Potential Risk of Isoflavones: Toxicological Study of Daidzein

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The Potential Risk of Isoflavones: A Toxicological Study of Daidzein Supplementation in Piglets Yi Xiao, Xiangbing Mao, Bing Yu, Jun He, Jie Yu, Ping Zheng, Zhiqing Huang, and Daiwen Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00677 • Publication Date (Web): 10 Apr 2015 Downloaded from http://pubs.acs.org on April 14, 2015

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Safety of a Dietary Daidzein

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The Potential Risk of Isoflavones: A Toxicological Study of Daidzein Supplementation in

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Piglets

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Xiao Yi, Mao Xiangbing, Yu Bing, He Jun, Yu Jie, Zheng Ping, Huang Zhiqing, Chen Daiwen*

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Key Laboratory of Animal Disease-Resistance Nutrition, Animal Nutrition Institute, Sichuan

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Agricultural University, Chengdu, China, 611130

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* Corresponding author. Tel.:+86 835 288 2088; fax: +86 835 288 2088. E-mail address:

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[email protected]


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ABSTRACT As a main component of soy isoflavones, daidzein is rich in soy-derived products, which are

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widely used as feed ingredients in farm animals. However, little research has been conducted on

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the side effects of dietary daidzein, especially in young animals. In this study, the safety of

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daidzein was evaluated. Results show that ingesting 400 mg/kg of dietary daidzein for 70 days (d)

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is associated with a lower average daily weight gain (ADG) (kilogram) (0.47 ± 0.03 vs. 0.54 ±

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0.04, P < 0.05) and a higher splenic damage index (1.00 ± 1.10 vs. 0.00 ± 0.00, P < 0.05) in

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young pigs compared with control. Female pigs receiving 200 mg/kg and 400 mg/kg daidzein

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showed reduced serum testosterone levels (ng/L) in d 35 and 70 compared with the control group

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(D 35: 246 ± 74 and 224 ± 20 vs. 362 ± 48, P < 0.05; D 70: 252 ± 38 and 219 ± 77 vs. 374 ± 38,

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P < 0.05). Daidzein residue (µg/kg) in pig livers increased (243 ± 80 vs. 142 ± 47, P < 0.05, d 70).

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These results suggest that dietary supplements of 400 mg/kg of daidzein negatively affect the

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weight gain and splenic morphology of pigs.

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Key words: daidzein, piglets, health, residue, safety


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INTRODUCTION

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Phytoestrogens, especially soy isoflavones, bind to estrogen receptors α and β due to their

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structural similarity with mammalian oestrogens1, 2. They have received much attention for their

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antioxidant properties and protective effects in hormone-related diseases3, 4. Isoflavones play a

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vital part in people's life for their abundance in soy products mostly in the forms of glycosides5.

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For instance, about 25% of infant formula on the shelves in the US is based on soy protein6.

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However, concerns have been voiced regarding the relationship between isoflavones to immunity,

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thyroid disease and nutrition. The issue of feeding human infants with soy-based formulas has

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received considerable debate7-9. The possible negative outcomes of longer-term use of isoflavones

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has received scant study, with controversial results.

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Isoflavones are in a class of organic compounds and biomolecules related to the flavonoids,

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including daidzin, glycitin, genistin, daidzein, glycitein and genistein10. Daidzein and genistein

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are found in high concentrations in plasma after soy consumption11. Most studies have focused on

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the effect of total isoflavones, with only a few focusing on the individuals effect of daidzein or

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genistein, both of which can be treated chemically to a purity of 90% or above12. Some studies

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show that daidzein or genistein can be added separately to animal feed for some certain benefits.

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For example, low doses of daidzein have been show to improve the egg laying of hens (10 mg/kg

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feed)13 and ducks (5 mg/kg feed)14. When adding to sows (1 mg/kg body weight), daidzein

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improves the meat quality and skeletal muscle cellularity of their progeny15. Moreover, daidzein

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may provide beneficial protection against particular immune challenge in weaned pigs (50

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mg/d)16. Therefore, daizein has been proposed as a potential additive to animal feed. Although

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both daidzein and total isoflavones do not appear to be highly toxic or lethal, even at high doses, ACS Paragon Plus Environment

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(1000 mg/kg feed for daidzein and 1047 mg/kg feed for total isoflavones)17, 18, few studies have

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evaluated the safety of dietary daidzein, especially in young animals. In fact, our literature review

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found no such studies on young pigs. Therefore, this study fills an important gap in the literature

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regarding the effects of higher daidzein doses and residues on certain animals. In addition,

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research on potential risks to human health for those who consume these animal products is also

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necessary.

