Electrochemiluminescence Arrays for Studies of Metabolite-related Toxicity

Abstract

This article reviews recent progress from our laboratory in electrochemiluminescence (ECL) arrays designed for screening toxicity-related chemistry of chemical and drug candidates. Cytochrome P450s and metabolic bioconjugation enzymes convert lipophilic chemicals in our bodies by oxidation and bioconjugation that can lead to toxic metabolites. DNA can be used as an easily measurable toxicity-related endpoint, targeting DNA oxidation and addcut formation with metabolites. ECL using guanosines in the DNA strands as co-reactants have been used in high throughput arrays utilizing DNA-enzyme films fabricated layer-by-layer. This review describes approaches developed to provide new high throughput ECL arrays to aid in toxicity assessment for drug and chemical product development.

Keywords: Cytochrome P450s; DNA Arrays; DNA Damage; Electrochemiluminescence; Metabolites; Toxicity Chemistry.

Fig. 1

CVs of microsomal LbL films cyt P450 1A2 with and without CPR on PG electrodes. (A) Background subtracted CVs of {a} CPR films at 0.3 Vs⁻¹ with no cyt P450, and cyt P450 1A2/CPR films at scan rates {b} 0.1, {c} 0.2, and {d} 0.3 Vs⁻¹. (B) Background subtracted CVs of cyt P450 1A2 film with no CPR {a} polyion film control; 0.1 Vs⁻¹ and {b} Cyt P450 films at 0.1 and {c} 0.2 Vs⁻¹. (C) Digitally simulated CVs corresponding to {a} reversible electron transfer for only CPR film at 0.3 Vs⁻¹, and {b to d} the E_rCE_o-model using parameters in Scheme 2 for cyt P450 1A2/CPR films at scan rates {b} 0.1, {c} 0.2, and {d} 0.3 Vs⁻¹ showing excellent agreement with experimental CVs in Figure 1A. (D) Influence of scan rate on oxidation (blue circles) and reduction (red diamonds) peak potentials for cyt P450 1A2/CPR films with theoretical peak potentials (lines) simulated using the E_rCE_o model. Reproduced with permission from ref. [35] American Chemical Society, copyright 2011.

Fig. 2

ECL array results for oxygenated 25 μM B[a]P in pH 7.4 phosphate buffer with electrochemical activation of cyt P450s at −0.65 V vs. Ag/AgCl (0.14 M KCl): (a and b) Reconstructed ECL array data from Ru^IIPVP/enzyme/DNA spots for reaction times 0–90 s. Control spots contained cyt P450 1B1 without (a) and with (b) EH subjected to the same reaction conditions as above without activation of cyt P450s. (c and d) Influence of enzyme reaction time on ECL increase for (c) cyt P450 1B1, and (d) human liver microsomes (HML). Controls for cyt P450s (green) and cyt P450s+EH (purple) behaved equivalently without substrate or with substrate but no activation of cyt P450s. Reproduced from ref. [18] with permission of the Royal Society of Chemistry, copyright 2013.

Fig. 3

ECL-derived data related to nucleotide oxidation. (a) Reconstructed, recolorized ECL image showing ECL for polyG and DNA treated with Fenton′s reagent in pH 7.2 PBS for time in min. (top image). Controls are polynucleotides treated only with buffer, with only FeSO₄ or with only H₂O₂. Graphs show relative %ECL increase for (b) polyG and (c) DNA. (d) UPLC-MS/MS for ECL array calibration showing measured ratio [8-oxodG]/([dG]+[8-oxodG]) vs. reaction time with Fenton′s reagent. (e) Array calibration as % ECL increase vs. ratio [8-oxo dG]/([dG]+[8-oxodG]). Reproduced from ref. [40] with permission American Chemical Society, Copyright 2016.

Scheme 1

Predominant DNA adduct formation sites at adenine and guanine.

Scheme 2

Ribbon structure of (A) human cyt P450 1A2 and (B) the role of cyt P450s and selected bioconjugation enzymes in human metabolism.

Scheme 3

E_rCE_o simulation model for CVs of cyt P450s+ CPR.

Scheme 4

ECL pathways using DNA as co-reactant, G=guanine.

Scheme 5

Schematic representation of sensor system for ECL detection, showing microwell-patterned PG array electrode the DNA-enzyme-Ru-PVP spots with Ag/AgCl reference (i) and Pt-wire counter electrodes (ii). For ECL readout, the device is placed into a dark box (iii) and connected to a potentiostat that applies the necessary voltage while a charge coupled device camera (CCD) detects the light (iv). A computer is interfaced for data capture and analysis.

Scheme 6

ECL microfluidic array assembly into flow cell: (a), underside view of reference and counter electrode wires in the top poly(methylmethacrylate) (PMMA) plate: (b) pyrolytic graphite (PG) chip with printed microwells in (c). The first row is shown containing 1 μL water droplets. The reaction system is operated by connection to a syringe pump. Reproduced with permission from ref. [18] The Royal Society of Chemistry, copyright 2013.

Scheme 7

Metabolic reactions of benzo[a]pyrene leading to DNA-reactive metabolite BPDE.

Scheme 8

Experimental design for 64-nanowell ECL chip with films of organ-derived enzymes and Ru^IIPVP/DNA for metabolic toxicity assays of test compounds. Symbols: HLC, H=human, L=liver, C=cytosol, Lu=lung, I=intestine, K=kidney, number-letter such as 3A4 indicate supersomes containing the denoted single cyt P450 manifold. Reproduced from ref. [38] with permission of the Royal Society of Chemistry, copyright 2015.

Scheme 9

Structure of [Os(bpy)₂(phen-benz-COOH)]²⁺.