Title

Detection of chemically induced DNA damage in thin films using a voltammetric probe

Date of Completion

January 2004

Keywords

Chemistry, Analytical

Degree

Ph.D.

Abstract

Electrochemical biosensors for rapid and inexpensive toxicity screening of chemicals have been developed. Chemically induced DNA damage caused by lipophilic pollutants and drugs metabolized by mammalian liver cytochrome P450 enzymes represents a major toxicity pathway. In many cases, the mechanism of causing diseases involves the damage of DNA and other essential biomolecules. Therefore, methods for detecting such DNA damage could serve as effective screens in vitro for the toxicity of new chemicals at an early stage in their commercial development. ^ The first generation of such biosensors was achieved by casting a mixture of double stranded (ds)-DNA and ionomer (Eastman AQ) on pyrolytic graphite (PG) electrodes. The sensor was evaluated with the model carcinogen styrene oxide. DNA damage caused by incubation of the sensor films with styrene oxide was detected with derivative square wave voltammetry (d-SWV). ^ The sensitivity of the detection method was significantly improved in a second generation sensor with the use of a metal complex probe, tris(2,2-bipyridyl)cobalt(III) [Co(bpy)33+] that binds reversibly to ds-DNA and with a weaker binding to damaged DNA. A layer-by-layer technique was used to build the sensor films in this method. DNA damage by styrene oxide can be detected with significant signals after only 2-min incubation, roughly corresponding to 0.05% damage or 5 damaged bases per 10000 in polyion/DNA films by square wave voltammetry (SWV). ^ A great leap was accomplished with third generation sensors. Enzymes were incorporated in the sensor film assembly to mimic enzyme activation of relatively nontoxic chemicals to genotoxic metabolites in the human liver. In the presence of hydrogen peroxide, myoglobin or cytochrome P450cam in the sensor films oxidized styrene to form styrene oxide. The generated styrene oxide damaged DNA in the same film. This damage was detected with square wave voltammetry using Co(bpy)33+ probe. ^ High performance liquid chromatography (HPLC) coupled with electrospray tandem mass spectrometry (ES-MS/MS) was used to confirm DNA adducts formation under analogous conditions to those used for electrochemical sensors. DNA adduct formation rates were also compared for 3 alkylating agents, styrene oxide, methyl methanesulfonate and diepoxybutane. ^