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Biochemical and Molecular Actions of Nutrients

Dietary Soy Protein Isolate and Isoflavones Modulate Hepatic Thyroid Hormone Receptors in Rats¹

Chao Wu Xiao^*,**,2, Mary R. L’Abbé^*, G. Sarwar Gilani^*, Gerard M. Cooke^,**, Ivan H. Curran and Suzanne A. Papademetriou^*

^* Nutrition Research Division, Toxicology Research Division, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, ON, Canada K1A 0L2 and ^** Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada

²To whom correspondence should be addressed. E-mail: chaowu__xiao{at}hc-sc.gc.ca.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Thyroid hormone receptors (TRs) are regulators of many genes involved in cholesterol and lipid metabolism. The purpose of this study was to examine the effect of soy protein isolate (SPI) and isoflavones on hepatic TRs in rats. In Expt. 1, Sprague-Dawley rats were fed diets containing either casein or alcohol-washed SPI with or without isoflavone supplementation (5–1250 mg/kg diet) for 70, 190, and 310 d. The offspring (F1) were fed the same diets as their parents (F0). In Expt. 2, Sprague-Dawley rats were fed diets containing casein or casein plus isoflavones (50–400 mg/kg diet) for 120 d. The mRNA and protein contents of the hepatic TRs were measured by semiquantitative RT-PCR and Western blot, respectively. TR1, TR2, and TRß2 contents were not affected by SPI. However, the content of the 52-kDa TRß1 protein, the major isoform present in the liver, was markedly increased by dietary SPI in both sexes of F0 and F1 compared with casein. The supplemental isoflavones had no effect on TRß1, whereas the high doses of isoflavones (250 and 1250 mg/kg diet) reduced the hepatic TR1 protein content in F1 male rats on d 28. SPI had no effect on total T3 and T4 levels. However, higher dose of supplemental isoflavones markedly increased T4 level in female rats. Overall, this study demonstrates for the first time that SPI upregulates hepatic TRß1 expression, and that isoflavones reduce the hepatic TR1 level in young male rats. The SPI-induced TRß1 may play a role in mediating the hypocholesterolemic and lipid-lowering actions of soy protein.

KEY WORDS: • rats • soy protein isolate • isoflavones • thyroid hormone receptors

Soy products are attracting more and more attention because of their beneficial effects on chronic diseases such as cardiovascular diseases, atherosclerosis, and type II diabetes. Interest in the clinical application of soy protein is increasing, especially after the approval of the food labeling health claims for soy protein in the prevention of coronary heart disease by the U.S. FDA (1).

Dietary supplementation with soy improves cardiovascular disease risk factors by lowering plasma total cholesterol, LDL cholesterol, and triglyceride concentrations and reducing the ratio of LDL to HDL in both normal and type II diabetic animals and humans (2–4). However, the components of soybeans that may contribute to the hypocholesterolemic and lipid-lowering properties of soy have not been well characterized. Isoflavones, the major phytoestrogens in soy, were shown to be hypolipidemic in both cynomolgus monkey (5) and humans (6). Ingestion of ethanol extract rich in isoflavones increases the abundance of hepatic mRNA for cholesterol 7-hydroxylase (CYP7a)³ and LDL receptors in rats, which play important roles in cholesterol catabolism (7). However, other studies demonstrated that soy protein rather than isoflavones contributes to the lipid-lowering and hypocholesterolemic properties of soy (8–14). Moreover, soy protein enhances the expression of the LDL receptor in hypercholesterolemic type II diabetic patients (15), animals (16,17), and cultured human hepatoma cells (18–20).

Increased thyroxine (T4) levels were proposed as the putative mechanism responsible for the hypocholesterolemic effect of soy protein (9) because T4 levels and plasma cholesterol and triglyceride concentrations were negatively correlated in gerbils (8), rats (21), and hamsters (22) fed soy protein–based diets. However, this notion seems inconsistent with the results obtained from clinical studies (23). For example, soy intake significantly decreased the plasma cholesterol concentration, but did not increase the level of T4 in postmenopausal women with type II diabetes (24). In addition, although the soy protein isolate (SPI) and soy protein concentrates are both hypocholesterolemic in hamsters, only SPI increases T4 levels. These results indicate that modulation of thyroid hormone status may not be the only mechanism responsible for the cholesterol-lowering action of soy protein (25).

