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Article

Chronic Alcohol Consumption Induces Genomic but Not p53-Specific DNA Hypomethylation in Rat Colon^{1 ,2 ,3}

Sang-Woon Choi^*, Felix Stickel, Hyun Wook Baik^*, Young-In Kim^**, Helmut K. Seitz and Joel B. Mason^*,4

^* Vitamin Bioavailability Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston 02111; Department of Gastroenterology, Stiftsklinik Augustinum, München, D-81375, Germany; ^** Division of Gastroenterology, Department of Medicine, St. Michael's Hospital and University of Toronto, Toronto, ON, M5S 1A8, Canada; Department of Medicine, Salem Hospital, University of Heidelberg, Heidelberg, 69121, Germany; and Divisions of Gastroenterology and Clinical Nutrition, Department of Medicine, New England Medical Center, Tufts University School of Medicine, Boston 02111

⁴To whom correspondence and reprint requests should be addressed.

	ABSTRACT

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Alcohol consumption has been implicated as an etiologic agent in colorectal carcinogenesis, but the mechanism by which alcohol enhances the development of colorectal cancer is not yet known. Recent reports indicate that alcohol consumption can diminish cellular S-adenosylmethionine levels, thus possibly altering normal patterns of DNA methylation, a phenomenon that is mediated by S-adenosylmethionine and whose abnormalities are observed in colonic neoplasia. This study investigated the effect of chronic alcohol consumption on genomic DNA methylation of rat colonic epithelium and methylation of the p53 tumor suppressor gene, abnormalities of which have been implicated in colonic carcinogenesis. Two groups of rats (n = 10/group) were pair-fed either an alcohol-containing or an isocaloric control Lieber-DeCarli diet for 4 wk. The extent of genomic DNA methylation was assessed by incubating the extracted DNA with [³H]S-adenosylmethionine and Sss1 methyltransferase. Gene-specific methylation was assessed by using semiquantitative polymerase chain reaction (PCR). Tritiated methyl uptake by colonic DNA (which is inversely correlated with genomic methylation) from alcohol-fed rats was 57% less than that in control DNA (P < 0.05). However, gene-specific DNA methylation, both in the p53 gene (exons 5–8) and in the ß-actin gene, a control gene, did not differ between the two groups. In conclusion, this study indicates that chronic alcohol consumption produces genomic DNA hypomethylation in the colonic mucosa. This may constitute a means by which carcinogenesis is enhanced, although further studies are required to establish causality.

KEY WORDS: • alcohol • DNA methylation • colorectal cancer • folate • rats

	INTRODUCTION

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Diet is one of the most important environmental factors in the etiology of colorectal cancer (Sandler et al. 1993 ). Diets high in animal fat or red meat and low in fruits and vegetables appear to increase the risk of this malignancy (Ghadirian et al. 1997 ). Alcohol consumption also has been regarded as an important dietary factor enhancing colorectal carcinogenesis, although epidemiologic studies on the association of alcohol intake with colorectal cancer have shown that this effect exists most consistently with distal, rather than proximal disease (Breslow and Enstrom 1974 , Stemmermann et al. 1990 , Wu et al. 1987 ). Animal studies utilizing various carcinogens that induce colonic neoplasia have also indicated that chronic alcohol ingestion stimulates colonic carcinogenesis (Seitz et al. 1984 and 1990 ), thus supporting such a relationship.

The results of recent studies have demonstrated that alcohol may decrease DNA methylation in hepatic tissue through its antagonistic action on folate metabolism and/or methionine synthetase and by decreasing S-adenosylmethionine (AdoMet)⁵ production (Barak et al. 1993 , Hidiroglou et al. 1994 , Trimble et al. 1993 ). Folate is an essential cofactor in the production of AdoMet, the primary methyl donor for DNA, and methionine is a precursor of AdoMet (Appling 1991 , Chiang et al. 1996 ). Low levels of AdoMet, therefore, may reduce methylation of DNA (Wainfan et al. 1989 , Pogribny et al. 1995b ). Garro et al. (1991) , for example, observed that alcohol diminishes hepatic AdoMet and is capable of causing DNA hypomethylation of fetal liver.

