Procainamide, but notN-Acetylprocainamide, Induces Protein Free Radical Formation on Myeloperoxidase: A Potential Mechanism of Agranulocytosis

Abstract

Procainamide (PA) is a drug that is used to treat tachycardia in post-operative patients or for long term maintenance of cardiac arrythmias. Unfortunately, its use has also been associated with agranulocytosis. Here we have investigated the metabolism of PA by myeloperoxidase (MPO) and the formation of an MPO protein free radical. We hypothesized that PA oxidation by MPO/H 2 O 2 would produce a PA cation radical that, in the absence of a biochemical reductant, would lead to the free-radical oxidation of MPO. We utilized a novel anti-DMPO antibody to detect DMPO (5,5-dimethyl-1-pyrroline N-oxide) covalently bound to protein, which forms only by the reaction of DMPO with a protein free radical. We found that PA metabolism by MPO/H 2 O 2 induced the formation of DMPO-MPO, which was inhibited by MPO inhibitors and ascorbate. N-acetyl-PA did not cause DMPO-MPO formation, indicating that the unsubstituted aromatic amine was more oxidizable. PA had a lower calculated ionization potential than N-acetyl-PA. The DMPO adducts of §To whom correspondence should be addressed: Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, 111 TW Alexander Dr., Research Triangle Park, NC USA, 27709. T: (919) 541-5197, F: (919) 541-1043, sirakia@niehs.nih.gov.. Abbreviations: PA, procainamide; NAPA, N-acetyl-procainamide; MPO, myeloperoxidase; ESR, electron spin resonance; DTPA, diethylenetriaminepentaacetic acid; MNP, 2-methyl-2-nitrosopropane; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; ABAH, 4-aminobenzoic acid hydrazide; G/GO,Glucose / glucose oxidase. MPO metabolism, as analyzed by electron spin resonance spectroscopy, included a nitrogen-centered radical and a phenyl radical derived from PA, either of which may be involved in the free radical formation on MPO. Furthermore, we also found protein-DMPO adducts in MPO-containing, intact human promyelocytic leukemia cells . MPO was affinity-purified from HL-60 cells treated with PA/H 2 O 2 and was found to contain DMPO using the anti-DMPO antibody. Mass spectrometry analysis confirmed the identity of the protein as human MPO. These findings were also supported by the detection of protein free radicals with electron spin resonance in the cellular cytosolic lysate. The formation of an MPO protein free radical is believed to be mediated by free radical metabolites of PA, which we characterized by spin trapping. We propose that drug-induced free radical formation on MPO may play a role in the origin of agranulocytosis. NIH Public AccessProcainamide (PA) is a sodium channel blocker which stabilizes electrical conduction in the heart. It is used for treating atrial tachycardia and is specifically recommended for atrial fibrillation of the Wolff-Parkinson-White syndrome (1). Unfortunately, agranulocytosis, an idiosyncratic adverse drug reaction, is associated with the use of procainamide (PA). This condition is characterized by severe neutropenia, where the absolute neutrophil count decreases below 500/uL (2,3), which greatly increases the risk of b...

Immuno-spin trapping from biochemistry to medicine: Advances, challenges, and pitfalls. Focus on protein-centered radicals

“…Since the production of the anti-DMPO antibody [23], a number of protein radicals have been detected by DMPO-based IST [21, 32], including myoglobin [23, 34–36], hemoglobin [22, 35, 37–38], cytochrome c [39], neuroglobin [40], Cu, Zn-superoxide dismutase (SOD1) [41–43] and Mn-SOD [44], catalase [33], myeloperoxidase [45–46], eosinophil peroxidase [47], lactoperoxidase [48–49], microglobulin [50], albumin [43, 51], thyroid peroxidase (TPO)[25], α-lactalbumin [52] and Ras GTPase [53]. Oxidants used to generate these radicals included H 2 O 2 , peroxynitrite, Cu 2+ /H 2 O 2 , hypochlorous acid, tert -butylhydroperoxide, peroxymonocarbonate, and reactive species generated from ketoprofen plus UV-A, riboflavin plus UV-A irradiation and nano-TiO 2 plus UV-A irradiation.…”

“…Protein-DMPO radical adducts have been studied using IST in models of amyotrophic lateral sclerosis or Luc Gerig’s disease [57], idiosyncratic agranulocytosis [45–46, 58–60], host-parasite interaction [61–62], sepsis [63–65], LPS-induced lung injury [63], ischemia/reperfusion (I/R) [39, 66–70], asthma [43, 47], diabetes [71–72] and obesity [73–74]. These studies may provide a molecular mechanism of tissue dysfunction in toxicology and pathology.…”

Hematotoxicity: In Vitro and Ex Vivo Compound Profiling

“…Some of the potential mechanisms of non-antiproliferative compounds leading to bone marrow toxicity include mitochondrial dysfunction [5,6], aromatic hydrocarbon receptor (AhR) activation, receptormediated, altered receptor expression [7] and reactive intermediates [8,9], but this list may grow with additional research as the mechanism(s) leading to bone marrow toxicity is still unknown for many compounds and will require significant amount of effort to elucidate. The next paragraphs briefly describe these potential mechanisms.…”

“…In addition to oxygen free radicals, other compounds such a clozapine, olanzapine and procainamide induce reactive intermediates [8,9]. Clozapine and olanzapine bioactivation is thought to occur through a nitrenium ion [20]; however clozapine but not olanzapine induce toxicity to neutrophils.…”

Proteomic profile of aminoglutethimide-induced apoptosis in HL-60 cells: Role of myeloperoxidase and arylamine free radicals

“…The activation of MPO requires H 2 O 2 as a cofactor, which then carries out two cycles of arylamine oxidation which produces arylamine free radicals [3]. There have been many studies demonstrating arylamine free radical reactions with biomolecules, including DNA, protein, glutathione, NAD(P)H, ascorbate [4][5][6][7][8][9]. However, in-depth studies http://dx.doi.org/10.1016/j.cbi.2015.06.020 0009-2797/Ó 2015 Elsevier Ireland Ltd. All rights reserved.…”