Thyroid hormone receptors (TRs), members of the nuclear receptor superfamily, are mediators of the thyroid hormone effect. Four TR isoforms (TR1, TR2, TRß1, and TRß2) have been identified to date (26). Contrary to other nuclear receptors, TRs can bind to the thyroid hormone response element of target genes (26), without the necessity for ligand binding, and repress gene transcription. Furthermore, TRs are key regulators of many genes that are involved in cholesterol and lipid metabolism, and also play important roles in the regulation of growth, differentiation, development, and carcinogenesis. However, whether soy protein and soy-derived isoflavones have any effect on the expression of hepatic TRs is unknown.

The objective of present study was to examine the effect of alcohol-washed SPI and supplemental isoflavones on the expression of hepatic TRs in rats.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Chemicals and reagents.

Alcohol-washed SPI (Pro Fam 930) and Novasoy soy isoflavone concentrate were purchased from Archer Daniels Midland Company. Casein protein (90% purity) was from ICN. Agarose, Tris, and phenylmethylsulfonyl fluoride (PMSF) were from Sigma Chemical. Enhanced chemiluminescence Western blotting detection kits were obtained from Amersham. Nitrocellulose membranes, acrylamide (electrophoresis grade), N, N'-methylene-bis-acrylamide, ammonium persulfate, dithiothreitol, glycine, goat anti-rabbit IgG (H+L)-horseradish peroxidase (HRP) conjugated antibody, goat anti-mouse IgG (H+L)-HRP conjugated antibody, and Bio-Rad protein assay kits were purchased from Bio-Rad Laboratories. X-Ray film was from Eastman Kodak Company. Rabbit polyclonal anti-chicken TR1, goat anti-human TR2, mouse monoclonal anti-human TRß1, and goat anti-human TRß2 antibodies were from Santa Cruz Biotechnology. TRIzol reagent and M-MLV reverse transcriptase were from Invitrogen Life Technologies. Taq DNA polymerase was purchased from New England Biolabs. Total triiodothyronine (T3) and total T4 ELISA kits were from Alpha Diagnostic International.

Animals and diets.

Animal experimental protocols were approved by the Health Canada-Ottawa Animal Care Committee, and all animal handling and care followed the guidelines of the Canadian Council for Animal Care. The rats were kept in an environmentally controlled room with a 12-h light:dark cycle and killed by exsanguination through cardiac puncture under general anesthesia with isoflurane. Tissue samples were collected, immediately frozen in liquid nitrogen, and stored at -80°C until analysis.

Experiment. 1.

Pubertal Sprague-Dawley rats (Charles River) were pair housed in disposable stainless steel cages and randomly divided into 6 groups (35 males and 35 females per dose group) as the parental generation (F0). After acclimation, starting at 50 d of age, rats had free access to 6 diets (Table 1) and water until they were killed. All 6 diets were formulated according to the specifications for the AIN93G diet (27) except that in diets 2–6, casein was replaced by alcohol-washed SPI (Pro Fam 930). In addition, diets 3–6 were supplemented with increasing amounts of isoflavones from Novasoy isoflavone concentrate. The body weight and food consumption were recorded weekly. The actual total isoflavone content including genistein, daidzein, and glycitein was determined by Waters HPLC linear gradient with UV detection monitored at 254 nm (28) and shown in Table 1.

Experiment. 2.

Weaning male and female Sprague-Dawley rats (Charles River; 45 ± 6 g) were housed individually in disposable stainless steel cages and randomly divided into 5 groups (10 males and 10 females per dose group). After acclimation, rats had free access to 5 casein-based diets and water for 120 d. All 5 diets were formulated according to the specifications for the AIN93G diet (27) and were then supplemented with 0, 50, 100, 200, and 400 mg isoflavones/kg diet from Novasoy isoflavone concentrate (Table 2). At the end of the feeding period, the rats were killed and tissues were collected.

