Paper Title
Nicking Assay and Oxygen Consumption Assay as New Useful Tools For Analysis Oxidoreductive Effects of Novel HNO Donors in Thrombosis Rats

Abstract
Destabilization of DNA in living organisms by molecular oxygen in solid and liquid tissues may lead to damages different part of cells like platelets mitochondrial membrane which can be one of the factors in neurodegenerative diseases formation and disturbances in thrombosis. These diseases are formed by intermediate molecules of oxygen like superoxide and lipid peroxidation processes. These molecules can oxidize DNA bases in a mitochondrial DNA (mtDNA) to either reactive compounds such as methylated or oxidised base pairs to exocyclic ethano and propano adducts-which may be a risk factor for development of various pathologies, including malignancy. Either processes of dysfunction platelets mitochondrial membrane and solid tissues may induce disturbances in thrombosis by a different mechanism of DNA repair, what affect the premature cancer changes in tumour critical genes. These changes may lead to the different types of cancer through the stage of DNA base modification. Understanding the genotoxic properties of endogenous DNA damage as etheno DNA adducts, such as 1,N6-ethenoadenine (etA), 3,N4-ethenocytosine (etC), N2,3-ethenoguanine (etG) and 1,N2-ethenoguanine (etG) and repair pathways, is fundamental to understanding the mechanisms of diseases that depend on chronic inflammation such as cancer or neurodegenerative diseases, and aging processes. Due to the large range of these products and pleiotropic action, knowledge about the molecular mechanisms of thrombosis is still fragmentary. We studied oxidoreductive effects triggered modulation of repair activity in the differents liquid and solid tissues in thrombotic Wistar rats. Animals were divided into five groups (10 pieces in a group) were treated HNO donors (nirtroxyles) each one individually; Cl-PA, Br-PA, F-PA, CH3-PA, and ASA at doses of 1, 3 or 10 umol/kg. compared to the control group (NAIVE-healthy rat without thrombosis). Then the levels of potential redox damage in tissues caused by nitroxyl treatment were measured using methods nicking assay and oxygen consumption assay. After all nitroxyles treatment in all analysed samples we found different level of DNA damages reconised by DNA glycosylases in nicking assay method. The level for etC were the lovest in comparsion to etA and 8oxoG were the level was 2 times higher. After treatment with Cl-PA, Br-PA, F-PA, CH3-PA, ASA. No differences were observed in repair rate of 1,N6-ethenoadenine (εA), 3,N4-ethenocytosine (εC) and 8-oxoguanine (8-oxoG) in tissues of these rats. A different stimulating effect of both doses of CL-PA, BR-PA, F-PA, CH3-PA, ASA was observed in oxygen consumption assay. Lower dose of CL-PA, BR-PA, F-PA, CH3-PA, ASA at 1 umol/kg increased (P<0.01) hypoxia in the all analysed tissue and a higher dose 3 or 10 umol/kg increased (P<0.01) hyperoxia in it in the all analysed group compared to the control group. This may be due to increased oxidative stress and imbalance in DNA repair. Keywords - Endothelium, Ischemia Reperfusion, Oxidative Stress, Oxygen Consumption Assay, Nicking Assay, Thrombosis.