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Therefore, this study investigated the health effects of daidzein feed supplementation for piglets

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and pork consumers. We assessed the no observed adverse effect level (NOAEL) for daidzein use

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in pigs in the growth stage and determined likely levels of background exposure to daidzein for

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pork consumers. The objectives of this research were to 1) conduct a safety evaluation of dietary

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daidzein in terms of animal toxicology using a model of weanling pigs, and 2) test the hypothesis

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that dietary supplementation of daidzein increases accumulation in the body tissues of pigs, in

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order to evaluate of the safety of daidzein use in animal feed under current conditions.

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MATERIALS AND METHODS

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The experimental protocol used in the following experiments was reviewed and approved by

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the Animal Experimental Committee of Sichuan Agricultural University. The experiment

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described here was conducted at the Animal Experiment Center of Sichuan Agricultural

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University.

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Chemicals. The daidzein added to diet in this experiment was synthetic, meeting a purity of

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98%, obtained from Guanghan Biochemical Products Co. (Deyang, China). The synthetic

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standards (glycitein, genistein, daidzein, equol) and β-Glucuronidase (129316 units/mL of

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glucuronidase activity and 775 units/mL of sulfatase activity) were obtained from Sigma-Aldrich ACS Paragon Plus Environment

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Co. (St. Louis, Mo., USA). All other reagents used for residue measurement were of HPLC-grade.

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Deionized water was used throughout.

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Experimental Design and Animal Management. Ninety-six crossbred [(Large White ×

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Landrace) × Duroc] weaned pigs (23 ± 2 d postfarrowing; 48 intact males and 48 females) were

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sorted by body weight (BW) and sex and randomly allotted to dietary treatment in a 70-d

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experiment [phase 1 (d 1-35), 4 treatments; 6 replicates (pens) per treatment; 4 pigs per pen;

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phase 2 (d 36-70), 4 treatments; 6 replicates (pens) per treatment; 3 pigs per pen]. Average initial

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BW was 6.4 ± 0.1 kg. Diets were arranged as follows: 1) The NRC (1998) standard commercial

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feed without any soy source were used as the control group (CTL), 2) CTL + 40 mg/kg of

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daidzein (the manufacturer’s recommended dose in young pigs), 3) CTL + 200 mg/kg of daidzein,

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and 4) CTL + 400 mg/kg of daidzein. Feed intake and BW measurements were obtained at the

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beginning of the experiment and weekly thereafter for the duration to determine average daily

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weight gain (ADG) and average daily feed intake (ADFI).

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All pigs were housed in a temperature-controlled room with continuous lighting. Each pen

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contained a single nipple waterer and a single self-feeder to facilitate ad libitum access to water

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and feed.

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Sample Collection. One pig was randomly selected from each pen to obtain blood samples

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collected from jugular veins on d 35 and d 70 before weighing (14 h after feeding), 10 mL of

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blood was collected into EDTA-coated pediatric Microvette tubes (Sarstedt AG and Co.,

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Numbrecht, Germany) for hematological analysis. Another 10 mL of blood from each pig was

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centrifuged at 3000 × g for 15 min to obtain serum, which was stored at -20°C until needed for

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the determination of blood markers and hormonal levels. Then the selected pigs were slaughtered ACS Paragon Plus Environment

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by a lethal injection of sodium pentobarbital to obtain samples of small intestinal segment,

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mesenteric lymph, muscle and organs (liver, heart, kidney, and spleen). The middle sections (4 cm)

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of duodenum, jejunum and ileum were isolated and kept in 10% formalin for microscopic

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assessment of mucosal morphology. About 5 cm3 of sample in regular shape from each organ was

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kept in the same way for morphological assessment. About 10 g of semitendinosus and liver

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samples were collected from each pig and kept at -20°C for residue analysis of isoflavones.

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Hormone Levels and Blood Parameters. Serum estradiol (E2), testosterone (T), growth

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hormone (GH), type-1 insulin like growth factor (IGF-1), triiodothyronine (T3), and

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tetraiodothyronine (T4) were assayed using commercially available swine ELISA kits (BioSource

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International Inc., Camarillo, CA). The minimum detectable levels were 0.8 µg/L for GH, 10 µg/L

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for IGF-1, 1.8 pmol/L for estradiol, 20 ng/L for testosterone, 3 ng/mL for T3 and 10 ng/mL for T4.

