Trapping Methylglyoxal by Genistein and Its Metabolites in Mice

Article pubs.acs.org/crt

Trapping Methylglyoxal by Genistein and Its Metabolites in Mice Pei Wang, Huadong Chen, and Shengmin Sang* Laboratory for Functional Foods and Human Health, Center for Excellence in Post-Harvest Technologies, North Carolina Agricultural and Technical State University, North Carolina Research Campus, 500 Laureate Way, Kannapolis, North Carolina 28081, United States S Supporting Information *

ABSTRACT: Increasing evidence supports dicarbonyl stress such as methylglyoxal (MGO) as one of the major pathogenic links between hyperglycemia and diabetic complications. In vitro studies have shown that dietary flavonoids can inhibit the formation of advanced glycation end products (AGEs) by trapping MGO. However, whether flavonoids can trap MGO in vivo and whether biotransformation limits the trapping capacity of flavonoids remain virtually unknown. In this study, we investigated whether genistein (GEN), the major soy isoflavone, could trap MGO in mice by promoting the formation of MGO adducts of GEN and its metabolites. Two different mouse studies were conducted. In the acute study, a single dose of MGO and GEN were administered to mice via oral gavage. In the chronic study, MGO was given to mice in drinking water for 1 month and then GEN was given to mice for 4 consecutive days via oral gavage. Two mono-MGO adducts of GEN and six mono-MGO adducts of GEN phase I and microbial metabolites were identified in mouse urine samples from these studies using liquid chromatography/electrospray ionization tandem mass spectrometry. The structures of these MGO adducts were confirmed by analyzing their MSn (n = 1−4) spectra as well as by comparing them with the tandem mass spectra of authentic standards. All of the MGO adducts presented in their phase II conjugated forms in mouse urine samples in the acute and chronic studies. To our knowledge, this is the first in vivo evidence to demonstrate the trapping efficacy of GEN in mice and to show that the metabolites of GEN remain bioactive.

■

INTRODUCTION Accumulating evidence indicates that the formation of advanced glycation end products (AGEs) is one of the major pathogenic links between hyperglycemia and diabetes-related complications.1 Of all of the AGE precursors, methylglyoxal (MGO) is known to contribute significantly to the formation of intracellular MGO-derived AGEs, such as Nε-carboxyethyllysine (CEL) and methylglyoxal lysine dimer (MOLD).2−4 MGO can react with proteins to produce AGEs, mainly through lysine, arginine, and cysteine residues. This process affects different proteins such as insulin, hemoglobin, and growth factors,5−7 and it damages DNA, resulting in cellular toxicity.8 It has also been reported that plasma levels of MGO in diabetic patients (2.2−3.8 μM) are significantly higher than those in healthy volunteers (0.4−1.0 μM).9 In addition, many food products and beverages, such as cookies, breads, honey, coffee, wine, beer, and carbonated soft drinks, as well as cigarette smoke, represent exogenous sources of MGO.10 Furthermore, numerous animal studies have reported that MGO is a key contributor to the production of AGEs and impairs insulin signaling, GLUT transporters, kinases, and endothelial cells.11−13 Thus, scavenging reactive dicarbonyl species is an effective strategy to prevent protein modifications and AGE formation.14−16 Previous studies from our laboratories have demonstrated that dietary flavonoids, such as (−)-epigallocatechin-3-O-gallate (EGCG) from tea, phloridzin and phloretin from apples, and © 2016 American Chemical Society

genistein (GEN, 4′,5,7-trihydroxyisoflavone; Figure 1) from soybeans, can effectively trap reactive α-dicarbonyl compounds and inhibit the formation of AGEs in vitro.17−21 Our results have also shown that the A ring is the critical active site in flavonoids that contributes to their MGO trapping efficacy.20 However, whether flavonoids can trap MGO in vivo remains unresolved. Many factors affect the translation of in vitro findings to the in vivo setting. One of these factors is the systemic bioavailability of flavonoids, which may result in therapeutic variability and, in some cases, therapeutic failure. Following their ingestion, the effects of the absorbed flavonoids may depend on their conversion by phase I/phase II host enzymes and/or the activity of the intestinal microbiota. Furthermore, the ability of the flavonoid metabolites to retain the MGO trapping efficacy requires further investigation. The main objective of this study was to investigate the in vivo MGO trapping efficacy of GEN, one of the dominant dietary isoflavones present in soybeans and plant-derived foods,22 and its metabolites in mice.

