Mutation Research/Fundamental and MolecularMechanisms of Mutagenesis
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rape seed polyphenols and curcumin reduce genomic instability events in aransgenic mouse model for Alzheimer’s disease
hilip Thomasa,1, Yan-Jiang Wangb,1, Jin-Hua Zhongb, Shantha Kosarajuc, Nathan J. O’Callaghana,in-Fu Zhoub,∗, Michael Fenecha,∗
CSIRO Human Nutrition, PO Box 10041, Adelaide BC, Adelaide, SA 5000, AustraliaDepartment of Human Physiology and Centre for Neuroscience, Flinders University, GPO Box 2100, Adelaide, SA, AustraliaCSIRO Food Science Australia, 671 Snydes Rd., Private Bag 16, Werribee, Victoria 3030, Australia
r t i c l e i n f o
rticle history:eceived 2 June 2008eceived in revised form 22 October 2008ccepted 24 October 2008vailable online 6 November 2008
The study set out to determine (a) whether DNA damage is elevated in mice that carry mutations in theamyloid precursor protein (APP695swe) and presenilin 1 (PSEN1-dE9) that predispose to Alzheimer’sdisease (AD) relative to non-transgenic control mice, and (b) whether increasing the intake of dietarypolyphenols from curcumin or grape seed extract could reduce genomic instability events in a transgenicmouse model for AD. DNA damage was measured using the micronucleus (MN) assay in both buccalmucosa and erythrocytes and an absolute telomere length assay for both buccal mucosa and olfactory bulbtissue. MN frequency tended to be higher in AD mice in both buccal mucosa (1.7-fold) and polychromaticerythrocytes (1.3-fold) relative to controls. Telomere length was significantly reduced by 91% (p = 0.04) andnon-significantly reduced by 50% in buccal mucosa and olfactory bulbs respectively in AD mice relativeto controls. A significant 10-fold decrease in buccal MN frequency (p = 0.01) was found for AD mice feddiets containing curcumin (CUR) or micro-encapsulated grape seed extract (MGSE) and a 7-fold decrease(p = 0.02) for AD mice fed unencapsulated grape seed extract (GSE) compared to the AD group on control
diet. Similarly, in polychromatic erythrocytes a significant reduction in MN frequency was found for theMGSE cohort (65.3%) (p < 0.05), whereas the AD CUR and AD GSE groups were non-significantly reducedby 39.2 and 34.8% respectively compared to the AD Control. A non-significant 2-fold increase in buccalcell telomere length was evident for the CUR, GSE and MGSE groups compared to the AD control group.Olfactory bulb telomere length was found to be non-significantly 2-fold longer in mice fed on the CUR dietcompared to controls. These results suggest potential protective effects of polyphenols against genomic
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instability events in differ
. Introduction
Alzheimer’s disease (AD) is associated with elevated rates ofxidative stress that contribute to increased rates of genomic insta-ility events such as an increased frequency in micronucleus (MN)ormation, chromosomal aberrations, and alterations in telomereength and levels of apoptosis [1–3]. The characteristic hallmarks of
D involve the abnormal deposition of proteins leading to amyloidlaques and neurofibrillary tangles in the brain and a progressive
oss of cognitive function [4,5]. These pathological features havelso been linked with oxidative stress [6,7]. It has been suggested
hat both plaques and tangles are produced in order to protectensitive areas of the brain from the effects of oxidative relatednjury [6]. However, other studies have shown that plaques mayroduce reactive oxygen species (ROS) when they form complexesith redox active metals such as copper and iron [8,9]. Free radical
eneration results from normal metabolic processes, in which theevels are maintained within normal homeostatic boundaries byn elaborate endogenous antioxidant system. However, when lev-ls of free radicals exceeds the normal clearing capacity of the cellsxidative stress follows resulting in potential cellular and genomeamage [10]. Damage to the genome could lead to altered geneosage and gene expression as well as contribute to the risk of
ccelerated cell death in neuronal tissues. DNA damage events suchs micronuclei formation, which are biomarkers of chromosomealsegregation and fragility have been found to be elevated in
oth lymphocytes and buccal cells from individuals suffering fromD [3,11]. It has recently been shown that individuals with mild
ognitive impairment (MCI) as well as more advanced AD have a-fold increase in DNA damage levels and oxidized bases in their
eucocytes compared with age matched controls not clinically diag-osed with AD [12]. This is suggestive that oxidative stress occurs
n the early stages of the disease, as MCI individuals progress to ADith an estimated probability of 50% within 4 years [12,13], sug-
esting MCI may be representative of early AD and that oxidativetress may directly contribute to disease pathology. Oxidative stressas also been shown to be related to telomere shortening, whichas also been identified in both lymphocytes and buccal cells ofatients clinically diagnosed with AD [14,15].
Recent studies have suggested the positive effects of dietaryntioxidants as an aid in potentially reducing somatic cell and neu-onal damage by free radicals [16,17]. Curcumin (CUR) is the yellowhenolic compound in the Indian curry spice turmeric. This spice
s used as a food preservative in India where the incidence of AD inndividuals aged between 70 and 79 years of age is 4.4-fold lesshan that in the United States [18]. CUR has been shown to acts an antioxidant through modulation of glutathione (GSH) levels19] and possesses anti-inflammatory properties possibly mediatedy inhibition of interleukin-8 release [19] as well as the ability toeduce Amyloid-beta (A�) 42 toxicity by preventing the formationf �42 oligomers leading to amyloid plaque formation [16,20,21].
Grape seed extract (GSE) contains a number of polyphenolsncluding proanthocyanidins and procyanidins [22,23]. They haveeen shown to be powerful free radical scavengers, possess anti-
nflammatory properties, reduce apoptosis and prevent hydrogeneroxide (H2O2) induced chromosomal damage in human lym-hoblastoid cells [24–26]. It has been shown that their free radicalcavenging capacity is 20 times more effective than vitamin End 50 times more effective than vitamin C [27]. It has alsoeen reported in rat models that cells are protected against A�oxicity when treated with GSE compared to unprotected neu-ons, possibly as a result of its antioxidant or chelating properties28,29].
The aims of this study were (a) to investigate whether DNA dam-ge events are increased with time to a greater extent in AD miceelative to controls and (b) to determine the potential protectiveffects of natural products rich in polyphenols on genomic insta-ility events in a transgenic mouse model for AD. We investigatedhe effects of CUR and GSE polyphenols on DNA damage by testinghe mice over a 9 month dietary treatment period utilizing bothbuccal micronucleus cytome assay, an erythrocyte micronucleusssay and by determining telomere length in both buccal cells andrain olfactory bulb (OB) tissue.
. Materials and methods
.1. Mouse model
The study involved the use of a double transgenic mouse model (B6C3-g(APPswePSEN1dE9)85Dbo/J (stock No. 004462) Jackson Laboratory, Maine, USA)hat expresses a chimeric mouse/human amyloid precursor protein (Mo/HuPP695swe) and a mutant presenilin 1 (PSEN1-dE9) that are both directed to cen-
ral nervous system (CNS) neurons. These mutations are both associated with early
i
2
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able 1ge at start of feeding intervention and gender ratio of mice in study groups.
WT CON, N = 20 AD CON, N = 10
GE (days) (mean ± SD) 119.9 ± 21.04 116.1 ± 25.15ender ratio (male:female) 10:10 6:4
here were no significant differences in gender ratio or age between cohorts.T CON, wild type control.
