Health effects of dietary oxidized tyrosine and dityrosine administration

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Health effects of dietary oxidized tyrosine and dityrosine administration in mice with nutrimetabolomic strategies Yuhui Yang, Hui Zhang, Biao Yan, Tianyu Zhang, Ying Gao, Yonghui Shi, and Guowei Le J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02003 • Publication Date (Web): 25 Jul 2017 Downloaded from http://pubs.acs.org on July 26, 2017

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Journal of Agricultural and Food Chemistry

Health Effects of Dietary Oxidized Tyrosine and Dityrosine Administration in Mice with Nutrimetabolomic Strategies Yuhui Yang†, Hui Zhang†, Biao Yan†, Tianyu Zhang†, Ying Gao†, Yonghui Shi†,‡, Guowei Le†,‡,* †

The Laboratory of Food Nutrition and Functional Factors, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China

‡

The State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China

Corresponding author * Guowei Le: Tel, 0510-85917789; Fax, +86-510-85869236; E-mail, [email protected].

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ACS Paragon Plus Environment


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ABSTRACT: This study aims to investigate the health effects of long-term dietary oxidized

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tyrosine (O-Tyr) and its main product (dityrosine) administration on mice metabolism. Mice

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received daily intragastric administration of either O-Tyr (320 µg/kg body weight), dityrosine

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(Dityr, 320 µg/kg body weight) or saline for consecutive six weeks. Urine and plasma

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samples were analyzed by NMR-based metabolomics strategies. Body weight, clinical

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chemistry, oxidative damage indexes and histopathological data were obtained as

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complementary information. O-Tyr and Dityr exposure changed many systemic metabolic

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processes, including reduced choline bioavailability, led to fat accumulation in liver, induced

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hepatic injury and renal dysfunction, resulted in changes in gut microbiota functions, elevated

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risk factor for cardiovascular disease, altered amino acid metabolism, induced oxidative stress

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responses, and inhibited energy metabolism. These findings implied that it is absolutely

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essential to reduce the generation of oxidation protein products in food system through

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improving modern food processing methods.

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KEYWORDS: oxidized tyrosine, dityrosine, health effects, metabolism, metabonomics

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INTRODUCTION

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Proteins are major ingredients of most foods (particularly many high-protein foods, such as

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meat, eggs, beans and milk products) in a healthy diet. Meanwhile food proteins are targets

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for reactive oxygen species (ROS), other radical and non-radical species, which makes them

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vulnerable to be oxidized in the process of food processing, storage, cooking, and food

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consumption during the subsequent digestion phases in gastrointestinal tracts.1 However, this

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question was largely overlooked for several decades while the oxidation of other food

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components, such as lipids, was studied in depth.2 The protein oxidation leads to many

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structural changes including loss of sulfhydryl groups, cleavage of peptide bonds, formation

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of cross-links and protein carbonyls, and modification of amino acids.3 In vitro, oxidized

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protein products can be easily found in food system, and leading to a reduced food

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digestibility and nutritional value.4 The past researches about food proteins is typically

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concentrated on the quantity, quality and bioavailability, the effects of consumption of dietary

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oxidized protein products on human nutrition and health aspects are typically ignored.

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Currently, the increasing interest among food scientists in this field has led to highlight the

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influence that consumption of oxidized protein products may have on human nutrition and

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health. The last decade has showed epidemiological evidence of a positive association

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between high consumption of red meat or processed meat and the risk of developing a series

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of diseases, including cardiovascular disease, diabetes, and colorectal cancer.5-6 Milk powder

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containing oxidized protein products led to lower growth response and caused eczema and

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dry stool in infants.7 Our past research has shown that dietary oxidized casein exposure

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caused oxidized injury by impairing antioxidant defense system, and induced fibrosis injury

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in mice liver and kidney by accumulating advanced oxidation protein products (AOPPs) and

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changing the related gene expressions of fibrosis.8 Several previous studies have also

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demonstrated that dietary sources of oxidized amino acids may be used for the de novo

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synthesis of proteins such as enzymes and structural components in cells, and potentially

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contributing to cell malfunction and apoptosis and disease status in body.9-11

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Tyrosine (Tyr) is one of the most easily oxidized amino acids in food proteins, with

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representative oxidative products including dityrosine (Dityr),12 which is widely detected in

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food system and identified as universal biomarkers of protein oxidation.13-14 Our previous

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studies have shown that 24-weeks exposure to dietary oxidized tyrosine (O-Tyr) can lead to

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oxidative stress, inflammation and fibrosis in rat liver and kidney.15-16 In addition, our

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previous experiments have also provided evidence that Dityr, which is the major oxidized

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product in O-Tyr (accounting for 22%), may be responsible for the O-Tyr-induced kidney

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injury.16 We have also found O-Tyr-induced oxidative injury in the pancreas potentially

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resulted in the disruption of glucose metabolism. And Dityr is responsible for O-Tyr-induced

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pancreatic injury.17 Moreover, our research has also demonstrated that Dityr exposure causes

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novel object recognition deficits in mice.18 What's more, our study has also showed that

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one-week O-Tyr and Dityr exposure can enhance energy metabolism, induce oxidative stress

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and alter gut microbiota functions.19 However, the exact mechanisms of the potential

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deleterious effects of long-term dietary O-Tyr and Dityr exposure are still not fully

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understood.

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Metabolomics in combination with multivariate pattern recognition analysis methods is a

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powerful systems biological tool which is well suited to facilitate the understanding of global

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metabolic variations in animals and humans in responses to changes in nutrition, genetics,

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environment, and gut microbiota.20-22 Multivariate pattern recognition analysis methods, such

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as principal component analysis (PCA), partial least-squares discriminant analysis (PLS-DA)

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and orthogonal partial least-squares discriminant analysis (OPLS-DA), are employed to

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reduce the interpretational challenge from large data sets.23-24 This strategy can be used to

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identify experimental groups with similar metabolic patterns, and discriminate significant

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different metabolites in these experimental groups.25 1H nuclear magnetic resonance (NMR)

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spectroscopy is one of the main methods used in metabolomics researches. And 1H NMR

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spectroscopy of tissues extracts and biological fluids generates global biochemical profiles of

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small molecular compounds (molecular weight less than 1000) that are regulated in responses

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to multifarious stresses to keep homeostasis. Recently, there are many studies that have

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demonstrated the 1H NMR-based metabonomics approach can be considered as an emerging

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and promising field of science with a level of information that outweighs conventional

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analytical methods in the aspects of elucidating systemic metabolic responses to dietary

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intervention and their unrecognized mechanisms.21, 26-29

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In the current study, we hypothesized long-term O-Tyr and Dityr exposure can induce

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global metabolic changes in mouse urine and serum. To verify this hypothesis, 1H

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NMR-based metabonomics approach combined with multivariate pattern recognition analysis

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methods, traditional clinical chemistry analysis, oxidative damage indexes and oxidative

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stress status analysis and histopathological analysis were employed to investigate the effects

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of long-term O-Tyr and Dityr exposure on systemic endogenous metabolites of mice urine

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and plasma. The purpose of this investigation was to explore the potential deleterious effects

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of long-term dietary O-Tyr and Dityr exposure in mice, which could be very important for

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food safety assessment, and modern food processing methods and human dietary habits

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improvement.

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MATERIALS AND METHODS

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Materials

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Tyr was acquired from Sigma Chemical Co. (St. Louis, MO, USA). O-Tyr sample was

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produced in our lab based on our previous studies.15-16 The method of HPLC-MS

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chromatogram was used in qualitative analysis of O-Tyr products. The results demonstrated

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that the O-Tyr products used in our study mainly contain Tyr, Dityr and 3-nitrotyrosine

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(3-NT), whose chemical structures were shown in our past research.15 Quantitative analysis

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of Dityr content in O-Tyr sample was performed in our previous study. We found Dityr

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accounts for 22% in the total O-Tyr products.16 Dityr was purchased from Xiamen Huijia

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Biotechnology Co., Ltd. (Xiamen, China). Phosphate buffer was prepared with

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K2HPO4•3H2O and NaH2PO4•2H2O for their good solubility and low-temperature stability, as

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previously reported.30 D2O (99.9% in D) and TMSP [3-(trimethylsilyl) propionic-(2,2,3,3-d4)

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acid sodium salt] were purchased from Cambridge Isotope Laboratories (MA, USA). ELISA

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kits of AOPPs, Dityr and 3-NT were obtained from Xiamen Huijia Bioengineering Institute

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(Xiamen, China). ELISA kit of choline was purchased from BioVision, Inc. (BioVision,

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USA). ELISA kit of very low density lipoprotein (VLDL), and detection kits of total

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antioxidant capacity (T-AOC), malondialdehyde (MDA), reduced glutathione (GSH),

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oxidized glutathione (GSSG), alanine aminotransferase (ALT), aspartate aminotransferase

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(AST), total protein (TP), plasma creatinine (Cre), triglyceride (TG), cholesterol (CHO) and

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low density lipoprotein (LDL) were all purchased from Nanjing Jiancheng Bioengineering

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Institute (Nanjing, China).

