Prolonged coenzyme Q10 treatment in Down syndrome patients, effect on DNA oxidation

Neurobiology of Aging 33 (2012) 626.e1– 626.e8 www.elsevier.com/locate/neuaging

Prolonged coenzyme Q10 treatment in Down syndrome patients, effect on DNA oxidation Luca Tianoa,*, Lucia Padellab, Lucia Santorob, Paola Carnevalib, Federica Principia, Francesca Brugèa, Orazio Gabriellib, Gian Paolo Littarrua b

a Department of Biochemistry Biology and Genetics, Polytechnic University of Marche, Ancona, Italy Clinic of Pediatrics, Department of Clinical Sciences, Polytechnic University of Marche, Azienda Ospedali Riuniti, Presidio Salesi, Ancona, Italy

Received 5 October 2010; received in revised form 22 February 2011; accepted 25 March 2011

Abstract Oxidative stress is known to play a relevant role in Down syndrome (DS) and its effects are documented from embryonic life. Oxidative DNA damage has been shown to be significantly elevated in Down syndrome patients, and this has been indicated as an early event promoting neurodegeneration and Alzheimer type dementia. The aim of this study was to investigate the efficacy of coenzyme Q10 (CoQ10) in delaying the effect of oxidative damage in these patients. In our previous study we demonstrated a mild protective effect of CoQ10 on DNA, although the treatment was unable to modify the overall extent of oxidative damage at the patient level. Possible limitations of the previous study were: time of treatment (6 months) or spectrum of DNA lesions detected. In order to overcome these limitations we planned a continuation of the trial aimed at evaluating the effects of CoQ10 following a prolonged treatment. Our results highlight an age-specific reduction in the percentage of cells showing the highest amount of oxidized bases, indicating a potential role of CoQ10 in modulating DNA repair mechanisms. © 2012 Elsevier Inc. All rights reserved. Keywords: Down syndrome; Oxidative DNA damage; Comet assay; Coenzyme Q10

1. Introduction Oxidative stress in Down syndrome (DS) depends on altered handling of reactive oxygen species due to an abnormal pattern of expression associated with the trisomy (Conti et al., 2007) as well as mitochondrial dysfunction found in DS cells from embryonic life (Busciglio et al., 1995). Although oxidative stress implications in DS phenotype are well documented (Capone, 2001; Jovanovic et al., 1998; Pallardó et al., 2006; Zana et al., 2007) the relevance of oxidative DNA damage in the syndrome is highly debated. Studies of oxidative modifications to nucleic acids in DS have been primarily associated with Alzheimer type

* Corresponding author at: Department of Biochemistry Biology and Genetics, Polytechnic University of Marche, Via Ranieri, 60100 Ancona, Italy. Tel.: ⫹390712204394; fax: ⫹390712204398. E-mail address: [email protected] (L. Tiano). 0197-4580/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2011.03.025

dementia, a distinctive and severe hallmark of the syndrome that typically becomes evident in all patients by age 30 (Contestabile et al., 2010). A body of evidence supports the occurrence of chronic oxidative injury at the neuronal level that could imply a risk factor for subsequent neurodegeneration in aged DS patients (Iannello et al., 1999; Nunomura et al., 2001; Subba Rao, 2007). However these observations are in contrast with other reports pointing out the lack of significant elevations in 8-OH-dG content of postmortem aged DS brain tissues (Seidl et al., 1997). These data together with the observations of decreased levels in 8-OH-dG in neurons developing neurofibrillary tangles led to the conclusion that oxidative DNA damage is an early event in the pathogenesis of both Alzheimer’s disease (AD) and DS, and it decreases with disease progression and more compromised histopathology (Zana et al., 2007), suggesting compensatory intracellular mechanisms that reduce oxidative stress. In nonneuronal tissues there are few indications