D CON, Alzheimer’s mouse on control diet.D CUR, Alzheimer’s mouse on curcumin diet.D GSE, Alzheimer’s mouse on grape seed extracts diet.D MSE, Alzheimer’s mouse on micro-encapsulated grape seed extracts diet.
earch 661 (2009) 25–34
nset AD. The “humanized” Mo/HuAPP695swe transgene allows the mice to secretehuman A� peptide. The included “swedish” mutations (K595N/M596L) elevate
he amount of A� peptide produced from the transgene by favoring processinghrough the beta-secretase pathway. The Mo/HuAPP695swe protein and the trans-enic mutant human presenilin protein (PSEN1-dE9) are both immunodetectedn whole brain homogenates [30,31]. Further information about strain develop-
ent and gene expression can be obtained from the Jackson laboratory websitettp://jaxmice.jax.org/strain/004462.html. All mice used in this experiment weref a mixed F2 background resulting from a cross between heterozygous transgenicnimals and C57BL/6J wild type mice (stock No. 005864).
These transgenic mice develop amyloid plaques in the hippocampus and cortext 4 months and in the thalamus at 6 months. At 9 months there is significant amyloidresent in the cortex, hippocampus and thalamus [32,33].
.2. Genotyping
All animals prior to receipt were genotyped at the Jackson laboratory forg(PSEN1) and Tg(APPswe) and all subsequent animals that were bred in houseere genotyped prior to the start of the study using standard protocols outlined by
he Jackson laboratory [34].
.3. Study design
Approval for this 9-month study was obtained from CSIRO Human Nutritionnd Flinders University Animal Ethics committees. The experimental design wasparallel dietary intervention involving four different dietary treatments using the6C3-Tg(APPswePSEN1dE9)85Dbo/J mouse, with dietary intervention starting at ange of 3–4 months (Table 1). The design of the study was intended to determinehether grape seed extract is as effective as curcumin in reducing genomic instabil-
ty pathology observed in AD, and whether micro-encapsulation of GSE, which mayodify absorption kinetics, enhances the observed effects. Normal mice (wild type
ittermates) with the same genetic background, but lacking defective APP and PSEN1enes were included as an added control to identify the point in time when buccalnd blood differences between normal and Alzheimer’s mice become evident, ando test the hypothesis that the Alzheimer’s prone mice exhibited elevated rates ofNA damage. The study design is outlined in Fig. 1.
The 3-month-old mice were randomly grouped with good balance in age, sexnd breeding pairs (Table 1). All animals were housed in the Flinders Medical Centrenimal holding facility, 2–3 per cage in standard cages ∼600 cm2 (150–200 cm2 pernimal), males and females were housed separately and were genotyped prior tohe commencement of the study following breeding from the originally acquiredransgenic model. All groups were kept in the same room on a 14/10 h light dark cyclend had free access to food and water ad libitum. Food consumption and bodyweightata was collected by the staff at the animal facility after 3 and 9 months dietaryreatment.
.4. HPLC analysis of polyphenols in GSE
Grape seed extract (Vinlife N05010) was purchased from Tarac Technologies P.L.nd was characterized by high performance liquid chromatography (HPLC) withoutny further extraction. Lyophilized extract obtained from grape seeds was dissolvedn 80% methanol acidified with 0.1% HCl to obtain final concentration of 2.0 mg/ml.0 �l was injected into the HPLC column for the analysis of polyphenolic compounds.PLC analysis was carried out according to methods described previously [35]. Ana-
ytical HPLC was run at 25 ◦C and monitored at 280 nm (hydroxybenzoic acids andavanols), 320 nm (hydroxycinnamic acids, stilbenes) and 370 nm (flavonols). Theobile phase consisted of 2% acetic acid in water (solvent A) and 1.0% acetic acid
n water and acetonitrile (50:50, v/v, solvent B). The flow rate was 1 ml min−1 . Theollowing gradient program was used: from 10 to 24% solvent B (20 min), from 24o 30% B (20 min), from 30 to 55% B (20 min), from 55 to 100% B (15 min), 100% B
socratic (8 min), from 100 to 10% B (2 min). The total run time was 85 min.
.5. Diets
All diets were prepared by Specialty Feeds, Glen Forrest, Western Australia. Theontrol diet consisted of 95% standard AIN-93G rodent diet comprising of 39.7% corn
P. Thomas et al. / Mutation Research 661 (2009) 25–34 27
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ig. 1. Study design showing mouse groups, dietary groups, schedule and samplingIN-93G diet (i.e. AD CON). AD CON was the control group for determining the effecnd AD MGSE groups respectively).
tarch, 20% casein (vitamin free), 13.2% dextrin, 10% sucrose, 7% soybean oil, 5% pow-ered cellulose, 3.5% AIN-93G mineral mix, 1% AIN-93G vitamin mix, 0.3% l-cysteine,.25% Choline bitartrate, 0.001% t-Butylhydroquinone and 5% maize starch [36]. Dietconsisted of 95% AIN-93G, 4.93% maize starch and 0.07% Curcumin (Sigma, Cato.: C1386, Australia). Diet 3 consisted of 95% AIN-93G, 3% maize starch and 2% freerape seed extract. Diet 4 consisted of 95% AIN-93G and 5% encapsulated grape seedxtract. Grape seed extract and curcumin at the nominated doses have been usedn previous studies in mice and rats without any indication of causing toxic and/orrritant effects [16,37]. Enough diet was ordered for the duration of the study andoused at the Flinders Medical Centre animal holding facility.
.6. Buccal cell collection
Buccal cells were collected from all animals after 3 and 9 months of dietaryreatment. Prior to sample collection the mouse was anaesthetized by isofluraneVeterinary companies of Australia Pty Ltd., NSW, Australia). The buccal cells wereollected by swabbing the inner cheeks with a thin cotton-tipped stick with 2 mmiameter buds (aluminium shaft 0.9 mm × 150 mm, Applimed SA, Chatel St. Denis,witzerland). Both cheeks were sampled and the head of the applicator placed inside1.5 ml eppendorf containing 1 ml of buccal cell buffer 0.01 M Tris–HCL (Sigma T-253), 0.1 M EDTA tetra sodium salt (Sigma E5391), 0.02 M sodium chloride (Sigma5886) and revolved to dislodge the cells. Cells were spun in a microfuge (Centrifuge415R, eppendorf) at 1800 rpm for 10 min. The supernatant was removed leavingpproximately 150 �l and replaced with 1 ml of buccal cell buffer. The cells werepun and washed twice more to remove DNAses and bacteria which may hamperhe quality of slide preparation and scoring. Cells were not homogenized or filteredo as not to lose cells unnecessarily. Buffer was removed leaving ∼120 �l. 6 �l ofMSO (5%) was added to the cell suspension to reduce cell clumping, agitated anddded to cytospin cups and spun at 600 rpm for 5 min in a cytocentrifuge (Shandonytospin 3). Slides were then air dried for 10 min and fixed in ethanol:acetic acid3:1) for 10 min prior to staining. The nuclei were counterstained with propidiumodide (Sigma P-4170) (0.05 �g/ml in 4× SSC) (Standard saline citrate, 0.15 M sodiumhloride, Sigma S9888/0.015 M sodium citrate, Sigma S4641) with 0.06% Tween 20Sigma P1379) for 3 min at room temperature in the dark and cover-slipped in anti-ade solution and kept at −20 ◦C until analysis could be performed. Slides werecored using a Nikon E600 microscope equipped with a triple band filter (Dapi, FITCnd rhodamine) at 1000× magnification.
