Mutagenesis, Vol. 15, No. 2, 177-184,
March 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Cytogenetic genotoxicity of anti-herpes purine nucleoside analogues in CHO cells expressing the thymidine kinase gene of herpes simplex virus type 1: comparison of ganciclovir, penciclovir and aciclovir
Institute for Antiviral Chemotherapy, Medical Faculty, Friedrich Schiller University of Jena, Nordhäuser Strasse 78, D-99089 Erfurt and 1 Division of Applied Toxicology, Institute of Toxicology, Medical Faculty, University of Mainz, Mainz, Germany
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Abstract |
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The three anti-herpes nucleoside analogues ganciclovir, penciclovir and aciclovir were investigated as to their recombinogenic [sister chromatid exchange (SCE) inducing] and clastogenic activity in CHO cells expressing the thymidine kinase gene of HSV-1, which is a precondition of therapeutic activity of these drugs. The compounds were applied for the duration of one cell cycle and cytogenetic end-points were measured between 0 and 42 h after exposure. Although the nucleoside analogues are quite similar with respect to chemical structure, they differ basically in their genotoxic potency, aberration types induced as well as the time course of chromosomal damage. Aciclovir induced SCEs and chromosomal aberrations immediately after exposure but only in a concentration range much higher than that reached in blood plasma during anti-herpes therapy. The direct genotoxic activity is explained by the obligate chain terminating property of aciclovir upon incorporation into genomic DNA. On the other hand, genotoxic damage caused by ganciclovir and penciclovir is of the delayed type requiring at least one post-exposure cell cycle for its expression. Unlike aciclovir, ganciclovir is an extremely potent inducer of SCEs and chromosome breaks and translocations at concentrations far below those impairing the proliferative activity and triggering apoptosis of the target cells (as shown by our previous investigation). Penciclovir is essentially devoid of genotoxic activity. It induces SCEs only at cytotoxic/apoptotic concentrations, is only weakly clastogenic and induces premature chromosome condensation which appears to result from uncoupling of karyokinesis and cytokinesis. The genotoxic activity of ganciclovir is explained as due to repair processes triggered in the second post-exposure replication cycle at the sites of nucleoside analogue incorporation into genomic DNA. The findings have considerable implications with respect to the use of ganciclovir or other antiviral drugs in suicide gene therapy of malignant diseases.
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Introduction |
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Anti-herpetic drugs, most of them purine nucleoside analogues, have been of considerable interest for several reasons: (i) herpesvirus infection or reactivation, which usually shows a mild course in immunocompetent patients, may be life-threatening in immunocompromised patients (patients with AIDS, with malignant diseases or under immunosuppressive chemotherapy, such as transplant recipients), and the number of these patients is increasing; (ii) one of the anti-herpes drugs, ganciclovir (GCV), is presently in clinical trials in combination with transduction of the herpes simplex virus thymidine kinase gene (HSVtk), as a `suicide gene', for the treatment of malignant tumours (suicide gene strategy) (Moolten, 1986
; Culver et al., 1992; Oldfield et al., 1993). In this setting, HSVtk expression in the transduced tumour cells leads to activation of the prodrug GCV and triggers the apoptotic pathway not only in metabolically competent cells but also in adjacent HSVtk– cells via gap junctional transfer of the ultimately toxic metabolite, GCV triphosphate (`bystander effect') (Freeman et al., 1993; Hamel et al., 1996; Mesnil et al., 1996; Dilber et al., 1997). The principle of antiviral activity of these agents, aciclovir (ACV), GCV and penciclovir (PCV), relies on the fact that herpesviruses (herpes simplex virus, varicella zoster virus and cytomegalovirus) encode their own nucleoside kinases which have a much lower substrate specificity than their cellular counterparts. Therefore, they are able to monophosphorylate certain nucleoside analogue drugs whereas cellular nucleoside kinases cannot do so or only to a very limited extent. The resulting analogue monophosphates are metabolized, by cellular kinases, to the respective triphosphates, which show distinctly lower molar inhibitory constants (Ki values) for herpesvirus-encoded DNA polymerases than for cellular DNA polymerases. This second step of antiviral selectivity causes obligate chain termination in the case of ACV and, thus, cessation of virus production. On the other hand, in contrast to ACV, which has only one hydroxyl group in its acyclic `sugar' moiety, GCV and PCV possess two hydroxyl groups and can be internally incorporated into the growing DNA chain.Their mode of antiviral action is less well understood so far (for reviews see Darby, 1994; De Clercq, 1994, 1995).