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Animals, Experimental Design and Sample Collections

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The experiment was approved by the Animal Care and Use Committee of the Animal

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Nutrition Institute of Jiangnan University and was performed according to the Chinese

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guidelines for animal welfare and experimental protocol. Thirty SPF female Kunming mice

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(aged approximately 4 weeks, weighted 25 ± 1 g) were purchased from the Suzhou

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University (Suzhou, China) and housed in an environmentally controlled facility (temperature

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20-24 °C, relative humidity 40-60%, 12 h light and dark cycle) at Jiangnan University Center

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for Animal Experiment (Wuxi, China). All mice had free access to the same standard diet and

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drinking water during the whole experiment. After 1 week of acclimation, all mice were

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randomly divided into three groups: (a) the control group (n = 10), which received daily

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intragastric administration of physiological saline solution (0.9% NaCl) for consecutive 6

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weeks; (b) the O-Tyr group (n = 10), which received daily intragastric administration of

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O-Tyr (320 µg/kg body weight) for consecutive 6 weeks; (c) the Dityr group (n = 10), which

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received daily intragastric administration of Dityr (320 µg/kg body weight) for consecutive 6

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weeks. The dosage chosen for O-Tyr and Dityr were based on our past researches.15-16, 18, 31

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The body weight of each mouse was recorded every three days during the 6 weeks of

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experimental period.

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Each mouse urine sample was collected into ice-cooled tubes containing 30 µL of sodium

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azide solution (1.0% w/v) from day 41 to day 42 of the overall exposure period (from 08:00

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a.m. to 08:00 a.m. of the next day). At the end of a 42 d exposure period, all mice were fasted

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for 12 h to avoid a postprandial effect on plasma metabolites, and sacrificed following

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anaesthesia (intraperitoneal injection of sodium pentobarbital). Blood samples were collected

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(10:00 a.m.-12:00 a.m.) into Eppendorf tubes containing sodium heparin as anticoagulant.

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After kept at 4 °C for 30 min, plasma samples were obtained by centrifugation at 3500 g for

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15 min at 4 °C. All urine and plasma samples were stored at −80 °C until ready for NMR

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spectroscopy analysis, traditional clinical chemistry and oxidative stress status analysis. Liver

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and kidney tissues from each mouse were immediately collected and weighed, washed in

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sterile 0.9% (w/v) NaCl solution and cut into two parts, one for the analysis of histopathology,

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and the remaining parts were stored at −80 °C for the other assays.

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Determination of Oxidative Damage

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Protein oxidative damage was determined by the formation of oxidized protein products

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(AOPPs, Dityr and 3-NT) concentrations, using the kits as described by the manufacturer’s

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instructions. The lipid membrane damage was observed by formation of products of lipid

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peroxidation (MDA) concentration, using the kits as described by the manufacturer’s

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instructions.

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Determination of Oxidative Stress Status

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ROS, T-AOC and GSH/GSSG levels were measured as indicators of oxidative stress status.

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The activitiy of T-AOC and GSH/GSSG levels in plasma, liver and kidney tissues were

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determined using kits as described by the manufacturer’s instructions. ROS level was

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measured in the whole blood, liver and kidney tissues by luminol-dependent

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chemiluminescence assay as described by Kobayashi et al..32 ROS production was expressed

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as relative light units (RLUs).

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Plasma and Liver Assays

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The activities of AST and ALT, and the contents of TP and Cre were determined using kits as

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described by the manufacturer’s instructions.

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Liver lipids were extracted as previously reported method with a little modification.33

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Briefly, mouse liver tissue (100 mg) was homogenized with 1.5 mL of isopropanol. After

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supersonic treatment, the homogenate was centrifuged at 2000 g, 15 min. Subsequently, the

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supernatant was used with a kit for measuring TG and CHO.

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Histological Analysis of the Liver and Kidney Tissues

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Histology analysis of liver and kidney tissues were performed by hematoxylin and eosin

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staining (H&E) according to previously described methods.34 In order to elucidate whether

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O-Tyr and Dityr exposure lead to lipids accumulation in mice liver, we measured the lipids

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content using the oil red O stain technique.35 The images were acquired using CX31 RTSF

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microscope (Olympus Corporation, Tokyo, Japan) and Image-Pro Plus version 7.0 (Media

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Cybernetics). The stained tissue sections were evaluated at 200× magnification by a

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pathologist blind to the treatment groups by using 5 fields per section.36

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Quantitative Analysis of LDL, VLDL and Choline

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The levels of LDL and VLDL in plasma were quantitatively determined using kits as

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described by the manufacturer’s instructions. Choline level was quantitatively measured

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fluorometrically in plasma and liver tissue using a choline quantification kit according to the

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manufacturer’s procedure.

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Sample Preparation and 1H NMR Spectroscopy

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The plasma and urine samples were prepared according to the methods of previously reported

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with a little modification.37 100 µL plasma was mixed with 500 µL phosphate buffer saline

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solution (0.9% NaCl, 45 mM K2HPO4/NaH2PO4, PH 7.4, 100% D2O). The phosphate buffer

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was used because of their good solubility and low-temperature stability.30, 38 Approximately

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550 µL of each sample was shifted to 5 mm NMR tube for NMR detection after vortex

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mixing and centrifugation (11000 g, 4 °C and 10 min). 100 µL urine was mixed with 450 µL

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D2O and 55 µL of phosphate buffer saline solution (1.5 M K2HPO4/NaH2PO4, PH 7.4, 100%

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D2O) containing 0.1% 3-(trimethylsilyl) propionic-(2,2,3,3-d4) acid sodium salt (TSP) as a

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chemical shift reference (δ 0.00 ppm). After vortex mixing and centrifugation (11000 g, 4 °C

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and 10 min), the supernatant (550 µL) was transferred into 5 mm NMR tube for subsequent

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NMR detection. NMR spectral analysis of all urine and plasma samples was conducted by

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Wuhan Zhongke Metaboss Technology CO., LTD (Wuhan, China). The 1H NMR spectra of

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all samples were acquired on a Bruker Avance II 600 MHz NMR spectrometer (Bruker

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Biospin, Germany) with a 5 mm broadband observe probe operating at a proton frequency of

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600.58 MHz and a temperature of 298 K. The detection parameters of urine and plasma

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samples were set on the basis of the previous study.39 The NMR signals of water molecules

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and macromolecules (proteins and lipoproteins) resonances in all plasma samples were

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suppressed by Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence [recycle delay−90°−

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(τ–180°–τ)n–acquisition]. The 1H NMR spectra of urine samples were obtained using the first

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increment of nuclear overhauser effect spectroscopy (NOESY) pulse sequence [recycle delay

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(RD)−90°−t1−90°−tm−90°−acquisition] with water presaturation. For assignment purposes,

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selected samples of plasma and urine.

