Glyoxal and methylglyoxal: Autoxidation from dihydroxyacetone and polyphenol cytoprotective antioxidant mechanisms

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Glyoxal and methylglyoxal: Autoxidation from dihydroxyacetone and polyphenol cytoprotective antioxidant mechanisms

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Hoyin Lip 1, Kai Yang 1, Stephanie L. MacAllister, Peter J. O’Brien ⇑ Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada

a r t i c l e

i n f o

Article history: Available online xxxx Keywords: Glyoxal Methylglyoxal Oxidative stress Autoxidation Protein carbonylation Polyphenols

a b s t r a c t Previously, this laboratory had shown that fructose and its downstream metabolites can be enzymatically metabolized to form glyoxal and methylglyoxal. Fructose metabolites, glycoaldehyde, glyceraldehyde and hydroxypyruvate had also been shown to be autoxidizable. However, whether fructose is capable of autoxidation or not has not been shown before. Over a period of six days, fructose was shown to cause protein carbonylation and autoxidize to form dicarbonyls. Dihydroxyacetone, a fructose metabolite, was shown to readily cause protein carbonylation and autoxidize to form dicarbonyls under standard conditions (37 °C, pH 7.4, PBS buffer). Oxidative stress condition (Fenton’s reaction) further potentiated the amount of protein carbonylation and dicarbonyls formation. This study investigated the natural polyphenols for their ability to protect against glyoxal- and methylglyoxal-induced cytotoxicity, reactive oxygen species formation and improved mitochondrial membrane potential maintenance. The polyphenols investigated were gallic acid, methyl gallate, ethyl gallate, propyl gallate, rutin and curcumin. The polyphenols were assayed using primary and GSH-depleted hepatocytes. The polyphenols were also investigated for their rescuing ability by adding the polyphenols 30 min after the toxin. Interestingly, the polyphenols showed a greater hepatoprotection when toxins were pre-incubated for 30 min. However, rutin was an exception and was less protective when rescuing hepatocytes. One explanation was that rutin metabolites may scavenge reactive oxygen species more effectively than rutin itself. A general relationship was observed between the length of the alkyl chain and the protectiveness of the polyphenol. The longer the alkyl group attached to the gallate compound, the more cytoprotective the polyphenol was. However, the reverse was true for reactive oxygen species scavenging ability. Also, the gallates with longer alkyl groups were less able to maintain the mitochondrial membrane potential. Ó 2012 Published by Elsevier Ireland Ltd.

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1. Introduction

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In diabetes mellitus patients, abnormal glucose metabolism leads to glucose intolerance and hyperglycemia. The excessive amount of sugars overwhelms the endogenous antioxidant and detoxifying systems. Under hyperglycemic conditions, the formation of toxic metabolites such as glyoxal and methylglyoxal increases [1]. These reactive dicarbonyls promote oxidative stress and can cause a number of cellular damages including covalent modification of amino and thiol groups of proteins to form advanced glycation endproducts (AGEs) [2,3]. AGEs have been implicated in the pathogenesis of diabetic complications such as atherogenesis, nephropathy and cataractogenesis [4,5]. Clinically, a number of therapeutic agents have been used to target the AGEs

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⇑ Corresponding author. Address: Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada M5S 3M2. Tel.: +1 416 978 2716; fax: +1 416 978 8511. E-mail address: [email protected] (P.J. O’Brien). 1 These authors are co-first authors.

directly or indirectly. Aminoguanidine prevented AGE formation at an early stage by scavenging precursors such as glyoxal [6,7]. Metformin is a biguanide compound that is currently used to control type 2 diabetes [8]. Metformin has also been shown to trap glyoxal [9]. Other effective agents used included hydralazine, pyridoxamine, penicillamine, and N-acetyl-cysteine which also target reactive dicarbonyls [10]. The consumption of fructose has increased significantly in the Western diet during the past three decades largely due to the introduction of high fructose corn syrup (HFCS) [11,12]. Increased fructose consumption has been linked to many health concerns such as metabolic syndrome and obesity. In particular, high fructose consumption leads to hepatic de novo lipogenesis, which subsequently contributes to increase visceral fat and insulin resistance, making fructose an important player in the pathogenesis of diabetes [13]. Fructose and several of its metabolites can also form reactive dicarbonyls such as glyoxal, which carbonylates proteins and forms AGEs [14]. methylglyoxal is formed from the metabolism of glucose and the metabolism of ketone bodies from acetone

0009-2797/$ - see front matter Ó 2012 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.cbi.2012.11.013

Please cite this article in press as: H. Lip et al., Glyoxal and methylglyoxal: Autoxidation from dihydroxyacetone and polyphenol cytoprotective antioxidant mechanisms, Chemico-Biological Interactions (2012), http://dx.doi.org/10.1016/j.cbi.2012.11.013

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[15]. glyoxal and methylglyoxal, the a-oxoaldehydes, are argininedirected glycating agents, forming hydroimidazolones which are important AGEs quantitatively with relatively short half-lives (12–60 days) [16]. The AGEs are involved in the macro and micro vascular complications in diabetic patients as they accumulate at the sites of vascular complications such as renal glomeruli, retina and peripheral nerves [17]. AGEs can also exerts their physiological effects by binding to cell surface receptors such as receptors of advanced glycation end products (RAGE), as well as the macrophage scavenger receptors ScR-II and CD-36 [18]. AGE–RAGE interactions could also lead to oxidative stress conditions and radical formation as activation of RAGE indirectly activates pro-inflammatory transcriptional factor NF-jB and generates reactive oxygen species (reactive oxygen species) from activated NADPH oxidase [19,20]. Previously demonstrated by this laboratory, fructose-induced hepatotoxicity increased more than 100-fold in the presence of a nontoxic concentration of H2O2 so as to mimic H2O2 levels formed by NADPH oxidase (NOX) released by activated immune cells such as neutrophils, eosinophils and macrophages [21]. The increased hepatotoxicity was mainly due to the enhanced conversion of fructose and fructose metabolites form glyoxal and also the synergistic hepatotoxicity between glyoxal and H2O2 [22,23]. Glyoxal can also readily be formed from the autoxidation of glyceraldehyde, glycoaldehyde and hydroxypyruvate in the absence of enzymes [23]. However, it is unclear whether the glyoxal formation from fructose and its metabolite dihydroxyacetone are enzyme-dependent. Understanding the relative contribution of autoxidation or enzymatic reaction to glyoxal formation could help improve the development of more effective therapeutic agents for diabetes. Various clinical studies have attempted to improve the clinical outcome of diabetic patients by introducing dietary and life style changes. A number of natural polyphenols have been demonstrated to have had beneficial effects on diseases such as cancer, cardiovascular diseases and inflammation. Gallic acid is found in various plants and has been implicated in a variety of herb-based therapy for hyperglycemia [24]. Methyl gallate, ethyl gallate and propyl gallate are esters of the gallic acid group. Ethyl gallate and propyl gallate are added to foods containing unsaturated oil to prevent oxidation [25,26]. Curcumin from turmeric and rutin from a variety of fruits and leaves were also investigated [27]. Curcumin was shown to have anti-cancer capability through mechanisms which disrupt the mitochondrial membrane potential [28,29]. The polyphenols have been shown to have reactive oxygen species-scavenging capability in addition to anti-cancer potential [30–34]. The hepatoprotective and rescue ability of gallic acid and its gallate derivatives, rutin and curcumin against methylglyoxal and glyoxal were investigated in physiological and GSH-depleted hepatocytes. In addition, reactive oxygen species formation and the mitochondrial membrane potential for each of the protective agents was analyzed. In this study, the polyphenols were ranked in the order of preventing hepatotoxicity, reactive oxygen species-scavenging capability and maintaining the mitochondrial membrane potential.