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regarding the level of DNA damage and oxidative modifications of DNA in DS (Pallardó et al., 2006). These reports suggest an increased susceptibility to oxidative damage in lymphocyte DNA from DS patients (Maluf and Erdtmann, 2001), in particular in pediatric age (Morawiec et al., 2008; Tiano et al., 2009). Despite the pivotal role played by oxidative stress in DS-related pathologies and hence the potential of antioxidant therapies, aimed at minimizing and delaying the complications of trisomy 21, no scientifically proven diet or drug is yet available for the prevention and therapy against oxidative stress in DS (Zana et al., 2007). In recent years our research unit through the joint effort of biochemists, pediatricians and patients’ families, has been involved in evaluating clinical and biochemical effects of coenzyme Q10 (CoQ10) in DS patients. CoQ10 is a bioactive quinone produced by our cells, ubiquitous in the organism, endowed with 2 major functions in mitochondrial bioenergetics and as a lipophilic antioxidant (Littarru et al., 2010). It was chosen as an intervention molecule in light of its role both in supporting mitochondrial dysfunction (Estornell et al., 1992) and counteracting oxidative DNA damage (Tomasetti et al., 2001), conditions, known to characterize DS a clinical setting and underlying neurological deterioration, leading to Alzheimer-type dementia. In the last 3 decades only a few of the published supplementation studies were randomized trials and none of them were specifically designed to evaluate antioxidant therapy (Miles et al., 2007). Recently we published the results of a randomized clinical trial aimed at verifying the effect of CoQ10 treatment on DNA damage in pediatric DS patients, where patients were treated with either 4 mg/kg per day of CoQ10 or placebo for 6 months (Tiano et al., 2009). Results highlighted a mild protective effect on the main population of lightly damaged cells, although no significant overall effect was observed at the patient level. Possible limitations of the study were identified in the limited treatment time as well as the type of DNA lesion detected. Here we describe the follow-up of the study where the entire cohort agreed to participate. Thirty patients, after a washout period of 3 months, were treated for 20 months with 4 mg/kg CoQ10 per day. The aim of the study was to evaluate the efficacy of a prolonged treatment in minimizing oxidative stress and damage to DNA in peripheral blood leukocytes of DS patients. In particular, biochemical endpoints included a higher resolution of DNA damage obtained through the use of DNA repair enzymes, Fpg and Endo III in association with the comet assay, as well as the evaluation of plasma total antioxidant capacity and allantoin levels, a product of nonenzymatic oxidation of uric acid (UA). 2. Methods 2.1. Subjects This study was approved by the Ethical Committee of the Marche Polythecnic University and Regional Hospital

Table 1 Demographic and clinical features of DS patients at the study entry

n (male) Age (years) BMI (kg/m2) Chol (mg/dL) CoQ10/Chol (nmol/L/mmol/L)

Total

5–12 Years

13–17 Years

17 10 ⫾ 0.8 19.7 ⫾ 1.2 154 ⫾ 9 111 ⫾ 7

11

6 14.8 ⫾ 0.3 22.8 ⫾ 0.8 149 ⫾ 5 124 ⫾ 6

9 ⫾ 0.4 20.2 ⫾ 0.8 169 ⫾ 6 115 ⫾ 6

Key: BMI, body mass index; Chol, cholesterol; CoQ10, coenzyme Q10; DS, Down syndrome.