Although all of the functions of DNA methylation are not yet understood, it appears to be an important mechanism for modulating gene expression and for stabilizing areas of the genome (Bestor and Tycko 1996 , Counts and Goodman 1994 ). Alterations in DNA methylation, which are among the most consistent molecular changes observed in carcinogenesis, could induce the expression of oncogenes, silence the expression of tumor suppressor genes or make critical tumor suppressor genes more susceptible to damage (Laird and Jaenisch 1996 , Versteeg 1997 ). In colorectal cancer, there is ample evidence to demonstrate an association between DNA hypomethylation and colorectal carcinogenesis, both genomically (Feinberg et al 1988 , Feinberg and Vogelstein 1983a , Goelz et al. 1985 ) and at the sites of selected protooncogenes and tumor suppressor genes (Kane et al. 1997 , Makos et al. 1992 ). Recently, we reported that dimethylhydrazine (DMH), a colon-specific chemical carcinogen, which alters the AdoMet level and methyl transferase activity preceding the histologic appearance of dysplasia, induces exon-specific p53 hypomethylation in rat colon, demonstrating that such effect is also observed in this rodent model of colonic carcinogenesis (Kim et al. 1996 ). This rodent model of colon cancer also has been shown to be modulated by alcohol ingestion (Hamilton et al. 1987 , Seitz et al. 1984 ) and therefore appears to be a suitable model with which to examine the interactions of alcohol and colonic carcinogenesis.

The aim of this study was to determine whether chronic ingestion of alcohol could lead to either genome-wide or p53-specific DNA hypomethylation in rats, thereby supporting DNA hypomethylation as a mechanism by which alcohol enhances the development of colorectal cancer. The p53 tumor suppressor gene was selected for the study because it is commonly found to be mutated in colorectal cancer and is thought to play an integral role in the transition from dysplasia to invasive cancer (Baker et al. 1989 , Remvikos et al. 1997 ). Dietary manipulations in laboratory animals have previously been observed to induce p53-specific DNA hypomethylation in rat colonic mucosa (Kim et al. 1997 , Pogribny et al. 1995a ). We therefore hypothesized that similar effects might be produced by chronic alcohol ingestion.

	MATERIALS AND METHODS

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Animal and diets.

This study was approved by the Animal Care and Use Committee of the USDA Human Nutrition Research Center on Aging at Tufts University. Male Sprague-Dawley rats (n = 20; 130 g, Charles River, Wilmington, MA) at 8 wk of age were randomly assigned to two groups (n = 10/group). The experimental group was fed a Lieber-DeCarli liquid diet containing 36% of total energy as ethanol, yielding a concentration of 6.2% (v/v). The ethanol was gradually introduced over a 2-wk adaptation period (Lieber et al. 1989 ). In the control diet, alcohol was replaced by an isocaloric amount of maltodextrin (Purina Test Diets, Richmond, IN, codes LD 106 and LD 106A-1, respectively). Both diets contained 16.4% of energy as protein and 35% of energy as fat. In the control diet, 48.6% of energy appeared as carbohydrates; in the alcoholic diet this figure was 12.6%. An accurate description of the diet nutrient composition is shown in Table 1 . The folate concentration of the diet was 2 mg/L, which is equivalent to 8 mg/kg in a dry diet on a per daily consumption basis. The daily requirement of folate in rats has been defined variously to be between 2 and 8 mg/kg in a dry diet (American Institute of Nutrition 1977 , Reeves et al. 1993 ). Succinylsulfathiazole (SST), a sulfonamide antibiotic, was added at a concentration of 3.3 g/kg to prevent additional folate synthesis by intestinal bacteria, thereby enabling us to control precisely the folate status of the rats, a dietary factor that we have shown previously to be an important determinant of p53 methylation (Kim et al. 1997 ). The SST concentration used in our experiment is equivalent to adding 1% SST to a solid diet, a maneuver that has been used extensively to control folate status predictably in rodents (Walzem and Clifford 1988 ). Each rat from the alcohol group was matched by weight with another individual from the control group and pair-fed. Control rats were fed the same amount of liquid diet that had been consumed by their partner rats from the alcohol group the day before. Rats were housed individually in wire-bottomed stainless steel cages to minimize coprophagy. Water was not given because the liquid diet provided a physiologic amount of fluid. Body weights were recorded twice a week.

