Potential of short chain fatty acids to modulate the induction of DNA damage and changes in the intracellular calcium concentration by oxidative stress in isolated rat distal colon cells

S.L. Abrahamse, B.L. Pool-Zobel and G. Rechkemmer1

Institute of Nutritional Physiology, Federal Research Centre for Nutrition, Haid-und-Neu-Straße 9, D-76131 Karlsruhe, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Short chain fatty acids (SCFA) are considered to be beneficial fermentation products in the gut by exerting trophic effects in non-transformed colon cells and by slowing proliferation and enhancing differentiation in colonic tumour cells. We have studied the further effects of SCFA on cellular events of early carcinogenesis, genotoxicity and cytotoxicity in rat distal colon cells. Cytotoxicity was assessed by measuring trypan blue exclusion and by determining the H2O2-induced changes in intracellular calcium concentration ([Ca2+]i) using a fluorospectrophotometer and the calcium-sensitive fluorescent dye Fura-2. The microgel electrophoresis technique (COMET assay) was used to assess oxidative DNA damage. Individual SCFA and physiological SCFA mixtures were investigated for their potential to prevent DNA and cell damage induced by H2O2. For this, freshly isolated colon cells were treated with H2O2 (100–500 µM) and 6.25 mM SCFA. We have found 100–500 µM H2O2 to cause a fast initial increase in [Ca2+]i, whereafter the levels gradually further increased. Addition of SCFA did not affect [Ca2+]i nor did it reduce the H2O2-induced increase in [Ca2+]i. Butyrate and acetate were able to reduce the induction of DNA damage by 100, 200 and 500 µM H2O2, respectively. In contrast, i-butyrate and propionate were ineffective. The degree of reduction of DNA damage for the two protective SCFA was similar. Physiological mixtures containing acetate, propionate and butyrate in ratios of 41:21:38 or 75:15:10 that are expected to arise in the colon after fermentation of resistant starches and pectin, respectively, did not show significant antigenotoxic effects. The major difference between butyrate and acetate, on one hand, and i-butyrate and propionate, on the other hand, is that the former compounds are utilized best as energy sources by the colon cells. Therefore, our results on antigenotoxicity coupled with the findings on [Ca2+]i homeostasis indicate that molecular effects on the energy system render these non-transformed, freshly isolated colon cells to be less susceptible to H2O2.

Abbreviations: AM, acetoxymethyl ester; DMSO, dimethyl sulphoxide; GST, glutathione S-transferase; PBS, phosphate-buffered saline; SCFA, short chain fatty acids.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Large bowel cancer, especially in colon and rectum, is frequent in Western Europe, North America and Australia. The major risk factors involved in the development of these cancers are of genetic and dietary origin. However, causal associations obtained in epidemiological studies between large bowel cancer development and diet are controversial and still subject to debate (1). Similarly, complexities arise in the assessment of cancer-preventing food components (2,3).

Recently, dietary fibres have been put forward as cancer-protective food components (4). The theory is that dietary fibre may protect against colon cancer through secondary events resulting from the fermentation of carbohydrates by the microflora. These will lead to faecal bulking, increased speed of colonic transit, increase in nitrogen metabolism, increased bacterial load in the colon, acidification and, finally, to the production of short chain fatty acids (SCFA) (4,5). Butyrate, one of the major SCFA, is considered to be beneficial due to its trophic effects as an essential nutrient to the colon epithelium. Suggested mechanisms of cancer prevention at a cellular level have been reported to be the promotion of differentiation, induction of apoptosis and inhibition of proliferation in colon tumour cell lines (69). These mechanisms are classified as suppressing activities of cancer-preventing agents (10). Support for these concepts stems from various animal and cell culture experiments reviewed elsewhere (6,9,11). Recently, additional evidence has accumulated showing butyrate to induce glutathione S-transferases (GSTs) (12). GST is a detoxifying enzyme system that provides defence against carcinogens and oxidative stress compounds (13,14). Also, butyrate was found to modulate c-myc expression by post-translational degradation of the mRNA (15). c-myc belongs to a family of proto-oncogenes and their gene products are involved in cell growth (16). Both effects were observed in Caco-2 cells, a human colon carcinoma cell line, and are considered to have protective effects in terms of cancer prevention (10).

