Accumulating evidence suggests that increased reactive
oxygen species (ROS) production has a role in inducing
senescence of human dermal
fibroblasts (HDFs) via the activation
of DNA damage and redox signaling pathways [1]. Unfortunately,
ROS-reducing antioxidant agents are only marginally effective in
delaying or preventing cellular senescence, even at a high
concentration [2,3]. We evaluated endogenous antioxidant
systems to better understand these
findings. Glutathione (GSH)
is the most abundant thiol-containing tripeptide that plays a
major role in eliminating H2O2 through a GSH peroxidase-
catalyzed reactions [4]. Moreover, GSH reduces oxidized proteins
through glutathionylation, thereby regulating the intensity and
duration of redox signaling. Thus, intracellular GSH level are
critical for maintaining redox homeostasis. Measuring GSH level
in live cells is challenging owing to the irreversible reaction of
fluorescent probes with GSH. We developed a new coumarin-
based GSH probe, FreSHtracer, which reversibly reacts with GSH
accompanied by spectral shifts in UV absorption (520 to 430 nm)
and
fluorescence emission (F580 to F510), allowing ratiometric
measurement of GSH in live cells [5]. Here, we monitored the
changes in GSH levels in HDFs during senescence and evaluated
the effect of antioxidants on GSH levels by measuring the F510/F580
ratio (FR).