“A.O. Ospedali Riuniti”, and performed with the subjects or family’s written informed consent. The DS patients were included if they had an age ranging between 5 and 17 years. Demographic and clinical features of patients at the study entry are summarized in Table 1. Exclusion criteria were the presence of autoimmune diseases (hypothyroidism, celiac disease, diabetes), pharmacological therapy or vitamin supplementation, and congenital cardiovascular disease. Thirty patients were assessed as eligible and enrolled in the study. Twenty-eight subjects completed the study: 2 patients dropped out for personal reasons. All the patients received CoQ10 (Mitoquinone, Pharma Nord, Vejle, Denmark) orally at doses of 4 mg/kg per day for 20 months. Patients were monitored every 6 months in concomitance with routine day-hospital visits at the local pediatric clinic. On these occasions hematological analysis was performed. At each visit a new supply of CoQ10 was given to the patients and compliance was checked counting residual soft-gels from the previous visit. Blood samples were collected at study entry and after 20 months of therapy. In order to evaluate age-dependent variations associated with changes in the endocrine status characterizing the onset of puberty data were stratified according to patient age namely 2 subgroups were considered: prepubertal (age 5–12; n ⫽ 20) and pubertal (age 13–17; n ⫽ 8). 2.2. Uric acid and allantoin determination Human blood was collected in heparinized tubes. Plasma was separated from blood cells by centrifugation at 3000g for 10 minutes, transferred to a fresh tube and stored at ⫺80 °C until required. Before analysis, plasma samples were thawed at room temperature and deproteinized with 1/25 volume of 4.6 mol/L HClO4 then centrifuged for 10 minutes at 12,000 rpm and ultrafiltered through a 0.2 ␮m filter. A 20 ␮L sample of plasma ultrafiltrate was injected into a 250 ⫻ 4 mm Supelcosil LC18-DB analytical column from Supelco. This column was protected by a Supelguard LC18DB precolumn. The mobile phase was 5 mmol/L potassium phosphate buffer, pH 4.6 and 5% methanol at a flow rate of 0.8 mL per minute. UA was detected at 254 nm. Under these conditions a fraction covering the retention time range 2.9 –5.5, where allantoin is known to be eluted, was collected, and then evaporated to dryness at 40 °C under vacuum. The residue was reconstituted in 100 ␮L of

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0.12 mol/L NaOH and incubated for 20 minutes at 100 °C. After adding 100 ␮L of 1 mol/L HCl of 3 mmol/L dinitrophenylhydrazine in 1 mol/L HCl, heating at 100 °C continued for 5 minutes. After cooling the derivative samples, a 20 ␮L volume was injected onto a Supelcosil LC18-DB analytical column (Supelco, Bellefonte, PA, USA). The mobile phase was 70% vol/vol 30 mmol/L sodium citrate/27.7 mmol/L sodium acetate buffer, pH 4.75, and 30% vol/vol methanol at a flow rate of 0.8 mL per minute. Detection was performed at 360 nm wavelength. 2.3. Plasma and cellular CoQ10 CoQ10 levels were assayed using a dedicated high-performance liquid chromatography (HPLC) system with electrochemical detector (ECD) by Shiseido Co Ltd. (Tokyo, Japan). Mobile phases were as described (Sekine et al., 2005). Pump 1 and 2 were Model 3001, auto sampler Model 3033, switch valve Model 3012, concentration column Capcel I Pak C8 DD and separation column Capcel I Pak C18 AQ, all from Shiseido Co Ltd. A peculiarity of the system was the use of a postseparation reducing column (CQ; 20 ⫻ 2.0 mm inner diameter) capable of fully reducing the peak of oxidized CoQ10. The oxidation potential for electrochemical detector was 650 mV. Fifty microliters of plasma or counted cell suspension in phosphate-buffered saline (PBS) were extracted by adding 250 ␮L of isopropanol, vortexed and centrifuged at 12,000 rpm for 2 minutes, 40 ␮L of propanolic extracts were directly injected in the HPLC column. Plasma and cellular levels of CoQ10 were expressed as ␮g/mL and ng/106 cells respectively. 2.4. Plasma oxygen radical absorbing capacity The antioxidant activity of plasma was assessed with the oxygen radical absorbing capacity (ORAC) assay according to the method described by Gillespie (Gillespie et al., 2007). Briefly, 150 ␮L of 0.08 ␮mol/L fluorescein dissolved in 75 mmol/L PBS was added in each well of a 96-well solid black microplate, followed by 25 ␮L of plasma previously diluted (1:200). After a 10-minute incubation in the dark, 25 ␮L of 147 mmol/L 2,2’-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH) was rapidly added to each well by using a repetitive multichannel pipette and the microplate was immediately placed in a microplate reader (Synergy HT, Bio-tek, Winooski, VT, USA). Fluorescence was recorded every 120 seconds for 3 hours, using an excitation wavelength of 485/20 nm and an emission filter of 528/20 nm. The net area under the fluorescence decay curve (AUC) for each compound was obtained by data processing using KC4 software (Synergy HT, Bio-tek, Winooski, VT, USA). To determine antioxidant activity, a 10 mmol/L stock solution of trolox was used as standard to construct a calibration curve in the range 0 to 200 ␮mol/L. The results were expressed as the concentration of trolox equivalent to the area under the fluorescence decay curve of each plasma sample.

