Oxidative stress and oxidative DNA damage is characteristic for mixed Alzheimer disease/vascular dementia

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Journal of the Neurological Sciences 266 (2008) 57 – 62 www.elsevier.com/locate/jns

Oxidative stress and oxidative DNA damage is characteristic for mixed Alzheimer disease/vascular dementia Daniel Gackowski a , Rafal Rozalski a , Agnieszka Siomek a , Tomasz Dziaman a , Krzysztof Nicpon b , Maciej Klimarczyk c , Aleksander Araszkiewicz c , Ryszard Olinski a,⁎ a

Department of Clinical Biochemistry, Nicolaus Copernicus University, Collegium Medicum in Bydgoszcz, Karlowicza 24, 85-092 Bydgoszcz, Poland b E. Warminski City Hospital, Szpitalna 19, Bydgoszcz, Poland c Department of Psychiatry, Nicolaus Copernicus University, Collegium Medicum in Bydgoszcz, Kurpinskiego 19, 85-094 Bydgoszcz, Poland Received 21 June 2007; received in revised form 29 August 2007; accepted 30 August 2007 Available online 20 September 2007

Abstract Oxidative DNA damage may contribute to neuronal cell loss and may be involved in pathogenesis of some neurodegenerative diseases. We assessed the broad spectrum of oxidative DNA damage biomarkers and antioxidants in mixed Alzheimer disease/vascular dementia (MD) and in control patients. The amount of the products of oxidative DNA damage repair (8-oxo-2′-deoxyguanosine and 8-oxoguanine) excreted into urine and cerebrospinal fluid (CSF) was measured by gas chromatography/mass spectrometry with HPLC pre-purification. The level of 8-oxo-2′deoxyguanosine in leukocytes' DNA, antioxidant vitamins and uric acid concentrations in blood plasma were analyzed by the mean of HPLC technique. For the first time we demonstrated oxidative DNA damage on the level of whole organism and in CSF of MD patients. Urinary excretion of oxidative DNA damage repair products were higher in patients with MD than in the control group. The level 8-oxoguanine in cerebrospinal fluid of MD patients almost doubled the level found in the control group. Also the concentrations of ascorbic acid and retinol in plasma were reduced in MD patients. Oxidative stress/DNA damage is an important factor that may be involved in pathogenesis of mixed dementia. It is likely that treatment of these patients with antioxidants may slow down the progression of the disease. © 2007 Elsevier B.V. All rights reserved. Keywords: Mixed dementia; Alzheimer disease; Oxidative stress; Oxidative damage to DNA; Antioxidants; Cerebrospinal fluid

1. Introduction The high rate of oxygen consumption per unit mass of tissue renders the brain especially vulnerably to the delete-

Abbreviations: CSF, cerebrospinal fluid; AD, Alzheimer disease; MD, mixed Alzheimer disease/vascular dementia; 8-oxodG-8-oxo-7,8-dihydro2′-deoxyguanosine; 8-oxoGua-8-oxo-7,8-dihydroguanine; VaD, vascular dementia; NER, nucleotide excision repair; BER, base excision repair; MRI, magnetic resonance imaging; CT, computed tomography; HPLC, highperformance liquid chromatography; GC/MS, gas chromatography/mass spectrometry; ROS, reactive oxygen species. ⁎ Corresponding author. Tel.: +48 525853744; fax: +48 525853771. E-mail address: [email protected] (R. Olinski). 0022-510X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2007.08.041

rious effects of oxidative stress, which can arise from the overproduction of reactive oxygen species (ROS) and/or from a deficiency of the antioxidant defense systems. It is possible that oxidative stress is an important factor that may be involved in pathogenesis of neurodegenerative diseases. This theory derives from the fact that ROS are implicated in neurotoxicity of amyloid beta peptides. It has been demonstrated that the peptides themselves spontaneously generate free radicals. There is considerable evidence that oxidative stress occurs in neurodegenerative diseases [1,2], and increased 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) levels in DNA isolated from post mortem brain tissues as well as from leukocytes of Alzheimer disease (AD), Parkinson disease and mild cognitive impairment have been found