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2. Materials and methods

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2.1. Chemicals

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Glyoxal, methylglyoxal, 1-bromoheptane, gallic acid, methyl gallate, ethyl gallate, propyl gallate, rutin hydrate, curcumin, 20 ,70 -dichlorofluorescin diacetate, rhodamine 123, sodium phosphate monobasic, anhydrous sodium borate, Girard’s T reagent was purchased from Sigma–Aldrich Corp. (Oakville, ON, CAN). Type II Collagenase was purchased from Worthington (Lakewood, NJ).

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2.2. Animals treatment and hepatocyte preparation

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Male Sprague–Dawley rats weighing 275–300 g (Charles River Laboratories) were housed in ventilated plastic cages with PWI8 – 16 hardwood bedding with 12 air changes per hour and 12 h of light period (lights on at 08:00 h). Relative humidity is 50–60% and temperature ranges from 21 to 23 °C. Normal standard chow diet and water ad libitum were fed to the animals. Hepatocytes were isolated from rats as described by Moldeus and cowokers [35]. Isolated hepatocytes (106 cells/mL) (10 mL) were suspended in Krebs–Henseleit buffer (pH 7.4) containing 12.5 mM HEPES in rotating 50 mL round-bottomed flasks under 95% O2/5% CO2 conditions in a water bath maintained at 37 °C for 30 min [35].

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2.3. Cell viability

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Cell viability was assessed microscopically via plasma membrane disruption determined by 0.1% w/v trypan blue exclusion test [36]. Viability was determined every 30 min during the 3 h incubation. Cell viability of at least 80–90% must be achieved before use. The cells were allowed 30 min to acclimatize before initiating experiments. Glyoxal and methylglyoxal was prepared immediately prior to use and was added to the hepatocytes before or after addition of protective agents. Pre-incubation with 1-bromoheptane to deplete hepatocyte GSH, glyoxal and methylglyoxal is indicated in each table. A lethal concentration of glyoxal and methylglyoxal was also used to cause 50% cytotoxicity at 2 h (LD50, 2 h). Stock solutions of chemicals were made up in H2O or dimethylsulfoxide (DMSO).

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2.4. Reactive oxygen species (reactive oxygen species) formation

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Hepatocyte reactive oxygen species generation was determined by adding dichlorofluorescin diacetate (DCFD) to hepatocyte incubate. DCFD enters the cell and was hydrolyzed to form non-fluorescent dichlorofluorescin. Dichlorofluorescin reacted with reactive oxygen species to form a highly fluorescent dichlorofluorescin which effluxes the cell [37]. One milliliter of sample is withdrawn at 46 and 90 min time point. These samples were centrifuged at 50g for 1 min. The supernatant was aspirated. The cells were resuspended in H2O and 1.6 lM DCFD [38]. The samples were incubated for 10 min at 37 °C. The samples were measured at 480 nm excitation and 520 emission wavelengths.

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2.5. Mitochondrial membrane potential assay

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Rhodamine 123 accumulates selectively in mitochondria by facilitated diffusion. When mitochondrial membrane potential is decreased, the amount of rhodamine 123 that enters the cell was decreased. Five hundred microliters of sample was withdrawn at 46 and 90 min. The samples were centrifuged at 1000 rpm for 1 min. The supernatant was aspirated. The cell pellet was resuspended in 2 mL of Krebs–Henseleit buffer with 1.5 lM rhodamine 123 and incubated at 37 °C for 10 min. The hepatocytes were separated by centrifugation and the amount of rhodamine 123 in the supernatant was measured fluorimetrically at 480 nm excitation and 520 nm emission wavelengths. The capacity of mitochondrial uptake of rhodamine 123 was calculated as the difference in fluorescence intensity between control and treated cells [39].

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2.6. Detection of protein carbonyl content of bovine serum albumin

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Total protein bound carbonyl content of bovine serum albumin were measured using 2,4-dinitrophenylhydrazine (DNPH). Bovine serum albumin (2 mg/mL) was prepared in 100 mM sodium phosphate buffer, pH 7.4. Samples were incubated over a 6 day period. Time points taken were 15 min, 60 min, 120 min, day 1, day 3 and

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day 6. At each time point, 0.5 mL of sample were withdrawn and was incubated with 0.5 mL of DNPH (0.1% w/v) in 2 N HCl for 1 h at room temperature in the dark. One milliliter of TCA (20% w/v) was added to stop the reaction and precipitate the protein. The sample was centrifuged at 1000 rpm for 1 min. The supernatant was aspirated and DNPH was removed by washing the pellet with 0.5 mL of ethyl acetate:ethanol (1:1) solution three times. After washing, the pellets were dried with nitrogen and dissolved in 1 mL of Tris-buffered 8.0 M guanidine–HCl, pH 7.2. The samples were read spectrometrically at 370 nm [40].

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2.7. Determination of reactive dicarbonyls

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Dicarbonyl concentrations were determined using Girard’s reagent T. Stocks of 50 lL were added to 120 mM sodium phosphate monobasic buffer, pH 7.5, to a total of 1 mL sample mixture. Fifty microliters of samples were added to 0.95 mL of 120 mM sodium borate buffer, pH 9.3, every hour for 3-h incubation period. The mixture was vortexed and allowed 10 min to achieve equilibrium. Two hundred microliters of this mixture was added to 800 lL of 100 mM Girard’s reagent T. The amount of dicarbonyl formation was detected spectrophotometrically at 326 nM [41,42]. The concentration of dicarbonyl was determined using a standard curve.

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2.8. Statistical analysis

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The statistical significance of hepatocyte treatments was determined by one way analysis of variance (ANOVA), with post hoc Tukey’s analysis using SPSS. Values were considered statistically significant when p < 0.05. Results from three independent experiments presented as mean ± SE.

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3. Results

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3.1. Autoxidation of fructose and dihydroxyacetone

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Dihydroxyacetone was readily autoxidized by Fenton’s reaction to form glyoxal and methylglyoxal in standard (37 °C, pH 7.4, PBS

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buffer) and oxidative stress conditions (Fenton’s reaction) as shown in the bovine serum album protein carbonylation assay and the Girard assay. In the bovine serum albumin assay, dihydroxyacetone resulted in significant protein carbonylation in standard and oxidative stress conditions compared to bovine serum albumin alone (Table 1). Fructose showed significant protein carbonylation (Table 1). The Fenton’s control condition showed significant protein carbonylation. However, the addition of fructose to Fenton’s condition did not increase protein carbonylation when compared to the Fenton’s control condition (Table 1). Fructose showed significant protein carbonylation under oxidative condition (Table 1). However, the amount of protein carbonylation between the control oxidative stress condition and the fructose under oxidative stress condition showed little differences (Table 1). In the Girard assay, dihydroxyacetone showed significant formation of glyoxal and methylglyoxal. Fructose showed significant formation on day 1 and onward (Table 2). However, the level of dicarbonyls produced was significantly less than the level produced by dihydroxyacetone (Table 2). Fructose was able to autoxidize into glyoxal and methylglyoxal under Fenton’s-induced oxidative stress condition. However, fructose produced significantly less dicarbonyls than dihydroxyacetone [43].

