Toxicity Screening by Electrochemical Detection of DNA Damage by Metabolites Generated In Situ in Ultrathin DNA−Enzyme Films

Abstract

Rapid detection of DNA damage could serve as a basis for in vitro genotoxicity screening for new organic compounds. Ultrathin films (20−40 nm) containing myoglobin or cytochrome P450_cam and DNA grown layer-by-layer on electrodes were activated by hydrogen peroxide, and the enzyme in the film generated metabolite styrene oxide from styrene. This styrene oxide reacted with double stranded (ds)-DNA in the same film, mimicking metabolism and DNA damage in human liver. DNA damage was detected by square wave voltammetry (SWV) by using catalytic oxidation with Ru(bpy)₃²⁺ (bpy = 2,2‘-bipyridine) and by monitoring the binding of Co(bpy)₃³⁺. Damaged DNA reacts more rapidly than intact ds-DNA with Ru(bpy)₃³⁺, giving SWV peaks at ∼1 V versus SCE that grow larger with reaction time. Co(bpy)₃³⁺ binds more strongly to intact ds-DNA, and its SWV peaks at 0.04 V decreased as DNA was damaged. Little change in SWV signals was found for incubations of DNA/enzyme films with unreactive organic controls or hydrogen peroxide. Capillary electrophoresis and HPLC−MS suggested the formation of styrene oxide adducts of DNA bases under similar reaction conditions in thin films and in solution. The catalytic SWV method was more sensitive than the Co(bpy)₃³⁺ binding assay, providing multiple measurements over a 5 min reaction time.