The effects of ozone exposure on the viability and function of cultured human cell lines

Abstract

Ozone exposure (Oз ) has been shown to have systemic biological effects, including dose- dependent oxidative stress and adaptation. However, the mechanisms of these effects remain elusive. The aims of the current study were to i) establish appropriate conditions for in vitro Oз, acute and repeated exposures, utilising cultured human epithelial (HeLa) cells, and ii) investigate effects of acute and repeated Oз exposure on cell viability. The involvement of enzymatic pathways in observed cellular adaptation was investigated by including selective enzyme inhibitors and observing changes in DNA integrity with Oз exposures. Cultured HeLa cells were exposed to Oз -saturated Krebs-Henseleit solution using various dosing regimes, including acute (0-55 minutes) and repeated exposures (4 x 5 minute Oз exposure every 4 hours, followed by overnight incubation, 25 minutes Oз, re-exposure and 0, 4, 8 or 12 hours incubation). Thereafter cell viability was determined utilising the trypan blue. MTT and DNA-fragmentation assay. Oз exposures were also performed in the presence or absence of ME10092 (xanthine oxidase and NFкβ inhibitor), Z-DQMD-FMK (caspase-3 and -6 inhibitor) and (-)-deguelin (Akt inhibitor). According to the trypan blue test, acute Oз exposure compromised cell membrane integrity. while the MTT test indicated only a slight reduction in mitochondria1 function. A repeated exposure regime consisting of multiple small dose exposures followed by a single high dose exposure was associated with a protective adaptation in cell membrane integrity. This was reversed by inhibition of Akt, caspase-3, xanthine oxidase and NFкβ. Repeated Oз exposures increased DNA integrity and repair capacity. In conclusion, the current data suggest that acute in vitro Oз exposure decreases HeLa cell membrane integrity, with no significant effect on mitochondria1 function. Importantly, regime specific multiple exposures to Oз induces an adaptive response, whereby cell plasticity is upregulated. The latter adaptive effect is associated with the modulation of apoptotic and anti-apoptotic pathways.