DNA from colonic mucosal scrapings was extracted by a conventional technique using a lysis buffer containing proteinase K followed by phenol, chloroform, and isoamyl alcohol organic extraction. The purified DNA was dialyzed against 0.1X Tris-EDTA buffer (1 mmol/L Tris-HCl, 0.1 mmol/L EDTA, pH 8.0) by using a conventional drop dialysis method. The size of DNA estimated by agarose gel electrophoresis was 20 kb in all instances. No RNA contamination was detected on agarose gel electrophoresis. The final preparations had an A260/280 ratio ~1.8. The purified DNA was stored at -70°C until the methylation assay.

Plasma and hepatic folate concentration.

Plasma folate concentrations were determined by a microtiter plate assay using Lactobacillus casei (Tamura 1990 ). Hepatic concentrations were measured by the same microbial assay after extraction of tissue folates in 10 vol of fresh extraction buffer (5 mmol/L ß-mercaptoethanol and 0.1 mol/L sodium ascorbate in 0.1 mol/L {bis[2-hydroxyethyl] imino} tris [hydroxymethyl]-methane, pH 7.85), respectively, followed by treatment with chicken pancreas conjugase to convert all of the polyglutamates to their corresponding diglutamate derivatives (Tamura 1990 , Wilson and Horne 1982 ). The latter method has been used extensively to assess tissue folate concentration quantitatively (Kim et al. 1995 and 1996 ).

Hepatic AdoMet and AdoHcy concentrations.

Hepatic AdoMet and AdoHcy concentrations were measured by HPLC with UV detection using the method as described by Fell et al. (1985) with modification (Miller et al. 1994 ).

Pyridoxal 5'-phosphate.

Serum and liver pyridoxal 5'-phosphate (PLP) levels were measured by the tyrosine decarboxylase method as described by Camp et al. (1983) ; samples were deproteinated before the assay with trichloroacetic acid precipitation.

Genomic DNA methylation.

The methylation status of CpG sites in genomic DNA was determined by the in vitro methyl acceptance capacity of DNA using [³H-methyl]AdoMet as a methyl donor and a prokaryotic CpG DNA methyltransferase, as previously described (Cravo et al. 1994 , Kim et al. 1995 ). The manner in which this assay is performed produces a reciprocal relationship between the endogenous DNA methylation status and the exogenous ³H-methyl incorporation. Briefly, 2 µg of DNA were incubated in 185 kBq of[³H-methyl] AdoMet (New England Nuclear, Boston, MA), 4 U of Sss1 methylase (New England Biolabs, Beverly, MA), 1 x Sss1 buffer (50 mmol/L NaCl, 10 mmol/L Tris-HCl, 10 mmol/L EDTA, 1 mmol/L dithiothreitol, pH 8.0) in a total volume of 50 µL methylation mixture for 3 h at 37°C. Sss1 methylase was denatured by heating at 65°C for 20 min. The incubation mixtures were applied onto discs of Whatman DE-81 ion exchange filters (Fisher Scientific, Springfield, NJ) using a vacuum filtration apparatus; the discs were then washed with 0.35 mol/L Na₂HPO₄ for 45 min. The discs were dried at 95°C for 30 min, and the resulting radioactivity of the DNA retained in the discs was measured by scintillation counting using a nonaqueous scintillation fluor. All analyses were done in duplicate.

Quantitative HpaII-polymerase chain reaction assay for gene specific methylation.

The methylation status of the p53 gene and ß-actin gene was assessed using semiquantitative polymerase chain reaction (PCR), utilizing primers flanking the HpaII cleavage sites (CCGG) within the genes, as previously described (Pogribny et al. 1995b , Singer-Sam et al. 1990 ). HpaII is a restriction endonuclease that cannot cut CCGG if the internal cytosine is methylated (Nelson and McClelland 1991 ). HpaII-induced strand breaks at nonmethylated CCGG sites halt the progression of Taq polymerase during PCR amplification. Therefore, quantitative recovery of ³²P-labeled PCR product amplified over primer-defined exons after treatment with HpaII is directly proportional to the degree of methylation at CCGG sites.