■

MATERIALS AND METHODS

Materials. GEN, MGO (40% in water), DMSO, and sulfatase from Aerobacter aerogenes and β-glucuronidase from Helix aspersa were purchased from Sigma (St. Louis, MO, USA). Mono-MGO adducts of Received: January 5, 2016 Published: February 16, 2016 406

DOI: 10.1021/acs.chemrestox.5b00516 Chem. Res. Toxicol. 2016, 29, 406−414

Article

Chemical Research in Toxicology

Figure 1. Structures of GEN and its phase I and microbial metabolites and their MGO adducts. GEN were synthesized in our laboratory.19 HPLC-grade solvents and other reagents were obtained from VWR International (South Plainfield, NJ, USA). LC-MS-grade solvents were obtained from Thermo Fisher Scientific (Pittsburgh, PA, USA). Animals. Experiments with mice were conducted according to a protocol approved by the Institutional Animal Care and Use Committee of the North Carolina Research Campus (no. 12-012). Female C57BL/6J mice were purchased from the Jackson Laboratory and acclimated for at least 1 week before being randomly assigned to different experimental groups. The mice were housed (5 mice/cage) and maintained in air-conditioned quarters with a room temperature of 20 ± 2 °C, relative humidity of 50 ± 10%, and a light−dark cycle of 12:12 h (7 am to 7 pm). The mice were allowed free access to water and were fed an AIN-93G diet. Animal Studies. Acute Study. Ten mice were divided into a control group and an acute MGO and GEN-treated group and were kept in metabolic cages (5 mice/cage). For the MGO and GENtreated group, each mouse received a single dose of GEN (400 mg/kg body weight in DMSO) via oral gavage. Ten minutes later, MGO solution (1.0 g/kg body weight in water) was administrated to the mice via oral gavage. The mice in the control group received DMSO followed by water. Twenty-four hour mouse urine samples were collected and stored at −80 °C before analysis. Chronic Study. Ten mice were divided into two groups: one control group and one MGO-treated group. In the MGO-treated group, MGO (0.96% MGO) was administered in the drinking water for 1 month. The mice were then treated with GEN by oral gavage (130 mg/kg body weight in DMSO) for 4 consecutive days. The mice in the control group were fed with water for 1 month and then treated with vehicle (DMSO). After the final treatment of GEN, urine samples were collected at 2, 4, 6, and 24 h. Urine Sample Preparation. Enzymatic deconjugation was performed as described previously with slight modification.23 In brief, duplicate mouse urine samples (100 μL) were prepared in the presence of β-glucuronidase (250 U) and sulfatase (3 U) for 2 h at 37 °C. The reaction was stopped with an equal volume of acetonitrile (1% acetic acid) and centrifuged at 16 100g for 10 min at 4 °C. The supernatant was transferred to a new tube and dried under nitrogen gas, and the residue was reconstituted in 200 μL of 75% aqueous methanol with 0.1% acetic acid for further LC-MS analysis.

LC-MS Analysis. LC-MS was performed with a Thermo-Finnigan Spectra system consisting of an Ultimate 3000 degasser, an Ultimate 3000 RS pump, an Ultimate 3000 RS autosampler, an Ultimate 3000 RS column compartment, and an LTQ Velos Pro ion trap mass spectrometer (Thermo Electrom) equipped with an electrospray ionization (ESI) interface. Chromatographic separation was performed using a Gemini C18 column (5 μm, 3.0 mm i.d. × 150 mm; Phenomenex, Torrance, CA, USA). The mobile phase consisted of 5% aqueous methanol with 0.1% formic acid (mobile phase A) and 95% aqueous methanol with 0.1% formic acid (mobile phase B). The gradient elution was performed for 40 min at a flow rate of 0.3 mL/ min. The gradient was initiated at 0% B and held constant for 5 min, followed by a linear increase to 35% from 5 to 10 min, to 55% from 10 to 15 min, to 85% from 15 to 25 min, to 100% from 25 to 30 min, and then held constant for 5 min. The column was then re- equilibrated with 0% B for 5 min. The injection volume of each sample was 10 μL. The LC eluent was introduced into the ESI interface. The positive ion polarity mode was set for the ESI source with the ion spray voltage at approximately 3.6 kV. Nitrogen gas was used as the sheath gas at a flow rate of 34 arb and the aux gas at a flow rate of 10 arb. Optimized parameters, including temperature (310 °C), voltage of the capillary (45 V), and voltage of the tube lens offset (120 V), were tuned using authentic GEN. Selected ion monitoring (SIM) mode was used to search genistein and its metabolites. For MS-MSn (n = 2−4) analysis, collision induced dissociation (CID) was conducted using an isolation width of 1.2 Da and the normalized collision energy of 35 values. The mass range was measured from 50 to 800 m/z. Data acquisition and analysis were performed using Xcalibur 2.0 (Thermo Electron; San Jose, CA, USA).