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Heparinized whole blood was assayed for white blood cell count, red blood cell count,

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hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, hemoglobin

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concentration and platelet count on an automated hematology analyzer (Sysmex XT 1800, Roche

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Diagnostics). Serum was assayed for the total protein, albumin, globulin, total bilirubin, alanine

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aminotransferase, aspartate aminotransferase, alkaline phosphatase, urea nitrogen and glucose by

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an automatic biochemical analyzer (Hitachi 7180, Japan). Antioxidant indices, including

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glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), malondialdehyde (MDA) and

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total anti oxygenic capability (T-AOC) were determined with commercially available reagent kits

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(Nanjing Jiancheng Bioengineering Institute, China). All measurements were done in duplicate at

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minimum.

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Intestinal Morphology and Organ/Tissue Histopathology. Organ (liver, heart, spleen and ACS Paragon Plus Environment

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kidney) samples, mesenteric lymph samples and intestinal segments were embedded in paraffin

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wax. Cross sections (6 µm thick) of each sample were stained with hematoxylin and eosin. A

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biological microscope with built-in digital camera was used (DM BA300, Motic, China). Organ

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and mesenteric lymph segments were observed at 100× to 400× magnifications to determine the

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morphological structures. The villus height and crypt depth of the intestinal segments were

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determined at 40× magnification to measure the lengths of 10 well-oriented intact villi and their

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associated crypt according to the methods of Touchette et al. (2002)19. The villus height-to-crypt

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depth ratio (VCR) was calculated.

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All sections were reviewed and graded in a blinded manner by a well-experienced vet from the

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pathology laboratory for histopathological analysis. For intestine, liver, kidney, heart, spleen and

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lymphoglandulae mesentericae, qualitative scorings with respective scores and grading standards

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(Table S1, see the Supplemental Information for details) were used to evaluate the health status of

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the pigs. The highest score of the pathological change observed would be the score of the section.

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For example, if in a field a pathological change score of 1 and another score of 2 were observed at

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the same time, the score of this section is 2. The average value of the scores of all the sections in

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one group would be the viscera damage indices of the treatment, ranging from 0-3.

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Distribution of Isoflavones in Body Tissues. Tissues from pigs (liver and semitendinosus

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muscle) were thawed at room temperature. Tissues (3-5 g depending on the type and amount of

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sample available) weighed with an electronic analytical balance (Hengping FA2004, Shanghai,

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China) with an accuracy of 1/10000 g were diluted with 10 mL ammonium acetate buffer solution

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(pH 5.0), homogenized with a Polytron System PET 2100 (Kinematica AG, Lucerne, Switzerland),

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then combined with 80 µL hydrolytic enzyme (β-Glucuronidase from Helix pomatia). The sample ACS Paragon Plus Environment

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mixture solution was incubated at 37°C for 8 h in a forma orbital shaker (Shanzhi TS-1102C,

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Shanghai, China). Then 10 ml of acetonitrile (Merck, Germany) and 10 mL of ethyl acetate

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(Merck, Germany) were added in sequence. After being sufficiently mixed with a vortex shaker

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(V-5, qilinbeier, Haimen, China), the samples were centrifuged at 10,000 × g for 10 min at 4°C.

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All of the supernatant was transferred to clean Erlenmeyer flasks. The Remaining isoflavones in

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the precipitate were extracted two more times with 20 ml of ethyl acetate each time to the same

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Erlenmeyer flasks. The extracts were concentrated to dry in a rotary evaporator at a negative

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pressure then dissolved with 3 mL of 50% (v/v) methanol in deionized water. The solutions were

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centrifuged at 10,000 × g for 10 min at 4°C and subsequently filtered through a 0.22-µm nylon

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membrane prior to analysis by UPLC/MS with the chromatographic conditions as follows:

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chromatographic column: thermoelectric C18.24 µm 2.4 × 50 mm (thermo); mobile phase: (A)

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water, (B) acetonitrile + methanol = 1 + 1; column temperature: 35°C; sample size: 5 µL. Using

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an electrospray ion source, negative ion mode (ESI-), multiple reaction monitoring (MRM) mode.

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The synthetic standards (glycitein, genistein, daidzein, equol) were obtained from Sigma-Aldrich.

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Three linear calibration curves (low, medium and high) with a minimum of 4 concentrations per

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curve were prepared according to the procedure of Gilani et al. (2011)18 for glycitein, genistein,

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daidzein and equol, respectively, and only calibration curves with high linearity from all analytes

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(R2 > 0.998) in the expected calibration range were used.

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Statistics. Data were compared between all of the 4 groups by subjecting them to ANOVA for a

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randomized complete block design by using the GLM procedure (SAS Inst. Inc., Cary, NC).

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Statistical differences among treatments were separated by Duncan’s multiple range tests. Results

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were expressed as mean ± standard deviation (SD). Probability values less than 0.05 were used as ACS Paragon Plus Environment

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the criterion for statistical significance. The pen served as the experimental unit in the analyses.

