(DCH) in rats, and its effect on oxidative stress and ... - ACS Publications

Oct 10, 2018 - Results indicated that DCH is more easily absorbed in rats than HMF. ... oxidative-stress-inducing agents instead of antioxidant agents...
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Article Cite This: J. Agric. Food Chem. 2018, 66, 11451−11458

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Absorption of 1‑Dicysteinethioacetal−5-Hydroxymethylfurfural in Rats and Its Effect on Oxidative Stress and Gut Microbiota Qianzhu Zhao,†,‡ Juanying Ou,†,‡,§ Caihuan Huang,‡ Ruixia Qiu,‡ Yong Wang,‡ Fu Liu,‡ Jie Zheng,*,‡ and Shiyi Ou*,‡ ‡

Department of Food Science and Engineering, Jinan University, Guangzhou, Guangdong 510632, People’s Republic of China Food and Nutritional Science Program, School of Biological Sciences, The University of Hong Kong, Pok Fu Lam, Hong Kong, People’s Republic of China

J. Agric. Food Chem. 2018.66:11451-11458. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 10/31/18. For personal use only.

§

S Supporting Information *

ABSTRACT: The absorption of a 5-hydroxymethylfurfural (HMF)−cysteine adduct, 1-dicysteinethioacetal−5-hydroxymethylfurfural (DCH), and its effect on antioxidant activity and gut microbiota were investigated. Results indicated that DCH is more easily absorbed in rats than HMF. Serum DCH concentrations were 15−38-fold of HMF concentrations from 30 to 180 min after intragastrical administration at the level of 100 mg/kg of body weight, and 2.7−4.5% of absorbed DCH was converted to HMF. The malondialdehyde content in the plasma, heart, liver, and kidneys significantly increased after drug (100 mg/kg of bw) administration for 1 week, suggesting that HMF and DCH were oxidative-stress-inducing agents, instead of antioxidant agents, in rats. HMF and DCH also modulated gut microbiota. HMF promoted the growth of Lactobacillus, Tyzzerella, Enterobacter, and Streptococcus. DCH increased the ratio of Firmicutes/Bacteroidetes and promoted the growth of Akkermansia, Shigella, and Escherichia while inhibiting the growth of Lactobacillus. KEYWORDS: 5-hydroxymethylfurfural, adduct, pharmacokinetics, oxidative stress, microbiota

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INTRODUCTION

The European Food Safety Authority (EFSA) established a threshold of concern of 0.54 mg/day for the intake of furan derivatives used as flavoring agents in Europe.16 However, the average intake level of HMF was estimated at 4−30 mg per person per day and even up to 350 mg/day from the beverages made from dried plums. 17 According to the Spanish estimation, HMF intake in children was higher than that in the adult population (14 versus 10 mg/day).7,18 HMF contains a highly reactive aldehyde functional group. HMF produced in foods can react with amino acids via the Michael addition to form various adducts during the thermal processing of foods. Previously, we identified one, three, and four adducts in the reaction models of HMF with glycine, lysine, and cysteine, respectively.19 More recently, Hamzalıoğlu and Gökmen detected three different adducts (nine in total) in each of the following reaction models (at 50 °C and pH 3.5 for 7 days): HMF−arginine, HMF−cysteine, and HMF−lysine.20 These amino acid−HMF adducts can be formed during the digestion of HMF-containing foods.1,19−21 Thus, the adducts of HMF−amino acids ubiquitously exist in thermally processed foods and in the gastrointestinal tracts after intake of these foods. However, their toxicity and pharmacokinetics are seldom investigated. In our previous research, we prepared 1-dicysteinethioacetal−5-hydroxymethylfurfural (DCH), a HMF−cysteine adduct formed between HMF and cysteine during gastric digestion. In