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[3–5]. Decreased repair of DNA damage could be involved in this process and it has been found that OGG1 level (the glycosylase involved in oxidative DNA damage removal) in patients with AD is significantly lower than in those in the control cases. The most popular way of exploring oxidative DNA damage on the level of the whole organism is the determination of 8-oxo-7,8-dihydroguanine (8-oxoGua) in DNA of surrogate tissues such as leukocytes. Instead of measuring damage in specific cells, with concomitant problems such as artefact formation, a whole-body burden of oxidative stress may be assessed by the measurement of urinary excretion of 8-oxoGua, and its deoxynucleoside equivalent 8-oxodG. It is generally accepted that 8-oxoGua and 8-oxodG excised from DNA by cellular repair is exported into the urine (as well as other extracellular fluids) without further metabolism [6,7]. Several glycosylases, which specifically recognize and remove 8-oxoGua in human cells, have been recently described, whereas the route by which 8-oxodG arises in extracellular matrices is less clear. Therefore, urinary assays which measure 8-oxoGua should reflect glycosylase activities. It is possible that the presence of the modified nucleoside in urine presumably represents nucleotide excision repair (NER) [8–10]. Alternatively, 8-oxodG in urine could derive from sanitization of the cellular nucleotide pool by MTH1 (Mut T Homolog 1) directed pathway [11]. However, it should be remembered that the products of NER and MTH both require further processing to result in 8-oxodG. The analysis of 8-oxoGua in extracellular fluids presents particular difficulties, and until now there has been no reliable assay for its detection. Recently, we used the method involving high-performance liquid chromatography (HPLC) pre-purification followed by gas chromatography with isotope dilution mass spectrometric detection (GC/MS) for the determination of 8-oxodG and 8-oxoGua in human urine. It was demonstrated that the amounts of both compounds in urine do not depend on diet and can give information about the formation rate of 8-oxoGua in cellular DNA [8,9,12]. Excretion of 8-oxodG repair products into urine represents the average rate of oxidative DNA damaging the whole body. Therefore, an increased level may represent not only changes in the rate of oxidative DNA damage but also alteration in the rate of repair. If possible the excretion rate should be combined with measurement of 8-oxodG in cellular DNA to study the question of rates of repair versus rates of damage. AD and vascular dementia (VaD) are the most common causes of age-related dementia and in most cases they are considered as separate disorders. However, some evidence suggests that particularly in elderly patients there is considerable overlap between AD and VaD [13,14]. This coexistence of both pathologies is named mixed dementia (MD) and vascular pathology occurs in from 25% to more than 50% of AD cases [15]. The present study was conducted in patients with mixed AD/VaD, which can be defined as cognitive decline suffi-

cient to impair independent functioning in daily life resulting from the cerebrovascular pathology, documented either by clinical criteria and/or neuroimaging findings, and the control group comprised healthy subjects who were matched with age, smoking status and dietary habits. To assess the meaning of oxidative DNA damage in development of MD in the present study, for the first time, the broad spectrum of oxidative DNA damage biomarkers was examined: i) the amount of the products of oxidative DNA damage repair (8-oxodG and 8-oxoGua) excreted into urine and cerebrospinal fluid (CSF), ii) the background level of 8-oxodG in leukocytes' DNA. Since antioxidant vitamins and uric acid are effective free radical scavengers, and can protect DNA from ROS concentration, these compounds were also determined in blood plasma. 2. Materials and methods 2.1. Subjects Patients had Mini-Mental State Examination (MMSE) scores of between 1 and 20 inclusive, indicating presence of dementia [16]. Patients met criteria for AD according to the Diagnostic and Statistical Manual, 4th edition (DSM-IV), confirmed by neuropsychological, neurological and psychiatric examination, verified by neuroimaging (MRI and/or CT) by showing the cortical atrophy. Cerebrovascular lesions (cerebral infarctions, multiple lacunar infarctions and/or ischemic periventricular leukoencephalopathy) were confirmed by neuroimaging (MRI and/or CT). This group is comprised by 8 males and 10 females with a median age of 75 years (range 53–84). The control groups (n = 33, 14 males and 19 females) were chosen in such a way that the following criteria matched the patient group: eating habits, age and body weight. The control group and the patient group have similar smoking status. The study was approved by the medical ethics committee of The L. Rydygier Medical University, Bydgoszcz, Poland, (in accordance with Good Clinical Practice, Warsaw 1998) and all the patients gave informed consent. 2.2. Urine, CSF and blood collection Spot-urine samples were collected. The creatinine concentration was measured by Jaffe method. Samples of CSF (up to 5 ml) were taken at lumbar puncture with the patient in the lateral position. Venous blood samples were taken using Vaccutainer system with sodium heparin. Urine, plasma and CSF were stored at − 85 °C for up to 3 months. 2.3. Urine and CSF analysis 0.5 nmol of [15N3, 13C] 8-oxoGua, 0.05 nmol of [15N5] 8oxodG and 10 μl of acetic acid were added to 2 ml of human urine or 4 ml of CSF. CSF was freeze-dried in the SpeedVac system and then reconstituted in 0.5 ml of MilliQ-grade

