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Elucidating Organ-Specific Metabolic Toxicity Chemistry from Electrochemiluminescent Enzyme/DNA Arrays and Bioreactor
Dhanuka P Wasalathanthri1, Dandan Li1, Donghui Song2
1Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States National University of Ireland at Galway, Ireland.
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 termed genotoxic. 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.
Insights
This study introduces a new method to assess chemical genotoxicity by examining metabolites from various organs. The approach links DNA damage rates to specific enzymes and metabolic pathways, improving chemical safety assessments.
Area of Science:
- Toxicology and Pharmacology
- Metabolomics
- Genetics
Background:
- Human toxic responses are often linked to metabolism, with organs beyond the liver converting chemicals into reactive, potentially genotoxic metabolites.
- Traditional studies focus on liver metabolism, overlooking the genotoxic contributions of other organs like the lungs, kidneys, and intestines.
Purpose of the Study:
- To develop and validate a comprehensive approach for evaluating chemical genotoxicity pathways stemming from metabolites generated in situ by a wide array of organ-specific enzymes.
- To investigate the formation of nucleobase-metabolite adducts and identify the enzymes responsible for producing reactive metabolites.
Main Methods:
- Utilized a microfluidic array with a 64-nanowell chip for simultaneous enzyme reactions and electrochemiluminescent (ECL) detection of DNA damage.
- Employed a complementary method using DNA/enzyme-coated magnetic beads in 96-well plates for LC-MS/MS detection of nucleobase-metabolite adducts.
- Applied enzymes from liver, lung, kidney, and intestinal sources in both the array and magnetic bead assays.
Main Results:
- Identified specific nucleobase adducts resulting from DNA damage and pinpointed enzymes, such as cytochrome P450s, responsible for generating reactive metabolites.
- Characterized the influence of bioconjugation and compared the dynamics of enzyme suites across different organs.
- Established correlations between DNA damage rates measured by the cell-free array and organ-specific cell-based DNA damage.
Conclusions:
- The combined DNA/enzyme microarray and LC-MS/MS approach offers a powerful and efficient means to explore organ-specific metabolic genotoxicity pathways for diverse chemicals.
- This methodology facilitates a deeper understanding of chemical toxicity mechanisms and aids in the safety assessment of drugs and environmental compounds.
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