Mutagenesis, Vol. 16, No. 2, 127-132,
March 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Effects of styrene-7,8-oxide over p53, p21, bcl-2 and bax expression in human lymphocyte cultures
1 Departamento de Biología Celular y Molecular, Facultad de Ciencias and 2 Instituto de Ciencias de la Salud, Universidade da Coruña, Campus da Zapateira s/n, 15071, A Coruña, Spain
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Abstract |
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Styrene is one of the most important organic chemicals in use today. The highest human exposures to styrene take place by inhalation during the production of fibreglass-reinforced plastics. Styrene is oxidized by hepatic cytochrome P450 to styrene-7,8-oxide (SO), an epoxide that has been shown to induce chromosome aberrations, sister chromatid exchanges and micronuclei in many cell systems. In this work, the effect of SO on the expression of some genes involved in the cell cycle and apoptosis regulation in human white blood cells was studied. Lymphocyte cultures from four donors were exposed to 50 and 200 µM SO, 1% DMSO being the control. Aliquots of the cultures were taken at six different time points (30, 36, 42, 48, 60 and 72 h), total mRNA was extracted in each one of them and RT–PCR was carried out to analyze the expression of the genes p53, p21, bcl-2 and bax. Moreover, a cytokinesis block assay was performed to estimate cell proliferation kinetics by calculating the cytokinesis block proliferation index (CBPI), and to evaluate the number of cells undergoing apoptosis. Furthermore, apoptotic events were detected by the DNA fragmentation assay. In our results, a high interindividual variation in the expression of the studied genes was observed. Expression curves obtained for the four genes, together with the data from the CBPI and apoptotic cells scored, suggest that exposure to high levels of SO may induce a delay in the cell cycle, probably directed to allowing repair systems to act on the genotoxic damage produced, more than driving cells towards programmed cell death.
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Introduction |
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Styrene is one of the most important organic chemicals in use today, and styrene polymers are among the four major polymers of commercial interest, along with polyethylene, polypropylene and polyvinyl chloride. Styrene is used at a level of ~40% as a reactive diluent in unsaturated polyester resins, most of which are reinforced with fibreglass (Pffäffli and Säämänen, 1993). The highest occupational exposures to styrene occur during the production of glass-reinforced polyester products, especially with large items such as boats that involve manual lay-up or spraying operations, where ingestion occurs mainly via inhalation of styrene vapour (Miller et al., 1994
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Styrene is oxidized by cytochrome P450-dependent monooxygenases to the primary genotoxic metabolite styrene-7,8-oxide (SO), an epoxide with the capacity for DNA binding (Norppa and Sorsa, 1993) and is the main in vivo mutagenic metabolite of styrene (Bond, 1989). SO has been shown to induce chromosome aberrations, sister chromatid exchanges and micronuclei in human lymphocytes, Chinese hamster cells and Allium cepa (Scott and Preston, 1994). SO can be converted to styrene-glycol by hydration via epoxide hydrolase and excreted in urine mainly as mandelic and phenylglyoxylic acids, all the intermediate metabolites are non-genotoxic (Sumner and Fenell, 1994).
It is now evident that DNA or chromosome damage is only one of several critical events that happen following exposure to xenobiotic agents (Green and Reed, 1998). When genotoxic stress is induced to a cell, changes in the expression of several critical genes are produced. The most relevant of those genes is p53, also called the `guardian of the genome'. The p53 tumour-suppressor gene encodes for a 393 amino acid nuclear protein that functions as a transcription factor important in the detection and repair of DNA damage (Ellis et al., 1997). Following DNA damage the p53 protein rapidly accumulates and becomes activated. Activation of p53 has two outcomes: cell cycle arrest in G1, allowing repair to take place, or apoptosis, if DNA damage is too extensive to be repaired (for review see Ko and Prives, 1996; May and May, 1999).
The cyclin-dependent kinase inhibitor p21 protein is a critical effector of the p53-mediated G1 arrest in response to DNA damage. It associates with a cyclin/cdk/PCNA complex and inhibits kinase activity, thus blocking cell cycle progression into S-phase (Gartel and Tyner, 1999).