.7. Scoring criteria for murine buccal micronucleus assay
The murine buccal micronucleus cytome assay we developed classifies buc-al cells into categories that distinguish between “normal” cells and cells that areonsidered “abnormal”, based on nuclear morphology and is based on the same prin-
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sf
s. WT CON group was the control for comparison of WT and AD mice on the sameurcumin, GSE and MGSE as additives in AIN-93G diet in AD mice (AD CUR, AD GSE
iples developed for the human buccal cytome assay as described previously [11,38].ome of these abnormal nuclear morphologies are thought to be indicative of DNAamage, e.g. micronucleus formation. Images showing distinct cell populations ascored in the assay are shown in Fig. 2. The different cell types scored include.
.8. Normal basal cells (Fig. 2a)
These are the cells from the basal layer. The nucleus is usually oval or roundn shape and uniformly stained. The nuclear to cytoplasm ratio is larger than thatn typical normal differentiated buccal cells. Basal cells are smaller in size whenompared to differentiated buccal cells. No other DNA containing structures apartrom the nucleus are observed in these cells.
.9. Normal differentiated cells (Fig. 2b)
These cells have a uniformly stained nucleus which is usually oval or round inhape and a nuclear to cytoplasmic ratio smaller than that of basal cells. No otherNA containing structures apart from the nucleus are observed in these cells.
.10. Basal and differentiated cells with micronuclei (Fig. 2c and d)
These cells are characterized by the presence of both a main nucleus and one orore smaller nuclei called micronuclei. The micronuclei are usually round or oval in
hape and their diameter may range between 1/3 and 1/16 the diameter of the mainucleus. Cells with micronuclei usually contain only one micronucleus. It is possibleut rare to find cells with more than 2 micronuclei. The nuclei in micronucleatedells usually have the morphology of nuclei in normal cells although occasionallyhe nuclei may appear to have diminished DNA content as observed in karyolyticells. The micronuclei must be located within the cytoplasm of the cells. The pres-nce of micronuclei is indicative of chromosome loss or fragmentation occurringuring previous nuclear division. Micronuclei were scored only in basal and normalifferentiated cells.
.11. Karyolytic cells (Fig. 2e–g)
Early karyolysis (Fig. 2e): these cells show the first signs of diminished DNA con-
ent but still have an apparent nuclear membrane. It is unclear at this time whetherhis group is representative of early apoptotic or necrotic pathways. This would needo be determined in future studies using specific cell surface markers.
Mid karyolysis (Fig. 2f): these cells have severe DNA depletion in the nucleus buttill have an apparent nuclear membrane. The nuclear staining in these cells is veryaint.
28 P. Thomas et al. / Mutation Research 661 (2009) 25–34
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ig. 2. Types of cells scored in the buccal micronucleus cytome assay. Cells wereells scored because cytoplasm is actually clearly visible when viewed through micepresents a normal differentiated cell, (c) represents a basal cell with micronuclei (aryolysis, (f) represents mid karyolysis, and (g) represents late karyolysis.
Late karyolysis (Fig. 2g): in these cells the nucleus is completely depleted of DNAnd apparent as a ghost-like image that has weak to negative fluorescent staining.hese cells may also appear to have no nucleus.
.12. Scoring method
1000 cells were scored per mouse for the presence of the described cell types.ucleated differentiated cells and basal cells were scored for micronuclei and their
cores were combined to give the overall incidence of micronucleated cells.
.13. Blood collection for micronucleated erythrocyte assay
Blood was collected from the tail at 3 months and from the right atrium duringost mortem after 9 months of dietary treatment. The cell preparation for this assayas the same at both time points. 500 �l of RPMI-1640 media was added to a mul-
ivette tube (Sarstedt ref No.: 15.1673). 20 units of heparin were added to each tube
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d with propidium iodide. Images shown under represent cytoplasmic staining ine. Arrows highlight presence of micronuclei. (a) Represents a buccal basal cell, (b)
rrowed, (d) represents a differentiated cell with MN (arrowed), (e) represents early
o prevent clotting. 10,000 U lyophilized heparin (Sigma, cat no. H3393-10KU) waseconstituted with 1 ml of 0.9% saline and 2 �l (20 units) added to each multivetteube containing media. The mouse was anaesthetized with isoflurane and the endf the tail removed (∼1 mm), 50 �l of blood was collected, added to the heparinizededia and thoroughly mixed. Within a biosafety hazard cabinet 30 �l was pipet-
ed onto a clean slide and a blood film prepared by running a coverslip along theength of the slide. The slide was air dried for 10 min and then fixed in methanol for0 min.
.14. Acridine orange staining
A stock solution of Acridine orange (Sigma A-6014) was prepared in phos-hate buffer saline (120 mM sodium chloride, 2.7 mM potassium chloride, 10 mMotassium phosphate monobasic, 10 mM sodium phosphate dibasic, pH 7.4) at aoncentration of 10 mg/ml. A 1:250 dilution was prepared in 4× Standard Salineitrate (0.30 M sodium chloride, 0.030 M sodium citrate, pH 7.4) to give a final con-
P. Thomas et al. / Mutation Research 661 (2009) 25–34 29
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d(detected. The level of proanthocyanidins (436.6 mg Catechin Eq/gDW) were also detected. The total polyphenolic content measuredwas 592.5 mg/g DW. Total phenolic content was obtained by theFolin-Ciocalteu method.
ig. 3. (a and b) Showing immature polychromatic erythrocytes with micronucleus,rythrocyte without micronuclei in same field of view. Cells were stained with acrid
entration of 40 �g/ml (200 �l of 10 mg/ml Acridine orange in 50 ml of 4× SSC).lides were stained for 4 min, rinsed briefly in 2× SSC and coverslipped prior tonalysis using a fluorescent microscope. Immature erythrocytes, i.e. polychromaticrythrocytes (PCE) were identified by their orange–red color, mature erythrocytesy their green–brown color and micronuclei by their yellow color (Fig. 3a–d). Therequency of PCE’s in a total of 2000 erythrocytes was determined as well as theumber of micronucleated PCE’s in 1000 PCE’s scored at both 3 and 9 months ofietary treatment (Table 3).
.15. DNA isolation for telomere length determination
The isolation of DNA from buccal cells and olfactory bulbs of the mouse brainere performed under conditions that minimise in vitro oxidation as previouslyescribed [14].
.16. Real time PCR for absolute quantitation for telomere length
The quantitative real time amplification of the telomere sequence was per-ormed as described by O’Callaghan et al. with modifications [39]. Briefly Cyclingonditions (for both telomere and 36B4 amplicons) were: 10 min at 95 ◦C, fol-owed by 40 cycles of 95 ◦C for 15 s, 60 ◦C for 1 min. Primers for the 36B4eaction consisted of (forward primer, 5′-ACT GGT CTA GGA CCC GAG AAG-3′)nd (reverse primer, 5′-TCA ATG GTG CCT CTG GAG ATT-3′ Geneworks, Adelaide,ustralia).
