Oxidative stress due to elevated production and levels of cellular reactive oxygen species is an important factor in numerous human disorders. Cigarette smoke is both a source and an inducer of cellular oxidative stress and has been implicated in the development and progression of smoking-related diseases including chronic obstructive pulmonary disease, cancer and cardiovascular disease. We describe the development and utilisation of an in vitro oxidative stress assay in which we monitored the levels of the intracellular antioxidant glutathione (GSH) in human bronchial epithelial (H292) cells.
Methods: GSH levels were measured using the GSH Glo assay (Promega) in confluent monolayers of H292 cells. Cigarette smoke total particulate matter (TPM) from University of Kentucky 3R4F reference cigarettes was trapped on a Cambridge filter pad, eluted in DMSO and added to cell media for 1 h at 37 °C prior to measuring GSH levels.
Results: TPM caused a concentration-dependent decrease in GSH levels when compared to cells treated with media alone. We examined the variability of cellular GSH levels in response to a number of experimental factors (TPM batch, cell passage number, assay plate, intra-plate replicates and experimenter). Statistically significant variation was observed for several parameters. However, as the level of variation was less than 10% of the mean it was not deemed to be biologically significant. We also examined the effects of TPM obtained from combustible reduced toxicant prototype (RTP) cigarettes compared to that obtained from appropriately-matched commercial control cigarettes. Statistical analyses showed that when using TPM derived from RTP cigarettes, lowering of GSH levels was less apparent compared to TPM derived from control cigarettes.
Conclusion: Monitoring GSH levels in vitro is a relevant oxidative stress assay which demonstrates acceptable levels of data variability and is suitable for the assessment of the biological effects of RTP particulate phase smoke extracts.