Grape seed polyphenols and curcumin reduce genomic instability events in a transgenic mouse model for Alzheimer's disease

Mutation Research 661 (2009) 25–34

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Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journal homepage: www.elsevier.com/locate/molmut Community address: www.elsevier.com/locate/mutres

Grape seed polyphenols and curcumin reduce genomic instability events in a transgenic mouse model for Alzheimer’s disease Philip Thomas a,1 , Yan-Jiang Wang b,1 , Jin-Hua Zhong b , Shantha Kosaraju c , Nathan J. O’Callaghan a , Xin-Fu Zhou b,∗ , Michael Fenech a,∗ a b c

CSIRO Human Nutrition, PO Box 10041, Adelaide BC, Adelaide, SA 5000, Australia Department of Human Physiology and Centre for Neuroscience, Flinders University, GPO Box 2100, Adelaide, SA, Australia CSIRO Food Science Australia, 671 Snydes Rd., Private Bag 16, Werribee, Victoria 3030, Australia

a r t i c l e

i n f o

Article history: Received 2 June 2008 Received in revised form 22 October 2008 Accepted 24 October 2008 Available online 6 November 2008 Keywords: Polyphenol DNA damage Alzheimer’s disease Micronuclei Buccal

a b s t r a c t The study set out to determine (a) whether DNA damage is elevated in mice that carry mutations in the amyloid precursor protein (APP695swe) and presenilin 1 (PSEN1-dE9) that predispose to Alzheimer’s disease (AD) relative to non-transgenic control mice, and (b) whether increasing the intake of dietary polyphenols from curcumin or grape seed extract could reduce genomic instability events in a transgenic mouse model for AD. DNA damage was measured using the micronucleus (MN) assay in both buccal mucosa and erythrocytes and an absolute telomere length assay for both buccal mucosa and olfactory bulb tissue. MN frequency tended to be higher in AD mice in both buccal mucosa (1.7-fold) and polychromatic erythrocytes (1.3-fold) relative to controls. Telomere length was significantly reduced by 91% (p = 0.04) and non-significantly reduced by 50% in buccal mucosa and olfactory bulbs respectively in AD mice relative to controls. A significant 10-fold decrease in buccal MN frequency (p = 0.01) was found for AD mice fed diets 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 the MGSE cohort (65.3%) (p < 0.05), whereas the AD CUR and AD GSE groups were non-significantly reduced by 39.2 and 34.8% respectively compared to the AD Control. A non-significant 2-fold increase in buccal cell 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 diet compared to controls. These results suggest potential protective effects of polyphenols against genomic instability events in different somatic tissues of a transgenic mouse model for AD. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved.

1. Introduction Alzheimer’s disease (AD) is associated with elevated rates of oxidative stress that contribute to increased rates of genomic instability events such as an increased frequency in micronucleus (MN) formation, chromosomal aberrations, and alterations in telomere length and levels of apoptosis [1–3]. The characteristic hallmarks of AD involve the abnormal deposition of proteins leading to amyloid plaques and neurofibrillary tangles in the brain and a progressive loss of cognitive function [4,5]. These pathological features have also been linked with oxidative stress [6,7]. It has been suggested

∗ Corresponding authors. Tel.: +61 8 3038880; fax: +61 8 303 8899. E-mail addresses: Zhou0010@flinders.edu.au (X.-F. Zhou), [email protected] (M. Fenech). 1 These authors contributed equally to this work.

that both plaques and tangles are produced in order to protect sensitive areas of the brain from the effects of oxidative related injury [6]. However, other studies have shown that plaques may produce reactive oxygen species (ROS) when they form complexes with redox active metals such as copper and iron [8,9]. Free radical generation results from normal metabolic processes, in which the levels are maintained within normal homeostatic boundaries by an elaborate endogenous antioxidant system. However, when levels of free radicals exceeds the normal clearing capacity of the cells oxidative stress follows resulting in potential cellular and genome damage [10]. Damage to the genome could lead to altered gene dosage and gene expression as well as contribute to the risk of accelerated cell death in neuronal tissues. DNA damage events such as micronuclei formation, which are biomarkers of chromosome malsegregation and fragility have been found to be elevated in both lymphocytes and buccal cells from individuals suffering from AD [3,11]. It has recently been shown that individuals with mild

0027-5107/$ – see front matter. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2008.10.016

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P. Thomas et al. / Mutation Research 661 (2009) 25–34

