title: Reduction of sulfamethoxazole hydroxylamine (SMX-HA) by the mitochondrial amidoxime reducing component (mARC).
abstract: Under high dose treatment with sulfamethoxazole (SMX)/trimethoprim (TMP), hypersensitivity reactions occur with a high incidence. The mechanism of this adverse drug reaction is not fully understood. Several steps in the toxification pathway of SMX were investigated. The aim of our study was to investigate the reduction of sulfamethoxazole hydroxylamine (SMX-HA) in this toxification pathway, which can possibly be catalyzed by the mARC-containing N-reductive enzyme system. Western blot analyses of subcellular fractions of porcine tissue were performed with antibodies against mARC-1, mARC-2, cytochrome b5 type B, and NADH cytochrome b5 reductase. Incubations of porcine and human subcellular tissue fractions and of the heterologously expressed human components of the N-reductive enzyme system were carried out with SMX-HA. mARC-1 and mARC-2 knockdown was performed in HEK-293 cells. Kinetic parameters of the heterologously expressed human protein variants V96L, A165T, M187 K, C246S, D247H, and M268I of mARC-1 and G244S and C245W of mARC-2 and N-reductive activity of 2SF, D14G, K16E, and T22A of cytochrome b5 type B were analyzed. Western blot analyses were consistent with the hypothesis that the mARC-containing N-reductive enzyme system might be involved in the reduction of SMX-HA. In agreement with these results, highest reduction rates were found in mitochondrial subcellular fractions of porcine tissue and in the outer membrane vesicle (OMV) of human liver tissue. Knockdown studies in HEK-293 cells demonstrated that mARC-1 and mARC-2 were capable of reducing SMX-HA in cell metabolism. Investigations with the heterologously expressed human mARC-2 protein showed a higher catalytic efficiency toward SMX-HA than mARC-1, but none of the investigated human protein variants showed statistically significant differences of its N-reductive activity and was therefore likely to participate in the pathogenesis of hypersensitivity reaction under treatment with SMX.
authors.affiliation: Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, Christian-Albrechts-University of Kiel , Gutenbergstrasse 76, D-24118 Kiel, Germany.
authors.forename: Gudrun
authors.initials: G
authors.lastname: Ott
authors.forename: Birte
authors.initials: B
authors.lastname: Plitzko
authors.forename: Carmen
authors.initials: C
authors.lastname: Krischkowski
authors.forename: Debora
authors.initials: D
authors.lastname: Reichmann
authors.forename: Florian
authors.initials: F
authors.lastname: Bittner
authors.forename: Ralf R
authors.initials: RR
authors.lastname: Mendel
authors.forename: Thomas
authors.initials: T
authors.lastname: Kunze
authors.forename: Bernd
authors.initials: B
authors.lastname: Clement
authors.forename: Antje
authors.initials: A
authors.lastname: Havemeyer
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D055162:Biocatalysis; D002851:Chromatography, High Pressure Liquid; D042966:Cytochrome-B(5) Reductase / Q000378:metabolism; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013420:Sulfamethoxazole / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D013552:Swine
chemical_list: D024101:Mitochondrial Proteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C059873:sulfamethoxazole hydroxylamine; D015786:Cytochromes b5; D010088:Oxidoreductases; C554553:MTARC1 protein, human; C554552:mitochondrial amidoxime reducing component 2, human; D042966:Cytochrome-B(5) Reductase; D013420:Sulfamethoxazole