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RESULTS

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Weight Gain and Feed Intake. In phase 1, there was no significant difference in average daily

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gain between the 4 groups. However, pigs fed 200 mg/kg of daidzein tended to have a greater

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ADG (P = 0.082) compared with those in CTL. In phase 2, ADG of pigs was decreased (P = 0.03)

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by 400 mg/kg of daidzein supplementation when compared with CTL. No differences were found

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in feed consumption (average daily feed intake, ADFI) throughout the whole experimental period

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(Figure 1). No gender differences were found in ADG or ADFI.

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Hormone Levels and Blood Parameters. There were no effects (P > 0.05) of daidzein

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treatments on the content of triiodothyronine, tetraiodothyronine, IGF-1, GH or estradiol in the

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serum of pigs in both phase 1 and phase 2 (Table 1). However, gilts receiving 200 mg/kg and 400

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mg/kg daidzein had a reduced (P = 0.02, phase 1; P = 0.004, phase 2) serum testosterone level

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compared with pigs in CTL.

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Hematology analysis revealed that HCT, MCV and MCH in pigs was significantly increased at

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the dose of 400 mg/kg of daidzein (P < 0.05) while other hematological parameters were not

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changed after 35 days of treatment (Table 2). It is shown that 200 mg/kg of daidzein

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supplementation also increased HCT (P < 0.05) of pigs compared with CTL. Beyond that, no

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significant differences in other tested hematological parameters were noted after a 35-day

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treatment. There was no significant differences between all the parameters in phase 2. We also

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investigated the effects of daidzein on some serum biochemical indices, however there was no

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significant differences (data now shown). Gender differences were not found in any of the blood

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parameters. ACS Paragon Plus Environment

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Pigs receiving 200 mg/kg daidzein had greater (P < 0.05) SOD activity and lower (P < 0.05)

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MDA content in serum compared with CTL in phase 1 (Table 3). However, T-AOC and GSH-PX

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were not affected by treatments. There were no significant differences between treatments on

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antioxidant activities in phase 2 (data not shown). And besides, there were no gender differences

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found in the antioxidant indices tested.

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Intestinal Morphology and Organ/Tissue Histopathology. Histological evaluation showed

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no effect of dietary supplementation of daidzein on villus height of the small intestine. However,

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pigs fed 200 mg/kg and 400 mg/kg of daidzein had smaller (P < 0.05) crypt depth and greater (P

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< 0.05) VCR in the duodenum compared with that in CTL on d 35 (Figure 2). However, intestinal

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morphology was not effected by daidzein in phase 2 (data not shown). No gender differences

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were observed in villus height, crypt depth or VCR.

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According to the histology score (organ damage index), there were few observable effects of 40

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mg/kg and 200 mg/kg of dietary daidzein supplementation on the morphology of all the organs

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and tissues examined in both phase 1 and 2. However, 400 mg/kg of daidzein supplementation

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could cause mild damage (P < 0.05) to the spleens of weanling pigs by d 70 (Figure 3) (this

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effect was not obvious on d 35, data not shown). Meanwhile, 400 mg/kg of daidzein had a trend

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to increase (P = 0.08) the histology score of livers at the end of phase 2. Histology score shows no

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evident gender differences.

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Distribution of Isoflavones in Body Tissues. The results (Figure 4) indicate that the amount

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of daidzein deposited in the liver and muscle of pigs was not affected (P > 0.05) by dietary

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daidzein supplementation for 35 days. In view of the fact that trace amounts of the soy

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isoflavones detected in diet (data not shown), we also monitored levels of glycitein and genistein ACS Paragon Plus Environment

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in animal tissues. On d 35, the contents of glycitein and genistein in the liver were decreased by

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ingesting daidzein. Genistein level in the muscle (semitendinosus) was also decreased by 400

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mg/kg dietary daidzein supplementation compared with CTL. On the 70th day of this experiment,

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dietary daidzein supplementation improved the content of daidzein (P < 0.05) in pigs’ liver

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compared with that in the control group. Similar to the result on the 35th day, the content of

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glycitein in the liver was decreased by 200 mg/kg or 400 mg/kg of dietary daidzein

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supplementation compared with CTL on d 70. In addition, the contents of equol tended to be

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enhanced in livers (P = 0.09) and muscles (P = 0.1) of pigs by dietary daidzein supplementation

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in phase 2. It was shown that there were greater distributions of isoflavones in liver than in

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muscle in both phase 1 and phase 2. Meanwhile, in any of the 4 groups, the level of daidzein in

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the liver on d 70 was higher (P < 0.01) than on d 35. No gender differences were found in tissue

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residues of isoflavones.

