INTRODUCTION
Ultraviolet (UV) radiation generates hydrogen peroxide
(H2O2) and other reactive oxygen species (ROS) that
may result in damage to DNA, RNA, lipids, and proteins
as well as cell death.1-4 Reactive oxygen species are an
important inducer of human skin aging, carcinogenesis, and
inflammation5,6 and are generated both exogenously, including
environmental (pollution, smoking, UV radiation) factors, and
endogenously, including normal metabolic processes, such as
mitochondria-based cytochrome P450 cycling, which is linked
to intrinsic aging.7 Oxidative damage to cells that is induced
by ROS is highly regulated by a variety of factors, including
cellular metabolism and repair in addition to environmental,
hormonal, nutritional, and toxicological contributions.8
With increasing evidence suggesting a central role of ROS in cell
death, it is logical to investigate novel antioxidant agents that protect
skin against ROS-induced toxicity. Little is known regarding the
role of ROS in human skin cell necrosis or the ability of caffeine to
protect against necrosis. Caffeine is a naturally occurring nutraceutical
described to have antioxidant and carcinogenic effects.9-14
The goal of this study was to evaluate the ability of caffeine
to prevent normal human skin fibroblast cell death in an in
vitro model of acute ROS exposure. We found that caffeine
prevents necrosis in human skin fibroblasts independent of
antioxidant mechanisms.
MATERIALS AND METHODS
Caffeine Preparation
Caffeine was prepared as a 1 mM stock solution as previously
described,15 followed by 1:10 serial dilutions to 0.1 and 0.01
mM final concentrations.
Cell Culture
Human skin fibroblasts (AG14135; Coriell Institute for Medical
Research, Camden, NJ) were grown and treated in culture, as previously
described.16 Briefly, plated cells were washed twice with
Dulbecco's modified Eagle medium ([DMEM], Gibco; Invitrogen,
Carlsbad, CA), followed by a 4-hour incubation with various concentrations
of caffeine (1, 0.1, and 0.01 mM) in a 95:5% air:carbon
dioxide (CO2.) humidified incubator at 37°C. After incubation, plated
cells were washed twice with DMEM, followed by incubation
with H2O2 (1.2 mM) for 30 or 120 minutes, or no H2O2 for controls.
After incubation, plated cells were prepared for mitochondrial
membrane potential or apoptosis/necrosis assays.
Morphology and Analysis of Absolute Cell Counts
Determination of cell morphology after H2O2 challenge was performed
using defined gates based on forward and side scatter, as
previously described.16 Determination of absolute cell counts after
H2O2 challenge was performed using Flow-Count Fluorospheres
(Beckman Coulter, Fullerton, CA), as previously described.16
Apoptosis and Necrosis
Flow cytometry was used to analyze apoptotic and necrotic cell
death, as previously described.16 Briefly, apoptosis and necrosis
were distinguished by the combination of labeling of annexin V
(AV) and propidium iodide ([PI]; BD Biosciences Pharmingen, San
Jose, CA). AV-PI- was defined as viable cells; AV+PI- was defined as
early apoptosis; AV-PI+ was defined as early necrosis; and AV+PI+
was defined as late stage cell death, either by apoptosis or necrosis.
Reactive Oxygen Species
Intracellular ROS were labeled by mitochondrial uptake of dihydrorhodamine
123 and measured by flow cytometry, as