5-Hydroxymethylfurfural (HMF) is an intermediate product in the Maillard reaction and caramelization. HMF can be formed through the reaction between sugars and amino acids in thermally processed food or via the acidic dehydration of carbohydrates.1 Instant coffee (up to 6.2 g/kg) and caramelcontaining foods (110−9500 mg/kg) have the highest HMF content, followed by dried fruits (up to 2200 mg/kg), breads (up to 410 mg/kg), and cookies (0.5−74.5 mg/kg).2−4 Although HMF has favorable biological effects, including antihypoxic, in vitro antioxidant, anti-allergic, and anti-sickling effects,4−6 it also has genotoxic, mutagenic, carcinogenic, DNA-damaging, organotoxic, and enzyme inhibitory effects.4 HMF can be converted into the genotoxicant, 5-sulfoxymethyfurfural (SMF), by the enzyme sulfotransferase, which is distributed in nearly all of the organs of animals and humans, especially in the liver, kidneys, and colon.7,8 SMF showed mutagenicity toward Salmonella typhimurium and substantial DNA damage in the renal cells of mice.9,10 HMF and SMF are carcinogenic agents in mice and induce aberrant crypt foci, adenomas, and tumors in the colon, kidneys, and skin.4 HMF has cytotoxic effects at high concentrations, causing irritation in the mucous membranes, skin, eyes, and upper respiratory tract11 and liver and renal damage in mice.12 The incubation of cell line V79 and Caco-2 with HMF at 50 and 120 mM decreases cellular glutathione (GSH) levels,13 indicating that HMF depletes antioxidants in vivo. HMF inhibits nuclear DNA replication by inhibiting polymerase γ and terminal deoxynucleotidyl transferase14 and causes the mortality of bees and their larvae.15 © 2018 American Chemical Society

Received: Revised: Accepted: Published: 11451

August 8, 2018 October 9, 2018 October 10, 2018 October 10, 2018 DOI: 10.1021/acs.jafc.8b04260 J. Agric. Food Chem. 2018, 66, 11451−11458

Article

Journal of Agricultural and Food Chemistry

The liver, heart, and kidney samples were rinsed in ice-cold saline for the removal of the blood cells, and then 10% (w/v) tissue homogenate was prepared in ice-cold saline with a glass homogenizer. The protein content (bicinchoninic acid method, PC0020), malondialdehyde (MDA, A003-2), total superoxide dismutase (TSOD, A001-2), and GSH (A061-1) were measured according to the instructions of the manufacturer of the commercial kits (Nanjing Jiancheng Biological Engineering Research Center, Nanjing, China). Glutathione S-transferase (GST) was measured with a test kit purchased from AmyJet Scientific, Inc. (Wuhan, China). Determination of HMF, DCH, and SMF. Before determination, tissues, blood, and feces were pretreated according to the method described by Heller et al. and Pastoriza de la Cueva et al.7,25 For the liver, heart, and kidneys, 0.2 g of rat tissue was homogenized with 1.0 mL of 10% methanol solution, sonicated for 5 min, and then centrifuged at 12000g for 20 min at 4 °C. To remove proteins, 200 μL of the obtained supernatant was mixed with 40 μL of trichloroacetic acid (TCA, 30%) and vortexed for 1 min. The mixture was centrifuged at 15000g for 10 min; HMF, DCH, and SMF in the supernatant were determined with HPLC−MS/MS. The blood samples were centrifuged at 12000g for 20 min at 4 °C; 100 μL of the supernatant was mixed with 20 μL of TCA (30%) and vortexed for 1 min. The mixture was centrifuged at 15000g for 10 min; HMF, DCH, and SMF were determined in the supernatant. Fecal samples at 0.1 g were homogenized with 1.0 mL of 1% TCA, sonicated for 15 min, and then centrifuged at 15000g for 10 min at 4 °C. HMF, DCH, and SMF were determined in the supernatant. HPLC−MS/MS analysis was performed using a Shimadzu Nexera X2 (Shimadzu Corporation, Kyoto, Japan) consisting of a DGU-20A degassing unit, two LC-30AD pumps, a SIL-30AC autosampler, a CTO-20AC column oven, and a SPD-20A ultraviolet/visible (UV/ vis) detector linked to a CBM-20A module controller interfaced to a Shimadzu triple quadruple LCMS-8045 mass spectrometer (Shimadzu Corp., Kyoto, Japan). The pretreated samples were filtered through a 0.45 μm syringe nylon membrane, and 4 μL of sample was injected into the liquid chromatography−mass spectrometry (LC−MS) system and analyzed with an Agilent InfinityLab Poroshell 120 ECC18 column (100 × 3 mm, 2.7 μm, Agilent Technologies, Inc., Santa Clara, CA, U.S.A.). The temperature of the column oven was set to 40 °C. The mobile phase consisted of 0.1% formic acid aqueous solution as solvent A and 0.1% formic acid in acetonitrile as solvent B. The binary eluting gradient program was as follows: 5% B at 0.3 mL/min for 0−6.5 min, 30% B at 0.4 mL/min for 6.5−15 min, and 5% B at 0.3 mL/min for 15.0−22 min. Electrospray ionization mass spectroscopy (ESI−MS) analysis was performed in positive-ion mode for HMF and DCH and in a negative ion mode for SMF detections. The electrospray source, heat block, and desolvation temperatures were set at 300, 450, and 250 °C, respectively. The flow rates of nebulizing gas, drying gas, and heating gas were kept at 3, 10, and 10 L/min, respectively. A multiple reaction monitoring (MRM) method was devised for detection and quantification of HMF using the fragmentation transitions m/z = 127.10 → 81.10 (collision energy of 17 eV) and m/z = 127.10 → 109.05 (14 eV), respectively; DCH, m/z = 351.40 → 212.10 (22 eV) and m/z = 351.40 → 230.05 (18 eV), respectively; and SMF, m/z = 204.90 → 80.9 (19 eV) and m/z = 204.90 → 95.90 (22 eV), respectively. HMF, DCH, and SMF were quantified by calibration curves of external standards. The linear regressions were obtained for HMF (r2 = 0.9997; 1.04−520 ppb), DCH (r2 = 0.9990; 10−2000 ppb), and SMF (r2 = 0.9973; 2−50 ppb). Fecal Bacterial DNA Extraction. The fecal bacterial DNA of each sample was extracted with a TIANamp stool DNA kit (Tiangen, Beijing, China), following the instructions of the manufacturer. 16S rRNA gene PCR amplification and sequencing was performed by Guangzhou Gene Denovo Co. (Guangzhou, China), as we previously described.26 Statistical Analysis. Data were expressed as the mean ± standard error. The statistical significance of the results was evaluated using one-way analysis of variance (ANOVA). Duncan’s multiple range was