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DISCUSSION

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Previous studies show that 20 or 40 mg/kg of dietary daidzein has no effect on the growth of

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mice20, but that 1000 mg/kg significantly decreases the body weight of female rats17. This

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suggests that there is a dose effect in terms of growth, regardless of gender. As mentioned above,

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the structure and function of daidzein resembles that of steroid estrogens. In many animals,

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including pigs, low concentration of daidzein bind weakly to estrogen receptors. However,

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daidzein at higher levels may act as anti-estrogen by antagonizing the binding of potent natural

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estrogens to its receptors21, 22. Studies show that estrogen affects weight gain in human and

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murines23, 24. Therefore, we speculate that the mechanism for daidzein’s effects on pig growth

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relates to this feature. Further research is needed to elucidate the growth axis of daidzein/estrogen ACS Paragon Plus Environment

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receptors.

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Nevertheless, it is unclear whether other hormones related to growth are linked to daidzein’s

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effects in regulating weight gain. Some studies show that daidzein promotes levels of

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growth-regulating hormones in breeding poultry (GH and tetraiodothyronine)25, 26 and intact male

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piglets (IGF-1)27. Other research indicates the inhibitory effect of daidzein in vitro and in vivo14, 28.

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Beyond these debates, our results show that daidzein is not effective enough to alter these

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hormones related to growth. This suggests that the function of daidzein in regulating growth is not

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through these hormones. Furthermore, studies show that endogenous estradiol is not affected by

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daidzein ingestion29, which accords with our results.

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Other studies indicate that dietary genistein (20 mg/kg/day) decreases testosterone plasma

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levels of male rats, while the same doses of daidzein have no effect in this regard29. However, our

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results reveal the inhibitory effect of higher concentrations (200 and 400 mg/kg) of daidzein on

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the serum testosterone content for female pigs. This suggests that gender and dosage may be

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critical factors for daidzein in terms of regulating testosterone. This effect is also evident in

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porcine ovaries, suggesting that daidzein may disturb reproductive processes in pigs22. An in vitro

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study showed that daidzein increases StAR and 3β-HSD-1 mRNA levels, thereby increasing

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testosterone production in cultured mouse leydig cells in the presence of the chorionic

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gonadotropic hormone30. Alexa et al. (2014) reported that daidzein alone increases testosterone

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production in porcine ovarian cells, but that in the presence of follicle-stimulating hormone (FSH),

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daidzein prevents the FSH-stimulated release of testosterone31. In fact, the endocrine system is a

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complex regulatory system involving various interactions between hormones. Therefore, the

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mechanisms for the testosterone-reducing effect of daidzein in female piglets and how it plays a ACS Paragon Plus Environment

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role in regulating steroid hormone metabolism in vivo warrant further study.

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Numerous studies reveal daidzein’s reactivity with active oxygen species. Therefore, it appears

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to have antioxidant potential via a radical scavenging mechanism4, 32. Our results indicate the

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efficacy of daidzein in piglets (d 35) in an oral dose of 200 mg/kg, suggesting that the protective

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effects of daidzein against oxidation are more pronounced in piglets than growing pigs. A

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decrease in MDA suggests decreased lipid peroxidation, a process which is initiated by free

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radical reactions33. However, in our study, a decrease in MDA may be also associated with an

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increase in the activity of total SOD. It is not yet clear whether daidzein develops its antioxidant

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potential via a direct radical scavenging mechanism or by improving the activity of other

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antioxidant enzymes such as SOD. Therefore, additional functional analyses are required to reveal

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how daidzein affects the free-radical scavenging system.

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In this study, we originally evaluated the effect of daidzein on intestinal morphology. Results

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show an increasing proliferation of duodenal mucosa by 200 mg/kg of daidzein. As previously

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noted, the same dose of daidzein tended to improve the ADG of pigs. This may be partly due to

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the increased VCR in the duodenum, since the absorption capacity of the small intestine is

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affected by VCR34. Some studies show that increases in SOD lead to a decrease in intestinal crypt

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depth35, which accords with our results. Including soybean meal (5 g/d) in the diets of

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early-weaned pigs has been found to decrease villus height and increase crypt depth, leading to

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transient hypersensitivity and subsequently poor growth performance36. However, there is no

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evidence to indicate that this effect mainly stems from soy isoflavones.