H-1H total correlation spectroscopy and 1H-1H correlation spectroscopy were obtained from

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All 1H NMR spectrums processing in this study were performed according to the method of

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Liu et al.40 These spectra were first manually adjusted for possible phase and baseline

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deformation. Plasma spectra were calibrated to the chemical shift (δ 1.33 ppm) of the methyl

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group in lactate, whereas urine spectra were calibrated to the chemical shift of TSP peak (δ

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0.00 ppm). All the 1H NMR spectral regions of 0.5-9.5 ppm were separated and integrated

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into small regions with an equal width of 0.01 ppm using Mestrenova 6.1.1 software

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(Mestrelab Research S.L., Spain). The spectral signals of all urine and plasma samples

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containing water peak (δ 4.68-5.16 ppm) were excluded to avoid the effects of incomplete

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water suppression. Additionally, the spectral regions from urea peak (δ 5.70-6.00 ppm) were

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removed from urine spectra. Signals normalization to total area of the spectrum was

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performed for urine and plasma spectra before multivariate pattern recognition analysis. The

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peaks of 1H NMR spectra are assigned to definite metabolites according to the published

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literature39, 41-46 and existing databases (the Human Metabolome Data Base),47 and they are

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validated by two-dimensional NMR spectra on selected samples.48

H NMR Spectral Data Processing and Statistical Analysis

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Multivariate pattern recognition analysis was performed with reference to the previously

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reported methods.49-51 The normalized 1H NMR datasets from plasma and urine samples were

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imported into software package SIMCA-P 13.0 (Umetrics, Umea, Sweden) for multivariate

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pattern recognition analysis, respectively. Initially, PCA was carried out as unsupervised

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clustering to identify any trends or outliers in the data sets. Subsequently, PLS-DA was used

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as supervised clustering to further explore the difference among the control, O-Tyr and Dityr

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groups mice. The model parameters of R2X (indicating the total explained variation) and Q2

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(showing the model predictability) were used to estimate the quality of the model. R2 and Q2

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all greater than 0.50 indicate that the model is robust and fit. Leave-one-out cross validation

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and the permutation test (200 cycles) were performed to evaluate the reliability of the model.

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Furthermore, OPLS-DA was also used as supervised pattern recognition approach to

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maximize the separation between the experiment group mice and the control group mice.

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X-matrix is the scaled NMR data and Y-matrix is the group information in this model. In

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order to explore the potential variables contributing to the difference between both groups,

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we carried out an S-plot for the OPLS-DA model used to define metabolites significantly

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contributing to the separation of the experiment group and the control group. Meanwhile the

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variable importance in the projection (VIP) values of all variables in this model were

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acquired to show their contribution to the difference of the two groups. VIP values of

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variables greater than 1.00 were regarded significant in discriminating between the two

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groups.52-53 Moreover, statistical analysis (one-way analysis of variance and Tukey’s test) was

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also performed to verify those significant contributing variables from OPLS-DA model using

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SPSS 17.0 software. P < 0.05 was considered to indicate statistical significance. Only those

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variables that meet the two criteria (VIP > 1.00 and P < 0.05) are ultimately confirmed as

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potential biomarkers.

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All other experimental data are expressed as the means ± standard error of the means

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(SEM). Statistical significance was defined by a one-way analysis of variance and Tukey’s

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test, using the software of SPSS 17.0. P < 0.05 was regarded as significant statistical

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significance.

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RESULTS AND DISCUSSION 12


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Effects of O-Tyr and Dityr Exposure on Body Weight Gain in Mice

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We compared female mice from the three experimental groups with initial body weight at the

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age of 5 weeks and finish body weight at the age of 11 weeks (Figure 1A). There were no

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significant differences in body weight of mice among the control, O-Tyr and Dityr groups at

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the beginning of the experiment. The three groups mice all showed a progressive increasing

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tendency in body weight over the 6 weeks experimental period. However, from the 39th day

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to the finish of the experimental protocol, Dityr exposure significantly elevated (P < 0.05) the

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body weight of mice compared to the control group, and also significantly increased (P < 0.05)

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the body weight gain of mice at the end of the experiments (Figure 1B). Compared with the

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control group, the body weight gain of mice in the O-Tyr group significantly increased at the

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finish of the experiment (P < 0.05). O-Tyr exposure showed an increasing tendency of the

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body weight of mice, but there were no statistical significance between the two groups (P >

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0.05).

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Effects of O-Tyr and Dityr Exposure on Oxidative Damage in Mice

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To evaluate the oxidative damage of proteins and lipids in vivo, the concentrations of AOPPs

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(Figure 2A), Dityr (Figure 2B), 3-NT (Figure 2C) and MDA (Figure 2D) were determined in

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the liver and kidney of the different groups. Compared with control group, O-Tyr exposure

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significantly increased (P < 0.05) the concentrations of AOPPs in liver and kidney, Dityr in

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kidney, 3-NT in liver, and MDA in liver and kidney. Dityr exposure significantly elevated (P

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< 0.05) the concentrations of AOPPs in liver and kidney, Dityr in liver and kidney, MDA in

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liver and kidney.

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Effects of O-Tyr and Dityr Exposure on Oxidative Stress Status in Mice

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ROS (Figure 3A) level in the whole blood, liver and kidney tissues, and T-AOC (Figure 3B)

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and GSH/GSSG (Figure 3C) levels in plasma, liver and kidney tissues were assayed to

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evaluate the oxidative stress status in mice. We observed a significant increase (P < 0.05) in

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the levels of ROS in the whole blood, liver and kidney in the O-Tyr group mice as compared

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to the control group mice. Dityr exposure significantly increased (P < 0.05) the levels of ROS

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in the whole blood, liver and kidney in comparison with the control group mice. Compared

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with control group, O-Tyr exposure significantly reduced (P < 0.05) the levels of T-AOC and

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GSH/GSSG in plasma and liver, and GSH/GSSG in kidney. Dityr exposure significantly

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declined (P < 0.05) the levels of T-AOC and GSH/GSSG in plasma, liver and kidney.

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Effects of O-Tyr and Dityr Exposure on Hepatotoxicity and Hepatic Steatosis in Mice

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Histopathology analysis of liver was performed to examine O-Tyr and Dityr exposure

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induced hepatocellular damage. The control group mice (Figure 4A) showed a normal liver

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lobular architecture and cell structure. The O-Tyr (Figure 4B) and Dityr (Figure 4C) groups

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mice showed, respectively, loss of normal liver architecture, and microvesicular steatosis

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within hepatocytes, and the phenomenon is characterized by the presence of many small lipid

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droplets within the affected cells, and the nucleus of the involved hepatocyte typically

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remains centrally located. The result of staining with Oil-Red O further confirmed the

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presence of lipid droplets (red area) within hepatocytes (Figure 4D-F). Compared with the

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control group, we found that O-Tyr and Dityr exposure significantly elevated (P < 0.05) liver

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weight (Figure 4G), and the levels of hepatic triglycerides and cholesterol (Figure 4H). The

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O-Tyr and Dityr groups mice also showed significant increase (P < 0.05) in plasma ALT

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(Figure 4I) and AST (Figure 4J) activities and decrease (P < 0.05) in plasma TP (Figure 4K)

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level.

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Effects of O-Tyr and Dityr Exposure on Nephrotoxicity in Mice

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Representative renal photomicrographs stained with H&E taken from the control, O-Tyr and

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Dityr groups mice are shown in Figure 5A-C. The control group (Figure 5A) showed a

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normal-appearing glomeruli and tubules. The O-Tyr (Figure 5B) and Dityr (Figure 5C)

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groups showed that, respectively, glomerular atrophy, focal tubular dilatation and

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simplification with intraluminal shedding of cytoplasmic debris. Compared with the control

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group, O-Tyr exposure significantly increased (P < 0.05) kidney weight (Figure 5D), and the

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plasma level of Cre (Figure 5F). Dityr exposure significantly increased (P < 0.05) kidney

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weight (Figure 5D), kidney index (Figure 5E), and the plasma level of Cre (Figure 5F).

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Effects of O-Tyr and Dityr Exposure on the Levels of VLDL, LDL and Choline in Mice

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Compared with the control group, O-Tyr exposure significantly decreased (P < 0.05) the

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levels of VLDL (Figure 6A) and choline (Figure 6C) in mice plasma, and choline in mice

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liver. Dityr exposure significantly reduced (P < 0.05) the levels of VLDL, LDL (Figure 6B)

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and choline in mice plasma, and choline in mice liver.

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Effects of O-Tyr and Dityr Exposure on the Levels of Systemic Endogenous Metabolites

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of Urine and Plasma from Mice

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The typical urine and plasma 1H NMR spectra obtained from randomly selected three groups

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mice are illustrated in Figure 7. A total of 74 metabolites were unambiguously assigned for

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urine spectra (Figure 7A-C), 45 metabolites were unambiguously assigned for plasma spectra

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(Figure 7D-F). The chemical shifts and peak multiplicities, and the corresponding 1H NMR

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signal multiplicities of these specific metabolites are listed in Table 1. In order to acquire

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more detailed information about the metabolite variations in the experimental group mice,

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multivariate pattern recognition analysis methods were further performed on the urine and

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plasma 1H NMR data in mice.