However, several inconsistencies prevail since experiments with non-transformed colon cells from animal and human biopsies have shown that butyrate induces proliferation rather than suppressing it (9,17). Thus, these results indicate that butyrate can be beneficial to non-transformed cells by trophic mechanisms, but put them at a higher risk of mutations by increasing the cell proliferation rate, since DNA replication can fix DNA damage as mutations. DNA damage may arise as a result of attack by exogenous agents or from endogenous stress factors such as products of lipid peroxidation (18). However, recently the lack of butyrate has been shown to increase apoptosis in non-transformed colon cells of the guinea-pig by eliminating suppression of bax gene expression (19).

Since primary cancer chemoprevention should pertain to protecting non-transformed cells from being initiated, and since the role of butyrate or other SCFA in this process is controversial and not known, we have performed a series of experiments in colon cells freshly isolated from the rat distal colon to elucidate other possible protective mechanisms. This paper will report on the continuation of those studies aimed at determining: (i) antigenotoxic effects of n-butyrate, i-butyrate, acetate and propionate (or physiologically relevant mixtures thereof) against H2O2-induced DNA damage; and (ii) modulation of H2O2-induced change in intracellular calcium concentration ([Ca2+]i) by SCFA, in freshly isolated non-transformed rat colonic epithelial cells.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
All chemicals were purchased from Merck (Darmstadt, Germany) unless otherwise stated. Bromo-A23187, Fura-2 acetoxymethyl ester (AM) and pentapotassium salt, the calcium calibration kit (with 1 mM MgCl2), ionomycin and pluronic acid were from Molecular Probes (Leiden, The Netherlands). RPMI 1640 medium and phosphate-buffered saline solution (PBS) without Ca2+ and Mg2+ were from Life Technologies (Eggenstein/Leopoldshafen, Germany). Sodium salts of butyrate and acetate were from Boehringer Ingelheim Bioproducts (Heidelberg, Germany). Iso-butyric acid and propionate were obtained from Sigma (Deisenhofen, Germany). All compounds were dissolved in the respective media. The pH of the solutions was measured routinely and adjusted to 7.2 prior to use.

Rat colon epithelial cells
Rat distal colon cells were isolated by collagenase digestion, as described previously (20). The whole isolation procedure took <1 h, and cells were divided into aliquots and distributed to the various laboratories to allow immediate simultaneous determination of cytotoxicity, genotoxicity and [Ca2+]i levels in the same batches of cells.

Cellular calcium determination
[Ca2+]i was determined using Fura-2 and a Deltascan Model 4000 fluorospectrophotometer (Photon Technology International, Wedel/Holstein, Germany). Cells were suspended at a concentration of 2x106 cells/ml in RPMI 1640 medium containing 5 µM Fura-2/AM, 0.2% (v/v) dimethyl sulphoxide (DMSO) and 0.025% (w/v) pluronic acid. After incubation for 30 min at room temperature, extracellular dye was removed by three subsequent centrifugation steps at 80 g for 3 min in RPMI 1640 medium. After the final centrifugation, 5x106 cells were resuspended in 2.5 ml RPMI 1640 medium supplemented with 1 mM CaCl2 and transferred to a quartz cuvette. The cuvette was inserted in a water-jacketed holder within the fluorospectrophotometer, set to maintain a constant temperature of 37°C inside the cuvette. Fura-2 fluorescence was measured at 340 and 380 nm excitation wavelengths (bandwidth 2.0 nm) with fluorescence emission measured at 490 nm wavelength (bandwidth 5.0 nm). The fluorescence intensities at each excitation wavelength were collected for 40 ms at a rate of 0.5 Hz, and the background fluorescence of unloaded cells was subtracted. Five 340:380 intensity ratio values that had been collected during 10 s were averaged. The corrected 340:380 intensity ratio was calibrated to [Ca2+]i using the equation published by Grynkiewicz et al. (21). Rmax, Rmin and Sf were determined in vitro using Fura-2 and a calibration kit. The dissociation constant (Kd) was determined in vivo using the following protocol. Fura-2-loaded cells were washed three times with PBS without Mg2+ and Ca2+ supplemented with 1 mM CaCl2 and 2 mM EGTA. The free Ca2+ concentration was determined using the calcium calibration kit and was 180 ± 20 nM. Subsequently, the cells were transferred to the fluorospectrophotometer and the 340:380 ratio was determined after addition of 10 µM ionomycin or bromo-A23187. The Kd of Fura-2 in the cells was obtained from the 340:380 ratio measured under these conditions, assuming that [Ca2+]i = [Ca2+]o, and from the Rmax, Rmin and Sf values of the in vitro calibration.