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3.2. Hepatoprotection and rescue against glyoxal with antioxidants in rat hepatocytes

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The protective ability of antioxidants against glyoxal was assessed in freshly isolated rat hepatocytes. Five millimolars of glyoxal (LD50) significantly increased hepatotoxicity and reactive oxygen species formation at 2 h (Table 3; p < 0.05). The polyphenols showed significant hepatoprotection, decreased reactive oxygen species formation and improved maintenance of the mitochondrial membrane potential against 5 mM glyoxal. The order of hepatoprotection by the polyphenols at 2 h was: rutin > methyl gallate > ethyl gallate > propyl gallate = gallic acid > curcumin (Table 3). The rank order for preventing reactive oxygen species formation at 90 min was: rutin > propyl gallate > ethyl gal-

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Table 1 Autoxidation of fructose and dihydroxyacetone into glyoxal and methylglyoxal measured with BSA protein carbonylation. BSA protein carbonylation (nM)

BSA (2 mg/mL) +Fentons +10 mM DHA +Fentons +10 mM Fructose +Fentons

15 min

60 min

120 min

Day 1

Day 2

Day 3

5.21 ± 0.22 10.76 ± 0.61a 13.00 ± 1.47a 14.74 ± 3.37a,b 5.46 ± 0.35 9.64 ± 1.59a,c

5.34 ± 0.10 12.21 ± 1.35a 13.55 ± 1.96a 21.67 ± 2.17a,b,d 5.68 ± 0.66 11.52 ± 1.63a,c

5.47 ± 0.54 12.17 ± 1.39a 13.73 ± 2.03a 26.27 ± 0.77a,b,d 5.55 ± 0.11 12.29 ± 1.63a,c

5.91 ± 0.29 21.02 ± 2.92a 32.76 ± 1.42a 75.55 ± 1.45a,b,d 6.96 ± 0.71 15.87 ± 1.57a,c

5.32 ± 0.14 19.62 ± 4.06a 28.92 ± 3.57a 57.12 ± 2.00a,b,d 6.64 ± 1.03 15.85 ± 1.46a,c

5.85 ± 0.21 14.24 ± 2.74a 21.67 ± 0.99a 41.17 ± 2.50a,b,d 6.08 ± 1.16 16.47 ± 0.95a,c

Fenton-induced oxidative condition generated with 200 lM FeII/EDTA + 1 mM H2O2. a Significant as compare to control (p < 0.05). b Significant as compare to 10 mM DHA (p < 0.05). c Significant as compare to 10 mM Fructose (p < 0.05). d Significant as compare to 10 mM Fructose + Fentons (p < 0.05). Table 2 Autoxidation of fructose and dihydroxyacetone into glyoxal and methylglyoxal measured using the Girard assay. Glyoxal (lM)

+10 mM DHA +Fentons +10 mM Fructose +Fentons

15 min

60 min

120 min

Day 1

Day 3

Day 6

0.93 ± 0.04b,c,d 2.72 ± 0.08a,b,d 0.04 ± 0.01a,c 0.01 ± 0.00a,c

2.62 ± 0.11b,c,d 6.45 ± 0.07a,bd 0.01 ± 0.00a,c 0.01 ± 0.00a,c

10.45 ± 0.05b,c,d 14.57 ± 0.10a,b,d 0.17 ± 0.05a,c,d 0.02 ± 0.01a,c

3.41 ± 0.06b,c,d 5.55 ± 0.09a,b,d 0.72 ± 0.05a,c 1.62 ± 0.06b,c

2.29 ± 0.03b,c,d 5.06 ± 0.05a,b,d 0.21 ± 0.03a,c,d 1.07 ± 0.03b,c

1.04 ± 0.05b,c 3.66 ± 0.05a,b,d 0.20 ± 0.02a,c,d 0.66 ± 0.02b,c

Fenton-induced oxidative condition generated with 200 lM FeII/EDTA + 1 mM H2O2; Control is sodium phosphate monobasic buffer pH 7.4. a Significant as compare to 10 mM DHA (P < 0.05). b Significant as compare to 10 mM Fructose (p < 0.05). c Significant as compare to 10 mM DHA + Fentons (p < 0.05). d Significant as compare to 10 mM Fructose + Fentons (p < 0.05).

Please cite this article in press as: H. Lip et al., Glyoxal and methylglyoxal: Autoxidation from dihydroxyacetone and polyphenol cytoprotective antioxidant mechanisms, Chemico-Biological Interactions (2012), http://dx.doi.org/10.1016/j.cbi.2012.11.013

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Table 3 Protective effects of polyphenol against glyoxal in rat hepatocytes. Treatment/time point

Control 5 mM Glyoxal 0.2 mM Gallic acid 0.2 mM Gallic acid (30 min)c 0.2 mM Methyl gallate 0.2 mM Methyl gallate (30 min)c 0.2 mM Ethyl gallate 0.2 mM Ethyl gallate (30 min)c 0.2 mM Propyl gallate 0.2 mM Propyl gallate (30 min)c 0.2 mM Rutin 0.2 mM Rutin (30 min)c 4 lM Curcumin 4 lM Curcumin (30 min)c

Cytotoxicity (% trypan blue taken)

ROS (F.I. units)

% MMP

60 min

120 min

180 min

90 min

90 min

26 ± 3 38 ± 2 24 ± 2 25 ± 3 25 ± 5 23 ± 4 28 ± 4 25 ± 2 29 ± 5 25 ± 3 26 ± 5 23 ± 4 31 ± 3 32 ± 3

33 ± 3 59 ± 3a 47 ± 3b 41 ± 2b 40 ± 2b 30 ± 2b 42 ± 2b 35 ± 4b 47 ± 4b 40 ± 2 31 ± 2b 44 ± 5b 48 ± 2b 42 ± 3b

40 ± 3 93 ± 3a 75 ± 5 72 ± 6 71 ± 4b 77 ± 5b 78 ± 7 75 ± 6 79 ± 6 76 ± 5b 70 ± 4b 77 ± 2 83 ± 6 82 ± 8

96 ± 3 224 ± 6a 114 ± 2b 135 ± 3b 105 ± 3b 92 ± 5b 101 ± 4b 90 ± 5 94 ± 4b 87 ± 5b 90 ± 5b 94 ± 4b 136 ± 7b 134 ± 7b

100 ± 6 55 ± 5a 84 ± 5b 83 ± 4b 86 ± 4b 84 ± 5 85 ± 5b 84 ± 6b 84 ± 6b 83 ± 3b 85 ± 7b 82 ± 8b 82 ± 6b 81 ± 4b

Isolated rat hepatocytes (106 cells/mL) were incubated at 37 °C in rotating round bottom flasks with 95% O2 and 5% CO2 in Krebs–Henseleit buffer (pH 7.4). Hepatotoxicity, ROS and MMP were determined as described in method section. Mean ± SEM. for three separate experiments are given. a Significant as compared to control (p < 0.05). b Significant as compared to 5 mM glyoxal (p < 0.05). c Cells are incubated with glyoxal for 30 min before addition of antioxidants. The addition of glyoxal marks the initiation of the 3 h experiment period.

