英-维生素CE、GSPE保护DNA氧化

bl tr d ois J a Laboratoire de Physico-Toxicochimie des Syste`mes Naturels, UMR 5472, CNRS Universite´ Bordeaux I, 33405 Talence Cedex, France oxodG. These results suggest that a vitamin E and A and GSPE enriched-diets have a protective effect on oxidative DNA damage limited In vitro studies have shown that procyanidins extracted from grape seeds have remarkable free radical scavenging activities (Da Silva et al., 1991) and can significantly delay the oxidation of low-density lipoprotein and lipid-contain- ing membranes induced by radical generators or metal ions (Mazur et al., 1999; Teissedre et al., 1996). Abbreviations: dG, 20-deoxyguanosine; GSPE, grape seed proanthocy- anidin extract; 8-OxodG, 8-oxo-7, 8-dihydro-2 0-deoxyguanosine; 8-Oxod- GTP, 8-oxo-7, 8-dihydro-20-deoxyguanosine-50-triphosphate; 8-OxoGua, 8-oxo-7, 8-dihydroguanine; 8-OxoGuo, 8-oxo-7, 8-dihydroguanosine. * Corresponding author. Tel.: +33 5 40 00 22 56; fax: +33 5 40 00 87 19. E-mail address: b.morin@ism.u-bordeaux1.fr (B. Morin). Available online at www.sciencedirect.com Food and Chemical Toxi to rat leukocytes. � 2007 Elsevier Ltd. All rights reserved. Keywords: Oxidative DNA damage; 8-OxodG; Rats; Fat-soluble-vitamins; Proanthocyanidin; Grape seed extract 1. Introduction Diets that are rich in plant have been associated with a decreased risk for specific disease processes and certain chronic diseases. In addition to essential macronutrients and micronutrients, the flavonoids in a variety of plant foodstuffs may have health-enhancing properties (Santos- Buelga and Scalbert, 2000). Proanthocyanidins are natu- rally occurring compounds widely available in fruits, vege- tables, seeds, flowers and bark (Lazarus et al., 1999). They are a class of phenolic compounds which take the form of oligomers or polymers of polyhydroxy flavan-3-ol units, such as (+)-catechin and (�)-epicatechin (Porter, 1986). Grape seeds are particularly rich sources of proanthocyani- dins, and only the procyanidin-type of proanthocyanidins have been detected in the seeds (Santos-Buelga et al., 1995; Fuleski and Ricardo da Silva, 1997). b Bio-Tox, BP 34, 21 Avenue du Ge´ne´ral de Castelnau, 33886 Villenave d’Ornon Cedex, France c Laboratoire Le´sions des Acides Nucle´iques, DRFMC/SCIB UMR-E No. 3 CEA-UJF, Grenoble, 17 Avenue des Martyrs, F-38054 Cedex 9, France Received 27 November 2006; accepted 8 October 2007 Abstract This study reports the effect of the fat-soluble vitamin A or vitamin E and grape seed proanthocyanidin extract (GSPE) on oxidative DNA damage estimated by 8-oxo-7, 8-dihydro-2 0-deoxyguanosine (8-oxodG) contents in urine and leukocyte of rats. Little is known about the antioxidant potency of dietary anthocyanidins and consequently, the aim of this study was to establish whether anthocyanidins could act as putative antioxidant micronutrients. Seven groups of male Sprague-Dawley rats were fed during 47 days with the following diets: a basic diet, two deficient vitamin A or E diets, two supplemented vitamin A or E diets and two supplemented diets enriched with two doses of grape seed proanthocyanidin extract. At the end of the diet intervention period, 24 h, urine and blood were collected. The levels of 8-oxodG in leukocytes rats were significantly lower in the supplemented vitamin A, E and GSPE diet groups with respect to the control group. However, consumption of a-tocopherol, vitamin A or GSPE had no effect on the excretion of the oxidised nucleoside 8- Effect of dietary fat-solu proanthocyanidin-rich ex oxidative DNA Be´ne´dicte Morin a,*, Jean-Franc¸ Carine Badouard c, 0278-6915/$ - see front matter � 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2007.10.011 e vitamins A and E and act from grape seeds on amage in rats Narbonne a, Daniel Ribera b, ean-Luc Ravanat c www.elsevier.com/locate/foodchemtox cology 46 (2008) 787–796 An in vivo study using rabbits has shown that a proan- thocyandin-rich extract from grape seeds