Effect of dietary fat-soluble vitamins A and E and proanthocyanidin-rich extract from grape seeds on oxidative DNA damage in rats

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Food and Chemical Toxicology 46 (2008) 787–796 www.elsevier.com/locate/foodchemtox

Effect of dietary fat-soluble vitamins A and E and proanthocyanidin-rich extract from grape seeds on oxidative DNA damage in rats Be´ne´dicte Morin a,*, Jean-Franc¸ois Narbonne a, Daniel Ribera b, Carine Badouard c, Jean-Luc Ravanat c a

c

Laboratoire de Physico-Toxicochimie des Syste`mes Naturels, UMR 5472, CNRS Universite´ Bordeaux I, 33405 Talence Cedex, France b Bio-Tox, BP 34, 21 Avenue du Ge´ne´ral de Castelnau, 33886 Villenave d’Ornon Cedex, France 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 8oxodG. These results suggest that a vitamin E and A and GSPE enriched-diets have a protective effect on oxidative DNA damage limited 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 (SantosBuelga and Scalbert, 2000). Proanthocyanidins are natu-

Abbreviations: dG, 2 0 -deoxyguanosine; GSPE, grape seed proanthocyanidin extract; 8-OxodG, 8-oxo-7, 8-dihydro-2 0 -deoxyguanosine; 8-OxodGTP, 8-oxo-7, 8-dihydro-2 0 -deoxyguanosine-5 0 -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: [email protected] (B. Morin). 0278-6915/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2007.10.011

rally occurring compounds widely available in fruits, vegetables, 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 proanthocyanidins, and only the procyanidin-type of proanthocyanidins have been detected in the seeds (Santos-Buelga et al., 1995; Fuleski and Ricardo da Silva, 1997). 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-containing membranes induced by radical generators or metal ions (Mazur et al., 1999; Teissedre et al., 1996).

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An in vivo study using rabbits has shown that a proanthocyandin-rich extract from grape seeds increases antioxidative activity in plasma (Yamakoshi et al., 1999). To date most of the studies on the antioxidant ability of flavan-3ols 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 polyphenols 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 treated with radical generators (Caderni et al., 2000; Bomser et al., 1999). Similarly, a recent study has shown that an anthocyanidin rich extract decreases indices of lipid peroxidation 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 enhance susceptibility to oxidative damage. 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-oxo7, 8-dihydro-2 0 -deoxyguanosine (8-oxodG) in blood and urine as noninvasive biomarkers for later studies in human.

rial Institute) medium was obtained from Gibco (Invitrogen Corporation, 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 acetate), a vitamin E-deficient diet Vit E (n = 12; control diet without atocopherol 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 supplemented 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-surOrge, France) in a granule form. A commercially available, dried, powdered 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. Materials and methods 2.1. Chemicals Nuclease P1, RNase IIIA, RNase T1, Triton X-100, NaCl, deferrioxamine 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-

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.

Table 1 Control and experimental diets composition Ingredient a

Diet mix Mineral mixb Vitamin mixc a-Tocopherol acetate (mg/g) Retinyl acetate (IU/g) GSPE (mg/g)

Control

920 70 10 0.05 10 0

Experimental diets Vit E

Vit E+

Vit A

Vit A+

GSPE1

GSPE2

920 70 10 0 10 0

920 70 10 5 10 0

920 70 10 0.05 0 0

920 70 10 0.05 200 0

920 70 10 0.05 10 0.04

920 70 10 0.05 10 0.4

Values are g/kg diet dry wt, unless otherwise indicated. a UAR 211A: the diet mixture provides the following amounts (g/kg diet1): casein, 230; dextrose, 380; corn starch, 200; cellulose, 60; stearic acid, 30; glycerol, 10; onagrine oil, 10. b UAR 205B: the salt mixture provides the following amounts (g/kg diet1): Ca, 7; K, 0.42; Na, 2.8; Mg, 0.7; Fe, 0.21; P, 5.425; trace elements: Mn, 0.056; Cu, 0.0087; Zn, 0.031; Co, 0.00006; I, 0.00034. c UAR 200: the vitamin mixture provides the following amounts (mg/kg diet1): cholecalciferol, 0.0625; thiamin, 20; riboflavin, 15; panthotenic acid, 68; pyridoxine, 10; inositol, 150; cyanocobalamine, 0.05; menadione, 40; nicotinic acid, 99; paraaminobenzoic acid, 49; folic acid, 5; biotin, 0.3; choline, 1360.