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Previous studies on the effect of daidzein or isoflavones on organ development have mainly

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focused on reproductive issues. Findings show that daidzein or isoflavones at low concentrations ACS Paragon Plus Environment

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have no significant effect on reproductive organs in hens (50 mg/kg daidzein) and mice (2 mg

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daidzein + 5 mg genistein/kg body weight/day)37, 38. To our knowledge, our study is the first to

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evaluate the long-term effects (35 and 70 days) of dietary daidzein at the high level of 400 mg/kg

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on pig organs. We employed histopathological scoring to identify the effects of dietary daidzein

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on pig organs. Our results suggest that ingesting high doses of daidzein for 70 d may increase

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spleen damage in pigs. This may compromise the ADG of pigs. However, the mechanisms

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involved are not clearly understood, and hence the biochemical and toxic responses of daidzein

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deserve further study.

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Previous studies on the tissue distribution of isoflavones in animals are limited. Urpi-Sarda et

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al. found that the major metabolites recovered in ewe tissues (liver and mammary glands) after

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consumption of red clover silage were equol and daidzein39. Gilani et al. studied tissue

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distribution of isoflavones in pigs following a daily dose of 2.3 g of total isoflavones. They found

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higher levels of daidzein, equol, genistein and glycitein in livers, compared with the CTL

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containing no isoflavones. The same effect was found in mammary gland extraparenchymal tissue

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of pigs for only daidzein and equol18. Our literature review found no studies on build-up in tissues

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when daidzein is added alone.

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In our study, it was not surprising that daidzein was the metabolite found in the greatest

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amounts in pig livers. Unlike the findings of Urpi-Sarda and Gilani, we found that equal levels in

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both liver and muscle were low. The production of equol differs between species40 and age stages

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(in a rat study)41. These levels were not affected by the amount of daidzein ingested, which was

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possibly due to the pigs being too young to produce equol themselves.

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Another notable finding from our study is that genistein and glycitein displayed an opposite ACS Paragon Plus Environment

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distribution from that of daidzein in pig tissue when the level of dietary daidzein was increased.

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Reports had shown that the fraction of daidzein absorbed is greater than that of genistein42, 43.

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Thus, we can speculate that daidzein acts as a competitive inhibitor against genistein and glycitein

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when they are deposited in animal tissue. Further studies will elucidate the absorption approach

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and deposition method of daidzein and other isoflavones, as well as the interaction between them

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in terms of build-up in animal tissue.

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Another concern is that isoflavones may have a cumulative effect44, so that the biological

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effect is evident only after longer durations. Our findings indicate that daidzein may have a

295

cumulative effect over time in terms of build-up in body tissues, as indicated by daidzein levels in

296

pig livers on d 35 and d 70. This suggests a link between daidzein levels in animal products and

297

the duration of ingestion.

298

In conclusion, this study on the biological activity of daidzein in pigs depends on dosage and

299

the age of the pigs, as well as the duration of treatment. It may be a feasible option to give up to

300

200 mg/kg of daidzein to weanling pigs without negative consequences. As for the effects on

301

consumer health, our findings reveal that the residual content of daidzein in pork products were

302

low to raise safety concerns.

303

CONFLICTS OF INTEREST

304 305

The authors declare that there are no conflicts of interest. ACKNOWLEDGMENTS

306

This work was financially supported by the Scientific and Technical Support Program of the

307

“Twelfth Five-Year Plan’’, Feed Quality and Safety Supervision Project of Ministry of Agriculture

308

(2012), PR China. ACS Paragon Plus Environment

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309

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310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351

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development of reproductive organs in female mice. J. Toxicol. Environ. Health 2012, 75, 649-660. 39. Urpi-Sarda, M.; Morand, C.; Besson, C.; Kraft, G.; Viala, D.; Scalbert, A.; Besle, J.-M.; Manach, C., Tissue distribution of isoflavones in ewes after consumption of red clover silage. Arch. Biochem. Biophys. 2008, 476, 205-210. 40. Gu, L.; House, S. E.; Prior, R. L.; Fang, N.; Ronis, M. J.; Clarkson, T. B.; Wilson, M. E.; Badger, T. M., Metabolic phenotype of isoflavones differ among female rats, pigs, monkeys, and women. J. Nutr. 2006, 136, 1215-1221. 41. Sepehr, E.; Cooke, G. M.; Robertson, P.; Gilani, G. S., Effect of glycosidation of isoflavones on their bioavailability and pharmacokinetics in aged male rats. Mol. Nutr. Food Res. 2009, 53, S16-S26. 42. King, R. A.; Bursill, D. B., Plasma and urinary kinetics of the isoflavones daidzein and genistein after a single soy meal in humans. Am. J. Clin. Nutr. 1998, 67, 867-872. 43. Setchell, K. D.; Faughnan, M. S.; Avades, T.; Zimmer-Nechemias, L.; Brown, N. M.; Wolfe, B. E.; Brashear, W. T.; Desai, P.; Oldfield, M. F.; Botting, N. P., Comparing the pharmacokinetics of daidzein and genistein with the use of 13C-labeled tracers in premenopausal women. Am. J. Clin. Nutr. 2003, 77, 411-419. 44. Murkies, A.; Lombard, C.; Strauss, B.; Wilcox, G.; Burger, H.; Morton, M., Dietary flour supplementation decreases post-menopausal hot flushes: effect of soy and wheat. Maturitas 1995, 21, 189-195.