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2.5. Platelets isolation Platelets were isolated by centrifuging whole blood at 90g for 15 minutes to obtain a platelet-enriched plasma. Platelets were counted by means of an automated cell counter (Coulter Microdiff18, Brea, CA, USA) and counted pellets were obtained by centrifugation at 1500g for 10 minutes and immediately frozen at ⫺80 °C for further analysis. 2.6. Lymphocyte isolation and cryopreservation Lymphocytes were isolated by stratification over Lymphoprep (Frasenius Kabi, Oslo, Norway) and centrifugation at 693g for 20 minutes at 4 °C. After 2 washes cell suspensions in PBS were cryopreserved with an equal volume of freezing mix containing human serum albumin (HSA) 5%; HSA 20%, dimethyl sulfoxide (DMSO) (3:1:1). Cryovials were chilled slowly (in boxes of expanded polystyrene) to ⫺80 °C. After completing the freezing process vials were transferred to liquid nitrogen for long-term storage. At the time of analysis, cells were rapidly thawed by immersion in a water bath at 37 °C, and immediately supplemented with an equal volume of a solution containing Eudextran (Frasenius Kabi, Oslo, Norway) and HSA 5% (1:1). 2.7. Comet assay 2.7.1. Preparation of the slides Dehibernated cells were resuspended in low melting agarose (LMA) at 0.7% and stratified on an HT Trevigen slide (Trevigen, Gaithersburg, MD, USA). Each slide was able to host 20 low melting agarose spots, each of them containing 1500 cells. Control and gamma-irradiated bovine lymphocytes as reference cells relative to low, mid, and high DNA damage levels were also embedded on the microscope slides. After agarose solidification cells were immersed in an alkaline lysis solution (pH 10) containing NaCl (2.5 mol/L), Na2 ethylenediaminetetraacetic acid sodium salt (EDTA) (0.1 mol/L), Tris-HCl (10 mmol/L), DMSO 10%, Triton x-100 1%, and kept at 4 °C for at least 1 hour. 2.7.2. Enzymatic treatment for detection of oxidative DNA damage After lysis the slides were washed twice for 15 minutes with enzyme buffer (40 mmol/L 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES), 0.1 mol/L KCl, 0.5 mmol/L EDTA, and 1% human serum albumin at pH 8) at 4 °C. Fifty microliters of enzyme buffer (used as control) or buffer containing Endo III or Fpg (dilution 1:3000) (kindly donated by Dr A Collins) was dropped onto each spot. The slides were then placed in a moist box and incubated at 37 °C for 45 minutes. 2.7.3. Electrophoresis Prior to electrophoresis, the slides were incubated for 20 minutes in the dark at 4 °C in alkaline electrophoresis buffer

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Table 2 Variation of coenzyme Q10 levels (in plasma and platelets), total antioxidant capacity, uricemia and allantoin/uric acid ratio after 20 months of treatment with coenzyme Q10 in the total DS patients and considering age-specific subgroups T0 T4 p 5–12 Years T0 T4 p 13–17 Years T0 T4 p

