title: Isolation and structural identification of a direct-acting mutagen derived from N-nitroso-N-methylpentylamine and Fenton's reagent with copper ion.
abstract: N-Nitrosodialkylamines show their mutagenicity by forming α-hydroxynitrosamines in the presence of rat S9 mix in the Ames assay. The hydroxyl radical derived from Fe(2+)-H(2)O(2) (Fenton's reagent) with Cu(2+) activates N-nitrosamines, with an alkyl chain longer than a propyl constituent, to a direct-acting mutagen. The reactivity of Fe(2+)-Cu(2+)-H(2)O(2) on nitrosamines in relation to their metabolic activation is not fully characterized. Here, we report the identification of the direct-acting mutagen derived from N-nitroso-N-methylpentylamine (NMPe) in the presence of Fe(2+), Cu(2+), H(2)O(2) and nitric oxide (NO), which is a product of nitrosamine metabolism. A dichloromethane extract of the NMPe reaction mixtures was fractionated by silica gel column chromatography several times and by a preparative high performance liquid chromatography (HPLC); we obtained white crystals as a product. The direct-acting mutagen that was isolated was provisionally identified as 5-ethyl-5-nitro-1-pyrazoline 1-oxide by (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy, infrared (IR) spectroscopy and X-ray crystallography. To confirm the structure of the mutagen, the authentic compound was synthesized from 2-nitrobutene and diazomethane, followed by N-oxidation with m-chloroperoxybenzoic acid. The (1)H NMR spectral data from the direct-acting mutagen that was synthesized was identical to the data from the isolated mutagen. Furthermore, the authentic 5-ethyl-5-nitro-1-pyrazoline 1-oxide was mutagenic in Salmonella typhimurium TA1535. The results showed that 5-ethyl-5-nitro-1-pyrazoline 1-oxide was a direct-acting mutagen derived from the reaction of NMPe and Fe(2+)-Cu(2+)-H(2)O(2)-NO.
authors.affiliation: Kyoritsu University of Pharmacy, Shibakoen 1-5-30, Minato-ku, Tokyo 105-8512, Japan.
authors.forename: Motofumi
authors.initials: M
authors.lastname: Miura
authors.forename: Keiko
authors.initials: K
authors.lastname: Inami
authors.forename: Masafumi
authors.initials: M
authors.lastname: Yoshida
authors.forename: Kentaro
authors.initials: K
authors.lastname: Yamaguchi
authors.forename: Tadahiko
authors.initials: T
authors.lastname: Mashino
authors.forename: Masataka
authors.initials: M
authors.lastname: Mochizuki
mesh_terms: D003300:Copper / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D005296:Ferrous Compounds / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry*; D007477:Ions / Q000737:chemistry; D008958:Models, Molecular; D015394:Molecular Structure; D009153:Mutagens / Q000737:chemistry* / Q000302:isolation & purification; D009154:Mutation; D009569:Nitric Oxide / Q000737:chemistry; D009602:Nitrosamines / Q000737:chemistry* / Q000302:isolation & purification; D012486:Salmonella typhimurium / Q000235:genetics*; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
chemical_list: D005296:Ferrous Compounds; D007477:Ions; D009153:Mutagens; D009602:Nitrosamines; D009569:Nitric Oxide; D003300:Copper; C014102:N-methyl-N-nitrosoaniline; D006861:Hydrogen Peroxide