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Elucidating Organ-Specific Metabolic Toxicity Chemistry from Electrochemiluminescent Enzyme/DNA Arrays and Bioreactor Bead-LC-MS/MS.

Wasalathanthri DP, Li D, Song D, Zheng Z, Choudhary D, Jansson I, Lu X, Schenkman JB, Rusling JF - Chem Sci (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department 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.

No MeSH data available.


Related in: MedlinePlus

Single reaction monitoring (SRM) LC-MS chromatogram for m/z transition 299–152 monitoring formation of O6-pyridyloxybutyl-guanine (4, Scheme 4) adducts from biocolloid reactors (enzyme color code on bottom) featuring (a) human organ microsomes, (b) cyt P450 supersomes; (c) and (d) are total DNA adducts found (pmol {μg of protein}–1 {mM of NNK}–1) after reactions with 150 μM NNK at pH = 7.4 for 18 h, (c) human organ microsomes, (d) cyt P450 supersomes.
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fig2: Single reaction monitoring (SRM) LC-MS chromatogram for m/z transition 299–152 monitoring formation of O6-pyridyloxybutyl-guanine (4, Scheme 4) adducts from biocolloid reactors (enzyme color code on bottom) featuring (a) human organ microsomes, (b) cyt P450 supersomes; (c) and (d) are total DNA adducts found (pmol {μg of protein}–1 {mM of NNK}–1) after reactions with 150 μM NNK at pH = 7.4 for 18 h, (c) human organ microsomes, (d) cyt P450 supersomes.

Mentions: Amounts of individual DNA adducts generated from bioactivation of test compounds with human tissue enzymes were measured by LC-MS/MS to complement ECL array results. Magnetic beads (1 μm) decorated with the same DNA/enzyme films used in the ECL array served as biocolloid reactors in a 96-well filter plate high-throughput platform to generate DNA adducts, followed by hydrolysis of the DNA, filtration and LC-MS/MS quantitation of the individual nucleobase adducts.26 7-Methylguanosine was used as an internal standard. Single reaction monitoring (SRM) chromatograms for the characteristic mass transition 299–152 of the O6-pyridyloxybutyl-guanine adduct (4, m/z = 299) from reaction products of NNK metabolized by human organ tissue fractions (Fig. 2a) and reactions with cyt P450 supersomes in (Fig. 2b) on the enzyme/DNA beads.


Elucidating Organ-Specific Metabolic Toxicity Chemistry from Electrochemiluminescent Enzyme/DNA Arrays and Bioreactor Bead-LC-MS/MS.

Wasalathanthri DP, Li D, Song D, Zheng Z, Choudhary D, Jansson I, Lu X, Schenkman JB, Rusling JF - Chem Sci (2015)

Single reaction monitoring (SRM) LC-MS chromatogram for m/z transition 299–152 monitoring formation of O6-pyridyloxybutyl-guanine (4, Scheme 4) adducts from biocolloid reactors (enzyme color code on bottom) featuring (a) human organ microsomes, (b) cyt P450 supersomes; (c) and (d) are total DNA adducts found (pmol {μg of protein}–1 {mM of NNK}–1) after reactions with 150 μM NNK at pH = 7.4 for 18 h, (c) human organ microsomes, (d) cyt P450 supersomes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4364445&req=5

fig2: Single reaction monitoring (SRM) LC-MS chromatogram for m/z transition 299–152 monitoring formation of O6-pyridyloxybutyl-guanine (4, Scheme 4) adducts from biocolloid reactors (enzyme color code on bottom) featuring (a) human organ microsomes, (b) cyt P450 supersomes; (c) and (d) are total DNA adducts found (pmol {μg of protein}–1 {mM of NNK}–1) after reactions with 150 μM NNK at pH = 7.4 for 18 h, (c) human organ microsomes, (d) cyt P450 supersomes.
Mentions: Amounts of individual DNA adducts generated from bioactivation of test compounds with human tissue enzymes were measured by LC-MS/MS to complement ECL array results. Magnetic beads (1 μm) decorated with the same DNA/enzyme films used in the ECL array served as biocolloid reactors in a 96-well filter plate high-throughput platform to generate DNA adducts, followed by hydrolysis of the DNA, filtration and LC-MS/MS quantitation of the individual nucleobase adducts.26 7-Methylguanosine was used as an internal standard. Single reaction monitoring (SRM) chromatograms for the characteristic mass transition 299–152 of the O6-pyridyloxybutyl-guanine adduct (4, m/z = 299) from reaction products of NNK metabolized by human organ tissue fractions (Fig. 2a) and reactions with cyt P450 supersomes in (Fig. 2b) on the enzyme/DNA beads.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department 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.

No MeSH data available.


Related in: MedlinePlus