Detection of DNA damage
Isolated rat colonic epithelial cells were incubated at a density of 2x106 cells/ml RPMI 1640 medium for 15 min supplemented with 6.25 mM SCFA (individuals or mixtures) from 100x concentrated stock solution in 10 µl 0.9% (w/v) NaCl. The medium was removed by centrifugation for 6 min at 200 g and replaced with medium containing 0–1000 µM H2O2 [100x concentrated stock solutions in 10 µl 0.9% (w/v) NaCl]. After further 15 min incubation at 37°C in a shaking water-bath, the cells were checked for viability with trypan blue as previously described in detail (22). Microgel electrophoresis (COMET assay) was basically performed according to the original protocol of Singh et al. (23). For this, cells were suspended in 75 µl low melting point agarose [0.7% (w/v) in PBS at pH 7.4, 37°C] and distributed onto microscopy slides pre-coated with 0.5% (w/v) normal melting point agarose. After solidification of the agar, slides were submersed in a lysis solution [1% (w/v) N-lauroyl sarcosin (Na-salt), 10% (v/v) DMSO, 100 mM Na2EDTA, 1% (v/v) Triton X-100, 2.5 mM NaCl and 10 mM Tris] for 60 min to remove proteins. The slides were placed in an electrophoresis chamber containing alkaline buffer (1 mM Na2EDTA in 300 mM NaOH) for DNA unwinding. After 20 min the current was switched on and electrophoresis was carried out at 25 V, 300 mA for 20 min. The slides were removed from the alkaline buffer and washed three times for 5 min with neutralization buffer (0.4 M Tris–HCl, pH 7.5). Slides were stained with 20 µg/ml ethidium bromide (100 µl/slide). All steps, beginning with the isolation of the enterocytes, were conducted under red light. Duplicate replicas were run for each experimental point of one experiment and each experiment was performed at least 3–4 times on independent days with colon cells of different animals. Using an image analysis system (Perceptive Instruments, Halstead, UK), image lengths were evaluated from 101 cells per slide, two slides per experimental condition and divided into classes according to degree of DNA damage (class 0, <35 µm; class 1, 35–70 µm; class 2, 70–110 µm; class 3, >110 µm). The percentage of cells in classes 1–3 was multiplied by the respective class numbers and added to yield one value per experimental point (image length units). This evaluation is an analogy to the `arbitrary units' based on `tail intensity' according to Collins et al. (24).

Statistical evaluation
Results are reported as means ± standard error (SE) unless otherwise stated. Data were analysed using GraphPad software (San Diego, CA). On comparison of two experimental groups, either paired or unpaired Student's t-test was used.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effects of H2O2 on cell viability
The effects of H2O2 on cell viability, as measured using trypan blue, and cell yield are summarized in Table IGo. Exposure of cells to up to 500 µM H2O2 for up to 30 min did not result in a decrease in the total number of cells, nor did it result in a decrease in the percentage of viable cells. These results indicate that H2O2 is not cytotoxic at the conditions used during these experiments.


