The effects of ozone exposure on markers of celllar resilience in cultured human epithelial HeLa cells

Abstract

Ozone is a natural occurring gas with strong oxidising properties. While its biological effects are associated with toxicity, it has also been used for therapeutic purposes. In biological systems ozone can elicit dose-dependent oxidative stress, which may induce adaptation. The mechanisms for these effects remain illusive. Previous studies in our laboratory indicated that repetitive exposure of cultured human epithelial (HeLa) cells to ozone may induce cytoprotective mechanisms (adaptation), which may include the up-regulation of anti-apoptotic pathways. The aim of the current study was to determine the effects of single and repetitive exposure of HeLa cells to ozone on the expression of genes that encode for anti-apoptotic (Akt, Bcl2, CREB and NFKB) and pro-apoptotic (Bax, caspase 3 and 8) proteins, as well as the corresponding protein expression levels. Cultured HeLa cells were exposed to control or ozone-saturated glucose-free Krebs-Henseleit solution. Exposures consisted of 4 x 5-minute exposures every four hours, followed by a 16-hour incubation in normal culture medium and then a 25-minute exposure. Cells were then lysed immediately (0 h) or after 8 hours (8 h) incubation in normal culture medium. The relative expression of genes encoding for pro-apoptotic factors and anti-apoptotic factors was then determined with quantitative real-time RT-PCR (RT2-PCR). Protein expression was also investigated for Akt, Akt phospho specific, CREB, CREB phospho specific, pro-caspase 3 and caspase 3 cleaved protein with Western blot after the respective ozone exposure regimens and 8 h after incubation in normal culture medium. When measuring gene expression at 0 h, genes encoding for Bcl-2, CREB and caspase 3 were down-regulated by ozone administered 4x5 minutes every 4 hours, but these returned to pre-treatment values at 8 h. Importantly, cells treated with ozone for 4 x 5 minutes every 4 hours plus a single 25 minute exposure to ozone 16 hours later, showed at 8 h an up-regulation of the genes encoding for the anti-apoptotic factors Akt, and CREB, while corresponding cells at 8 h that treated with ozone either for a single 25-minute exposure or a 4 x 5 minutes plus a single 25 minute exposure showed an up-regulated expression of caspase 3. The Western blot analysis of the proteins showed no significant differences or trends of protein expression for any of the corresponding treatment groups at 8h. The current gene expression data suggest that up-regulation of Akt and CREB (anti-apoptotic) may be involved in the adaptation of cultured human epithelial HeLa cells after repetitive exposure to ozone. While it up-regulation of caspase 3 (pro-apoptotic) has also been demonstrated, its role and significance in ozone-elicited adaptation is unclear.