Abstract
Ionizing radiation produces reactive oxygen species (ROS) leading to cellular DNA damage. Therefore, patients undergoing radiation therapy or first responders in radiological accident scenarios could both benefit from the identification of specifically acting pharmacological radiomitigators. The synthetic triterpenoid bardoxolone-methyl (CDDO-Me) has previously been shown to exert antioxidant, anti-inflammatory and anticancer activities in several cell lines, in part by enhancing the DNA damage response. In our study, we examined the effect of nanomolar concentrations of CDDO-Me in human peripheral blood mononuclear cells (PBMC). We observed increased cellular levels of the antioxidative enzymes heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase (quinone1) and mitochondrial superoxide dismutase 2 by immunoblotting. Surprisingly, we found increased intracellular ROS-levels using imaging flow-cytometry. However, the radiation-induced DNA double-strand break (DSB) formation using the γ-H2AX + 53BP1 DSB focus assay and the cytokinesis-block micronucleus assay both revealed, that nanomolar CDDO-Me pre-treatment of PBMC for 2 h or 6 h ahead of X irradiation with 2 Gy did neither significantly affect γ-H2AX + 53BP1 DSB foci formation nor the frequency of micronuclei. CDDO-Me treatment also failed to alter the nuclear division index and the frequency of IR-induced PBMC apoptosis as investigated by Annexin V-labeled live-cell imaging. Our results indicate that pharmacologically increased cellular concentrations of antioxidative enzymes might not necessarily exert radiomitigating short-term effects in IR-exposed PBMC. However, the increase of antioxidative enzymes could also be a result of a defensive cellular mechanism towards elevated ROS levels.
Highlights
Ionizing radiation (IR) induces biological effects, amongst others, via the generation of intracellular reactive oxygen or nitrogen species (ROS, RNS) such as hydroxyl radicals, which is considered to represent a major cause for post-irradiation cell damage (Azzam et al 2012; Maier et al 2016; Riley 1994)
We investigated reactive oxygen species (ROS) generation within peripheral blood mononuclear cells (PBMC) by imaging flow cytometry using the fluorochrome DCF as ROSindicator
DCF fluorescence signal intensity was significantly increased in all treatment groups when irradiating H2DCF-labeled PBMC with 2 Gy (Fig. 2a, b)
Summary
Ionizing radiation (IR) induces biological effects, amongst others, via the generation of intracellular reactive oxygen or nitrogen species (ROS, RNS) such as hydroxyl radicals, which is considered to represent a major cause for post-irradiation cell damage (Azzam et al 2012; Maier et al 2016; Riley 1994). Both acute and chronic biological effects of IR. Mitochondrial SOD2 has been shown to be essential for aerobic life by its capability to convert the physiological superoxide byproduct of the electron-transport respiratory chain to peroxide (Son et al 2013; Dinkova-Kostova and Talalay 2010; Lebovitz et al 1996)
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