Although the hitherto licensed drugs ACV, GCV and PCV (in the form of its oral prodrug, famciclovir) have passed the routine genotoxicity screening necessary for approval, only little data on this topic have been published (for a review see Thust et al., 1996).
We have checked several purine nucleoside analogues for induction of sister chromatid exchange (SCE) and structural chromosome aberrations in genetically unmodified Chinese hamster V79 cells and observed a high genotoxic potency with respect to GCV, whereas ACV and PCV were essentially inactive (Thust et al., 1996). During the course of this study we realized that a relevant assessment of possible genetic risks posed by these drugs would require a target system that is metabolically competent, i.e. reflects the metabolic capacity of herpesvirus-infected cells. By means of stable transfection of the HSVtk gene into CHO cells we have generated such a system. This appears to be very suitable to address questions of genotoxicity and induction of apoptosis as well as their mutual relationship. Here we report our findings on the induction of genotoxic damage by three antiviral drugs in this target system.
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Materials and methods |
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Chemicals
GCV [9-(1.3-dihydroxy-2-propoxymethyl)-guanine, CymeveneTM] and ACV [9-(2-hydroxyethoxy)-guanine] were purchased from Syntex Arzneimittel GmbH (Aachen, Germany) and the Wellcome Foundation (London, UK), respectively. PCV (9-[4-hydroxy-3-hydroxybut-1-yl]-guanine) was a gift from SmithKline Beecham (Hamburg, Germany). GCV and ACV were directly dissolved in complete culture medium. PCV was dissolved in a small volume of 0.3 N NaOH and then diluted in complete culture fluid. If necessary, the medium was neutralized with 0.2 N HCl.
Cells
The CHO cells were grown in Ham's F12/Dulbecco's minimal essential medium (1:1) containing 10% inactivated neonatal bovine serum. The generation and properties of transfectants expressing the tk gene of HSV-1 have been described in detail (Thust et al., 2000). From a panel of HSVtk-expressing cell lines, clone TM8 CHO 3-C2 (in the following designated CHO HSVtk+) was used for the studies presented here. Cells were routinely cultured in medium containing 1.5 mg/ml G418, which was omitted during the experiments.
SCE and clastogenicity assays
Asynchronously growing cells were seeded at a density of 3x105 cells/25 cm2 flask. One day later, the experiments were started. Drug exposure lasted for 14 h, followed by careful rinsing and cultivation for recovery periods of different lengths in agent-free medium. Cultures for SCE detection received 10 µg/ml 5-bromodeoxyuridine (BrdU). Cells were harvested and fixed at different times after BrdU labelling and drug exposure. The following treatment schedules were applied: 0 h recovery (14 h pre-labelling with BrdU + 14 h drug exposure in the presence of BrdU); 14 h recovery (14 h drug + BrdU exposure followed by 14 h drug-free BrdU post-labelling); 28 h recovery (14 h drug exposure + 28 h culture in BrdU-containing drug-free medium); 42 h recovery (14 h drug exposure + 14 h culture in normal medium + 28 h BrdU post-labelling). Chromosome preparations were made in the usual manner after trypsinization of the cultures. For metaphase arrest, colcemid (0.05 µg/ml) was added during the last 3 h prior to preparation. SCEs were visualized by a modified FPG technique (Wolff and Afzal, 1996) or by reverse SCE staining (Takayama and Sakanishi, 1977). Thirty stemline metaphases per sample were enumerated for SCE induction. The significance of the SCE rates was checked using Student's t-test. Clastogenicity was evaluated in 100 metaphases/sample of preparations stained by the conventional Giemsa technique. Aberration types scored are given in Tables I–III. CHO cells show a series of fragile sites on several chromosomes, especially on the long arm of the X chromosome (Xq), which may impose gaps or breaks, even in untreated cultures (Slijepcevic and Natarajan, 1995; Hilliard et al., 1998). Therefore, alterations in these chromosomal regions were not considered in the evaluation. The mitotic index (MI) was calculated from the metaphase/interphase ratio in 10 arbitrarily chosen microscopical fields at low magnification (100x) of the chromosome preparations. All exposure conditions were checked at least twice in independent experimental series and gave highly consistent data.