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Table I. Effects of different concentrations of H2O2 on cell yield and viability
 
Effects of H2O2 ± SCFA on intracellular calcium levels
Trypan blue staining is known to be a rather insensitive method to detect cell injury. Therefore, we studied changes in [Ca2+]i during exposure to H2O2. Figure 1AGo shows the effects of 200 µM H2O2 on [Ca2+]i in rat colonic epithelial cells. On addition of H2O2, there is a prompt rise in [Ca2+]i. Subsequently, [Ca2+]i further increases at a slower rate. Similar results, although not strictly concentration related, were seen for 500 and 1000 and, to a lesser extent, for 100 µM H2O2. The effects of 6.25 mM SCFA on [Ca2+]i and the H2O2-induced increase in [Ca2+]i are shown in Figure 1BGo–D. None of the three investigated SCFA influenced [Ca2+]i levels, nor did they modulate the increase in [Ca2+]i induced by 200 µM H2O2.



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Fig. 1. Time-dependent increase of [Ca2+]i in freshly isolated rat distal colon cells on addition of SCFA and H2O2. (A) Changes in [Ca2+]i during control experiments ({circ}) and after addition of 200 µM H2O2 ({bullet}). (B) Changes in [Ca2+]i after the addition of 6.25 mM butyrate ({circ}) or 6.25 mM butyrate and 200 µM H2O2 ({bullet}). (C) Changes in [Ca2+]i after the addition of 6.25 mM acetate ({circ}) or 6.25 mM acetate and 200 µM H2O2 ({bullet}). (D) Changes in [Ca2+]i after the addition of 6.25 mM propionate ({circ}) and 6.25 mM propionate and 200 µM H2O2 ({bullet}). Data are presented as the increase in [Ca2+]i ({Delta} [Ca2+]i) in comparison with the [Ca2+]i levels at 1 min before addition of SCFA ± H2O2, which was 144 ± 18 nM (n = 30). Values are means ± SE of 3–5 experiments.

 
DNA breaks by H2O2 and modulation by SCFA
Using the single cell microgel electrophoresis technique, we investigated the genotoxic effects of H2O2 by measuring H2O2-induced DNA damage. Table IIGo summarizes the data for cells that have been exposed to different concentrations of H2O2 for 15 and 30 min. A significant decrease in intact cells and a concomitant increase in cells with >70 µm image length are observed at 200 µM H2O2 and more. Moreover, median image length is significantly increased with increasing concentrations and increasing duration of incubation concentration.


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Table II. Dose- and time-dependence of the genotoxic effects of H2O2 in rat colonic epithelial cells
 
In order to study protection by SCFA against cytotoxic and genotoxic effects of H2O2, we developed the following protocol. Cells were pre-incubated for 15 min with 6.25 mM SCFA. Thereafter cells were exposed to 0, 100, 200 or 500 µM H2O2 for 15 min. Table IIIGo summarizes the effects of cells pre-treated with 6.25 mM of individual SCFA and the control group receiving NaCl. Subsequent exposure to 100–500 µM H2O2 results in a significant decrease in cell damage of cells pre-treated with acetate and butyrate. For the presentation of these results, we chose the parameter `image length units' since acetate as well as butyrate are distinctly effective in reducing the proportion of cells with high image lengths in addition to the absolute proportion of damaged cells. This reduction of the proportion of cells with high image lengths is clearly apparent, as shown in Figure 2Go. Pre-treatment with i-butyrate and propionate did not result in an increase of intact cells, nor did it reduce the overall image lengths of the cells.


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Table III. Effects of pre-incubation with short chain fatty acids on H2O2-induced DNA damage in rat colonic epithelial cells
 


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Fig. 2. Effects of pre-incubation with 6.25 mM acetate (A) and butyrate (B) on H2O2-induced DNA damage in rat colonic epithelial cells. Solid line, image length distribution of control cells without SCFA pre-incubation; dotted line, effect of pre-incubation with SCFA. Each curve shows values for all evaluated cells of three independent experiments (total of 606 cells).