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water. Isotopic purity of the applied standards was 97.6% and 99.7% respectively. An isotopically labeled standard of 8-oxoGua was the kind gift of Dr M. Dizdaroglu from the National Institute of Standard and Technology, Gaithersburg, USA. The standard of 8-oxodG was prepared from [15N5] dGTP according to the procedure described by Bialkowski [17] with addition of an alkaline phosphatase digestion step. After centrifugation of urine and concentrated CSF (2000 ×g, 10 min.), supernatant was filtered through a Millipore GV13 0.22 μm syringe filter and 500 μl of this solution was injected onto HPLC system. Urine and CSF HPLC purification of 8-oxoGua and 8oxodG was performed according to the method described by Gackowski et al. and Rozalski et al. [12,18]. GC/MS analysis was performed according to the method described by M. Dizdaroglu [19], adapted for additional [15N5] 8-oxoGua analyses (m/z 445 and 460 ions were monitored; these ions represent the masses of characteristic ions of the base shifted in the mass spectra according to the extent of labeling). 2.4. Isolation of leukocytes from venous blood, DNA isolation and 8-oxodG determination in DNA isolates Venous blood samples from the patients were collected. The blood was carefully applied on top of Histopaque 1119 solution (Sigma, St. Louis, MO), and leukocytes were isolated by centrifugation according to the procedure described by the manufacturer. Quantification of 8-oxodG in DNA isolates was described previously [20]. 2.5. Determination of plasma vitamins A, E, C and uric acid concentration by HPLC Quantification of vitamin E (α-tocopherol), vitamin A (retinol), vitamin C (ascorbic acid) and uric acid by HPLC technique was described previously [20]. 2.6. Statistical analysis The STATISTICA (version 6.0) computer software (StatSoft, Inc, Tulsa, OK, USA) was used for the statistical analysis. Student-t test (for variables with normal distribution) and U Mann–Whitney's test (for variables with non-parametric distribution) were carried out. For normal distribution, variables were analyzed by the Kolmogorov–Smirnov test with Lillefor's correction. Statistical significance was considered at p b 0.05. 3. Results General characteristic of the analytical parameters of the study groups is described in Table 1. The median values of 8-oxoGua and 8-oxodG in urine samples and level of 8-oxodG in DNA isolated from leukocytes of control group were 9.06 nmol/mmol creatinine and 2.02 nmol/mmol creatinine, and 6.50 8-oxodG per 106 dG


Table 1 General characteristic of the analytical parameters Control group 8-oxodG/106dG in leukocytes

7.47 ± 2.95 6.50 (3.31–9.51) N = 33 Ascorbic acid [μmol/l] 47.13 ± 24.33 44.75 (30.56–61.78) N = 31 Uric acid [μmol/l] 258.35 ± 77.39 251.46 (203.38–290.88) N = 33 Retinol [μmol/l] 2.62 ± 0.94 2.46 (2.09–2.85) N = 33 α-Tocopherol [μmol/l] 25.33 ± 12.48 23.47 (16.17–32.56) N = 33 Urinary 8-oxoGua 11.54 ± 9.23 [nmol/mmol creatinine] 9.06 (6.41–12.76) N = 32 Urinary 8-oxodG 2.11 ± 1.07 [nmol/mmol creatinine] 2.02 (1.29–2.63) N = 32 8-oxoGua in CSF [nmol/l] 0.31 ± 0.10 0.27 (0.24–0.39) N = 18 8-oxodG in CSF [nmol/l] 0.26 ± 0.11 0.24 (0.16–0.41) N = 18

