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Nutrition and Cancer

Dietary Folate and Selenium Affect Dimethylhydrazine-Induced Aberrant Crypt Formation, Global DNA Methylation and One-Carbon Metabolism in Rats¹

U.S. Department of Agriculture, Grand Forks Human Nutrition Research Center, Grand Forks, ND 58202-9034

²To whom correspondence should be addressed. E-mail: davisci{at}mail.nih.gov.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Several observations suggest a role for DNA methylation in cancer pathogenesis. Although both selenium and folate deficiency have been shown to cause global DNA hypomethylation and increased cancer susceptibility, the nutrients have different effects on one-carbon metabolism. Thus, the purpose of this study was to investigate the interactive effects of dietary selenium and folate. Weanling, Fischer-344 rats (n = 23/diet) were fed diets containing 0 or 2.0 mg selenium (as selenite)/kg and 0 or 2.0 mg folate/kg in a 2 x 2 factorial design. After 3 and 4 wk of a 12-wk experiment, 19 rats/diet were injected intraperitoneally with dimethylhydrazine (DMH, 25 mg/kg) and 4 rats/diet were administered saline. Selenium deficiency decreased (P < 0.05) colonic DNA methylation and the activities of liver DNA methyltransferase and betaine homocysteine methyltransferase and increased plasma glutathione concentrations. Folate deficiency increased (P < 0.05) the number of aberrant crypts per aberrant crypt foci, the concentration of colonic S-adenosylhomocysteine and the activity of liver cystathionine synthase. Selenium and folate interacted (P < 0.0001) to influence one-carbon metabolism and cancer susceptibility such that the number of aberrant crypts and the concentrations of plasma homocysteine and liver S-adenosylhomocysteine were the highest and the concentrations of plasma folate and liver S-adenosylmethionine and the activity of liver methionine synthase were the lowest in rats fed folate-deficient diets and supplemental selenium. These results suggest that selenium deprivation ameliorates some of the effects of folate deficiency, probably by shunting the buildup of homocysteine (as a result of folate deficiency) to glutathione.

KEY WORDS: • selenium • folate • rats • DNA methylation • one-carbon metabolism

Colorectal cancer is the fourth most common type of cancer and the second most common cause of cancer deaths in the United States (1). Dietary factors are thought to play prominent roles in the causation of colorectal cancer. Several epidemiologic studies have suggested an inverse association between dietary folate and the risk for colorectal cancer (2–8). For example, Giovannucci et al. (2) showed that prolonged use of supplements containing folic acid significantly reduced the risk of developing colorectal cancer in the 89,000 participants in the Nurse’s Health Study. In animal studies, a folate-deficient diet increased the incidence of aberrant crypt foci and colonic adenomas, but only if the deficient diet was begun before the initiation/promotion phases of carcinogenesis (9–11). Aberrant crypt foci (ACF)² are putative preneoplastic lesions that have been detected in human colon resections and in experimental animals treated with chemical carcinogens (12,13). Studies in humans have shown that colonic ACF are precursor lesions from which adenomas and adenocarcinomas will develop (14,15).

The main biochemical role of folate is the transfer of one-carbon moieties (16). Folate functions in DNA synthesis and repair, and in methylation, by providing methylene and formyl groups for the synthesis of thymidine and purines and methyl groups for the synthesis of S-adenosylmethionine (SAM). SAM is the methyl donor for DNA methylation reactions. DNA methylation is an important epigenetic mechanism exerting control on gene expression (17,18). In eukaryotic cells, only small regions of the genome containing repeat CpG residues (CpG islands) are methylated. These CpG islands are localized predominantly in the promoter regions of genes, and methylation is thought to control gene transcription (18). Several observations implicate a role for DNA methylation in cancer pathogenesis. Abnormal methylation patterns have been detected early in the development of cancer, including colorectal cancer (19–21). These changes consist mainly of global hypomethylation, regional DNA hypermethylation and overexpression of DNA methyltransferase 1 (22). Folate deficiency can cause hypomethylation within a highly conserved region of the p53 tumor suppressor gene, where the majority of the genetic alterations implicated in the development of several neoplasms have occurred (21,23,24).

