Effect of Diphenyleneiodonium Chloride on Intracellular Reactive Oxygen Species Metabolism with Emphasis on NADPH Oxidase and Mitochondria in Two Therapeutically Relevant Human Cell Types

Abstract

Reactive oxygen species (ROS) have recently been recognized as important signal transducers, particularly regulating proliferation and differentiation of cells. Diphenyleneiodonium (DPI) is known as an inhibitor of the nicotinamide adenine dinucleotide phosphate oxidase (NOX) and is also affecting mitochondrial function. The aim of this study was to investigate the effect of DPI on ROS metabolism and mitochondrial function in human amniotic membrane mesenchymal stromal cells (hAMSCs), human bone marrow mesenchymal stromal cells (hBMSCs), hBMSCs induced into osteoblast-like cells, and osteosarcoma cell line MG-63. Our data suggested a combination of a membrane potential sensitive fluorescent dye, tetramethylrhodamine methyl ester (TMRM), and a ROS-sensitive dye, CM-H2DCFDA, combined with a pretreatment with mitochondria-targeted ROS scavenger MitoTEMPO as a good tool to examine effects of DPI. We observed critical differences in ROS metabolism between hAMSCs, hBMSCs, osteoblast-like cells, and MG-63 cells, which were linked to energy metabolism. In cell types using predominantly glycolysis as the energy source, such as hAMSCs, DPI predominantly interacted with NOX, and it was not toxic for the cells. In hBMSCs, the ROS turnover was influenced by NOX activity rather than by the mitochondria. In cells with aerobic metabolism, such as MG 63, the mitochondria became an additional target for DPI, and these cells were prone to the toxic effects of DPI. In summary, our data suggest that undifferentiated cells rather than differentiated parenchymal cells should be considered as potential targets for DPI.

Mitochondrial ROS driven by NOX4 upregulation promotes hepatocellular carcinoma cell survival after incomplete radiofrequency ablation by inducing of mitophagy via Nrf2/PINK1

“…Further inhibition of NADPH oxidase activity by DPI or VAS2870 attenuated mtROS production, demonstrating an upstream role of NADPH oxidase in mtROS activation. Recent studies have also reported crosstalk between these two producers [39,[48][49][50]. Indeed, a previous study showed that the mitochondrial inner membrane was still intact, and there was no significant change in the composition of electron transport chain complexes in isolated mitochondria after acute heat treatment [26].…”

Eosinophil extracellular traps in asthma: implications for pathogenesis and therapy

“…Diphenylammonium chloride (DPI) and N-acetylcysteine (NAC) are NADPH oxidase inhibitors and ROS scavengers, respectively, both of which reduce intracellular ROS levels [ 115 , 116 ].Silveira et al found that DPI and NAC treatment reduced the number of inflammatory cells and ROS levels in the lungs of OVA-challenged mice, in addition to reducing airway levels of released EETs [ 117 ]. However, as mentioned above, not all stimuli are dependent on the PAD4 mechanism, and the necessity of NADPH enzymes for EETs formation is controversial.…”

Inhibition of phase-1 biotransformation and cytostatic effects of diphenyleneiodonium on hepatoblastoma cell line HepG2 and a CYP3A4-overexpressing HepG2 cell clone

“…Its chemical nature makes DPI a potent inhibitor of flavin bearing oxidoreductases, which are generally an integral element of electron transport chains. DPI have a wide spectrum of known cellular targets including CPR [13,15,23], NADPH oxidase (NOX) [24][25][26][27][28][29][30][31], mitochondrial respiratory chain complex I (NADH ubiquinone oxidoreductase) [28,[32][33][34], and different types of nitric oxide synthase [13,35]. It is assumed that DPI inhibition is achieved by covalent modification of flavin and/or heme prosthetic groups within enzymes based on radical formation.…”