development of new pharmaceuticals. During a pre-clinical safety evaluation programme the cytogenetic test (HPLT) was carried out on seven different compounds: two ß-lactam ester antibiotics, one ß-lactam antibiotic salt, three quinoline derivatives and one indole derivative. One cytogenetic test in CHL cells was also performed using an eighth compound. All tests were performed in both the absence and presence of a rat liver exogenous metabolic activation system (S9 mix). Mitomycin C and cyclophosphamide were used as positive controls in the absence and presence of S9, respectively. The Comet assay was performed on these novel compounds in both the absence and presence of metabolic activation (S9 mix) using either HPLT (for the first seven compounds) or CHL cells (for the eighth compound). Methyl methanesulphonate and cyclophosphamide, well-known genotoxic compounds, were used to assess the sensitivity and reproducibility of the methods (Miyamae, 1997).
The experiments were performed following the recommendations of the International Comet Assay Workshop (Anderson and Plewa, 1998) concerning cell viability, gel concentration, cell density, lysing solution (pH and ingredients of lysing solution), alkaline unwinding conditions (pH, voltage, current and duration), neutralization, stain imaging conditions, scoring and cell selection criteria (Tice et al., 2000).
In most experiments, a 3 h exposure time in both the absence and presence of metabolic activation was used. However, two experiments using ß-lactam esters 1 and 2 investigated different exposure times (0.25, 0.5, 1, 2 and 3 h) in order to determine the effect on Comet induction.
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Materials and methods |
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Cells
Human peripheral blood was obtained by venepuncture from healthy male human volunteers and was supplied in heparinized containers by the Clinical Pharmacology Unit, GlaxoSmithKline SpA (Verona, Italy). Male volunteers were used throughout. All donors were required to confirm that they were non-smokers, were not suffering from any infection or undergoing medication and had not been exposed to X-rays within the previous 6 months.
The CHL fibroblasts were cultured in Eagle's MEM supplemented with 10% fetal bovine serum and L-glutamine (200 mM). Cells (~4000/ml, counted with a Coulter counter Z1) were seeded in flasks with 5 ml of culture medium. Different concentrations of each compound were added to 1-day-old monolayer cultures incubated in the dark at 37°C, 5% CO2.
Test chemicals
The 10 test chemicals evaluated in our studies are listed in Table I. The concentrations of the novel drugs are expressed as active moiety (i.e. in terms of free acid). All chemicals were initially tested at a range of doses comparable to those used for the chromosomal aberration test using human lymphocytes. Subsequently we had to test higher doses to identify the range of doses in which a dose-related Comet relationship could be found.
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Methyl methanesulphonate, a direct-acting genotoxin, and cyclophosphamide, a drug requiring metabolic activation, were tested to assess the basic performance of the in vitro Comet assay. Two independent experiments were performed with each of these compounds to confirm the reproducibility of the results. The other eight test drugs were supplied by Pharmaceutical Research & Development, GlaxoSmithKline SpA.
Each drug was dissolved in a suitable solvent (Table I
).
Rat liver S9 mix
All standard in vitro assays were performed in the absence and presence of a rat liver exogenous metabolic activation system (S9 mix). The post-mitochondrial fraction (S9) was derived from livers of male Wistar rats (supplied by Charles River Italia SpA, Calco, Italy), aged 6–9 weeks (prepared by the Genetic Toxicology Laboratory, Glaxo SmithKline SpA). The animals were pre-treated with a combination of phenobarbitone sodium (administered i.p. at 80 mg/kg) and ß-naphthoflavone (administered i.p. at 8 mg/kg) for 3 consecutive days prior to killing (Elliott et al., 1992). The S9 fraction was prepared essentially as described by Maron and Ames (1983) and stored for up to 6 months at –80°C. The S9 fraction was used at 2% (final concentration in medium) in conjunction with a NADPH generating system, the composition of which has been described previously (Gatehouse and Delow, 1979).
In vitro cytogenetic analysis using HPLT
Seven new chemical entities were evaluated for in vitro clastogenicity using HPLT.
