Detection of early gene expression changes by differential display in the livers of mice exposed to dichloroacetic acid

Sheau-Fung Thai, James W. Allen, Anthony B. DeAngelo, Michael H. George and James C. Fuscoe1,

National Health and Environmental Effects Research Laboratory, Environmental Carcinogenesis Division, US Environmental Protection Agency, Research Triangle Park, NC 27711, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Dichloroacetic acid (DCA) is a major by-product of water disinfection by chlorination. Several studies have demonstrated the hepatocarcinogenicity of DCA in mice when administered in drinking water. The mechanism of DCA carcinogenicity is not clear and we speculate that changes in gene expression may be important. In order to analyze early changes in gene expression induced by DCA treatment we used the differential display method. Mice were treated with 2 g/l DCA in drinking water for 4 weeks. Total RNAs were obtained from livers of both control and treated mice for analysis. Of ~48 000 bands on the differential display gels representing an estimated 96% of RNA species, 381 showed differences in intensity. After cloning and confirmation by both reverse-northern and northern analyses, six differentially expressed genes were found. The expression of five of these genes was suppressed in the DCA-treated mice while one was induced. After sequencing, four genes were identified and two were matched to expressed sequence tags through the BLAST program. These genes are alpha-1 protease inhibitor, cytochrome b5, stearoyl-CoA desaturase and carboxylesterase. Stearoyl-CoA desaturase was induced ~3-fold in the livers of DCA-treated mice and the other three genes were suppressed approximately 3-fold. Stearoyl-CoA desaturase, cytochrome b5 and carboxylesterase are endoplasmic reticulum membrane-bound enzymes involved in fatty acid metabolism. The expression pattern of four of these genes was similar in DCA-induced hepatocellular carcinomas and the 4 week DCA-treated mouse livers. The expression of stearoyl-CoA desaturase and one of the unidentified genes returned to control levels in the carcinomas. Understanding the roles and interactions between these genes may shed light on the mechanism of DCA carcinogenesis.

Abbreviations: DCA, dichloroacetic acid; DD, differentially displayed; EST, expressed sequence tag; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MDD, mean daily dosage.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The introduction of chlorine into public drinking water supplies has produced a dramatic decrease in water-borne disease outbreaks. Studies done in the 1970s revealed that chlorine can react with the humic and fulvic acids found in the surface water, forming potentially toxic chlorinated by-products (1,2). Dichloroacetic acid (DCA) is a major by-product of such reactions, with concentrations in finished drinking water ranging from 33 to 160 µg/l (2). Several studies have demonstrated the hepatocarcinogenicity of DCA in mice when administered in drinking water (35). In these studies, DCA appeared to function as a complete carcinogen, although the mechanism of carcinogenicity is not clear. The extremely high dose at which DCA was shown to be active as a rodent carcinogen (1–5 g/l) (3,6,7) and the finding of only limited genotoxicity (810) suggests that the mechanism(s) responsible for carcinogenesis at these high doses may not be operating at lower, environmentally relevant exposure levels. We speculate that changes in gene expression may play a role in the carcinogenicity of DCA at high doses, and that by identifying these particular genes and their products, insights will be gained into the mechanism of DCA carcinogenesis. This, in turn, will enhance the ability to estimate cancer risks at environmental exposure levels of DCA.