comparison to HMF, DCH showed a considerably lower absorption rate and lower cytotoxicity in Caco-2 cells.1 However, decreased absorption of the adduct in vivo is not yet proven. This research aims to investigate the absorption and pharmacokinetics of DCH and its effect on the oxidative stress in rats, using HMF as the positive control. Given that the bioaccessibility of HMF is only 60−80% in rats22 and approximately 40% HMF in dried fruits is not absorbed in humans,23 the unabsorbed part of HMF reaches the colon. Thus, another goal of this research is to investigate the modulation effect of DCH and HMF on gut microbiota.

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MATERIALS AND METHODS

Reagents. HMF (98%) and L-cysteine (99%) were purchased from Aladdin Reagent Database, Inc. (Shanghai, China). SMF was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). DCH (99.9%) was prepared as we previously reported.1 Animal Groups and Experimental Design. The 7-week-old Sprague Dawley (SD) male rats (150−160 g) and diets (18% protein, 60% carbohydrate, and 12% fat) were purchased from the Experimental Animal Center of Guangdong Province (certificate number SCXK 2016-0167, Guangzhou, China). The animals were acclimatized for 7 days in an environment-controlled room at 21 ± 2 °C and relative humidity of 75% with a 12 h light−dark cycle. A first study was performed to assess the absorption and distribution of DCH and HMF in rats after their intragastrical administration at the level of 100 mg/kg of body weight (bw). Animals were fasted overnight (12 h). Tail blood was collected in 30, 60, 120, 150, and 180 min after DCH and HMF were intragastrically administered. HMF, DCH, and SMF were determined in the blood using the high-performance liquid chromatography−tandem mass spectrometry (HPLC−MS/MS) method after pretreatment. The animals were anesthetized with pelltobarbitalum natricum and sacrificed at 180 min; heart, liver, and kidneys were removed to determine HMF, DCH, and SMF contents. We performed the second study to investigate the effect of DCH and HMF on oxidative stress and gut microbiota after the administration of different doses of DCH and HMF for 1 week. The animals were divided into five groups (n = 6, with three rats in a cage): group 1, rats without administration of drugs; groups 2 and 3, rats administered with 20 and 100 mg kg−1 of bw day−1 of HMF (HMF 20 and HMF 100), respectively; and groups 4 and 5, rats administered with 20 and 100 mg kg−1 of bw day−1 of DCH (DCH 20 and DCH 100), respectively. The drugs were administered intragastrically every morning for 1 week. The animals (fasted overnight, 12 h) were weighed and then sacrificed after they were anesthetized with pelltobarbitalum natricum. For the biochemical estimations, plasma samples were obtained by centrifuging the blood samples of the rats at 4000g and 4 °C for 20 min. The heart, liver, and kidneys of each rat were homogenized in phosphate-buffered saline (PBS) at 4 °C for protein determination and biochemical assessments. HMF and DCH were determined in the colon contents and feces after pretreatment, and microbiota was determined in the feces using the 16S rRNA gene polymerase chain reaction (PCR) amplification and sequencing method. All of the animals were maintained in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of People’s Republic of China. All of the procedures were conducted according to the protocol (20180507023) approved by the Institutional Animal Care and Use Committee of the Laboratory Animal Center of Jinan University. Biochemical Assessment. Triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels of rats were determined with a 7180 model automatic biochemistry analyzer (Hitachi, Tokyo, Japan) according to our previous research.24 11452