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late > methyl gallate > gallic acid > curcumin (Table 3). The polyphenols showed significant protection against the collapse of the mitochondrial membrane potential caused by glyoxal. To assess the rescue effect of the antioxidants, freshly isolated rat hepatoctyes were pre-incubated with glyoxal for 30 min before the addition of antioxidants. The addition of glyoxal marked the initiation of the experiment. The polyphenols showed significant hepatoprotection, decrease in reactive oxygen species formation and improved mitochondrial membrane potential maintenance. The rank order for hepatoprotection at 2 h for hepatoprotection was: methyl gallate ethyl gallate > propyl gallate > gallic acid = curcumin > rutin (Table 3). At 90 min, the reactive oxygen species data show a significant decrease in preventing reactive oxygen species formation. The rank order was however different for hepatoprotection at 2 h: propyl gallate > ethyl gallate > methyl gallate > rutin > curcumin > gallic acid (Table 3). The polyphenols showed significant protection against the collapse of the mitochondrial membrane potential caused by glyoxal. 3.3. Hepatoprotection and rescue by antioxidants against methylglyoxal in rat hepatocytes The protective ability of antioxidants against methylglyoxal was assessed in freshly isolated rat hepatocytes. Fifteen millimolars of methylglyoxal significantly increased cytotoxicity at 2 h (LD50). The polyphenols also significantly decreased cytotoxicity, reactive oxygen species formation and improved mitochondrial membrane potential maintenance. At 2 h the rank order for hepatoprotection was: rutin > curcumin > methyl gallate = ethyl gallate > propyl gallate > gallic acid (Table 4). The ranking for prevention of reactive oxygen species formation at 90 min was: propyl gallate > ethyl gallate = gallic acid > methyl gallate > curcumin (Table 4). The polyphenols showed significant protection against the collapse of the mitochondrial membrane potential caused by methylglyoxal. To assess the rescue effectiveness of the antioxidants against methylglyoxal, freshly isolated rat hepatocytes were pre-incubated with glyoxal for 30 min before adding the antioxidants. The addition of glyoxal marked the start of the experiment. The polyphenols significantly decreased cytotoxicity, reactive oxygen species formation and improved mitochondrial membrane potential maintenance. The rank order of the rescue effect of antioxidants at 2 h for hepatoprotection was: methyl gallate > ethyl gallate > propyl gallate > rutin > gallic acid > curcumin (Table 4). At 90 min, the

rank order for reactive oxygen species data was different from the rank order for hepatoprotection at 2 h, propyl gallate > ethyl gallate = methyl gallate > gallic acid > rutin > curcumin (Table 4). The polyphenols showed significant protection against the collapse of the mitochondrial membrane potential caused by methylglyoxal. The rescuing effect of antioxidants against methylglyoxal was not as prominent. Only methyl gallate, rutin and curcumin showed decreased cytotoxicity. The overall trend was similar to that for glyoxal. As the length of the alkyl side chain increased cytotoxicity increased. Reactive oxygen species scavenging ability was increased, and improved mitochondrial membrane potential maintenance.

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3.4. Polyphenols protect against glyoxal and methylglyoxal in GSHdepleted hepatocytes

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The rat hepatocytes were depleted of GSH via pre-incubation with 1-bromoheptane for 30 min. Glyoxal was added after the pre-incubation period and the addition of glyoxal marked the start of the 3 h experiment. In the GSH-depleted cells treated with glyoxal, all the polyphenols showed significant hepatoprotection, significant reactive oxygen species scavenging and improved mitochondrial membrane potential maintenance. The order of hepatoprotection at 2 h by the polyphenols was different than in non-GSH depleted hepatocytes: methyl gallate > ethyl gallate > rutin > propyl gallate > gallic acid = curcumin (Table 5). The ranking order for preventing reactive oxygen species formation at 90 min was: rutin > propyl gallate > ethyl gallate > methyl gallate (Table 5). Gallic acid and curcumin were not effective at scavenging ROS. The polyphenols showed significant protection against the collapse of the mitochondrial membrane potential caused by glyoxal. To assess the ability of antioxidants for protecting against methylglyoxal in GSH-depleted hepatocytes the same procedure was used. The polyphenols significantly prevented methylglyoxal-induced cytotoxicity, scavenged reactive oxygen species and improved mitochondrial membrane potential maintenance. The rank order for hepatoprotection at 2 h was different from that for methylglyoxal: methyl gallate > ethyl gallate > propyl gallate = rutin > gallic acid > curcumin (Table 5). However, curcumin was able to scavenge reactive oxygen species. At 90 min, the rank order for the scavenging reactive oxygen species data was: propyl

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Cytotoxicity (% trypan blue taken)

Control 15 mM Methylglyoxal 0.2 mM Gallic acid 0.2 mM Gallic acid (30 min)c 0.2 mM Methyl gallate 0.2 mM Methyl gallate (30 min)c 0.2 mM Ethyl gallate 0.2 mM Ethyl gallate (30 min)c 0.2 mM Propyl gallate 0.2 mM Propyl gallate (30 min)c 0.2 mM Rutin 0.2 mM Rutin (30 min)c 4 lM Curcumin 4 lM Curcumin (30 min)c

ROS (F.I. units)

% MMP

60 min

120 min

180 min

90 min

90 min

24 ± 2 46 ± 4a 34 ± 6 34 ± 4 29 ± 5 28 ± 6 30 ± 4 34 ± 3 35 ± 5 34 ± 6 25 ± 3b 30 ± 5 36 ± 5 26 ± 3b

34 ± 3 56 ± 5a 43 ± 4b 41 ± 2b 34 ± 3b 32 ± 4b 34 ± 5b 33 ± 6 40 ± 4b 38 ± 6 30 ± 5b 40 ± 4b 32 ± 5b 41 ± 6b

41 ± 3 78 ± 5a 49 ± 5b 46 ± 3b 51 ± 3b 46 ± 4b 54 ± 3b 46 ± 5b 53 ± 4b 48 ± 6b 47 ± 4b 51 ± 5b 61 ± 6b 56 ± 4b

97 ± 4 139 ± 6a 95 ± 5b 97 ± 4b 96 ± 3b 84 ± 6b 95 ± 5b 84 ± 4b 90 ± 7b 74 ± 6b 90 ± 4b 109 ± 5b 103 ± 6b 111 ± 5b

100 ± 4 64 ± 5a 85 ± 4b 85 ± 5b 84 ± 6b 84 ± 5b 84 ± 4b 82 ± 3b 81 ± 4b 81 ± 4b 84 ± 2b 81 ± 4b 78 ± 2b 80 ± 5b

Isolated rat hepatocytes (106 cells/mL) were incubated at 37 °C in rotating round bottom flasks with 95% O2 and 5% CO2 in Krebs–Henseleit buffer (pH 7.4). Hepatotoxicity, ROS and MMP were determined as described in method section. Mean ± SEM. for three separate experiments are given. a Significant as compared to control (p < 0.05). b Significant as compared to 15 mM methylglyoxal (p < 0.05). c Cells are incubated with methylglyoxal for 30 min before addition of antioxidants. The addition of methylglyoxal marks the initiation of the 3 h experiment period.

