Issue 2, 2009
From the journal:

Molecular BioSystems

Human cyt P450 mediated metabolic toxicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) evaluated using electrochemiluminescent arrays†

Sadagopan Krishnan,a   Eli G. Hvastkovs,c   Besnik Bajrami,a   John B. Schenkmanb  and  James F. Rusling*ab  

* Corresponding authors

a Department of Chemistry, University of Connecticut, Storrs, USA
E-mail: james.rusling@uconn.edu

b Department of Pharmacology, University of Connecticut, Farmington, USA

c Department of Chemistry, East Carolina University, Greenville, USA

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 NNKmetabolites formed upon H2O2-enzymatic activation were captured as DNAadducts 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 ≈ 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.

Graphical abstract: Human cyt P450 mediated metabolic toxicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) evaluated using electrochemiluminescent arrays

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Article information

DOI
https://doi.org/10.1039/B815910F
Article type
Paper
Submitted
12 Sep 2008
Accepted
12 Nov 2008
First published
12 Dec 2008

Mol. BioSyst., 2009,5, 163-169

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Human cyt P450 mediated metabolic toxicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) evaluated using electrochemiluminescent arrays

S. Krishnan, E. G. Hvastkovs, B. Bajrami, J. B. Schenkman and J. F. Rusling, Mol. BioSyst., 2009, 5, 163 DOI: 10.1039/B815910F

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