| Summary: | Monocyte cells can be damaged when exposed to reactive oxidative species during
inflammatory events within the body. This research project has examined in detail the
cellular events associated with free radical damage induced death of monocyte cells,
using the water soluble peroxyl radical generator 2, 2' -azobis(2-methylpropionamidine)
dihydrochloride (AAPH) as a model of inflammation induced oxidative stress.
The human monocyte-derived cell line THP-1 was incubated with 10mM AAPH
in Earle's Balanced Salt Solution at 37°C for up to 24 hours. Protein hydroperoxide
formation was observed to occur after a six hour lag, period which corresponded to the
time in which most of the intracellular glutathione was lost from the cell. Cell viability
loss, measured by the MTT reduction, was also observed after six hours of incubation
while no caspase-3 activation was observed. A control ethanol treatment confirmed that
caspase-3 could be activated within the THP-1 cells. Flow cytometry analysis failed to
show phosphatidylserine exposure due to the AAPH treatment but DNA staining by
propidium iodide confirmed the loss of viability seen with the MTT cell viability assay.
This data strongly suggests that THP-1 cells undergo AAPH induced necrosis as a result
of cellular damage including GSH loss and protein hydroperoxide formation.
Microscopic examination of the cells identified many of the morphological
characteristics of necrosis in the AAPH treated cells.
Studies on the effect of AAPH on U937 cells were unsuccessful due to the
possible presence of intracellular infection, resulting in an increased oxidant stress in
the untreated cells.
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