Table 5 Protective effect of polyphenols against glyoxal and methylglyoxal in GSH-depleted rat hepatocytes. Treatment/time point

Cytotoxicity (% trypan blue taken) 60 min

120 min

180 min

ROS (F.I. units)

% MMP

90 min

90 min 100 ± 4

Control GSH depleted hepatocytes 0.8 mM Glyoxal 0.2 mM Gallic acid 0.2 mM Methyl gallate 0.2 mM Ethyl gallate 0.2 mM Propyl gallate 0.2 mM Rutin 4 lM curcumin

25 ± 1

32 ± 2

36 ± 2

98 ± 6

44 ± 4a 34 ± 5 34 ± 4 33 ± 4 31 ± 2 29 ± 3b 28 ± 4b

62 ± 3a 47 ± 2b 37 ± 2b 41 ± 4b 45 ± 3b 42 ± 4b 47 ± 5b

89 ± 2a 63 ± 7b 58 ± 5b 61 ± 2b 68 ± 4b 52 ± 2b 69 ± 5b

166 ± 6a 93 ± 4b 84 ± 4b 82 ± 4b 80 ± 6b 78 ± 5b 157 ± 3

61 ± 4a 86 ± 4b 85 ± 5b 82 ± 4b 80 ± 6b 84 ± 3b 79 ± 3b

3 mM Methylglyoxal 0.2 mM Gallic acid 0.2 mM Methyl gallate 0.2 mM Ethyl gallate 0.2 mM Propyl gallate 0.2 mM Rutin 4 lM curcumin

54 ± 5a 40 ± 6 33 ± 4c 36 ± 5c 39 ± 4 33 ± 4c 30 ± 3c

69 ± 4a 50 ± 6c 40 ± 5c 44 ± 3c 48 ± 5c 48 ± 2c 51 ± 2c

84 ± 5a 82 ± 5 78 ± 2 78 ± 3 76 ± 3 82 ± 6 79 ± 4

135 ± 3a 79 ± 5c 77 ± 6c 72 ± 4c 71 ± 5c 72 ± 3c 78 ± 5c

52 ± 4a 84 ± 5c 83 ± 4c 82 ± 5c 81 ± 5c 82 ± 3c 80 ± 4c

Isolated rat hepatocytes (106 cells/mL) were incubated at 37 °C in rotating round bottom flasks with 95% O2 and 5% CO2 in Krebs–Henseleit buffer (pH 7.4). Hepatotoxicity, ROS and MMP were determined as described in method section. Mean ± SEM. for three separate experiments are given. In GSH-depleted cells, cells were pre-incubated with 1-bromoheptane for 30 min before addition of glyoxal or methylgloxal and antioxidants. a Significant as compared to control (p < 0.05). b Significant as compared to 0.8 mM glyoxal (p < 0.05). c Significant as compared to 3 mM methylglyoxal (p < 0.05).

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gallate > ethyl gallate = rutin > methyl gallate > curcumin > gallic acid (Table 5). The polyphenols showed significant protection against the collapse of the mitochondrial membrane potential caused by methylglyoxal.

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4. Discussion

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Consumption of fructose has significantly increased over the past three decades as a result of the addition of HFCS to the diet [11,12]. High fructose diets are associated with glucose intolerance, obesity, fatty liver, diabetes and non-alcoholic steatohepatitis [23]. Previously, fructose and its metabolites had been shown to generate glyoxal through enzymatic metabolism [14,21,23]. Under oxidative conditions, it was determined that fructose cytotoxicity was significantly increased in hepatocytes [14]. Dihydroxyacetone is part of the fructose metabolic pathway and can be further

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metabolized to form glyoxal and methylglyoxal [14,23,45]. Dihydroxyacetone and fructose metabolites have been shown to cause DNA damage and glycate human serum albumin in vitro [45]. However, whether dihydroxyacetone can autoxidize to glyoxal and methylglyoxal has not been investigated. This study investigated the autoxidation of fructose and dihydroxyacetone under standard and oxidative stress conditions. The Fenton’s reaction was used to generate oxidative stress conditions by producing hydroxyl radicals [46]. The bovine serum assay and the Girard-T assay were employed to investigate the sugars ability to cause protein carbonylation and to form dicarbonyls respectively. In the bovine serum albumin assay, it was found that fructose did not cause protein carbonylation under standard condition, but caused protein carbonylation under oxidative stress condition (Table 1). In the Girard assay, it was determined that fructose and dihydroxyacetone autoxidized to form glyoxal and methlyglyoxal in standard and oxidative stress conditions. The concentration of

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glyoxal decreased after day one in all treatments (Table 2) because at atmospheric conditions the half-life of glyoxal is one day [48]. It was also found that the amount of glyoxal formed from fructose autoxidation was significantly less than the amount formed from dihydroxyacetone autoxidation in all conditions (Table 2). The discrepancy between the two assays can be explained by comparing the effect of the Fenton’s condition alone against fructose and dihydroxyacetone in oxidative conditions. Under the Fenton’s conditions, protein carbonylation occurred. However, this protein carbonylation was due to the protein oxidation by the oxidative stress induced by the Fenton’s reaction that generated reactive oxygen species [47]. When comparing the amount of protein carbonylation formed under the Fenton’s condition and fructose under oxidative stress condition, there were similar levels of protein oxidation. In addition, there were significantly less dicarbonyl formation detected by the Girard assay for fructose in standard and oxidative stress conditions in comparison to dihydroxyacetone in both conditions. Thus, it can be concluded that fructose autoxidized into low levels of glyoxal and methylglyoxal and the majority of the protein carbonylation was due to iron-induced protein oxidation. However, it is important to note that the enzymatic pathway for fructose metabolism remains an important step in the generation of glyoxal and methylglyoxal as it is the downstream metabolites of fructose that generate glyoxal and methylglyoxal enzymatically and non-enzymatically [14]. The results are in agreement with Manini et al. findings which show that fructose can be autoxidized to glyoxal and methylglyoxal under iron-induced oxidative stress condition [43]. However the difference in the level of dicarbonyl formation for fructose was significantly different from that formed by dihydroxyacetone [43]. The protection with polyphenols against glyoxal and methylglyoxal was investigated in rat hepatocytes. Gallic acid and its related gallate derivatives are known to be a strong antioxidant, antiinflammatory, anti-mutagenic and anti-cancer compounds (Fig. 1) [30,32,33,50]. The antioxidative property of the gallate compounds is related to its lipophilicity [30,50]. These gallate compounds also elicited cytotoxicity at high concentrations due to interaction with the mitochondrial membrane [50,51]. The gallates with longer alkyl groups are toxic to hepatocytes due to their ability to cause mitochondrial uncoupling [49]. Rutin, the glycosidic form of quercetin, is a polyphenolic flavonoid with antioxidative and anticancer capabilities [51]. Rutin is also a protector of mitochondrial function [52]. The last polyphenol investigated was curcumin. Curcumin is the principal curcuminoid of the Indian spice turmeric [53]. Curcumin is a potent antioxidant and anti-cancer compound [28,29]. At high concentrations, curcumin is able to induce apoptosis both in cancer and primary cells [28,55]. The cytotoxicity of curcumin is likely due to its ability to uncouple the mitochondria, activate the caspase-3 and activate the mitochondrial apoptotic pathway [28]. Additionally, curcumin induces the release of cytochrome c from the mitochondria [28]. At lower concentrations, curcumin demonstrated the ability to inhibit cytochrome c release and caspase 3 activation in mouse embryonic stem cells [53]. Also, curcumin can trap methylglyoxal at 1:1 ratios [55]. The level of hepatoprotection was determined with the trypan blue cytotoxicity, reactive oxygen species and mitochondrial membrane potential. The antioxidants significantly protected the rat hepatocytes against glyoxal under physiological conditions in terms of cytotoxicity, prevention of reactive oxygen species formation and maintaining the mitochondrial membrane potential (Table 3). The rankings are described in Sections 3.2 and 3.3. As expected, as the length of the alkyl chains increased, cytotoxicity increased due to its disruption of the mitochondrial function. Also, as the length of the alkyl chain increased, reactive oxygen species scavenging ability also increased [30,50]. Curcumin did not protect