Plasma T3 and T4 concentrations were measured using commercial ELISA kits according to the manufacturer’s instructions. Briefly, 50 µL (for T3) or 25 µL (for T4) plasma was added to microtiter plate wells precoated with anti-T3 or anti-T4 antibodies, and then incubated with T3-HRP or T4-HRP conjugates for 1 h before the wells were washed. HRP substrate was added into each well and incubated for 30 min. The absorbance was read at 450 nm in a microplate reader (Model 3550-UV, Bio-Rad). The concentrations of T3 and T4 were calculated using standard curves.

Protein extraction and Western blot analysis.

Rat tissues were homogenized completely in 0.2–1 mL lysis buffer (pH 7.4) containing NaCl (150 mmol/L), SDS (3.5 µmol/L), sodium deoxycholate (11.6 µmol/L), NP-40 (16.6 µmol/L) in PBS, and protease inhibitors [PMSF (1 mmol/L), aprotinin (1.5 µmol/L), sodium orthovanadate (1 mmol/L)] using an Ultra Turrax T8 homogenizer (VWR). The samples were centrifuged (15,000 x g, 20 min) and the supernatant was retained and stored at -80°C. The protein content of the extracts was determined with the Bio-Rad DC Protein Assay Reagent. Total protein samples (40 µg) were mixed with loading buffer, resolved by 12% SDS-PAGE, and electrotransferred (30 V, overnight) onto nitrocellulose membranes. The total protein on the nitrocellulose membranes was stained with Ponceau S solution and scanned using a regular scanner (EPSON perfection 1250). After blocking for 1 h with nonfat milk powder (5%) in Tris-buffered saline (10 mmol/L Tris, 150 mmol/L NaCl; TBS) and Tween-20 (0.05%; TBS-T), membranes were incubated for 3 h with primary antibodies in TBS-T containing nonfat milk powder, and subsequently with HRP-conjugated secondary antibody (1:500010,000) in TBS-T with milk powder at room temperature for 45 min. Immunoreactivity was detected by chemiluminescence autoradiography in accordance with the manufacturer’s instructions, and the images were scanned. The intensities of the protein bands of interest and the Ponceau-stained proteins were determined densitometrically using Scion Image. All of the Ponceau-stained protein bands from the same sample were selected and measured together by the software. The intensities of the target proteins were normalized by the respective Ponceau-stained total protein (29).

Quantitation of TR1 and TRß1 mRNA.

Total RNA was isolated from rat liver samples with TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. Total RNA (500 ng) was reverse transcribed for cDNA synthesis. One tenth of the cDNA synthesized was then amplified with the following primers: rat TR1 [forward: 5'-CTGCTGCATGGAGATCATGT-3' (1275–1294), reverse: 5'-TCACCAAACTGCTGCTCAAG-3' (1781–1762)]; rat TRß1 [forward: 5'-GGTGCATTGAAGAATGAGCA-3' (389–408), reverse: 5'- TCTTGATGAGCTCCCATTCC-3' (920–901)]; rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [forward: 5'- GGC ATT GCT CTC AAT GAC AA-3' (1741–1760), reverse: 5'- CCT GTT GTT ATG GGG TGT GG-3' (1991–1972)]. Rat TR1 and TRß1 PCR cycle conditions were 94°C for 5 min, 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min for 30 cycles, 72°C for 10 min. The PCR cycle conditions for GAPDH were 94°C for 5 min, 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min for 25 cycles, 72°C for 10 min. Samples were resolved on 2% agarose gels and visualized with ethidium bromide. TR1 and TRß1 mRNA levels were normalized against their respective GAPDH mRNA content.

Statistical analyses.

Results are expressed as means ± SEM. Effects of treatment on plasma total T3 and T4 concentrations were analyzed using 3-way factorial ANOVA, which included the main effects of diet, feeding period, and gender, as well as the interactions of diet x feeding period, gender x feeding period, and gender x diet. The dietary effects on food consumption, body and liver weights as well as TR1 and TRß1 protein contents shown in Figures 1a and b, 2, and 3a and b were analyzed by 1-way ANOVA. The effects of feeding period and diets on TRß1 protein were assessed by 2-way factorial ANOVA, which included the main effects of diet, feeding period, and the diet x feeding period interaction. Differences between individual means were determined by Fisher’s least significant difference test. A probability of P < 0.05 was considered to be significant. Data were analyzed using Statistica Version 6.1 (StatSoft).