B. Morin et al. / Food and Chemical Toxicology 46 (2008) 787–796 A sensitive HPLC assay was adapted from a recent article (Taibi and Nicotra, 2002) and validated for a-tocopherol, retinol and retinyl palmitate 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 foilcovered 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, the working standards solution were prepared as follows: a-tocopherol, 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 chloroform 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 autosampler vial. Forty microlitres was injected into the HPLC system for 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 reversephase 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 (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 Polymorphprep (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 centrifuged at 700g for 20 min at 20 C and the supernatant was discarded.

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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 leukocyte 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 vigorous agitation and made up to 4.7 ml with buffer. SDS (300 ll) 10% 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 chloroform: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 several times. The tube was left on ice for 10 min to facilitate DNA precipitation. Ethanol was then removed and DNA washed with 10 ml of icecold 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 thermostated 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 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

B. Morin et al. / Food and Chemical Toxicology 46 (2008) 787–796

2.11. Statistical analysis Results are expressed as means ± SD Student’s t-test was used to evaluate differences between the control group and other groups; p < 0.05 was accepted as significant. Tests for data normality and analyses of variance were first carried out.

35

mean food consumption (g/day)

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 purpose, each urine sample was diluted with an equal volume of the mobile phase spiked with (15N5)-8-oxodG (8-oxodG M+5) and (2-amino-7, 915N3, 8-13 C)-8-oxoGuo (8-oxoGuo M+4) prepared as previously described (Stadler et al., 1994; Ravanat et al., 2000). The LOD was determined around 30 fmol injected corresponding to a 3 nM urinary concentration for both the oxidised 2 0 -deoxyribonucleoside and its corresponding ribonucleosides. Creatinine in urine was measured colorimetrically using a creatinine kit (Sigma–Aldrich, St Quentin Fallavier, France).

control VitE-

30

VitA-

25 20 15 10 5 0 1

2

3

5

6

4

5

6

control

35

VitE+ 30

VitA+

25 20 15 10 5 0 1

2

3

weeks

3. Results

The initial body weight of rats from the seven dietary groups was the same (49 ± 5 g). When euthanized there was a significant difference (p < 0.05) in body weight among the vitamins A and E deficiency diet groups and the control (Table 2). As expected, these animals were in a vitamin subdeficiency status. No significant difference 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 proanthocyanidin intakes of rats fed each diet Experimental Body group weight gain (g)

Vitamin E intake (g/kg/day)

Vitamin A Proanthocyanidin intake intake (g/kg/day) (IU/kg/day)

Control Vit E Vit E+ Vit A Vit A+ GSPE1 GSPE2

1.42 ± 0.06 – 136.0 ± 5.5 1.32 ± 0.16 1.32 ± 0.06 1.19 ± 0.18 1.31 ± 0.07

0.28 ± 0.01 0.26 ± 0.01 0.27 ± 0.01 – 5.30 ± 0.24 0.24 ± 0.03 0.26 ± 0.01

316 ± 21 289 ± 24* 327 ± 24 283 ± 25* 311 ± 11 306 ± 18 319 ± 15

p < 0.05 vs control.