The resultant amplification produces a value that is equivalent to telomereequence length (kb) per 20 ng of total genomic DNA which is equivalent to telom-re sequence length (kb) per 3472 mouse diploid genomes. The telomere sequenceength (kb) per diploid genome can then be derived from this value by dividing by472.
.17. Statistical analysis
One-way ANOVA analysis was used to determine the significance of differ-nces between groups for all measurements, whereas pair wise comparison ofignificance was determined using the parametric Tukey’s test if distribution of
alues is Gaussian, or the non-parametric Kruskal–Wallis test if distribution of val-es was not Gaussian. Comparisons between two groups were performed usinghe Mann–Whitney non-parametric t-test if values were not Gaussian or paramet-ic t-test if values were found to be Gaussian in distribution (Graphpad PRISMGraphpad Inc., San Diego, CA)). p-Values <0.05 were considered to be signifi-ant.
FaatTa
ture erythrocyte with micronucleus, and (d) polychromatic erythrocyte and maturerange.
. Results
.1. HPLC analysis of GSE
By HPLC analysis we detected compounds only at 280 nm (Fig. 4)hich is the wavelength at which phenolic acids (e.g. gallic acid)
nd flavanols (e.g. catechins) are apparent. The level of proantho-yanidins was calculated as a difference between the total peakt 280 nm and the area of individual peaks that represent theonomers.Through spectral characteristics and comparison with stan-
ards, three main compounds gallic acid (49 mg/g dry weightDW)), catechin (41 mg/g DW), and epicatechin (66 mg/g DW) were
ig. 4. HPLC profile of phenolic compounds in GSE. Analytical HPLC was run at 25 ◦Cnd monitored at 280 (hydroxybenzoic acids and flavanols), 320 (hydroxycinnamiccids, stilbenes) and 370 nm (flavonols). HPLC has detected compounds at 280 nm. Athis wavelength phenolic acids (gallic acid) and flavanols (catechins) were detected.he level of proanthocyanidins was calculated as a difference between the total peakrea at 280 nm and the area of individual peaks that represent the monomers.
30 P. Thomas et al. / Mutation Research 661 (2009) 25–34
Table 2Food consumption (g/animal/week) and bodyweights (g).
WT CON, N = 20 AD CON, N = 10 AD CUR, N = 10 AD GSE, N = 10 AD MGSE, N = 10 One-way ANOVA, p value
* p < 0.05 when compared to AD CON.** p < 0.01 When compared to AD CON.
*** p < 0.001 when compared to AD CON.
.2. Food consumption
Food consumption (mg/animal/week) was measured at 3 and 9onths for a period of 1 week. Food consumption data for all groups
fter 3 and 9 months of dietary treatment is shown in Table 2.At 3 months there was no significant difference in the amount
f food consumed between the WT control and AD control group.here was a significant reduction in food consumption for mice onurcumin, GSE and MGSE diets compared to the AD control group.
At 9 months there was no significant difference in the amountf food consumed between the WT control and AD controlroup. No significant differences occurred between the AD con-rol group and any of the cohorts on the curcumin, GSE and MGSEiets.
.3. Bodyweight
Bodyweight data for all groups after 3 and 9 months of dietaryreatment is shown in Table 2.
At 3 months there was no significant difference in bodyweightetween the WT control and AD control groups. A significant reduc-ion in bodyweight occurred in AD CUR mice compared to the AD
ontrol group (p < 0.01).
At 9 months there was a significant increase in bodyweight forhe AD control mice compared to WT control mice (p < 0.05). Noignificant differences in bodyweight occurred between AD controlnd the AD CUR, AD GSE and AD MGSE diet groups.
1Adcg
able 3NA damage endpoints measured after 3 and 9 months dietary treatment.
* p < 0.05 when compared to the AD control group.** p < 0.01 when compared to the AD control group.a p < 0.05 when compared to the WT control group.
. Buccal micronucleus cytome assay
The results for the buccal micronucleus cytome assay at monthsand 9 are summarized and illustrated in Table 3.
.1. Basal cells
There were no significant differences in basal cell frequencyetween the WT Control and AD control group at both the 3nd 9 months time points. There were no significant differencesn basal cell frequency between the AD control group and cur-umin, GSE and MGSE groups at both the 3 and 9 months timeoints.
.2. Micronucleated cells (combined basal and differentiatedicronucleated cells)
After 3 months there was no significant difference in the fre-uency of micronucleated cells between the WT Control and ADontrol group and between the AD control and the curcumin,SE and MGSE dietary groups. After 9 months a non-significant
.7-fold increase in micronucleated buccal cells occurred in theD control compared to the WT control group. A significantecrease in micronucleated cells (p < 0.05) occurred in both theurcumin and MGSE dietary groups compared to the AD controlroup.
After 3 months dietary intervention there were no significantifferences in the frequencies of early, mid or late karyolytic cellsetween the WT control and the AD control groups. A significanteduction in mid karyolytic cells occurred in the MGSE dietaryroup compared to the AD control group (p < 0.05).
After 9 months dietary intervention there were no significantifferences in the frequencies of early, mid or late karyolytic cellsetween the WT control and the AD control groups or between theD control and curcumin, GSE and MGSE dietary groups.
. Whole blood micronucleus erythrocyte assay
Results for this assay at 3 and 9 months post-treatment arehown in Table 3.
.1. Micronucleated PCE
At 3 months there were no significant differences in the fre-uency of micronucleated PCE between the WT control and ADontrol group. There was a trend for increased MN in PCE’s as miceot older but increases were not significantly different betweenand 9 months. After 9 months dietary intervention there were
o significant differences in the frequency of micronucleated PCEetween the WT control and AD control group. The frequency oficronucleated PCE was significantly reduced (65.3%) for the ADGSE cohort (p < 0.05), whereas the AD Cur and AD GSE groupsere non-significantly reduced by 39.2 and 34.8% respectively
ompared to the AD control.
.2. Micronucleated non-polychromatic erythrocytes
No significant differences occurred in the micronucleated non-olychromatic erythrocyte frequency between the WT Con and ADon at both 3 and 9 months time points. No significant differencesccurred between the AD control and the AD Cur, AD GSE and MGSEohorts at both 3 and 9 months.
.3. Polychromatic erythrocyte percentage
There were no significant differences in polychromatic erythro-yte percentage between the WT control and the AD control groupst both 3 and 9 months time points. No significant differencesccurred between the AD Con and AD Cur, AD GSE and AD MGSEohorts at both 3 and 9 months time points.
.4. Correlation between micronuclei in buccal cells andolychromatic erythrocytes
Combined data for all cohorts showed no significant correla-ion (r = 0.09, p = 0.28) between the combined MN for basal andifferentiated buccal cells and micronuclei in the polychromaticrythrocytes.
. Telomere length at 9 months
.1. Olfactory bulb brain tissue
The results for absolute telomere length in olfactory bulb tissueor all the cohorts sampled are shown in Table 3. A non-significant-fold decrease in absolute olfactory bulb telomere length was evi-ent between the AD control and WT control groups. The AD Curohort had a non-significant 2-fold increase in telomere length
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arch 661 (2009) 25–34 31
ompared to the AD control group, whereas both the AD GSEnd MGSE cohorts showed a non-significant reduction in telomereength when compared to the AD control cohort.