Each colonic DNA sample was digested with HpaII (New England Biolabs) at a final concentration of 5 U/µg DNA at 37°C for 3 h in a buffer consisting of 10 mmol/L Bis Tris Propane-HCl, 10 mmol/L MgCl₂ and 1 mmol/L dithiothreitol, pH 7.0. After digestion, the incubation mixture was heated at 65°C for 25 min to denature HpaII before PCR amplification. The control for each DNA sample was incubated in the identical mixture except without HpaII. Each 0.25-µg DNA sample was amplified in a total volume of 50 µL PCR incubation mixture containing 100 pmol of each primer (for exons 5–8 p53 gene: sense primer 5'-ACTCAATTTCCCTCAATAAG and antisense primer 5'-TCTCTTTGCACTCCCTGGGG; for exons 2–3 of the ß-actin primer: sense primer 5'-GTGCCTTGATAGTTCGCCATGG and antisense primer 5'-GGTCACCCAGGATACTGACCTGG), 0.2 mmol/L of each dNTP, PCR buffer (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl and 0.1g/L gelatin). The samples were denatured at 94°C for 5 min in a thermal cycler before the addition of 2.5 U AmpliTaq DNA polymerase and 148 kBq [³²P]dCTP (New England Nuclear). The PCR amplification mixture was denatured at 94°C for 30 s, annealed at 55°C for 30 s, and extended at 72°C for 40 s for a total of 30 cycles in the thermocycler. Amplified PCR products were separated on 3% NuSieve agarose gel and stained with ethidium bromide. The radiolabled single band representing the amplified target locus was punched out from the gel, transferred to scintillation vials in 2 mL of distilled water and melted by microwave heating. ³²P incorporation was measured by scintillation counter using nonaqueous scintillation cocktail. The relative extent of internal cytosine methylation at the CCGG sequences within the specific site of each DNA sample was assessed by comparing the radioactivity of the HpaII-treated product with that of the control, non-HpaII-treated product.

Statistical analysis.

The significance of the compared mean values was determined using two-tailed Student's t test with a P-value of <0.05 considered significant (Systat 5 for the Macintosh, Evanston, IL). All results in the text are reported as means ± SD.

	RESULTS

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Body weights of rats.

Because the alcohol-fed rats had a lower food intake during the 2-wk adaptation period in which alcohol was gradually added to the normal liquid diet, they had a lower mean weight than the controls at the start of the experimental feeding period (212.2 ± 8.6 g vs. 233.7 ± 11.5 g, P < 0.05). To correct this weight difference, the paired food intake of control rats was reduced by 15% during wk 3 of pair-feeding. After 3 wk of the experimental period, the mean weights of groups were no longer significantly different and precise pair-feeding was reinstituted (Fig. 1 ).

View larger version (15K): [in this window] [in a new window]   Figure 1. Growth curves for rats that were pair-fed either an alcohol-containing or an isocaloric Lieber-DeCarli diet for 4 wk. Values are mean weights ± SD, n = 10. Asterisks reflect those time points at which the mean weights were significantly different (P < 0.05). Pair-feeding was slightly modified between the wk 2 and 3 time points as described in the text.

    Pyridoxal 5'-phosphate. Alcohol-fed rats had an ~33% lower plasma PLP concentration (P = 0.02) and a 16% lower hepatic PLP concentration (P = 0.03) than controls (Table 2) . In contrast, hepatic PLP concentration expressed per milligram protein did not differ between the two groups.

    Genome-wide DNA methylation. The in vitro methyl acceptance capacity of alcohol-fed rat DNA was 2.63 ± 0.39 kBq/2 µg DNA, and that of control DNA was 1.67 ± 0.22 kBq/2 µg DNA (mean ± SD). Genomic DNA methylation was significantly decreased in the colonic DNA from alcohol-fed rats compared with the control group (P < 0.05).

Quantitative HpaII- PCR assay for the gene specific DNA methylation.