The bcl-2 family proteins are involved in the control of apoptosis and can either function as inhibitors (e.g. bcl-2, bcl-xL, bag) or promoters (e.g. bax, bcl-xS, bad, bak) of cell death (Burger et al., 1998). Some of these proteins physically interact with each other and form homo- and heterodimers (e.g. bcl-2/bcl-2, bcl-2/bax and bax/bax). It was proposed that bax homodimers promote apoptosis and that the bax-mediated cell death is counteracted by bcl-2/bax heterodimerization (Andreeff et al., 1999). p53 can positively regulate bax gene expression and is involved in negative regulation of bcl-2 gene expression (Lotem and Sachs, 1999). Therefore, p53 status may determine the vulnerability of cells to apoptotic stimuli by modulation of the bcl-2/bax complex.
In this work, human lymphocyte cultures were exposed to SO in order to study its effect on mRNA expression of some genes involved in cell cycle and apoptosis regulation, such as p53, p21, bcl-2 and bax, by reverse-transcription–polymerase chain reaction (RT–PCR). Moreover, a cytokinesis block assay was performed to obtain an index of cell proliferation kinetics and to evaluate the number of cells undergoing apoptosis (Kirsch-Volders et al., 1997), in addition to detecting apoptosis by the use of a DNA fragmentation assay.
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Materials and methods |
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Lymphocyte cultures
Whole blood (0.5 ml) obtained by venipunction from two male and two female healthy, non-smoking donors (D1–D4) aged between 23 and 30 years were added to 5 ml of culture medium (Chromosome kit P, Euroclone) and maintained at 37°C. After 24 h, 50 µl of SO solutions in DMSO were added to the cultures to obtain final concentrations of 50 and 200 µM SO, 1% DMSO being the solvent control, and cultures with no treatment being the negative controls. SO concentrations assayed were chosen to ensure that at least the higher one produced genotoxic effects on the basis of the results obtained by Norppa et al. (1981) and Norppa et al. (1983) that describe significant increments in the frequency of chromosomal aberrations and sister-chromatid exchanges, with respect to the controls, in lymphocytes treated with 200 and 150 µM SO, respectively. Aliquots of 750 µl were extracted from each culture at six different times: 30, 36, 42, 48, 60 and 72 h.
mRNA extraction
Total mRNA was extracted from each aliquot (QuickPrep® Micro mRNA Purification Kit, Amersham Pharmacia Biotech) following the manufacturer's recommendations. The final product was aliquoted and stored at –80°C until the analysis.
RT–PCR
For first-strand cDNA synthesis 8 µl mRNA were added to 5 µl Bulk First-Strand cDNA Reaction Mix, 1 µl 200 mM dithiothreitol and 1 µl 0.2 µg/µl pd(N)6 primer (First-Strand cDNA Synthesis Kit, Amersham Pharmacia Biotech). The reaction was carried out at 37°C for 1 h.
For each gene examined, standard curves of cycle number were generated to determine the optimum cycle number within the linear range for PCR amplification. For all genes examined this was determined to be 32 cycles, except for ß2-microglobulin that was 30 cycles. ß2-microglobulin RT–PCR was performed in order to equalize the results of gene expression with ß2-microglobulin mRNA content observed in the same samples. Six microlitres of cDNA were used for RT–PCR with specific primers for ß2-microglobulin, p53, p21 and bcl-2, and 3 µl cDNA for amplification of bax. Reactions were performed in a 15 µl reaction mix containing 5 mM MgCl2, 0.4 mM dNTPs, 1 µM primers, 4% DMSO (only for p53 and bax) and 0.5 U of Taq DNA polymerase. The rounds of amplification preceded by an initial 5 min, 95°C denaturation were undertaken in a Techne Progene thermocycler, according to the following reaction conditions: 45 s at 95°C, 35 s at 56°C and 1 min at 72°C for ß2-microglobulin; 45 s at 95°C, 35 s at 65°C and 1 min at 72°C for p53 and bcl-2; 45 s at 95°C, 30 s at 63°C and 50 s at 72°C for p21; and 45 s at 95°C, 45 s at 71°C and 1 min 15 s at 72°C for bax.