DOI: 10.1021/acs.jafc.8b04260 J. Agric. Food Chem. 2018, 66, 11451−11458

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Journal of Agricultural and Food Chemistry used to analyze differences between the means. Statistical significance was set at the p < 0.05 level.

Table 1. HMF and DCH Contents (μg/g of Protein) in the Heart, Liver, and Kidneys after the Intragastric Administration of 100 mg/kg of bw of DCH and HMF at 180 min

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RESULTS AND DISCUSSION Absorption and Distribution of HMF and DCH in Rats. The acute oral LD50 values of HMF were 3100 and 1910 mg/kg of bw in rats and mice, respectively.17 In the present study, in order not to induce acute toxicity of rats, we used an a relatively lower dose (20 and 100 mg/kg of bw) of HMF (or DCH) in comparison to the dose (1500 mg/kg bw) used by Pastoriza de la Cueva et al.7 DCH, HMF, and SMF can be well-determined using HPLC−MS/MS; their retention time was 3.0, 4.9, and 2.7 min, respectively (Figure S1 of the Supporting Information). In the HMF-administered rats, the serum HMF concentration reached a peak at 120 min and then decreased again below the concentration determined after the first 30 min (top panel of Figure 1). A trace amount of SMF (0.04−0.09 μg/L) was

heart HMF administration

DCH administration

HMF content DCH content HMF content DCH content

0.4 ± 0.2 ND

liver 0.1

kidneys 1.2 ± 0.3

ND

ND

0.2 ± 0

1.0 ± 0.4

1.1 ± 0.5

5.0 ± 1.3

3.4 ± 1.8

36.5 ± 4.8

DCH concentrations were 15−38-fold of HMF concentrations at different times after drug administration. It was different from the situation that we found in Caco-2 cells, in which DCH was less absorbed than HMF.1 The possible reason is that Caco-2 cells do not always express an adequate amount of transporters or enzymes and exhibit a much lower permeability coefficient than the small intestine for some drugs, such as araloside.27 In comparison to HMF, DCH had a higher concentration in the tissues, especially in the kidneys, after administration of each (Table 1). Therefore, DCH were more likely to be absorbed and accumulated in tissues of different organs than HMF. A trace amount of SMF (0−0.04 μg/L; data not shown) was detected in the serum. The detected SMF was possibly metabolized from HMF that was converted from DCH. The present research found that 2.7% (30 min) to 4.5% (150 min) of DCH was converted to HMF, as shown in Figure 1, which confirmed our previous finding in Caco-2 cells (a total of 3.31% conversion).1 Surprisingly, the released amount (concentration) of HMF from DCH in serum reached as high as that from the same dose of HMF administration at 120 min and even higher afterward (Figure 1). Thioacetals are inert toward nucleophiles and reductive conditions.28 In the chemical synthesis, its deprotection to the parent carbonyl compounds is difficult;29 the deprotection can only be undertaken through the addition of an oxidizing reagent or catalysis by Brønsted acids.29 These transformation conditions do not occur in the serum of rats. Cytochrome P450 may contribute to the transformation of DCH to HMF. Cytochrome P450 is an ubiquitous enzyme family involved in the metabolism of xenobiotics; they metabolize approximately 90% of the marketed drug compounds.30 The oxidation process may follow dealkylation (heteroatom release), as described by Guengerich.31 Antioxidant or Oxidative Stress Effect of HMF and DCH. Oxidative stress is an excessive production of reactive oxygenated species that cannot be counteracted by the action of antioxidants as well as a perturbation of the cell redox balance.32 Oxidative degradation of lipids of the cell membrane is usually expressed in terms of the MDA content. In the living organisms, superoxide dismutase (SOD), catalase, and glutathione peroxidase are the enzyme defense systems against oxidative stress. GSH is an antioxidant and a cytoprotectant that can scavenge hydrogen peroxide, hydroxyl anion, and chlorinated oxidants.32,33 Oxidative stress is associated with an increased production of oxidizing species or a significant decrease in the effectiveness of antioxidant defenses, such as GSH, and the activity of SOD, catalase, and glutathione peroxidase.33 The antioxidative stress effects refer to the ability