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PCA was initially carried out on the urine and plasma spectral data (Figure 8A and Figure

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9A, respectively). These results showed that separations from the control, O-Tyr and Dityr

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groups mice were non-significant in their metabolic urine and plasma profiles. Therefore,

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PLS-DA was performed to investigate the inherent differences among the three experimental

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groups. The urine and plasma samples from three different groups were distinctly divided and

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classified into three obvious clusters showed in the PLS-DA score plot (Figure 8B and Figure

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9B, respectively). The validated models indicated that the two models was robust and no over

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fitting (Figure 8C and Figure 9C, respectively). Furthermore, the urine and plasma metabolic

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changes in the three groups mice were analyzed by using OPLS-DA (Figure 8D, F and Figure

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9D, F, respectively). These metabolites responsible for a significant contribution to the

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obvious separation of the experiment group and the control group mice were displayed in the

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corresponding S-plot (Figure 8E, G and Figure 9E, G, respectively). According to the VIP and

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P values of assigned metabolites (VIP > 1 and P < 0.05), the significant different metabolites

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were presented in Table 2 and Table 3, respectively.

326

Compared with the control group, O-Tyr exposure significantly increased the urinary levels

327

of succinimide, trimethylamine-N-oxide (TMAO), phenylacetyglycine, indoxyl sulfate,

328

N1-methyl-2-pyridone-5-carboxamide (2-PY) and N1-methyl-4-pyridone-5-carboxamide

329

(4-PY), and decreased the urinary levels of citrate, sarcosine, α-ketoglutarate, creatinine,

330

choline, glycine, fumarate, hippurate and nicotinate (P < 0.05, Table 2). Moreover, O-Tyr

16


Page 16 of 60

Page 17 of 60


331

exposure significantly elevated the plasma levels of trimethylamine, creatine, creatinine and

332

TMAO, and reduced the plasma levels of VLDL, valine, propionate, isobutyrate,

333

3-hydroxybutyrate, lipids, lysine, pyruvate, citrate, albumin, choline and phosphatidylcholine.

334

(P < 0.05, Table 3).

335

Compared with the control group, Dityr exposure significantly increased the urinary levels

336

of trimethylamine, TMAO, allantoin, indoxyl sulfate, 4-PY and nicotinamide N-oxide, and

337

decreased the urinary levels of citrate, sarcosine, α-ketoglutarate, creatinine, choline,

338

phenylacetate, cis-aconitate, fumarate, hippurate and nicotinate, compared with the control

339

group (P < 0.05, Table 2). As well as Dityr exposure significantly elevated the plasma levels

340

of trimethylamine, creatinine, TMAO and α-glucose, and reduced the plasma levels of LDL,

341

VLDL, valine, isobutyrate, 3-hydroxybutyrate, lipids, lactate, glutamate, pyruvate, albumin,

342

choline, phosphatidylcholine and betaine (P < 0.05, Table 3).

343

Related Metabolic Pathways Analysis

344

In this study, our results supported the hypothesis that long-term O-Tyr and Dityr exposure

345

can cause systemic metabolic changes in mice serum and urine. After O-Tyr and Dityr

346

exposure for 6 weeks, 15 and 16 metabolites in urine (respectively), and 16 and 17

347

metabolites in plasma (respectively) were significantly and differentially changed, and were

348

found to play an important role in choline metabolism, gut microbiota metabolism, fat

349

metabolism, amino acid metabolism, oxidative stress responses and energy metabolism.

350

O-Tyr and Dityr Exposure Reduced Choline Bioavailability, Led to Fat Accumulation in

351

Liver, and Induced Liver Injury

352

A new and intriguing observation is that O-Tyr and Dityr exposure changed the pathways of

17



353

dietary choline metabolism, led to fat accumulation in liver, and then caused liver injury.

354

Dietary choline is utilized by three major pathways, including a sym-xenobiotic pathway and

355

two pure mammalian pathways:54 1) choline is broken down by gut microbiota to form

356

methylamines (trimethylamine, TMAO and dimethylamine), 2) choline is also converted to

357

betaine and sarcosine, resulting in the generation of creatine and creatinine, 3) choline is also

358

used to synthesize phosphatidylcholine. In this study, compared with control group, the

359

decreased plasma choline, phosphatidylcholine and betaine levels, and increased plasma

360

trimethylamine and TMAO levels implied that O-Tyr and Dityr exposure reduced choline

361

bioavailability. In support of this notion, the decreased levels of choline and sarcosine, and

362

increased levels of trimethylamine and TMAO in urine, and the reduced levels of choline in

363

plasma and liver with the kit method of quantitative determination. Choline-deficient diets

364

have been definitely connected with hepatic steatosis, which is reversible by choline

365

intravenous administration.55 We showed here that lower plasma phosphatidylcholine levels

366

in the O-Tyr and Dityr groups mice compared with control group mice can be explained by

367

reduced bioavailability of choline in body. The reason is that dietary choline was converted

368

into more methylamines by gut microbiota, with subsequently eliminating through the urine.

369

This underlying mechanism thus mimics a choline-deficient diet. This declined choline

370

bioavailability may lead to the inability to synthesize phosphatidylcholine, which is essential

371

for the assembly and secretion of VLDL,

372

liver.57 The result is consistent with the decrease in levels of VLDL and LDL in plasma

373

(NMR and kit detection, respectively), the increase in levels of TG and TC in liver, and the

374

increase in liver weight in mice. In addition, through histopathological analysis, we found

56

and subsequent accumulation of lipids in mice

18


Page 18 of 60

Page 19 of 60


375

that H&E staining and Oil-Red O staining distinctly showed lipid droplets are emerged in the

376

hepatocytes from the O-Tyr and Dityr groups mice, strongly revealing that O-Tyr and Dityr

377

exposure induced steatosis and liver injury in mice. Even more intriguing, O-Tyr and Dityr

378

exposure induced hepatic lipids accumulation, which was accompanied by reduced

379

concentration of lipids in mice plasma. These findings seemed to be a common metabolic

380

response to a number of hepatotoxicity compounds, such as aflatoxin-B1,58 acetaminophen59

381

and ethionine.60 The hepatic lipidosis caused by O-Tyr and Dityr exposure may also be due to

382

the declined changes in lipids transport from hepatic tissue to the plasma as in the case of the

383

hepatic lipids accumulation induced by allyl formate,61 and perfluorododecanoic acid.62 Liver

384

injury induced by O-Tyr and Dityr exposure were further proved by significantly elevated

385

AST, ALT and TP levels in plasma. This finding is in agreement with our past study that

386

24-weeks exposure to oxidized tyrosine induces hepatic fibrosis in sprague-dawley rats

387

model.15

388

O-Tyr and Dityr Exposure Resulted in Changes in Gut Microbiota Functions, and

389

Elevated Risk Factor for Cardiovascular Disease

390

Choline and its metabolic product (betaine), which are methyl donors, are metabolically

391

related to the pathways of transmethylation including the synthesis of the risk factor

392

(homocysteine) of cardiovascular disease. Deficiencies of choline and betaine have been

393

demonstrated to generate epigenetic variations in genes linked to atherosclerosis.63-64 The

394

identified pathway (dietary choline and phosphatidylcholine → intestinal-flora-formed TMA

395

→ hepatic flavin monooxygenase-formed TMAO) shows a unique additional nutritional

396

contribution to the pathogenesis of cardiovascular disease

19


that correlates with


Page 20 of 60

397

phosphatidylcholine and choline metabolism, a responsible role for the intestinal

398

microflora.65 In our study, compared with control group, the increase in contents of

399

trimethylamine and TMAO in plasma and urine suggested that O-Tyr and Dityr exposure

400

resulted in disturbances in gut microbiota functions, and elevated risk factor for

401

cardiovascular disease. Gut microbial communities lately also has been involved in the

402

development of some metabolic phenotypes, such as obesity, diabetes, cardiovascular disease,

403

and changes in immune responses.57,

404

investigate and report a relation among O-Tyr and Dityr, intestinal microflora and

405

cardiovascular disease risk. These findings imply that a reasonable intervention of probiotics

406

may serve as a protection measures during excess intake of oxidative proteins. Interestingly,

407

generation of TMAO can be changed by probiotics intervention.68

65-67

To our knowledge, this study is the first to

408

O-Tyr and Dityr exposure also led to variations in the gut microbiota related plasma

409

metabolites (including propionate and isobutyrate), and urinary metabolites (including