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Results |
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General observations
When CHO HSVtk+ cells were exposed to the drugs for one cell cycle (14 h), concentrations of
1 µM GCV, ~4 µM PCV and >100 µM ACV triggered apoptosis (Thust et al., 2000). This gave a coarse orientation for the cytogenetic studies. In order to obtain measurable effects, much lower concentrations had to be used in the case of GCV and similar or distinctly higher ones for PCV and ACV. Under these conditions, treatment of CHO HSVtk+ cells with the three anti-herpes purine nucleoside analogues caused changes in cell morphology which became visible ~24 h after pulse treatment for one cell cycle. There was a distinct increase in size of cells and nuclei and, at later post-exposure times, the nuclei became fragmented (Figure 1A–C). This phenomenon was most dramatic after treatment with PCV and ACV but was much less conspicuous after GCV exposure, even at apoptotic concentrations. Immediately after exposure to PCV and ACV the mitotic index decreased strongly and recovered thereafter. The generally lower mitotic indices observed after longer post-exposure periods (Tables I–III
) might be caused by cell cycle arrest (Tomicic et al., in preparation).
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Another consequence of drug exposure common to the three antivirals was the strong inhibition of condensation of chromosome Xq (Figure 1D
). This was observed immediately after treatment and affected, depending on drug concentration, up to two thirds of all metaphases. This effect closely resembles the inhibition of condensation of genetically inactive chromatin caused by 5-azacytidine analogues, which has been suggested to be reliant on selective inhibition of DNA methylation (Haaf, 1995). Such a property is so far unknown for the nucleoside analogues studied here.
SCE induction
Neither GCV nor PCV caused a significant increase in SCE rates immediately after drug exposure for 14 h, but after longer post-exposure periods a steady rise in SCE frequency was observed with both drugs (Figure 2A and B). With a GCV concentration of 0.1 µM, which is well below that which diminishes the plating efficiency of the target cells, and recovery periods of 28 and 42 h, GCV-induced SCE rates reached the limit of countability (mean values 
120 SCEs/cell). Higher concentrations led to a strong cell cycle delay preventing sister chromatid differential staining (Figure 2A). A no-effect threshold was not found. Thus, even a GCV concentration as low as ~1 nM still induced a statistically significant SCE response (data not shown). It appears that GCV is an extremely potent recombinogenic agent in metabolically competent target cells. This contrasts with the activity of PCV in the SCE assay.
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PCV, when checked within a maximum post-exposure period of 42 h, induced a distinct increase in SCE frequency only in the dose range which is cytotoxic/apoptotic (1.1–3.3 µM) and maximum values were only four times the control rate (~32 versus 7–8 SCEs/cell; Figure 2B
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ACV was tested in the SCE assay up to a concentration of 1 mM, which is at the upper limit of water solubility. Contrasting with GCV and PCV, ACV induced an increase in SCE frequency immediately after exposure and reached a peak of ~72 SCEs/cell at 28 h post-exposure. Fourteen hours later, the SCE values declined to the range of background rates in CHO cells surviving treatment with high concentrations of ACV (Figure 2C).