The procedure was carried out according to the recommendations of the UKEMS Guidelines Committee (Scott et al., 1983, 1990). Duplicate whole blood cultures (phytohaemagglutinin-stimulated human peripheral blood from healthy male donors) were prepared in Iscove's medium for each test concentration. Treatment took place 48 h after culture initiation and lasted for 24 h in the absence of S9 mix and 3 h in the presence of S9 mix. The spindle poison colcemid (final concentration 0.2 µg/ml in medium) was added 2 h prior to harvest, which took place 72 h after culture initiation. After metaphase preparation, the chromosomes were stained with Giemsa and a maximum of 400 metaphase cells for solvent and untreated control cultures and of 200 metaphase cells for each treatment group were analysed for chromosome damage. All slides were coded and scored blind in random order.
Aberrations were classified according to Savage (1976) into chromosome and chromatid type damage, with a further division into deletions and exchanges. Displaced and undisplaced fragments separated by a non-staining region equal to or greater than the width of the chromatid were scored as deletions. Non-staining regions of less than the chromatid width were scored as gaps. Prior to chromosomal analysis the mitotic indices (MI) for each culture were estimated based upon 1000 lymphocytes.
In vitro cytogenetic analysis using CHL cells
ß-Lactam ester 3 was tested for in vitro clastogenicity in CHL fibroblasts (Ishidate, 1987).
The protocol using this cell line was essentially the same as that used for HPLT. One-hundred well-spread metaphases were scored for the highest concentration and the solvent control. Only 50 metaphases were scored for the cultures treated with the positive control. All slides were coded and scored blind in random order.
Aberrations were classified according to Savage (1976), as described above.
In vitro Comet assay in human whole blood
In the absence of metabolic activation, cultures were set up by adding 0.8 ml of heparinized blood to 4.2 ml of Iscove's medium containing L-glutamine (200 mM). In the presence of metabolic activation, cultures were set up by adding 0.8 ml of heparinized blood to 3.2 ml of Iscove's medium and 1 ml of S9 mix. The S9 mix contained 100 µl/ml S9 fraction and the final concentration of S9 fraction in the medium was 2%.
Solvent controls were treated with either 1% (v/v) DMSO, 1% ethanol or 10% (v/v) water for irrigation.
Compound and solvent control cultures were incubated at 37°C, 5% CO2 for 3 h in both the presence and absence of S9 mix, after which the cultures were washed with 10 ml of Ca2+- and Mg2+-free phosphate-buffered saline (PBS) to remove S9 mix and the compounds. After centrifugation the cells were resuspended in 5 ml of PBS. Then, 40 µl of cell suspension was added to 260 µl of low melting point agarose (LMA) at a final concentration of 0.5% at 45°C and 75 µl of this suspension was pipetted onto frosted glass microscope slides pre-coated with a layer of 0.5% normal melting point agarose. Finally, 75 µl of LMA was quickly added. The gels were allowed to solidify for 5 min in the refrigerator. The cells were lysed at 4°C in the dark (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH adjusted to 10 using NaOH, with 1% Triton X-100 and 10% DMSO added fresh) for at least 1 h. Subsequently, the DNA was allowed to unwind for 40 min in the alkaline electrophoresis buffer (200 mM EDTA, 10 M NaOH, pH 
13) and subjected to electrophoresis in the same buffer for 30 min at 25 V (0.7 V/cm) and 300 mA. Optimum lysis and electrophoretic conditions were determined previously (data not shown). Afterwards the slides were removed, neutralized with 3x5 min washes with Tris (0.4 M, pH adjusted to 7.5 using HCl) and stained with 45 µl of 20 µg/ml propidium iodide (Andrew et al., 1990).
Supplementary tests using ß-lactam esters 1 and 2
To determine the repair kinetics of DNA strand breaks in human lymphocytes, cells were incubated at 37°C in appropriate culture medium and samples taken at 15 and 30 min and 1, 2 and 3 h after treatment for analysis by Comet assay.
In vitro Comet assay in CHL cells
Different concentrations of the compounds were added to 1-day-old monolayer cultures incubated in the dark at 37°C, 5% CO2 for 3 h in both the absence and presence of metabolic activation. Subsequently, the cultures were washed with 10 ml of PBS to remove S9 mix and the compound and a cell suspension prepared using a trypsin (0.05% w/v) and EDTA (0.02% w/v) solution incubated at 37°C for ~3 min. A previous report using a microgel assay indicated that, in contrast to trypsin, which may increase DNA fragmentation, a trypsin + EDTA solution minimizes DNA damage (Monteith and Vanstone, 1995). After centrifugation the cells were resuspended in 1 ml of MEM.