■

RESULTS Identification of the Major Phase I and Microbial Metabolites of GEN in the Acute Study. It has been reported that GEN can be metabolized by the cytochrome P450s to hydroxylated metabolites (6-, 8-, and 3′-hydroxyGEN)24 and by the gut microbiota to dihydrogenistein (DGEN), 5-hydroxy-equol (5-OH-equol), and 6′-hydroxy-Odemethylangolensin (6′-OH-DMA).25,26 In the present study, all of the potential metabolites in the urine samples collected 407

DOI: 10.1021/acs.chemrestox.5b00516 Chem. Res. Toxicol. 2016, 29, 406−414

Article

Chemical Research in Toxicology

Figure 2. (A−E) SIM chromatograms and ESI-MS2 (positive ion) spectra of GEN and its metabolites.

vitro study, we purified one of the two peaks and identified it as the MGO adduct conjugated at C-8 of the A ring of GEN using NMR, and we proposed that the other peak was the C-6 conjugated adduct of GEN.19 Therefore, we identified the minor peak (tR 19.2) as 8-MGO-GEN (MM-GEN-1) and the major peak (tR 18.7) as 6-MGO-GEN (MM-GEN-2) (Figure 1). The potential mass fragmentation pathways of MM-GEN-1 are proposed in Figure 4A. Identification of MGO Adducts of GEN Phase I and Microbial Metabolites in the Acute Study. Among the six identified phase I and microbial metabolites, DGEN, orobol, 5OH-equol, and 6′-OH-DMA shared the same A ring structure with that of GEN. We recently reported that the A ring is the critical active site of flavonoids contributing to their MGO trapping efficacy in vitro.20 We hypothesized that these four metabolites should retain the MGO trapping effect of GEN in vivo. Thus, we searched for the potential mono- and di-MGO adducts of these four metabolites in the urine samples collected in the acute study, including mono-MGO DGEN (MM-DGEN, m/z 345), mono-MGO orobol (MM-orobol, m/z 359), monoMGO 5-OH-equol (MM-5-OH-equol, m/z 331), mono-MGO 6′-OH-DMA (MM-6′-OH-DMA, m/z 347), di-MGO DGEN (DM-DGEN, m/z 417), di-MGO orobol (DM-orobol, m/z 431), di-MGO 5-OH-equol (DM-5-OH-equol, m/z 403), and di-MGO 6′-OH-DMA (DM-6′-OH-DMA, m/z 419). The mono- and di-MGO adducts of 6-OH-GEN (MM-6-OH-GEN, m/z 359; DM-6-OH-GEN, m/z 431) and 8-OH-GEN (MM-8OH-GEN, m/z 359; DM-8-OH-GEN, m/z 431) were also placed on the SIM list in case of any omission. As we hypothesized, we were able to detect the mono-MGO adducts of DGEN, orobol, 5-OH-equol, and 6′-OH-DMA but not those of 6-OH-GEN and 8-OH-GEN. In addition, we were unable to

from the acute MGO and GEN-treated mice were assessed using SIM mode, including 6-hydroxy-GEN (6-OH-GEN, m/z 287), 8-hydroxy-GEN (8-OH-GEN, m/z 287), 3′-hydroxyGEN (orobol, m/z 287), DGEN (m/z 273), 5-OH-equol (m/z 259), and 6′-OH-DMA (m/z 275). After an acute GEN and MGO challenge, DGEN (retention time (tR) 19.6 min), orobol (tR 19.5 min), 5-OH-equol (tR 17.7 min), 6-OH-GEN (tR 18.9 min), 8-OH-GEN (tR 18.3 min), and 6′-OH-DMA (tR 20.6 min) were detected as the phase I and microbial metabolites of GEN in mouse urine. All six metabolites were identified by comparing their mass spectra (Figure 2) with those reported in the literature.24,27−30 The mass spectra of 6-OH-GEN and 8OH-GEN are shown in Figure S1. Identification of the MGO Adducts of GEN in the Acute Study. In our in vitro study, we identified both monoand di-MGO adducts of GEN in the reaction between MGO and GEN.31 Therefore, we searched both the mono- and diMGO adducts of GEN in the mouse samples collected in the acute study. However, we observed only two peaks (tR 18.7 and 19.2 min) that corresponded to the mono-MGO adduct of GEN (molecular ion m/z 343 [M + H]+) obtained from the positive ESI-MS (Figure 3A). Both peaks were 72 mass units higher than that of GEN (m/z 270), which indicated that they were mono-MGO adducts of GEN (the molecular weight of MGO is m/z 72). This finding was further confirmed by the observation that both peaks included a fragment ion that had lost one MGO unit (m/z 271), and the tandem mass spectra of this fragment ion, m/z 271 (MS3 271/343) (Figure 3B, C), were almost identical to those of GEN (MS2 271) (Figure 2A). In addition, both peaks had similar tandem mass spectra of their major fragment ions m/z 325 (MS3 325/343) and m/z 297 (MS4 297/325/343) (Figure 3B, C). In our previous in 408

