title: Aminoglutethimide-induced protein free radical formation on myeloperoxidase: a potential mechanism of agranulocytosis.
abstract: Aminoglutethimide (AG) is a first-generation aromatase inhibitor used for estrogen-dependent breast cancer. Unfortunately, its use has also been associated with agranulocytosis. We have investigated the metabolism of AG by myeloperoxidase (MPO) and the formation of an MPO protein free radical. We hypothesized that AG oxidation by MPO/H2O2 would produce an AG cation radical that, in the absence of a biochemical reductant, would lead to the 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 AG metabolism by MPO/H2O2 induced the formation of DMPO-MPO, which was inhibited by MPO inhibitors and ascorbate. Glutethimide, a congener of AG that lacks the aromatic amine, did not cause DMPO-MPO formation, indicating the necessity of oxidation of the aniline moiety in AG. When analyzed by electron spin resonance spectroscopy, we detected a phenyl radical adduct, derived from AG, 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 (HL-60). MPO was affinity-purified from HL-60 cells treated with AG/H2O2 and was found to contain DMPO. 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 one of two free radical drug metabolites of AG, one of which was characterized by spin trapping with 2-methyl-2-nitrosopropane. These results are the first demonstration of MPO free-radical detection by the anti-DMPO antibody that results from drug oxidation. We propose that drug-dependent free radical formation on MPO may play a role in the origin of agranulocytosis.
authors.affiliation: Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, 111 Alexander Drive, Research Triangle Park, North Carolina 27709, USA. sirakia@niehs.nih.gov
authors.forename: Arno G
authors.initials: AG
authors.lastname: Siraki
authors.forename: Marcelo G
authors.initials: MG
authors.lastname: Bonini
authors.forename: JinJie
authors.initials: J
authors.lastname: Jiang
authors.forename: Marilyn
authors.initials: M
authors.lastname: Ehrenshaft
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Mason
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000380:Agranulocytosis / Q000139:chemically induced / Q000378:metabolism*; D000616:Aminoglutethimide / Q000737:chemistry / Q000494:pharmacology*; D000814:Aniline Compounds / Q000737:chemistry / Q000378:metabolism; D047072:Aromatase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D001205:Ascorbic Acid / Q000737:chemistry / Q000378:metabolism; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003497:Cyclic N-Oxides / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D004797:Enzyme-Linked Immunosorbent Assay; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism*; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D005984:Glutethimide / Q000737:chemistry / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D009589:Nitrogen Oxides / Q000737:chemistry / Q000378:metabolism; D009603:Nitroso Compounds / Q000737:chemistry / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism*; D013053:Spectrophotometry
chemical_list: D000814:Aniline Compounds; D047072:Aromatase Inhibitors; D003497:Cyclic N-Oxides; D005609:Free Radicals; D009589:Nitrogen Oxides; D009603:Nitroso Compounds; D000616:Aminoglutethimide; C034343:4-aminobenzhydrazide; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D005984:Glutethimide; D009195:Peroxidase; C039900:nitroxyl; D005947:Glucose; C034526:tert-nitrosobutane; D001205:Ascorbic Acid