cognitive impairment (MCI) as well as more advanced AD have a 2-fold increase in DNA damage levels and oxidized bases in their leucocytes compared with age matched controls not clinically diagnosed with AD [12]. This is suggestive that oxidative stress occurs in the early stages of the disease, as MCI individuals progress to AD with an estimated probability of 50% within 4 years [12,13], suggesting MCI may be representative of early AD and that oxidative stress may directly contribute to disease pathology. Oxidative stress has also been shown to be related to telomere shortening, which has also been identified in both lymphocytes and buccal cells of patients clinically diagnosed with AD [14,15]. Recent studies have suggested the positive effects of dietary antioxidants as an aid in potentially reducing somatic cell and neuronal damage by free radicals [16,17]. Curcumin (CUR) is the yellow phenolic compound in the Indian curry spice turmeric. This spice is used as a food preservative in India where the incidence of AD in individuals aged between 70 and 79 years of age is 4.4-fold less than that in the United States [18]. CUR has been shown to act as an antioxidant through modulation of glutathione (GSH) levels [19] and possesses anti-inflammatory properties possibly mediated by inhibition of interleukin-8 release [19] as well as the ability to reduce Amyloid-beta (A␤) 42 toxicity by preventing the formation of ␤42 oligomers leading to amyloid plaque formation [16,20,21]. Grape seed extract (GSE) contains a number of polyphenols including proanthocyanidins and procyanidins [22,23]. They have been shown to be powerful free radical scavengers, possess antiinflammatory properties, reduce apoptosis and prevent hydrogen peroxide (H2 O2 ) induced chromosomal damage in human lymphoblastoid cells [24–26]. It has been shown that their free radical scavenging capacity is 20 times more effective than vitamin E and 50 times more effective than vitamin C [27]. It has also been reported in rat models that cells are protected against A␤ toxicity when treated with GSE compared to unprotected neurons, possibly as a result of its antioxidant or chelating properties [28,29]. The aims of this study were (a) to investigate whether DNA damage events are increased with time to a greater extent in AD mice relative to controls and (b) to determine the potential protective effects of natural products rich in polyphenols on genomic instability events in a transgenic mouse model for AD. We investigated the effects of CUR and GSE polyphenols on DNA damage by testing the mice over a 9 month dietary treatment period utilizing both a buccal micronucleus cytome assay, an erythrocyte micronucleus assay and by determining telomere length in both buccal cells and brain olfactory bulb (OB) tissue. 2. Materials and methods 2.1. Mouse model The study involved the use of a double transgenic mouse model (B6C3Tg(APPswePSEN1dE9)85Dbo/J (stock No. 004462) Jackson Laboratory, Maine, USA) that expresses a chimeric mouse/human amyloid precursor protein (Mo/Hu APP695swe) and a mutant presenilin 1 (PSEN1-dE9) that are both directed to central nervous system (CNS) neurons. These mutations are both associated with early

onset AD. The “humanized” Mo/HuAPP695swe transgene allows the mice to secrete a human A␤ peptide. The included “swedish” mutations (K595N/M596L) elevate the amount of A␤ peptide produced from the transgene by favoring processing through the beta-secretase pathway. The Mo/HuAPP695swe protein and the transgenic mutant human presenilin protein (PSEN1-dE9) are both immunodetected in whole brain homogenates [30,31]. Further information about strain development and gene expression can be obtained from the Jackson laboratory website http://jaxmice.jax.org/strain/004462.html. All mice used in this experiment were of a mixed F2 background resulting from a cross between heterozygous transgenic animals and C57BL/6J wild type mice (stock No. 005864). These transgenic mice develop amyloid plaques in the hippocampus and cortex at 4 months and in the thalamus at 6 months. At 9 months there is significant amyloid present in the cortex, hippocampus and thalamus [32,33]. 2.2. Genotyping All animals prior to receipt were genotyped at the Jackson laboratory for Tg(PSEN1) and Tg(APPswe) and all subsequent animals that were bred in house were genotyped prior to the start of the study using standard protocols outlined by the Jackson laboratory [34]. 2.3. Study design Approval for this 9-month study was obtained from CSIRO Human Nutrition and Flinders University Animal Ethics committees. The experimental design was a parallel dietary intervention involving four different dietary treatments using the B6C3-Tg(APPswePSEN1dE9)85Dbo/J mouse, with dietary intervention starting at an age of 3–4 months (Table 1). The design of the study was intended to determine whether grape seed extract is as effective as curcumin in reducing genomic instability pathology observed in AD, and whether micro-encapsulation of GSE, which may modify absorption kinetics, enhances the observed effects. Normal mice (wild type littermates) with the same genetic background, but lacking defective APP and PSEN1 genes were included as an added control to identify the point in time when buccal and blood differences between normal and Alzheimer’s mice become evident, and to test the hypothesis that the Alzheimer’s prone mice exhibited elevated rates of DNA damage. The study design is outlined in Fig. 1. The 3-month-old mice were randomly grouped with good balance in age, sex and breeding pairs (Table 1). All animals were housed in the Flinders Medical Centre animal holding facility, 2–3 per cage in standard cages ∼600 cm2 (150–200 cm2 per animal), males and females were housed separately and were genotyped prior to the commencement of the study following breeding from the originally acquired transgenic model. All groups were kept in the same room on a 14/10 h light dark cycle and had free access to food and water ad libitum. Food consumption and bodyweight data was collected by the staff at the animal facility after 3 and 9 months dietary treatment. 2.4. HPLC analysis of polyphenols in GSE Grape seed extract (Vinlife N05010) was purchased from Tarac Technologies P.L. and was characterized by high performance liquid chromatography (HPLC) without any further extraction. Lyophilized extract obtained from grape seeds was dissolved in 80% methanol acidified with 0.1% HCl to obtain final concentration of 2.0 mg/ml. 10 ␮l was injected into the HPLC column for the analysis of polyphenolic compounds. HPLC analysis was carried out according to methods described previously [35]. Analytical HPLC was run at 25 ◦ C and monitored at 280 nm (hydroxybenzoic acids and flavanols), 320 nm (hydroxycinnamic acids, stilbenes) and 370 nm (flavonols). The mobile phase consisted of 2% acetic acid in water (solvent A) and 1.0% acetic acid in water and acetonitrile (50:50, v/v, solvent B). The flow rate was 1 ml min−1 . The following gradient program was used: from 10 to 24% solvent B (20 min), from 24 to 30% B (20 min), from 30 to 55% B (20 min), from 55 to 100% B (15 min), 100% B isocratic (8 min), from 100 to 10% B (2 min). The total run time was 85 min. 2.5. Diets All diets were prepared by Specialty Feeds, Glen Forrest, Western Australia. The control diet consisted of 95% standard AIN-93G rodent diet comprising of 39.7% corn

Table 1 Age at start of feeding intervention and gender ratio of mice in study groups.