– – – – – 1.0 ± 0.1 10.5 ± 0.6

mean food consumption (g/day)

3.1. Growth and antioxidant intake

*

4

weeks

mean food consumption (g/day)

790

35

control

30

GSPE1 GSPE2

25 20 15 10 5 0 1

2

3

4

5

6

weeks

Fig. 1. Mean food consumption of rats fed each diet.

rats was the lowest (45 ± 4 g). Vitamins A, E and proanthocyanidin 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, retinyl 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 lowest concentrations we encountered were well above the limits of detection (0.46 and 0.02 lM, respectively).

B. Morin et al. / Food and Chemical Toxicology 46 (2008) 787–796 Table 3 Relation between antioxidant supplies and plasmatic vitamin status

8

Vitamin Retinyl palmitate (nM)

a-Tocopherol (lM)

2.1 ± 0.1 2.6 ± 0.1** 2.0 ± 0.2 0.12 ± 0.06** 2.2 ± 0.1* 2.2 ± 0.2 2.1 ± 0.1

51 ± 2 43 ± 4* 35 ± 4* nd 668 ± 114** 48 ± 6 18 ± 5**

12.4 ± 1.4 0.7 ± 0.1** 42.2 ± 4.4* 17.9 ± 1.2** 12.5 ± 0.6 11.5 ± 1.8 9.8 ± 0.8*

6

6

Retinol (lM)

8-oxodG /10 dG

Diet

791

Control Vit E Vit E+ Vit A Vit A+ GSPE1 GSPE2

nd: not detectable. * p < 0.05 vs control. ** p < 0.01 vs control.

Retinyl palmitate plasmatic concentration was, in most cases, determined to be higher than the LOD of our method except when a vitamin A-deficient diet was used. Vitamin A deficiency was confirmed by determining the content of retinol in plasma. Vitamin A restriction produced subclinical plasma retinol concentrations (0.12 lmol/l) and negligible retinyl palmitate plasmatic concentrations compared to the control group (Table 3). A higher proportion of plasmatic a-tocopherol was present in vitamin A-deficient rats compared to vitamin A-adequate and supplemented rats. With the vitamin A-supplemented diet, the plasma vitamin A level only showed a minor, although significant, increase at the end of the study in comparison to rats fed a standard diet. In this case, most of the plasma vitamin A was in the form of retinyl ester. The dietary content of vitamin E is correlated with the level of plasmatic a-tocopherol (Table 3). The vitamin Esupplemented diet did not modify the plasmatic level of retinol but decreased the retinyl palmitate level (31% vs control). Dietary deficiency of vitamin E induced a limited plasmatic increase of retinol (+22% vs control). These results are in contrast with those of Westendorf et al., 1990 in beef steers and in chicken (Frigg and Broz, 1984). For rats receiving 0.04 mg/g of grape seed proanthocyanidin extracts (GSPE1), the plasmatic level of fat-soluble vitamins A and E was not significantly different from control (Table 3). The higher administration of GSPE (0.4 mg/ g diet) induced a decrease of plasmatic a-tocopherol and retinyl palmitate.

3.3. HPLC/EC analysis of 8-oxodG in leukocytes A 8-oxodG mean background level in rat leukocytes of 3.4 lesions/106 dG was measured for the control group. The protective abilities of GSPE, vitamins E and A against oxidative DNA damage in rat leukocytes were assessed. Pre-treatment of rats with GSPE (0.04 and 0.4 mg/g diet), vitamin E (5 mg/g diet) or vitamin A (200 IU/g diet) decreased 8-oxodG level in leukocytes by, respectively, 27%, 20%, 65% and 63% compared to the control group (Fig. 2). No dose dependent protection ability

4

*

*

*

**

2

0

Control Vit E-

Vit E+

Vit A-

Vit A+ GSPE1 GSPE2

* P<0.01, significant difference from control group ** P<0.05, significant difference from control group

Fig. 2. Effect of vitamin E, vitamin A and GSPE on 8-oxodG formation in leukocytes of rats.