Mixed dementia


8.67 ± 3.81 7.44 (5.49–10.92) N = 14 23.94 ± 17.48 21.26 (4.89–35.43) N = 15 215.40 ± 61.62 215.30 (173.98–269.91) N = 18 2.00 ± 0.85 2.09 (1.61–2.73) N = 18 26.16 ± 9.77 27.93 (17.43–32.43) N = 18 14.64 ± 9.49 12.19 (10.26–14.46) N = 14 3.48 ± 2.43 2.44 (1.94–4.95) N = 15 0.57 ± 0.42 0.46 (0.24–0.51) N=8 0.22 ± 0.11 0.23 (0.11–0.32) N=8

0.3894 a





0.0378 a

0.0496 a



Data expressed as mean ± SD, median and interquartile range. a U-Mann–Whitney test.

molecules, respectively. Increases in the level of these parameters over the control level were observed in the case of patients with MD. The median values were 12.19 nmol/ mmol creatinine for 8-oxoGua, 2.44 nmol/mmol creatinine for 8-oxodG, and 7.44 8-oxodG per 106 dG molecules for 8oxodG in cellular DNA. The increases in urinary excretion rates were statistically significant. The median endogenous levels of vitamin C in the plasma of the control group and MD patients reached the values of 44.75 μmol/l and 21.26 μmol/l respectively. The difference was statistically significant. Retinol concentrations were significantly reduced in the plasma of MD when compared with the control group. The median values were 2.09 μmol/ l and 2.46 μmol/l respectively. No statistically significant differences among the study groups have been found in αtocopherol concentration where the median values were 23.47 μmol/l (control group) and 27.93 μmol/l (MD). The median level of 8-oxoGua in CSF samples in control group and MD patients reach the respective values of 0.27 and 0.46 nmol/l. The difference was statistically significant. For 8-oxodG in CSF no differences were found between the study groups. Respective median values were: 0.24 (MD) and 0.23 nmol/l (control group).


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4. Discussion Cellular damage due to oxidative stress has been proposed to contribute to the pathophysiology of some neurodegenerative diseases. Cumulative damage to DNA may contribute to progressive neuronal cell loss as unrepaired DNA damage is responsible for apoptosis and an increase in oxidative DNA damage was detected in the brain of AD and Parkinson disease [4,21]. In our study, for the first time the broad spectrum of oxidative DNA damage biomarkers i.e. urinary excretion of the modified base and nucleosides and also the level of 8oxodG in leukocytes' DNA was analyzed. All these parameters were higher in patients with mixed dementia than in the control group (however the increase of 8-oxodG in leukocytes' DNA didn't reach statistical significance). In good agreement with this finding the concentration of vitamin C in blood was also reduced in MD patients when compared with the control group (Table 1), while no changes in the concentration of α-tocopherol was detected. Vitamin C is a major aqueous-phase antioxidant. It should also be remembered that vitamin C acts in synergy with tocopherol by regenerating tocopheroxyl radical to tocopherol [22]. One plausible explanation of the above-presented results of vitamins C and E analyses are the data of reports which demonstrate the sequential consumption of vitamins C and E as a result of the disease-dependent intensification of oxidative stress. It was shown that during free radical mediated oxidation a decrease in vitamin E concentration can only be seen after the complete consumption of vitamin C [23]. The sequential consumption of these antioxidants was also shown by the use of ESR spectroscopy [24]. Vitamin C is found at 10 fold higher level in the brain than plasma, emphasizing the significant role for this antioxidant in central nervous system [25]. Indeed, vitamin C may be a critical cofactor of dopamine beta-hydroxylase and also may protect membrane phospholipids acting as a scavenger of ROS. It has been found that vitamin C can cross the bloodbrain barrier via the GLUT1 receptor, in its oxidized form and is retained in brain tissue in the form of ascorbic acid [26]. Therefore, it is possible that decreasing blood concentration can also be responsible for a decrease in vitamin C concentration in the brain. In our study we also found decreased concentration of vitamin A in patients with MD in comparison with the control group (Table 1). The simple explanation of the changes in the vitamin's concentration might be the differences in the eating habits between the studied groups. However, it is noteworthy that the members of the studied groups were chosen randomly and, according to the interview, the groups were constituted in such a way that their members could match eating habits and living conditions (see M&M Section). Therefore, it is rather unlikely that the different concentration of the vitamins in their blood was entirely a result of lifestyle. Presumably, severe oxidative stress resulting in the production of ROS is responsible for the consumption of the antioxidant vitamins.