The protocol using this cell line was essentially the same as that used for the HPLT.
Comet scoring
For each dose at least 100 cells, 50 cells from each of two replicate slides, were examined with a fluorescence microscope connected to a Comet Assay II image analysis system (Perceptive Instruments, UK). DNA damage was quantified as tail moment (defined as the DNA product in the tail and the mean migration distance in the tail), tail length (the distance from the peak head position to the end of the tail) and tail intensity (per cent of DNA migrating into the tail).
Cell viability
At first viability was determined on the basis of the percentage apoptosis (Fairbairn et al., 1995) in the cells examined for the following compounds: ß-lactam esters 1 and 2, the indole derivative and quinoline derivative 1. DNA pieces migrate more freely into the tail of the comet with increasing numbers of breaks and at the extreme (apoptotic cells) the head and the tail are well separated. In line with Fairbairn et al. (1995), we considered that comets having a well-separated head and tail indicate apoptotic cells. Subsequently, for the remaining compounds and for the positive controls cyclophosphamide and methyl methanesulphonate, cell viability was determined using fluorescein diacetate and ethidium bromide (Pfuhler and Wolf, 1996). Viable cells fluoresced green, whereas dead cells were indicated by orange stained nuclei.
To avoid false positive responses with substances of unknown hazard, the viability of the cells used in the in vitro Comet assay should exceed 75% (Anderson and Plewa, 1998; Henderson et al., 1998).
Statistical analysis
For the cytogenetic tests each of the treatment groups (including the positive control groups) was compared separately to the solvent control group using a one-sided individual comparison test. Each 2x2 contingency table was analysed using Fisher's exact test (Armitage, 1971). This analysis was carried out twice: firstly, using the numbers of all aberrant cells, including those containing chromatid and chromosome gaps; secondly, on the same data but excluding cells containing only gaps.
For the Comet assay the significance of the effect of each treatment dose versus the solvent control was evaluated by Dunnett's test. This test was performed on tail moment only and treatment groups were considered significantly different from the solvent control group at P < 0.05.
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Results |
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The Comet assay was performed on eight compounds using a 3 h exposure time in both the absence and presence of metabolic activation (S9 mix) using either whole human blood (for the first seven compounds) or CHL cells (for the eighth compound) and on methyl methanesulphonate and cyclophosphamide (Figures 1 and 2
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In vitro cytogenetic results in HPLT and CHL cells in comparison with the Comet assay are shown in Tables IV and V
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Secondarily, the antibiotic ß-lactam esters 1 and 2 were re-tested using additional exposure times in order to assess which allowed the identification of maximum Comet induction.
ß-Lactam antibiotic ester 1
Results of the HPLT assay (Table II)
In the absence of S9 mix this compound induced statistically significant increases in aberration frequency compared with the solvent control at doses of 191 and 383 µg/ml. These doses reduced the MI by 30 and 54%, respectively. In the presence of S9 mix the compound had no clastogenic activity.
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Results of the Comet assay (Table III
)
In the absence of S9 mix a significant increase in tail moment at the maximum dose of 766 µg/ml was observed. In the presence of S9 mix no increase in tail moment was observed at any dose.
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No evidence of overt toxicity was observed on the basis of percentage apoptosis.
ß-Lactam antibiotic ester 2
Results of the HPLT assay (Table II)
In the absence of S9 mix this compound induced statistically significant increases in aberration frequency compared with the solvent control at doses of 15.6 and 62.5 µg/ml. These doses reduced the MI by ~22 and 50%, respectively. This effect disappears in the presence of metabolic activation.
Results of the Comet assay (Table III)
In the absence of S9 mix a significant dose-related increase in tail moment at doses of 200 and 400 µg/ml was observed. In the presence of S9 mix no effects were seen at the concentrations used.
No evidence of overt toxicity was observed on the basis of percentage apoptosis.
ß-Lactam antibiotic ester 3
Results of the CHL assay (Table II)
In the absence of S9 mix this compound was clastogenic at a dose of 62.5 µg/ml. This dose reduced the MI by ~70%. In the presence of S9 mix the compound induced statistically significant increases in aberration frequency at a dose of 250 µg/ml. This dose did not reduce the MI.