Figure 1. Serum concentration of HMF and DCH in rats after intragastric administration of 100 mg/kg of bw of (top) HMF and (bottom) DCH. The bars represented standard errors (n = 6). Values with different letters (capital letters for HMF and lowercase letters for DCH) in a curve are significantly different (p < 0.05).

detected in the serum (data not shown), and an extremely low HMF level was detected in the heart, liver, and kidneys (Table 1). However, no DCH was detected in the serum of the heart, liver, and kidneys (top panel of Figure 1 and Table 1) after HMF administration. Similar to HMF, the serum DCH concentration of DCHadministered rats also reached a peak at 120 min and then slowly decreased; its concentration in the serum still maintained twice the serum concentration that was determined after the first 30 min (bottom panel of Figure 1). In comparison to HMF, DCH was more easily absorbed; serum 11453

DOI: 10.1021/acs.jafc.8b04260 J. Agric. Food Chem. 2018, 66, 11451−11458

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Journal of Agricultural and Food Chemistry of a cell overcoming small perturbations and regaining its original state.34 HMF has been reported to show in vitro antioxidant and antiproliferative activities and increase the GSH level as well as the activity of SOD, catalase, and glutathione peroxidase in the alcoholic liver of mice.6,35 Thus, in this study, the SOD activity and GSH and MDA contents were determined in the plasma, heart, liver, and kidneys of rats after drug administrated at two levels for 1 week. The results in Figure 2 shows that HMF and DCH administration at 20 mg/kg of bw significantly increased the GSH level and SOD activity in the plasma, heart, liver, and kidneys. When the administration level of HMF increased to 100 mg/kg of bw, the level of GSH and SOD decreased to or even lower than that of the control in the plasma, heart, liver, and kidneys, respectively (Figure 2). Increasing the administration level of DCH to 100 mg/kg of bw also significantly decreased the level of GSH and SOD in comparison to that in the rats HMF-administrated with 20 mg/kg of bw, but the GSH level in the plasma and kidneys and SOD activity in the three tissues were still higher than those of the control (Figure 2). Oxidative stress induces an increase in the level of SOD, catalase, and GSH in people with diabetes and lead exposure.36 Thus, under oxidative-stress-induced conditions (such as those induced by drugs and toxins), an increase in the activity of oxidative stress defense system is an adaptive mechanism against reactive oxygen species.37 Among the biological targets of oxidative stress, lipids in the cell membrane are the most involved class of biomolecules. Lipid oxidation generates numerous secondary products, mainly aldehydes.38 MDA is the principal and most studied product of unsaturated fatty acid peroxidation, which acts as an indirect determinant of lipid peroxidation in cells.4 Figure 2 shows that HMF administration at a level of 100 mg/kg of bw significantly increased the MDA content in the plasma, heart, liver, and kidneys, which confirmed the finding from Li et al.35 They administered mice with HMF at 7.5, 15, and 30 mg/kg of bw and found that HMF increased the hepatic MDA content in a dose-dependent manner. Specifically, the hepatic MDA content increased by 43% when the HMF administration level was increased from 7.5 to 30 mg/kg of bw. DCH was highly effective in elevating the MDA content in the plasma and in the three organs at high administration levels (Figure 2). Especially in the liver and kidneys, the MDA content in DCHtreated rats was significantly higher than that in HMF-treated rats, suggesting that DCH-derived HMF as well as DCH itself co-contributed to the elevation of the MDA level. We speculate that an increase in the antioxidant (SOD and GSH) contents in the plasma and tissues after HMF and DCH administration is an adaptive mechanism against oxidative stress induced by HMF and DCH, instead of an antioxidant activity produced by HMF and DCH. Furthermore, a high dose (100 mg/kg) of drug administration may destroy the generation of antioxidant systems in rats and decrease the SOD activity and GSH level. Changes in GST activity also proved our speculation. GSTs are crucial xenobiotic-metabolizing enzymes. Their overexpression responds to the production of reactive oxygen species induced by drugs, such as thiopurine.39 Results in Figure 3 indicated that GST activity sharply increased after administering 20 mg/kg of HMF or DCH.