The decreased amount of uric acid (Table 1) also supports this assumption. Summing up, the study results of this part suggest that the patients with MD experienced severe oxidative DNA damage/oxidative stress on the level of the whole body reflected in elevated level of all the parameters representing oxidative DNA damage. Distinct reduction of the level of vitamin C underlines the importance of this antioxidant for MD patients. Cerebrospinal fluid filters and disposes degraded cellular biomolecules from the brain. Therefore, the level of 8-oxoGua and 8-oxodG in CSF should reflect oxidative stress/ DNA damage in the central nervous system while the amount of modified base/nucleoside excreted into urine should represent the same phenomenon in every tissue/organ of the organism [27]. In good agreement with the above-presented data the level of 8-oxoGua in CSF of MD patients almost doubled the level found in the control group (Table 1). This indicates that the brain in MD patients experiences oxidative damage to DNA higher than that found at the whole-body level, pointed out at significance of oxidative DNA damage in the target organ (compare about 80% increase in CSF with about 30% elevation in urine) . The interesting hypothesis was recently put forward that explains the massive death of neurons in AD. It has been proved that neurons that are about to die (like in the case of AD) are those that have been attempting to re-enter the cell cycle and synthesize DNA. Spurious accumulation of DNA damage in neurons, however, may result in triggering cell death. Moreover, it has been demonstrated, in experiments with mouse model of AD that all neurons that re-entered into S-phase had oxidized DNA structures [10,28]. This may also apply to other neurodegenerative diseases. In fact, a growing body of evidence implicates oxidative stress as being involved in at least the propagation of cellular injury that leads to neuropathology in many neurodegenerative diseases (for review see [29]). Our data are consistent with the aforementioned hypothesis and for the first time demonstrate severe oxidative DNA damage in the brain of MD patients. For 8-oxodG in CSF no differences were found between the study groups. There is a possibility that the cerebrovascular lesions are responsible for the oxidative stress observed in MD patients. Therefore, the changes of analyzed parameters would be rather a consequence of the features characteristic for MD. However, it is less likely because the lesions are rather subtle and restricted to the small regions of the brain, while oxidative stress symptoms in this group were observed also on the level of the whole body (Table 1). It has been documented that ROS play important roles in a wide variety of vascular pathologies including atherosclerosis. Although there are several enzymatic systems that can produce ROS, the potential major sources of vascular ROS include NADPH-dependent oxidases [30]. Interestingly, NADPH oxidase-dependent ROS production in endothelial cells can be abrogated with vitamin C [31]. Thus, it is possible that vitamin C deficiency detected in MD patients in several ways may influence oxidative stress detected in MD group.

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More importantly, recently it has been found that NADPH oxidase is responsible for cerebrovascular dysregulation induced by amyloid beta peptide [32]. It should also be remembered that antioxidant vitamins may have biological activities that are separate from their direct antioxidant effect, e.g. they can regulate changes in gene expression [33]. Therefore, changes in their concentrations may also affect genome functions in this way. In this context it is interesting to note that NADPH oxidase expression and in turn its ROS formation capacity may be regulated by redox sensitive mechanism dependent on vitamin C concentration [31]. Currently available medication provides only modest clinical benefits once a patient has developed AD. [15]. However, unlike AD, early treatment intervention may slow the course of MD [34]. Our results suggest that simple treatment of these patients with antioxidant vitamins may likely present a strategy for preventing/slowing progression of the disease.




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