In order to identify specific genes whose transcription was induced or suppressed by DCA treatment, we used the RNA differential display method (11,12). This method allows comparison of the expression levels of genes in one cell population with another cell population. Pairs of primers are used to amplify partial cDNA sequences, using reverse transcription and PCR, from the two different cell populations. The primers are typically a 3' oligo dT-containing sequence targeting the 3'-end of mRNAs and an arbitrary short primer that allows amplification of a subset of mRNAs. Side-by-side comparison of reaction products from the two cell populations on sequencing gels is then used to identify differentially expressed genes. The advantage of this method is that new and novel genes may be identified. This method has been used successfully to clone genes that were differentially expressed in neoplastic cells (1316) and in chemically treated cells (1720).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and DCA treatment
Three-week-old male B6C3F1 strain mice were purchased from Charles River Laboratories (Portage, MI). Maintenance and treatment of mice in the EPA animal facility was in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee. After 1 week in quarantine, the animals (28 days old) were weighed and randomly distributed into groups. Experimental animals were administered 2 g/l DCA (CAS No. 79-43-6; Aldrich, Milwaukee, WI) in drinking water. Control animals received deionized filtered water. All animals received their respective drinking water ad libitum. The preparation and administration of DCA solutions for this study have been previously published (3). The dosage (2 g/l) was chosen because of its ability to cause liver tumors in B6C3F1 mice (3,6,7). Water consumption and body weights were recorded weekly during the treatment time (4 weeks). Mean daily dosage (MDD) for each of the 4 weeks of treatment was calculated as water intake for that week (ml)x2.0 g DCA/l/average weight (kg)/7 days. Mean daily dosage for the entire experiment was the average of the MDD for the 4 weeks. At the end of 4 weeks, animals were killed by asphyxiation with carbon dioxide. Livers were removed, weighed and homogenized immediately in Tri-Reagent (Molecular Research Center, Cincinnati, OH) and total RNA was extracted according to the manufacturer's instructions.

Differential display
Total RNA samples from livers of both control and DCA-treated mice were analyzed by the differential display method (11,12) using RNAimage kits (GenHunter, Nashville, TN). In short, total RNA was treated with DNase I (MessageClean kit; GenHunter) and three pools of first-strand cDNA were created using the three one-base anchored oligo-dT primers provided. cDNA from each pool was PCR amplified (40 cycles) with [{alpha}-33P]dATP using the same one-base anchored primer and one of the 80 arbitrary primers provided in the RNAimage kit. In total, 240 primer pair combinations were used (three one-base anchored primersx80 arbitrary primers). PCR products were electrophoresed on a 6% polyacrylamide/7 M urea DNA sequencing gel with samples from control and treated animals in adjacent wells. Gels were dried (Bio-Rad model 583 gel dryer; Bio-Rad, Hercules, CA) and exposed to Biomax MR film (Eastman Kodak Company, Rochester, NY). The exposed X-ray film was aligned with the gel and a gel slice corresponding to a band of DCA-altered intensity (at least 3-fold) was excised. The gel slice was rehydrated in 100 µl deionized H2O and boiled for 10 min. After brief centrifugation, the supernatant was transferred to a new tube and the cDNA fragments ethanol precipitated in the presence of 0.5 mg/ml glycogen. The resultant DNA pellet was resuspended in 10 µl deionized water. A 4 µl aliquot of cDNA was re-amplified under the same conditions (without [{alpha}-33P]dATP) as the first PCR reaction in a final volume of 40 µl. After purification from the agarose gel using a gel extraction kit (Qiagen, Valencia, CA), re-amplified cDNAs were cloned into the plasmid pCR2.1 (TA Cloning kit; Invitrogen, Carlsbad, CA) for reverse-northern analysis.

Reverse-northern analysis
Four or five clones from the potentially differentially expressed cDNA in each gel slice were evaluated by reverse-northern analysis. cDNA fragments (amplified with primers flanking the pCR2.1 cloning site) were denatured, neutralized and spotted onto duplicate Nytran SuperCharge membranes (Schleicher & Schuell, Keene, NH) using the Minifold II slot-blot system (Schleicher & Schuell). Membranes were then hybridized to 32P-labeled probes prepared from total RNA from the livers of control or DCA-treated mice using a ReversePrimeTM kit (GenHunter). Hybridization was done in ExpressHybTM hybridization solution (Clontech, Palo Alto, CA) following the manufacturer's suggested conditions (60°C for 3 h). Clones that showed differential intensity were then used as probes for northern analysis.