The nutrient selenium also affects colon cancer susceptibility and DNA methylation. Rats fed selenium-deficient diets had significantly hypomethylated liver and colon DNA compared with rats fed diets supplemented with selenite or selenomethionine (25). Selenium-deficient rats also formed more carcinogen-induced aberrant crypts (26,27). Thus, alterations in DNA methylation may help explain the increased tumorigenesis associated with selenium deficiency.

Although both selenium and folate deficiency result in global DNA hypomethylation and increased cancer susceptibility, these nutrients influence one-carbon metabolism differently. Folate deficiency increases plasma homocysteine concentrations and decreases SAM to S-adenosylhomocysteine (SAH) ratios (28), whereas selenium deficiency decreases plasma homocysteine concentrations and slightly increases SAM to SAH ratios in the liver (27,29). Increased plasma homocysteine concentrations were recently hypothesized to be a risk factor for cancer and a new potential tumor marker (30). Thus, the purpose of this study was to investigate the interactive effects of dietary selenium and folate on aberrant crypt formation, DNA methylation and one carbon-metabolism. A second objective was to compare DNA methylation and one carbon-metabolism in the liver vs. the colon for a subgroup of rats.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Animals and diets.

Weanling male Fischer-344 rats (n = 92) were purchased from Sasco (Omaha, NE). All rats were housed individually in stainless steel wire-bottomed cages in a room with controlled temperature and light. Rats had free access to demineralized water and purified diet. The basal diet was an amino acid–based diet formulated to be low in selenium and folate. The diets contained (on a per kg basis): 186.1 g amino acid mixture, 35 g selenium-deficient mineral mixture, 10 g folate-deficient vitamin mixture, 100 g corn oil, 50 g Alphacel fiber (ICN Pharmaceuticals, Costa Mesa, CA), 2.5 g choline bitartrate, 466.4 g sucrose and 150 g cornstarch. The vitamin and mineral mixtures had a composition similar to those of the AIN-93 diet (31). In a 2 x 2 factorial design, the basal diet was supplemented with 0 or 2 mg selenium/kg diet as sodium selenite (by analysis, the diets contained <3 µg and 1.99 mg selenium/kg, respectively) and with 0 or 2 mg folate/kg as folic acid. No antibiotics were used so that only a moderate folate deficiency would be obtained. These diets were considered to be either deficient or supplemental in selenium and either deficient or adequate in folate because the recommendations for dietary selenium and folate in the AIN-93 diet are 0.15 and 2.0 mg/kg diet, respectively (31). Supplemental rather than adequate dietary selenium was chosen to maximize potential differences between the dietary treatments. After 3 wk of consuming the experimental diets, 19 rats/diet were given two interperitoneal injections, separated by 1 wk, of dimethylhydrazine (DMH; 25 mg/kg body). The remaining rats (n = 4/diet), were injected with saline. Rats consumed the same diets for an additional 8 wk.

This study was approved by the Animal Care Committee of the Grand Forks Human Nutrition Research Center. The rats were maintained in accordance with the guidelines for the care and use of laboratory animals.

Sample collection.

Food was withheld overnight (at least 12 h) before rats were anesthetized with xylazine (Rompon, Mobay, Shawnee, KS) and ketamine (Ketaset, Aveco, Fort Dodge, IA) and killed by exsanguination. Blood was collected by cardiac puncture into syringes containing EDTA such that the final concentration was 1 g EDTA/L blood. Hematologic indices were determined with a Coulter S + IV hematology analyzer (Coulter Electronics, Hialeah, FL). For aberrant crypt analysis (n = 15/diet, all administered DMH), the colon and rectum were removed, flushed with 9 g/L NaCl, opened longitudinally and fixed flat between paper towels in 700 mL/L ethanol and stored at 4°C before analysis. For the remaining rats (n = 8/diet, 4 administered DMH and 4 administered saline), the colon was opened longitudinally and the mucosa was scraped off with a microscope slide. Aliquots were stored at -70°C before analysis of DNA methylation, DNA methyltransferase activity, SAM and SAH.

Analysis of aberrant crypt foci.