against DNA oxidative damage was demonstrated by GSPE. We showed in the vitamin A- and E-deficient rats, lower circulating levels of vitamins A and E and high levels of 8oxodG in leukocytes DNA with respect to the control animals. 3.4. HPLC/MS/MS analysis of 8-oxodG and 8-oxoGuo excretion in urine At the end of diets administration 24 h urines were collected for the determination of 8-oxodG and 8-oxoGuo by HLPC/MS/MS. The concentrations of the oxidised guanine species in control rat urine were: 8-oxoGuo, 78 ± 50; 8-oxodG, 10 ± 4 nmol/l (n = 12). These findings are similar to those reported previously. Some of the recent published levels of urinary 8-oxodG excretion are presented in Table 4. For instance, Fraga et al., 1990 using HPLC–EC with immunoaffinity pre-purification column reported that the 8-oxodG urinary output for the 2-months old Fisher rats was 481 ± 163 pmol/d/kg BW. To the best of our knowledge no experimental data are available to date on 8-oxodG excretion, measured by HPLC/MS/MS, in rat. In the groups fed the control, vitamin E- and A-deficient diets, the excretion of 8-oxodG was, respectively, 512 ± 185, 510 ± 452, 656 ± 262 pmol/24 h/kg BW. In the groups fed vitamins E, vitamin A, GSPE1 and GSPE2 supplemented diets, the excretion of 8-oxodG was, respectively, 402 ± 137, 493 ± 103, 1223 ± 547, 462 ± 222 pmol/ 24 h/kg BW. A large inter-individual variability of urinary excretion of 8-oxodG (relative SD ranging from 20% to 45% except for the group with vitamin E-deficient diet which shows a higher variation 86%) and 8-oxoGuo (relative SD ranging from 33% to 62%) was observed for most dietary groups as previously described in humans (Renner et al., 2000) and also in rats (Loft et al., 1998; Fraga et al., 1990). Oxidative DNA damage as measured by urinary

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B. Morin et al. / Food and Chemical Toxicology 46 (2008) 787–796

Table 4 Published levels of urinary 8-oxodG in rats Author

Sex/strain

Age

Method

Fraga et al. (1990)

Male Fischer

Park et al. (1992)

Male Fischer

2 Months 24 Months 3–6 Months

Topp et al. (2000) Lengger et al. (2000) Loft et al. (1998)

Male Wistar

Ns

Immunoaffinity column/HPLC-ED Immunoaffinity column/HPLC-ED HPLC-ED

Male Fischer Female Fischer Male Wistar Male Fischer Male Fischer

Ns Ns 2 Months 3 Months 2 Months

Sakamoto et al. (2003) Toraason et al. (1999) This study

HPLC-ED ELISA kit HPLC-ED HPLC/MS/MS

Mean ± SDa 481 ± 163 165 ± 66 370 ± 63 (30) 414 ± 227 (27) 333 ± 47 (7) 403 ± 150 (6–10) 752 ± 80 (6–10) 384 ± 91 pmol/mg creatinine (16) 383 (6) 512 ± 185 (12)

Ns: not specified. a Results are expressed as pmol/day/kg body weight unless otherwise indicated. Into brackets are the number of replicates when available.

Table 5 Effect of vitamin E, vitamin A and GSPE on urinary 8-oxodG and 8oxoGuo levels determined by HPLC/MS/MS Diet

