title: Human cyt P450 mediated metabolic toxicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) evaluated using electrochemiluminescent arrays.
abstract: Electrochemiluminescent (ECL) arrays containing polymer ([Ru(bpy)(2)(PVP)(10)](2+), PVP = polyvinylpyridine), DNA, and selected enzymes were employed to elucidate cytochrome (cyt) P450 dependent metabolism of the tobacco specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Bioactivated NNK metabolites formed upon H(2)O(2)-enzymatic activation were captured as DNA adducts and detected simultaneously from 36 spot arrays by capturing and quantifying emitted ECL with an overhead CCD camera. Increased ECL emission was dependent on NNK exposure time. Of the enzymes tested, the activity toward NNK bioactivation was cyt P450 1A2 > 2E1 > 1B1 approximately chloroperoxidase (CPO) > myoglobin (Mb) in accordance with reported in vivo studies. Cyt P450/polyion films were also immobilized on 500 nm diameter silica nanospheres for product analysis by LC-MS. Analysis of the nanosphere film reaction media provided ECL array validation and quantitation of the bioactivated NNK hydrolysis product 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) confirming production of reactive metabolites in the films. Chemical screening in this fashion allows rapid clarification of enzymes responsible for genotoxic activation as well as offering insight into cyt P450-related toxicity and mechanisms.
authors.affiliation: Department of Chemistry, University of Connecticut, Storrs, CT 06269-3060, USA.
authors.forename: Sadagopan
authors.initials: S
authors.lastname: Krishnan
authors.forename: Eli G
authors.initials: EG
authors.lastname: Hvastkovs
authors.forename: Besnik
authors.initials: B
authors.lastname: Bajrami
authors.forename: John B
authors.initials: JB
authors.lastname: Schenkman
authors.forename: James F
authors.initials: JF
authors.lastname: Rusling
mesh_terms: D002074:Butanones / Q000633:toxicity; D002273:Carcinogens; D002711:Chloride Peroxidase / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism / Q000502:physiology*; D018736:DNA Adducts; D004563:Electrochemistry / Q000379:methods*; D005821:Genetic Techniques; D006801:Humans; D006868:Hydrolysis; D049449:Luminescence; D013058:Mass Spectrometry / Q000379:methods; D008956:Models, Chemical; D009211:Myoglobin / Q000737:chemistry; D009602:Nitrosamines / Q000633:toxicity*; D011725:Pyridines / Q000633:toxicity
chemical_list: D002074:Butanones; D002273:Carcinogens; D018736:DNA Adducts; D009211:Myoglobin; D009602:Nitrosamines; D011725:Pyridines; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; D003577:Cytochrome P-450 Enzyme System; D002711:Chloride Peroxidase; C482281:4-hydroxy-1-(3-pyridyl)-1-butanone