Independent enzymatic control of oxygen and H2O2 in cultured cells

Abstract

Aims: Oxidative stress has been identified as a major pathogenic factor in many liver diseases including alcoholic and non-alcoholic liver disease, hereditary hemochromatosis, and chronic hepatitis C. To mimic such conditions in vitro, oxidants such as H2O2 or redox-cycling drugs such as doxycyclin or antimycin A are routinely applied to cultured cells. However, these experiments are typically performed at air oxygen levels of 21%. This is hyperoxic as compared to the normal tissue environment considered to be at ca. 1–2% O2. We here aimed at developing an enzymatic system that allows to independently control oxygen and H2O2 levels in cultured cells. Methods and Results: We use glucose oxidase (GOX) to rapidly remove oxygen from the culture medium and we show that oxygen tensions are only determined by the GOX activity and the medium volume (diffusion distance). Catalase (CAT) was added to control H2O2 levels. Thus, varying Cat levels at a fixed GOX activity of kGOX=1.5×10–7 M/s H2O2 allowed the generation of non- to toxic H2O2 concentration (0.01µM to 10µM) under conditions of sustained hypoxia of 2% oxygen. Intracellular H2O2 levels were validated using the fusion protein YFP-OxyR that is based on the bacterial H2O2 sensor. Using heptoma cells (HepG2 and Hep3B), we finally demonstrate ROS-mediated toxicity and upregulation of redox-active transcription factors such as hypoxia inducible factor 1 alpha despite pronounced hypoxia. Conclusion: Enzymatic and independent control of H2O2 and oxygen with the GOX/CAT system is a novel in vitro approach to study defined oxidative stress conditions under hypoxia, a typical pathological condition in chronic liver disease.