**2.2.1 Patients**

352 Etiology and Pathophysiology of Parkinson's Disease

In the patients with PD (Table 1), the levels of 8-oxo2dG in peripheral blood lymphocytes

In the PD patients (Table 2) disease progress from stage I to IV (according to the scale of Hoehn and Yahr) resulted in higher level of 8-oxo2dG in DNA (p<0.05) also observed

> **Stage II (34-81 years)**

nonparametric of Mann-Whitney test for unlinked variables was used. Differences

8-oxo2dG 17.4 ± 16.9 20.5 ± 14.2 25.2 ± 22.7 23.2 ± 12.8\* Table 2. Levels of oxidative DNA damage (8-oxo2dG/dG x 10-5), as related to the stage of the PD according to the scale of Hoehn and Yahr. Results are expressed as means ± SD. The

Pharmacotherapy with L-dopa (Table 3) affected the level of 8-oxo2dG (p<0.01), as

8-oxo2dG 13.7 ± 7.6 19.3 ± 17.3 22.6 ± 18.0\*\*

pharmacotherapy with L-dopa (+) in the patients with PD. Results are expressed as means ± SD. The nonparametric of Mann-Whitney test for unlinked variables was used. Differences

Our results indicated that, L-dopa can modify the level of oxidative DNA damage (8 oxo2dG) in the peripheral blood cells of PD patients. On the other hand, it is interesting that in PD a significant increase in DNA damage has been observed in the IVth stage of the disease development (according to Hoehn and Yahr), even so 8-oxo2dG levels are increased between the stages I and III of the disease evolution. It seems that in PD the reason for increasing levels of oxidative process altered nucleic acids is thought to involve overproduction of free radicals as well as decreased levels of enzymatic and non-enzymatic antioxidants and less effective repair mechanisms. In AD patients have been found to contain lowered activity of specific 8-oxoguanine glycosylase 1, OGG1, and more oxidative DNA damage which might induce of apoptosis (Dorszewska et al., 2005, 2009a, 2010).

Table 3. Levels of oxidative DNA damage (8-oxo2dG/dG x 10-5), as related to

**Patients with PD L-dopa (-) (34-79 years)** 

**Patients with PD (34-81 years)** 

> **Stage IV (56-78 years)**

**Patients with PD L-dopa (+) (35-81 years)** 

**Stage III (46-81 years)** 

were significantly increased (p<0.05), as compared to the controls.

**(22-76 years)** 

between stages I and III, and a tendency to further decrease in stage IV.

8-oxo2dG 13.7 ± 7.6 21.8 ± 17.8\* Table 1. Levels of DNA oxidative damage (8-oxo2dG/dG x 10-5) in the PD patients and in control group. Results are expressed as a means ± SD. The nonparametric of Mann-Whitney test for unlinked variables was used. Differences significant at \*p<0.05, as compared to the

**Parameter Controls** 

**(35-79 years)** 

significant at \*p<0.05, between stages I and IV of PD.

**(22-76 years)** 

significant at \*\*p<0.01, as compared to the controls.

**Parameter Stage I** 

compared to the healthy controls.

**Parameter Controls** 

**2.1.3 Results** 

controls.

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 800-1500 mg/day).

The control group included 27 individuals, 19 women and 8 men, 35-73 years of age (mean age: 54.0±10.7 years).

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 Yahr.

None of the control subjects had verifiable symptoms of dementia or any other neurological disorders and smoking, and drinking habits.

A Local Ethical Committee approved the study and the written consent of all patients or their caregivers was obtained.

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

*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

Oxidative DNA Damage and the Level of Biothiols, and L-Dopa Therapy in Parkinson's Disease 355

p53 0.52 ± 0.37 0.19 ± 0.11\*\* 0.30 ± 0.15 Bax 0.60 ± 0.50 0.12 ± 0.06\*\*\* 0.14 ± 0.08\*\*\* Bcl-2 1.20 ± 0.77 0.15 ± 0.06\*\*\* 0.16 ± 0.10\*\*\* Bax/Bcl-2 1.46 ± 3.77 1.00 ± 0.64 1.26 ± 0.97\* PARP 2.12 ± 0.83 0.82 ± 0.36\*\*\* 2.36 ± 1.08(\*\*\*) 85-kDa 0.42 ± 0.80 0.22 ± 0.13 0.58 ± 0.32\*\*\*(\*\*) Table 5. Level of p53, Bax, Bcl-2, PARP proteins and of 85-kDa protein subunit in peripheral blood lymphocytes in PD patients untreatment L-dopa (-) and treatment L-dopa (+), and in the control group. Apoptotic proteins represent % of area of immunoreactivity bonds. Results are expressed as a means ± SD. The nonparametric test of Kruskal-Wallis was used. Differences significant at \*p<0.05; \*\*p<0.01; \*\*\*p<0.001 as compared to the controls, and (\*\*)p<0.01; (\*\*\*)p<0.001 between PD patients untreatment L-dopa (-) and treatment L-dopa (+). However, (Table 6) long-term (more than 5 years) therapy of L-dopa in PD patients probably leads to apoptosis, because elevated levels of Bax:Bcl-2 ratio (p<0.05 as compared

