There is increasing evidence supporting a causal role of oxidatively damaged DNA in neurodegeneration during the natural aging process and neurodegenerative diseases such as Parkinsons and Alzheimers. tiron, catalase, bathocuproine or methional to the dopamine/Cu(II) reaction mixture resulted in a substantial decrease ( 90%) in oxidation DNA product levels, indicating a role of singlet oxygen, superoxide, H2O2, Cu(I) and Cu(I)OOH in their formation. While the addition of N- 0.05. Results Detection of Oxidatively Damaged DNA All catecholamine neurotransmitters and their congeners (30 M) tested (Figure 1), in the presence of Cu(II) (30 M CuCl2) and ST-DNA (300 g/ml), resulted, in addition to 8-oxodG, the formation of a S/GSK1349572 inhibitor DNA decomposition pattern containing nearly a dozen unidentified oxidation DNA products (Figure 2) as detected by 32P-postlabeling coupled with a PEI-cellulose TLC [38]. The decomposition pattern was chromatographically identical for all neurotransmitters and their congeners tested (not shown). The characterization of these DNA decomposition products as oxidation DNA products is based on their ability to migrate under low-salt conditions, while DNA adducts formed from the covalent binding of quinone metabolites of catecholamine neurotransmitters, such as dopamine, to DNA require high-salt and high-urea concentrations to displace them from the origin during thin-layer chromatography [30]. An identical DNA decomposition pattern (Figure 3) was also found upon comparison with a Fenton-type reaction known to generate hydroxyl radicals from H2O2 (30 M) with CuCl2 (30 M) in the presence of ST-DNA (300 g/ml) further supporting oxidative based characterization of these decomposition products. Open in a separate window Figure 1 Structure of catecholamine neurotransmitters and their congeners. Open in a separate window Figure 2 Representative autoradiographs of S/GSK1349572 inhibitor 32P-labeled DNA items caused by auto-oxidation of dopamine (30 M) and Cu2+ (30 M)-mediated activation of dopamine (30 M) and H2O2 (30 M). Particular circumstances are referred to in text. The same DNA decomposition design pursuing auto-oxidation and Cu(II)-mediated oxidation of dopamine was discovered and visualized with much longer exposure moments. Oxidation DNA items were solved by two-directional polyethyleneimine (PEI)-cellulose TLC (D1 = 45 mM sodium phosphate, pH 5.8/1 M formic acidity onto a 6 cm Whatman no. 17 paper wick; D2 = 100 mM sodium phosphate, 6 pH.0/10% acetonitrile (v/v). OR, origins. Many of these oxidation DNA items detected were discovered to become oxidatively customized dinucleotides since these DNA items co-migrated with oxidation DNA items formed by result of CuCl2 and specific dinucleotides (for instance dApG, dGpT, etc.). Chromatographic identification from the DNA-derived as well as the dinucleotide-derived items was set up in two different solvent systems (data S/GSK1349572 inhibitor not really proven). Unidentified oxidation DNA item amounts mediated by Cu(II)-activation from the catechol neurotransmitters and their congeners ranged from 80 to 383 oxidative items/106 nucleotides with dopamine leading to the highest amounts (Body 4). For evaluation purposes 8-oxodG amounts were also assessed and ranged from 37 to 172 per 106 nucleotides with epinephrine leading to the highest amounts pursuing Cu(II)-mediated catalysis. The Fenton-type result of H2O2 (30 M) with CuCl2 (30 M) led to 20-fold lower oxidation DNA item amounts than dopamine with CuCl2 beneath the same circumstances. In the lack of copper, unidentified oxidation DNA item amounts ranged from 5 to 30 oxidative items/106 nucleotides for the average person neurotransmitters and their analogs while 8-oxodG amounts ranged from 6 to 13 adducts/106 S/GSK1349572 inhibitor nucleotides (data not really proven), indicating their potential for auto-oxidation. Open in a separate window Physique 4 Oxidation DNA product levels of catecholamine neurotransmitters and their congeners (30 M) following incubation with DNA (300 g/ml) in the presence and absence Rabbit Polyclonal to IL-2Rbeta (phospho-Tyr364) (auto-oxidation) of CuCl2 (30 M). Vehicle used was 1% DMSO. DNA product levels were decided as the mean of 3 to 4 4 replicates SE In order to determine if copper was the only transition metal which could catalyze formation of these oxidation DNA products from catecholic neurotransmitters, dopamine was reacted with either Cu(I) (CuCl), Cu(II) (CuCl2), Fe(II) (FeSO4), or Fe(I) (FeCl3) (30 M) in the presence of DNA (300 g/ml) and 10 mM Tris HCl,.