Triterpenoid CDDO-Me induces ROS generation and up-regulates cellular levels of antioxidative enzymes without induction of DSBs in human peripheral blood mononuclear cells

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.

Keywords: Antioxidant; Bardoxolone-methyl; CDDO-me; DNA damage; Micronucleus assay; Radioprotective activity; γ-H2AX foci analysis.

Fig. 1

a Molecular structure of synthetic oleanane triterpenoid Bardoxolone-methyl or CDDO-Me (2-cyano-3,12-dioxooleane-1,9(11)-dien-28-oic acid methyl ester). b Incubation of PBMCs with nanomolar CDDO-Me for 6 h induced cellular accumulation of the antioxidative enzymes HO-1, NQO1 and SOD2 as shown by immunoblotting (n = 3). DMSO served as control (0 nM CDDO-Me)

Fig. 2

Cellular ROS activity was measured by ImageStream™ flow cytometry using the fluorogenic dye DCF. a PBMCs were treated with 0 nM (DMSO), 20 nM or 50 nM CDDO-Me for 5.5 h before DCFDA-labeling for 0.5 h and subsequent irradiation (sham versus 2 Gy). ROS induction by 2 Gy significantly increased DCF fluorescence intensity. b Representative pictures of PBMCs show differential DCF fluorescence intensity levels (Ch2) and corresponding brightfield microscopy (Ch4). c ROS activity appeared to be significantly elevated by 20 nM CDDO-Me treatment under both, irradiation and sham conditions. Statistics: one way analysis of variance, Bonferroni post-hoc test, ***p < 0.001; n.s. not significant; n > 11.000 (error bars: SEM)

Fig. 3

a IF staining for DSB-indicating γ-H2AX (green) + 53BP1 (red) foci. Colocalizing colors (reddish/yellow) indicate DSB foci (examples arrowed) in PBMC nuclei (blue) 6 h after 2 Gy irradiation versus control (0 Gy). b γ-H2AX + 53BP1 DSB foci frequencies in human PBMCs after 2 or 6 h pre-incubation with nanomolar concentrations of CDDO-Me showed no significant changes when fixing cells 6 h or 24 h post IR. DMSO served as control (0 nM CDDO-Me). Statistics: one way analysis of variance, Bonferroni post-hoc test, n.s. not significant for p > 0.05; n = 100 cells per sample (error bars: SE)

Fig. 4

MNi frequencies in PBMC after pre-treatment with CDDO-Me (a 2 h/20 nM; b 6 h/20 nM; c 2 h/50 nM; d 6 h/50 nM). Error bars: standard deviations of four independent experiments. DMSO served as control (0 nm CDDO-Me). e Representative DAPI-stained binucleated human peripheral blood lymphocyte (harboring one, two and three MNi, arrows) scanned with Metafer4 platform

Fig. 5

Nuclear division index (NDI) of PBMC after incubation with CDDO-Me (a 2 h/20 nM; b 6 h/20 nM; c 2 h/50 nM; d 6 h/50 nM). DMSO served as control (0 nM CDDO-Me). Error bars: standard deviations of four independent experiments

Fig. 6

a Viability of PBMCs after incubation with CDDO-Me (20 nM/50 nM; administration 6 h before irradiation). Cellular ATP content as a measure of cell viability was determined using the Cell Titer Glo™ Assay. b Induction of apoptosis was monitored in the live cell imaging IncuCyte S3 system by quantification of Annexin V-positive cells (green) and subsequent normalization considering confluency. Time-dependent increase of Annexin V-positive cells (green; arrows) is shown in representative pictures. No significant changes were measurable between the treatment groups at the respective incubation time points. Statistics: one way analysis of variance, Bonferroni post-hoc test, n.s. not significant for p > 0.05; n = 3 (error bars: SD)