increases antiox- idative activity in plasma (Yamakoshi et al., 1999). To date most of the studies on the antioxidant ability of flavan-3- ols and procyanidins have been focused on lipids as substrates for oxidation. The effect of flavan-3-ols and procyanidins on the oxidation of DNA has received less attention (Wei and Frenkel, 1993; Ottaviani et al., 2002). Nevertheless, the few in vivo studies have established that their consumption decreases DNA damage in human (Simonetti et al., 2002) and rats. For instance, wine poly- phenols given orally to rats were shown to limit DNA oxidative damage in colon mucosal cells (Giovannelli et al., 2000; Lodovici et al., 2000), in hepatic cells (Casalini et al., 1999) and reduced the number of tumours in rats enhance susceptibility to oxidative damage. rial Institute) medium was obtained from Gibco (Invitrogen Corpora- tion, UK). Lymphoprep was obtained from Nycomed Pharma (Oslo, Norway). Acetonitrile, methanol and dichloromethane were HPLC grade and obtained from ICS Nationale (Belin Beliet, France). 2.2. Animal study design and sample collection Eighty-four weaning male Sprague-Dawley rats (OFA strain, 38–59 g) were purchased from IFFA CREDO (L’Arbresle – France). Prior to experimental treatment, rats were acclimatized for at least 3–5 days and provided with food and water ad libitum. Exposure was performed at Phycher Bio-de´veloppement (Pessac, France) under quality assurance and good laboratory practices. Each group of animals was fed one of the seven diets (Table 1) for 47 days. A control diet group Control (n = 12; 0.05 mg a-tocopherol acetate/ g diet dry weight, 10 IU retinyl acetate/g diet dry weight, no GSPE), a vitamin A-deficient diet Vit A� (n = 12; control diet without retinyl ace- ts Vit 920 70 10 5 10 0 �1): �1) 788 B. Morin et al. / Food and Chemical Toxicology 46 (2008) 787–796 In this study, we compared, in healthy male rats, the protective abilities of dietary GSPE and vitamins A and E against oxidative DNA damage as measured by 8-oxo- 7, 8-dihydro-2 0-deoxyguanosine (8-oxodG) in blood and urine as noninvasive biomarkers for later studies in human. 2. Materials and methods 2.1. Chemicals Nuclease P1, RNase IIIA, RNase T1, Triton X-100, NaCl, defer- rioxamine mesylate, sodium dodecyl sulfate retinol, a-tocopherol, retinyl palmitate, phosphate buffered saline and isoamyl alcohol were obtained from Sigma–Aldrich Chimie SARL (St Quentin Fallavier, France). Alkaline phosphatase and proteinase K were obtained from Roche Diagnostic (Mannheim, Germany). RPMI-1640 (Roswell Park Memo- Table 1 Control and experimental diets composition Ingredient Control Experimental die Vit E� Diet mixa 920 920 Mineral mixb 70 70 Vitamin mixc 10 10 a-Tocopherol acetate (mg/g) 0.05 0 Retinyl acetate (IU/g) 10 10 GSPE (mg/g) 0 0 Values are g/kg diet dry wt, unless otherwise indicated. a UAR 211A: the diet mixture provides the following amounts (g/kg diet glycerol, 10; onagrine oil, 10. b UAR 205B: the salt mixture provides the following amounts (g/kg diet 0.056; Cu, 0.0087; Zn, 0.031; Co, 0.00006; I, 0.00034. treated with radical generators (Caderni et al., 2000; Bom- ser et al., 1999). Similarly, a recent study has shown that an anthocyanidin rich extract decreases indices of lipid perox- idation and DNA damage in vitamin E depleted rats (Ramirez-Tortosa et al., 2001). However, most of the work concerning the antioxidant abilities of procyanidins in vitro and in vivo have been undertaken with organisms under oxidative stress conditions (induced by radical generators, UV, metal ions, antioxidant deficient diets. . .) in order to c UAR 200: the vitamin mixture provides the following amounts (mg/kg diet� pyridoxine, 10; inositol, 150; cyanocobalamine, 0.05; menadione, 40; nicotinic a tate), a vitamin E-deficient diet Vit E� (n = 12; control diet without a- tocopherol acetate), a vitamin A-supplemented diet Vit A+ (n = 12; 200 IU retinyl acetate/g diet dry weight), a vitamin E-supplemented