AGE (days) (mean ± SD) Gender ratio (male:female)

WT CON, N = 20

AD CON, N = 10

AD CUR, N = 10

AD GSE, N = 10

AD MGSE, N = 10

119.9 ± 21.04 10:10

116.1 ± 25.15 6:4

111.5 ± 21.04 5:5

119.9 ± 21.04 5:5

119.9 ± 21.04 6:4

There were no significant differences in gender ratio or age between cohorts. WT CON, wild type control. AD CON, Alzheimer’s mouse on control diet. AD CUR, Alzheimer’s mouse on curcumin diet. AD GSE, Alzheimer’s mouse on grape seed extracts diet. AD MSE, Alzheimer’s mouse on micro-encapsulated grape seed extracts diet.

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Fig. 1. Study design showing mouse groups, dietary groups, schedule and sampling points. WT CON group was the control for comparison of WT and AD mice on the same AIN-93G diet (i.e. AD CON). AD CON was the control group for determining the effects of curcumin, GSE and MGSE as additives in AIN-93G diet in AD mice (AD CUR, AD GSE and AD MGSE groups respectively).

starch, 20% casein (vitamin free), 13.2% dextrin, 10% sucrose, 7% soybean oil, 5% powdered cellulose, 3.5% AIN-93G mineral mix, 1% AIN-93G vitamin mix, 0.3% l-cysteine, 0.25% Choline bitartrate, 0.001% t-Butylhydroquinone and 5% maize starch [36]. Diet 2 consisted of 95% AIN-93G, 4.93% maize starch and 0.07% Curcumin (Sigma, Cat No.: C1386, Australia). Diet 3 consisted of 95% AIN-93G, 3% maize starch and 2% free grape seed extract. Diet 4 consisted of 95% AIN-93G and 5% encapsulated grape seed extract. Grape seed extract and curcumin at the nominated doses have been used in previous studies in mice and rats without any indication of causing toxic and/or irritant effects [16,37]. Enough diet was ordered for the duration of the study and housed at the Flinders Medical Centre animal holding facility. 2.6. Buccal cell collection Buccal cells were collected from all animals after 3 and 9 months of dietary treatment. Prior to sample collection the mouse was anaesthetized by isoflurane (Veterinary companies of Australia Pty Ltd., NSW, Australia). The buccal cells were collected by swabbing the inner cheeks with a thin cotton-tipped stick with 2 mm diameter buds (aluminium shaft 0.9 mm × 150 mm, Applimed SA, Chatel St. Denis, Switzerland). Both cheeks were sampled and the head of the applicator placed inside a 1.5 ml eppendorf containing 1 ml of buccal cell buffer 0.01 M Tris–HCL (Sigma T3253), 0.1 M EDTA tetra sodium salt (Sigma E5391), 0.02 M sodium chloride (Sigma S5886) and revolved to dislodge the cells. Cells were spun in a microfuge (Centrifuge 5415R, eppendorf) at 1800 rpm for 10 min. The supernatant was removed leaving approximately 150 ␮l and replaced with 1 ml of buccal cell buffer. The cells were spun and washed twice more to remove DNAses and bacteria which may hamper the quality of slide preparation and scoring. Cells were not homogenized or filtered so as not to lose cells unnecessarily. Buffer was removed leaving ∼120 ␮l. 6 ␮l of DMSO (5%) was added to the cell suspension to reduce cell clumping, agitated and added to cytospin cups and spun at 600 rpm for 5 min in a cytocentrifuge (Shandon cytospin 3). Slides were then air dried for 10 min and fixed in ethanol:acetic acid (3:1) for 10 min prior to staining. The nuclei were counterstained with propidium iodide (Sigma P-4170) (0.05 ␮g/ml in 4× SSC) (Standard saline citrate, 0.15 M sodium chloride, Sigma S9888/0.015 M sodium citrate, Sigma S4641) with 0.06% Tween 20 (Sigma P1379) for 3 min at room temperature in the dark and cover-slipped in antifade solution and kept at −20 ◦ C until analysis could be performed. Slides were scored using a Nikon E600 microscope equipped with a triple band filter (Dapi, FITC and rhodamine) at 1000× magnification. 2.7. Scoring criteria for murine buccal micronucleus assay The murine buccal micronucleus cytome assay we developed classifies buccal cells into categories that distinguish between “normal” cells and cells that are considered “abnormal”, based on nuclear morphology and is based on the same prin-

ciples developed for the human buccal cytome assay as described previously [11,38]. Some of these abnormal nuclear morphologies are thought to be indicative of DNA damage, e.g. micronucleus formation. Images showing distinct cell populations as scored in the assay are shown in Fig. 2. The different cell types scored include. 2.8. Normal basal cells (Fig. 2a) These are the cells from the basal layer. The nucleus is usually oval or round in shape and uniformly stained. The nuclear to cytoplasm ratio is larger than that in typical normal differentiated buccal cells. Basal cells are smaller in size when compared to differentiated buccal cells. No other DNA containing structures apart from the nucleus are observed in these cells. 2.9. Normal differentiated cells (Fig. 2b) These cells have a uniformly stained nucleus which is usually oval or round in shape and a nuclear to cytoplasmic ratio smaller than that of basal cells. No other DNA containing structures apart from the nucleus are observed in these cells. 2.10. Basal and differentiated cells with micronuclei (Fig. 2c and d) These cells are characterized by the presence of both a main nucleus and one or more smaller nuclei called micronuclei. The micronuclei are usually round or oval in shape and their diameter may range between 1/3 and 1/16 the diameter of the main nucleus. Cells with micronuclei usually contain only one micronucleus. It is possible but rare to find cells with more than 2 micronuclei. The nuclei in micronucleated cells usually have the morphology of nuclei in normal cells although occasionally the nuclei may appear to have diminished DNA content as observed in karyolytic cells. The micronuclei must be located within the cytoplasm of the cells. The presence of micronuclei is indicative of chromosome loss or fragmentation occurring during previous nuclear division. Micronuclei were scored only in basal and normal differentiated cells. 2.11. Karyolytic cells (Fig. 2e–g) Early karyolysis (Fig. 2e): these cells show the first signs of diminished DNA content but still have an apparent nuclear membrane. It is unclear at this time whether this group is representative of early apoptotic or necrotic pathways. This would need to be determined in future studies using specific cell surface markers. Mid karyolysis (Fig. 2f): these cells have severe DNA depletion in the nucleus but still have an apparent nuclear membrane. The nuclear staining in these cells is very faint.