CoQ10 pl ␮g/mL

CoQ10 plt ng/106 cells

ORAC ␮mol/L eq trolox

UA ␮mol/L

All/UA

0.44 ⫾ 0.03 2.19 ⫾ 0.24 ⬍ 0.001

0.13 ⫾ 0.01 0.36 ⫾ 0.04 ⬍ 0.001

14.3 ⫾ 1.7 13.4 ⫾ 1.5 0.32

259 ⫾ 13 240 ⫾ 15 0.68

0.02 ⫾ 0.002 0.02 ⫾ 0.003 0.65

0.44 ⫾ 0.04 2.4 ⫾ 0.33 ⬍ 0.001

0.13 ⫾ 0.02 0.35 ⫾ 0.06 0.0017

14.1 ⫾ 2.1 12.9 ⫾ 1.7 0.21

233 ⫾ 13 215 ⫾ 15 0.35

0.02 ⫾ 0.002 0.02 ⫾ 0.003 0.8

0.42 ⫾ 0.06 1.68 ⫾ 0.38 0.03

0.15 ⫾ 0.03 0.35 ⫾ 0.08 0.04

14.9 ⫾ 5.0 15.2 ⫾ 4.1 0.38

321 ⫾ 30 321 ⫾ 42 0.38

0.016 ⫾ 0.004 0.008 ⫾ 0.002 0.1

Results are expressed as mean ⫾ standard error of the mean (SEM). Significance reported using Wilcoxon signed rank test. Key: CoQ10, coenzyme Q10; ORAC, oxygen radical absorbing capacity; pl, plasma; plt, platelets; UA, uric acid.

(1 mmol/L EDTA and 300 mmol/L sodium hydroxide, pH ⬎13) to produce single-stranded DNA and to express alkalilabile sites as single-strand breaks (unwinding). The singlestranded DNA in the gels were electrophoretically processed under alkaline (pH ⬎13) conditions to produce comets at 1 V/cm, neutralized in Tris-HCl pH 7.5 for 5 minutes, dehydrated in methanol for 2 minutes, and finally dried at 60 °C for 2 minutes and stored for microscopic examination. Before analysis, slides were stained with ethidium bromide (20 ␮g/mL) and viewed under fluorescent light using an Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan) connected to a PC. Observations were performed at a magnification of 200⫻. 2.7.4. Computer-aided comet analysis Thirty randomly acquired images for each patient in duplicate (15 for each spot) were processed using custom-made software that enable automatic identification of the comets, greatly reducing operator-dependent variability. 2.8. Statistical methods Because the distribution of the comet assay data resulted as strongly skewed, a nonparametric approach of analysis was followed at the patient level. Results are reported as % of DNA in the comet tail or tail intensity (TI) for each cell expressed as means of the median TI ⫾ standard error of the mean (SEM) at different experimental moments (study entry and 20 months). Moreover, cells were ranked into classes of damage according to threshold values defined through the use of reference cells exposed to different radiation doses, and the percentage of cells in each class was calculated. The comparison of DNA damage extent at different experimental moments was performed using Wilcoxon signed rank test. A sample size of 30 patients was calculated as having a probability of 81% to detect a treatment difference at a 2-sided 5% significance level considering the minimal detectable variation in tail intensity of damaged cells of 13% and a standard

deviation of the same parameter in the studied population of 12%. Pearson correlation coefficients and their significance levels were calculated for linear regression analysis. All the statistical analyses were performed using SAS 9.1 (SAS, Cary, NC, USA).