Northern analysis
Total RNA (15 µg) from each liver sample were electrophoresed on a 1% denaturing agarose gel. RNA was transferred onto Nytran SuperCharge membrane using the TurboBlotter System (Schleicher & Schuell) and RNA was cross-linked to the membrane using the UV StratalinkerTM 1800 (Stratagene, La Jolla, CA). Membranes were hybridized to 32P-labeled cDNA probes (High-Prime Nucleic Acid Labeling System; Roche Molecular Biochemicals, Indianapolis, IN). Hybridization was carried out in ExpressHybTM hybridization solution following the manufacturer's suggested conditions (68°C for 3 h). After washing, membranes were exposed to a PhosphorImager cassette and analyzed with ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The same membrane was stripped and hybridized to the mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene to verify that the same amount of RNA was electrophoresed in each lane. In addition, ethidium bromide stained rRNA in the electrophoresed samples was used to ensure equal amounts of RNA on the gels.

DNA sequencing and sequence analysis
Clones that hybridized to differentially expressed RNAs on northern blots were sequenced using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit and ABI PRISM 377 Sequencer (Perkin Elmer, Norwalk, CT). Sequences were compared with published sequences in GenBank using the program BLAST (21) (National Center for Biotechnology Information).

mRNA levels of differentially expressed clones in DCA-induced hepatocellular carcinomas
Hepatocellular carcinomas were induced in male B6C3F1 mice exposed to 3.5 g/l DCA in the drinking water for 93 weeks. The conditions of dosing solution preparation, animal husbandry and treatment, and histopathological identification of tumors were as previously described (6). Mice were then killed and liver carcinomas were immediately flash-frozen, total RNA was extracted with Tri-Reagent and northern analysis was performed as described earlier.


    Results
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 Materials and methods
 Results
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 References
 
Eight mice were used in these experiments, four in the control group and four in the DCA-treated group (Table IGo). After calculating the average mean daily dosage (MDD; mg/kg/day), total RNA from two mice with similar MDD (nos 5 and 8) were used for differential display analysis. Total RNA from two control mice (nos 2 and 3) with weight gain similar to the DCA-treated mice were used as controls. At the time of death, no lesions were apparent in the livers of either control or treated mice. However, the livers of treated mice were significantly larger than the control mice as a result of periportal cytoplasmic hepatocellular vacuolization associated with increased amounts of periodic acid-Schiff positive staining material morphologically consistent with glycogen accumulation. No other histopathology was observed. The ratio of liver weight to total body weight for DCA-treated mice was ~150% of control mice at the time of death.


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Table I. Weight gain and average daily dose of DCA
 
Differential display
Two hundred and forty differential display reactions were carried out for each of the total liver RNA samples isolated from mice numbers 2, 3, 5 and 8. For each primer pair, ~200 cDNA fragments were visible on the sequencing gel. Therefore, ~48 000 cDNA fragments were examined for each RNA sample. Comparison of the relative intensities of control to treated samples suggested that 381 of the 48 000 fragments were candidates for differentially expressed genes. A typical differential display gel illustrating differences in band intensities is shown in Figure 1Go. Of 381 partial cDNA bands that were re-amplified and cloned, 97 clones showed differential intensity with reverse-northern analysis.



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Fig. 1. Differential display gel. Total RNA was isolated from livers of DCA-treated (+) or control (–) mice. Differential display reactions and gels were done according to Materials and methods. Arrows point to bands that were induced or suppressed by DCA treatment.

 
Northern analysis of differentially expressed clones
When hybridized to RNA from control and DCA-treated livers, 64 of the 97 clones did not hybridize to any detectable RNAs, and 27 of them hybridized to RNAs with similar intensities, indicating no differential expression. Six of the clones tested showed differential mRNA levels in control and treated samples, and were designated DD (differentially displayed) clones. Northern blots for these six genes are shown in Figure 2Go. One (clone 19.2) of the six genes was induced by DCA treatment while the other five (clones 11.4, 6B.5, 111.3, 9a.3 and 8.4) were all suppressed. The levels of induced or suppressed expression were consistently 2–3-fold (average of three experiments). Band intensities were normalized to the signals from GAPDH (data not shown).