8-OxodG pmol/24 h

pmol/mg creatinine

pmol/d/kg BW

Control Vit E Vit E+ Vit A Vit A+ GSPE1 GSPE2

189 ± 70 178 ± 176 143 ± 51 212 ± 80 164 ± 32 397 ± 172 159 ± 72

18.8 ± 7.8 15.9 ± 13.8 12.2 ± 3.6 22.6 ± 9.1 16.1 ± 3.5 37.3 ± 15.3 14.4 ± 5.8

512 ± 186 510 ± 453 403 ± 138 656 ± 263 493 ± 104 1223 ± 548 462 ± 223

Diet

8-OxoGuo nmol/24 h

pmol/mg creatinine

nmol/d/kg BW

1.6 ± 0.9 1.1 ± 0.6 1.3 ± 0.7 0.9 ± 0.5 0.7 ± 0.2 1.1 ± 0.6 1.4 ± 0.7

146 ± 87 106 ± 66 108 ± 54 91 ± 57 73 ± 24 108 ± 66 129 ± 62

Control Vit E Vit E+ Vit A Vit A+ GSPE1 GSPE2

4.2 ± 2.5 3.3 ± 1.9 3.6 ± 1.9 2.7 ± 1.6 2.2 ± 0.7 3.4 ± 1.8 4.1 ± 2.2

excretion of 8-oxodG was not significantly influenced by plasma concentration of antioxidants (a-tocopherol, retinol) and by a GSPE supplemented diet regardless of the method of expressing excretion: 24 h excretion, 24 h excretion body weight corrected or per mg creatinine (Table 5). The mean level of 8-oxoGuo in the urine samples of the control group was 4.2 ± 2.5 nmol/24 h/kg BW. It is interesting to note that the highest urinary level of 8-oxoGuo was found for the animals fed the control diet. Nevertheless, no significant changes have been found between the different dietary treatments. 4. Discussion Fruit and vegetable intake is associated to a reduced risk of cancer and cardiovascular disease. While these protective effects have been primarily attributed to antioxidants such as vitamins C, E, A and b-carotene or mineral micronutrients, flavonoid may also play a role. We planned this study

to verify whether other putative dietary nutrients, such as a commercially available grape seed proanthocyanidin extract can protect against oxidative damage. VITISOL grape seed extract contains oligomeric and polymeric proanthocyanidins (>95%). It is important to note that the actual partitioning of the flavonoids in cellular compartments is poorly understood. Concerning the occurrence of oligomers higher than dimers, their presence in human/rat plasma has not been reported. The apparent lack of trimers and larger oligomers could be attributed, at least in part to a significant gastric digestion of the oligomers combined with limited gut absorption of these molecules. In addition, the bioavailability of these compounds is rather poor. They are rapidly conjugated by phase II detoxication reactions and levels of free flavonoids in human plasma are very low (Williamson et al., 2005). The exact mechanisms of flavonoid absorption and metabolism remain uncertain and appear to depend on the type of flavonoid. Nonetheless, consumption of these compounds has been shown to have beneficial health effect and can contribute to in vivo antioxidant defences (Manach and Donovan, 2004). The protective effect of proanthocyandin could be due to the chelation of redox active metals, as well as to the trapping of some reactive oxygen species. There are two different approaches to evaluate the in vivo protective effect on oxidative DNA damage. First, steady-state damage can be measured when DNA is isolated from cells and tissues and analysed for base damage products. Such level presumably reflects the balance between damage and DNA repair. Hence a rise in steadystate oxidative DNA damage could be due to increased damage and/or decreased repair. Second, 8-oxodG is eliminated from DNA in urine by a repair via base excision and nucleotide excision repair mechanisms, leading to the release of the nucleobase 8-oxoGua and the deoxynucleoside 8-oxodG, respectively. It has been shown that 8oxodG is not metabolised but virtually quantitatively excreted in urine and that 8-oxodG in the diet does not contribute to urinary 8-oxodG (Helbock et al., 1998; Park et al., 1992; Cooke et al., 2005). The amount of the