The p53 gene specific methylation did not differ between the alcohol and control groups. In the HpaII-PCR assay for the p53 gene, the ratio of digested PCR product/undigested PCR product approached 1.0 in both the alcohol and control groups (0.98 ± 0.12 vs. 0.860 ± 0.04, respectively, P > 0.1). The fact that HpaII predigestion produced little change in amplification indicates that most of the CCGG sites in exons 5–8 of p53 were fully methylated in both groups. In contrast, the ratio in the ß-actin assay was ~0.3 in both the alcohol and control groups (0.30 ± 0.10 vs. 0.36 ± 0.09, respectively, P > 0.1). This indicates that the ß-actin gene was less than fully methylated at CCGG loci, a phenomenon that is commonly observed in constituitively expressed genes.

	DISCUSSION

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In this rat model of chronic ethanol ingestion, we observed genomic undermethylation of colonic DNA in the alcohol-fed rats compared with control rats. The means by which ethanol ingestion produces this effect cannot be definitively determined from our data. However, we did observe that concentrations of AdoMet, the compound that provides methyl groups for DNA methylation and that is present in large quantities in the liver (Corrales et al. 1990 ), was significantly diminished in the hepatic tissue of the alcohol-fed rats. Also, the diminished concentration of hepatic AdoMet was accompanied by an increase in the hepatic AdoHcy concentration as well as a significant reduction in the AdoMet/AdoHcy ratio (P < 0.01). The latter value is important because it is thought to reflect methylation capability more accurately than an isolated assessment of either the AdoMet or AdoHcy concentration (Garcea et al. 1989 , Hoffman et al. 1980 ). It is therefore likely that the limited availability of AdoMet, as well as the inhibitory effect of excess AdoHcy (Cox et al. 1977 , DeCabo et al. 1995 ), played roles in producing the genomic undermethylation that we observed.

Our observation of diminished methylation capability is entirely consistent with earlier studies in ethanol-fed laboratory animals (Barak et al. 1993 , Trimble et al. 1993 ). Although the means by which ethanol alters the AdoMet/AdoHcy ratio are not clear, there are several possible pathways by which this might occur. Barak et al. (1987) also observed that chronic alcohol ingestion in rats diminished tissue levels of AdoMet, whereas it increased AdoHcy concentrations; these investigators reported that this effect was a result of impaired activity of the methionine synthase enzyme. Trimble et al. (1993) proposed that ethanol metabolism releases an excess of free radicals and acetaldehyde, thereby stimulating the cell to redirect homocysteine to the catabolic transulfuration pathway in order to replenish glutathione, thus diminishing AdoMet production by the alternative transmethylation pathway. In addition, the cell attempts to sustain methionine availability through futile cycles of choline oxidation, thereby wasting additional methyl groups. Methyltetrahydrofolate is a substrate for methionine synthesis; thus, a depletion of folate could impair AdoMet synthesis. In this study, no reduction in systemic folate status was observed, although we cannot exclude the possibility that aberrations in the distribution of folate coenzymes, rather than total folate concentrations, might be responsible for the decreased AdoMet levels. A recent study showed that such shifts occur in the livers of alcoholic animals in the absence of changes in total folate concentration (Hidiroglou et al. 1994 ). Finally, there are also observations in humans (Cravo et al. 1996 ) as well as in this study (see Table 2 ) that indicate that vitamin B-6 status is impaired in chronic alcohol ingestion. Because vitamin B-6 is a necessary cofactor for serine hydroxymethylase in the synthesis of 5,10-methylenetetrahydrofolate, this could impair the availability of methyl groups available for methionine synthesis.

Regardless of how chronic ethanol ingestion produces genomic undermethylation of the colonic mucosa, it may have some implications regarding the mechanism(s) by which chronic alcohol exposure increases the risk of colorectal cancer. Vogelstein and colleagues established some time ago that genomic undermethylation of DNA was both a very early event in human colorectal carcinogenesis as well as one that was present in a very consistent fashion (Feinberg and Vogelstein 1983a ). Cravo et al.(1994) reported that genomic DNA hypomethylation is also present in the normal colonic mucosa of individuals who harbor colonic neoplasms, indicating that the appearance of DNA undermethylation may even precede histologic evidence of dysplasia. The phenomenon has also been observed to occur in carcinogen-induced rodent models of colorectal cancer, underscoring the consistency of its association with colorectal carcinogenesis (Kim et al. 1996 ). DNA methylation plays important roles in determining gene expression (Counts and Goodman 1994 ) and protects the genome from damage by endonucleases (Antequeera et al. 1990 , Wolf and Migeon 1985 ); interruptions in either of these crucial roles could enhance the likelihood of carcinogenesis. Although genomic DNA hypomethylation has not been definitively established to play a mechanistic role in colorectal carcinogenesis, our observations provide a possible explanation for how ethanol enhances colorectal carcinogenesis.