.2. Buccal cell absolute telomere length
The results for absolute telomere length in buccal cells for allhe cohorts sampled are shown in Table 3. A significant decreasep = 0.04) in absolute buccal telomere length was evident in the ADontrol when compared to the WT control. The AD Cur, AD GSEnd AD MGSE polyphenol cohorts tended to have longer telomereshen compared to the AD control group but significance was not
chieved.
.3. Correlation between buccal cell and olfactory bulb telomereength
Combined data for all cohorts did not correlate between buccalell and olfactory bulb telomere length (r = −0.12, p = 0.23).
There were no significant gender effects observed within any ofhe cohorts for any of the parameters investigated.
. Discussion
Oxidative stress (OS) is thought to play a key role in the earlytages of AD pathology [1,12,40]. OS results from an imbalanceetween free radical formation, and the counteractive endogenousntioxidant defense systems such as superoxide dismutase (SOD),atalase (CAT) and glutathione peroxidase (GPx). Reactive oxygenpecies (ROS) are damaging to cells leading to the oxidation ofssential cellular components such as proteins and lipids, leadingo eventual genomic instability events such as MN formation andelomere shortening [2,3].
Results from this study show differences in biomarkers for DNAamage between the transgenic AD control group and the WT con-rol groups. A buccal micronucleus cytome approach was used andhowed an increase in MN frequency for both control groups overhe duration of the 9-month study. WT controls showed an over-ll 8% increase whereas the AD control group showed an overallncrease in MN frequency of 20%. After 9 months a non-significant.7-fold increase in MN frequency was apparent in the transgenicD buccal mucosa compared to the WT group. Differences werepparent in the MN frequency between AD control mice and theD groups with an increased dietary polyphenol intake. The resultsuggest a potential protective effect from a polyphenol-enrichediet as the MN frequency in buccal cells was reduced compared tohe control diets. The curcumin cohort showed a 72% decrease in theuccal MN frequency over the course of the study whereas the GSEnd MGSE cohorts showed an overall decrease of 9 and 84% respec-ively. There was a significant 10-fold decrease (p = 0.01) in buccal
N frequency at 9 months between the AD control group and bothhe curcumin and MGSE cohorts and a significant 7-fold decreaseor the GSE cohort (p = 0.02). The frequencies of micronuclei ascored in the assay were found not to be significantly differentetween basal cells (0.31%) and differentiated cells (0.38%). This
s the first time that buccal cells have been used to investigatehe effects of polyphenols on MN frequency in a transgenic mouse
odel for AD and suggest that studies in other models and humansight be feasible with this approach.Similarly in whole blood a 24% increase in micronucleated
olychromatic erythrocytes occurred in the WT control group com-ared to a 50% increase in the AD controls during the study timerame. After 9 months the AD control had a non-significant 1.3-foldncrease in MN frequency compared to the WT control. Differ-nces in MN frequency were also apparent between the AD control
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nd polyphenol diet groups. All the polyphenol groups showedreduction in MN frequency between the 3 and 9 months sam-
le points. However, no significant positive correlation for MNrequency between these tissues was evident (r = 0.09, p = 0.28).hese results suggest that the polyphenol diets may be protectiven reducing the MN frequency in both buccal mucosa and wholelood, at the levels investigated confirming the results of earlier initro studies involving human lymphocytes [26,41,42].
Taken together, these data suggest that the AD control cohortsend to have an increased rate of DNA damage in different tissueseyond the rate of normal ageing. It is possible that had samplingccurred at a later stage of disease progression these initial trendsay prove to be significantly different. These findings are similar to
he results in human buccal mucosa and lymphocytes which showsimilar trend for increased MN frequency in untreated AD cases
ompared to age matched controls [3,11].It has been shown that in AD the olfactory system inclusive
f the olfactory bulb (OB) is severely damaged which results inmpaired olfaction and is now considered part of the early clinicalhenotype of AD [43]. It has also been demonstrated by immuno-istochemistry that the OB contains both neurofibrillary tanglesnd amyloid deposits, characteristic hallmarks of AD [44]. Theres a close relationship existing between cortical degenerative andlfactory changes and suggests that olfactory bulb involvement isne of the earliest events in the degenerative process to occur inD [43]. Previous studies have shown that PSEN 1 mRNA and beta-myloid precursor protein mRNA is expressed in the developing ratlfactory and vestibulocochlear systems [45].
Telomere length was investigated as a biomarker of genomicnstability in both buccal cells and OB brain tissue, as telomereength has been shown to be compromised in AD and under con-itions of OS [15,46]. In buccal cells a significant (p = 0.04) 11-foldeduction in telomere length was found in the AD control groupompared to the WT control. This reduction in buccal telomereength in the AD control cohort may be due to increased lev-ls of OS, or a higher turnover rate of cells. These results are ingreement with the findings from a human study where telom-re length was found to be shorter in AD buccal cells comparedo age and gender matched controls [14]. All polyphenol cohortshowed a non-significant 2-fold increase in buccal cell telomereength compared to the AD control cohort and a non-significant 4.3-old decrease compared to the WT cohort. This would suggest thatolyphenols, possibly through their effective scavenging of ROS andctivation of endogenous defense mechanisms, may provide someegree of protection towards telomere maintenance in the buc-al mucosa at the levels investigated. However the study was notowered sufficiently to detect significant effects in this tissue.
Telomere length within the OB brain tissue of AD controls wasound to be non-significantly reduced by 2-fold compared to the
T control group. Both the GSE and MGSE polyphenol group wereound to have a non-significant 34 and 25% reduction in brainelomere length compared to the AD control group. This slighteduction may be explained as a result of sampling differences.f samples from the olfactory bulb were to contain increased lev-ls of proliferative cells then it would be expected that the overallelomere length would be reduced following cellular division. AD
ice that were fed a curcumin rich diet were found to have a 2-foldncrease in brain telomere length compared to the AD control group,nd were found to be only 4% shorter than the WT control cohort.t would appear, based on these results, that curcumin is probably
ore beneficial in maintaining telomere length within the olfac-ory bulb than either GSE or MGSE. This may be related to the facthat curcumin has the ability to induce expression of genes involvedn the cellular stress response network [47]. Curcumin can activateeme oxygenase-1 (HO-1) which increases cellular resistance to
oowdc
earch 661 (2009) 25–34
xidative stress and phase II enzyme expression in neural tissuehrough the activation of the transcription factor Nrf2, affording sig-ificant protection to neurons exposed to oxidative stress [47–50].urthermore, curcumin and GSE have been shown to inhibit theormation of amyloid oligomers and prevents fibril formation thatead to the formation of amyloid plaques [16,20,28,51]. Beta amy-oid has been shown to form complexes with copper and iron toroduce hypochlorous acid that increase the levels of OS withinhe brain [8,9]. Both Curcumin and GSE act as a metal chelatorinding these metals, possibly reducing amyloid levels resulting
n lower levels of oxidative stress [52,53]. Similarly, future stud-es involving telomere length should be undertaken to investigatehe effect of polyphenol diets on WT control mice to determinehether the same changes in brain pathology manifest themselvesnder similar dietary conditions.