Figure 2. Effect of HMF and DCH on GSH, SOD, MDA, and GST in the plasma, heart, liver, and kidneys and on serum lipids after drug administration for 1 week. Values [means ± standard deviation (SD); n = 6] with different letters are significantly different (p < 0.05).

Hyperlipidemia is another characteristic of oxidative stress. However, DCH and HMF showed no influential effect on serum lipid in rats after 1 week of intake. Modulation of HMF and DCH in Gut Microbiota. HMF can be metabolized by intestinal bacteria, such as Escherichia 11454

DOI: 10.1021/acs.jafc.8b04260 J. Agric. Food Chem. 2018, 66, 11451−11458

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Journal of Agricultural and Food Chemistry

Figure 3. Effects of DCH and HMF on gut microbiota: (A) phylum, (B) class, (C) order, and (D) family. Values (means ± SD; n = 6) with different letters are significantly different (p < 0.05).

Table 2. OTU and Different Estimator Parameters for α Diversity in the Gut Microbiota of Rats after Oral Administration of DCH and HMF for 7 Daysa OTU Shannon Simpson

CK

DCH 20

DCH 100

HMF 20

HMF 100

829 ± 34 b 5.98 ± 0.32 a 0.96 ± 0.01 a

787 ± 101 b 6.25 ± 0.53 a 0.96 ± 0.02 a

697 ± 73 a 6.07 ± 0.38 a 0.95 ± 0.03 a

815 ± 64 b 6.31 ± 0.1 a 0.97 ± 0.0 a

772 ± 65 a 6.14 ± 0.36 a 0.96 ± 0 a

Values (means ± SD; n = 6) with different letters in a row are significantly different (p < 0.05).

a

sp. 88, Bacillus sp. 46, and Bacteroides sp.,40 and HMF in foods negatively affected the growth of Lactobacilli, Escherichia, and Shigella.41,42 To test the modulation effect of HMF and DCH on the gut microbiota, in the present study, we determined the microbiota in feces after 1 week of drug administration using the 16S rRNA gene PCR amplification and sequencing method.

An operational taxonomic unit (OTU) is used to estimate the total bacterial species.26 The results in Table 2 indicated that administration of DCH and HMF at 100 mg/kg of bw significantly decreased bacterial species in the feces of rats (Table 2). Shannon and Simpson diversity indices were used for the estimation of the richness and diversity of bacteria. The administration of HMF and DCH at two levels did not influence the values of these parameters (Table 2). 11455

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Journal of Agricultural and Food Chemistry Table 3. Effect of DCH and HMF on the Number of Reads at the Genus Levela CK Akkermansia Bacteroides Prevotellaceae UCG-001 Lachnospiraceae NK4A136 group Alloprevotella Parasutterella Lactobacillus Blautia Alistipes Escherichia−Shigella Ruminococcus 1 Prevotellaceae NK3B31 group Desulfovibrio Bifidobacterium Oscillibacter Dorea Coprostanoligenes group Xylanophilum group Lachnoclostridium Parabacteroides Streptococcus Coriobacteriaceae UCG002 Ruminiclostridium 9 Ruminiclostridium Roseburia Anaerotruncus Helicobacter Butyricimonas Intestinimonas Rikenella Tyzzerella Adlercreutzia Aerococcus Enterorhabdus

7120 5674 7712 3140 3997 2069 2515 1952 2178 805 895 1508 533 676 1364 187 714 682 643 565 115 383 238 419 232 292 142 182 105 93 85 48 163 47