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P. Thomas et al. / Mutation Research 661 (2009) 25–34

Fig. 2. Types of cells scored in the buccal micronucleus cytome assay. Cells were stained with propidium iodide. Images shown under represent cytoplasmic staining in cells scored because cytoplasm is actually clearly visible when viewed through microscope. Arrows highlight presence of micronuclei. (a) Represents a buccal basal cell, (b) represents a normal differentiated cell, (c) represents a basal cell with micronuclei (MN) arrowed, (d) represents a differentiated cell with MN (arrowed), (e) represents early karyolysis, (f) represents mid karyolysis, and (g) represents late karyolysis. Late karyolysis (Fig. 2g): in these cells the nucleus is completely depleted of DNA and apparent as a ghost-like image that has weak to negative fluorescent staining. These cells may also appear to have no nucleus. 2.12. Scoring method 1000 cells were scored per mouse for the presence of the described cell types. Nucleated differentiated cells and basal cells were scored for micronuclei and their scores were combined to give the overall incidence of micronucleated cells.

to prevent clotting. 10,000 U lyophilized heparin (Sigma, cat no. H3393-10KU) was reconstituted with 1 ml of 0.9% saline and 2 ␮l (20 units) added to each multivette tube containing media. The mouse was anaesthetized with isoflurane and the end of the tail removed (∼1 mm), 50 ␮l of blood was collected, added to the heparinized media and thoroughly mixed. Within a biosafety hazard cabinet 30 ␮l was pipetted onto a clean slide and a blood film prepared by running a coverslip along the length of the slide. The slide was air dried for 10 min and then fixed in methanol for 10 min. 2.14. Acridine orange staining

2.13. Blood collection for micronucleated erythrocyte assay Blood was collected from the tail at 3 months and from the right atrium during post mortem after 9 months of dietary treatment. The cell preparation for this assay was the same at both time points. 500 ␮l of RPMI-1640 media was added to a multivette tube (Sarstedt ref No.: 15.1673). 20 units of heparin were added to each tube

A stock solution of Acridine orange (Sigma A-6014) was prepared in phosphate buffer saline (120 mM sodium chloride, 2.7 mM potassium chloride, 10 mM potassium phosphate monobasic, 10 mM sodium phosphate dibasic, pH 7.4) at a concentration of 10 mg/ml. A 1:250 dilution was prepared in 4× Standard Saline Citrate (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

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Fig. 3. (a and b) Showing immature polychromatic erythrocytes with micronucleus, (c) mature erythrocyte with micronucleus, and (d) polychromatic erythrocyte and mature erythrocyte without micronuclei in same field of view. Cells were stained with acridine orange.

centration of 40 ␮g/ml (200 ␮l of 10 mg/ml Acridine orange in 50 ml of 4× SSC). Slides were stained for 4 min, rinsed briefly in 2× SSC and coverslipped prior to analysis using a fluorescent microscope. Immature erythrocytes, i.e. polychromatic erythrocytes (PCE) were identified by their orange–red color, mature erythrocytes by their green–brown color and micronuclei by their yellow color (Fig. 3a–d). The frequency of PCE’s in a total of 2000 erythrocytes was determined as well as the number of micronucleated PCE’s in 1000 PCE’s scored at both 3 and 9 months of dietary treatment (Table 3). 2.15. DNA isolation for telomere length determination The isolation of DNA from buccal cells and olfactory bulbs of the mouse brain were performed under conditions that minimise in vitro oxidation as previously described [14]. 2.16. Real time PCR for absolute quantitation for telomere length The quantitative real time amplification of the telomere sequence was performed as described by O’Callaghan et al. with modifications [39]. Briefly Cycling conditions (for both telomere and 36B4 amplicons) were: 10 min at 95 ◦ C, followed by 40 cycles of 95 ◦ C for 15 s, 60 ◦ C for 1 min. Primers for the 36B4 reaction consisted of (forward primer, 5 -ACT GGT CTA GGA CCC GAG AAG-3 ) and (reverse primer, 5 -TCA ATG GTG CCT CTG GAG ATT-3 Geneworks, Adelaide, Australia). The resultant amplification produces a value that is equivalent to telomere sequence length (kb) per 20 ng of total genomic DNA which is equivalent to telomere sequence length (kb) per 3472 mouse diploid genomes. The telomere sequence length (kb) per diploid genome can then be derived from this value by dividing by 3472.