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modified base/nucleoside should represent the average rate of DNA damage in the whole body and may be an indicator of oxidative stress at the level of the organism. These facts have led to the use and general interpretation of urinary 8-oxodG as a noninvasive marker of oxidative DNA damage by ROS in nutritional studies (Moller et al., 2003; Halliwell, 1998; Verhagen et al., 1995; van Poppel et al., 1995). However, it is possible that some or all of the 8oxodG excreted in urine may not arise from DNA but from either cell death or dGTP in the DNA precursor pool of nucleotides. An enzyme has been described which hydrolyses deoxyGTP containing oxidised guanine to prevent its incorporation into DNA (Mo et al., 1992). The previous method, for analysing 8-oxodG in urine by HPLC–EC (Loft et al., 1992; Bogdanov et al., 1999), required a complicated set-up and multiple runs per sample and was limited to 8-oxodG analysis. The HPLC/MS/MS method developed previously for urine sample (Ravanat et al., 1998; Weimann et al., 2002) is fast, reliable and allows the measurement of two and potentially more oxidative DNA products. The main drawback of the method is that the equipment is expensive. So, in this study, we decided to measure the level of oxidised nucleoside both in DNA isolated from rat leukocytes by HPLC/EC and in urine using HPLC/MS/MS analysis. It is worth noting that HPLC with amperometric detection, which is in theory less sensitive than coulometric detection, is able to detect very low level of damage (ESCODD, 2002b). The sensitivity of our HPLC–EC method allowed the measurement of about 0.6 lesion/106 dG. The values obtained in this study were on average from 1.9 to 5.6 8-oxodG/106 dG. The method is then suitable for measurement of 8oxodG in DNA leukocytes of rats. The described HPLCMS/MS method is a sensitive tool for the determination of the urinary excretion of 8-oxodG even at the low level of non-exposed animals. A 3 nM 8-oxodG and 8-oxoGuo limit of detection is sensitive enough because 8-oxodG and 8-oxoGuo concentrations in urine from rats in our study were on average from 8 to 19 nM for 8-oxodG and from 35 to 91 nM for 8-oxoGuo. In the field of 8-oxodG analysis, a technical difficulty is to avoid artefactual oxidation of nucleosides during DNA isolation from cells. There is indeed convincing evidence that substantial oxidation of guanine occurs during preparation of samples for HPLC analysis. The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up in 1997 in order to identify the sources of artefacts, to develop standards and reliable techniques and to reach a consensus on the true background level of damage in normal cell. The aim of this European laboratory network was to compare and validate different methods of measuring 8-oxodG in pure DNA as well as in DNA isolated from cells or tissues (ESCODD, 2002a, 2002b, 2003; ESCODD et al., 2005; Riis Jensen et al., 2002). The laboratory was a member of ESCODD and so special care was taken to reduce artefactual production of 8oxodG during DNA extraction. The extraction protocol

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used in this study was previously designed to minimise this effect and was successfully used during the ESCODD program. This protocol allowed us to measure 8-oxodG background level in rat leukocytes of 3.4 lesions/106 dG (Fig. 1). It is interesting to note that this value is in the range recommended by ESCODD and is very close to the median reported by the latest ESCODD trial (ESCODD et al., 2005) of 4.2 8-oxodG/106 dG in lymphocytes from healthy young men. We are not aware of other published values on 8-oxodG level in rat leukocytes. In rat lymphocytes about 12 8oxodG/106 dG were measured using HPLC/EC (Toraason et al., 1999). In agreement with previous studies, we showed an increase in the level of 8-oxodG in the DNA samples isolated from leukocytes of the vitamin E- and A-deficient diet when compared to the control group. Antioxidant vitamins can protect biomolecules against free radicals attack. Endogenous concentration of retinol, a-tocopherol and retinyl palmitate were determined in plasma. In the present study, plasma levels of vitamin A were responsive to alterations in plasma antioxidant concentrations. With a vitamin A-deficient diet, there was a corresponding decrease in plasma retinol and retinyl palmitate. However, despite no change in vitamin E content between the diets, plasma concentrations of vitamin E were higher for the Vit A diet. The results were consistent with previous work showing that vitamin A deficiency could increase plasma vitamin E levels (Crissey et al., 1998). The interrelationship between dietary vitamins A and E status has been documented. High dietary levels of vitamin A have been shown to depress vitamin E status in rats, chickens, and dairy cattle (Blakely et al., 1991; Eicher et al., 1997; Frigg and Broz, 1984). The mechanism(s) responsible for this antagonistic effect remains unexplained. There is evidence indicating that vitamin A may interfere with vitamin E absorption. The diets supplemented with vitamin A or vitamin E were compared to diets enriched with a grape seed proanthocyanidin extract. Our results clearly show that vitamin supplementation exert a protective effect against DNA damage. When compared to other antioxidants, the dietary proanthocyanidins isolated from grape seeds exert a DNA protection in the same order than dietary vitamins E and A at the doses that were used in our study. It should however be pointed out that the level of vitamin E (5000 mg/kg diet) in enriched diets was higher than those of proanthocyanidins (40 and 400 mg/kg diet). Bagchi et al. (1998) have reported that grape seed proanthocyanidin extract provides significantly greater protection against free radicals and free radical-induced lipid peroxidation and DNA damage than vitamins C, E and b-carotene using similar vitamin and GSPE doses. Although proanthocyanidins are effective antioxidant in vitro, there are only a limited number of studies in humans and also in rats that have addressed the efficacy of these compounds as antioxidants in vivo as previously discussed in the introduction section. The original contribution of this study was to assess the antioxidant

