Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia

Abstract

In vivo, mammalian cells reside in an environment of 0.5-10% O 2 (depending on the tissue location within the body), whilst standard in vitro cell culture is carried out under room air. Little is known about the effects of this hyperoxic environment on treatment-induced oxidative stress, relative to a physiological oxygen environment. In the present study we investigated the effects of long-term culture under hyperoxia (air) on photodynamic treatment. Upon photodynamic irradiation, cells which had been cultured long-term under hyperoxia generated higher concentrations of mitochondrial reactive oxygen species, compared with cells in a physioxic (2% O 2 ) environment. However, there was no significant difference in viability between hyperoxic and physioxic cells. The expression of genes encoding key redox homeostasis proteins and the activity of key antioxidant enzymes was significantly higher after the long-term culture of hyperoxic cells compared with physioxic cells. The induction of antioxidant genes and increased antioxidant enzyme activity appear to contribute to the development of a phenotype that is resistant to oxidative stress-induced cellular damage and death when using standard cell culture conditions. The results from experiments using selective inhibitors suggested that the thioredoxin antioxidant system contributes to this phenotype. To avoid artefactual results, in vitro cellular responses should be studied in mammalian cells that have been cultured under physioxia. This investigation provides new insights into the effects of physioxic cell culture on a model of a clinically relevant photodynamic treatment and the associated cellular pathways.

Hypoxia and metabolic inhibitors alter the intracellular ATP:ADP ratio and membrane potential in human coronary artery smooth muscle cells

“…Although cell culture is typically carried out at an atmospheric condition with an oxygen concentration of 20-21%, we are aware that cells in vivo reside under considerably lower oxygen tension, named physiologically relevant O 2 concentration, also known as physioxia (Carreau et al, 2011). It has been reported that standard in vitro cultured cells experience a constant state of oxidative stress due to hyperoxic environment (Ferguson et al, 2018). Therefore, most of the contemporary experiments using in vitro cell culture are conducted with oxidatively stressed cells.…”

“…Therefore, most of the contemporary experiments using in vitro cell culture are conducted with oxidatively stressed cells. Culture in hyperoxic conditions has conferred a protective resilience to many kinds of oxidative stressors, which is thought to be an artefact arising from the current culture leading to poor translation of investigations from in vitro to in vivo (Ferguson et al, 2018;Lin et al, 2020). On the contrary, the extent of antioxidant enzyme activities and redox homeostasis are examined to be closer to in vivo conditions if the cells are pre-conditioned under physioxia (Pastore et al, 2001;Shindo et al, 1994).…”

Docosahexaenoic Acid Attenuates Mitochondrial Alterations and Oxidative Stress Leading to Cell Death Induced by Very Long-Chain Fatty Acids in a Mouse Oligodendrocyte Model

“…The results observed with oxidative stress are obtained under conventional conditions of cell culture which transiently presents hyperoxia. Consequently, these results should be verified in models close to the conditions found in vivo, since cell cultures under these "conventional" conditions have certain limitations [14][15][16].…”

Irreversible oxidative post-translational modifications in heart disease

“…A crucial point not considered by most studies employing in vitro systems is the lower in vivo concentration of oxygen as compared to air. Physioxic conditions are tissue dependent but concentrations of 0.5-10% oxygen are commonly reported [121], which suggests a 2-to 40-fold overabundance of oxygen under commonly used cell culture conditions. The effect of long-term hyperoxic culturing on RNOS levels and expression of redox defense systems was recently investigated by Ferguson et al A significant increase in RNOS levels and a plethora of adaptions to the hyperoxic conditions as compared to physioxia were found, including different levels of lipid peroxidation and increased antioxidant enzyme expression levels and activities [121].…”