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Fig. 2. Northern analysis of differentially displayed cDNA clones. Six cDNA clones that showed differential expression were used as probes for northern analysis. RNA gels were run according to Materials and methods. From left to right, the first two lanes contain liver RNA from control mice and the next two lanes contain liver RNA from DCA-treated mice. Probes for each gel were as indicated at the bottom.

 
Sequence analysis
These six DD clones were sequenced and examined for homology to known genes in the NCBI/GenBank DNA sequence database using the BLAST program. As shown in Table IIGo, three of these clones were almost completely homologous (>97%) to known mouse genes, i.e. mouse alpha-1 protease inhibitor 3, mouse stearoyl-CoA desaturase exon 6 and mouse carboxylesterase genes. Clone 11.4 is highly homologous (92%) to the rat cytochrome b5, indicating it may be the murine cognate of this gene. Two of the six clones did not correspond to any known genes but shared significant homology to mouse expressed sequence tags (ESTs). Therefore, the identity and function of these two genes are not known.


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Table II. Differentially expressed genes
 
Expression of DD clones in DCA-induced hepatocellular carcinomas
Total RNA from 10 hepatocellular carcinomas (from 10 different mice treated with 3.5 g/l DCA for 93 weeks) were analyzed together with total RNA from short-term (2 g/l for 4 weeks) DCA-treated mouse livers. Four of the six DD genes identified in this study were expressed at similar levels in the tumors and in the 4 week DCA-treated mouse livers (Figure 3Go). The two clones that showed a different pattern are stearoyl Co-A desaturase and clone 8.4, with the level of expression of stearoyl Co-A desaturase in the carcinomas being similar to that in the control livers. Clone 8.4 hybridized to two RNA species; the larger RNA was expressed at similar levels in the carcinomas and the control livers while the smaller species was expressed at similar levels in the carcinomas and the 4 week DCA-treated livers.



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Fig. 3. Northern analysis of differentially expressed genes in hepatocellular carcinomas compared with 4 week DCA-treated samples. Total RNA (15 µg) was electrophoresed and hybridized to probes prepared from the six differentially displayed cDNA clones as described in the Materials and methods. Lanes 1 and 2, RNA from control animals as in Figure 2Go, lanes 1 and 2. Lanes 3 and 4, RNA from 4 week DCA-treated animals as in Figure 2Go, lanes 3 and 4. Lanes 5–14, RNA from 10 different hepatocellular carcinomas.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The differential display method was used to analyze early DCA-induced changes in gene expression in mouse livers. The novel aspect of the differential display method is the possibility of isolating new genes on a systematic basis. Indeed, the expression of two ESTs, in addition to four known genes, was found to be altered by 4 week exposure of mice to DCA in drinking water.

It has been estimated that when 80 arbitrary primers are used in combination with 3 one-base anchored primers, ~96% of the 10 000 expressed mRNA species will have been analyzed (22). Of the estimated 48 000 fragments visualized on the differential display gels, 381 showed differences in intensity between controls and DCA-treated samples. Of these 381 cDNA fragments, 97 showed differential intensity in reverse-northern analysis. Upon northern analysis, 64 clones failed to detect any transcripts (no bands) and 27 clones detected specific RNAs with similar expression levels in control and DCA-treated mouse livers. Six of 97 clones ultimately showed a difference in mRNA levels. The false positive rate is 93.8% (91/97), which is similar to that reported by Harris et al. (23), but somewhat higher than other published data (50–70%) (15,20). Thus, although the method was found to be relatively simple and reproducible, a significant disadvantage was the high rate of false positives.

These data suggest that the dosage and length of time of DCA treatment used in this study did not have a great effect on the overall gene expression pattern in the mouse livers, although this dose is carcinogenic when continued over the life of the mice (6). The expression of five of the six differentially expressed genes was suppressed by ~2–3-fold in the livers of DCA-treated mice while one gene was induced ~3-fold. Because the RNA samples were from whole livers, these changes in RNA levels may result from either changes in most of the cells or very large changes in only a subset of the cells. For example, small changes in gene expression in polyploid hepatocytes would result in a proportionally larger absolute change than the same change in a 2N cell. Future studies, using these cDNAs as probes on liver tissue sections may resolve this question. If the former proves to be correct, then the liver cancer may result from small changes in the mRNA levels or other more subtle changes (e.g. changes in post-transcriptional modifications or protein localizations).