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412

FIGURE CAPTIONS

413

Figure 1. Effects of increasing concentrations of daidzein on performance of pigs in phase 1 (d

414

1-35) and 2 (d 36-70). (A) Average daily weight gain (ADG) of repeated dose daidzein-treated

415

piglets with an initial average BW of 6.4 kg. (B) Average daily feed intake (ADFI) of repeated

416

dose daidzein-treated piglets. Each bar represents the mean ± SD of 6 experimental units (pens).

417

CTL, control group fed a basal diet. 40/200/400 DA, 40/200/400 mg/kg daidzein supplemented to

418

CTL. *Significantly different from control at P < 0.05. NS, not significant (P > 0.05).

419

Figure 2. Intestinal morphology of repeated dose daidzein-treated piglets. (A)-(C) Villus height

420

and crypt depth of duodenum, jejunum and ileum, respectively on d 35. (D) The villus

421

height-to-crypt depth ratio (VCR) of duodenum, jejunum and ileum. Each bar represents the mean

422

± SD of 6 experimental units (pens) (n = 6). CTL, control group fed a basal diet. 40/200/400 DA,

423

40/200/400 mg/kg daidzein supplemented to CTL. Bars with different letters are significantly (P

424

< 0.05) different from each other. NS, not significant (P > 0.05).

425

Figure 3. Organ damage indices of repeated dose daidzein-treated pigs on d 70. (A) - (D) The

426

damage degree of liver, heart, kidney and spleen of pigs in the 4 treatments, respectively. The

427

damage indices ranges from 0-3 (0: completely healthy; 1: mild injury; 2: moderate injury; 3:

428

severe injury (not shown in the graphs). Each dot represents the organ damage score of an

429

individual pig (n = 6). Data are also expressed as mean ± SD. CTL, control group fed a basal diet.

430

40/200/400 DA, 40/200/400 mg/kg daidzein supplemented to CTL. Bars with different letters are

431

significantly (P < 0.05) different from each other. NS, not significant (P > 0.05).

432

Figure 4. Concentrations of several isoflavones (µg/kg) in livers and muscles (semitendinosus) of

433

repeated dose daidzein-treated pigs on d 35 and d 70. (A) - (D) Residual Amount of daidzein,

434

Paragon Plus Environment genistein, glycitein and equol inACS tissues, respectively. Each bar represents the mean ± SD of 6

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435

experimental units (pens) (n = 6). CTL, control group fed a basal diet. 40/200/400 DA,

436

40/200/400 mg/kg daidzein supplemented to CTL. Bars with different letters are significantly (P

437

< 0.05) different from each other. NS, not significant (P > 0.05).


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Table 1. Serum hormone levels of repeated dose daidzein-treated pigs on d 35 and d 70.1 Dietary daidzein, mg/kg Item2

control

40

200

400

D 35 T3

14.8 ± 0.13

15.0 ± 0.85

16.0 ± 3.18

14.7 ± 0.075

T4

121 ± 19.7

117 ± 21.9

132 ± 12.5

110 ± 10.0

IGF-1

150 ± 9.24

148 ± 27.7

157 ± 8.01

148 ± 36.9

GH

77.0 ± 14.0

88.8 ± 12.0

90.7 ± 18.0

81.9 ± 7.60

T-m

296 ± 72.8

346 ± 168

334 ± 167

320 ± 64.6

T-f

362 ± 47.7a

324 ± 37.6ab

246 ± 74.5bc

224 ± 20.2c

E2-m

38.4 ± 2.00

44.3 ± 6.43

42.3 ± 5.98

38.3 ± 2.46

E2-f

37.5 ± 0.53

39.1 ± 3.45

36.3 ± 2.10

40.8 ± 14.3

D 70 T3

15.1 ± 0.42

15.7 ± 2.54

15.4 ± 1.07

14.8 ± 0.23

T4

100 ± 13.0

101 ± 13.1

100 ± 23.5

85.0 ± 9.98

IGF-1

151 ± 38.4

150 ± 39.8

159 ± 24.3

148 ± 15.0

GH

80.8 ± 14.9

83. 8 ± 20.5

91.8 ± 16.3

69.5 ± 21.0

T-m

272 ± 98.1

225 ± 13.8

245 ± 6.43

238 ± 57.4

T-f

374 ± 37.6a

344 ± 43.8ab

252 ± 37.6bc

219 ± 76.6c

E2-m

37.6 ± 3.17

39.9 ± 6.59

38.8 ± 0.61

38.2 ± 3.34

E2-f

36.2 ± 3.14

42.9 ± 3.28

40.6 ± 7.51

40.7 ± 1.55

a-c

1

Means in the same row with different superscripts differ (P < 0.05).