410

hippurate, phenylacetate and phenylacetyglycine). Gut microbiota break down dietary

411

nondigestible fibers to form short-chain fatty acids (such as propionate and isobutyrate) that

412

can be utilized by the host as energy sources or/and as precursors for fatty acids synthesis.66

413

Hippurate is the common metabolic product between intestinal microflora and host.69 Thus,

414

an alteration in the excretion of these compounds indicates a change in the metabolic function

415

of intestinal microflora. Phenylacetate is derived from phenylalanine via the function of

416

intestinal

417

phenylacetylglycine.70 Previous researches demonstrate that the changed levels of

418

phenylacetate and phenylacetyglycine are indicated in the disturbance of gut microbiota

microflora,

and

phenylacetate

is

conjugated

20


with

glycine

to

form

Page 21 of 60


419

metabolism.71-72 Mammalian metabolism is significantly affected by the complex gut

420

microbiota. In the current study, O-Tyr exposure decreased the levels of propionate and

421

isobutyrate in plasma, and the level of hippurate in urine, and increased the level of

422

phenylacetyglycine in urine. Dityr exposure reduced the level of isobutyrate in plasma, and

423

the levels of hippurate and phenylacetate in urine. Variations in these metabolites are

424

attributed to the reduced number and/or changed activities of intestinal microflora. The

425

intestinal microflora is closely related to the function and structure of the intestine, and the

426

immune system of host.73 Therefore, the changes in intestinal microflora caused by the

427

exposure of O-Tyr and Dityr can potentially affect host health status.

428

O-Tyr and Dityr Exposure Induced Renal Dysfunction

429

Because the kidney organ is particularly specialized in generating a metabolically rich and

430

plentiful biofluid (urine), it is not amazing to find that most metabonomics studies about

431

organs functions have concentrated on the kidney.74 Many previous studies have described or

432

assessed urine and plasma biomarkers associated with renal damage and dysfunction.75-77 One

433

common feature to most these researches is the substantial increase showed in both urine and

434

plasma contents of TMAO (an osmolyte mention above). In this study, compared with control

435

group, the increased plasma and urinary TMAO levels indicated O-Tyr and Dityr exposure

436

induced nephrotoxicity. Supporting evidence for kidney injury and dysfunction could also be

437

found from the glomerular atrophy, focal tubular dilatation and simplification with

438

intraluminal shedding of cytoplasmic debris in kidney tissue noted in the histopathological

439

analysis. In addition, the excretion of urine creatinine was significantly decreased, which

440

suggested O-Tyr and Dityr exposure induced glomerular filtration function injury. This is

21



Page 22 of 60

441

consistent with the increased levels of creatine and creatinine in plasma. Obvious renal injury

442

and dysfunction in mice was also proved by the elevated plasma Cre level noted in the

443

traditional clinical biochemical analysis. This finding is in agreement with our previous

444

studies that 24-weeks O-Tyr administration causes renal fibrosis and dysfunction by inducing

445

oxidative stress and inflammation in rat.16

446

O-Tyr and Dityr Exposure Altered Amino Acid Metabolism

447

We found that the decreased plasma valine, lysine, glutamate and albumin levels, and the

448

decreased urinary glycine level, together with the reduced plasma TP level through traditional

449

clinical chemistry method, implied that O-Tyr and Dityr exposure affected amino acid

450

metabolism and inhibited protein synthesis. The reason is that amino acids are precursors to

451

protein synthesis.78-79

452

The spontaneous processes of deamidation, isomerization, and racemization in proteins

453

cause cellular accumulation of altered residues, result in the loss of the functional capacities

454

of the cell, and appear to be major pathways of structural damage in cellular proteins. The

455

structural alterations observed in proteins due to the three processes are linked by a common

456

succinimide intermediate, which can be hydrolyzed to a mixture of normal and isomerized

457

proteins.80 In this study, O-Tyr exposure significantly elevated the level of succinimide in

458

mice urine, implied that O-Tyr exposure caused structural damage in cellular proteins.

459

O-Tyr and Dityr Exposure Induced Oxidative Stress Responses

460

O-Tyr and Dityr exposure elevated urinary level of indoxyl sulfate, which is a circulating

461

uremic toxin, and is considered as an important marker for oxidative stress.

462

implied that O-Tyr and Dityr exposure induced oxidative stress responses in agreement with

22


81

This result

Page 23 of 60


463

previous reports that suggested elevation of urinary allantoin level as an indicator for

464

oxidative stress.82-83 In support of this view, O-Tyr and Dityr exposure increased urinary level

465

of allantoin, which is generated via nonenzymatic methods through high contents of ROS.40

466

Furthermore, O-Tyr exposure increased the contents of urinary 2-PY and 4-PY, and decreased

467

the level of urinary nicotinate, and Dityr exposure elevated the contents of urinary 4-PY and

468

nicotinamide N-oxide, and reduced the level of urinary nicotinate. These findings also

469

supported the notion that O-Tyr and Dityr exposure induced oxidative stress responses in

470

mice. Because nicotinate is a constituent of nicotinamide adenine dinucleotide (NAD) related

471

to intracellular respiration to oxidize energy substrates. And 4-PY, 2-PY and nicotinamide

472

N-oxide are oxidative products of nicotinate in liver. The variations of these nicotinate

473

metabolites are probably also indications for changed oxidative stress responses. Previous

474

reports suggest that the altered contents of nicotinate, 2-PY and 4-PY are shown in the

475

changes of oxidative stress responses.40, 84 In addition, our past study has also found that

476

one-week O-Tyr and Dityr exposure induced oxidative stress responses and elevated

477

metabolism of vitamin-B3 in mice.19 Moreover, The elevated levels of AOPPs, Dityr and

478

3-NT (as markers of proteins oxidative damage), and MDA (as markers of lipids oxidative

479

damage), and reduced the activity of T-AOC and level of GSH/GSSG, combine with the

480

increased level of ROS also supports O-Tyr and Dityr exposure induced oxidative stress

481

responses in mice.

482

O-Tyr and Dityr Exposure Induced Changes in Energy Metabolism

483

O-Tyr and Dityr exposure inhibited energy metabolism. Glucose is a major energy substrate

484

in animals and humans, and can be broken down by a series of glycolytic reactions to produce

23



485

pyruvate.85 Pyruvate can be transformed into acetyl coenzyme A, which is transported into

486

the tricarboxylic acid cycle (TCA) in the presence of oxygen.86 In the present study,

487

compared with control group mice, the increased plasma α-glucose level, and decreased

488

plasma pyruvate level indicated that O-Tyr and Dityr exposure inhibited glycolysis. Lactate,

489

which is generated from pyruvate and reduced form of nicotinamide adenine dinucleotid

490

(NADH) by lactate dehydrogenase, is strongly associated with energy metabolism.87 The

491

decreased levels of lactate in body may be related to an elevated rate of gluconeogenesis in

492

liver and a decreased rate of anaerobic glycolysis. The decrease in the urinary concentrations

493

of citrate, α-ketoglutarate, cis-aconitate and fumarate (TCA intermediates) showed that O-Tyr

494

and Dityr exposure down-regulated the rate of the TCA. O-Tyr exposure caused the depletion

495

of citrate in plasma also supported that O-Tyr exposure suppressed the rate of the TCA cycle.

496

3-hydroxybutyrate is a product of fatty acids β-oxidation in the mitochondria.88 The

497

decreased plasma 3-hydroxybutyrate level showed that O-Tyr and Dityr exposure suppressed

498

the β-oxidation of fatty acids, which may imply an elevated fat accumulation in the body.

499

This is consistent with Dityr exposure led to the significantly increased body weight, and

500

O-Tyr exposure had an increased tendency in the body weight of mice. Taken together,

501

6-weeks O-Tyr and Dityr exposure inhibited energy metabolism in mice. However, in our

502

previous research, we found one-week O-Tyr and Dityr exposure enhanced energy

503

metabolism (including promoted fatty acids oxidation, glucose-alanine cycle, glycolysis and

504

TCA) in mice.19 The possible reason for this difference is that long-term O-Tyr and Dityr

505

exposure induced multiple organ injuries, and then led to disorder of metabolic function.