No toxicity was observed.
Results of the Comet assay (Table III)
In the absence of metabolic activation dose-related increases in tail moment were observed at doses of 500 and 1000 µg/ml. In the presence of S9 mix a significant increase in tail moment at the maximum dose of 1000 µg/ml was also noted.
The viability of the cells was >90%.
ß-Lactam antibiotic salt
Results of the HPLT assay (Table II)
This compound was not clastogenic in either the absence or presence of an in vitro metabolic activation system (rat liver S9 mix). The maximum test concentration analysed for the 3 h treatment period was limited to 3400 µg/ml in both the absence and presence of S9 mix. This dose, equivalent to 10 mM, is recommended by current guidelines (Scott et al., 1990; OECD, 1997). The maximum concentration analysed for the continuous treatment period was restricted to 1700 µg/ml, due to cytotoxic effects of the compound. This dose reduced the MI by 51%.
Results of the Comet assay (Table III)
No dose-related increase in tail moment in either the absence or presence of S9 mix was noted up to the maximum dose of 3400 µg/ml.
The viability of the cells was >90%.
The indole derivative
Results of the HPLT assay (Table II)
In the absence of S9 mix this compound at a dose of 850 µg/ml induced statistically significant increases in the aberration frequency compared with the solvent control. This dose reduced the MI by 57%. In the presence of S9 mix this compound induced statistically significant increases in aberration frequency at doses of 425 and 850 µg/ml. These doses reduced the MI by 42 and 62%, respectively.
Results of the Comet assay (Table III)
In the absence of S9 mix a significant increase in tail moment was observed at the maximum dose of 3400 µg/ml. In the presence of S9 mix a dose-related increase in tail moment was noted at doses of 1700 and 3400 µg/ml.
No evidence of overt toxicity was observed on the basis of percentage apoptosis.
Quinoline derivative 1
Results of the HPLT assay (Table II)
In the absence of S9 mix this compound induced statistically significant increases in aberration frequency compared with the solvent control at a dose of 122 µg/ml. This dose reduced the MI by 50%. In the presence of S9 mix an increase in aberration frequency, of borderline statistical significance, was noted at a dose of 244 µg/ml. This dose reduced the MI by 49%.
Results of the Comet assay (Table III)
In the absence of metabolic activation a significant increase in tail moment was observed at the maximum dose of 1000 µg/ml. No evidence of overt toxicity was observed on the basis of apoptosis. In the presence of S9 mix no dose-related increase in tail moment was noted up to 500 µg/ml. At 1000 µg/ml a strong decrease in the cell population, probably due to a haemolytic effect, was observed.
Quinoline derivative 2
Results of the HPLT assay (Table II)
This compound induced statistically significant increases in aberration frequency at a dose of 104 µg/ml in the absence of S9 mix and at a dose of 420 µg/ml in the presence of S9 mix. These doses reduced the MI by ~41 and 47%, respectively.
Results of the Comet assay (Table III)
A statistically significant increase in tail moment was observed at the maximum dose of 1680 µg/ml, both in the absence and presence of metabolic activation.
The viability of the cells was >90%.
Quinoline derivative 3
Results of the HPLT assay (Table II)
This compound has a clastogenic effect in vitro at a dose of 156.2 µg/ml in the absence of S9 mix and at a dose of 1250 µg/ml in the presence of S9 mix. These doses reduced the MI by 46 and 49%, respectively.
Results of the Comet assay (Table III)
In the absence of metabolic activation a significant dose-related increase in tail moment was observed at doses of 2500, 3500 and 4500 µg/ml. In the presence of S9 mix an increase in tail moment was observed at the maximum dose of 4500 µg/ml.
The viability of the cells was 90% in the presence and 89% in the absence of metabolic activation.
Time–course study Comet assay with ß-lactam antibiotic esters nos 2 and 1
These experiments were carried out using whole human blood, in triplicate for ß-lactam ester 2 and in duplicate for ß-lactam ester 1, in the absence of metabolic activation.
The individual tail moment values for different sampling times (0.25, 0.5, 1, 2 and 3 h) and each dose for both compounds are shown in Tables VI and VII.
