**2.1.2 Determination of 8-oxo2dG**

*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 precipitated with 98% ethanol.

*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 UV detection (HPLC/EC/UV).

*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 (Olsen et al., 1999).

Oxidative DNA Damage and the Level of Biothiols, and L-Dopa Therapy in Parkinson's Disease 353

It seems that analysis of the level of oxidative stress (8-oxo2dG) may be represented targets

**2.2 Influence of L-dopa treatment on the level of apoptotic factors in peripheral blood** 

At the neuropathological studies, PD is mainly characterized by neuronal intracellular inclusions named Lewy's bodies with α-synuclein. These inclusions are now known to be comprised of filamentous polymers of α-synuclein, which may generate oxidative stress in the brain of PD patients. It could results from several mechanisms, such as depletion of antioxidants, defects in mitochondrial electron transport, neurotoxin exposure, and excessive oxidation of dopamine in the patients given L-dopa. Conway et al. (2001) showed that dopamine or L-dopa inhibits the fibrillization of α-synuclein filaments by stabilization

However Alves Da Costa et al. (2002) showed that α-synuclein drastically lowered caspase-3 activity and p53 protein expression, and transcriptional activity, proteins controlled the apoptotic cascade. Blandini et al. (2004), Dorszewska et al. (2009b) and Iwashita (2004) showed that, apoptotic proteins such as: Bcl-2 family proteins and PARP are involved in the

The aim of the study was to estimate the levels of p53, and PARP proteins, and 85 kDa fragment, and two Bcl-2 family proteins: Bcl-2 and Bax in peripheral lymphocytes of patients with PD and in control group. The attention was also paid to L-dopa

The studies were conducted on 45 patients with PD, among their 22 patients, including 9 women and 13 men, aging 41-79 years (mean age: 58.0±10.7 years) awaited L-dopa treatment and 23 patients, including 11 women and 12 men aging 45-81 years (mean age: 68.0±8.6 years) were treated with L-dopa preparations in daily doses (up to 5 years treatment to 500 mg/day, 5-10 year treatment 500-800 mg/day, and over 10 year treatment

The control group included 27 individuals, 19 women and 8 men, 35-73 years of age (mean

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 proteins.* Blood was gradiated onto gradisol L at a 1:1 ratio and centrifuged, followed by collection of the interphase which was then rinsed in PBS buffer (0.9% NaCl in phosphate buffer) and centrifuged. The obtained lymphocyte precipitate was rinsed with radioimmunoprotein assay (RIPA) buffer (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% IGEPAL CA-630, 0.05% SDS, and 1% sodium deoxycholate), supplemented with a

**2.2.2 Estimation of p53, Bax, Bcl-2, PARP proteins and 85-kDa subunit** 

for diagnosis of PD and therapy in future.

of their structure.

pathogenesis of PD as well.

pharmacotherapy in PD.

**2.2.1 Patients** 

800-1500 mg/day).

age: 54.0±10.7 years).

disorders and smoking, and drinking habits.

their caregivers was obtained.

Yahr.

**lymphocytes of Parkinson's disease patients** 