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abilities of vitamins and proanthocyanidin in vivo without any induction by a radical generator or other external factors producing oxidative stress on cells. Simonetti et al. have shown that procyanidins from Vitis vinifera seeds provide a reduction of 8-oxodG level in human lymphocytes. In the present study, no significant correlation was found between the level of 8-oxodG in leukocytes DNA and the urinary excretion of the modified nucleoside (data not shown, r = 0.07). This was consistent with the results recently published in human (Folinski et al., 2003). The urinary excretion of 8-oxodG showed wide inter-individual variation, so the power to detect a possible effect of the diet was reduced. The fact that urinary 8-oxodG level did not change when levels in cellular DNA altered, may imply that most of the 8-oxodG in urine did not arise from cellular DNA. It is therefore possible that some or all of the 8oxodG excreted in urine may arise from either cell death or dGTP in the DNA precursor pool of nucleotides. A recent report on damaged DNA lesions in urine proposed arguments which suggest the involvement of cell death to be minimal (Cooke et al., 2005). The concentrations of the oxidised guanine species in rat urine fed with control diet were: 8-oxodG, 512 ± 185 pmol/d/kg; 8-oxoGuo, 4.2 ± 2.5 nmol/d/kg (n = 12). The ratio of the oxidised nucleoside/oxidised deoxynucleoside is 1/8. This is in agreement with the work of Ames et al. who found a concentration ratio of 1:8 of 8oxodG:8-oxoGuo in rat urine quantified by an immunoaffinity pre-purification, HPLC-electrochemical method (Park et al., 1992). The levels of 8-oxodG in urine have mainly been measured by HPLC–EC. To our knowledge the result on the relative level of the modified guanine species measured by HPLC/MS/MS was only reported by Poulsen’s laboratory in human urine (8-oxoGua: 136 nmol/24 h; 8-oxoGuo: 48 nmol/24 h; 8-oxodG: 28 nmol/24 h (Weimann et al., 2001, 2002, 2004). Recent results have highlighted the higher sensitivity to oxidation of RNA relatively to DNA (Hofer et al., 2005) and therefore levels of 8-oxoGuo, that could be easily determined in urine samples by HPLC–MS/MS measurement (Weimann et al., 2002), might be a better biomarker of oxidative stress than its corresponding 2 0 -deoxyribonucleoside. However, an important inter-individual variability in the measured levels of both nucleosides is observed and this probably prevents determination of significant variation between the different dietary treatments. Such results strongly suggest that the measurement of urinary levels of oxidised nucleosides as potential biomarkers of oxidative stress is questionable. The determination of the amount of the oxidised base (8-oxoGua) might be more appropriated. However, in our hands, the HPLC–MS/ MS determination of 8-oxoGua in urine samples was poorly reproducible, most probably due to the poor solubility of the free base. Therefore, the free base 8-oxoGua, that is expected to be the major repair end-product of 8-oxodG in DNA, was not monitored in the present study.