The RT–PCR products were loaded onto 2% agarose gels, electrophoresed at 60 V, stained with 0.5 mg/l ethidium bromide and then photographed under UV illumination (Figure 1). EcoRI- and HindIII-digested 
DNA (0.25 µg/µl, Boehringer Mannheim) was used for quantification and as the molecular weight standard. Quantification of the bands was carried out by densitometry using the Gelworks program (gelwld 2.51) and expressed as arbitrary units (AU).
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Primers
The sequences of the paired primers used in this study were: p53 5'-CTG GCC CCT GTC ATC TTC TG-3' and 5'-CCG TCA TGT GCT GTG ACT GC-3'; p21 5'-TGA GCG ATG GAA CTT CGA CT-3' and 5'-GAC AGT GAC AGG TCC ACA TGG-3'; bcl-2 5'-AAG CCG GCG ACG ACT TCT-3' and 5'-GGT GCC GGT TCA GGT ACT CA-3'; bax 5'-ATG GAC GGG TCC GGG GAG-3' and 5'-ATC CAG CCC AAC AGC CGC-3'; ß2-microglobulin 5'-CCA GCA GAG AAT GGA AAG TC-3'and 5'-GAT GCT GCT TAC ATG TCT CC-3'.
Cytokinesis block assay
In order to obtain an index of cell proliferation kinetics and to assess morphologically cells undergoing apoptosis, lymphocyte cultures from the same donors were established as described previously. Treatments were performed 24 h after culture initiation and were prolonged until the end of the culture. After 44 h, 10 µl Cyt-B previously dissolved in DMSO at 3 mg/ml was added at a final concentration of 6 µg/ml. Cultures were harvested at 72 h by centrifugation at 800 r.p.m. for 10 min. As described by Surrallés et al. (1992), to eliminate red cells and preserve lymphocyte cytoplasmic membranes a mild hypotonic solution (0.075 M KCl at 4°C) was added to the cultures, followed by immediate centrifugation. Next, a methanol:acetic acid (3:1) solution was gently added. This fixation step was repeated twice and the resulting cells were resuspended in a small volume of fixative solution and carefully placed onto clean slides. Finally, the slides were stained with DAPI 5 µg/ml. All cultures were performed in duplicate.
Prior to scoring all slides were coded. Scoring was carried out by a single individual (B.L.) using a Leica DM-RXA microscope equipped with a 100 W mercury lamp at 400x magnification with a DAPI filter. Five hundred lymphocytes (250 from each duplicate culture) were scored to evaluate the number of cells with one, two, three or four nuclei, or undergoing apoptosis. The cytokinesis block proliferation index (CBPI), an index for measuring cell proliferation kinetics that indicates the average number of cell cycles undergone by a given cell, was calculated following the formula: CBPI = [MI + 2MII + 3(MIII + MIV)]/total, where MI–MIV represent the number of cells with one to four nuclei (Surrallés et al., 1995).
The guidelines for scoring apoptotic cells were followed according to Fenech et al. (1999). Briefly, cells showing chromatin condensation with intact cytoplasmic and nuclear boundaries as well as cells exhibiting nuclear fragmentation into smaller nuclear bodies within an intact cytoplasm/ cytoplasmic membrane were classified as apoptotic. Figure 2 shows the morphology of a normal binucleated cell and an apoptotic binucleated cell exhibiting the typical nuclear bodies.