View larger version (37K): [in this window] [in a new window]   FIGURE 1 Hepatic TRß1 protein content in rats fed diets containing either casein or alcohol-washed SPI in the absence or presence of increasing amounts of supplemental ISF (5, 50, 250, and 1250 mg/kg diet) for 70 d (a and b) or fed diets containing either casein (C) or alcohol-washed SPI (S) for 70, 190, and 310 d (c and d). The images shown are representative of three replicates. Values are mean ± SEM, n = 3. Means with different letters differ, P < 0.01.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In Expt. 1, food consumption did not differ among the dietary groups. By the end of the treatment periods, the body and liver weights were not different among the dietary groups in male rats. However, in female rats, body and liver weights were lower (P < 0.05) in those fed the diet containing the highest dose of supplemental isoflavones (1250 mg/kg diet). In Expt. 2, food consumption and final body weight of rats did not differ among the various dietary groups.

Plasma T3 and T4 concentrations in F0 rats.

Plasma total T3 concentrations decreased with the increased length of the feeding period in both male and female rats fed diets containing casein and SPI alone (P < 0.01). Females had lower T3 level than males (P < 0.01). Dietary effect on total T3 concentrations was not significant (P > 0.05) except that the males fed the diet containing supplemental isoflavones (250 mg/kg diet) for 70 d had lower T3 levels (P < 0.05, Table 3).

Hepatic TR protein content in F0 rats.

Neither SPI nor supplemental isoflavones had any significant effect on hepatic TR1, TR2, and TRß2 protein levels in the rats fed diets containing either casein or SPI in the absence or presence of increasing amounts of supplemental isoflavones for 70 d (data not shown), whereas all 5 of the SPI-based diets markedly increased TRß1 protein content in both female (Fig. 1a) and male rats (Fig, 1b) compared with the casein-based diet (P < 0.01). The supplemental isoflavones had no additional effect compared with SPI alone. In addition, increasing amounts of isoflavones (50, 100, 200, and 400 mg/kg diet) added into the casein-based diet in Expt. 2 had no effect on any of the 4 TRs in either sex (data not shown).

Hepatic TRß1 protein levels were consistently elevated by dietary SPI in both male and female rats fed SPI-based diet for 70, 190, and 310 d (P < 0.01; Fig. 1c and d). The effect of feeding period was not significant. TR1, TR2, and TRß2 protein contents were unchanged by dietary SPI throughout the study compared with the casein control diet (data not shown).

Hepatic TR1 and TRß1 mRNA abundances.

The hepatic TR1 and TRß1 mRNA abundances measured by semiquantitative RT-PCR in rats fed the experimental diets for 70, 190, and 310 d were not affected by dietary SPI throughout the feeding study in both female and male rats (data not shown).

Hepatic TR protein content in F1 rats.

SPI had no significant effect on TR1, TR2, and TRß2 expression at d 28, 70, 120, and 240 of age in both F1 female and male rats (data not shown). However, higher doses of supplemental isoflavones (250 and 1250 mg/kg diet) in SPI-based diets attenuated the expression of TR1 in F1 males at d 28 compared with casein alone (P < 0.01, Fig. 2).

View larger version (23K): [in this window] [in a new window]   FIGURE 2 Hepatic TR1 protein content in F1 male rats fed diets containing either casein or alcohol-washed SPI in the absence or presence of increasing amounts of supplemental isoflavones ISF (50, 250, and 1250 mg/kg diet) after weaning and killed at 28 d of age. The image shown is representative of three replicates. Values are mean ± SEM, n = 3. Means with different letters differ, P < 0.01.