5. Conclusions This study suggests that the 8-oxodG level in leukocytes is a more appropriate biomarker in nutritional studies than urinay 8-oxodG. Indeed, a strong correlation was obtained between 8-oxodG level in rat leukocytes and supplemented diet with vitamins A and E as well as with a grape seed proanthocyanidins extract. GSPE demonstrated significant protective ability against oxidative damage in leukocytes DNA of healthy rat subjects. Acknowledgments This work was supported by grants from the French Ministry of Research and Agriculture (AQS No. 99P0347) and the Aquitaine region (P3AN No. 20010442). Many thanks to Berkem society (Gardonne, France) which provided the GSPE extract. References Bagchi, D., Garg, A., Krohn, R.L., Bagchi, M., Bagchi, D.J., Stohs, S., 1998. Protective effects of grape seed proanthocyanidins and selected antioxidants against TPA-induced hepatic and brain lipid peroxidation and DNA fragmentation, and peritoneal macrophage activation in mice. Gen. Pharm. 30, 771–776. Blakely, S.R., Mitchell, G.V., Jenkins, M.Y., Grundel, E., Whittaker, P., 1991. Canthaxanthin and excess vitamin A alter alpha-tocopherol, carotenoid and iron status in adult rats. J. Nutr. 10, 1649–1655. Bogdanov, M.B., Bcal, M.F., Douglas, R.M., Griffin, R.M., Matson, W.R., 1999. A carbon column based LCEC approach to routine 8-hydroxy-2 0 -deoxyguanosine measurements in urine and other biological matrices. Free Rad. Biol. Med. 29, 601–608. Bomser, J.A., Singletary, K.W., Wallig, M.A., Smith, M.A., 1999. Inhibition of TPA-induced tumor promotion in CD-1 mouse epidermis by a polyphenolic fraction from grape seeds. Cancer Lett., 151–157. Caderni, G., De Filippo, C., Luceri, C., Salvadori, M., Giannini, A., Biggeri, A., 2000. Effects of black tea, green tea and wine extracts on intestinal carcinogenesis induced by azoxymethane in F344 rats. Carcinogenesis 21, 1965–1969. Casalini, C., Lodovici, M., Briani, C., Paganelli, G., Remy, S., Cheynier, V., Dolora, P., 1999. Effect of complex polyphenols and tannins from red wine (WCPT) on chemically induced oxidative DNA damage in the rat. Eur. J. Nutr. 3, 190–195. Cooke, M.S., Evans, M.D., Dove, R., Rozalski, R., Gackowski, D., Siomek, A., Lunec, J., Olinski, R., 2005. DNA repair is responsible for the presence of oxidatively damaged DNA lesions in urine. Mutat. Res. 574, 58–66. Crissey, S.D., McGill, P., Simeone, A.M., 1998. Influence of dietary vitamins A and E on serum a- and c-tocopherols, retinol, retinyl palmitate and carotenoid concentrations in Humboldt penguins (Spheniscus humboldti). Comp. Biochem. Biophys. 121, 333–339. Da Silva, J.M., Darmon, N., Fernandez, Y., Mitjavila, S., 1991. Oxygen free radical scavenger capacity in aqueous models of different procyanidins from grape seeds. J. Agric. Food Chem. 39, 1549–1552. Danelisen, I., Palace, V., Lou, H., Singal, P.K., 2002. Maintenance of myocardial levels of vitamin A in heart failure due to adriamycin. J. Mol. Cell. Cardiol. 34, 789–795. Eicher, S.D., Morrill, J.L., Velazco, J., 1997. Bioavailability of a-tocopherol fed with retinol and relative bioavailability of D-atocopherol or DL-a-tocopherol acetate. J. Dairy Sci. 80, 393–399. ESCODD (European Standards Committee on Oxidative DNA Damage), 2002a. Inter-laboratory validation of procedures for measuring 8-oxo-7,

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