**1. Introduction**

348 Etiology and Pathophysiology of Parkinson's Disease

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Y., Hong, J.S. and Zhang, J. (2005). Aggregated alpha-synuclein activates microglia: a process leading to disease progression in Parkinson's disease. *FASEB J,* Vol. 19, Parkinson's disease (PD) is a chronic and progressive neurological disorder characterized by resting tremor, rigidity, and bradykinesia, affecting at least 1% of individuals above the age of 65 years. Parkinson's disease is a result of degeneration of the dopamine-producing neurons of the *substantia nigra*. Available therapies in PD will only improve the symptoms but not halt progression of disease. The most effective treatment for PD patients is therapy with L-3,4 dihydroxy-phenylalanine (L-dopa) (Olanow, 2008). As indicated in literature reports, L-dopa therapy leads to motor fluctuations and disabling involuntary movements called L-dopainduced dyskinesia (Carta et al., 2006; Obeso et al., 2008). Literature reports indicate also that long-term administration of L-dopa in PD patients may not only alter arginine (Arg) levels but may also lead to increased concentrations of homocysteine (Hcy), the factor responsible for development of atherosclerosis and dysfunction of nigral endothelial cells (Muller et al., 1999). Methylenetetrahydrofolate reductase (MTHFR) represents enzyme involved in remethylation of Hcy to methionine (Met). The C667T transition in *MTHFR* results in Ala>Val substitution in position 226 and, as a consequence, in 50 % decrease in the enzyme activity, and thus in an increased concentration of Hcy (Frosst et al., 1995). The study of

Yasui et al. (2000) indicated that the TT genotype might be linked to pathogenesis of PD, particularly when the level of folates is low. Moreover, L-dopa metabolism via Omethylation by catechol-O-methyl-transferase (COMT) using S-adenosyl-L-methionine (SAM) leads to increase Hcy levels, hyper-Hcy (O'Suilleabhain et al., 2004a). A percentage 10-30% of PD patients exhibits hyper-Hcy. Hyper-Hcy in PD has been associated with affective and cognitive impairment, dementia, dyskinesia, and vascular disease (O'Suilleabhain et al., 2004b; Rogers et al., 2003; Zoccolella et al., 2006, 2009).

The exact mechanism of development and progression of PD pathology is not clear. It is known, that a complex interplay of multiple environmental and genetic factors has been involved in pathogenesis of PD and it is possibly that PD represents rather a syndrome but not a single disorder. Moreover, is likely that in pathogenesis of PD there are several mechanisms involved, such as: oxidative stress, mitochondrial dysfunction, DNA damage, protein aggregation, neuroinflammation, excitotoxicity, apoptosis and loss of trophic factors. The most probably is that all factors are represented targets for PD therapy.

Oxidative DNA Damage and the Level of Biothiols, and L-Dopa Therapy in Parkinson's Disease 351

The contribution of L-dopa therapy to oxidative damage and apoptosis in peripheral cells in

The aim of the study was to estimate the degree of oxidative damage to DNA (marker: 8-

The studies were conducted on 98 patients with PD, including 37 women and 61 men aging 34-81 years (mean age: 60.8±10.7 years). Among the patients with PD, 27 patients (9 women and 18 men) awaited L-dopa treatment (patients' age: 34-79 years) and the remaining 71 individuals, 28 women and 43 men (patients' age: 35-81 years) were treated with L-dopa preparations in daily doses (up to 5 years treatment to 500 mg/day, 5-10 year treatment 500-

Control group included 50 individuals, 34 women and 16 men, 22-76 years of age (mean age:

Patients with PD were diagnosed using the criteria of UK Parkinson's Disease Society Brain Bank (Litvan et al., 2003), however stage of disease according to the scale of Hoehn and

None of the control subjects had verifiable symptoms of dementia or any other neurological

A Local Ethical Committee approved the study and the written consent of all patients or

*Isolation of DNA.* DNA was isolated from peripheral blood lymphocytes by fivefold centrifugation in a lytic buffer, containing 155 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4, in the presence of buffer containing 75 mM NaCl, 9 mM Na2EDTA , pH 8.0, and sodium dodecyl sulfate and proteinase K (Sigma, St. Louis, MO). Subsequently, NaCl was added, the lysate was centrifuged, and DNA present in the upper layer was

*Enzymatic hydrolysis of DNA to nucleosides.* DNA was hydrolyzed to nucleosides using P1 nuclease (Sigma), for 2 h at 37oC in 10 mM NaOAc, pH 4.5. The solution was buffered with 100 mM Tris-HCl, pH 7.5. Subsequently, the DNA was hydrolyzed with alkaline phosphatase (1U/µl; Roche, Germany) for 1 h at 37 oC and the obtained nucleosides mixture was applied to high-pressure liquid chromatography system with both electrochemical and

*Estimation of 8-oxo2dG.* To determine 8-oxo2dG level, the nucleosides mixture was applied to the HPLC/UV system (P580A; Dionex, Germany) coupled to an electrochemical detector (CoulArray 5600; ESA, USA). Nucleosides were separated in a Termo Hypersil BDS C18 (250 x 4.6 x 5µm) column (Germany). The system was controlled, and the data were collected and processed using Chromeleon software (Dionex, Germany). The results were expressed as a ratio of oxidized nucleosides in the form of 8-oxo2dG to unmodified 2'dG

**2.1 Influence of L-dopa treatment on the level of 8-oxo-2dG in peripheral blood** 

oxo2dG) in PD patients before and during treatment with L-Dopa, and in controls.

PD patients is not clear, and is still debated.

**2.1.1 Patients** 

44.6±16.2 years).

Yahr.

**lymphocytes of Parkinson's disease patients** 

800 mg/day, and over 10 year treatment 800-1500 mg/day).

disorders and smoking, and drinking habits.

their caregivers was obtained.

precipitated with 98% ethanol.

UV detection (HPLC/EC/UV).

(Olsen et al., 1999).

**2.1.2 Determination of 8-oxo2dG** 