Fig. 1. Chemical structures of the polyphenols investigated. Gallic acid and the gallate derivatives and rutin can be found in tea leaves. Curcumin is the main compound found in the spice turmeric.

against methylglyoxal in contradiction with the literature; however, this can be explained by the 1:1 scavenging ratio of methylglyoxal [54,56]. The curcumin concentration used in this study was not equimolar with the methylglyoxal used because concentrations as low as 24 lM could activate apoptotic pathway in cells [54]. Furthermore, the rescuing ability of each polyphenols was assessed. Glyoxal and methylglyoxal were pre-incubated for 30 min before addition of the antioxidants. The order of hepatoprotection was similar for glyoxal and methylglyoxal (Tables 3 and 4). Interestingly, the rescue with the polyphenols achieved greater hepatoprotection (Tables 3 and 4). A plausible explanation is that the antioxidants do not scavenge glyoxal or methylglyoxal directly but rather targets downstream effects such as generation of reactive oxygen species or disruption of the mitochondrial membrane potential [30,32,33,53,56,58]. Another explanation is that the polyphenols had innate hepatotoxicity leading to increased hepatotoxicity when hepatocytes were treated for a longer period [50]. Curcumin was an exception in the order of hepatoprotection, where curcumin demonstrated greater hepatoprotection against methylglyoxal than glyoxal. Curcumin is a methylglyoxal scavenger and its ability to scavenge methylglyoxal is related to its structural properties [55]. Methylglyoxal is trapped between the

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two keto-carbon groups at C10 [55]. Another exception was rutin which demonstrated less hepatoprotection when rescuing against glyoxal and methylglyoxal (Tables 3 and 4). Although rutin is an antioxidant, metabolites of rutin such as quercetin also elicit antioxidative properties [59]. The results of this study have shown that the metabolites of rutin could be better antioxidants than rutin. Thus, simultaneously adding rutin and the toxins allowed rutin to be converted into its more potent metabolites. Curcumin was consistently placed last in the rankings as well. Gallic acid was found to be cytotoxic and does not follow the trend of increasing alkyl chain length and hepatotoxicity. Gallic acid has three adjacent phenolic hydroxyl groups that was linked with its hepatoxicity [59]. Lastly, the effect of GSH-depletion and the protection against glyoxal and methylglyoxal with the antioxidants was investigated. 1-Bromoheptane was used as the agent to deplete GSH irreversibly [56]. The depletion of GSH promotes oxidative stress and compromised the detoxification of many electrophilic compounds, including glyoxal and methylglyoxal [56]. This study demonstrated that the antioxidant ability of the polyphenols. Polyphenols are vulnerable to oxidation by thiols, such as GSH, which compromise their antioxidant ability [60]. The polyphenols reduced oxidative stress and alleviated some hepatotoxicity in GSH-depleted hepatocytes. Hepatotoxicity was greater in comparison with hepatocytes without GSH depletion because GSH is a co-factor for glyoxalase which detoxifies glyoxal and methylglyoxal [15].

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5. Summary

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In summary, fructose and dihydroxyacetone are part of the same metabolic pathway that generates glyoxal and methylglyoxal. However, fructose metabolites can generate glyoxal or methylglyoxal through enzymatic pathways and autoxidation. Dihydroxyacetone, a metabolite of fructose, can also be autoxidized into glyoxal or methylglyoxal in physiological and oxidative conditions. However, it is important to remember the importance of the enzymatic pathway since this is the crucial step in which fructose is able to generate metabolites that can form glyoxal or methylglyoxal. The second part of the paper, a variety of polyphenols was investigated for their abilities to protect against glyoxal and methylglyoxal. Cytotoxicity, reactive oxygen species and mitochondrial membrane potential assays were used to evaluate each of these polyphenols. It was found that as the length of the alkyl side chain cytotoxicity increased; reactive oxygen species scavenging ability increased; and mitochondrial membrane potential decreased. This relationship was demonstrated in primary cells, GSH-depleted cells and in cells rescued with the polyphenols. Lastly, it is interesting to note that the polyphenols fared better as an antidote than as a scavenger. The polyphenols do not scavenge glyoxal or methylglyoxal directly, but they target downstream effects the toxins produced such as reactive oxygen species formation and decreasing mitochondrial membrane potential. In the future, there will be investigations on the protective abilities of the polyphenols to determine if these mechanisms are enzyme-dependent. Understanding, their mechanisms of protections can lead to the production of novel therapeutic agents that targets the early AGE pathways or downstream effects of AGEs.

519

Conflict of interest statement

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The authors report no conflict of interest.

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Uncited references

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[44,57].

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Acknowledgement

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This research was funded by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada.

