Validation of Assays for Reactive Oxygen Species and Glutathione in Saccharomyces cerevisiae during Microgravity Simulation

Abstract

ABSTRACT The effects of spaceflight on yeast have high concordance with agents that induce a very low intracellular redox state and induce a massive efflux of glutathione. These results raise important issues. Can the reduced redox state during spaceflight be reproduced and modulated in ground-based simulations? Will this allow definition of unique drug pathways as a low redox potential state mirrors the electrophilic properties of mitochondria where many drugs are metabolized? Unfortunately, assays for redox status and its major cellular determinant—glutathione—are diverse and often cell-type-specific. Currently, an accepted redox probe set for yeast studies is not available. This paper validates fluorescent probes for glutathione and reactive oxygen status in yeast to support mechanistic studies of microgravity and drug metabolism. The plethora of fluorescent reagents for reactive oxygen species and glutathione makes head-to-head comparisons of all the alternatives impractical. These reagents measure the physiological milieu of reactive oxygen species and diverse thiols, rather than specific individual molecules. We report that in yeast, monochlorobimane (mBCL) and 2’,7’-dichlorodihydrofluorescein diacetate (DC-FDA) are suitable for fluorometric and flow cytometry studies of glutathione and reactive oxygen species, respectively. Both dyes have low background fluorescence, predictable loading, good retention, and are not acutely toxic to Saccharomyces cerevisiae. Both dyes show concordance with other fluorescent and biochemical assays of reactive oxygen species.