DOI: 10.1021/acs.chemrestox.5b00516 Chem. Res. Toxicol. 2016, 29, 406−414

Article

Chemical Research in Toxicology

Figure 3. (A) SIM chromatogram and (B, C) ESI-MSn (n = 2−4; positive ion) spectra of MM-GEN.

Figure 4. Potential fragmentation pathways of MM-GEN-1, MM-orobol, MM-DGEN-1, and MM-5-OH-equol-1.

409

DOI: 10.1021/acs.chemrestox.5b00516 Chem. Res. Toxicol. 2016, 29, 406−414

Article

Chemical Research in Toxicology

Figure 5. (A) SIM chromatogram and (B, C) ESI-MSn (n = 2−4; positive ion) spectra of MM-DGEN.

under positive mode), which was 72 mass units higher than that of 5-OH-equol (m/z 259), indicating that they are the monoMGO adducts of 5-OH-equol. They also contained the fragment ion (m/z 259) that had lost one MGO molecule, and the tandem mass spectra of this fragment ion (MS3 259/ 331) were almost identical to that of 5-OH-equol (MS2 259). Our results indicate that these two peaks had almost identical mass fragments (Figures 6B, C and 2D), and the potential fragmentation pathways are presented in Figure 4C. We tentatively assigned these two peaks as MM-5-OH-equol-1 (tR 16.4 min) and MM-5-OH-equol-2 (tR 15.8 min) (Figure 1). The proposed fragmentation pathways of MM-5-OH-equol-1 are presented in Figure 4C. Using a similar approach, we identified the mono-MGO adduct of orobol (MM-orobol) and 6′-OH-DMA (MM-6′-OHDMA) (Figure 1). We were able to detect only one peak for each of them (Figure 7A, B), and both contained the fragment ion that had lost one MGO molecule (m/z 287 for orobol and m/z 275 for 6′-OH-DMA). The tandem mass spectra of m/z 287 (MS3 287/359) and m/z 275 (MS3 275/347) were almost identical to those of orobol (MS2 287) and 6′-OH-DMA (MS2 275) (Figures 7A, B and 2B, E), respectively. Identification of the Metabolites of GEN and the MGO Adducts of GEN and Its Phase I and Microbial Metabolites in the Chronic Study. To further confirm our

detect any di-MGO adducts in the urine samples collected from acute MGO and GEN-treated mice. In the SIM chromatogram of m/z 345 [M + H]+ (molecular ion of the mono-MGO adduct of DGEN), two peaks (tR 17.4 and 17.9 min) were detected in the urine samples collected in the acute study (Figure 5A). Both peaks were 72 mass units higher than that of DGEN (m/z 272), which indicated that they are the mono-MGO adduct of DGEN. Both peaks contained the fragment ion m/z 273 [M + H − 72]+, suggesting the loss of one MGO (m/z 72) molecule. The MS3 spectrum of this fragment ion m/z 273 (MS3 273/345) was almost identical to the MS2 spectrum of DGEN (Figures 5B, C and 2C). In addition, both peaks were 2 mass units higher than that of MM-GEN, and a mass difference of 2 Da was also evident for the fragments at m/z 327, 299, 281, 271, 243, and 181 versus the fragments of MM-GEN at m/z 325, 297, 279, 269, 241, and 179. Taken together, these findings indicate that these two metabolites are the mono-MGO adducts of DGEN, and similar to the mono-MGO adducts of GEN, we tentatively assigned the minor peak (tR 17.9 min) as MM-DGEN-1 and the major peak (tR 17.4 min) as MM-DGEN-2 (Figure 1). The proposed fragmentation pathways of MM-DGEN-1 are presented in Figure 4B. Similarly, two new peaks (tR 15.8 and 16.4 min) were observed at m/z 331 (mono-MGO adduct of 5-OH-equol 410

