Mitochondrial probes for oxidative stress. (360G-Wellcome-075614_Z_04_A)

Mitochondrial probes for oxidative stress Interest has recently been focused on the damage caused to mitochondria by reactive oxygen species (ROS) such as hydroxyl (HO·), peroxyl (HO2·) and superoxide (O2·) radicals. Such damage appears to be causal in the universally experienced, but poorly understood, process of ageing. Thus, the extension of lifespan when there is calorie restriction in the diet of rats appears to arise at least in part from reduced oxidative damage to mitochondria. Furthermore, murine life span can be extended by overexpression of catalase targeted to mitochondria, where the increased removal of hydrogen peroxide will reduce ROS production and consequent damage. Oxidative stress is also important in a range of pathologies including the accelerated atherosclerosis associated with diabetes. Mitochondria are particularly susceptible to oxidative stress because much of the intracellular production of ROS arises in these organelles, particularly when a large proton motive force is generated by mitochondria respiring, but not producing ATP. Understanding the role and significance of ROS generated in mitochondria in ageing and pathologies where oxidative stress appears important has been hampered by a lack of mitochondria-targeted probes that specifically detect radicals. The aim of this research is to develop such generally useful probes and to use them to test the hypothesis that increased production of ROS in mitochondria is a major contributor to oxidative damage during hyperglycaemia (important in diabetes). The only technique that observes radicals directly and to the exclusion of all non-radical species is electron paramagnetic spectroscopy (EPR). The technique is very versatile and is useful in studies extending from simple chemical reactions in the test tube to the observation of radicals in vivo. Thus, the probes to be developed are for use with EPR spectroscopy. The specific objectives are to: (i) Design and synthesise new hydroxylamine H-atom transfer probes for EPR spectroscopy. (ii) Characterisation of their chemical reactivity with radicals and the longevity of the EPR signal produced in the presence of ascorbic acid. (iii) Determination of uptake into isolated mitochondria and whole cells. (iv) Application of probes to investigate the role of mitochondrial dysfunction in oxidative damage in hyperglycaemia (and to protect cells against cellular ageing).