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

4891 a 2126 a 2394 b 523 a 2590 c 1579 a 630 b 719 a 656 b 130 a 310 b 300 b 184 a 349 a 244 c 67 a 154 a 190 a 236 b 176 ab 60 a 324 a 160 a 86 a 171 a 155 a 38 ab 66 a 52 a 25 a 39 ab 20 a 58 c 24 a

DCH 20 9289 4587 2304 3003 2137 2204 1423 2538 1989 1685 568 698 1245 483 664 2026 675 603 522 469 1168 378 384 288 256 258 154 185 141 136 130 79 54 66

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

DCH 100

7987 ab 1152 a 1256 a 1212 a 857 ab 1579 a 474 a 432 a 493 b 892 b 207 a 138 a 472 b 524 a 180 a 478 b 271 a 213 a 178 b 115 a 939 d 403 a 326 a 170 a 106 a 114 a 50 ab 71 a 31 ab 34 b 60 bc 31 ab 18 a 18 a

15356 4656 1876 2955 1822 2600 1582 1761 2074 1317 1121 715 920 788 615 560 730 596 339 647 770 544 269 343 397 235 103 235 107 153 73 91 91 67

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

5051 b 1560 a 867 a 1409 a 449 a 1097 a 146 a 533 a 396 b 369 ab 316 bc 360 a 89 b 423 ab 200 a 585 a 197 a 299 a 194 a 208 ab 415 c 382 ab 55 a 171 a 176 a 49 a 46 a 50 a 27 a 37 b 27 a 39 b 13 b 27 a

HMF 20 4828 4636 3378 4938 1629 2333 1702 1918 1753 709 1149 734 927 821 1047 407 520 747 531 567 306 425 703 295 404 332 301 204 172 76 137 103 25 69

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

5273 a 944 a 1137 a 1478 b 695 a 1731 a 371 ab 519 a 351 ab 434 a 191 bc 273 a 181 b 499 ab 309 b 192 a 154 a 255 a 173 b 187 ab 269 b 75 a 286 b 141 a 222 a 138 a 58 c 48 a 53 b 23 a 48 c 38 b 15 a 28 a

HMF 100 4357 4513 6487 2690 3662 3639 4151 1949 1338 1046 1386 1187 993 1413 607 385 693 538 888 698 371 809 421 448 208 252 195 201 167 144 141 121 28 114

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3875 a 1180 a 2817 b 425 a 850 bc 1982 a 1520 c 1012 a 308 a 450 ab 213 c 259 b 235 b 497 b 235 a 227 a 113 a 287 a 277 c 155 b 310 b 190 b 218 a 160 a 56 a 82 a 85 b 55 a 37 b 10 b 28 c 30 b 16 a 30 b

Values (means ± SD; n = 6) with different letters in a row are significantly different (p < 0.05).

a

Firmicutes, Bacteroidetes, Clostridia, Proteobacteria, and Actinobacteria are the dominant bacteria detected in rat feces (Figure 3A). The administration of DCH significantly increased the number of reads for Proteobacteria and Verrucomicrobia but decreased the number of reads for Bacteroidetes. Moreover, it decreased the ratio of Bacteroidetes/Firmicutes, an indicator for weight loss.26 The administration of HMF (100 mg/kg of bw) increased the number of reads for Bacteriodetes, Acinobacteria (100 mg/kg of bw), and Proteobacteria (20 and 100 mg/kg of bw). However, it also decreased the ratio of Bacteroidetes/ Firmicutes after 20 mg/kg of bw of HMF administration (Figure 3A). At the class level, DCH (100 mg/kg of bw) significantly increased the number of reads for Verrucomicrobiae and Betaproteobacteria while significantly decreasing the number of reads for Bacteroidia. HMF (100 mg/kg of bw) only significantly increased the number of reads for Betaproteobacteria (Figure 3B). At the order level, DCH (100 mg/kg of bw) significantly decreased the number of reads for Bacteroidales while significantly increasing Verrucomicrobiales. HMF significantly increased the number of reads for Burkholderiales (Figure 3C). At the family level, DCH significantly decreased the number of reads for Prevotellaceae and Rikenellaceae (20 and 100 mg/kg of bw) but significantly