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DNA fragmentation assay
For DNA fragmentation analysis, after extracting the six aliquots of 750 µl at the different time points, the remaining lymphocytes from each culture were harvested and incubated at 60°C for 1 h in lysis buffer (50 mM Tris–HCl, 100 mM EDTA, 150 mM NaCl, 1.25% SDS and 0.3 mg/ml proteinase K). DNA was extracted with 500 µl phenol:chloroform:isoamylalcohol (25:24:1) and then with 500 µl chloroform:isoamylalcohol (24:1). DNA was precipitated in ethanol and resuspended in TE. Samples were electrophoresed on a 1.5% agarose gel at 50 V, stained with 0.5 mg/l ethidium bromide and photographed under UV illumination.
Genotyping
Genomic DNA of the donors was isolated from whole blood as described by Sambrook et al. (1989). Glutathione S-transferase (GST) genetic polymorphism was determined for GSTM1 and GSTT1 genes as reported elsewhere (Hirvonen et al., 1996). Genotypes were clasified as null (both alleles deleted) or positive (at least one undeleted allele).
Statistical analysis
Statistical evaluation was conducted using the non-parametric Kruskal–Wallis test for multi-group analysis and the Mann–Whitney test for comparison between two groups. P < 0.01 or P < 0.05 was considered significant. Analyses were performed using the SPSS statistical package, version 9.0.
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Results |
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Amplification of p53, p21, bcl-2, bax and ß2-microglobulin mRNA by RT–PCR gave fragments of 242, 210, 258, 455 and 267 bp, respectively. Untreated cultures provided expression curves similar to solvent-treated cultures (data not shown); thus, expression curves from 1% DMSO-exposed cells were considered as controls in order to compare with expression curves from SO-exposed cells. Expression curves of each one of the genes studied in all the donors are represented in Figure 3
. Control curves for p53 stayed constant in D2 and D4, and increased slightly in D1 and D3 at 48 h. Levels of p21 remained quite constant for all donors, except an initial increase and subsequent decrease for D2. For bcl-2 and bax only D4 exhibited variable expression with time.
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In cultures exposed to 50 µM SO, expression of all D1 genes was similar to controls. In D2 and D3 cultures, expression increased at the final phase of the culture (starting from 54 h) for every gene, as well as for bcl-2 and bax from D4.
All genes from D1 cultures treated with 200 µM SO expressed levels higher than controls and 50 µM SO cultures. A sudden increase in p53 was observed at 42 h in D2 and D4, and expression of all the other genes showed a progressive increment starting from that time. Only D3 cells exposed to 200 µM SO showed levels close to the control, more so than those from the 50 µM SO culture.
The CBPI of the cultures of every donor exposed to 1% DMSO and 50 µM SO was near 2, and did not differ significantly from each other (Table I). On the contrary, all cultures treated with 200 µM SO showed a CBPI significantly lower (P < 0.01) than the other two treatments, indicating a delay in the kinetics of the cell cycle.
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Table II
shows the number of apoptotic cells detected in all cultures from D1 to D4. Apoptotic cells found in each one of D1 and D3 SO-treated cultures were not significantly elevated with respect to control cultures; however, for D2, both SO treatments (50 and 200 µM) induced a significant increment (P < 0.01 and P < 0.05, respectively) and only D4 cultures exposed to 200 µM SO significantly enhanced (P < 0.01) the number of apoptotic cells over control cultures.
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Figure 4
represents the detection of apoptosis by DNA fragmentation assay in 72 h cultures. As shown, we were unable to detect the DNA ladder pattern of approximately 200 bp integer multiples, indicative of apoptotic events, in any of the 72 h cultures analyzed.
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Genotype analysis showed D1 as GSTM1 positive and GSTT1 null, D2 as positive for both genes, and D3 and D4 as GSTM1 null and GSTT1 positive.
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Discussion |
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Figure 3
shows the levels of mRNA corresponding to p53, p21, bcl-2 and bax expression in all cultures from donors D1 to D4. Gene expression of controls (1% DMSO) was more stable with time in D2 than in the other donor cultures. In D1 and D3 an increase in p53 expression was observed at 48 h, coinciding with the second cell cycle after culture initiation. This observation is in accordance with the statement that activation of p53 during a normal cell cycle is part of its function as a checkpoint protein: in the absence of DNA damage, MDM-2 would inactivate p53, allowing cell division to develop normally (Lassus et al., 1996).