DOI: 10.1021/acs.chemrestox.5b00516 Chem. Res. Toxicol. 2016, 29, 406−414

Article

Chemical Research in Toxicology

Figure 6. (A) SIM chromatogram and (B, C) ESI-MSn (n = 2−3; positive ion) spectra of MM-5-OH-equol.

findings in the acute study, we conducted a chronic MGO treatment study in mice. MGO was supplied in the drinking water for 1 month, and then GEN was administered by oral gavage at a lower dose (130 mg/kg) than the dose used in the acute study (400 mg/kg) for 4 consecutive days. Urine samples were collected at different time points (2, 4, 6, and 24 h). Next, we searched all of the potential phase I and microbial metabolites of GEN and the MGO adducts of GEN and its metabolites using LC-MS. Similar to the findings in the acute study, we were able to detect GEN, its six metabolites, and the mono-MGO adducts of GEN, DGEN, and 5-OH-equol (Table 1). The two minor MGO-adducts, MM-orobol and MM-6′OH-DMA, were not detected in the urine samples in the chronic study. More importantly, the microbial-derived metabolites of GEN and their mono-MGO adducts (DGEN, 5-OH-equol, 6′-OH-DMA, MM-DGEN-1, MM-DGEN-2, MM-5-OH-equol-1, and MM-5-OH-equol-2) were found in the urine samples collected at later time points (Table 1).

mono- and di-MGO adducts.20 The critical question is whether this simple trapping chemical reaction will occur in vivo because the in vivo conditions are quite different from those employed in the in vitro studies. Many factors may affect the reaction, such as the oxygen pressure, the pH, and the presence of other endogenous and exogenous compounds. These criteria are further complicated by the extensive metabolism of flavonoids. Whether the biotransformation of flavonoids affects their trapping ability remains an intriguing question. To tackle these questions, we conducted two mouse studies to determine whether GEN and its metabolites could trap MGO in mice. Using the LC-MS conditions from our in vitro study,19 we assessed the formation of the mono- and di-MGO adducts of GEN in mouse urine samples in acute and chronic studies. We were able to identify the mono-MGO but not the di-MGO adducts of GEN from both studies. Our results clearly indicated that a chemical reaction between MGO and GEN could occur in vivo. In contrast to the in vitro analysis, in the in vivo reaction, the mono-MGO adducts could not further react with MGO, at least not in sufficient quantities to result in detectable amounts of di-MGO adducts under our LC-MS conditions. To determine whether the phase I and microbial metabolites of GEN retained the trapping efficacy of GEN, we searched for the metabolites of GEN and the MGO adducts of these metabolites in mouse urine samples. Our results showed that GEN is extensively metabolized in mice. Six GEN metabolites

■

DISCUSSION The results of our previous investigations have revealed that dietary flavonoids, such as EGCG, phloretin, phloridzin, and GEN, can efficiently trap MGO under physiological conditions (pH 7.4 and 37 °C), inhibiting the formation of AGEs.17−19,32 We also demonstrated that the A ring of flavonoids is the active site that contributes to their MGO trapping efficacy: MGO can react with the two unsubstituted carbons in the A ring to form 411

DOI: 10.1021/acs.chemrestox.5b00516 Chem. Res. Toxicol. 2016, 29, 406−414

Article

Chemical Research in Toxicology

Figure 7. SIM chromatograms and ESI-MSn (n = 2−4; positive ion) spectra of MM-orobol (A) and MM-6′-OH-DMA (B) and potential fragmentation pathways of MM-6′-OH-DMA (B).