506

In conclusion, long-term O-Tyr and Dityr exposure caused obvious metabolic alterations in

24


Page 24 of 60

Page 25 of 60


507

mice urine and plasma. These changes indicated that O-Tyr and Dityr exposure reduced

508

choline bioavailability, led to fat accumulation in liver, induced liver injury, induced renal

509

dysfunction, resulted in changes in gut microbiota functions, and elevated risk factor for

510

cardiovascular disease, altered amino acid metabolism, induced oxidative stress responses

511

and changed energy metabolism. These findings have important implications in food and

512

nutrition research in humans and animals. And this study would contribute to further

513

understanding of the mechanisms underlying the effects of O-Tyr and Dityr exposure on

514

animals and human health, and provide vital information for the assessment of food safety

515

and improvement of human dietary habits. Future studies may be directed toward a

516

mechanistic understanding of the relationships between O-Tyr and Dityr exposure and

517

cardiovascular disease.

518

ABBREVIATIONS USED

519

O-Tyr, oxidized tyrosine; Dityr, dityrosine; ROS; reactive oxygen species; AOPPs, advanced

520

oxidation protein products; PCA, principal component analysis; PLS-DA, partial

521

least-squares discriminant analysis; O-PLS-DA, orthogonal partial least-squares discriminant

522

analysis; 1H NMR, 1H nuclear magnetic resonance; 3-NT, 3-nitrotyrosine; T-AOC, total

523

antioxidant capacity; MDA, malondialdehyde; GSH, reduced glutathione; GSSG, oxidized

524

glutathione; H&E, hematoxylin and eosin staining; ALT, alanine aminotransferase; AST,

525

aspartate aminotransferase; TP, total protein; Cre, plasma creatinine; TG, triglyceride; CHO,

526

cholesterol; TSP, 3-(trimethylsilyl) propionic-(2,2,3,3-d4) acid sodium salt; VIP, variable

527

importance in the projection; LDL, low density lipoprotein; VLDL, low density lipoprotein;

528

4-PY, N1-methyl-4-pyridone-5-carboxamide; 2-PY, N1-methyl-2-pyridone-5-carboxamide;

25



529

TMAO, trimethylamine-N-oxide; GPC, glycerophosphorylcholine; TCA, tricarboxylic acid

530

cycle; NAD, nicotinamide adenine dinucleotide; NADH, reduced form of nicotinamide

531

adenine dinucleotid.

532

ACKNOWLEDGMENTS

533

We thank all of participants in this study for their constant efforts and assistances, and

534

especially thank Wuhan Zhongke Metaboss Technology CO., LTD for technical support in

535

NMR dection.

536

AUTHOR INFORMATION

537

Corresponding Author

538

* Guowei Le: Tel, 0510-85917789; Fax, +86-510-85869236; E-mail, [email protected].

539

Author Contributions

540

Yang Y. and Zhang H. are contributed equally to this work.

541

Funding

542

This work received financial support from "Collaborative innovation center of food safety

543

and quality control in Jiangsu Province", the National Natural Science Foundation of China

544

(No. 31571841), State Key Laboratory of Food Science and Technology of Jiangnan

545

University in China (No. SKLF-ZZB-201609) and the China Postdoctoral Science

546

Foundation (No. 2015M571669).

547

Notes

548

The authors declare no competing financial interest.

549 550

26


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551

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37



793

FIGURE CAPTIONS

794

Figure 1. Effects of O-Tyr and Dityr exposure on body weight (A) of mice were analyzed

795

every three days. Body weight gain (B) of mice was measured from the 5th week until the

796

11th week in the three groups. Data were presented as means ± SEM for all groups (n = 10).

797

Significance levels at P < 0.05 were considered to indicate statistical significance. * indicates

798

significantly different between the O-Tyr and the control groups (P < 05). # indicates

799

significantly different between the Dityr and the control groups (P < 05).

800

Figure 2. Protein oxidation, lipid peroxidation. The effects of O-Tyr and Dityr exposure on

801

the levels of AOPPs (A), Dityr (B), 3-NT (C) and MDA (D) in mice liver and kidney. Data

802

were presented as means ± SEM for all groups (n = 10). Significance levels at P < 0.05 were

803

considered to indicate statistical significance.

804

Figure 3. Oxidative stress status. The effects of O-Tyr and Dityr exposure on the levels of

805

ROS in mice blood, liver and kidney (A). The effects of O-Tyr and Dityr exposure on the

806

activity of T-AOC (B) and GSH/GSSG (C) level in mice plasma, liver and kidney. Data were

807

presented as means ± SEM for all groups (n = 10). Significance levels at P < 0.05 were

808


809

Figure 4. Lipid accumulation in mice liver. Representative hepatic photomicrographs

810

(hematoxylin and eosin staining, magnification ×200) in mice acquired from the control (A),

811

O-Tyr (B) and Dityr (C) groups. Representative hepatic photomicrographs (Oil Red O

812

staining, magnification ×200) in mice acquired from the control (D), O-Tyr (E) and Dityr (F)

813

groups. The effects of O-Tyr and Dityr exposure on liver weight and liver index (G) in mice,

814

liver index (%) was calculated as: (liver weight/body weight) × 100. The effects of O-Tyr and

38


Page 38 of 60

Page 39 of 60


815

Dityr exposure on the levels of triglyceride and cholesterol (H) in mice liver. The effects of

816

O-Tyr and Dityr exposure on the plasma levels of ALT (I), AST (J) and TP (K) in mice. Data

817

were presented as means ± SEM for all groups (n = 10). Significance levels at P < 0.05 were

818


819

Figure 5. Representative renal photomicrographs (hematoxylin and eosin staining,

820

magnification ×200) in mice obtained from the control (A), O-Tyr (B) and Dityr (C) groups.

821

The effects of O-Tyr and Dityr exposure on kidney weight (D) and kidney index (E) in mice,

822

kidney index (%) was calculated as: (kidney weight/body weight) × 100. The effects of O-Tyr

823

and Dityr exposure on the plasma level of Cre (F) in mice. Data were presented as means ±

824

SEM for all groups (n = 10). Significance levels at P < 0.05 were considered to indicate

825

statistical significance.

826

Figure 6. Quantitative analysis of LDL, VLDL and choline. The effects of O-Tyr and Dityr

827

exposure on the levels of VLDL (A) and LDL (B) in mice plasma. The effects of O-Tyr and

828

Dityr exposure on the levels of choline (C) in mice plasma and liver. Data were presented as

829

means ± SEM for all groups (n = 10). Significance levels at P < 0.05 were considered to

830

indicate statistical significance.

831

Figure 7. Representative 600 MHz 1H NMR spectra of urine obtained from mice in the

832

control (A), O-Tyr (B) and Dityr (C) groups, as well as plasma obtained from mice in the

833

control (D), O-Tyr (E) and Dityr (F) groups. The dashed boxes were perpendicularly enlarged

834

16 and 8 times in the spectra of urine, 64 times in the plasma. The keys for metabolites are

835

given in Table 1.

836

Figure 8. PCA score plots (A: R2X = 0.815, Q2 = 0.760), PLS-DA score plots (B: R2X =

39



837

0.821, R2Y = 0.638, Q2 = 0.530), PLS-DA validation plots (C: permutation number, 200

838

cycles), OPLS-DA (D: R2X = 0.842, R2Y = 0.903, Q2 = 0.700; F: R2X = 0.869, R2Y = 0.953,

839

Q2 = 0.825) and corresponding S-plot (E and G, respectively) based on the 1H NMR spectra

840

of urinary metabolites obtained from the control (5-Point star), O-Tyr (Circle) and Dityr

841

(Diamond) groups. One urine sample from the O-Tyr group was removed because it

842

positioned outside the Hotelling’s T2 elipse on the score plot.

843

Figure 9. PCA score plots (A: R2X = 0.678, Q2 = 0.561), PLS-DA score plots (B: R2X =

844

0.756, R2Y = 0.698, Q2 = 0.589), PLS-DA validation plots (C: permutation number, 200

845

cycles), OPLS-DA (D: R2X = 0.720, R2Y = 0.904, Q2 = 0.621; F: R2X = 0.785, R2Y = 0.863,

846

Q2 = 0.778), and corresponding S-plot (E and G, respectively) based on the 1H NMR spectra

847

of plasma metabolites obtained from the control (5-Point star), O-Tyr (Circle) and Dityr

848

(Diamond) groups.