diet Vit E+ (n = 12; 5 mg a-tocopherol acetate/g diet dry weight), a low GSPE sup- plemented diet GSPE1 (n = 12; 0.04 mg/g diet dry weight) and a high GSPE supplemented diet GSPE2 (n = 12; 0.4 mg/g diet dry weight). Each diet was obtained from the U.A.R. Factory (Villemoison, Epinay-sur- Orge, France) in a granule form. A commercially available, dried, pow- dered GSPE (VITISOL� batch no. 2724) was kindly provided from the Berkem Society (Gardonne, France) and was added to the control diet by U.A.R. Rats were randomly divided into seven groups and housed three per wire cage. Food consumption and body weight were recorded weekly all through the study. Clinical signs were checked daily. After 47 days, rats were housed individually in metabolic cages and 24 h urine were collected and stored at �80� until analysed. At the end of the urine collection, the rats were anesthetized with sodium pentobarbital and blood was sampled by cardiac puncture and drawn into heparin vacutainers to prevent coagulation. At sacrifice all rats were healthy. Blood of three rats from the same diet group were combined and divided into two fractions. One fraction (8 ml) was used to obtain the plasma and the remaining (20–25 ml) was used to the leukocytes isolation. Each fraction was kept on ice and used within 2 h. 2.3. Plasmatic vitamin determination Blood (8 ml) was centrifuged at 1500g for 15 min at 4 �C. Plasma was removed and aliquoted into 1 ml plastic tubes, snap-frozen in liquid nitrogen and stored at �80 �C until vitamin analysis. E+ Vit A� Vit A+ GSPE1 GSPE2 920 920 920 920 70 70 70 70 10 10 10 10 0.05 0.05 0.05 0.05 0 200 10 10 0 0 0.04 0.4 casein, 230; dextrose, 380; corn starch, 200; cellulose, 60; stearic acid, 30; : Ca, 7; K, 0.42; Na, 2.8; Mg, 0.7; Fe, 0.21; P, 5.425; trace elements: Mn, 1): cholecalciferol, 0.0625; thiamin, 20; riboflavin, 15; panthotenic acid, 68; cid, 99; paraaminobenzoic acid, 49; folic acid, 5; biotin, 0.3; choline, 1360. the working standards solution were prepared as follows: a-tocopherol, vigorous agitation and made up to 4.7 ml with buffer. SDS (300 ll) 10% several times. The tube was left on ice for 10 min to facilitate DNA pre- ical (70:20:10, v/v/v) as eluant, at a flow rate of 1.2 ml/min. The UV detector was programmed to monitor retinol at 325 nm from 0 to 4 min, tocopherol at 292 nm from 4 to 8 min and retinyl palmitate at 325 nm from 8 to 5 min. Each vitamin was quantified on the basis of peak area using the calibration curves previously obtained from the standard solution. Detection limit tested for a-tocopherol, retinol and retinyl palmitate, assuming that the signal-to-noise ratio should be at least three was found to be 0.46, 0.02 and 0.014 lM, respectively. 2.7. Leukocyte preparation To the anticoagulated whole blood sample (20–25 ml) in 50 ml Falcon tubes, an equal volume of RPMI medium (20–25 ml) was added. The solution was carefully layered onto an equal volume of Polymor- phprep (Nycomed Pharma AS, Norway) with a density of 1.113 in two 50 ml Falcon tubes and centrifuged at 600g for 70 min at 20 �C without breaking. A mixture of all leukocytes (lymphocytes, granulocytes and monocytes) was collected from the interface layer and made up to 50 ml with RPMI medium. The cells were spun down at 700g for 20 min at 20 �C with breaking and the supernatant was removed. Red blood cells in the pellet were lysed by adding 4 ml of sterile water for 15 s. PBS (2 ml) was then added to stop the lysis. The solution was transferred to a 15 ml Falcon tube and made up to 15 ml with PBS. The leukocytes were analysis. 