Three of the differentially expressed genes, stearoyl-CoA desaturase, carboxylesterase and cytochrome b5, are endoplasmic reticulum membrane-bound enzymes involved in fatty acid metabolism. Stearoyl-CoA desaturase and cytochrome b5 (a heme-containing electron transporter) are both necessary for the conversion of stearoyl-CoA to oleoyl-CoA by the introduction of a double bond. Since stearoyl-CoA desaturase is induced by DCA treatment while cytochrome b5 is suppressed, it is unclear how this lipid desaturation might be directly involved in DCA-induced carcinogenesis. Cytochrome b5 has additional roles in the formation of bile salts (in the liver) for the digestion and absorption of dietary lipids and in the biosynthesis of steroid hormones (in the adrenal glands). Such alterations in lipid metabolism may be related to the ability of DCA to specifically activate pyruvate dehydrogenase (24), which catalyzes the production of acetyl CoA and thus dramatically increases the flux through the citric acid cycle. This cycle is the hub of aerobic metabolism and alterations in its regulation would be expected to have a significant impact on the metabolism of lipids, carbohydrates and amino acids. In fact, DCA-treated mice have been shown to become hypolipidemic, have increased glycogen deposition in the liver and have severely reduced serum cholesterol levels (4,23,24; DeAngelo, unpublished data). In addition, Kandala et al. (27) have found increased expression of a gene involved in mitochondrial electron transport, which would be expected to be up-regulated in response to increased acetyl CoA production. The changed expression of these three genes may therefore be secondary to DCA's effect on the citric acid cycle.

Alternatively, stearoyl-CoA desaturase is also a key regulator of cellular cholesterol homeostasis (28) and membrane fluidity (29) whose alteration has been implicated in a variety of disease states, including cancer (30). In addition, stearoyl-CoA desaturase has been found to be induced by hepatic carcinogens, such as clofibrate and gemfibrozil, that may act through a peroxisome proliferation mechanism (31). DCA has been shown to be an inducer of peroxisome proliferation in the livers of mice (32), as well as in mouse hepatocytes (33), although this property is not believed to be the dominant mechanism for the hepatocarcinogenic property of DCA. Induction of stearoyl-CoA desaturase may therefore be the result of the peroxisome proliferation property of DCA.

Carboxylesterases comprise a group of serine hydrolases with at least 20 genetically distinct loci in mice. It appears that they play a role in detoxification of foreign compounds with ester or amino bonds (34,35) and also in regulating some membrane lipid concentrations (36). A carboxylesterase has also been cloned by the differential display method from the livers of mice treated with phenobarbitone, another non-genotoxic mouse hepatocarcinogen (19). However, phenobarbitone induced carboxylesterase expression while DCA suppressed it. Thus, the significance of this gene in the carcinogenic process is unclear.

Alpha-1 protease inhibitor is an acute phase protein (37,38). Individuals who are homozygous mutant for the alpha-1 protease inhibitor gene usually develop pulmonary emphysema and they also have a higher chance of developing hepatocellular carcinoma, although the increased cancer risk is thought not to be due to lack of enzyme activity but rather to inappropriate expression of a mutant form of the enzyme (39). However, when the activity of alpha-1 protease inhibitor is inhibited, the activity of matrix metalloproteinases is increased (40). Matrix metalloproteinases not only have a direct role in tumor invasion by facilitating extracellular matrix degradation, but also have an important role in maintaining the tumor micro-environment and thus promoting tumor growth (41). Therefore, in DCA-treated mice with alpha-1 protease inhibitor expression reduced, the invasiveness of tumors may be facilitated by the higher expression levels of metalloproteinases.