The fixed colon and rectum were stained with 1 g/L methylene blue in 0.1 mol/L sodium phosphate buffer (pH 7.4). Aberrant crypt foci and the total number of aberrant crypts were scored without knowledge of the dietary treatment by using a dissecting microscope to visualize the aberrant crypt foci (26).

Genomic DNA methylation.

To assess the methylation status of CpG sites in genomic DNA, the in vitro methyl acceptance capacity of DNA was determined using [³H-methyl]SAM as a methyl donor and a prokaryotic CpG Dnmt1 (25). The endogenous DNA methylation status is reciprocally related to the exogenous ³H-methyl incorporation. Briefly, DNA (2 µg) was incubated with 185 kBq of [³H-methyl]SAM (Amersham Life Science, Piscataway, NJ), 4 U of Sss1 methyltransferase (New England Biolabs, Beverly, MA), 1X Sss1 buffer (50 mmol/L NaCl, 10 mmol/L Tris-HCl, 10 mmol/L EDTA and 1 mmol/L dithiothreitol, pH 8.0 (21). All analyses were in duplicate.

DNA methyltransferase activity.

Frozen liver (100 mg) or scraped colonic mucosa (50 mg) were homogenized with a Mark II Tissumizer (Tekmar, Cincinnati, OH) in1 mL lysis buffer (50 mmol/L Tris, pH 7.8, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 0.1 g/L sodium azide, 6 g/L phenylmethylsulfonyl fluoride, 100 mL/L glycerol and 10 mL/L Tween 80) as described previously (32). This suspension was passed through an 18-gauge needle, then through a 25-gauge needle. It was frozen at -70°C, and then thawed. The freeze-thaw cycle was repeated three times. The samples were stored at -70°C until analyzed. Protein concentrations were determined using the Bradford assay (Bio-Rad Laboratories, Richmond, CA).

DNA methyltransferase activity was determined by a modification of the procedure of Issa et al. (32). Briefly, 10 µg of cellular protein were mixed with 0.5 µg of synthetic DNA consisting of repeats of inosine-cytosine [poly d(I-C).d(I-C), Amersham, Arlington Heights, IL) and 3 µCi [³H-methyl]-SAM (2.16 kBq/mmol, Amersham) in a total volume of 20 µL. This solution was incubated at 37°C for 2 h (colon) or 4 h (liver). The reaction was terminated by adding 350 µL of a stop solution (10 g/L SDS, 2 mmol/L EDTA, 30 mg/L 4-aminosalicylate, 50 mL/L butanol, 125 mmol/L sodium chloride, 0.25 g/L carrier salmon testis DNA and 1 g/L proteinase K). After an additional 30 min of incubation at 37°C, DNA was purified by phenol/chloroform extraction and ethanol precipitation. RNA was removed by resuspension in 0.3 mol/L sodium hydroxide and incubation at 37°C for 45–60 min. The final solution was spotted onto Whatman GF/C filters. The filters were dried at 80°C, placed on a manifold, washed with 5 mL 50 g/L trichloracetic acid containing 10 g/L bovine serum albumin, then washed with 3 mL of 700 mL/L ethanol, dried again at 80°C, placed in scintillation cocktail, and counted with a scintillation counter. Results are expressed as disintegrations per minute per 10 µg cellular protein per 4 h incubation (liver) or 2 h incubation (colon).

Liver and colon SAM and SAH.

Portions of fresh liver and frozen colon were weighed and homogenized at 11,500 rpm in 0.4 mol/L HClO₄ by using a Mark II Tissumizer (Tekmar, Cincinnati, OH). Samples were centrifuged at 2000 x g at 4°C for 30 min. Each supernatant was filtered through a 0.45-µm filter and stored at -70°C until analysis. SAM and SAH were measured on a Shimadzu. LC-10 HPLC (Columbia, MD) equipped with a 250 x 4.6 mm Ultrasphere 5-µm C18 IP column (Phenomenex, Torrance, CA) according to the procedure of Wagner et al. (33)

Liver enzyme activities.