2.6. Chromatographic separation The samples were analysed using an Agilent HPLC system (model 1100 series) equipped with an autoinjector with a 100 ll injection loop, a quaternary pump and a spectrophotometer detector. We used a reverse- phase Lichrosphere 100RP18 column (5 lm particle size, 150 mm L · 4.6 mm ID) (Interchim, Montluc¸on, France). The HPLC mobile phase was acetonitrile/dichloromethane/methanol retinol and retinyl palmitate stock solutions were mixed 1/1/1 (v/v/v) and were diluted 10, 20, 40, 80 and 200 times with ethanol. In microtube, 150 ll of the working standard solutions were mixed with 50 ll chloro- form and 100 ll water, vortex mixed for 1 min, allowed to stand for 5 min and mixed for a further 1 min. After centrifugation at 2000g for 8 min at room temperature, the clear supernatant was transferred to an amber autosampler vial. Forty microlitres was injected into the HPLC system for analysis. 2.5. Sample preparation The extraction procedure was carried out in a room protected from direct sunlight. Plasma (100 ll) was mixed with 200 ll ethanol–chloroform (3:1, v/v), vortex mixed for 1 min, allowed to stand for 5 min and mixed for a further 1 min. After centrifugation at 2000g for 8 min at room temperature, the clear supernatent was transferred to an amber auto- sampler vial. Forty microlitres was injected into the HPLC system for A sensitive HPLC assay was adapted from a recent article (Taibi and Nicotra, 2002) and validated for a-tocopherol, retinol and retinyl palmi- tate in plasma using liquid–liquid extraction and UV detection. 2.4. Standards preparation Individual stock solution of commercial vitamins was prepared in ethanol and consisted in 2 mM of a-tocopherol, 223 lM of retinol and 25 lM of retinyl palmitate. These solutions were stored in aluminum foil- covered glass containers and kept at �20 �C. Absorbance was determined using a spectrophotometer and concentrations were calculated from the standard absorbance E (1 cm/1%): retinol, 1850 at 325 nm; a-tocopherol, 75.8 at 292 nm; retinyl palmitate, 975 at 325 nm. On the day of the assay, B. Morin et al. / Food and Chem centrifuged at 700g for 20 min at 20 �C and the supernatant was discarded. cipitation. Ethanol was then removed and DNA washed with 10 ml of ice- cold 70% ethanol, three times, ethanol being removed by aspiration. Finally, DNA was recovered by centrifugation, and dissolved into 10 mM Tris–HCl prior to DNA digestion. The DNA hydrolysis was performed with nuclease P1 and alkaline phosphatase as described previously (Ravanat et al., 2002, Protocol Dig-2). 2.9. HPLC–EC measurement of 8-oxodG in leukocytes For analysis, a Beckman series pump system equipped with a pulse damper, a cooling autosampler and a spectrophotometric detector, set at 254 nm, connected to a Kontron amperometric detector, was used. The electrochemical cell was equipped with a glassy carbon working electrode, operated at 650 mV vs an Ag/AgCl reference electrode. The system was operated at 0.5 nA full range detection. The HPLC separation was obtained on a Uptisphere ODB C18 column (5 lm particle size, 250 · 4.6 mm) equipped with a Uptisphere ODB C18 guard column (5 lm particle size, 50 · 4.6 mm) (Interchim, Montluc¸on, France). The mobile phase used for isocratic elution of 8-oxodG was composed of 50 mM ammonium acetate pH 5.5 containing 10% methanol at a flow rate of 0.8 ml/min and the injection volume was 100 ll. The concentration of dG was estimated from the UV peak and the concentration of 8-oxodG from the electrochemical signal using an external calibration. Results are expressed as the number of residues of 8-oxodG per 106 dG. The LOD was determined around 20 fmol injected corresponding to 0.6 8-oxodG/106 dG for 50 lg of injected DNA. 2.10. HPLC–MS/MS measurement of 8-oxodG and 8-oxoGuo in urine On-line HPLC–MS/MS measurements were carried out using an Agilent (Massy, France) 1100 HPLC system, equipped with a thermo- stated autosampler, a binary HPLC pumping system, an oven and a UV detector. Separations were performed using a reversed phase C18 (5 lm, 250 · 2 mm) column from Alltech (Deerfield, Ilinois, USA). The elution was added to the nuclear suspension to obtain a final concentration of 0.6%. At this stage, the pellet must be well dispersed otherwise SDS fails to lyse all the nuclear membrane. The tube is gently inverted several