Abstract

Glutathione is the most abundant of the low-molecular-mass molecules that provide reducing equivalents that protect cells from oxidative stress. We used immunoelectron microscopy to investigate glutathione distribution in normal and oxidatively stressed cells. Here, for the first time, we show that reduced glutathione is distributed relatively evenly throughout the cell, with the exception of the lumen of the rough endoplasmic reticulum, where little is detected. Oxidant exposure, either to 0.1 mM diamide or ethycrinic acid, eventually caused cellular glutathione depletion. However, despite entering a cell within seconds, both oxidants required hours to dramatically affect glutathione levels in the majority of cells in a population. Interestingly, cells within a homogeneous cell line population lost glutathione at different rates. Structural changes associated with oxidative stress, such as increased vacuolization and membrane blebbing, were correlated with glutathione depletion. Oxidant-exposed cells that appeared normal had higher glutathione levels than those within the same population that appeared stressed. The last reserves of cellular glutathione were found within mitochondria.