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References

526

[1] N. Shangari, W.R. Bruce, R. Poon, P.J. O’Brien, Toxicity of glyoxals – role of oxidative stress, metabolic detoxification and thiamine deficiency, Biochem. Soc. Trans. 31 (2003) 1390–1393. [2] N. Ahmed, Advanced glycation endproducts—role in pathology of diabetic complications, Diabetes Res. Clin. Pract. 67 (2005) 3–21. [3] P.J. Thornalley, A. Langborg, H.S. Minhas, Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose, Biochem. J. 344 (Pt. 1) (1999) 109–116. [4] P.J. O’Brien, A.G. Siraki, N. Shangari, Aldehyde sources, metabolism, molecular toxicity mechanisms and possible effects on human health, Crit. Rev. Toxicol. 35 (2005) 609–662. [5] A. Negre-Salvayre, C. Coatrieux, C. Inqueneau, R. Salvayre, Advanced lipid peroxidation end products in oxidative damage to proteins. Potential role in diseases and therapeutic prospects for the inhibitors, Br. J. Pharmacol. 153 (2008) 6–20. [6] A.S. Chen, T. Taguchi, M. Sugiura, Y. Wakasugi, A. Kamei, M.W. Wang, I. Miwa, Pyridoxalaminoguanidine adduct is more effective than aminoguanidine in preventing neuropathy and cataract in diabetic rats, Horm. Metab. Res. 36 (2004) 183–187. [7] P.J. Thornalley, Use of aminoguanidine (Pimagedine) to prevent the formation of advanced glycation endproducts, Arch. Biochem. Biophys. 419 (2003) 31–40. [8] T. Kiho, M. Kato, S. Usui, K. Hirano, Effect of buformin and metformin on formation of advanced glycation end products by methylglyoxal, Clin. Chim. Acta 358 (2005) 139–145. [9] J. Peyroux, M. Sternberg, Advanced glycation endproducts (AGEs): pharmacological inhibition in diabetes, Pathol. Biol. (Paris) 54 (2006) 405–419. [10] R. Mehta, L. Wong, P.J. O’Brien, Cytoprotective mechanisms of carbonyl scavenging drugs in isolated rat hepatocytes, Chem. Biol. Interact. 178 (2009) 317–323. [11] M.B. Vos, J.E. Kimmons, C. Gillespie, J. Welsh, H.M. Blanck, Dietary fructose consumption among US children and adults: the third national health and nutrition examination survey, Medscape J. Med. 10 (2008) 160. [12] G.A. Bray, S.J. Nielsen, B.M. Popkin, Consumption of high-fructose corn syrup in beverages may play a role in the epidemic of obesity, Am. J. Clin. Nutr. 79 (2004) 537–543. [13] L. Tappy, K.A. Le, C. Tran, N. Paquot, Fructose and metabolic diseases: new findings, new questions, Nutrition 26 (2010) 1044–1049. [14] K. Yang, D. Qiang, S. Delaney, R. Mehta, W.R. Bruce, P.J. O’Brien, Differences in glyoxal and methylglyoxal metabolism determine cellular susceptibility to protein carbonylation and cytotoxicity, Chem. Biol. Interact. 191 (2011) 322– 329. [15] D.L. Vander Jaqt, R.K. Hassebrook, L.A. Hunsaker, W.M. Brown, R.E. Royer, Metabolism of the 2-oxoaldehyde methylglyoxal by aldose reductase and by glyoxalase-I: roles for glutathione in both enzymes and implications for diabetic complications, Chem. Biol. Interact. 130–132 (2001) 548–562. [16] N. Ahmed, P.J. Thornalley, Advanced glycation endproducts: what is their relevance to diabetic complications, Obes. Metab. 9 (2007) 233–245. [17] P.J. Thornalley, S. Battah, N. Ahmed, N. Karachalias, S. Agalou, R. Babei-Jadidi, A. Dawnay, Quantitative screening of advanced glycation endproducts in cellular and extracellular proteins by tandem mass spectrometry, Biochem. J. 375 (2003) 581–592. [18] M. Busch, S. Franke, C. Ruster, G. Wolf, Advanced glycation end-products and the kidney, Eur. J. clin. Invest. 40 (2010) 742–755. [19] A.M. Schmidt, S.D. Yan, S.F. Yan, D.M. Stern, The multiligand receptor RAGE as a progression factor amplifying immune and inflammatory responses, J. Clin. Invest. 108 (2001) 949–955. [20] M.P. Wautier, O. Chappey, S. Corda, D.M. Stern, A.M. Schmidt, J.L. Wautier, Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE, Am. J. Physiol. Endocrinol. Metab. 280 (2001) E685–E694. [21] O. Lee, W.R. Bruce, Q. Dong, J. Bruce, R. Mehta, P.J. O’Brien, Fructose and carbonyl metabolites as endogenous toxins, Chem. Biol. Interact. 178 (2009) 332–339. [22] N. Shangari, T.S. Chan, M. Popovic, P.J. O’Brien, Glyoxal markedly compromises hepatocytes resistance to hydrogen peroxide, Biochem. Pharmacol. 71 (2006) 1610–1618. [23] K. Yang, C. Feng, H. Lip, W.R. Bruce, P.J. O’Brien, Cytotoxic molecular mechanisms and hepatoprotection by enzymic metabolism or autoxidation for glyceraldehyde, hydroxypyruvate and glycolaldehyde, Chem. Biol. Interact. 191 (2011) 315–321. [24] V. Gulati, I.H. Harding, E.A. Palombo, Enzyme inhibitory and antioxidant activities of traditional medicinal plants potential application in the management of hyperglycemia, BMC Complement. Altern. Med. 12 (2012), http://dx.doi.org/10.1186/1472-6882-12-77. [25] Final report on the amended safety assessment of propyl gallate. Int J Toxicol. 26 (2007) 89–118. [26] S.N. Mink, H. Jacobs, J. Gotes, K. Kasian, Z.Q. Cheng, Ethyl gallate, a scavenger of hydrogen peroxide that inhibits lysozyme-induced hydrogen peroxide

Please cite this article in press as: H. Lip et al., Glyoxal and methylglyoxal: Autoxidation from dihydroxyacetone and polyphenol cytoprotective antioxidant mechanisms, Chemico-Biological Interactions (2012), http://dx.doi.org/10.1016/j.cbi.2012.11.013

525

Q3

527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603

CBI 6747

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[27]

[28]

[29]

[30]

[31] [32]

[33]

[34]

[35] [36]

[37]

[38] [39] [40]

[41]

[42]