 
In the colon, SCFA occur as mixtures of acetate, butyrate and propionate. The relative concentrations are dependent on the types of fibre that are consumed. In this study we tested two mixtures of SCFA, which potentially arise in the colon after fermentation of resistant starches and pectin, for their ability to reduce H2O2-induced DNA damage. Table IVGo shows the results obtained after pre-incubation of cells for 15 min with a mixture of 6.25 mM SCFA containing acetate, propionate and butyrate at a relative concentration of 75:15:10. This combination of SCFA did not reduce the DNA damage induced by H2O2. Moreover, pre-incubation of cells with a mixture of 6.25 mM SCFA with relative acetate, propionate and butyrate concentrations of 41:21:38 also did not result in significant decrease of H2O2-induced DNA damage (Table IVGo).


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Table IV. Effects of pre-incubation with mixtures of SCFA on H2O2-induced DNA damage in rat colonic epithelial cells
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To our knowledge this is the first convincing demonstration of genotoxic events by H2O2 in freshly isolated colon cells. The H2O2 concentrations used in the present study are similar to those used by Baskar and Balasubramanian (25). However, these authors did not observe any alterations in parameters of cell viability and enzyme activities in rat colonocytes when using H2O2. The concentrations are also similar to those used by Watson et al. (26) and Sandström and Marklund (27) who found the human derived colon cell line HT-29 to be sensitive to oxidant injury by H2O2 with respect to cellular proliferation and DNA single strand breaks, respectively. Moreover, the H2O2 concentrations used in this study are known to increase prostaglandin secretion in the rat colon (28). H2O2 is a model compound for oxygen-related active compounds, free radical intermediates of oxidative stress or lipid peroxidation products. These factors may be a consequence of imbalanced nutritional status and are thus implicated in the aetiology of dietary-based colon-cancers. It is well known that oxidative stress induces increased [Ca2+]i levels, DNA damage and cell death. Our report uniquely compares the effective concentrations for these three parameters in the same batches of colon cells.

Accordingly, cell death is induced beyond 500 to 1000 µM H2O2. [Ca2+]i is affected at concentrations between 100 and 200 µM. These results demonstrate that cell death, as assessed by trypan blue exclusion, is not a sensitive measure of cell injury. This was also observed by Baker et al. (29,30), who reported changes in hexose monophosphate shunt activity, oxidized glutathione levels and energy charge in Caco-2 cells during exposure to oxygen metabolites, which arose from other compounds at concentrations that did not cause any change in trypan blue exclusion or 51Cr release. In the present study, we investigated changes in [Ca2+]i as a more sensitive parameter of cell injury. Under normal conditions, [Ca2+]i is in the region of 100 nM, whereas the extracellular calcium concentration is in the range of 1 mM. Regulation of [Ca2+]i depends on energy metabolism, the integrity of the plasma membrane, ion translocation systems and transmembrane signalling. Impairment of any of these will unbalance Ca2+ homeostasis, and may lead to an increase of cell injury or cell death (31,32).

Although our results demonstrate that [Ca2+]i is indeed a more sensitive indicator of H2O2-induced cell injury in rat colonic epithelial cells, DNA damage was shown to be the most sensitive parameter of oxidative stress. DNA damage begins at concentrations of 100 µM and less (data not shown) depending on the exposure time. In fact the 15 min exposure time is probably an underestimation of the actual damage that has occurred due to the rapid onset of repair (24). Recent studies show H2O2 to be active already at concentrations of 75 µM and lower following 5 min exposure on ice in human colon cells (data not shown).