3. Results 3.1. Oxidative status in plasma 3.1.1. Plasma and cellular coenzyme Q10 levels Treatment with 4 mg/kg per day of CoQ10 for 20 months produced a significant increase both in plasma (⫹ 502%, p ⬍ 0.01) and platelets (⫹ 275%; p ⬍ 0.01). In particular, patients at the end of supplementation presented mean plasma levels of 2.19 ␮g/mL and 0.36 ng/106 platelets (Table 2). Patients were monitored every 6 months for routine visits as described in section 2. Methods. No adverse effects were recorded. Two patients dropped out for personal reasons. Patient compliance was verified during each visit and, very rarely, missing doses were recorded accounting at the most (in a couple of cases) for 8% of the 6 months total dosage (15 missing doses). These noncompliances probably did not affect bioavaiability because all patients showed significant increases in plasma CoQ10. 3.1.2. Uricemia and plasma allantoin levels DS patients displayed UA levels above the normal range at study entry, as often reported in this pathology. Treatment with CoQ10 for 20 months did not affect plasma UA levels nor did it lower allantoin/UA ratio in the overall population. Nonetheless, an interesting decrease in allantoin/UA was observed in a subgroup of patients (n ⫽ 6; 2 samples were inadequate for the assay) 13 to 17 years old, from 0.016 ⫾ 0.004 to 0.008 ⫾ 0.002. This reduction however was not statistically significant, probably due to the limited number of patients in this subgroup. Total antioxidant capacity was not affected by CoQ10 treatment (Table 2).

L. Tiano et al. / Neurobiology of Aging 33 (2012) 626.e1– 626.e8 Table 3 Effect of CoQ10 on cellular DNA damage in all DS patients and considering age-specific subgroups TI % DNA

⫺Fpg ⫺Endo III

⫹Fpg

⫹Endo III

T0 T4 p 5–12 years T0 T4 p 13–17 years T0 T4 p

5.0% ⫾ 0.3% 5.0% ⫾ 0.2% 0.99

36% ⫾ 2% 34% ⫾ 3% 0.57

8% ⫾ 1% 8% ⫾ 1% 0.6

4.9% ⫾ 0.4% 4.6% ⫾ 0.2% 0.75

34.3% ⫾ 2.1% 35.9% ⫾ 4.2% 0.73

7.3% ⫾ 0.8% 6.9% ⫾ 0.7% 0.63

4.6% ⫾ 0.6% 5.0% ⫾ 0.5% 0.62

40.5% ⫾ 5.5% 28.7% ⫾ 5.0% 0.1

9.0% ⫾ 1.5% 9.4% ⫾ 1.9% 0.7

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old) CoQ10 treatment approached significance in reducing the percentage of cells showing the highest amount of oxidized purines with differences close to statistical significance (Class III; p ⫽ 0.08) associated with an increase of cells in Class II (Fig. 3). The small sample size of the pubertal subgroup most likely underlies limited significance. After Fpg treatment, which allows to detect oxidized purine bases, a remarkable inverse correlation was found, in the overall population, between cellular CoQ10 content and the percentage of cells in Class III (p ⫽ 0.008) and a direct correlation with percentage of cells in Class II (p ⫽ 0.008) (Fig. 4).

Results are expressed as mean ⫾ standard error of the mean (SEM). Significance reported using Wilcoxon signed rank test. Key: CoQ10, coenzyme Q10; DS, Down syndrome.

3.2. Oxidative DNA damage in lymphocytes 3.2.1. “Classical analysis” When considering the classical comet index % of DNA in the comet tail (tail intensity) in the total population studied, we did not observe any significant variations in the basal DNA damage values after 20 months of treatment with CoQ10 in terms of double strand breaks (DSBs), single strand breaks (SSBs), and oxidized bases (Table 3). 3.2.2. Age-specific differences in the distribution of cells among 3 classes of DNA damage Using gamma-irradiated lymphocytes as reference for low/mid/high levels of DNA damage, a calibration curve was constructed and used to rank cells among 3 different classes of DNA damage. This analysis allowed the identification of age-specific differences in the distribution of the cells according to the integrity of their DNA. In fact, the older group of patients (aged from 13 to 17 years old) showed a higher proportion of cells in Class I (lowest basal DNA strand breaks [SBs]) and a decreased percentage of cells in Class III (highest basal DNA SBs) although not statistically significant. No differences were found in the distribution of cells according to the levels of oxidized bases comparing the 2 age groups (Fig. 1). 3.2.3. Effect of CoQ10 on distribution of cells among classes of DNA damage Treatment with CoQ10 did not modify the distribution of cells among the 3 classes of damage in the overall studied population, either when considering basal DNA damage or oxidized bases (Fig. 2). However, significant modifications in the pattern of distribution were observed, following CoQ10 treatment, when considering age-specific differences in the amount of oxidized bases. Younger patients (aged from 5 to 12 years old) showed a significant reduction in the % of cells with the highest amount of oxidized pyrimidines (Class III; p ⫽ 0.04) and a concomitant increase of the % of cells in Class II. In older patients (aged from 13 to 17 years