3. Results 3.1. HPLC analysis of GSE By HPLC analysis we detected compounds only at 280 nm (Fig. 4) which is the wavelength at which phenolic acids (e.g. gallic acid) and flavanols (e.g. catechins) are apparent. The level of proanthocyanidins was calculated as a difference between the total peak at 280 nm and the area of individual peaks that represent the monomers. Through spectral characteristics and comparison with standards, three main compounds gallic acid (49 mg/g dry weight (DW)), catechin (41 mg/g DW), and epicatechin (66 mg/g DW) were detected. The level of proanthocyanidins (436.6 mg Catechin Eq/g DW) were also detected. The total polyphenolic content measured was 592.5 mg/g DW. Total phenolic content was obtained by the Folin-Ciocalteu method.

2.17. Statistical analysis One-way ANOVA analysis was used to determine the significance of differences between groups for all measurements, whereas pair wise comparison of significance was determined using the parametric Tukey’s test if distribution of values is Gaussian, or the non-parametric Kruskal–Wallis test if distribution of values was not Gaussian. Comparisons between two groups were performed using the Mann–Whitney non-parametric t-test if values were not Gaussian or parametric t-test if values were found to be Gaussian in distribution (Graphpad PRISM (Graphpad Inc., San Diego, CA)). p-Values <0.05 were considered to be significant.

Fig. 4. HPLC profile of phenolic compounds in GSE. Analytical HPLC was run at 25 ◦ C and monitored at 280 (hydroxybenzoic acids and flavanols), 320 (hydroxycinnamic acids, stilbenes) and 370 nm (flavonols). HPLC has detected compounds at 280 nm. At this wavelength phenolic acids (gallic acid) and flavanols (catechins) were detected. The level of proanthocyanidins was calculated as a difference between the total peak area at 280 nm and the area of individual peaks that represent the monomers.

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P. Thomas et al. / Mutation Research 661 (2009) 25–34

Table 2 Food consumption (g/animal/week) and bodyweights (g). WT CON, N = 20 Food consumption at 3 months (mean ± SD) Bodyweight at 3 months (mean ± SD) Food consumption at 9 months (mean ± SD) Bodyweight at 9 months (mean ± SD) a * ** ***

41.5 42.8 36.8 44.3

± ± ± ±

2.3 4.6 16.9 3.5

AD CON, N = 10 42.4 46.9 41.9 49.4

± ± ± ±

1.3 4.8 18.5 5.7a

AD CUR, N = 10 37.2 42.3 44.4 50.6

± ± ± ±

***

1.2 3.8 7.4 4.6

AD GSE, N = 10 39.5 40.3 39.2 48.5

± ± ± ±

**

1.5 4.4** 6.7 3.2

AD MGSE, N = 10 40.1 44.6 43.5 49.6

± ± ± ±

± 1.3 3.4 19.7 4.1

*

One-way ANOVA, p value <0.0001 0.012 0.618 0.0002

p < 0.05 when compared to WT CON. p < 0.05 when compared to AD CON. p < 0.01 When compared to AD CON. p < 0.001 when compared to AD CON.

3.2. Food consumption

4. Buccal micronucleus cytome assay

Food consumption (mg/animal/week) was measured at 3 and 9 months for a period of 1 week. Food consumption data for all groups after 3 and 9 months of dietary treatment is shown in Table 2. At 3 months there was no significant difference in the amount of food consumed between the WT control and AD control group. There was a significant reduction in food consumption for mice on curcumin, GSE and MGSE diets compared to the AD control group. At 9 months there was no significant difference in the amount of food consumed between the WT control and AD control group. No significant differences occurred between the AD control group and any of the cohorts on the curcumin, GSE and MGSE diets.

The results for the buccal micronucleus cytome assay at months 3 and 9 are summarized and illustrated in Table 3.

3.3. Bodyweight

4.2. Micronucleated cells (combined basal and differentiated micronucleated cells)

Bodyweight data for all groups after 3 and 9 months of dietary treatment is shown in Table 2. At 3 months there was no significant difference in bodyweight between the WT control and AD control groups. A significant reduction in bodyweight occurred in AD CUR mice compared to the AD control group (p < 0.01). At 9 months there was a significant increase in bodyweight for the AD control mice compared to WT control mice (p < 0.05). No significant differences in bodyweight occurred between AD control and the AD CUR, AD GSE and AD MGSE diet groups.

4.1. Basal cells There were no significant differences in basal cell frequency between the WT Control and AD control group at both the 3 and 9 months time points. There were no significant differences in basal cell frequency between the AD control group and curcumin, GSE and MGSE groups at both the 3 and 9 months time points.

After 3 months there was no significant difference in the frequency of micronucleated cells between the WT Control and AD control group and between the AD control and the curcumin, GSE and MGSE dietary groups. After 9 months a non-significant 1.7-fold increase in micronucleated buccal cells occurred in the AD control compared to the WT control group. A significant decrease in micronucleated cells (p < 0.05) occurred in both the curcumin and MGSE dietary groups compared to the AD control group.

Table 3 DNA damage endpoints measured after 3 and 9 months dietary treatment. WT CON

AD CON

AD CUR

AD GSE

AD MGSE

One-way ANOVA

Erythrocyte (3 month) MN PCE (mean ± SD) MN NCE (mean ± SD) PCE/2000 NCE (mean ± SD)

4.33 ± 1.44 6.62 ± 3.31 3.07 ± 0.51

4.60 ± 1.17 6.30 ± 3.86 3.30 ± 0.81

3.91 ± 1.97 5.36 ± 1.50 2.61 ± 0.34*

3.00 ± 1.55 5.90 ± 1.92 2.82 ± 0.31

4.30 ± 1.76 5.60 ± 2.76 3.24 ± 0.49

0.138 0.181 0.014

Erythrocyte (9 month) MN PCE (mean ± SD) MN NCE (mean ± SD) PCE/2000 NCE (mean ± SD)