The other two cDNA clones identified in this study were suppressed in DCA-treated samples and did not share homology with any known genes. They did, however, match certain ESTs. Clone 9a.3 matched an EST from mouse four-cell embryo cDNA (GenBank accession no. AU043077) (381 nucleotides sequenced, 100% match) and also an EST from dioxin-treated mouse liver cDNA (GenBank accession no. AW109986) (315 nucleotides sequenced, 99% match). Dioxin is an environmental contaminant that can cause chronic liver diseases including liver tumors. The other clone, clone 8.4, matched an EST in the Mouse Tumor Gene Index (GenBank accession no. AW209125; 212 nucleotides sequenced, 100% match) published by the Cancer Genome Anatomy Project (NCI) which identifies genes expressed during the development of mouse tumors. AW209125 is expressed in infiltrating mammary ductal carcinoma cells. Thus, two new potential biomarkers of DCA exposure have been identified.

When the expression of these six genes was analyzed in DCA-induced hepatocellular carcinomas from mice, it was found that four of them were altered in the same manner as in the 4 week DCA-treated mouse livers. The similar change in expression pattern of four of these genes (carboxylesterase, alpha-1 protease inhibitor, cytochrome b5 and clone 9a.3) indicates that they may be important for tumorigenesis and/or tumor growth. The two clones that showed a different expression pattern were stearoyl-CoA desaturase and clone 8.4. The expression of stearoyl-CoA desaturase in the hepatocellular carcinomas was similar to that in the livers of control mice. Thus, the expression of stearoyl-CoA desaturase returned to control levels in the tumors, suggesting that altered expression of this gene is not necessary for tumor growth throughout the whole period. Perhaps the altered expression occurs in the majority of liver cells that do not develop into the tumor, while expression is not altered in those cells that actually develop into a tumor. Clone 8.4 identifies two RNA species and it is not known whether these represent transcripts from two independent genes or if these represent differentially processed RNAs from the same gene. The expression of the smaller species is reduced in the carcinomas just as it is in the 4 week DCA-treated mouse livers and therefore mimics the expression pattern of the other four genes described above. The expression of the larger species in the carcinomas, however, is similar to that in the untreated controls (while suppressed in the 4 week DCA-treated mouse livers) and may therefore not play a role in the carcinogenesis of DCA. Further studies will be required to resolve the reason(s) for the different expression patterns.

In summary, the differential display method was used to analyze early changes in gene expression in the livers of DCA-exposed mice. Our results did not reveal any altered expression of genes commonly involved in genotoxicity pathways. Six cDNA fragments from genes whose expression patterns were altered in mouse livers in response to administration of the relatively non-genotoxic hepatocarcinogen DCA were cloned. Stearoyl Co-A desaturase, cytochrome b5 and carboxylesterase are related to lipid metabolism and might not be involved in the carcinogenesis of DCA. Cytochrome b5 is an electron transporter that participates in many different reactions and its role in carcinogenesis awaits further research. Alpha-1 protease inhibitor was found to be similarly altered in early pre-tumor DCA-exposed livers and in the DCA-induced hepatocellular carcinomas. This protein plays a role in tissue remodeling and tumor invasion which may be important for tumor growth in the later stage. The possible role of the other two unidentified genes in DCA carcinogenesis is also not clear at the present time.


    Notes
 
1 To whom correspondence should be addressed at present address: National Center for Toxicological Research, Division of Genetic and Reproductive Toxicology, HFT-120, 3900 NCTR Road, AR 72079, USA Email: jfuscoe{at}nctr.fda.gov Back


    Acknowledgments
 
We are grateful to Geremy W.Knapp for his expert assistance in the preparation of the liver samples. We also thank Drs Lynn Crosby and Marc Mass for critical review of the manuscript and helpful suggestions. This article has been reviewed by the US EPA and approved for publication. Approval does not signify that the contents necessarily reflect the views or policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 12, 2001; revised April 23, 2001; accepted April 27, 2001.