Fig. 1. Age-specific differences in the distribution of cells among 3 classes of DNA damage. Results are expressed as mean ⫾ standard error of the mean (SEM). Wilcoxon rank-sum test was applied but no statistically significant differences were found.

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trolled clinical trial (Tiano et al., 2009) where treatment for 6 months was shown not to produce, at the patient level, a significant protection in terms of DNA strand breaks. In this second phase we tried to address some limitations of that study mainly involving a short observational time frame and a limited type of DNA lesion detected. In relation to time of treatment, prolonging it from 6 to 20 months did not produce any significant variation on the overall extent of DNA damage at the patient level quantified as DNA strand breaks. In a study where lymphocytes from healthy volunteers had been supplemented with CoQ10 (either in vitro or in vivo) and challenged with 100 ␮mol/L H2O2 (Tomasetti et al., 2001), protection was evident, both in terms of decrease of DNA strand breaks and of increased SB repair capacity. This discrepancy might arise from the different type of oxidative stress experienced in vivo by our patients and in vitro in the mentioned report; moreover, altered SB repair capacity associated with the disease could be involved. In relation to the type of lesion, the use of repair enzymes allowed a higher resolution of DNA damage detection highlighting the presence of remarkably high levels of oxidized purines as reported also by Zana and Pallardó. In this study first of all we highlighted age-specific differences in the patients, probably due to compensatory responses associated

Fig. 2. Effect of coenzyme Q10 (CoQ10) on DNA damage pattern analysis. Results are expressed as mean ⫾ standard error of the mean (SEM). No statistically significant differences were found. Wilcoxon signed rank test was applied.

4. Discussion Treatment of pediatric DS patients with 4mg/kg per day CoQ10 for 20 months, produced a significant rise in its plasma levels not associated with any variations in the considered plasma oxidative stress-related indexes (ORAC and allantoin levels). Supplementation of DS pediatric patients with 10 mg/kg per day of ubiquinol has been shown to affect CoQ10 oxidative status in plasma correcting unbalanced ratio of CoQ oxidized/total (Miles et al., 2007). Unfortunately in our study it was not possible to reliably assess this index because time of sample processing exceeded 1 hour. Concerning oxidative DNA damage-related parameters, this study represents the prolongation of a shorter randomized double blinded con-

Fig. 3. Effect of coenzyme Q10 (CoQ10) on the distribution of oxidized pyrimidines and purines considering age-specific differences. Results are expressed as mean ⫾ standard error of the mean (SEM). Oxidized pyrimidines in younger patients (aged from 5 to 12 years old; n ⫽ 20; ⴱ p ⫽ 0.04). Oxidized purines in older patients (aged from 13 to 17 years old; n ⫽ 8, ⫹ p ⫽ 0.08). Statistical significance calculated using Wilcoxon signed rank test.