forms in urine. Without enzymatic deconjugation, we were barely able to detect GEN and its phase I and microbial metabolites as well as their corresponding MGO adducts, indicating that the MGO adducts are mainly in their phase II conjugated forms in mouse urine (data not shown). Furthermore, the MGO adducts of the microbial-derived metabolites of GEN could be detected only at later time points in the chronic study, especially those for MM-5-OH-equol (Table 1). This finding is consistent with the previous finding that the biotransformation pathway for GEN via DGEN to 5OH-equol and 6′-OH-DMA is catalyzed mainly by the gut microbiota.25,26 Apparently, the conversion of isoflavones by intestinal bacteria may impacts their biological effects. For example, the bacterial metabolite of daidzein, equol, exhibits a greater affinity for estrogen receptor β than that of its precursor.33 Because of the complexity of the human gut microbiota, the production of DGEN, 5-OH-equol, and 6′-OHDMA may vary among individuals. Furthermore, differences have been observed in the gut microbiota between human adults with type II diabetes and nondiabetic control subjects.34,35 Therefore, the composition of the gut microbiota may affect the trapping efficacy of GEN in humans. In summary, this is the first report to demonstrate that GEN and its metabolites have the ability to trap MGO in vivo to form the corresponding MGO adducts. This discovery is consistent with our previous in vitro finding that the A ring is the active site responsible for trapping reactive dicarbonyl species.20 Factors that affect the biotransformation of soy isoflavoins, such as the gut microbiota, may influence in vivo trapping activities. Our findings provide useful information and directions that may shed light on in vivo MGO trapping efficiency studies of other dietary flavonoids. It will be worthwhile to further

Table 1. Phase I and Microbial Metabolites of GEN as Well as MGO Adducts of GEN and Its Metabolites in Mouse Urine after Enzymatic Deconjugationa [M + H] GEN orobol 6-OH-GEN 8-OH-GEN DGEN 5-OH-equol 6′-OH-DMA MM-GEN-1 MM-GEN-2 MM-orobol MM-DGEN-1 MM-DGEN-2 MM-5-OH-equol-1 MM-5-OH-equol-2 MM-6′-OH-DMA a

271 287 287 287 273 259 275 343 343 359 345 345 331 331 347

+

tR (min)

acute mouse study (mouse urine)

chronic mouse study (mouse urine)

21.4 19.5 18.9 18.3 19.6 17.7 20.6 19.2 18.7 16.0 17.9 17.4 16.4 15.8 18.0

yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes

2, 4, 6, 24 2, 4, 6, 24 2, 4, 6, 24 2, 4, 6, 24 4, 6, 24 4, 6, 24 6, 24 2, 4 2, 4, 6, 24 N.D. 4, 6, 24 4, 6, 24 6, 24 6, 24 N.D.

tR, retention time; N.D., not detected.

were detected, and among the six metabolites, only metabolites with the same A ring of GEN (DGEN, orobol, 5-OH-equol, and 6′-OH-DMA) could trap MGO to form mono-MGO adducts, which is consistent with our previous in vitro finding that the A ring is the active site in flavonoids for trapping MGO.20 Hydroxylation at C-6 and C-8 of the A ring diminished the trapping efficacy of GEN. Both GEN and its phase I and microbial metabolites are subjected to phase II glucuronidation/sulfation. They are mainly in their conjugated 412