Aberrations in regional patterns of DNA methylation have also been observed in colonic carcinogenesis. Many investigations have observed regional hypomethylation of protooncogenes such as c-myc, k-ras, and H-ras in colon cancer (Feinberg and Vogelstein 1983b , Sharrad et al. 1992 ). In addition, a CpG-rich region of chromosome 17p, which is normally unmethylated, becomes increasingly methylated; this regional hypermethylation has been associated with the predisposition for allelic losses of chromosome 17p in colon cancer (Makos et al. 1992 ). Recently, we reported the induction of p53-specific colonic DNA hypomethylation in the hypermutable coding region (exons 5–8) of rats exposed to the combination of a colonic carcinogen (DMH) and a folate-deficient diet (Kim et al. 1996 ). In this study, we investigated the effects of chronic alcohol consumption on p53-specific DNA hypomethylation and we found no effect of the alcohol. Similarly, there was no effect on a control, housekeeping gene, ß-actin. HpaII sites (CCGG) of exons 5–8 in the p53 gene appeared to be fully methylated because the quantity of PCR product was essentially identical in the predigested and undigested incubations, i.e., the digested:undigested ratio was ~1. This is consistent with a previous report in human colonic cells indicating that all 46 CpG sites along exons 5–8 are completely methylated, regardless of whether cell transformation has occurred. Whether this is also true for the rat is not yet known. In contrast, the ß-actin gene showed a diminished quantity of PCR product after digestion with HpaII, suggesting that CCGG sites in ß-actin gene are less than fully methylated at CCGG loci. This is consistent with other reports indicating generalized hypomethylation within constituitively expressed "housekeeper" genes (Kafri et al. 1992 , Monk et al. 1987 ). Although our observations regarding methylation of the p53 gene are negative, we cannot exclude the possibility that alcohol exposure for >4 wk might change p53 gene-specific methylation, or that demethylation might occur at CpG sites other than CCGG loci. We also cannot exclude the possibility that alcohol consumption might induce p53 hypomethylation, but only in conjunction with other procarcinogenic factors such as DMH.

In summary, this study has demonstrated that substantial alcohol ingestion over a period of several weeks produces genomic DNA hypomethylation in the colonic mucosa of rats. Such an effect was not seen when the region of the p53 gene that is most closely linked to colonic carcinogenesis was examined. Further studies will be required to determine whether this alteration in DNA methylation plays a role in the means by which ethanol promotes carcinogenesis in the colon.

	FOOTNOTES

 
¹ Presented in part at the 17th International Congress of Biochemistry and Molecular Biology, August 24–29, 1997, San Francisco, CA and published in abstract form [Choi S. W., Stickel F., Baik H. W., Kim Y. I., Seitz J. B. & Mason J. B. (1997) Chronic alcohol consumption induces colonic DNA hypomethylation in the rat. FASEB. J. 11: A1255 (abs.)].

² Supported in part by a National Cancer Institute Grant (1 U01-CA 63812–01; J.B.M.), the American Institute for Cancer Research (J.B.M.), the U.S. Department of Agriculture, Agricultural Research Service (Contract 53–3K06–01; J.B.M.), and the Cancer Research Foundation of America (S.W.C.).

³ The contents of this publication do not necessary reflect the views or policies of the U.S. Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

⁵ Abbreviation used: AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; DMH, dimethylhydrazine; PLP, pyridoxal 5'-phosphate; SST, succinylsulfathiazole.

Manuscript received April 5, 1999. Initial review completed May 27, 1999. Revision accepted July 14, 1999.

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