title: Characterizing metabolic inhibition using electrochemical enzyme/DNA biosensors.
abstract: Studies of metabolic enzyme inhibition are necessary in drug development and toxicity investigations as potential tools to limit or prevent appearance of deleterious metabolites formed, for example, by cytochrome (cyt) P450 enzymes. In this paper, we evaluate the use of enzyme/DNA toxicity biosensors as tools to investigate enzyme inhibition. We have examined DNA damage due to cyt P450cam metabolism of styrene using DNA/enzyme films on pyrolytic graphite (PG) electrodes monitored via Ru(bpy)(3)(2+)-mediated DNA oxidation. Styrene metabolism initiated by hydrogen peroxide was evaluated with and without the inhibitors, imidazole, imidazole-4-acetic acid, and sulconazole (in micromolar range) to monitor DNA damage inhibition. The initial rates of DNA damage decreased with increased inhibitor concentrations. Linear and nonlinear fits of Michaelis-Menten inhibition models were used to determine apparent inhibition constants (K(I)*) for the inhibitors. Elucidation of the best fitting inhibition model was achieved by comparing correlation coefficients and the sum of the square of the errors (SSE) from each inhibition model. Results confirmed the utility of the enzyme/DNA biosensor for metabolic inhibition studies. A simple competitive inhibition model best approximated the data for imidazole, imidazole-4-acetic acid and sulconazole with K(I)* of 268.2, 142.3, and 204.2 microM, respectively.
authors.affiliation: Department of Chemistry and Institute of Materials Science, University of Connecticut, 55 N. Eagleville Road, Storrs, Connecticut 06269, USA.
authors.forename: Dominic O
authors.initials: DO
authors.lastname: Hull
authors.forename: Besnik
authors.initials: B
authors.lastname: Bajrami
authors.forename: Ingela
authors.initials: I
authors.lastname: Jansson
authors.forename: John B
authors.initials: JB
authors.lastname: Schenkman
authors.forename: James F
authors.initials: JF
authors.lastname: Rusling
mesh_terms: D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry; D015374:Biosensing Techniques; D019475:Camphor 5-Monooxygenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage / Q000187:drug effects; D004563:Electrochemistry; D004566:Electrodes; D004791:Enzyme Inhibitors / Q000737:chemistry*; D007093:Imidazoles / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D036103:Nanotechnology; D009942:Organometallic Compounds / Q000138:chemical synthesis / Q000737:chemistry; D011199:Potentiometry; D020058:Styrene / Q000737:chemistry / Q000633:toxicity
chemical_list: D004791:Enzyme Inhibitors; D007093:Imidazoles; D009942:Organometallic Compounds; C058901:tris(2,2'-bipyridine)ruthenium III; D020058:Styrene; D015082:2,2'-Dipyridyl; D004247:DNA; D019475:Camphor 5-Monooxygenase