Title

Electrochemical and electrochemiluminescent genotoxicity screening using DNA-enzyme based sensors

Date of Completion

January 2008

Keywords

Chemistry, Analytical|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

Bioactivation of lipophilic pollutants or drugs to electrophilic forms by phase I and II enzymes is a major source of genotoxicity. Detection methods of DNA damage using sensor format have been developed as the basis for genotoxicity screening. These biosensors feature thin films of DNA and metabolic enzymes immobilized by layer-by-layer (LbL) assembly on a pyrolytic graphite electrode. The enzymes in the films metabolize a procarcinogen, creating possibly reactive metabolites, which can form DNA adducts. For the detection of DNA damage, simultaneous square wave voltammetric (SWV) and electrochemiluminescent method was demonstrated with the corporation of electrochemiluminescence (ECL) generating metallopolymer [Ru(bpy)2PVP10]2+ in DNA/enzyme films. A standard voltammetry cell coupled with an optical fiber and photomultiplier tube was used for dual ECL/SWV measurement. Cytochrome P450cam and myoglobin were used as model monoxygenase enzymes of phase I to mimic in vivo processes by converting styrene to styrene oxide, which reacts with DNA nucleobases. In order to extend enzymatic bioactivation process beyond phase I, genotoxicity screening sensors that measure DNA damage from phase II metabolism of arylamines were evaluated. N-acetyltransferase (NAT) in the film catalyzed the conversion of the arylamine 2-aminofluorene (2-AF) to 2-acetylaminofluorene (2-AAF) by acetyl coenzyme A (AcCoA) dependent N-acetylation. DNA damage using catalytic voltammetric oxidation with Ru(bpy)32+ occurred more rapidly under weakly acidic conditions (pH 5.5 to 5.8) than at neutral pH, suggesting that genotoxicity from arylamine metabolism by NAT could be more significant in slightly acidic environments. Finally, two-enzyme biosensors of cyt P450 1A2 and NAT were demonstrated for genotoxicity screening from metabolites generated by sequential bioactivations of a heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP). DNA damage rate from metabolites of PhIP by cyt P450 1A2 mediated hydrogen peroxide-driven N-hydroxylation following NAT mediated acetylation were compared to only N-hydroxylation of PhIP and only N-acetylation of PhIP. These two-enzyme biosensors can estimate the roles of enzymes in multistep metabolic pathways of heterocyclic aromatic amines. These approaches provided valuable tools for drug and chemical product development and toxicity prediction. Future work will focus on high throughput biosensor arrays based on LbL spots with voltammetry and ECL methodology. ^