It has previously been shown that both curcumin and GSE areffective antioxidants and have contributory roles in protectingells from OS and resulting DNA damage [25,54]. It is thought thathe effectiveness of these compounds as free radical scavengersan be directly attributed to their structural characteristics. Thehenolic and methoxy groups on the curcumin benzene rings andhe 1,3-diketone system are thought to be important structuresor effective oxygen free radical scavenging [55,56]. GSE has beenhown to have a high number of conjugated structures between the-ring catechol groups and the 3-OH free groups of the polymerickeleton allowing these compounds to be effective free radical scav-ngers and metal chelators [41,57]. As GSE scavenges free radicals,he resulting aroxyl radical formed has been shown to be moretable than those generated from other polyphenolic compoundsotentially aiding in preventing further DNA damage [58].
Further mechanisms that have been attributed to both curcuminnd GSE that enhance their effectiveness as powerful antioxidantsnvolve the enhanced synthesis of the endogenous antioxidantnzymes SOD, CAT and GPx [42,59,60]. Reduced glutathione isnother endogenous antioxidant that regulates the expression ofntioxidant genes [19,61]. GSH levels and subsequent activity isaintained by the enzyme �-glutamylcysteine ligase catalytic sub-
nit (GCLC) which in turn is regulated by OS [62]. It has beenhown that both curcumin and GSE protect against GSH deple-ion by sequestering ROS and increases GSH synthesis by enhancingCLC expression [19,63].
A weak non-significant inverse correlation was found betweenelomere length in brain tissue and buccal mucosa (r = −0.15,= 0.24), indicating that it is not possible to use buccal cells as sur-
ogates for telomere length measures in the brain. At this staget is uncertain the contribution made by each cellular populationo overall telomere length. It would be interesting to perform fur-her studies using flow cytometry or Q-FISH to isolate populationsf cells to determine the contribution of both basal and differen-iated cells to overall telomere length. It has been shown in thistudy that a high proportion of buccal cells are under going vari-us stages of karyolysis which may distort the true telomere lengthf the progenitor basal cells by over representing senescent cellshat would be expected to contain shorter telomeres. Only futuretudies on progenitor basal cells will be able to determine whetherstronger correlation exists between these two tissues, which if
roven may lead to buccal cells potentially being used as a nonin-asive biomarker reflecting physiological changes within telomereength in the brains of premature ageing mouse models for AD.
The buccal micronucleus cytome assay also provided measures
f potential cell proliferation in relation to potential genotoxicityf the polyphenols at the levels investigated. Basal cell frequencyithin both the control groups showed a slight but non-significantecrease over the duration of the study. After 9 months the basalell frequency in the AD control group had decreased by 7% possi-
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ly indicating the initial stages of a decline in regenerative potentialith advancing age [64,65]. It would have been interesting to have
ampled the buccal mucosa at later time points, in order to deter-ine whether similar changes in basal cell frequency reflect those
bserved in earlier human studies which showed a sharp reductionn buccal basal cell frequency in AD cases compared to controls11,38]. All of the polyphenol cohorts did not show any markedhanges in the frequency of basal cells which may be possiblyndicative as a proliferation marker compared to controls suggest-ng that polyphenols at the dose used in the study had no cytotoxicr cytostatic effects. Similarly karyolytic cell frequency was notifferent between dietary groups after 9 months.
Upon completion of the 9-month study there were no signifi-ant differences for food consumption and/or bodyweight betweenhe WT control group and the AD control group or between theolyphenol cohorts and the AD control group. However at threeonths there was a significant reduction in both food consumption
nd bodyweight for the curcumin cohort compared to the AD con-rol group. Similarly, a significant reduction in food consumptionccurred for the GSE and MGSE cohort although no significant lossn bodyweight was recorded. This may have been attributable to aertain degree of unpalatability associated with the diet to whichhe animals may have possibly become habituated to with time,aving previously been fed the AIN-93G diet. No change in behaviorr mortality was noted. Previous studies have shown that increasedietary polyphenols had no adverse effects on bodyweight [29,66].
t would also be important to determine the effect of bodyweightnd food consumption on control WT mice when fed high polyphe-ol diets to observe any similar trends to those seen in the ADroups.
Although the study revealed differences in DNA damage mea-ures between the WT control and AD control mice, the lack ofignificance of the majority of biomarkers investigated may beue to the fact that they are not a sensitive enough indicatorf AD pathology in mice. Alternatively the study may have beenerminated prematurely before the selected biomarkers could sig-ificantly reflect changes in AD pathology, as shown in previousuman studies where the same battery of biomarkers were used inlinically diagnosed AD patients. As trends were apparent betweenhe WT control and AD control mice further studies would needo be performed to relate the stage of disease progression andhe significance and efficacy of the DNA damage biomarkers mea-ured. Future studies involving this mouse model and potentialolyphenol cohorts should be taken over a longer time frame with
ntermittent evaluation of cognitive impairment measures. Thisata together with levels of beta 42 and genomic instability eventsan then be used to determine the efficacy of polyphenols as poten-ial protective agents in AD pathology by relating the stage ofisease progression within this mouse model to its human coun-erpart whether it is mild cognitive impairment, early or late AD.n light of the non-significance of some of the biomarkers investi-ated between the control groups, it is important to perform repeatxperiments involving WT control mice with the various polyphe-ol diets used in this study. This will allow us to investigate potentialrotective effects between the AD control mice and WT mice inelation to genome instability markers.
In conclusion, these results are suggestive of a potential protec-ive effect of a polyphenol enriched diet in terms of reducing DNAamage events, such as micronuclei and telomere length in differ-nt somatic tissue types in this transgenic mouse model, with no
dverse effects on cell proliferation rates or cell death markers. Onlyuture experiments thoroughly investigating the effects of polyphe-ols on brain pathology in relation to genomic instability events canupport or refute the potential protective effects of polyphenols inD pathology.
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eferences
[1] P. Mecocci, M.C. Polidori, T. Ingegni, A. Cherubini, F. Chionne, R. Cecchetti, U.Senin, Oxidative damage to DNA in lymphocytes from AD patients, Neurology51 (1998) 1014–1017.
[2] N. Bresgen, G. Karlhuber, I. Krizbai, H. Bauer, H.C. Bauer, P.M. Eckl, Oxidativestress in cultured cerebral endothelial cells induces chromosomal aberrations,micronuclei, and apoptosis, J. Neurosci. Res. 72 (2003) 327–333.
[3] L. Petrozzi, C. Lucetti, R. Scarpato, G. Gambaccini, F. Trippi, S. Bernardini, P.Del Dotto, L. Migliore, U. Bonuccelli, Cytogenetic alterations in lymphocytes ofAlzheimer’s disease and Parkinson’s disease patients, Neurol. Sci. 23 (Suppl. 2)(2002) S97–S98.
[4] J. Gotz, F. Chen, J. van Dorpe, R.M. Nitsch, Formation of neurofibrillary tanglesin P301L tau transgenic mice induced by Abeta 42 Fibrils, Science 293 (2001)1491–1495.
[5] P.H. St. George-Hyslop, Piecing together Alzheimer’s, Sci. Am. 283 (2000)76–83.
[6] P.I. Moreira, M.A. Smith, X. Zhu, K. Honda, H.G. Lee, G. Aliev, G. Perry, Oxidativedamage and Alzheimer’s disease: are antioxidant therapies useful? Drug NewsPerspect. 18 (2005) 13–19.