View larger version (42K): [in this window] [in a new window]   FIGURE 3 Hepatic TRß1 protein content in F1 rats fed diets containing either casein or alcohol-washed SPI in the absence or presence of increasing amounts of supplemental isoflavones ISF (50, 250, and 1250 mg/kg diet) (a and b) after weaning and killed at 28 d of age or fed diets containing either casein (C) or alcohol-washed SPI (S) and killed at d 28, 70, 120, and 240 (c and d). The images shown are representative of three replicates. Values are mean ± SEM, n = 3. Means with different letters, P < 0.01.

TRß1 proteins were detected by Western blot as 52- and 55-kDa bands in the tissues of F0 female (Fig. 4a) and male (Fig. 4b) rats fed either casein- or SPI-based diets for 70 d. The 52-kDa protein was expressed mainly in the liver, whereas the 55-kDa protein was predominantly expressed in thyroid, heart, kidney, testis, and uterus. TRß1 proteins were not detectable in the brain. SPI significantly increased the 52-kDa TRß1 levels in the liver, but not in the other tissues measured, whereas the expression of the 55-kDa receptor in different tissues was not significantly affected by dietary SPI (data not shown).

View larger version (35K): [in this window] [in a new window]   FIGURE 4 Distribution of TRß1 proteins in different tissues collected from F0 rats fed diets containing either casein (C) or alcohol-washed SPI (S) for 70 d. Total protein was extracted and analyzed for TRß1 proteins by Western blot. The images shown are representative of three replicates for females (a) and males (b).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In the present study, we demonstrated for the first time that feeding diets containing alcohol-washed SPI significantly increased hepatic TRß1 protein content in both male and female rats in the parental and F1 generations compared with feeding a casein-based diet. Although the physiologic significance of this cellular response to SPI remains to be investigated, we hypothesize that the increased TRß1 protein induced by SPI may play a role in the regulation of cholesterol and lipid metabolism. It is known that TRß1 is a mediator of the thyroid hormone effect on CYP7a, a rate-limiting enzyme responsible for the conversion of cholesterol to bile acids (30,31), and also regulates the expression of other hepatic genes involved in cholesterol catabolism including those for the LDL receptor (32) and lecithin:cholesterol acyltransferase (33). In addition, the genes for lipogenic enzymes including malic enzyme (34), fatty acid synthase (35), and acetyl-CoA carboxylase (36) were shown to contain a thyroid hormone response element in their promoters and to be regulated by thyroid hormones. Hence, the hypocholesterolemic and lipid-lowering actions of soy protein may be mediated by increased hepatic TRß1 through regulation of cholesterol catabolism and lipogenesis. This notion is consistent with the previous observations in which dietary soy protein reduced lipid levels and attenuated the gene expression (37) and activities of hepatic lipogenic enzymes in both normal (38) and obese rats (37).

Dietary SPI had no significant effect on either plasma total T3 or T4 concentrations in this study, which is consistent with the results obtained in male rats (39). Supplementation with isoflavones in a SPI-based diet significantly increased the T4 level in female but not male rats. This appears to be different from what was observed in Balmir’s study, in which the T4 levels in male rats were elevated by isoflavones (39). Ingestion of either low or high amounts of isoflavones in postmenopausal women had no effect on either T3 or T4 levels (40). These discrepancies in the effect of isoflavones on thyroid hormones in different studies may be due to the different doses of isoflavones used and variation among the species. The effect of isoflavones on T4 production may be associated with their estrogenic properties, which may contribute to the increased sensitivity of the pituitary or thyroid gland to normal feedback mechanisms (41,42).

The goitrogenic effect of excessive soy intake had been reported in both iodine-deficient rodents (43–45) and infants fed soy flour–based formula without iodine fortification (46,47) in the 1930s to early 1960s. To prevent enlarged thyroids, the animals fed soy required almost twice as much iodine, an essential component of thyroid hormones, as animals not fed soy (44,48). However, the underlying mechanism is not well understood. Isoflavones were originally believed to be antithyroid components (49,50) because they inhibited thyroid peroxidase, the enzyme responsible for biosynthesis of the thyroid hormone. However, recent studies showed that components in defatted soybean other than isoflavones dramatically influenced the development of thyroid hyperplasia in iodine-deficient rats (51,52). Whether the SPI-induced TRß1 plays a role in mediating the antithyroid action of soy under conditions of iodine deficiency remains to be elucidated.

