Toxicity screening by electrochemical detection of DNA damage by metabolites generated in situ in ultrathin DNA-enzyme films.
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)(3)(2+) (bpy = 2,2'-bipyridine) and by monitoring the binding of Co(bpy)(3)(3+). Damaged DNA reacts more rapidly than intact ds-DNA with Ru(bpy)(3)(3+), giving SWV peaks at approximately 1 V versus SCE that grow larger with reaction time. Co(bpy)(3)(3+) 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)(3)(3+) binding assay, providing multiple measurements over a 5 min reaction time.
Zhou
Liping
L
Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269-3060, USA.
Yang
Jing
J
Estavillo
Carmelita
C
Stuart
James D
JD
Schenkman
John B
JB
Rusling
James F
JF
D015374:Biosensing Techniques / Q000379:methods; D019475:Camphor 5-Monooxygenase / Q000737:chemistry*; D018736:DNA Adducts / Q000737:chemistry; D004249:DNA Damage; D004563:Electrochemistry / Q000379:methods; D004852:Epoxy Compounds / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D009152:Mutagenicity Tests / Q000379:methods*; D009211:Myoglobin / Q000737:chemistry; D020058:Styrene / Q000737:chemistry
D018736:DNA Adducts; D004852:Epoxy Compounds; D009211:Myoglobin; D020058:Styrene; C013690:styrene oxide; D006861:Hydrogen Peroxide; D019475:Camphor 5-Monooxygenase