H. Lip et al. / Chemico-Biological Interactions xxx (2012) xxx–xxx signaling in vitro, reverses hypotension in canine septic shock, J. Appl. Physiol. 110 (2011) 359–374. N. Kamalakkannan, P.S. Prince, Antihyperglycaemic and antioxidant effect of rutin, a polyphenolic flavonoid, in streptozotocin-induced diabetic Wistar rats, Basic Clin. Pharmacol. Toxicol. 98 (2006) 97–103. L.D. Guo, X.J. Chen, Y.H. Hu, Z.J. Yu, D. Wang, J.Z. Liu, Curcumin inhibits proliferation and induces apoptosis of human colorectal cancer cells by activating the mitochondria apoptotic pathway, Phytother. Res. (2012), http:// dx.doi.org/10.1002/ptr.4731. W.H. Wang, I.T. Chiang, K. Ding, J.G. Chun, W.J. Lin, S.S. Lin, J.J. Hwang, Curcumin-induced apoptosis in human hepatocellular carcinoma J5 cells: critical role of Ca+2-dependent pathway, Evid. Based Complement. Altern. Med. (2012), http://dx.doi.org/10.1155/2012/512907. S. Umadevi, V. Gopi, S.P. Simma, A. Parthasarathy, S.M. Yousuf, V. Elangovan, Studies on the cardio protective role of gallic acid against AGE-induced cell proliferation and oxidative stress in H9C2 (2-1) cells, Cardiovasc. Toxicol. (2012), http://dx.doi.org/10.1007/s12012-012-9170-2. U. Subudhi, G.B. Chainy, Curcumin and vitamin E modulate hepatic antioxidant gene expression in PTU-induced hypothyroid rats, Mol. Biol. Rep., in press. T. Kalaivani, C. Rajasekaran, L. Mathew, Free radical scavenging, cytotoxic, and hemolytic activities of an active antioxidant compound ethyl gallate from leaves of Acacia nilotica (L.) Wild. Ex. Delile Subsp. Indica (Benth.) Brenan, J. Food Sci. 76 (2011) T144–T149. J.A. Crispo, M. Piché, D.R. Ansell, J.K. Eibl, I.T. Tai, A. Kumar, G.M. Ross, T.C. Tai, Protective effects of methyl gallate on H2O2-induced apoptosis in PC12 cells, Biochem. Biophys. Res. Commun. 393 (2010) 773–778. S. Criado, C. Allevi, N.A. Garcia, Visible-light-promoted degradation of the antioxidants propyl gallate and t-butylhydroquinone: mechanistic aspects, Redox Rep. 17 (2012) 131–138. P. Moldeus, J. Hogberg, S. Orrenius, Isolation and use of liver cells, Methods Enzymol. 52 (1978) 60–71. M.Y. Moridani, J. Pourahmad, H. Bui, A. Siraki, P.J. O’Brien, Dietary flavonoid iron complexes as cytoprotective superoxide radical scavengers, Free Radic. Biol. Med. 34 (2003) 243–253. H. Possel, H. Noack, W. Augustin, G. Keilhoff, G. Wolf, 2,7Dihydrodichlorofluorescein diacetate as a fluorescent marker for peroxynitrite formation, FEBS Lett. 416 (1997) 175–178. J. Pourahmad, P.J. O’Brien, A comparison of hepatocyte cytotoxic mechanisms for Cu2+ and Cd2+, Toxicology 143 (2000) 263–273. B.S. Andersson, T.Y. Aw, D.P. Jones, Mitochondrial transmembrane potential and pH gradient during anoxia, Am. J. Physiol. 252 (1987) C349–C355. D.P. Hartley, D.J. Kroll, D.R. Petersen, Prooxidant initiated lipid peroxidation in Isolated rat hepatocytes: detection of 4-hydroxynonenaland malondialdehyde-protein adducts, Chem. Res. Toxicol. 10 (1997) 895–905. P.A. Voziyan, T.O. Metz, J.W. Baynes, B.G. Hudson, A post-Amadori inhibitor pyridoxamine also inhibits chemical modification of proteins by scavenging carbonyl intermediates of carbohydrate and lipid degradation, J. Biol. Chem. 277 (2002) 3397–3403. R.E. Mitchel, H.C. Birnboim, The use of Girard-T reagent in a rapid and sensitive methods for measuring glyoxal and certain other alpha-dicarbonyl compounds, Anal. Biochem. 81 (1977) 47–56.

[43] P. Manini, P. La Pietra, L. Panzella, A. Napolitano, M. d’Ischia, Glyoxal formation by Fenton-induced degradation of carbohydrates and related compounds, Carbohydr. Res. 341 (2006) 1828–1833. [44] N. Shangari, P.J. O’Brien, The cytotoxic mechanism of glyoxal involves oxidative stress, Biochem. Pharmacol. 68 (2004) 1433–1442. [45] C. Seneviratne, G.W. Dombi, W. Liu, J.A. Dain, In vitro glycation of human serum albumin by dihydroxyacetone and dihydroxyacetone phosphate, Biochem. Biophys. Res. Commun. 417 (2012) 817–823. [46] C.Y. Feng, S. Wong, Q. Dong, J. Bruce, R. Mehta, W.R. Bruce, P.J. O’Brien, Hepatocyte inflammation model for cytotoxicity research: fructose or glycolaldehyde as a source of endogenous toxins, Arch. Physiol. Biochem. 115 (2009) 105–111. [47] L. Benov, A.F. Beema, Superoxide-dependence of the short chain sugarsinduced mutagenesis, Free Radic. Biol. Med. 34 (2003) 429–433. [48] R. Atkinson, Kinetics and mechanisms of the gas-phase reactions of the hydroxyl radical with organic compounds under atmospheric conditions, Chem. Rev. 85 (1985) 69–201. [49] C. Frey, M. Pavani, G. Cordano, S. Muñoz, E. Rivera, J. Medina, A. Morello, J. Diego Maya, J. Ferreira, Comparative cytotoxicity of alkyl gallates on mouse tumor cell lines and isolated rat hepatocytes, Comp. Biochem. Physiol. A: Mol. Integr. Physiol. 146 (2007) 520–527. [50] Y. Nakagawa, S. Tayama, Cytotoxicity of propyl gallate and related compounds in rat hepatocytes, Arch. Toxicol. 69 (1995) 204–208. [51] K.H. Janbaz, S.A. Saeed, A.H. Gilani, Protective effect of rutin on paracetamoland CCl4-induced hepatotoxicity in rodents, Fitoterapia 73 (2002) 557–563. [52] C. Carrasco-Pozo, M.L. Mizgier, H. Speisky, M. Gotteland, Differential protective effects of quercetin, resveratrol, rutin and epigallocatechin gallate against mitochondrial dysfunction induced by indomethacin in Caco-2 cells, Chem. Biol. Interact. 195 (2012) 199–205. [53] Y.D. Hsuuw, C.K. Chang, W.H. Chan, J.S. Yu, Curcumin prevents methylglyoxalinduced oxidative stress and apoptosis in mouse embryonic stem cells and blastocysts, J. Cell. Physiol. 205 (2005) 379–386. [54] C.C. Chen, M.S. Hsieh, Y.D. Hsuuw, F.J. Huang, W.H. Chan, Hazardous effects of curcumin on mouse embryonic development through a mitochondriadependent apoptotic signaling pathway, Int. J. Mol. Sci. 11 (2010) 2839–2855. [55] T.Y. Hu, C.L. Liu, C.C. Chiyau, M.L. Hu, Trapping of methylglyoxal by curcumin in cell-free systems and in human umbilical vein endothelial cells, J. Agric. Food Chem. (2012), http://dx.doi.org/10.1021/jf302188a. [56] S. Khan, P.J. O’Brien, 1-bromoalkanes as new potent nontoxic glutathione depletors in isolated rat hepatocytes, Biochem. Biophys. Res. Commun. 179 (1991) 436–441. [57] T.W. Wu, K.P. Fung, L.H. Zeng, J. Wu, H. Nakamura, Propyl gallate as a hepatoprotector in vitro and in vivo, Biochem. Pharmacol. 48 (1994) 416–422. [58] S. Pashikanti, D.R. de Alba, G.A. Boissonneault, D. Cervantes-Laurean, Rutin metabolites: novel inhibitors of nonoxidative advanced glycation end products, Free Radic. Biol. Med. 48 (2010) 656–663. [59] M. Inoue, R. Suzuki, N. Sakaguchi, Z. Li, T. Takeda, Y. Ogihara, B.Y. Jiang, Y. Chen, Selective induction of cell death in cancer cells by gallic acid, Biol. Pharm. Bull. 18 (1995) 1526–1530. [60] N.R. Unnadkat, R.J. Elias, Oxidative stability of ()-epigallocatechin gallate in the presence of thiols, J. Agric. Food Chem. 60 (2012) 10815–10821.

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