DOI: 10.1021/acs.chemrestox.5b00516 Chem. Res. Toxicol. 2016, 29, 406−414

Article

Chemical Research in Toxicology

(10) Nemet, I., Varga-Defterdarovic, L., and Turk, Z. (2006) Methylglyoxal in food and living organisms. Mol. Nutr. Food Res. 50, 1105−1117. (11) Dornadula, S., Elango, B., Balashanmugam, P., Palanisamy, R., and Kunka Mohanram, R. (2015) Pathophysiological insights of methylglyoxal induced type-2 diabetes. Chem. Res. Toxicol. 28, 1666− 1674. (12) Dhar, A., Dhar, I., Jiang, B., Desai, K. M., and Wu, L. (2011) Chronic methylglyoxal infusion by minipump causes pancreatic betacell dysfunction and induces type 2 diabetes in Sprague-Dawley rats. Diabetes 60, 899−908. (13) Crisostomo, J., Matafome, P., Santos-Silva, D., Rodrigues, L., Sena, C. M., Pereira, P., and Seica, R. (2013) Methylglyoxal chronic administration promotes diabetes-like cardiac ischaemia disease in Wistar normal rats. Nutr., Metab. Cardiovasc. Dis. 23, 1223−1230. (14) Wang, Y., and Ho, C. T. (2012) Flavour chemistry of methylglyoxal and glyoxal. Chem. Soc. Rev. 41, 4140−4149. (15) Peng, X., Ma, J., Chen, F., and Wang, M. (2011) Naturally occurring inhibitors against the formation of advanced glycation endproducts. Food Funct. 2, 289−301. (16) Shapiro, H. K. (1998) Carbonyl-trapping therapeutic strategies. Am. J. Ther. 5, 323−353. (17) Sang, S., Shao, X., Bai, N., Lo, C.-Y., Yang, C. S., and Ho, C.-T. (2007) Tea polyphenol (−)-epigallocatechin-3-gallate: a new trapping agent of reactive dicarbonyl species. Chem. Res. Toxicol. 20, 1862− 1870. (18) Shao, X., Bai, N., He, K., Ho, C.-T., Yang, C. S., and Sang, S. (2008) Apple polyphenols, phloretin and phloridzin: new trapping agents of reactive dicarbonyl species. Chem. Res. Toxicol. 21, 2042− 2050. (19) Lv, L., Shao, X., Chen, H., Ho, C.-T., and Sang, S. (2011) Genistein inhibits advanced glycation end product formation by trapping methylglyoxal. Chem. Res. Toxicol. 24, 579−586. (20) Shao, X., Chen, H., Zhu, Y., Sedighi, R., Ho, C.-T., and Sang, S. (2014) Essential structural requirements and additive effects for flavonoids to scavenge methylglyoxal. J. Agric. Food Chem. 62, 3202− 3210. (21) Zhu, Y., Zhao, Y., Wang, P., Ahmedna, M., Ho, C.-T., and Sang, S. (2015) Tea Flavanols block advanced glycation of lens crystallins induced by dehydroascorbic acid. Chem. Res. Toxicol. 28, 135−143. (22) Dixon, R. A., and Ferreira, D. (2002) Genistein. Phytochemistry 60, 205−211. (23) Wang, P., Chen, H., Zhu, Y., McBride, J., Fu, J., and Sang, S. (2015) Oat Avenanthramide-C (2c) is biotransformed by mice and the human microbiota into bioactive metabolites. J. Nutr. 145, 239−245. (24) Roberts-Kirchhoff, E. S., Crowley, J. R., Hollenberg, P. F., and Kim, H. (1999) Metabolism of genistein by rat and human cytochrome P450s. Chem. Res. Toxicol. 12, 610−616. (25) Kwak, H. S., Park, S. Y., Kim, M. G., Yim, C. H., Yoon, H. K., and Han, K. O. (2009) Marked individual variation in isoflavone metabolism after a soy challenge can modulate the skeletal effect of isoflavones in premenopausal women. J. Korean Med. Sci. 24, 867−873. (26) Matthies, A., Loh, G., Blaut, M., and Braune, A. (2012) Daidzein and genistein are converted to equol and 5-hydroxy-equol by human intestinal Slackia isoflavoniconvertens in gnotobiotic rats. J. Nutr. 142, 40−46. (27) Matthies, A., Clavel, T., Gütschow, M., Engst, W., Haller, D., Blaut, M., and Braune, A. (2008) Conversion of daidzein and genistein by an anaerobic bacterium newly isolated from the mouse intestine. Appl. Environ. Microb. 74, 4847−4852. (28) Bursztyka, J., Perdu, E., Tulliez, J., Debrauwer, L., Delous, G., Canlet, C., De Sousa, G., Rahmani, R., Benfenati, E., and Cravedi, J.-P. (2008) Comparison of genistein metabolism in rats and humans using liver microsomes and hepatocytes. Food Chem. Toxicol. 46, 939−948. (29) Hur, H.-G., Lay, J. O., Jr, Beger, R. D., Freeman, J. P., and Rafii, F. (2000) Isolation of human intestinal bacteria metabolizing the natural isoflavone glycosides daidzin and genistin. Arch. Microbiol. 174, 422−428.

examine whether GEN can prevent MGO toxicity in vivo, especially the formation of AGEs, by trapping MGO.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.5b00516. SIM chromatograms and ESI-MS2 (positive ion) spectra of 6-OH-GEN and 8-OH-GEN (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Tel: 704-250-5710. Fax: 704-250-5709. E-mail: ssang@ncat. edu or [email protected]. Funding

We gratefully acknowledge financial support from the USDA (grant 2012-67017-30175 to S. Sang). Notes

The authors declare no competing financial interest.