Cells from D1 treated with 50 µM SO show levels of expression for all genes studied very close to controls so exposure to this SO concentration does not seem to induce any change in these cells. However, cultures from D2 and D3 given the same treatment exhibit an increase in their p53 expression starting from 48 h. It could be indicative of a slow accumulation of genotoxic damage that leads to a rise in p53 levels (Ellis et al., 1997), and subsequently to a rise in p21 levels, since p53 acts by transactivating p21. However, this increase in p21 expression takes place only at the end of the culture time, so cell cycle arrest promoted by p21 cannot be manifested and the CBPI of the cultures does not differ significantly from control cultures.
Furthermore, in the cited cultures there is a progressive increase in bcl-2 and bax expression starting from 48 h. It is known that p53 upregulates bax expression and downregulates bcl-2 expression (Burger et al., 1998; Sheikh and Fornace, 2000), but Miturski et al. (1998) suggest that mechanisms other than p53 may play a role in the regulation of bcl-2 expression, so such mechanisms could be responsible for the rise in bcl-2 levels accompanying the rise in p53 expression. Several factors, including cell type, presence or absence of survival factors in the external environment, extent of DNA damage and levels of p53 are involved in the choice between cell cycle arrest and apoptosis (Wiman, 1997; May and May, 1999). Moreover, the probability of a cell surviving exposure to a DNA-damaging agent is dependent on the propensity of that cell to undergo apoptosis (Fenech et al., 1999). On the other hand, there are many reports that state that it is the ratio of bcl-2 to bax that determines sensitivity to apoptosis, rather than the absolute levels of either protein. In the case of D3 and D4 cultures exposed to 50 µM SO, the increase in expression of both genes goes in parallel, so we can suppose that there will not be apoptosis induction caused by SO exposure, as can be seen in morphological assessment of cells undergoing apoptosis in such cultures.
However, in D2 50 µM SO curves, the slope at final times seems to be slightly higher in bax expression than in bcl-2 expression. This may drive to an imbalance in bcl-2/bax rate, a prevalence of bax/bax homodimers over bcl-2/bax heterodimers and a promotion of apoptosis, which would explain the significant increase detected in the number of apoptotic cells scored in that culture.
An increase in p53 expression can be observed in D1 cells exposed to 200 µM SO that is maintained over control levels until culture end. This could be motivated by the DNA damage produced by SO, and as a consequence p21 transactivation is induced, its expression increases above the control and a delay in the cell cycle takes place, as reflected in the significant decrease in CBPI. Increases in bcl-2 and bax expression were also observed, but they remain parallel, so it is supposed that there will not be apoptotic events; namely, DNA damage induced by SO is not so extensive as not to be repaired.
The same increase in p53 expression takes place in D4 cells treated with 200 µM SO, but p21 transactivation occurs later. However, CBPI shows a significant delay in the cell cycle, but to a lesser extent than in D1. In the same culture, bax levels are above control levels, whereas bcl-2 levels are closer to controls. This imbalance in bcl-2/bax rate could explain the significant increment detected in apoptotic cells among these lymphocytes.
A p53 level increase was also observed in D2 cells exposed to 200 µM SO, but it occurs before the second cell cycle and then falls gently towards control levels. Nevertheless, this increase is enough to transactivate p21, which undergoes a fast and sudden rise that produces a delay in the cell cycle for repair to take place, as reflected in the significant decrease in CBPI. But, why is this increase in p53 expression not maintained until the end of the culture? It has been reported that, in response to various cytotoxic and genotoxic stresses, post-translational modifications increase p53 stability and lead to nuclear accumulation (Jiménez et al., 1999). Also, certain stress signals, such as treatment with several DNA damaging agents, have been shown to lead to a transient decrease of MDM-2 expression, which would allow p53 stabilization (Lohrum and Vousden, 1999). Thus, perhaps some kind of post-translational modification over p53 protein takes place that increases the p53 half-life, and the cells have no need to keep p53 transcription elevated with time. An elevation in bcl-2 and bax expression in the D2 culture treated with 200 µM SO may also be observed, but the bax curve has a steeper slope than the bcl-2 curve, potentially explaining the significant increment in the number of apoptotic cells scored in this culture.