Values of T3, T4, GH and IGF-1 are means ± SD (n = 6). Values of T-m, T-f, E2-m and E2-f are

means ± SD (n = 3). 2

T3 = triiodothyronine, µg/L, T4 = tetraiodothyronine, µg/L, IGF-1 = type-1 insulin like growth

factor, µg/L, GH = growth hormone, µg/L, T-m = testosterone level of male pigs, ng/L, T-f = testosterone level of female pigs, ng/L, E2-m = estradiol level of male pigs, pmol/L, E2-f = ACS Paragon Plus Environment

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estradiol level of female pigs, pmol/L.


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Table 2. Haematology and clinical biochemistry analysis of repeated dose daidzein-treated pigs on d35 and d 70.1 Dietary daidzein, mg/kg Item2

control

40

200

400

D 35 WBC

14.5 ± 3.45

14.7 ± 2.12

17.4 ± 4.51

16.9 ± 3.22

RBC

6.01 ± 0.37

6.00 ± 0.50

6.38 ± 0.42

6.22 ± 0.48

HGB

96.2 ± 6.91

100 ± 10.4

105 ± 8.17

105 ± 19.2

HCT

0.350 ± 0.016b 0.350 ± 0.036b 0.390 ± 0.16a

0.384 ± 0.0090a

MCV

58.1 ± 2.10b

58.4 ± 4.05b

61.4 ± 3.21ab

63.1 ± 2.73a

MCH

15.3 ± 1.43b

15.7 ± 1.41b

16.8 ± 0.54ab

17.3 ± 0.75a

MCHC 274 ± 14.3

271 ± 6.98

274 ± 6.69

274 ± 8.18

PLT

398 ± 46.4

440 ± 54.3

379 ± 62.5

438 ± 86.3

D 70 WBC

15.4 ± 1.05

20.1 ± 5.78

19.6 ± 2.41

17.4 ± 1.78

RBC

6.85 ± 0.51

6.77 ± 0.73

6.72 ± 0.47

6.86 ± 0.51

HGB

106 ± 7.79

107 ± 8.41

113 ± 7.77

113 ± 8.52

HCT

0.398 ± 0.025

0.392 ± 0.028

0.420 ± 0.029 0.413 ± 0.023

MCV

57.8 ± 1.49

58.0 ± 5.03

62.0 ± 2.90

60.1 ± 2.65

MCH

15.4 ± 0.48

15.8 ± 1.40

16.9 ± 0.94

16.6 ± 0.72

MCHC 267 ± 7.42

273 ± 3.76

273 ± 8.26

275 ± 9.97

PLT

469 ± 87.1

474 ± 133

532 ± 35.3

a, b

488 ± 178


1

Values are means ± SD (n = 6).

2

WBC = White Blood Count, 10^9/L, RBC = red blood cell count, 10^12/L, HGB = hemoglobin,

g/L, HCT = hematocrit, %, MCV = Mean Corpuscular Volume, fL, MCH = mean corpuscular hemoglobin, pg, MCHC = mean corpuscular hemoglobin concentration, g/L, PLT = blood platelet count, 10^9/L. ACS Paragon Plus Environment

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Table 3. Serum antioxidant levels of repeated dose daidzein-treated pigs on d35.1 Dietary daidzein, mg/kg Item2

control

GSH-PX 729 ± 127

40

200

400

795 ± 172

1022 ± 361

845 ± 244

SOD

18.4 ± 1.39b 18.8 ± 1.59b 21.0 ± 0.78a 19.6 ± 0.53ab

MDA

7.94 ± 2.05a 7.78 ± 1.83a 5.42 ± 1.44b 6.51 ± 0.95ab

T-AOC

10.4 ± 2.26

a, b

10.2 ± 3.02

11.2 ± 2.21

11.2 ± 3.02


1

Values are means ± SD (n = 6).

2

GSH-PX = glutathione peroxidase, U/mL, SOD = superoxide dismutase, U/mL, MDA =

malondialdehyde, nmol/mL, T-AOC = total antioxygenic capability, U/mL.


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Figure 1


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Figure 2


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Figure 3


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Figure 4


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Potential Risk of Isoflavones: Toxicological Study of Daidzein

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