40


Page 40 of 60

Page 41 of 60


TABLES Table 1. 1H NMR Data for Metabolites in Urine and Plasmaa keys

metabolites

δ1H (ppm) and multiplicity

moieties

samples

1

α-hydroxy-n-valerate

CH3, γCH2

0.86(t), 1.31(m)

U

2

valerate

δCH3, γCH2, βCH2, αCH2

0.88(t), 1.31(m), 1.61(m), 2.28(t)

U

3

α-hydroxybutyrate

CH3

0.89(t)

U

4

butyrate

CH3

0.92(t)

U

5

2-ketoisocaproate

CH3, CH, CH2

0.93(dd), 2.09(m), 2.59(m)

U

6

α-hydroxy-iso-valerate

δCH3

0.83(d), 0.97(d)

U

7

propionate

CH3, CH2

1.06(t), 2.18(q)

U, P

8

2-keto-isovalerate

CH3, CH2

1.11(d), 3.03(m)

U

9

isobutyrate

CH3

1.14(d)

U, P

10

ethanol

CH3, CH2

1.18(t), 3.66(q)

U, P

11

3-hydroxybutyrate

γCH3, αCH2, βCH

1.20(d), 2.28(dd), 2.42(dd)

U, P

12

methylmalonate

CH3, CH

1.24(d), 3.75(m)

U

13

lactate

βCH3, αCH

1.33(d), 4.13(q)

U, P

14

2-hydroxyisobutyrate

CH3

1.36(s)

15

alanine

βCH3, αCH

1.48(d), 3.77(q)

U, P

16

citrulline

γCH2, βCH2

1.56(m), 1.82(m)

U, P

17

putrescine

CH2, CH2-NH2

1.78(m), 3.06(t)

U

18

N-acetylglutamate

γCH2, CH3, βCH2

1.88(m), 2.04(s), 2.07(m)

U

19

acetate

CH3

1.92(s)

41


U

U, P


Page 42 of 60

20

acetamide

CH3

2.00(s)

U

21

acetone

CH3

2.24(s)

U

22

acetoacetate

CH3

2.3(s)

U, P

23

pyruvate

CH3

2.35(s)

U, P

24

succinate

CH2

2.41(s)

U

25

α-ketoglutarate

βCH2, γCH2

2.45(t), 3.01(t)

U

26

citrate

CH2

2.55(d), 2.68(d)

U, P

27

methylamine

CH3

2.62(s)

U, P

28

dimethylamine

CH3

2.73(s)

U

29

sarcosine

CH3, CH2

2.76(s), 3.65(s)

U

30

succinimide

CH2

2.79(s)

U

31

methylguanidine

CH3

2.83(s)

U

32

trimethylamine

CH3

2.88(s)

U, P

33

dimethylglycine

CH3

2.93(s)

U

34

creatine

CH3, CH2

3.04(s), 3.93(s)

U, P

35

creatinine

CH3, CH2

3.05(s), 4.05(s)

U, P

36

ethanolamine

CH2

3.13(t)

U

37

malonate

CH2

3.16(s)

U

38

choline

OCH2, NCH2, N(CH3)3

4.07(t), 3.53(t), 3.20(s)

U, P

39

phosphatidylcholine

N(CH3)3, OCH2, NCH2

3.22(s), 4.21(t), 3.61(t)

U, P

40

taurine

-CH2-S, -CH2-NH2

3.27(t), 3.43(t)

U, P

41

TMAO

CH3

3.28(s)

U, P

42


Page 43 of 60

42


phenylacetate

CH2,

2,6-CH,

4-CH, 3.55(s), 7.28(m), 7.29(m), 7.32(m)

U

3,5-CH 43

glycine

CH2

3.57(s)

44

p-hydroxyphenylacetate

6-CH, 2-CH, 3,5-CH

3.6(s), 6.87(d), 7.15(d)

45

phenylacetyglycine

2,6-CH,

3,5-CH,

U, P U

7-CH, 7.31(t), 7.37(m), 7.42(m), 3.68(s)

U

10-CH 46

guanidoacetate

CH2

3.80(s)

47

hippurate

CH2,

3,5-CH,

U

4-CH, 3.97(d), 7.57(t), 7.65(t), 7.84(d)

U

2,6-CH 48

inosine

8-CH, 2-CH, 1-CH, 2-CH, 8.24(s), 8.35(s), 6.05(d), 4.79(m), 3-CH, 4-CH, CH2

49

trigonelline

4.43(dd), 4.28(m), 3.87(dd)

2-CH, 4-CH, 6-CH, 5-CH, 9.12(s), 8.85(m), 8.83(dd), 8.19(m), CH3

50

N-methylnicotinamide

β-glucose

CH3, 5-CH, 4-CH, 6-CH, 4.48(s), 8.19(m), 8.91(m), 8.97(d),

α-glucose

1-CH, 2-CH, 3-CH, 4-CH, 4.65(dd), 3.25(dd), 3.49(t), 3.41(dd),

54

4-cresol glucuronide

allantoin

U, P

3.46(m), 3.73(dd), 3.90(dd)

1-CH, 2-CH, 3-CH, 4-CH, 5.24(d), 5-CH, 6-CH

53

U

9.28(s)

5-CH, 6-CH 52

U

4.44(s)

2-CH 51

U

3.54(dd),

3.71(dd),

U, P

3.42(dd), 3.84(m), 3.78(m)

CH3, 2, 6-CH, 3, 5-CH, 2.30(s), 7.05(d), 7.23(d), 5.03(d), 5-CH, 4-CH, 3-CH

3.61(m), 3.89(m)

CH

5.40(s)

43


U

U


Page 44 of 60

55

cis-aconitate

CH2, CH

3.12(s), 5.69(s)

U

56

urea

NH2

5.80(s)

U

57

4-PY

N-CH3, 3-CH, 2-CH, 6-CH

3.89(s), 6.71(d), 7.83(dd), 8.55(d)

U

58

2-PY

N-CH3, 3-CH, 4-CH,

3.64(s), 6.67(d), 7.96(dd),

U

6-CH

8.33(d)

59

homogentisate

6-CH, 5-CH

6.70(d), 6.76(d)

U

60

1-methylhistidine

4-CH, 2-CH

7.05(s), 7.78(s)

U, P

61

3-methylhistidine

4-CH, 2-CH

7.12(s), 7.67(s)

U

62

indoxyl sulfate

4-CH, 5-CH, 6-CH,

7.51(m), 7.22(m), 7.28(m),

U

7-CH, CH3

7.71(m), 7.37(s)

63

m-hydroxyphenylacetate 6-CH, 4-CH, 3-CH

6.92(m), 7.05(d), 7.27(t)

U

64

nicotinate

2,6-CH, 4-CH, 5-CH

8.62(d), 8.25(d), 7.50(dd)

U

65

guanine

CH

7.68(s)

U

66

4-aminohippurate

CH2

7.71(d)

U

67

nicotinamide N-oxide

5-CH, 6-CH, 2-CH, 4-CH

7.74(m), 8.12(m), 8.75(m), 8.49(m)

U

68

formate

CH

8.46(s)

69

4-HPPA

CH2, 3, 5-CH, 2, 6-CH

4.01(s), 6.88(m), 7.18(m)

70

2-HPPA

CH3, CH, 3, 5-CH, 2, 6-CH 1.37(d), 3.58(q), 6.89(m), 7.18(m)

U

71

trans-aconitate

CH2, CH

3.44(s), 6.60(s)

U

72

glycolate

CH2

3.96(s)

U

73

orotate

CH

6.19(s)

U

74

fumarate

CH, CH3

6.53(s)

U

44


U, P U

Page 45 of 60


75

LDL

CH3(CH2)n

0.88(m)

P

76

VLDL

CH3CH2CH2C=

0.90(t)

P

77

leucine

αCH, βCH2, γCH,

3.73(t), 1.72(m), 0.96(d),

P

δCH3

0.91(d)

αCH, βCH, βCH3,

3.68(d), 1.99(m), 1.01(d),

γCH2, δCH3

1.26(m), 1.47(m), 0.94(t)

78

isoleucine

P

79

valine

αCH3, βCH, γCH3

3.62(d), 0.99(d), 1.04(d)

P

80

lipids

CH2*CH2CO, CH2-C=C

1.29(m), 1.58(m), 2.02(m)

P

CH2-C=O, CH-O-CO

2.25(m), 2.77(m)

81

threonine

αCH, βCH, γCH3

1.32(d), 4.25(m), 3.58(d)

P

82

lysine

αCH, βCH2, γCH2, δCH2

3.77(t), 1.89(m), 1.73(m)

