Oxidative stress (OS), involved in many biological phenomena, such as aging, carcinogenesis and inflammation, is defined as an imbalance between reactive oxygen species (ROS) and organism’s antioxidant defenses. The consequences of this imbalance lead to the disruption of cellular redox homeostasis and oxidative damage to DNA, proteins and lipids.(1) One metric of the relative strengths of oxidizers implicated in causing pathogenic oxidative damage via ROS production is the effective redox potential experienced by the cells. Therefore, establishing the relationship between the strength of the oxidative action, characterized by redox potential, and the biomolecular consequences is critical to elucidating the thermodynamic framework of oxidative damage. Experiments with OS in the lab environment require subjecting the study object to a given level of oxidative bias relative to biosystem’s natural equilibrium, preferably in a well-defined, reproducible and quantitative fashion. Popular in vitro OS inducers include exogenous chemical agents (hydrogen peroxide, paraquat, menadione, and t-butyl hydroperoxide), ionizing radiation or heat shock. In addition to disrupting redox homeostasis, each of these agents will also have a specific chemical interaction with cell components, which have to be accounted for during interpretation.(2)

Here, we describe a novel way to create an OS environment for surface attached Chinese Hamster Ovary (CHO) cells by exposing them to an electrochemical potential gradient. With this technique we can apply a stable linear potential profile over a cell monolayer, thus providing an efficient method to screen live cell response to an exogenous redox background. It is based on a bipolar electrode configuration (3), adapted for a rectangular cell growth flask. A constant current is produced in cell media, thus generating an electric field and potential drop over the mammalian cell monolayer. Surface attached cells experience varying electrochemical potential, depending on their location along the current axis. Subsequently, cell viability gradients were produced within a range of treatment levels. The live-dead assay fluorescence image of the flask bottom slide shows the CHO cell condition following incubation under the redox potential gradient for a fixed time interval (see Figure). Images contain a rather uniform live, dead and a narrow changeover region ( magnified view in the lower panel of Figure ), consistent with a commencement of cell death at increasingly positive solution redox potential. The cell viability ratio was calculated from integrated pixel density using a custom ImageJ software macro. Therefore, the sigmoidal cell viability curve shape, produced when cells are exposed to the electrochemical potential gradient , is consistent with a two state conversion model and could be numerically characterized by a half maximum effective potential. This concept is widely used to describe dose-response curves in toxicology. Such numerical values would be useful to compare cell viability when subjected to oxidative stress in diverse experimental circumstances. That way, the higher effective potential value would indicate the increase in cell viability or the efficacy of the antioxidant.

We have compared CHO cell response when exposed to three hydrogen peroxide concentrations (20 mM, 50 mM, and 100 mM) with the exposure to electrochemically induced potential gradient. Redox potential values of these H₂O₂ preparations were measured in separate experiments 1h after hydrogen peroxide was diluted into the cell growth media. Remarkably, cell viability vs solution redox potential dependencies are very close suggesting that exogenous hydrogen peroxide causes CHO cell OS largely by a positive shift in the solution redox potential, at least in this concentration range.

Our test platform allows for direct imaging and analysis of cell viability, exposed to a range of electrochemical potentials. The slide vessel system can readily be adapted for other cellular assays such as ROS detection, apoptosis, etc. The system is particularly useful for providing a linear range of oxidative and reductive potentials in a single experiment. By replacing the vessel contents before during and after treatment a wide range of solution conditions can be tested and cell images can be acquired during oxidative treatment. This is particularly useful for in vitro testing of the effectiveness of additives such as anti-oxidants as it enables monitoring a real time cellular response. In addition, various cell types including cancer cell lines can be compared in their resistance to oxidative potentials and effectiveness of various drugs.

References

(1) Halliwell, B. Trends Pharmacol Sci 2013, 34, 301.

(2) Buettner, G. R.; Wagner, B. A.; Rodgers, V. G. J. Cell Biochem Biophys 2013, 67, 477.

(3) Fleischmann, M.; Ghoroghchian, J.; Rolison, D.; Pons, S. J Phys Chem-Us 1986, 90, 6392.