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Fig. 4. Correlation between % of cells with increased levels of oxidized purines and cellular coenzyme Q10 (CoQ10) content (n ⫽ 27; p ⫽ 0.008). Direct linear correlation between % of cells with oxidized purines with a mid level of damage and cellular CoQ10 content (n ⫽ 27; p ⫽ 0.008).

with a chronic exposure to stress conditions, leading to a reduction in DNA SB in adolescent patients (13–17 years) compared with younger ones (5–12 years), as suggested also by other authors (Pallardó et al., 2006; Zana et al., 2006). As proposed by Pallardó, endocrine effectors associated with the onset of puberty could be responsible for substantial changes in the antioxidant status and expression of longevity-related genes, which would at least partially explain the paradoxical compensatory mechanisms experienced in DS patients along with the transition to puberty and aging. Age-related differences probably do not affect the mechanism of repair of oxidized bases because the pattern of distribution of cells in relation to Fpg/Endo III-sensitive sites does not vary significantly in the 2 age groups. On the contrary, age-related differences should be taken into account considering the effect of CoQ10 on base oxidation indicating that treatment was able to affect the distribution of cells according to their content in oxidized bases: in the younger subjects treatment decreased the amount of oxidized pyrimidines, while in older ones it was effective in lowering the amount of cells with oxidized purines. Although the differences were only close to significant, this trend was also supported by a strong negative correlation linking cellular CoQ10 content and the amount of oxidized purines in the overall population. This effect in DS might be related both to CoQ10 bioenergetic activity, minimizing con-

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genital mitochondrial impairment, and to its antioxidant properties. In particular, ubiquinol could act directly as a scavenger of reactive oxygen species (ROS) as well as, in association with vitamin E, inhibiting lipid peroxide-induced DNA damage (Lim et al., 2004). In fact vitamin E has been shown to exert a protective effect on chromosomal injury in DS patients through an antioxidant mechanism (Pincheira et al., 1999). Moreover, the effect of CoQ10 might also be associated with a gene induction of DNA repair enzymes. To our knowledge only nucleotide excision repair (NER) systems have been investigated in DS (Raji and Rao, 1998) showing a decreased repair ability associated with the syndrome. These systems are not the major repair mechanisms involved in counteracting oxidative DNA damage. In fact the oxidatively damaged bases are preferentially repaired by enzymes of the base excision repair (BER) pathway (Loft et al., 2008). The absence of a measurable plasma antioxidant response, which could be masked by the characteristic hyperuricemia, together with unchanged levels of DNA SB harmonize with the hypothesis that CoQ10 acts not simply as a reactive oxygen species (ROS) scavenger, but possibly also stimulating oxidative damage repair enzymes. In fact, ubiquinol, the reduced form of coenzyme Q10, might affect the intracellular redox potential involved in the regulation of gene expression (Groneberg et al., 2005). Evaluation of the modulation of the redox status of CoQ10 in plasma and at the cellular level following supplementation, could have supported this hypothesis and will be taken into account for further studies. Agerelated differences in the activity of CoQ10 should be considered in relation to transition to puberty associated with estrogens production. Sex hormones are known to modulate antioxidant-related gene expression through transduction pathways involving nuclear translocation of NFkB (Borrás et al., 2005), a transcription factor influenced also by CoQ10-mediated changes in intracellular redox status (Schmelzer et al., 2007). Further studies regarding the interplay of these actors might provide useful information to describe molecular mechanism underlying age-related differences described. In conclusion, although at patient level, 20 months of treatment with 4 mg/kg per day CoQ10 did not affect the overall extent of DNA damage, the present work suggests that CoQ10 might act as a modulator of DNA repair mechanisms. Research in this respect warrants further investigation because it could represent a target for delaying neurodegenerative processes in adult Down syndrome patients. Limitations of the study that should be addressed in future research include the CoQ10 dosage used and the association of other mitochondrial nutrients. In particular, taking into account the dosage of CoQ10 used in neurological disorders and the promising results of Miles et al. (2007) in correcting CoQ10 oxidative status in DS patients, a dose of 10 mg/kg per day could produce more consistent results at the patient level. Moreover, a factorial design should allow the determination of CoQ10 effect alone or in association with other mitochondrial nutrients that could act synergistically in counteracting congenital mitochondrial dysfunction (Tar-

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