Cellular Pre-Adaptation to the High O₂ Concentration Used in Standard Cell Culture Confers Resistance to Subsequent...

The addition of hydrogen peroxide (H₂O₂) to cultured cells is widely used as a method to modulate redox-regulated cellular pathways, including the induction of programmed cell death in cell culture experiments and the testing of pro- and antioxidant compounds. Here, we assessed the effect on the cellular response to H₂O₂ of pre-adapting squamous cell carcinoma cells (A431) to the standard cell culture oxygenation of 18.6% O₂, compared to cells pre-adapted to a physiological skin O₂ concentration (3.0% O₂). We showed that cells pre-adapted to 18.6% O₂ resisted H₂O₂-induced cell death compared to cells pre-adapted to 3.0% O₂ for 96 h prior to treatment with H₂O₂. Moreover, the enzymatic activities of catalase and glutathione reductase, as well as the protein expression levels of catalase, were higher in cells pre-adapted to 18.6% O₂ compared to cells pre-adapted to 3.0% O_2. H₂O₂-resistant cells, pre-adapted to 18.6% O₂, exhibited increased nuclear Nrf-2 levels. It is concluded that A431 cells pre-adapted to standard cell culture oxygenation conditions resist H₂O₂-induced cell death. This effect may be related to their heightened activation of Nrf-2.