increased the number of reads for Lachnospiraceae (20 mg/kg of bw) and Verrucomicrobiaceae; HMF (100 mg/kg of bw) significantly increased the number of reads for Bacteroidales S 24 (100 mg/kg of bw), Ruminococcaceae (20 and 100 mg/kg of bw), Verrucomicrobiaceae (100 mg/kg of bw), Lactobacillaceae (100 mg/kg of bw) but significantly decreased the number of reads for Bacteroidaceae and Rikenellaceae (100 mg/kg of bw) (Figure 3D). The number of reads assigned to each genus was listed in Table 3. DCH (100 mg/kg of bw) significantly increased the number of reads for Akkermansia, Escherichia, Shigella, and Rikenella but significantly decreased the number of reads for Prevotellaceae UCG-001, Alloprevotella, Lactobacillus, Prevotellaceae NK3B31 group, and Oscillibacter. HMF (100 mg/kg of bw) significantly increased the number of reads for Lactobacillus, Ruminococcus 1, Desulfovibrio, Lachnoclostridium, Streptococcus, Coriobacteriaceae UCG-002, Intestinimonas, Rikenella, Tyzzerella, and Enterorhabdus but significantly decreased the number of reads for Alistipes, Oscillibacter, and Aerococcus (Table 3). Gut microbiota are related to human health. Akkermansia muciniphila has potential anti-inflammatory properties; a decrease in A. muciniphila has been observed in patients with inflammatory bowel diseases (mainly ulcerative colitis) and 11456

DOI: 10.1021/acs.jafc.8b04260 J. Agric. Food Chem. 2018, 66, 11451−11458

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Journal of Agricultural and Food Chemistry metabolic disorders.43 Tyzzerella, Tyzzerella 4, and Enterobacter are associated with increased cardiovascular disease.44 Shigella species invade the epithelial lining of the colon, resulting in severe inflammation and death of the cells lining the colon. Administration of DCH or HMF influenced the growth of these bacteria (Table 3), indicating that their intake might influence human health. Amino acid−HMF adducts are ubiquitously formed during thermal processing of foods and gastrointestinal tracts after food intake. This study presented the first investigation of the absorption and metabolism of one of the cysteine−HMF adducts, DCH. In this research, DCH was proven to be more easily absorbed than its parent foodborne contaminant (HMF). It could be transformed into HMF after absorption, and it could generate oxidative stress and modulate the gut microbiota. Some other toxic foodborne contaminants, especially acrolein and acrylamide, can more easily form adducts with amino acids.45,46 The findings in our present research indicated that food scientists should focus on the adduct formed between these contaminants and amino acids. Although HMF and DCH administration concentrations in the present study were within the usual range of similar studies,7 they were much higher than the regular HMF exposition of humans.7 To understand the real effects of HMF and DCH, lower doses of HMF and DCH over a longer term still need further investigation in future studies.

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sulfoxymethyfurfural; SOD, superoxide dismutase; TC, total cholesterol; TCA, trichloroacetic acid; TG, triglyceride

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b04260. Detemination of DCH (3.0 min), HMF (4.9 min), and SMF (2.7 min) in the serum using HPLC−MS/MS (Figure S1) (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*Telephone: +86-18148962369. Fax: +86-20-85226630. Email: [email protected]. *Telephone: +86-13640210646. Fax: +86-20-85226630. Email: [email protected]. ORCID

Yong Wang: 0000-0001-7547-1542 Jie Zheng: 0000-0001-9755-5595 Shiyi Ou: 0000-0002-6779-0858 Author Contributions †

Qianzhu Zhao and Juanying Ou contributed equally to this work. Funding

The present work was funded by the National Natural Science Foundation of China (Grants 31671957 and 31871902). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED DCH, 1-dicysteinethioacetal-5-hydroxymethylfurfural; GSH, glutathione; GST, glutathione S-transferase; HDL-C, highdensity lipoprotein cholesterol; HMF, 5-hydroxymethylfurfural; LDL-C, low -density lipoprotein cholesterol; MDA, malondaildehyde; MRM, multiple reaction monitoring; PBS, phosphate-buffered saline; ppb, parts per billion; SMF, 511457

DOI: 10.1021/acs.jafc.8b04260 J. Agric. Food Chem. 2018, 66, 11451−11458

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DOI: 10.1021/acs.jafc.8b04260 J. Agric. Food Chem. 2018, 66, 11451−11458