Clastogenicity
GCV is a very strong clastogen and induces a spectrum of aberrations which is typical for clastogens with delayed effects, i.e. chromatid and isochromatid breaks and chromatid exchange. Within the dose range tested, clastogenicity started 14 h after exposure and increased during prolonged post-exposure, thus reaching a maximum of 75% of cells with aberrations after 42 h at 0.4 µM GCV. This is a concentration that hardly diminished the plating efficiency of the target cells (Thust et al., 2000). Moreover, these clastogenic effects were not accompanied by a dramatic decrease in MI. Furthermore, GCV did not influence the frequency of polyploid metaphases (Table I).
On the other hand, PCV did not significantly induce `typical' chromosomal aberrations. Premature chromosome condensations (PCCs) were the predominant type of chromosomal damage provoked by PCV (Table II). These PCCs differ crucially from those which are observed at late recovery periods (at or after the second mitosis after treatment) with potent clastogens and are micronucleus derived due to non-disjunctions (Beek et al., 1980). Whereas micronucleus-derived PCCs occur in metaphases whose undamaged chromosomes regularly comprise the complete chromosomal complement of the host cells, the PCCs induced by PCV were accompanied by an incomplete number of normally condensed chromosomes (Figure 3A and B). In cytogenetic preparations from PCV-treated cultures that were stained for sister chromatid contrast, occasionally metaphases were observed whose chromosomes were highly different with respect to their replication stage but doubtlessly belonged to the same mitosis (Figure 3B). Furthermore, a distinct increase in polyploid metaphases was observed at later periods after PCV exposure (Table II).
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ACV induced dose-dependent clastogenicity directly after cell exposure, with a maximum at 28 h post-exposure. Aberration types were primarily chromatid breaks and translocations. At later periods, extremely aberrant metaphases (with >10 aberrations) predominated. The increase in polyploid metaphases was even more distinct after ACV exposure than after PCV treatment (Table III
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Discussion |
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The three nucleoside analogues investigated in the present study contain an unchanged guanine base and their intracellularly formed triphosphates are thought to compete with dGTP for incorporation into DNA (Coen, 1992
). Because the acyclic `sugar' moieties of the drugs are different, they may cause fundamentally different biological effects. The aliphatic side chain of ACV has only one hydroxyl group, similar to the 5-hydroxyl group of deoxyribose in natural nucleosides. It therefore leads to obligate chain termination upon incorporation into the DNA. Whether this happens in cellular DNA is still unknown. In theory, one molecule of ACV monophosphate might be added per replicon. In fact, significant incorporation of radiolabelled ACV in the genomic DNA of HSVtk-expressing human glioma cells has not been observed (Rubsam et al., 1998). We suggest that the genotoxic effects of ACV described here are due to chain termination, whereby attempts to bypass the sites of incorporation causing DNA replication blockage by recombination may lead to the formation of SCEs or cause DNA strand breaks (due to nuclease attack at stalled replication forks), leading to structural chromosomal aberrations. Chain termination in consequence of incorporation of ACV monophosphate would also explain why the majority of genotoxic effects occur immediately after drug exposure. Block of DNA replication has recently been proposed to be the major cause of induction of DNA breakage and aberrations for chemical mutagens (Kaina, 1998); a model which can be extended to these antiviral drugs. The relatively low clastogenic activity of ACV as compared with those of the other analogues is presumably due to the very short intracellular half-life of its triphosphate; 0.7 h (Vere Hodge and Perkins, 1989) versus 12–18 h for GCV triphosphate (Biron et al., 1985; Smee et al., 1985) or 10 h for PCV triphosphate (Vere Hodge and Perkins, 1989).