The activity of betaine-homocysteine methyltransferase (BHMT) was determined according to Finkelstein and Mudd (34) as modified by Xue and Snoswell (35). The substrate [methyl-³H]betaine was prepared according to Xue and Snoswell (35). Liver was prepared by homogenization (1 g liver/4 mL buffer) in 0.04 mol/L potassium phosphate buffer, pH 7.4. The homogenate was centrifuged at 18,000 x g for 15 min at 4°C; the supernatant was used for the assay. Methionine synthase (MS) activity was determined by the method of Sauer (36). For the MS assay, liver was prepared by homogenization (1 g liver/4 mL buffer) in 0.01 mol/L potassium phosphate buffer, pH 7.5, containing 1 mmol/L GSH. The homogenate was centrifuged at 40,000 x g for 30 min at 4°C, and the supernatant was used for the assay. The activity of cystathionine synthase (CBS) was determined according to a modified method of Suda et al. (37), which is based on the method of Mudd et al. (38) as described previously (29). These assays were conducted only on rats analyzed for DNA methylation and DNA methyltransferase activity.

Plasma homocysteine and glutathione (GSH).

Total homocysteine and GSH were determined in heparinized plasma by using HPLC according to the procedure of Durand et al. (39).

Selenium status.

Selenium concentrations in the plasma, liver and colon were determined by hydride-generation atomic absorption spectrometry according to a published procedure (40). Samples were prepared for analysis by predigestion in nitric acid and hydrogen peroxide, followed by high temperature ashing in the presence of MgNO₃ as an aid to prevent selenium volatization.

GSH peroxidase enzyme activity was determined by the coupled enzymatic method of Paglia and Valentine (41), which uses hydrogen peroxide as the substrate.

Plasma vitamin B-12 and folate.

Plasma vitamin B-12 and folate were determined by RIA (KVSP2, B-12; and KFSP, folate; Diagnostic Products, Los Angeles, CA).

Statistical analysis.

The data were initially analyzed by a three-way ANOVA (diet selenium, diet folate and drug treatment-DMH vs. saline) using the SAS general linear models program (SAS Version 8.02, SAS Institute, Cary, NC). Because drug treatment did not affect most of the variables, the data were also analyzed with a two-way ANOVA (diet selenium and diet folate). If no effect occurred, the nonsignificant effect of drug treatment is not listed. Because the colon DNA methyltransferase activity did not follow a normal distribution, data were logarithmically transformed before statistical analysis. These data are expressed as geometric means ± 1 SEM. Other data with unequal variances were not transformed before statistical analysis because the unequal variances did not affect the statistical results. Tukey’s contrasts were used to differentiate among means for variables (P < 0.05) affected by an interaction between selenium and folate. Because the number of aberrant crypts and aberrant crypt foci were assumed to follow a Poisson distribution, the data were analyzed using a generalized linear model (Genmod procedure, SAS). Bonferroni adjustments were used when comparing means to account for multiple comparisons in the analysis of aberrant crypts and aberrant crypt foci. Pearson correlations were used to determine the association between plasma homocysteine and liver enzymes and between colon and liver SAM, SAH and SAM/SAH ratios. Values are reported as means ± SEM.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Rats fed the selenium-deficient diet gained less (P < 0.0001) body weight than rats fed the selenium-supplemented diet (314 ± 3 vs. 335 ± 3 g, respectively). Dietary folate and carcinogen treatment did not affect the body weight of the rats (data not shown). Only a moderate folate deficiency occurred as evidenced by the lack of an effect of folate on growth of the rats.

Dietary selenium and folate interacted to affect hematological indices and plasma folate and vitamin B-12 concentrations (P < 0.03; Table 1). Rats fed the diets containing adequate folate and supplemental selenium had the highest hematocrits, hemoglobin, plasma folate and plasma vitamin B-12 concentrations. In contrast, only dietary selenium affected plasma GSH concentrations (P < 0.0001; Table 1) and indicators of selenium status (Table 2). However, dimethylhydrazine treatment reduced liver selenium concentrations (P < 0.02; 28.4 ± 0.4 vs. 31.1 ± 0.9 mmol/kg in rats injected with dimethylhydrazine and PBS, respectively). This effect was significant only in the selenium-supplemented rats. Carcinogen treatment did not affect RBC or liver GSH peroxidase activity.