3.65 ± 2.29 5.58 ± 1.90 3.53 ± 0.695

4.60 ± 1.17 5.60 ± 1.83 3.96 ± 0.845

2.81 ± 1.32 5.45 ± 1.91 3.95 ± 0.65

3.00 ± 1.54 4.36 ± 1.85 4.51 ± 1.77

1.60 ± 1.17** 4.00 ± 1.76 4.56 ± 1.41

0.004 0.11 0.064

Buccal assay (3 months) % Basal cells (mean ± SD) Buccal MN (mean ± SD)

1.67 ± 0.71 0.21 ± 0.41

1.59 ± 0.93 0.30 ± 0.57

1.08 ± 0.53 0.18 ± 0.39

1.61 ± 0.63 0.27 ± 0.45

1.11 ± 0.28 0.30 ± 0.57

0.06 0.862

Buccal assay (9 months) % Basal cells (mean ± SD) Buccal MN (mean ± SD)

1.37 ± 0.84 0.29 ± 0.50

1.27 ± 0.63 0.50 ± 0.51

0.98 ± 0.30 0.05 ± 0.22*

1.21 ± 0.36 0.18 ± 0.50

1.48 ± 0.48 0.05 ± 0.22*

0.439 <0.0001

Telomere length (9 months) Buccal cell (mean ± SD) Olfactory bulb (mean ± SD) * ** a

5.20 ± 9.16 30.52 ± 36.4

p < 0.05 when compared to the AD control group. p < 0.01 when compared to the AD control group. p < 0.05 when compared to the WT control group.

0.47 ± 0.70a 14.26 ± 15.0

1.25 ± 3.15 29.44 ± 56.8

1.13 ± 2.29 9.55 ± 5.81

1.15 ± 1.09 10.71 ± 17.1

p = 0.04 p = 0.02

P. Thomas et al. / Mutation Research 661 (2009) 25–34

4.3. Karyolytic cells After 3 months dietary intervention there were no significant differences in the frequencies of early, mid or late karyolytic cells between the WT control and the AD control groups. A significant reduction in mid karyolytic cells occurred in the MGSE dietary group compared to the AD control group (p < 0.05). After 9 months dietary intervention there were no significant differences in the frequencies of early, mid or late karyolytic cells between the WT control and the AD control groups or between the AD control and curcumin, GSE and MGSE dietary groups.

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compared to the AD control group, whereas both the AD GSE and MGSE cohorts showed a non-significant reduction in telomere length when compared to the AD control cohort. 6.2. Buccal cell absolute telomere length

5. Whole blood micronucleus erythrocyte assay

The results for absolute telomere length in buccal cells for all the cohorts sampled are shown in Table 3. A significant decrease (p = 0.04) in absolute buccal telomere length was evident in the AD control when compared to the WT control. The AD Cur, AD GSE and AD MGSE polyphenol cohorts tended to have longer telomeres when compared to the AD control group but significance was not achieved.

Results for this assay at 3 and 9 months post-treatment are shown in Table 3.

6.3. Correlation between buccal cell and olfactory bulb telomere length

5.1. Micronucleated PCE At 3 months there were no significant differences in the frequency of micronucleated PCE between the WT control and AD control group. There was a trend for increased MN in PCE’s as mice got older but increases were not significantly different between 3 and 9 months. After 9 months dietary intervention there were no significant differences in the frequency of micronucleated PCE between the WT control and AD control group. The frequency of micronucleated PCE was significantly reduced (65.3%) for the AD MGSE cohort (p < 0.05), whereas the AD Cur and AD GSE groups were non-significantly reduced by 39.2 and 34.8% respectively compared to the AD control. 5.2. Micronucleated non-polychromatic erythrocytes No significant differences occurred in the micronucleated nonpolychromatic erythrocyte frequency between the WT Con and AD Con at both 3 and 9 months time points. No significant differences occurred between the AD control and the AD Cur, AD GSE and MGSE cohorts at both 3 and 9 months. 5.3. Polychromatic erythrocyte percentage There were no significant differences in polychromatic erythrocyte percentage between the WT control and the AD control groups at both 3 and 9 months time points. No significant differences occurred between the AD Con and AD Cur, AD GSE and AD MGSE cohorts at both 3 and 9 months time points. 5.4. Correlation between micronuclei in buccal cells and polychromatic erythrocytes Combined data for all cohorts showed no significant correlation (r = 0.09, p = 0.28) between the combined MN for basal and differentiated buccal cells and micronuclei in the polychromatic erythrocytes. 6. Telomere length at 9 months 6.1. Olfactory bulb brain tissue The results for absolute telomere length in olfactory bulb tissue for all the cohorts sampled are shown in Table 3. A non-significant 2-fold decrease in absolute olfactory bulb telomere length was evident between the AD control and WT control groups. The AD Cur cohort had a non-significant 2-fold increase in telomere length