In contrast to ACV, there is extensive incorporation of GCV into the genomic DNA of HSVtk-expressing cells (St Clair et al., 1987; Rubsam et al., 1998; Thust et al., 2000). This incorporation is the main difference between GCV and ACV. It is important to note that during the incorporation cycle SCEs and aberrations were not induced by GCV. Here we propose that the clastogenicity induced by ACV is due to immediate chain termination, whereas that induced by GCV is due to replication inhibition in the post-incorporation cycle. Moreover, it should be noted that the high intracellular stability of GCV triphosphate, as compared with ACV triphosphate, may also play a role in its genotoxic potency, explaining the relatively high dose of ACV needed to elicit clastogenic effects comparable with GCV. As intracellularly formed GCV triphosphate, like other nucleotides, cannot freely cross the cell membrane, it forms an intracellular pool which is available for ongoing incorporation into genomic DNA. In consequence this may lead to potentiation of the biological activity of this drug. A similar situation could be assumed for PCV but not for ACV, whose triphosphate is rapidly decomposed. The delayed genotoxic and apoptotic activity of GCV, which is an essential precondition for the `multilog cytotoxicity' of the drug (Rubsam et al., 1998), suggests that processing and/or replication of the GCV-substituted template are decisive for its therapeutic efficacy.
Neither the experiments of Rubsam et al. (1998) on [3H]thymidine incorporation nor our own (M.Tomicic, unpublished findings) on incorporation of BrdU showed a distinct inhibition of DNA replication during or immediately after GCV exposure. According to Rubsam et al. (1998), at apoptotic GCV concentrations HSVtk-expressing cells traverse at least one complete cell cycle during and after drug exposure and then accumulate in early S phase, where they remain until death by apoptosis. Under similar conditions, by flow cytometry we detected a blockage in S phase that affected ~40% of the CHO HSVtk+ cells, while ~25% were blocked in G2/M phase (M.Tomicic, unpublished findings). The block at G2/M is probably the reason for the increase in size of cells and nuclei mentioned above. Nevertheless, after GCV exposure a considerable number of the HSVtk transfectants are able to pass mitoses, but with severe and presumably lethal chromosome damage, as shown here.
Recently, the action of GCV on several cell cycle checkpoints has been studied in HSVtk-expressing murine melanoma cells (Halloran and Fenton, 1998). It was found that inhibition of Cdc2/cyclin B activity led to an irreversible arrest at the G2/M checkpoint responsible for the demise of the cells, but not by apoptosis. The latter conclusion remains obscure because assays specific for apoptosis have not been conducted in this investigation, while all other studies published so far have confirmed that the mode of killing by GCV is programmed cell death.
It is known that GCV can form regular Watson–Crick base pairs with dC, but physicochemical investigations in GCV-containing synthetic oligodeoxynucleotides have shown that, although the B conformation of the DNA double helix remains undisturbed, thermal stability is reduced and, more importantly, significant distortions of the sugar–phosphate backbone occur at or near the site of GCV incorporation, leading to kinks in the helix (Marshalko et al., 1995; Foti et al., 1997). Presumably, alterations in the sugar–phosphate chain due to GCV incorporation impair the function of the replication complex and trigger repair processes that result in the formation of SCEs and chromosome aberrations.
With respect to their chemical structure, GCV and PCV are very alike. Both drugs contain two hydroxyl groups in the aliphatic side chain that are similar to the 5- and 3-hydroxyl groups of deoxyribose of natural nucleosides. But the ether oxygen in the side chain of GCV is replaced by a methylene group in PCV. Obviously, this subtle difference has profound consequences on biological activity. It has been surmised that both drugs exert their antiviral activities via mechanistically independent pathways, but details of the modes of action are so far essentially unknown and cannot be ascribed to the different monophosphorylating capacities of the respective herpesvirus-encoded nucleoside kinases (Vere Hodge and Cheng, 1993). While GCV is a highly potent drug for treatment of cytomegalovirus diseases, PCV is rather inefficient in this respect but highly effective in the therapy of varicella zoster virus.
