EPR detection of cellular and mitochondrial superoxide using cyclic hydroxylamines

Abstract

Superoxide (O2•) has been implicated in the pathogenesis of many human diseases but detection of the O2• radicals in biological systems is limited due to inefficiency of O2• spin trapping and lack of site-specific information. In this work we studied production of extracellular, intracellular and mitochondrial O2• in neutrophils, cultured endothelial cells and isolated mitochondria using new set of cationic, anionic and neutral hydroxylamine spin probes with various lipophilicity and cell permeability. Cyclic hydroxylamines rapidly react with O2• producing stable nitroxides and allowed site-specific O2• detection in intracellular, extracellular and mitochondrial compartments. Negatively charged 1-hydroxy-4-phosphono-oxy-2,2,6,6-tetramethylpiperidine (PP-H) and positively charged 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium (CAT1-H) detected only extramitochondrial O2•. Inhibition of EPR signal by SOD2 overexpression showed that mitochondria targeted mitoTEMPO-H detected intramitochondrial O2• both in isolated mitochondria and intact cells. Both 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) and 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CM-H) detected increase in cytoplasm O2• stimulated by PMA but only CM-H and mitoTEMPO-H showed increase in rotenone-induced mitochondrial O2•. These data show that new set of hydroxylamine spin probes provide unique information about site-specific production of O2• radical in extracellular or intracellular compartments, cytoplasm or mitochondria.

Production of superoxide in chloroplast thylakoid membranes

“…A new class of O 2 ÅÀ -detectors are cyclic hydroxylamines (CHAs), which become paramagnetic after being oxidized. Their rate constants of oxidation by O 2 ÅÀ are two orders of magnitude higher than the constants of O 2 ÅÀ trapping by such established spin traps as DMPO and EMPO [10]. The reaction product, nitroxide radical, which can be detected by ESR, is much more stable than spin adducts.…”

“…The partition coefficients of TMT-H and DCP-H in octanolphosphate buffer (pH 7.4) system are 35 and 0.05, respectively [10]. DCP-H unlike TMT-H has two carboxylic groups and at pH higher than 7 is negatively charged.…”

Lipotoxic brain microvascular injury is mediated by activating transcription factor 3-dependent inflammatory and oxidative stress pathways

“…Each treatment condition also contained 0.5 mM 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) spin trap (35). Spin traps are capable of rapidly trapping free radicals such as O 2 . to form more persistent radicals (spin adducts) detectable by electron paramagnetic resonance (EPR).…”

Imaging of Superoxide Generation in the Dopaminergic Area of the Brain in Parkinson’s Disease, Using Mito-TEMPO

“…10−12 In the present study, we applied the described methodology for visualization of superoxide production in the dopaminergic area of the brain of MPTP-treated mice, using mito-TEMPO. Mito-TEMPO is BBB-and cell-penetrating nitroxide derivative with prevalent mitochondrial accumulation, 26 superoxide scavenging properties 26,27 and comparatively high T 1 MRI contrast. This makes it a useful molecular sensor for detection of superoxide production in the dompaniergic area of the brain, using MRI ( Figure 1).…”