Modulation of intracellular GSH in THP-1 cells during oxidative stress induced by AAPH
Degree GrantorUniversity of Canterbury
Degree NameMaster of Science
The human monocyte-derived THP-1 cell line was incubated with 10mM AAPH in Earle’s Balanced Salt Solution at 37°C for up to 24 hours. Protein hydroperoxide formation occurred after an 8 hour lag phase which corresponded to glutathione loss observed in the cells. SDS-Page analysis confirmed protein degradation occurred after 6 hours. Cell viability measured by the MTT reduction assay also dropped after 8 hours. Reduction of intracellular glutathione levels using BSO caused reduction of the lag phase seen in protein hydroperoxide formation. Cell viability of BSO-treated cells was lower than control cells, indicating the initiation of apoptotic events. Flow cytometry analysis showed no difference between BSO-treated and control cells, indicating that GSH levels do not have an effect on the type of cell death observed in AAPH-induced oxidative damage on THP-1 cells. These results confirmed previous data in the lab suggesting THP-1 cells undergo AAPH-induced necrosis as a result of cellular damage, including the loss of GSH and the formation of protein hydroperoxides.