Combined data for all cohorts did not correlate between buccal cell and olfactory bulb telomere length (r = −0.12, p = 0.23). There were no significant gender effects observed within any of the cohorts for any of the parameters investigated. 7. Discussion Oxidative stress (OS) is thought to play a key role in the early stages of AD pathology [1,12,40]. OS results from an imbalance between free radical formation, and the counteractive endogenous antioxidant defense systems such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Reactive oxygen species (ROS) are damaging to cells leading to the oxidation of essential cellular components such as proteins and lipids, leading to eventual genomic instability events such as MN formation and telomere shortening [2,3]. Results from this study show differences in biomarkers for DNA damage between the transgenic AD control group and the WT control groups. A buccal micronucleus cytome approach was used and showed an increase in MN frequency for both control groups over the duration of the 9-month study. WT controls showed an overall 8% increase whereas the AD control group showed an overall increase in MN frequency of 20%. After 9 months a non-significant 1.7-fold increase in MN frequency was apparent in the transgenic AD buccal mucosa compared to the WT group. Differences were apparent in the MN frequency between AD control mice and the AD groups with an increased dietary polyphenol intake. The results suggest a potential protective effect from a polyphenol-enriched diet as the MN frequency in buccal cells was reduced compared to the control diets. The curcumin cohort showed a 72% decrease in the buccal MN frequency over the course of the study whereas the GSE and MGSE cohorts showed an overall decrease of 9 and 84% respectively. There was a significant 10-fold decrease (p = 0.01) in buccal MN frequency at 9 months between the AD control group and both the curcumin and MGSE cohorts and a significant 7-fold decrease for the GSE cohort (p = 0.02). The frequencies of micronuclei as scored in the assay were found not to be significantly different between basal cells (0.31%) and differentiated cells (0.38%). This is the first time that buccal cells have been used to investigate the effects of polyphenols on MN frequency in a transgenic mouse model for AD and suggest that studies in other models and humans might be feasible with this approach. Similarly in whole blood a 24% increase in micronucleated polychromatic erythrocytes occurred in the WT control group compared to a 50% increase in the AD controls during the study time frame. After 9 months the AD control had a non-significant 1.3-fold increase in MN frequency compared to the WT control. Differences in MN frequency were also apparent between the AD control

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and polyphenol diet groups. All the polyphenol groups showed a reduction in MN frequency between the 3 and 9 months sample points. However, no significant positive correlation for MN frequency between these tissues was evident (r = 0.09, p = 0.28). These results suggest that the polyphenol diets may be protective in reducing the MN frequency in both buccal mucosa and whole blood, at the levels investigated confirming the results of earlier in vitro studies involving human lymphocytes [26,41,42]. Taken together, these data suggest that the AD control cohorts tend to have an increased rate of DNA damage in different tissues beyond the rate of normal ageing. It is possible that had sampling occurred at a later stage of disease progression these initial trends may prove to be significantly different. These findings are similar to the results in human buccal mucosa and lymphocytes which show a similar trend for increased MN frequency in untreated AD cases compared to age matched controls [3,11]. It has been shown that in AD the olfactory system inclusive of the olfactory bulb (OB) is severely damaged which results in impaired olfaction and is now considered part of the early clinical phenotype of AD [43]. It has also been demonstrated by immunohistochemistry that the OB contains both neurofibrillary tangles and amyloid deposits, characteristic hallmarks of AD [44]. There is a close relationship existing between cortical degenerative and olfactory changes and suggests that olfactory bulb involvement is one of the earliest events in the degenerative process to occur in AD [43]. Previous studies have shown that PSEN 1 mRNA and betaamyloid precursor protein mRNA is expressed in the developing rat olfactory and vestibulocochlear systems [45]. Telomere length was investigated as a biomarker of genomic instability in both buccal cells and OB brain tissue, as telomere length has been shown to be compromised in AD and under conditions of OS [15,46]. In buccal cells a significant (p = 0.04) 11-fold reduction in telomere length was found in the AD control group compared to the WT control. This reduction in buccal telomere length in the AD control cohort may be due to increased levels of OS, or a higher turnover rate of cells. These results are in agreement with the findings from a human study where telomere length was found to be shorter in AD buccal cells compared to age and gender matched controls [14]. All polyphenol cohorts showed a non-significant 2-fold increase in buccal cell telomere length compared to the AD control cohort and a non-significant 4.3fold decrease compared to the WT cohort. This would suggest that polyphenols, possibly through their effective scavenging of ROS and activation of endogenous defense mechanisms, may provide some degree of protection towards telomere maintenance in the buccal mucosa at the levels investigated. However the study was not powered sufficiently to detect significant effects in this tissue. Telomere length within the OB brain tissue of AD controls was found to be non-significantly reduced by 2-fold compared to the WT control group. Both the GSE and MGSE polyphenol group were found to have a non-significant 34 and 25% reduction in brain telomere length compared to the AD control group. This slight reduction may be explained as a result of sampling differences. If samples from the olfactory bulb were to contain increased levels of proliferative cells then it would be expected that the overall telomere length would be reduced following cellular division. AD mice that were fed a curcumin rich diet were found to have a 2-fold increase in brain telomere length compared to the AD control group, and were found to be only 4% shorter than the WT control cohort. It would appear, based on these results, that curcumin is probably more beneficial in maintaining telomere length within the olfactory bulb than either GSE or MGSE. This may be related to the fact that curcumin has the ability to induce expression of genes involved in the cellular stress response network [47]. Curcumin can activate Heme oxygenase-1 (HO-1) which increases cellular resistance to