The estrogenic and antiestrogenic properties of soy isoflavones have been a concern for soy consumers. However, the potential estrogenic or antiestrogenic actions of soy protein have not been evaluated. It was shown that the thyroid hormone response element and the estrogen response element of the target genes share an identical half-site (53,54). This suggests that an increase in TRß1 induced by SPI may influence estrogen functions by competitive binding to the consensus estrogen response element of the target genes.

In the present study, two TRß1 subtypes (52 and 55 kDa) were detected in rat tissues. The 55-kDa receptor, present mainly in thyroid, heart, kidney, testis, and uterus, was not affected by SPI, whereas the protein content of the 52-kDa receptor, primarily present in liver, was significantly enhanced by dietary SPI. Liver is an important organ in controlling plasma cholesterol and triglyceride concentrations. This tissue-specific cellular response to SPI further supports our hypothesis that TRß1 may play important roles in mediating the lipid-lowering and hypocholesterolemic actions of soy proteins. In this study, the different doses of supplemental isoflavones had no effect on TRß1 expression in the presence of either casein or SPI.

Interestingly, the effect of SPI on TRs was isoform specific. SPI had no effect on TR1, TR2, and TRß2, whereas it increased TRß1. However, we found that compared with casein, higher doses of supplemental isoflavones (250 and 1250 mg/kg diet, Fig. 2) reduced the hepatic TR1 protein level in F1 male rats at d 28, but not at later ages. The mechanism(s) involved and the physiologic influence of this reduction are unclear. The component(s) in SPI that are responsible for the increase in hepatic TRß1 content and how they function remain to be identified. However, we showed in this study that SPI did not change the mRNA steady-state level of TRß1, suggesting that the bioactive component(s) in SPI may affect the process of TRß1 protein synthesis or degradation.

In conclusion, this study demonstrated for the first time that dietary SPI, but not supplemental isoflavones, significantly increased the 52-kDa TRß1 protein content in rat liver, and had no effect on total T3 and T4 levels. However, higher doses of supplemental isoflavones markedly increased T4 level in female rats. Induction of TRß1 by SPI might be a novel mechanism by which soybean bioactive components regulate the expression of genes involved in cholesterol and lipid metabolism. Further studies are required to understand the roles of the SPI-induced TRß1 in the regulation of cholesterol and lipid levels as well as in the prevention of chronic diseases such as cardiovascular disease.

	ACKNOWLEDGMENTS

 
The authors thank Elizabeth Krzykwa and Bridget Thompson for technical assistance.

	FOOTNOTES

 
¹ This is publication no. 589 of the Bureau of Nutritional Sciences. Presented in part at the 5th International Symposium on the Role of Soy in Preventing and Treating Chronic Disease, September 21–24, 2003, Orlando, FL [Xiao, C. W., L’Abbé, M. R., Gilani, G. S., Cooke, M. G., Curran, I. H. & Papademetriou, S. A. (2003) Dietary soy protein isolate and isoflavones modulate thyroid hormone receptor expression. G-1 (abs.)] and at the 46th Annual Meeting of the Canadian Federation of Biological Societies, June 11–15, 2003, Ottawa, Canada [Xiao, C. W., L’Abbé, M. R., Gilani, G. S., Cooke, M. G., Curran, I. H., Papademetriou, S. A. & Thompson, B. (2003) Soy protein isolates increase thyroid hormone receptor (TR)-ß1 and TR-regulated gene expression in rats. T40 (abs.)].

³ Abbreviations used: CYP7a, cholesterol 7-hydroxylase; F0, parental generation; F1, offspring generation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ISF, isoflavones; HRP, horseradish peroxidase; SPI, soy protein isolate; T3, triiodothyronine; T4, thyroxine; TBS-T, Tris-buffered saline and Tween-20; TR, thyroid hormone receptor.

Manuscript received 6 November 2003. Initial review completed 9 December 2003. Revision accepted 22 January 2004.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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