Post-translational modification of p53 protein might have also occurred in D3 cells exposed to 200 µM SO, since a significant reduction in CBPI can be observed, probably caused by the final increase produced in p21 expression. However, it must be taken into account that p21 expression following genotoxic stress can also be strongly modulated on the post-transcriptional level (Butz et al., 1998). As for bcl-2 and bax expression in this culture, both remain close to control levels and balanced, so an increase in apoptotic cells was not expected.
A DNA fragmentation assay was performed in order to detect apoptosis induction at the macromolecular level. This assay is based on the observation of a DNA ladder pattern of approximately 200 bp integer multiples, caused by activation of an endogenous endonuclease that cleaves DNA between the nucleosomes during the apoptotic process (Eastman, 1995). As can be seen in Figure 4, no DNA ladder was observed in any of the cultures assayed. This may seem to be in contrast to the results obtained for D2 and D4 cultures treated with 200 µM SO, where a significant increment in apoptotic cells has been found. However, it must be taken into account that the proportion of apoptotic cells was small (10/500 at most), and the DNA fragmentation produced in those cells may not be enough to be detected in a conventional agarose electrophoresis gel. In addition, reports of incidences in which cells have undergone the morphological features of apoptosis without the characteristic DNA fragmentation are increasing (McGahon et al., 1995); therefore, since apoptotic cells are morphologically highly distinctive and are easily distinguishable from viable cells, death assays should be coupled with direct morphological evaluation of the cell population. Moreover, the morphological assessment of apoptotic cells has been performed in 72 h cultures, so if apoptotic events have taken place in preceding culture times, they could not have been detected because at 72 h, apoptotic bodies coming from dead cells might have already been phagocytosed by their neighbours or by specialist phagocytes, since the apoptotic process is completed in a very short time (Wyllie et al., 1999).
With regard to the genetic polymorphism of the donors, no relationship could be observed between the data obtained for expression levels of genes studied and the GSTM1 or GSTT1 genotype. It has been described that individual variation in susceptibility to genotoxic chemicals depends not only on the activity of GSTs, but also on other metabolising enzymes and on the efficiency of DNA repair (Sasiadek et al., 1999). However, previous studies have reported that the lack of the GSTT1 gene (but not the lack of the GSTM1 gene) increases the genotoxic effects of SO in human whole-blood lymphocyte cultures, evaluated by means of sister-chromatid exchanges test (Uusküla et al., 1995; Ollikainen et al., 1998). However, the number of donors used for this work is too low to permit any relationship with GST polymorphisms to be determined.
In summary, the interpretation of the changes in the expression of the studied genes induced by exposure of cultured white blood cells to SO, and of the consequences over apoptosis induction and change in cell cycle kinetics, is complex, and the interindividual variation is considerable. Moreover, it must be taken into account that the fact of establishing the cultures with whole blood and not with an isolated cell type, T-lymphocytes as the most appropriate, may constitute the most important source of variation in this work. Future studies on gene expression must be conducted on a defined cell population rather than on whole blood. Nevertheless, in addition to the CBPI data and the morphological assessment of apoptotic cells in each of the cultures, it can be suggested that exposure to high levels of SO induces a delay in the cell cycle, probably directed to allow repair systems to act over the genotoxic damage caused, more than to drive cells towards programmed cell death.
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Acknowledgments |
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The excellent technical assistance of Luisa López Armesto is gratefully acknowledged. This work has been supported by a FPU fellowship from the Spanish Ministry of Education (to B.L.) and by a grant from the Xunta de Galicia (XUGA 10605B98).
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3 To whom correspondence should be addressed. Tel: +34 981 167000; Fax: +34 981 167065; Email: fina{at}udc.es
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Received on June 28, 2000; accepted on December 7, 2000.
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