View larger version (31K): [in this window] [in a new window]   FIGURE 1 Effect of dietary selenium and folate on total number of aberrant crypts and aberrant crypt foci in the colon and rectum of rats treated with dimethylhydrazine and fed amino acid–based diets supplemented with 0 or 2 mg/kg folate and 0 or 2 mg selenium/kg as selenite in a 2 x 2 factorial design. Values are means ± SEM, n = 15. ANOVA: selenium x folate, P < 0.0001. Means without common letters differ, P < 0.05.

View larger version (40K): [in this window] [in a new window]   FIGURE 2 Effect of dietary selenium and folate on plasma homocysteine concentrations in rats fed amino acid–based diets supplemented with 0 or 2 mg/kg folate and 0 or 2 mg selenium/kg diet in a 2 x 2 factorial design. Values are means ± SEM, n = 22–23. ANOVA; selenium, P < 0.001; folate, P < 0.0001; selenium x folate, P < 0.0001. Means without common letters differ, P < 0.05.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Folate deficiency and selenium deficiency appear to have opposite effects on homocysteine metabolism. There are two major metabolic reactions involving homocysteine, i.e., remethylation and transsulfuration (Fig. 3). In remethylation, homocysteine is converted to methionine by acquiring a methyl group from either N-5-methyltetrahydrofolate or from betaine, catalyzed by the enzymes MS and BHMT, respectively. In the transsulfuration pathway, homocysteine condenses with serine to form cystathionine in an irreversible reaction catalyzed by CBS. The activities of all three liver enzymes involved in the metabolism of homocysteine were significantly correlated with plasma homocysteine concentrations.

View larger version (18K): [in this window] [in a new window]   FIGURE 3 Simplified version of one-carbon metabolism involving DNA methylation. CH3, methyl group; CpG, cytosine-guanine dinucleotide sequence. Abbreviations in italics represent enzymes that were measured in the current study: BHMT, betaine homocysteine methyltransferase; CBS, cystathionine synthase; DNMT, DNA methyltransferase; MAT, methionine adenosyltransferase; MS, methionine synthase.

The decreased homocysteine concentrations observed during selenium deficiency were most likely the result of increased transsulfuration of homocysteine. Hill and Burk (43) reported that -glutamylcysteine synthetase activity in selenium-deficient rat liver was twice that of controls, resulting in increased plasma GSH. In our study, we show that rats fed the selenium-deficient diets had significantly increased plasma GSH concentrations. This effect was most pronounced when rats were fed a folate-deficient diet. Selenium deficiency did tend to increase activity of liver CBS, the first enzyme in the transsulfuration pathway.

Interestingly, the activity of CBS was significantly lower in rats fed the folate-deficient, selenium-supplemented diets compared with the other dietary treatments. Decreased CBS activity contributed to the very high plasma homocysteine observed in rats fed this diet. This is also the group of rats with the highest liver SAH concentrations and the lowest SAM/SAH ratio. Similarly, CBS knockout mice have elevated plasma homocysteine and decreased liver SAM/SAH ratios compared with wild-type mice (44). Also, SAH concentrations in these mice were significantly higher in homozygous mutant mice than in wild-type mice in all tissues examined; however, SAM concentrations responded in a tissue-specific manner (44). In the current study, folate deficiency significantly increased colon SAH but did not affect colon SAM concentrations.

Despite the fact that folate deficiency significantly increased colonic SAH concentrations, folate did not affect colonic DNA methylation. Other investigators also observed that folate deficiency does not induce significant genomic DNA hypomethylation in the colon despite increased SAH concentrations (45,46). This is a surprising finding considering that SAH is a potent inhibitor of most SAM-dependent methyltransferases including DNA methyltransferase (42) and that increased plasma and intracellular SAH levels were shown recently to be accurate predictors of genomic DNA hypomethylation (47,48). One explanation for the lack of correlation between colonic SAH and colonic genomic DNA methylation is that the range of changes in colonic mucosal SAH levels induced by dietary folate and selenium concentrations used in the present study is not sufficient to modulate colonic genomic DNA methylation (45). The present data do not rule out the possibility that the increases in SAH might have produced gene-specific hypomethylation of DNA (46) in the absence of genomic hypomethylation, as was observed previously in folate-deficient rats (23).

