Cellular redox responses under standard oxygen cell culture conditions compared with physioxia

The physiological oxygen environment in vivo (physioxia) typically ranges from 1.0-12.5% O₂. However, in vitro mammalian cell culture largely utilises an O₂ concentration ([O₂]) of 18.6% O₂ (which accounts for the partial pressure of water in the incubator) during the growth stage. As such, most mammalian cells are grown in a non-physiological [O₂] during in vitro cell culture. The effect of growing mammalian cells long term in 18.6% O₂ on the subsequent cellular response to redox-active compounds compared to cells grown in physioxia is not well understood. It was shown that human non-melanoma squamous cell carcinoma cells (A431) cultured long term under 18.6% O₂ were resistant to auranofin (thioredoxin reductase inhibitor), and H₂O₂-induced cell death compared to A431 cells grown in 3.0% O₂ (physioxia in human skin). A431 cells grown in 18.6% O₂ were also resistant to H₂O₂, mercaptosuccinic acid, and cumene hydroperoxide-induced lipid peroxidation compared to A431 cells grown in 3.0% O₂. Auranofin-induced ROS generation and oxidative stress was lower in A431 cells grown in 18.6% O₂ compared to A431 cells grown in 3.0% O₂ for 96 h under the same treatment conditions. Catalase and glutathione reductase enzyme activities were higher in A431 cells grown in 18.6% O₂ compared to A431 cells grown in 3.0% O₂ for 96 h. Additionally, the expression levels of nuclear Nrf-2 protein, and NAD(P)H quinone oxidoreductase (NQO-1) protein, were about two-fold higher in A431 cells grown in 18.6% O₂ compared to the levels in A431 cells grown in 3.0% O₂ for 96 h. Auranofin treatment did not induce NQO-1 protein expression in A431 cells grown in 18.6% O₂ whilst A431 cells grown in 3.0% O₂ for 96 h showed higher auranofin-induced NQO-1 protein expression levels. These data showed that A431 grown in 18.6% O₂ exhibited resistance to auranofin and H₂O₂-induced cell death compared to A431 cells grown in physioxia under the same treatment conditions. This resistance involves, in part, higher levels and activities of certain antioxidant proteins and enzymes as conferred by long term growth in standard cell culture [O₂] compared to physioxia. It is concluded that the in vitro testing of redox-active compounds on squamous cell carcinoma cells grown in 18.6% O₂ may yield artefactual in vitro data compared to such in vitro testing on A431 cells grown in physioxia. It is advised that the in vitro testing of redox-active compounds on human non-melanoma squamous cell carcinoma cells should be performed on such cells grown in physioxia for at least four days prior and not on such cells grown chronically in a standard cell culture [O₂].