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The effects of H 2 O 2  are widely studied in cell cultures and other  in vitro  systems. However, such investigations are performed with the assumption that H 2 O 2  concentration is constant, which may not properly reflect  in vivo  settings, particularly in redox-turbulent microenvironments such as mitochondria. Here we introduced and tested a novel concept of fluctuating oxidative stress. We treated C6 astroglial cells and primary astrocytes with H 2 O 2 , using three regimes of exposure – continuous, as well as fluctuating at low or high rate, and evaluated mitochondrial membrane potential and other parameters of mitochondrial activity – respiration, reducing capacity, and superoxide production, as well as intracellular ATP, intracellular calcium, and NF-κB activation. When compared to continuous exposure, fluctuating H 2 O 2  induced a pronounced hyperpolarization in mitochondria, whereas the activity of electron transport chain appears not to be significantly affected. H 2 O 2  provoked a decrease of ATP level and an increase of intracellular calcium concentration, independently of the regime of treatment. However, fluctuating H 2 O 2  induced a specific pattern of large-amplitude fluctuations of calcium concentration. An impact on NF-κB activation was observed for high rate fluctuations, whereas continuous and low rate fluctuating oxidative stress did not provoke significant effects. Presented results outline the (patho)physiological relevance of redox fluctuations.

Fluctuating vs. Continuous Exposure to H2O2: The Effects on Mitochondrial Membrane Potential, Intracellular Calcium, and NF-κB in Astroglia