■

ABBERVIATIONS AGEs, advanced glycation end products; CID, collision induced dissociation; DGEN, dihydrogenistein; EGCG, (−)-epigallocatechin-3-O-gallate; ESI, electrospray ionization; GEN, genistein; orobol, 3′-hydroxy-genistein; 8-OH-GEN, 8-hydroxygenistein; 6′-OH-DMA, 6′-hydroxy-O-demethylangolensin; 5OH-equol, 5-hydroxy-equol; 6-OH-GEN, 6-hydroxy-genistein; MGO, mehtylglyoxal; MM-DGEN, mono-MGO of dihydrogenistein; MM-GEN, mono-MGO of genistein; MM-6′-OHDMA, mono-MGO of 6′-hydroxy-O-demethylangolensin; MM5-OH-equol, mono-MGO of 5-hydroxy-equol; MM-orobol, mono-MGO of 3′-hydroxy-genistein; SIM, selected ion monitoring

■

REFERENCES

(1) Singh, R., Barden, A., Mori, T., and Beilin, L. (2001) Advanced glycation end-products: a review. Diabetologia 44, 129−146. (2) Maessen, D. E., Stehouwer, C. D., and Schalkwijk, C. G. (2015) The role of methylglyoxal and the glyoxalase system in diabetes and other age-related diseases. Clin. Sci. 128, 839−861. (3) Allaman, I., Belanger, M., and Magistretti, P. J. (2015) Methylglyoxal, the dark side of glycolysis. Front. Neurosci. 9, 23. (4) Angeloni, C., Zambonin, L., and Hrelia, S. (2014) Role of methylglyoxal in Alzheimer′s disease. BioMed Res. Int. 2014, 238485. (5) Jia, X., Olson, D. J., Ross, A. R., and Wu, L. (2006) Structural and functional changes in human insulin induced by methylglyoxal. FASEB J. 20, 1555−1557. (6) Gao, Y., and Wang, Y. (2006) Site-selective modifications of arginine residues in human hemoglobin induced by methylglyoxal. Biochemistry 45, 15654−15660. (7) Cantero, A. V., Portero-Otín, M., Ayala, V., Auge, N., Sanson, M., Elbaz, M., Thiers, J. C., Pamplona, R., Salvayre, R., and Nègre-Salvayre, A. (2007) Methylglyoxal induces advanced glycation end product (AGEs) formation and dysfunction of PDGF receptor-β: implications for diabetic atherosclerosis. FASEB J. 21, 3096−3106. (8) Ahmad, S., Dixit, K., Shahab, U., Alam, K., and Ali, A. (2011) Genotoxicity and immunogenicity of DNA-advanced glycation end products formed by methylglyoxal and lysine in presence of Cu2+. Biochem. Biophys. Res. Commun. 407, 568−574. (9) Khuhawar, M., Kandhro, A., and Khand, F. (2006) Liquid Chromatographic Determination of Glyoxal and Methylglyoxal from Serum of Diabetic Patients using Meso-Stilbenediamine as Derivatizing Reagent. Anal. Lett. 39, 2205−2215. 413

DOI: 10.1021/acs.chemrestox.5b00516 Chem. Res. Toxicol. 2016, 29, 406−414

Article

Chemical Research in Toxicology (30) Schoefer, L., Mohan, R., Braune, A., Birringer, M., and Blaut, M. (2002) Anaerobic C-ring cleavage of genistein and daidzein by Eubacterium ramulus. FEMS Microbiol. Lett. 208, 197−202. (31) Nagao, M., Fujita, Y., Sugimura, T., and Kosuge, T. (1986) Methylglyoxal in beverages and foods: its mutagenicity and carcinogenicity. IARC Sci. Publ., 283−291. (32) Li, X., Zheng, T., Sang, S., and Lv, L. (2014) Quercetin inhibits advanced glycation end product formation by trapping methylglyoxal and glyoxal. J. Agric. Food Chem. 62, 12152−12158. (33) Kinjo, J., Tsuchihashi, R., Morito, K., Hirose, T., Aomori, T., Nagao, T., Okabe, H., Nohara, T., and Masamune, Y. (2004) Interactions of phytoestrogens with estrogen receptors. ALPHA. and. BETA.(III). Estrogenic activities of soy isoflavone aglycones and their metabolites isolated from human urine. Biol. Pharm. Bull. 27, 185−188. (34) Larsen, N., Vogensen, F. K., Van Den Berg, F., Nielsen, D. S., Andreasen, A. S., Pedersen, B. K., Al-Soud, W. A., Sorensen, S. J., Hansen, L. H., and Jakobsen, M. (2010) Gut microbiota in human adults with type 2 diabetes differs from non-diabetic adults. PLoS One 5, e9085. (35) Qin, J., Li, Y., Cai, Z., Li, S., Zhu, J., Zhang, F., Liang, S., Zhang, W., Guan, Y., Shen, D., et al. (2012) A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature 490, 55−60.

414

DOI: 10.1021/acs.chemrestox.5b00516 Chem. Res. Toxicol. 2016, 29, 406−414