oxidative stress and phase II enzyme expression in neural tissue through the activation of the transcription factor Nrf2, affording significant protection to neurons exposed to oxidative stress [47–50]. Furthermore, curcumin and GSE have been shown to inhibit the formation of amyloid oligomers and prevents fibril formation that lead to the formation of amyloid plaques [16,20,28,51]. Beta amyloid has been shown to form complexes with copper and iron to produce hypochlorous acid that increase the levels of OS within the brain [8,9]. Both Curcumin and GSE act as a metal chelator binding these metals, possibly reducing amyloid levels resulting in lower levels of oxidative stress [52,53]. Similarly, future studies involving telomere length should be undertaken to investigate the effect of polyphenol diets on WT control mice to determine whether the same changes in brain pathology manifest themselves under similar dietary conditions. It has previously been shown that both curcumin and GSE are effective antioxidants and have contributory roles in protecting cells from OS and resulting DNA damage [25,54]. It is thought that the effectiveness of these compounds as free radical scavengers can be directly attributed to their structural characteristics. The phenolic and methoxy groups on the curcumin benzene rings and the 1,3-diketone system are thought to be important structures for effective oxygen free radical scavenging [55,56]. GSE has been shown to have a high number of conjugated structures between the B-ring catechol groups and the 3-OH free groups of the polymeric skeleton allowing these compounds to be effective free radical scavengers and metal chelators [41,57]. As GSE scavenges free radicals, the resulting aroxyl radical formed has been shown to be more stable than those generated from other polyphenolic compounds potentially aiding in preventing further DNA damage [58]. Further mechanisms that have been attributed to both curcumin and GSE that enhance their effectiveness as powerful antioxidants involve the enhanced synthesis of the endogenous antioxidant enzymes SOD, CAT and GPx [42,59,60]. Reduced glutathione is another endogenous antioxidant that regulates the expression of antioxidant genes [19,61]. GSH levels and subsequent activity is maintained by the enzyme ␥-glutamylcysteine ligase catalytic subunit (GCLC) which in turn is regulated by OS [62]. It has been shown that both curcumin and GSE protect against GSH depletion by sequestering ROS and increases GSH synthesis by enhancing GCLC expression [19,63]. A weak non-significant inverse correlation was found between telomere length in brain tissue and buccal mucosa (r = −0.15, p = 0.24), indicating that it is not possible to use buccal cells as surrogates for telomere length measures in the brain. At this stage it is uncertain the contribution made by each cellular population to overall telomere length. It would be interesting to perform further studies using flow cytometry or Q-FISH to isolate populations of cells to determine the contribution of both basal and differentiated cells to overall telomere length. It has been shown in this study that a high proportion of buccal cells are under going various stages of karyolysis which may distort the true telomere length of the progenitor basal cells by over representing senescent cells that would be expected to contain shorter telomeres. Only future studies on progenitor basal cells will be able to determine whether a stronger correlation exists between these two tissues, which if proven may lead to buccal cells potentially being used as a noninvasive biomarker reflecting physiological changes within telomere length in the brains of premature ageing mouse models for AD. The buccal micronucleus cytome assay also provided measures of potential cell proliferation in relation to potential genotoxicity of the polyphenols at the levels investigated. Basal cell frequency within both the control groups showed a slight but non-significant decrease over the duration of the study. After 9 months the basal cell frequency in the AD control group had decreased by 7% possi-

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bly indicating the initial stages of a decline in regenerative potential with advancing age [64,65]. It would have been interesting to have sampled the buccal mucosa at later time points, in order to determine whether similar changes in basal cell frequency reflect those observed in earlier human studies which showed a sharp reduction in buccal basal cell frequency in AD cases compared to controls [11,38]. All of the polyphenol cohorts did not show any marked changes in the frequency of basal cells which may be possibly indicative as a proliferation marker compared to controls suggesting that polyphenols at the dose used in the study had no cytotoxic or cytostatic effects. Similarly karyolytic cell frequency was not different between dietary groups after 9 months. Upon completion of the 9-month study there were no significant differences for food consumption and/or bodyweight between the WT control group and the AD control group or between the polyphenol cohorts and the AD control group. However at three months there was a significant reduction in both food consumption and bodyweight for the curcumin cohort compared to the AD control group. Similarly, a significant reduction in food consumption occurred for the GSE and MGSE cohort although no significant loss in bodyweight was recorded. This may have been attributable to a certain degree of unpalatability associated with the diet to which the animals may have possibly become habituated to with time, having previously been fed the AIN-93G diet. No change in behavior or mortality was noted. Previous studies have shown that increased dietary polyphenols had no adverse effects on bodyweight [29,66]. It would also be important to determine the effect of bodyweight and food consumption on control WT mice when fed high polyphenol diets to observe any similar trends to those seen in the AD groups. Although the study revealed differences in DNA damage measures between the WT control and AD control mice, the lack of significance of the majority of biomarkers investigated may be due to the fact that they are not a sensitive enough indicator of AD pathology in mice. Alternatively the study may have been terminated prematurely before the selected biomarkers could significantly reflect changes in AD pathology, as shown in previous human studies where the same battery of biomarkers were used in clinically diagnosed AD patients. As trends were apparent between the WT control and AD control mice further studies would need to be performed to relate the stage of disease progression and the significance and efficacy of the DNA damage biomarkers measured. Future studies involving this mouse model and potential polyphenol cohorts should be taken over a longer time frame with intermittent evaluation of cognitive impairment measures. This data together with levels of beta 42 and genomic instability events can then be used to determine the efficacy of polyphenols as potential protective agents in AD pathology by relating the stage of disease progression within this mouse model to its human counterpart whether it is mild cognitive impairment, early or late AD. In light of the non-significance of some of the biomarkers investigated between the control groups, it is important to perform repeat experiments involving WT control mice with the various polyphenol diets used in this study. This will allow us to investigate potential protective effects between the AD control mice and WT mice in relation to genome instability markers. In conclusion, these results are suggestive of a potential protective effect of a polyphenol enriched diet in terms of reducing DNA damage events, such as micronuclei and telomere length in different somatic tissue types in this transgenic mouse model, with no adverse effects on cell proliferation rates or cell death markers. Only future experiments thoroughly investigating the effects of polyphenols on brain pathology in relation to genomic instability events can support or refute the potential protective effects of polyphenols in AD pathology.

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