Glutathione, a tripeptide molecule, is the primary endogenous antioxidant in cells. It is abundant and participates in a wide array of protective antioxidant and detoxification mechanisms. Homeostatic mechanisms maintain glutathione predominantly in its reduced state (GSH); oxidation of GSH causes the accumulation of its oxidized dimer form GSSG. Thus, examining the ratio of GSH:GSSG is a good indicator of oxidative stress and redox maintenance. Many chronic diseases of aging have oxidative stress components that cause significant declines in GSH:GSSG, rendering cells and tissues more susceptible to further damage. Although the biochemical pathways that regulate glutathione balance are well-defined, the genetic regulation of this parameter is not, despite findings suggesting that glutathione balance is regulated by genetics in mammals. The current project identifies genes regulating glutathione balance in mice. This work may reveal novel targets for therapies that maintain glutathione balance in disease, which would have the potential in humans to improve patient treatment, prognosis, and quality of life. Our experimental strategy is to confirm that differences in glutathione balance exist between C57BL/6 (B6) and DBA/2 (D2) mice by quantifying GSH and GSSG concentrations and calculating GSH:GSSG in several tissues. We will then analyze recombinant inbred mouse strains produced from B6 x D2 crosses (BXD mice) in order to perform QTL analysis and find loci responsible for GSH:GSSG in mice. Concurrently, we will perform a strain survey involving 10 additional inbred strains, including 4 wild-derived strains that will contribute significant genetic variation to this study. Strains with very different GSH:GSSG will be crossed and F2 progeny will be produced; QTL analysis will then proceed to confirm the loci identified with the BXD mice. Aim 1 tests if genetic regulation o GSH:GSSG ratios (as well as absolute values of [GSH] and [GSSG]) in liver, kidney, heart, and brain causes significant variation among at least 10 recombinant inbred BXD lines of mice. If this hypothesis is correct, we will use complete RI line analysis in 60 additional BXD strains to identify loci and suggest candidate genes regulating glutathione balance. Aim 2 defines glutathione balance in liver, kidney, heart, and brain in each of 12 inbred strains of mice aged 4-6 months. The set of 12 strains includes 4 wild-derived strains to maximize genetic variance and the chance of finding genetic regulators of glutathione balance in mice. We will cross mouse strains with higher and lower GSH:GSSG to produce F2 progeny and identify regulatory loci. Aim 3 compares glutathione balance in liver, kidney, heart, and brain of inbred mice in middle-aged, 14- 15 month old mice, and in young adult 3-4 month old control mice from 12 genetically diverse inbred strains. We will cross strains with important differences in aging to identify loci responsible for this difference. In all aims, gene identification will suggest clinical treatments. PUBLIC HEALTH RELEVANCE: Glutathione is an endogenous cellular antioxidant that becomes dysregulated in a wide variety of chronic disease, for example, diabetes and Alzheimer's disease; maintaining the proper balance of glutathione may prevent or slow progression of these diseases. Glutathione status appears to be affected by genetics, but past research has focused on biochemical rather than genetic tools. This project will, for the first time, identify how genetic variation influences glutathione balance in mammals as part of a long-term effort to help determine the genetic susceptibility to a wide variety of chronic disease and guide future design of more effective clinical interventions.