Title

Oxidative voltammetric electrochemical analysis of chemically induced DNA damage

Date of Completion

January 2002

Keywords

Chemistry, Analytical

Degree

Ph.D.

Abstract

Fast and accurate toxicity screening of potentially new commercial organic compounds is in great demand as more and more new chemicals are generated every year. Chemically induced DNA damage by metabolites formed during catalytic oxidation of lipophilic pollutants and drugs in mammalian liver is a major toxicity pathway. Easy-to-construct prototype sensors featuring DNA films on electrode surfaces were developed to detect DNA damage in vitro by metabolites of drugs and pollutants. ^ The first DNA film type was constructed by casting mixtures of double stranded (ds)-DNA and ionomer (Eastman AQ or Nafion) on pyrolytic graphite (PG) electrodes. DNA damage caused by the known carcinogen styrene oxide upon incubation was detected directly using derivative square wave voltammetry (SWV). The integrated oxidative peak areas increased with incubation time. Films were then built on an oxidized PG surface using poly(diallydimethyl-ammonium) (PDDA) cations and ds-DNA by the layer-by-layer assembly method. These films gave improved detection of chemical damage to ds-DNA by styrene oxide. Catalytic oxidation using 50 μM Ru(bpy)32+ (bpy = 2,2-bipyridine) and SWV provided greater sensitive detection of DNA damage than direct SWV oxidation. The catalytic peaks increased linearly with incubation time in styrene oxide. Studies of DNA and polynucleotides in solutions and films suggested that oxidation of guanine and chemically damaged adenine in partially un-raveled, damaged DNA were the most likely contributors to the catalytic peak. This sensor scheme was further developed by coupling a myoglobin (Mb) or cytochrome (cyt) P450cam layer to the film to mimic enzyme activation of relatively nontoxic chemicals to genotoxic forms in the human liver. During bulk electrolysis in aerobic media, or reaction with peroxide, Mb/cyt P450cam converted styrene to styrene oxide that damaged DNA in the same film. This damage was then analyzed by catalytic SWV. The catalytic peak currents increased with reaction time. For all these reactions, only minor changes were detected during incubation with non-reactive toluene and in other appropriate controls. Capillary electrophoresis, high performance liquid chromatography and mass spectrometry results confirmed the formation of guanosine-styrene oxide and adenosine-styrene oxide adducts under the same experimental conditions used with the biosensors. ^