Another surprising result in the current study was the significantly increased liver and colon DNA methyltransferase activity when rats were fed a selenium-supplemented diet. The higher DNA methyltransferase activity was associated with increased colonic DNA methylation in selenium-supplemented rats. In contrast to the results in the current study, Fiala et al. (49) observed that sodium selenite, benzyl selenocyanate and 1,4-phenylenebis(methylene)selenocyanate (p-XSC) inhibited DNA methyltransferase extracted from nuclei of a human colonic carcinoma. p-XSC also inhibited the enzyme in HCT116 human carcinoma cells in culture at a concentration of 0–40 µmol/L. However, these studies investigated the effect of selenium on the activity of the purified enzyme in vitro at very high concentrations of selenium. The current study investigated the effect of selenium on the activity of the enzyme in vivo at physiologic concentrations of selenium. Future studies should investigate how and why selenium supplementation increases DNA methyltransferase activity.

In the current study, selenium deficiency significantly depressed global DNA methylation in the colon but not in the liver. Furthermore, the DNA was more hypomethylated in the colon than in the liver as evidenced by the higher incorporation of [³H-methyl] groups. This may be a result of the higher rate of cell proliferation in the colon than in the liver. Previous studies have suggested that hypomethylation may be a feature of proliferating cells because during DNA replication, the newly synthesized strand is not methylated. However, after cell replication, DNA methyltransferase recognizes the 5-methylcytosine at the CpG site on the parental strand as a signal to methylate the corresponding CpG site on the daughter strand (50). Rapidly proliferating cells have a relatively high proportion of hemimethylated sites in their DNA, and thus have relatively low total genomic DNA methylation, whereas differentiated cells have relatively stable methylation patterns (51).

In this study, dietary folate did not affect DNA methylation, and colonic DNA methylation was not associated with aberrant crypt formation. This is in contrast to current dogma, which suggests that low folate status may increase the risk of colon cancer through alterations in DNA methylation. However, colonic DNA methyltransferase activity was highest in rats fed folate-deficient diets and supplemental selenium (Table 3). This was also the group of rats with the highest number of aberrant crypts and aberrant crypt foci. Trasler et al. (52) observed that both DNA methyltransferase deficiency and folate deficiency can modulate intestinal tumor numbers in multiple intestinal neoplasia (Min) mice without concomitantly altering overall genomic DNA methylation. These results suggest that other mechanisms, in addition to changes in DNA methylation, are involved in the increased colon cancer susceptibility of folate-deficient animals. Examples might include induction of mutations and DNA damage.

Another interesting finding from the current study was the lack of correlation between liver and colon SAM, DNA methylation and DNA methyltransferase activity, thus suggesting that in future studies, these variables should be investigated in the tissue of interest rather than assuming that all tissues respond in the same manner.

In summary, results from the current study suggest that selenium deficiency and folate deficiency have opposite effects on plasma homocysteine concentrations. Selenium deficiency appears to ameliorate the large buildup of homocysteine as a result of folate deficiency and shunt it to GSH production. Future studies are required to evaluate whether the protective effect of selenium occurs between deficient and adequate or between adequate and supplemental selenium.

	ACKNOWLEDGMENTS

 
The authors thank Denice Schafer and her staff for care of the animals, Sheila Bichler and LuAnn Johnson for performing the statistical analysis and Laura Idso, Kim Baurichter and Thomas Zimmerman for technical assistance.

	FOOTNOTES

 
¹ The U.S. Department of Agriculture, Agriculture Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination.

³ Abbreviations used: ACF, aberrant crypt foci; BHMT, betaine homocysteine methyltransferase; CBS, cystathionine synthase; DMH, dimethylhydrazine; MAT, methionine adenosyltransferase; MS, methionine synthase, p-XSC, 1,4-phenylenebis(methylene)selenocyanate; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine.

Manuscript received 3 June 2003. Initial review completed 19 June 2003. Revision accepted 27 June 2003.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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