Evaluating Metabolite-Related DNA Oxidation and Adduct Damage from Aryl Amines Using a Microfluidic ECL Array

Abstract

Damage to DNA from the metabolites of drugs and pollutants constitutes a major human toxicity pathway known as genotoxicity. Metabolites can react with metal ions and NADPH to oxidize DNA or participate in S_N2 reactions to form covalently linked adducts with DNA bases. Guanines are the main DNA oxidation sites, and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) is the initial product. Here we describe a novel electrochemiluminescent (ECL) microwell array that produces metabolites from test compounds and measures relative rates of DNA oxidation and DNA adduct damage. In this new array, films of DNA, metabolic enzymes, and an ECL metallopolymer or complex assembled in microwells on a pyrolytic graphite wafer are housed in dual microfluidic chambers. As reactant solution passes over the wells, metabolites form and can react with DNA in the films to form DNA adducts. These adducts are detected by ECL from a RuPVP polymer that uses DNA as a coreactant. Aryl amines also combine with Cu²⁺ and NADPH to form reactive oxygen species (ROS) that oxidize DNA. The resulting 8-oxodG was detected selectively by ECL-generating bis(2,2'-bipyridine)-(4-(1,10-phenanthrolin-6-yl)-benzoic acid)Os(II). DNA/enzyme films on magnetic beads were oxidized similarly, and 8-oxodG determined by LC/MS/MS enabled array standardization. The array limit of detection for oxidation was 720 8-oxodG per 10⁶ nucleobases. For a series of aryl amines, metabolite-generated DNA oxidation and adduct formation turnover rates from the array correlated very well with rodent 1/TD₅₀ and Comet assay results.

Figure 1

Recolorized, reconstructed images of ECL for DNA oxidation (1A) and DNA adduct formation (1D) using 4ABP. For DNA oxidation microwells, DNA/enzyme films were reacted with 3 mM 4ABP in pH 7.4 PBS + NADPH + Cu²⁺ ions. Then, 2 mM Os(bpy)₂(phen-benz-COOH)]²⁺ is delivered to the microwell reactor and incubated, and 0.7 V vs Ag/AgCl is applied to generate ECL. For DNA adduct formation, wells containing RuPVP/Enzyme/DNA films were reacted with 4ABP in pH 7.4 PBS with bioelectronic activation of supersomal and microsomal enzymes at −0.65 V vs Ag/AgCl. 1.25 V vs Ag/AgCl was applied to generate ECL in a dark box. (B), (C), (E), and (F) show influence of enzyme reaction time on %ECL increase for these reactions. Control experiments lacked 4ABP or enzymes.

Figure 2

Reconstructed, recolorized ECL images for DNA oxidation caused by (A) AF, (D) o-anisidine, and (G) NA. DNA/enzyme films were reacted with 3 mM AF or anisidine or 5 mM NA in pH 7.4 PBS + NADPH regenerating system + Cu²⁺. Os(bpy)₂(phen-benz-COOH)]²⁺was delivered to the reactor and incubated, and 0.7 V was applied to obtain ECL. % ECL vs enzyme reaction time shown in (B), (E), (H) and (C), (F), (I) with different enzyme sources. Controls lack test compounds or enzymes.

Figure 3

Bar graphs showing (A) relative DNA oxidation rate and (B) relative DNA adduction rate as R ({μg of protein}⁻¹ min⁻¹ mM⁻¹) from exposure of 4-ABP, AF, anisidine, and NA to different cyt P450 sources in the array. Line graphs showing correlations of relative DNA damage rates (R) from human liver microsomes found by using the ECL array with the reciprocal of rodent liver TD₅₀ values and comet assay values (OTM/C in AU*mM⁻¹) from human urinary bladder cell lines for 4-ABP and NA and human peripheral lymphocytes for AF.^, (C) and (D) shows correlation with relative DNA oxidation rate and relative DNA adduction rates for 4-ABP, AF, anisidine, and NA.

Figure 4

UPLC/MS/MS results for DNA oxidation. (A) MRM chromatogram with mass transition m/z 268–152 indicating dG and m/z 284–168 indicating 8-oxodG for DNA/enzyme magnetic biocolloid reactors incubated with 4-ABP, Cu²⁺, and NADPH for 40 min followed by hydrolysis of the DNA. (B) Ratio of 8-oxodG to total dG from magnetic biocolloid reactors reacted with 4-ABP in the presence of Cu²⁺ and NADPH. (C) Calibration curve for sensor % ECL increase vs relative amount of 8-oxodG. (D) Influence of reaction time on 8-oxodG per 10⁶ bases formation when DNA/enzyme films were incubated with 4-ABP in the presence of Cu²⁺ and NADPH from array data. Control experiments contained Cu²⁺ and NADPH but no 4-ABP.

Scheme 1. Array Strategy for Screening Genotoxic Pathways Using (A) DNA/Enzyme Films, (B) Biocolloid Reactors, and (C) ECL Arrays^#

^# Metabolites are generated in DNA/enzyme films from reactant solutions by applying voltage to an array or by an NADPH regenerating system with the beads to activate cyt P450s. Reactive metabolites that were formed reacted with DNA to give DNA adducts, and they were detected by ECL from RuPVP. Oxidative DNA damage from a Cu²⁺-metabolite mediated redox pathway was detected by ECL using [Os(bpy)₂(phen-benz-COOH)]²⁺. Calibration for oxidation product 8-oxodG was established by using LC/MS/MS generated standards.

Scheme 2. Photographs of Fluidic Array Featuring Two PDMS Channels in a PMMA Housing^∇

^∇ Ag/AgCl reference and Pt counter wire electrodes are symmetrically placed along the lengths of the channels. (A) Fluidic reactor shown with dual syringe pump to deliver buffer and reactants solutions to the array. (B) Fluidic reactor showing channels and auxiliary electrodes. (C) PDMS slab showing two fluidic channels. (D) Pyrolytic graphite (PG) chip showing array of two rows of microwells.

Scheme 3. Schematic Representation of One Microwell Containing PDDA/Cyt P450 Source/PDDA/DNA Film^⠭

^⠭ (A) Represents enzyme bioactivation process in the presence of oxygenated substrate, NADPH and Cu²⁺ leading to 8-oxodG formation; (B) Reaction of 8-oxodG with [Os(bpy)₂(phen-benz-COOH)]²⁺ and ECL generation upon applying a potential of 0.7 V vs Ag/AgCl. liver.^,

Scheme 4

Major Metabolic Pathway of Aryl Amines Causing DNA Adduct Formation

Scheme 5

Proposed Pathway Involving Cu²⁺ and NADPH for ROS Formation Mediated by 4-ABP Leading to DNA Oxidation

Scheme 6

Proposed Mechanism of DNA Oxidation by Aminofluorene (AF) and 2- Naphthylamines (NA) in Redox Cycles with Cu²⁺ and NADPH^,

Scheme 7

Proposed Pathway for DNA Oxidation Caused by o-Anisidine, Cu²⁺, and NADPH^,