Protection of mESCs from oxidative stress-induced cell death by a novel class of mitochondrial-targeted, high-potency antioxidant

Donald McPhail1, Graeme Cook1, Scott Johnstone1, Kevin Docherty2. 1Antoxis Limited, Aberdeen, United Kingdom, 2University of Aberdeen, Aberdeen, United Kingdom.

In mammalian systems the main endogenous source of reactive oxygen species (ROS) is ascribed to the production of the superoxide radical anion in the mitochondria by electron leakage onto molecular oxygen from complex I and complex III of the electron transport chain (1). However, many other factors are known to contribute to ROS production including changes in oxygen tension (reperfusion effects), xenobiotic exposure, immune system activation, inflammatory responses and loss of cellular compartmentalisation(2). When oxidative stress exceeds the cellular antioxidant defence capacity a cascade of free radical-mediated reactions occur which result in damage to key biomolecules, including membrane lipids, ultimately leading to cell death. Despite being an area of intense research in conventional cell biology the implications of oxidative stress and antioxidant regulation have received much less attention in the stem cell field. This paper describes an oxidative stress assay, for use with mESCs, in which a steady source of ROS is generated in the cells by exposure to tert-butyl hydroperoxide. Loss of viability is measured colorometrically by the extent to which MTT (3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide) can no longer be reduced to Formazan by the cells. The assay has been applied to determine the protective effects of AO-1-530, a proprietary antioxidant molecule3,4, designed from a flavonoid scaffold and known to protect neuronal cells from oxidative stress. Addition of the compound to the cell medium almost fully blocked the 60% loss of viability observed in unprotected mESCs. Furthermore, once incorporated into the cell, the compound could confer a degree of protection extending to 24 hrs. Fluorescence studies indicate that the compound incorporates rapidly into the cell and targets the mitochondrial compartment which might explain its potency. Although developed for therapeutic use, AO-1-530 and analogues may have considerable potential to protect stem cell viability where procedures, including transplantation, or metabolic effects give rise to excess ROS production.

(1) Lin, M. T.; Beal, M. F. (2006) Nature, 443, 787-795.
(2) Halliwell, B.; Gutteridge, J. M. C. (1999) Free Radicals in Biology and Medicine; 3rd Ed.; OUP: Oxford.