P

83

N-acetyl glycoprotein

CH3

2.05(s)

P

84

O-acetyl glycoprotein

CH3

2.09(s)

P

85

glutamate

αCH, βCH2, γCH2

3.75(m), 2.12(m), 2.35(m)

P

86

methionine

αCH, βCH2, γCH2, S-CH3

3.87(t), 2.16(m), 2.65(t), 2.14(s)

P

87

glutamine

αCH, βCH2, γCH2

3.68(t), 2.15(m), 2.45(m)

P

88

albumin

Lysyl-CH2

3.02(s)

P

89

GPC

N-(CH3)3, OCH2, NCH2

3.23(s), 4.33(t), 3.51(t)

P

90

betaine

CH3, CH2

3.28(s), 3.90(s)

P

91

proline

βCH2, γCH2, δCH2

2.02-2.33(m), 2.00(m), 3.35(t)

P

92

ornithine

CH2, αCH

3.80(s), 3.79(t)

P

93

myo-Inositol

5-CH, 4,6-CH, 2-CH

3.30(t), 3.63(t), 4.06(t)

P

45



a

Page 46 of 60

94

phenylalanine

2,6-CH, 3,5-CH, 4-CH

7.32(m), 7.42(m), 7.37(m)

P

95

tyrosine

2,6-CH, 3,5-CH

7.19(dd), 6.90(d)

P

U, urine; P, plasma; s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; m, multiplet; 4-PY,

N1-methyl-4-pyridone-5-carboxamide;

2-PY,

N1-methyl-2-pyridone-5-carboxamide;

4-HPPA,

4-hydroxyphenylpyruvate; 2-HPPA, 2-(4-hydroxyphenyl)propanoate; LDL, low density lipoprotein; VLDL, low density lipoprotein; TMAO, trimethylamine-N-oxide; GPC, glycerophosphorylcholine.

46


Page 47 of 60


Table 2. Metabonomic Changes in Urine from the Control, O-Tyr and Dityr Groupsa metabolites

chemical

O-Tyr vs control

Dityr vs control

shift (δ)

FC

VIP

P value

Change

FC

VIP

P value

Change

citrate

2.68(d)

0.74

2.13

0.021

↓

0.68

1.95

0.018

↓

sarcosine

2.76(s)

0.80

1.29

0.034

↓

0.64

1.52

0.010

↓

succinimide

2.79(s)

1.29

1.06

0.049

↑

-

-

-

-

trimethylamine

2.88(s)

-

-

-

-

1.36

2.93

0.037

↑

α-ketoglutarate

3.01(t)

0.81

1.42

0.035

↓

0.75

1.31

0.036

↓

creatinine

3.07(s)

0.80

2.61

0.044

↓

0.78

3.36

0.028

↓

choline

3.20(s)

0.76

2.51

0.047

↓

0.72

2.92

0.021

↓

TMAO

3.28(s)

1.34

2.03

0.049

↑

1.38

1.99

0.046

↑

glycine

3.57(s)

0.73

2.27

0.022

↓

-

-

-

-

phenylacetate

3.55(s)

-

-

-

-

0.60

4.11

0.005

↓

phenylacetyglycine

3.68(s)

1.32

2.37

0.038

↑

-

-

-

-

allantoin

5.40(s)

-

-

-

-

1.47

2.48

0.031

↑

cis-aconitate

5.69(s)

-

-

-

-

0.76

1.09

0.020

↓

fumarate

6.53(s)

0.64

1.33

0.015

↓

0.78

1.15

0.046

↓

indoxyl sulfate

7.22 (m)

1.27

3.13

0.048

↑

1.33

3.41

0.032

↑

hippurate

7.84(d)

0.81

2.15

0.040

↓

0.55

4.56

0.011

↓

2-PY

8.33(d)

1.59

1.66

0.045

↑

-

-

-

-

4-PY

8.55(d)

1.55

1.94

0.038

↑

1.64

2.47

0.020

↑

nicotinate

7.50(dd)

0.70

1.62

0.029

↓

0.78

1.44

0.043

↓

47



nicotinamide

7.74(m)

-

-

-

-

1.37

Page 48 of 60

3.99

0.044

↑

N-oxide a

O-Tyr vs control, the O-Tyr group compared with the control group; Dityr vs control, the Dityr group

compared with the control group; FC, fold change was calculated as the ratio of the mean metabolite levels between experimental group and control group, FC > 1 indicates a relatively higher concentration, while FC < 1 means a relatively lower concentration present in experimental group as compared to the control group; VIP, variable importance in the projection; Metabolites with“↑/↓” means increased/decreased, “-” means the metabolite levels are the same as the control group; Significant differences are set at VIP > 1.00 and P < 0.05.

48


Page 49 of 60


Table 3. Metabonomic Changes in Plasma from the Control, O-Tyr and Dityr Groupsa metabolites

chemical

O-Tyr vs control

Dityr vs control

shift (δ)

FC

VIP

P value

Change

FC

VIP

LDL

0.88(m)

-

-

-

-

0.79

1.83

0.048

↓

VLDL

0.90(t)

0.78

2.53

0.044

↓

0.62

2.91

0.016

↓

valine

1.04(d)

0.80

1.05

0.043

↓

0.75

1.39

0.041

↓

propionate

1.08(t)

0.56

1.89

0.007

↓

-

-

-

-

isobutyrate

1.12(d)

0.81

1.30

0.039

↓

0.47

1.45

0.017

↓

3-hydroxybutyrate

1.21(d)

0.75

1.06

0.048

↓

0.77

1.28

0.049

↓

lipids

1.29(m)

0.80

2.86

0.030

↓

0.51

3.99

0.009

↓

lactate

1.33(d)

-

-

-

-

0.58

1.91

0.022

↓

lysine

1.89(m)

0.66

1.21

0.041

↓

-

-

-

-

glutamate

2.35(m)

-

-

-

-

0.75

1.33

0.026

↓

pyruvate

2.38(s)

0.80

2.01

0.047

↓

0.74

1.44

0.034

↓

citrate

2.54(d)

0.76

2.17

0.046

↓

-

-

-

-

trimethylamine

2.92(s)

1.34

1.23

0.045

↑

1.40

1.51

0.029

↑

albumin

3.02(s)

0.77

2.29

0.048

↓

0.74

2.13

0.040

↓

creatine

3.93(s)

1.55

2.21

0.041

↑

-

-

-

-

creatinine

3.05(s)

1.48

2.03

0.029

↑

1.87

2.26

0.010

↑

choline

3.20(s)

0.71

3.51

0.033

↓

0.49

2.48

0.009

↓

phosphatidylcholine

3.22(s)

0.76

1.87

0.049

↓

0.72

1.35

0.042

↓

TMAO

3.25(s)

1.42

1.78

0.038

↑

1.55

1.86

0.037

↑

49


P value Change


Page 50 of 60

betaine

3.28(s)

-

-

-

-

0.74

1.83

0.024

↓

α-glucose

5.26(d)

-

-

-

-

1.61

1.17

0.038

↑

a

O-Tyr vs control, the O-Tyr group compared with the control group; Dityr vs control, the Dityr group

compared with the control group; FC, fold change was calculated as the ratio of the mean metabolite levels between experimental group and control group, FC > 1 indicates a relatively higher concentration, while FC < 1 means a relatively lower concentration present in experimental group as compared to the control group; VIP, variable importance in the projection; Metabolites with“↑/↓” means increased/decreased, “-” means the metabolite levels are the same as the control group; Significant differences are set at VIP > 1.00 and P < 0.05.

50


Page 51 of 60


Figure 1 197x80mm (300 x 300 DPI)



Figure 2 209x177mm (300 x 300 DPI)


Page 52 of 60

Page 53 of 60


Figure 3 209x145mm (300 x 300 DPI)



Figure 4 209x255mm (300 x 300 DPI)


Page 54 of 60

Page 55 of 60


Figure 5 209x114mm (300 x 300 DPI)



Figure 6 129x159mm (300 x 300 DPI)


Page 56 of 60

Page 57 of 60


Figure 7 209x168mm (300 x 300 DPI)



Figure 8 196x251mm (300 x 300 DPI)


Page 58 of 60

Page 59 of 60


Figure 9 196x250mm (300 x 300 DPI)



TOC Graphic 82x44mm (300 x 300 DPI)


Page 60 of 60

Health effects of dietary oxidized tyrosine and dityrosine administration

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