[7] Y. Christen, Oxidative stress and Alzheimer disease, Am. J. Clin. Nutr. 71 (2000)621S–629S.
[8] A.I. Bush, Metal complexing agents as therapies for Alzheimer’s disease, Neu-robiol. Aging (2002) 1031–1038.
[9] A.I. Bush, The metallobiology of Alzheimer’s disease, Trends Neurosci. 26 (2003)207–214.
10] S.J. van Rensburg, J.M. van Zyl, F.C. Potocnik, W.M. Daniels, J. Uys, L. Marais, D.Hon, B.J. van der Walt, R.T. Erasmus, The effect of stress on the antioxidativepotential of serum: implications for Alzheimer’s disease, Metab. Brain Dis. 21(2006) 171–179.
11] P. Thomas, J. Hecker, J. Faunt, M. Fenech, Buccal micronucleus cytome biomark-ers may be associated with Alzheimer’s disease, Mutagenesis 22 (2007)371–379.
12] L. Migliore, I. Fontana, F. Trippi, R. Colognato, F. Coppede, G. Tognoni, B. Nuc-ciarone, G. Siciliano, Oxidative DNA damage in peripheral leukocytes of mildcognitive impairment and AD patients, Neurobiol. Aging 26 (2005) 567–573.
13] M. Flint, Beal oxidative damage as an early marker of Alzheimer’s disease andmild cognitive impairment, Neurobiol. Ageing 26 (2005) 585–586.
14] P. Thomas, N.J. O’Callaghan, M. Fenech, Telomere length in white blood cells,buccal cells and brain tissue and its variation with ageing and Alzheimer’sdisease, Mech. Ageing Dev. 129 (2008) 183–190.
15] T. Von, Zglinicki role of oxidative stress in telomere length regulation andreplicative senescence, Ann. N.Y. Acad. Sci. 908 (2000) 99–110.
16] G.P. Lim, T. Chu, F. Yang, W. Beech, S.A. Frautschy, G.M. Cole, The curry spicecurcumin reduces oxidative damage and amyloid pathology in an Alzheimertransgenic mouse, J. Neurosci. 21 (2001) 8370–8377.
17] J.A. Joseph, B. Shukitt-Hale, N.A. Denisova, D. Bielinski, A. Martin, J.J. McEwen,P.C. Bickford, Reversals of age-related declines in neuronal signal transduction,cognitive, and motor behavioral deficits with blueberry, spinach, or strawberrydietary supplementation, J. Neurosci. 19 (1999) 8114–8121.
18] M. Ganguli, V. Chandra, M.I. Kamboh, J.M. Johnston, H.H. Dodge, B.K. Thelma,R.C. Juyal, R. Pandav, S.H. Belle, S.T. DeKosky, Apolipoprotein E polymorphismand Alzheimer disease: The Indo-US Cross-National Dementia Study, Arch.Neurol. 57 (2000) 824–830.
19] S.K. Biswas, D. McClure, L.A. Jimenez, I.L. Megson, I. Rahman, Curcumin inducesglutathione biosynthesis and inhibits NF-kappaB activation and interleukin-8 release in alveolar epithelial cells: mechanism of free radical scavengingactivity, Antioxid. Redox Signal. 7 (2005) 32–41.
20] F. Yang, G.P. Lim, A.N. Begum, O.J. Ubeda, M.R. Simmons, S.S. Ambegaokar, P.P.Chen, R. Kayed, C.G. Glabe, S.A. Frautschy, G.M. Cole, Curcumin inhibits forma-tion of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloidin vivo, J. Biol. Chem. 280 (2005) 5892–5901.
21] J.M. Ringman, S.A. Frautschy, G.M. Cole, D.L. Masterman, J.L. Cummings,A potential role of the curry spice curcumin in Alzheimer’s disease, Curr.Alzheimer Res. 2 (2005) 131–136.
22] D. Bagchi, C.K. Sen, S.D. Ray, D.K. Das, M. Bagchi, H.G. Preuss, J.A. Vinson, Molec-ular mechanisms of cardioprotection by a novel grape seed proanthocyanidinextract, Mutat. Res./Fundam. Mol. Mech. Mutagen. 523–524 (2003) 87–97.
23] S.L. Nuttall, M.J. Kendall, E. Bombardelli, P. Morazzoni, An evaluation of theantioxidant activity of a standardized grape seed extract, Leucoselect®, J.Clin. Pharm. Ther. 23 (1998) 385–389.
24] Y. Feng, Y.M. Liu, J.D. Fratkins, M.H. LeBlanc, Grape seed extract suppresses lipidperoxidation and reduces hypoxic ischemic brain injury in neonatal rats, BrainRes. Bull. 66 (2005) 120–127.
25] M. Balu, P. Sangeetha, G. Murali, C. Panneerselva, Modulatory role of grape seedextract on age-related oxidative DNA damage in central nervous system of rats,Brain Res. Bull. 68 (2006) 469–473.
26] A. Sugisawa, S. Inoue, K. Umegaki, Grape seed extract prevents H(2)O(2)-induced chromosomal damage in human lymphoblastoid cells, Biol. Pharm.
Bull. 27 (2004) 1459–1461.
27] J. Shi, J. Yu, J.E. Pohorly, Y. Kakuda, Polyphenolics in grape seeds-biochemistryand functionality, J. Med. Food 6 (2003) 291–299.
28] M.H. Li, J.H. Jang, B. Sun, Y.J. Surh, Protective effects of oligomers of grape seedpolyphenols against beta-amyloid-induced oxidative cell death, Ann. N. Y. Acad.Sci. 1030 (2004) 317–329.
3 n Res
[
[
[
[
[
[[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
4 P. Thomas et al. / Mutatio
29] J. Deshane, L. Chaves, K.V. Sarikonda, S. Isbell, L. Wilson, M. Kirk, C. Grubbs, S.Barnes, S. Meleth, H. Kim, Proteomics analysis of rat brain protein modulationsby grape seed extract, J. Agric. Food Chem. 52 (2004) 7872–7883.
30] W.V. Nikolic, Y. Bai, D. Obregon, H. Hou, T. Mori, J. Zeng, J. Ehrhart,R.D. Shytle, B. Giunta, D. Morgan, T. Town, J. Tan, Transcutaneous beta-amyloid immunization reduces cerebral beta-amyloid deposits without T cellinfiltration and microhemorrhage, Proc. Natl. Acad. Sci. U.S.A. 104 (2007)2507–2512.
31] J.L. Jankowsky, D.J. Fadale, J. Anderson, G.M. Xu, V. Gonzales, N.A. Jenkins, N.G.Copeland, M.K. Lee, L.H. Younkin, S.L. Wagner, S.G. Younkin, D.R. Borchelt,Mutant presenilins specifically elevate the levels of the 42 residue {beta}-amyloid peptide in vivo: evidence for augmentation of a 42-specific {gamma}secretase, Hum. Mol. Genet. 13 (2004) 159–170.
32] D.R. Borchelt, T. Ratovitski, J. van Lare, M.K. Lee, V. Gonzales, N.A. Jenkins, N.G.Copeland, D.L. Price, S.S. Sisodia, Accelerated amyloid deposition in the brainsof transgenic mice coexpressing mutant presenilin 1 and amyloid precursorproteins, Neuron 19 (1997) 939–945.
