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Henry Ford

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Electrochemical study of DNA damaging by oxidation stress

Reactive oxygen species
Reactive oxygen species (ROS) are strictly regulated by compounds with radical scavenging and antioxidant activities called antioxidants. Disruption of this delicate balance between reactive oxygen species and their scavengers leads to the induction of oxidative stress, which may induce various pathological processes ended by death of an organism. The oxidative stress is associated with the ageing processes, carcinogenesis and a number of diseases, such as Alzheimer's disease, atherosclerosis and Parkinson's disease [1-4]. Oxidation stress is induced by both free radicals and compounds of non-radical type (H2O2, ozone, 1O2, HOCl). We can differentiate two basic groups of radicals inducing oxidative stress as reactive oxygen species – ROS (HO., H2O2, 1O2, O3, ROO., HO2.) and as reactive nitrogen species – RNS (NO., NO2.) [5]. Thiol and carbon centred radicals belong to additional possible sources of oxidative stress [6].

Effect of ROS on the cellular level
Increased levels of ROS directly damage DNA and can induce cascade of reactions, which can be terminated by apoptosis. This cascade has been intensively studied in numerous types of cells including neuronal ones, as it is shown in Fig. 1. Depending on the initial stress stimulus, activation of p53 occurs via various pathways that may intersect with each other upstream of p53 activation. Amount of this 393-aminoacid protein is kept at low concentration level by ubiquitine system degradation in unstressed cells [7, 8]. During cell stress (e.g. DNA damage induced by UV rays and/or alkylating agents) the degradation process of protein p53 is blocked and its intracellular concentration significantly increases. After that, the protein p53 is posttranslationally modified and subsequently oligomerized to tetramer, which has high affinity to DNA [9, 10]. Protein p53 is able to bind onto DNA specifically (by sequence specific bond) or nonspecifically e.g. via protein C-terminal domain [11]. Moreover, p53 exerts its deadly function by transactivation of pro-apoptotic target genes including those encoding Bax, the BH3-only proteins PUMA and Noxa, which translocate to mitochondria where they mediate disruption of the mitochondrial membrane potential and release of apoptotic factors including cytochrome c and AIF. Many transcriptional targets such as Peg3/Pw1, Siah-1a and SIVA act in a similar way by interacting with pro-apoptotic members of the Bcl-2 family at the level of mitochondria. In addition, p53 may promote cell death via transactivation of the death receptor Fas or up-regulation of APAF-1, which promotes caspase-dependent apoptosis after formation of the apoptosome with cytochrome c and caspase-9 (Fig. 1B). In addition to such transcriptional control of the cell death machinery, p53 can directly trigger apoptosis after translocation to mitochondria, a process that can occur in synapses (synaptic apoptosis) and may involve interactions with Bax or Bcl-xL. The origination of mutations, translational mistakes or subsequent inhibition of proteosynthesis is the results of these damages. Free radicals can damage not only DNA but also proteins and lipids, which are essential components of biomembranes [12]. Scheme of such damaging is shown in Fig. 2A. A model for iron misregulation and reactive oxygen species generation followed by gyrase inhibition and DNA damage formation is introduced. (a) Gyrase inhibitors (red triangles), such as norfloxacin and CcdB, target DNA-bound gyrase (yellow circles). Resulting complex induces double-stranded breakage and loss of chromosomal supercoiling by preventing strand rejoining by the gyrase enzyme. (b) Gyrase poisoning promotes the generation of superoxide (O2-), which (c) oxidatively attacks iron–sulphur clusters (three-dimensional cube depicts [4Fe–4S] cluster; iron and sulphur are shown as orange and blue circles, respectively); sustained superoxide attacks the iron–sulphur-containing proteins (light blue), which leads to functional inactivation (dark blue), destabilization and iron leaching. (d) Repetitious oxidation and repair of clusters, or redox cycling, promote iron misregulation and may serve to generate a cytoplasmic pool of 'free' ferrous (Fe2+) iron. (e) Ferrous iron, via the Fenton´s reaction, rapidly catalyses the formation of deleterious hydroxyl radicals (OH), which readily damage DNA, lipids and proteins; the Fenton´s reaction can thus take place at the destabilized iron–sulphur clusters or where free ferrous iron has accumulated. It can be suggested that reactive oxygen species are generated via an oxygen-dependent death pathway that amplifies the primary effect of gyrase inhibition and contributes to cell death following gyrase poisoning.

Podpořeno projekty: CEITEC CZ.1.05/1.1.00/02.0068

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