title: Elucidating Organ-Specific Metabolic Toxicity Chemistry from Electrochemiluminescent Enzyme/DNA Arrays and Bioreactor Bead-LC-MS/MS.
abstract: Human toxic responses are very often related to metabolism. Liver metabolism is traditionally studied, but other organs also convert chemicals and drugs to reactive metabolites leading to toxicity. When DNA damage is found, the effects are termedgenotoxic.Here we describe a comprehensive new approach to evaluate chemical genotoxicity pathways from metabolites formed in-situ by a broad spectrum of liver, lung, kidney and intestinal enzymes. DNA damage rates are measured with a microfluidic array featuring a 64-nanowell chip to facilitate fabrication of films of DNA, electrochemiluminescent (ECL) detection polymer [Ru(bpy)2(PVP)10]2+{(PVP = poly(4-vinylpyridine)} and metabolic enzymes. First, multiple enzyme reactions are run on test compounds using the array, then ECL light related to the resulting DNA damage is measured. A companion method next facilitates reaction of target compounds with DNA/enzyme-coated magnetic beads in 96 well plates, after which DNA is hydrolyzed and nucleobase-metabolite adducts are detected by LC-MS/MS. The same organ enzymes are used as in the arrays. Outcomes revealed nucleobase adducts from DNA damage, enzymes responsible for reactive metabolites (e.g. cyt P450s), influence of bioconjugation, relative dynamics of enzymes suites from different organs, and pathways of possible genotoxic chemistry. Correlations between DNA damage rates from the cell-free array and organ-specific cell-based DNA damage were found. Results illustrate the power of the combined DNA/enzyme microarray/LC-MS/MS approach to efficiently explore a broad spectrum of organ-specific metabolic genotoxic pathways for drugs and environmental chemicals.
authors: Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States National University of Ireland at Galway, Ireland.
authors: Dhanuka P
authors: DP
authors: Wasalathanthri
authors: Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States National University of Ireland at Galway, Ireland.
authors: Dandan
authors: D
authors: Li
authors: Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut 06269, United States.
authors: Donghui
authors: D
authors: Song
authors: Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States National University of Ireland at Galway, Ireland.
authors: Zhifang
authors: Z
authors: Zheng
authors: Department of Surgery, University of Connecticut Health Center, Farmington, Connecticut 06032, United States.
authors: Dharamainder
authors: D
authors: Choudhary
authors: Department of Cell Biology, University of Connecticut Health Center, Farmington, Connecticut 06032, United States.
authors: Ingela
authors: I
authors: Jansson
authors: Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut 06269, United States.
authors: Xiuling
authors: X
authors: Lu
authors: Department of Cell Biology, University of Connecticut Health Center, Farmington, Connecticut 06032, United States.
authors: John B
authors: JB
authors: Schenkman
authors: Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States National University of Ireland at Galway, Ireland ; Department of Cell Biology, University of Connecticut Health Center, Farmington, Connecticut 06032, United States.
authors: James F
authors: JF
authors: Rusling