Both the genotoxic and the cytotoxic effects of H2O2 were studied to investigate the protective effects of SCFA. SCFA studied here are connected to specific food components and physiological functions related to cancer prevention. In this context, the antigenotoxic properties of butyrate, acetate and physiological mixtures with high butyrate content are of special importance. The practical consequence of these findings is that this may be an important mechanism by which butyrate and acetate can prevent initiation of normal, non-transformed colonocytes. Both compounds, which acted protectively, may be utilized by colonocytes as energy sources (33,34). Thus by pre-treating the cells with these SCFA, the decrease in vulnerability against a subsequent H2O2 insult may be a reflection of enhanced cellular metabolism, including stimulation of DNA repair, antioxidant defence systems, energy turn over, etc. These endogenous protection mechanisms are expected to be more active in cells with complete metabolic activity than in quiescent ones. In fact, the suggestion has recently been extensively discussed that butyrate acts as a fuel and thus as a survival factor for colonic epithelial cells (35). Redegeld et al. have shown that the energy status of cells is critical for the induction of cell damage by oxidative stress (36). Using menadione as an inducer of oxidative stress, it was suggested that a depletion of ATP could be mediated by interference with glycolysis and protein breakdown to result in a lack of oxidizable substrate for ATP generation. Also, H2O2 can lead to a decrease in ATP in a dose-related fashion (37). Declines in ATP levels may increase vulnerability of cells, disturb the cellular ion homeostasis by reducing Na+-K+-ATPase activity and Ca2+-ATPases, and may lead to cytoskeleton disruption (31,32). Supplementation with metabolic substrates, such as pyruvate, oxalate and glutamine, may postpone menadione-induced ATP depletion and delay onset of cell death. It is conceivable that butyrate and acetate may protect against H2O2 in a similar fashion.

One other mechanism for the protective effect of butyrate against DNA damage that may be related to the mechanism of metabolic substrate supplementation is the protection against apoptosis. Apoptosis is known to occur both in the physiology and pathophysiology of gastrointestinal epithelium cells (38). Hass et al. have demonstrated that the absence of butyrate after the isolation of the colonic epithelium induces apoptosis (19). Addition of butyrate protected against the induction of apoptosis. This reduction of apoptosis was paralleled by a reduced increase in the expression of Bax proteins (19). We do not know whether or not apoptosis is initiated during the isolation procedure and subsequent incubations in our short-term in vitro experiments. The DNA breaks that are detected by the microgel electrophoresis assay may or may not stem from apoptotic cells, since the DNA of apoptotic cells at the onset of fragmentation might appear as comets. However, cells with excessive DNA fragmentation cannot be detected using the comet assay, as too many DNA breaks will result in the diffusion and subsequent disappearance of DNA into the agar. Thus, although this technique detects DNA breaks, it cannot be applied to study apoptosis. Also during the 30 min incubation protocol used here, no additional cytotoxic or genotoxic effects are induced. The cells retain both viability as well as basal levels of genetic damage. If apoptosis continues and if it is detectable as comets, there should be a higher yield of comets after 30 min in vitro incubation, which was not the case. Nevertheless, our results on the protective effects of butyrate against the H2O2-induced DNA damage and the ability of butyrate to reduce apoptosis of isolated colonic epithelium cells warrants further investigations using adequate techniques.

In mixtures of acetate, propionate and butyrate the protective effect of both compounds is not as apparent as for the single substances. These findings were unexpected on the basis of the effects found with acetate and butyrate alone. We do not have an explanation for these results. Obviously, there is an interaction between acetate, propionate and butyrate in these experiments. It is also clear that butyrate and acetate do not act additively for the parameter of antigenotoxicity. Otherwise, we would have observed protective effects for both mixtures. Also, the preferential utilization (34,33) and cellular uptake (39) of butyrate relative to propionate and acetate by colonocytes do not explain our observations. The preferential utilisation and cellular uptake of butyrate relative to propionate and acetate would result in less DNA damage after pre-incubation with the mixture containing the highest butyrate concentration.

The pre-treatment of colon cells with butyrate resulted in significant protection against oxidative DNA damage induced by H2O2. These results are another parameter pointing to the unique role of butyrate as a potential protective agent.


    Acknowledgments
 
This study was supported by a grant from the European Community (programs ECAIR-2-CT94-0933 and ECAIR-1-CT92-256), and the EDEN-Stiftung, Bad Soden, Germany. The authors wish to thank C.Csovcsics, M.Knoll, R.Lamberts and B.Mathony-Holschuh for excellent technical assistance and Mrs A.Lindner for competent secretarial work.


    Notes
 
1 To whom correspondence should be addressedEmail: gerhard.rechkemmer{at}bfe.uni-karlsruhe.de Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 4, 1998; revised December 2, 1998; accepted December 2, 1998.