**PD patients L-dopa (-) 41-79 years of age** 

> **PD patients L-dopa < 5 years**

p53 0.52 ± 0.37 0.30 ± 0.13 0.30 ± 0.16 Bax 0.60 ± 0.50 0.14 ± 0.09\*\* 0.15 ± 0.08\*\*\* Bcl-2 1.20 ± 0.77 0.19 ± 0.08\* 0.15 ± 0.11\*\*\* Bax/Bcl-2 1.46 ± 3.77 0.85 ± 0.69 1.40 ± 1.03\* PARP 2.12 ± 0.83 2.03 ± 1.00 2.48± 1.12 85-kDa 0.42 ± 0.80 0.44 ± 0.20 0.64 ± 0.35\*\* Table 6. Level of p53, Bax, Bcl-2, PARP proteins and of 85-kDa protein subunit in peripheral blood lymphocytes in PD patients treatment L-dopa less and more than 5 years, and in the control group. Apoptotic proteins represent % of area of immunoreactivity bonds. Results are expressed as a means ± SD. The nonparametric test of Kruskal-Wallis was used. Differences significant at \*p<0.05; \*\*p<0.01; \*\*\*p<0.001 as compared to the controls.

It seems that pharmacological treatment of PD patients with L-dopa has a major role in modulating of levels in lymphocytes of some apoptotic proteins, important for this process. Further investigation is thus requisite to analysis expression and mutations of genes encoding proteins important for effective repair and/or apoptosis in PD patients treatment

**3. Homocysteine and asymmetric dimethylarginine and L-dopa treatment in** 

Elevated Hcy level is a risk factor for vascular diseases, cognitive impairment and dementia, and neurodegenerative diseases (e.g. PD). It is also known that vascular dementia and cognitive impairment worsen the prognosis of PD patients, and it is important to minimize

**PD patients L-dopa (+) 45-81 years of age** 

> **PD patients L-dopa > 5 years**

**Parameter Controls** 

**Parameter Controls** 

with L-dopa.

**Parkinson's disease** 

**35-73 years of age** 

to the controls) and 85-kDa fragment (p<0.05 as compared to the controls).

protease inhibitor cocktail (Sigma) and homogenized in a mixture of RIPA with protease inhibitor cocktail (16:1) and 0.5 µl PSMF (Sigma) in isopropanol (10 mg/100 µl), centrifuged, and the obtained supernatant underwent further analysis (Ohnishi et al., 1996).

*Western Blot.* The Bax and Bcl-2 proteins were analyzed in 12% and p53, and PARP proteins were analyzed in 7.5% polyacrylamide gel. Equivalent amounts of protein (30 µg protein/lane) were loaded to the wells. The gel-separated proteins were electrotransferred to nitrocellulose filter in a semidry Western Blot analysis apparatus (Apelex, France). To estimate the levels of the PARP protein, the filters were exposed first to an anti-PARP monoclonal antibody (G-2-10, IgG, 0.05 ml, Sigma, USA), diluted 1:2000, while the p53, Bax, Bcl-2 proteins were identified using anti-p53 (IgG-2a, 200 µg/1.0 ml; Santa Cruz, USA), anti-Bax (IgG-2b, 200 µg/1.0 ml; Santa Cruz, USA) and anti-Bcl-2 (IgG-1, 200 µg/1.0 ml; Santa Cruz, USA) mouse monoclonal antibody, respectively, diluted 1:500.

Subsequently, individual sheets of nitrocellulose filter were incubated with the second antibody, goad antimouse IgG-HRP (200 µg/0.5 ml; Santa Cruz, USA) at a dilution of 1:2000. To stain immunoreactive bands, peroxidase BMB was added (BM blue POD substrate precipitation; Roche, Germany). The surface area of the immunoreactive bands was registered using a densitometer (GS-710; Bio-Rad, Hercules, CA) in the Quantity One System.