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ß-Lactam ester 2 The following concentrations were used: 15.6, 31.2, 62.5, 125 and 250 µg/ml. At all doses significant increases in tail moment values were observed immediately after treatment. At doses of 15.6–125 µg/ml primary DNA damage began to decrease 30 min after treatment, although significant increases in tail moment were observed at all sampling times at a dose of 250 µg/ml. In the three experiments at the maximum dose of 250 µg/ml, with 3 h pulse treatment, cell viability was always >80%.
ß-Lactam ester 1 The following concentrations were used: 48, 96, 192, 383 and 766 µg/ml. Up to a dose of 383 µg/ml DNA damage started at 15 min and decreased with time, returning to almost the control level at 3 h. Significant increases in tail moment were observed at all sampling times at a dose of 766 µg/ml. For all sampling times cell viability was >85% in both experiments at the maximum dose of 766 µg/ml.
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Discussion |
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Very high consistency was found between the results obtained in the cytogenetic and Comet assays carried out in HPLT and CHL cells in both the absence and presence of S9 mix. However, positive results in the Comet assay were always observed at higher doses than in the cytogenetic test. This might be due to the methodological or biological differences underlying the two assays. In the Comet assay leukocytes are tested for 3 h and analysed in G0 phase, whereas for the cytogenetic test chemicals are usually added to mitogen-stimulated lymphocytes for 24 h. Given the diversity of the test protocols, in order to further investigate this observation a time–course experiment was performed with the antibiotic ß-lactam esters 1 and 2. It is possible that DNA strand breaks are too short lived to be detected by the Comet assay under the experimental conditions (a 3 h exposure time) used. The aim of this study was to determine the DNA damage and repair kinetics in whole human blood using the in vitro Comet assay at 0.25, 0.5, 1, 2 and 3 h after treatment.
In our experiments the quantitative response was greater at the earlier sampling times (0.25–1 h) and tailed off up to 3 h after treatment. With the two ß-lactam esters the damage was more intense at the earlier sampling time and at dosages lower than those observed using the standard 3 h exposure time. Furthermore, the doses that showed positive results at the earlier time points were very similar to those obtained with the cytogenetic tests performed on the same compounds. Our data agree with that previously published on the analysis of repair kinetics in human lymphocytes exposed to UV and 
-radiation using the Comet assay (Frankenberg, 1989). These studies showed that the bulk of DNA repair occurred within the first 15 min and was essentially completed at 120 min after exposure (Singh et al., 1988). Other studies showed an initial rapid phase of repair, with >50% of the damage being repaired at 15 min, and a slower phase, with 14–24% of the damage remaining unrepaired after 60 min (Alapetite et al., 1999). Finally, the maximum DNA migration was observed after 30 min with lymphocytes, whereafter the tail length started to decline (Lankinem et al., 1996; Fortini et al., 1996).
It should also be noted that the two ß-lactam esters are lipophilic prodrugs with a theoretical partition coefficient (log10
) between 1-octanol and water of 1.7 and of 2.4, respectively. They could therefore quickly enter the cell, resulting in possible DNA damage after a few minutes incubation, which could then be repaired over the following 3 h (Comet negative). Nevertheless, misrepairing of the initial lesions, as strand breaks, may lead to the chromosome aberrations observed in the in vitro cytogenetic test (Pfeiffer et al., 2000).
Few studies have been performed to determine the optimal sampling time for the detection of primary DNA damage with the Comet assay. However, we feel that our data show a requirement for sampling times <3 h, especially for lipophilic prodrugs with a high octanol/water partition coefficient.
In connection with the reference mutagens, cyclophosphamide requires metabolic activation by cytochrome P450s to exert its genotoxic effects. An increase in DNA strand lesions was observed, but compared with methyl methanesulphonate, cyclophosphamide was less effective in the induction of strand lesions. However, cyclophosphamide also induces DNA crosslinks (Hengstler, 1997) and it is well known that crosslinking agents either inhibit or cause a reduction in induced DNA migration (Pfuhler and Wolf, 1996).
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2 To whom correspondence should be addressed. Tel: +39 04592 18824; Fax: +39 04592 18174; Email: elg62175{at}gsk.co.uk
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Received on April 23, 2001; accepted on November 19, 2001.
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