33] Y.J. Wang, A.N. Pollard, H.D. Zhou, J.H. Zhong, X.F. Zhou, Characterization of anAlzheimer’s disease mouse model bearing mutant genes of amyloid precur-sor protein and human presenilin, Proceeding of the Australian NeuroscienceSociety (Sydney), 2006, p. 150.
34] J. laboratory http://jaxmice.jax.org/strain/004462.html (Technical support).35] D. Kammerer, A. Claus, R. Carle, A. Schieber, Polyphenol screening of pomace
from red and white grape varieties (Vitis vinifera L.) by HPLC-DAD-MS/MS, J.Agric. Food Chem. 52 (2004) 4360–4367.
36] P.G. Reeves, Components of the AIN-93 Diets as Improvements in the AIN-76ADiet, J. Nutr. 127 (1997) 838S.
38] P. Thomas, S. Harvey, T. Gruner, M. Fenech, The buccal cytome and micronu-cleus frequency is substantially altered in Down’s syndrome and normal ageingcompared to young healthy controls, Mutat. Res. 638 (2007) 37–47.
39] N.J. O’Callaghan, V.S. Dhillon, P. Thomas, M. Fenech, A quantitative real-timePCR method for absolute telomere length, Biotechniques 44 (2008).
40] A. Nunomura, G. Perry, G. Aliev, K. Hirai, A. Takeda, E.K. Balraj, P.K. Jones, H.Ghanbari, T. Wataya, S. Shimohama, S. Chiba, C.S. Atwood, R.B. Petersen, M.A.Smith, Oxidative damage is the earliest event in Alzheimer disease, J. Neu-ropathol. Exp. Neurol. 60 (2001) 759–767.
41] J. Castillo, O. Benavente-Garcia, J. Lorente, M. Alcaraz, A. Redondo, A. Ortuno, J.A.Del Rio, Antioxidant activity and radioprotective effects against chromosomaldamage induced in vivo by X-rays of flavan-3-ols (Procyanidins) from grapeseeds (Vitis vinifera): comparative study versus other phenolic and organiccompounds, J. Agric. Food Chem. 48 (2000) 1738–1745.
42] M. Srinivasan, N. Rajendra Prasad, V.P. Menon, Protective effect of curcuminon gamma-radiation induced DNA damage and lipid peroxidation in culturedhuman lymphocytes, Mutat. Res. 611 (2006) 96–103.
43] S. Christen-Zaech, R. Kraftsik, O. Pillevuit, M. Kiraly, R. Martins, K. Khalili, J.Miklossy, Early olfactory involvement in Alzheimer’s disease, Can. J. Neurol.Sci. 30 (2003) 20–25.
44] T. Kovacs, N.J. Cairns, P.L. Lantos, beta-amyloid deposition and neurofibrillary
tangle formation in the olfactory bulb in ageing and Alzheimer’s disease, Neu-ropathol. Appl. Neurobiol. 25 (1999) 481–491.
45] M. Utsumi, K. Sato, H. Tanimukai, T. Kudo, M. Nishimura, M. Takeda, M. Tohyama,Presenilin-1 mRNA and beta-amyloid precursor protein mRNA are expressed inthe developing rat olfactory and vestibulocochlear systems, Acta Otolaryngol.118 (1998) 549–553.
[
[
earch 661 (2009) 25–34
46] L.A. Panossian, V.R. Porter, H.F. Valenzuela, X. Zhu, E. Reback, D. Masterman,J.L. Cummings, R.B. Effros, Telomere shortening in T cells correlates withAlzheimer’s disease status, Neurobiol. Aging 24 (2003) 77–84.
47] V. Calabrese, D. Boyd-Kimball, G. Scapagnini, D.A. Butterfield, Nitric oxide andcellular stress response in brain aging and neurodegenerative disorders: therole of vitagenes, In Vivo 18 (2004) 245–267.
48] V. Calabrese, G. Scapagnini, A. Ravagna, R.G. Fariello, A.M. Giuffrida Stella, N.G.Abraham, Regional distribution of heme oxygenase, HSP70, and glutathione inbrain: relevance for endogenous oxidant/antioxidant balance and stress toler-ance, J. Neurosci. Res. 68 (2002) 65–75.
49] K.D. Poss, S. Tonegawa, Reduced stress defense in heme oxygenase 1-deficientcells, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 10925–10930.
50] G. Scapagnini, C. Colombrita, M. Amadio, V. D’Agata, E. Arcelli, M. Sapienza,A. Quattrone, V. Calabrese, Curcumin activates defensive genes and protectsneurons against oxidative stress, Antioxid. Redox Signal. 8 (2006) 395–403.
51] K. Ono, K. Hasegawa, H. Naiki, M. Yamada, Preformed beta-amyloid fibrils aredestabilized by coenzyme Q10 in vitro, Biochem. Biophys. Res. Commun. 330(2005) 111–116.
52] L. Baum, A. Ng, Curcumin interaction with copper and iron suggests one possiblemechanism of action in Alzheimer’s disease animal models, J. Alzheimer’s Dis.6 (2004) 367–377 (discussion 369–443).
53] Y. Yilmaz, R.T. Toledo, Health aspects of functional grape seed constituents,Trends Food Sci. Technol. 15 (2004) 422–433.
54] R.A. Sharma, A.J. Gescher, W.P. Steward, Curcumin: the story so far, Eur. J. Cancer41 (2005) 1955–1968.
55] N. Sreejayan, M.N. Rao, Free radical scavenging activity of curcuminoids,Arzneimittelforschung 46 (1996) 169–171.
56] Sreejayan, M.N. Rao, Nitric oxide scavenging by curcuminoids, J. Pharm. Phar-macol. 49 (1997) 105–107.
57] J. Castillo, O. Benavente-Garcia, M.J. Del Bano, J. Lorente, M. Alcaraz, M.J. Dato,Radioprotective effects against chromosomal damage induced in human lym-phocytes by gamma-rays as a function of polymerization grade of grape seedextracts, J. Med. Food 4 (2001) 117–123.
58] O. Benavente-Garcia, J. Castillo, F.R. Marin, A. Ortuno, J.A. Del Rio, Uses andproperties of citrus flavonoids, J. Agric. Food Chem. 45 (1997) 4505–4515.
59] A.C. Reddy, B.R. Lokesh, Effect of dietary turmeric (Curcuma longa) on iron-induced lipid peroxidation in the rat liver, Food Chem. Toxicol. 32 (1994)279–283.
60] M. Balu, P. Sangeetha, D. Haripriya, C. Panneerselvam, Rejuvenation of antiox-idant system in central nervous system of aged rats by grape seed extract,Neurosci. Lett. 383 (2005) 295–300.
61] I. Rahman, W. MacNee, Regulation of redox glutathione levels and gene tran-scription in lung inflammation: therapeutic approaches, Free Radic. Biol. Med.28 (2000) 1405–1420.
62] I. Rahman, W. MacNee, Oxidative stress and regulation of glutathione in lunginflammation, Eur. Respir. J. 16 (2000) 534–554.
63] M.C.W. Myhrstad, H. Carlsen, O. Nordstrom, R. Blomhoff, J.O. Moskaug,Flavonoids increase the intracellular glutathione level by transactivation ofthe [gamma]-glutamylcysteine synthetase catalytical subunit promoter, FreeRadic. Biol. Med. 32 (2002) 386–393.
