title: Detection and characterization of DNA adducts of 3-methylindole.
abstract: The pneumotoxin 3-methylindole is metabolized to the reactive intermediate 3-methyleneindolenine which has been shown to form adducts with glutathione and proteins. Reported here is the synthesis, detection, and characterization of nucleoside adducts of 3-methylindole. Adducted nucleoside standards were synthesized by the reaction of indole-3-carbinol with each of the four nucleosides under slightly acidic conditions, which catalyze the dehydration of indole-3-carbinol to 3-methyleneindolenine. Following solid phase extraction, the individual adducts were infused via an electrospray source into an ion trap mass spectrometer for molecular weight determination and characterization of the fragmentation patterns. The molecular ions and fragmentation of the dGuo, dAdo, and dCyd adducts were consistent with nucleophilic addition of the exocyclic primary amine of the nucleosides to the methylene carbon of 3-methyleneindolenine. The apparent chemical preference of this addition lead primarily to dAdo and dGuo adducts, with substantially less of the dCyd adduct formed. No adduct with dThd was detected. The adducts were purified by HPLC and subsequent NMR analysis of the dGuo and dCyd adducts confirmed the proposed structures. Mass spectral fragmentation of the three adducts produced primarily two ions which were the result of the loss of either the 3-methylindole moiety or the sugar. On a triple quadrupole electrospray mass spectrometer, the neutral loss of the sugar, [M + H - 116](+), was utilized for selected reaction monitoring of the calf thymus DNA adducts, formed by incubations of 3-methylindole with various microsomes (rat liver, goat lung, and human liver). All three adducts were detected from each of the microsomal incubations, following extraction and cleavage of the DNA to the nucleoside level. The dGuo adduct was the primary adduct formed, with smaller amounts of the dAdo and dCyd adducts. Rat hepatocytes incubated with 3-methylindole produced the same three adducts, in approximately the same proportions, while no adducts were detected in untreated hepatocytes. Microsomal incubations in the presence of ([3-(2)H(3)]-methyl)indole confirmed the formation and identification of the adducts as well as the fragmentation patterns. These results demonstrate that bioactivated 3-methylindole forms specific adducts with exogenous or intact cellular DNA, and indicates that 3-methylindole may be a potential mutagenic and/or carcinogenic chemical.
authors.affiliation: Biochemical and Investigative Toxicology and Genetic Toxicology, Department of Safety Assessment, Merck Research Laboratories, WP319, West Point, PA 19486, USA.
authors.forename: K A
authors.initials: KA
authors.lastname: Regal
authors.forename: G M
authors.initials: GM
authors.lastname: Laws
authors.forename: C
authors.initials: C
authors.lastname: Yuan
authors.forename: G S
authors.initials: GS
authors.lastname: Yost
authors.forename: G L
authors.initials: GL
authors.lastname: Skiles
mesh_terms: D000818:Animals; D002273:Carcinogens / Q000737:chemistry* / Q000378:metabolism; D018736:DNA Adducts / Q000032:analysis* / Q000138:chemical synthesis; D008099:Liver; D009682:Magnetic Resonance Spectroscopy; D008861:Microsomes; D009705:Nucleosides; D051381:Rats; D012862:Skatole / Q000737:chemistry* / Q000378:metabolism; D018629:Spectrometry, Mass, Secondary Ion
chemical_list: D002273:Carcinogens; D018736:DNA Adducts; D009705:Nucleosides; D012862:Skatole