times to mix and incubate 10 min at 37 �C. Thereafter, 200 ll RNase IIIA (1 mg/ ml) in RNase buffer (10 mM Tris–HCl, 0.4 M NaCl, pH 8) and 10 ll RNase T1 (1 U/ll in RNase buffer) were added and incubated for 30 min at 37 �C. Proteinase K (1 mg) was then added prior to incubation for 30 min at 37 �C. The solution was cooled at room temperature and transferred to a stoppered glass tube. An equal volume of chloro- form:isoamyl alcohol (24:1) was added. After 15 s of vigorous shaking, the solution was centrifuged for 10 min at 2400g at 20 �C with no brake. The upper phase was collected, taking care not to disturb the cloudy interface. The chloroform and isoamyl alcohol extraction step was repeated with the aqueous phase. The aqueous layer was transferred to a 15 ml Falcon tube and the volume was measured. Y ml of 6 M NaCl (where Y = 0.311· measured volume) was added, vortexed for 10 s and centrifuged for 10 min at 200g at room temperature. The supernatant was carefully decanted in a 15 ml tube and cooled on ice for 5 min. Two volumes of cold ethanol were then added. DNA precipitation was achieved by gently inverting the tube 2.8. DNA extraction from leukocytes Three millilitres of lysis buffer (10 mM Tris–HCl, 0.4 M NaCl, 1 mM deferoxamine mesylate pH 8, 0.5% Triton X-100) was added to the leu- kocyte pellet. After agitation, the nuclei were collected by centrifugation at 1200g for 5 min at 4 �C and washed with 5 ml Triton-free lysis buffer. To the nuclear pellet, obtained by centrifugation (1200g for 5 min at 4 �C), 1 ml Triton-free buffer was added. The pellet was well dispersed by a Toxicology 46 (2008) 787–796 789 was achieved at a flow rate of 0.2 ml/min in the gradient mode, the column being maintained at 28 �C. The proportion of acetonitrile in 5 mM ammonium acetate (pH 6.5), starting from 0%, reached 3% within 5 min, and 12% within 25 min for the measurement of both 8-oxodG and its corresponding ribonucleoside, 8-oxoGuo. After the completion of the HPLC analysis (30 min), the column was reequilibrated with 100% ammonium acetate for 15 min before next injection. After addition of MeOH (0.1 ml/min) at the output of the UV detector, set at 260 nm, the mobile phase was directed onto a API3000 tandem mass spectrometer (Applied Biosystems) through a ‘‘Turbospray’’ electrospray source (Sciex, Thornil Canada) as described in details elsewhere for the 8-oxodG (Ravanat et al., 1998; Frelon et al., 2000). The same conditions were applied for the 8-oxoGuo analysis. The system was entirely controlled by Analyst software 1.2. Quantification of 8-oxdG and 8-oxoGuo was obtained by using isotopically labeled internal standards. For that pur- pose, each urine sample was diluted with an equal volume of the mobile rats was the lowest (45 ± 4 g). Vitamins A, E and proanth- ocyanidin intakes are shown in Table 2. The rats did not exhibit clinical symptoms. 3.2. Fat-soluble vitamin concentration in plasma The endogenous concentrations in plasma retinol, reti- nyl ester and a-tocopherol were consistent with values in healthy male rats reported in the literature (Danelisen et al., 2002; Henning et al., 1997). Plasma concentrations of a-tocopherol and retinol were high (Table 3) (relative to the limits of detection of our method) so that even the 1 2 3 4 5 6 weeks 1 2 3 4 5 6 weeks ea 0 5 10 15 20 25 30 35 m e an fo od c on su m pt io n (g/ da y) control VitE+ VitA+ 0 5 10 15 20 25 30 35 m ea n fo o d co ns um pt io n (g/ da y) control GSPE1 GSPE2 Fig. 1. Mean food consumption of rats fed each diet. 790 B. Morin et al. / Food and Chemical was observed among the other groups compared to the control animals. No significant change in weekly food intake was observed in the dietary group (Fig. 1). Although, the mean food consumption was lower for GSPE1 diet, the body weight gain was not significantly different from the control group (Table 2). The initial body weight of the GSPE1 diet Table 2 Body weight gain and vitamin E, vitamin A and