PCV triphosphate shows a 100-fold weaker interaction with herpesvirus DNA polymerases than ACV triphosphate (Earnshaw et al., 1992) and previous investigations did not demonstrate incorporation of PCV into viral or cellular DNA (Vere Hodge and Cheng, 1993; Darby, 1995). In our experiments in HSVtk-expressing CHO cells we observed an ~500-fold lower incorporation of PCV into the genomic DNA as compared with GCV. On the other hand, both drugs were almost equipotent with respect to induction of apoptosis in these cells but differed qualitatively and quantitatively as to chromosomal damage (Thust et al., 2000; this paper). As shown here, PCV caused a weak increase in SCE rates only in the cytotoxic/apoptotic dose range; it induced hardly any chromosome breaks or translocations. Premature chromosome condensation as the predominant chromatin alteration provoked by PCV suggests a mechanism that is crucially different from the genotoxic effects of GCV. PCCs are known to arise when cells containing asynchronous nuclei or nuclear fragments, due to either cell fusion or severe chromatin damage, enter mitosis (Sperling and Rao, 1974; Beek et al., 1980; Mackey et al., 1988; Ianzini and Mackey, 1997). In these cases the chromatin compartments most advanced with respect to cell cycle traverse form normally condensed chromosomes, whereas the lagging chromatin condenses prematurely, thus forming either single-stranded (G1 PCC) or double-stranded chromosomes (G2 PCC) or showing a pulverized appearance (S PCC). Whereas the degree of asynchrony between normally condensed chromosomes and PCCs in the same mitoses is rather small, i.e. less than one cell cycle, the chromosomes with and without sister chromatid differential contrast within the same metaphase obviously differ in their replication stage by more than one complete cell cycle (Figure 3B). To our knowledge, such a phenomenon has not been reported so far and we have never seen it before in our decades-long experience with the SCE assay and a large series of different chemicals. Presumably, both the PCCs and metaphases with asynchronous chromosomes are caused by the same mechanism. Based on the facts that penciclovir is only very weakly clastogenic and the observed PCCs are obviously constituents of the same chromosomal complements of damaged cells and not micronucleus derived, we conclude that PCV-induced PCCs arise from uncoupling of karyokinesis and cytokinesis. It is well known that damage to cell cycle checkpoints is important in causing apoptosis (for a review see Schimke et al., 1994). The mechanism by which PCV or its triphosphate disturb the coordination of cell cycle events, which appears to be the most prominent effect of this nucleoside analogue in metabolically competent cells, is still unknown and deserves further investigations.
The results presented here, together with our previous investigations in a HSVtk-expressing cell system (Thust et al., 2000), have shown that: (i) GCV is extensively incorporated into the genomic DNA of cells, whereas much lower incorporation was found for PCV; (ii) GCV is an extremely potent recombinogenic (SCE inducing) and clastogenic agent in the subtoxic dose range that corresponds to GCV levels in blood plasma in the clinical setting of anti-cytomegalovirus therapy while PCV is essentially inactive as to genotoxic effects under these circumstances; (iii) both GCV and PCV kill the HSVtk-expressing target cells at similar concentrations (1–4 µM). The main route of cell killing was apoptosis. The observation that PCV, at subtoxic concentrations, is devoid of genotoxic activity but triggers apoptosis at approximately equimolar concentrations to GCV leads us to recommend PCV as a safer alternative drug than GCV for the suicide gene therapy of malignant diseases. However, at toxic/apoptotic concentrations PCV causes chromatin damage, namely premature chromosome condensation, which differs crucially from the `typical' aberrations induced by GCV, i.e. chromatid and isochromatid breaks and translocations. In contrast to both these drugs, ACV is clastogenic directly after treatment of CHO HSVtk+ cells but at concentrations that are irrelevant to the clinical setting in the treatment of HSV infections or varicella zoster virus. We suggest that the similar and/or different genotoxic and apoptotic effects provoked by these, with respect to chemical structure, closely related antiviral drugs may yield important clues as to their modes of action, as well as to the relationship between chromosomal damage and programmed cell death in general.
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Acknowledgments |
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This research was supported by the Deutsche Forschungsgemeinschaft (grants TH 670/1-1 and KA 724/7-1 to R.T. and B.K.).
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2 To whom correspondence should be addressed. Tel: +49 361 7411707; Fax: +49 361 7411114; Email: thust{at}zmkh.ef.uni-jena.de
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References |
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Received on September 20, 1999; accepted on November 29, 1999.
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