Electrochemiluminescent arrays for cytochrome P450-activated genotoxicity screening. DNA damage from benzo[a]pyrene metabolites

Abstract

Arrays suitable for genotoxicity screening are reported that generate metabolites from cytochrome P450 enzymes (CYPs) in thin-film spots. Array spots containing DNA, various human cyt P450s, and electrochemiluminescence (ECL) generating metallopolymer [Ru(bpy)2PVP10]2+ were exposed to H2O2 to activate the enzymes. ECL from all spots was visualized simultaneously using a CCD camera. Using benzo[a]pyrene as a test substrate, enzyme activity for producing DNA damage in the arrays was found in the order CYP1B1 > CYP1A2 > CYP1A1 > CYP2E1 > myoglobin, the same as the order of their metabolic activity. Thus, these arrays estimate the relative propensity of different enzymes to produce genotoxic metabolites. This is the first demonstration of ECL arrays for high-throughput in vitro genotoxicity screening.

Figure 1

Characterization data for films: (a) Background-subtracted, smoothed reversible CV of cyt P450 1B1 on PG electrode at 50 mV/s in pH 7.1 phosphate buffer + 50 mM NaCl. Film composition on electrode was PDDA/DNA/cyt P450 1B1/DNA. (b) Influence of film layers added on QCM frequency decrease for enzymes used in this study. The first three film layers are omitted (RuPVP/DNA/RuPVP) for clarity. (c) Visible difference spectrum for immobilized PDDA/cyt P450cam film on glass microscope slide after reduction to the ferrous form and addition of CO showing the characteristic cyt P450 Fe^II-CO band near 450 nm.

Figure 2

(a) A 36-spot ECL array not exposed to procarcinogenic material showing the spot-to-spot consistency generated from each location on the PG block. Conditions: 10 mM sodium acetate, 0.15 M NaCl, pH 5.5, +1.25 V; 20 s. (b) A column chart showing the relative ECL intensity to spot A3 (upper right corner) from each 4-unit spot location on the PG block.

Figure 3

(a) CCD image of ECL array with 49 individual RuPVP/DNA/enzyme spots containing cyt P450 1B1. Boxes denote spots that were exposed to 0.5 mM H₂O₂ + 100 μM B[a]P for the labeled times (min). Controls on bottom were exposed to B[a]P or H₂O₂ only for increasing amounts of time from 1 to 7 min as viewed from right to left (not marked for clarity). (b) ECL ratio plot demonstrating the increase in ECL intensity vs time of enzyme reaction. Controls show ECL response vs time exposed to B[a]P only (blue squares), H₂O₂ only (green diamonds), and 0.5 mM H₂O₂ + 100 μM B[a]P + 30 μM of inhibitor αNF (purple triangles).

Figure 4

Comparison of ECL response of RuPVP/DNA/enzyme spots containing cyt P450 1B1 after denoted exposure time (min) to 0.5 mM H₂O₂ + 100 μM B[a]P (a) without and (b) with the addition of 30 μM inhibitor αNF.

Figure 5

(a) Reconstructed array image where multiple cyt P450 enzymes were present in the Ru/PVP films on a single PG array and exposed to 0.5 mM H₂O₂ + 100 μM B[a]P for increasing times (s). (b) Schematic showing the location of cyt P450 enzymes in each 4-spot image in Figure 7a.

Figure 6

Influence of B[a]P/H₂O₂ exposure time for cyt P450 enzymes located on same array. ECL ratio was normalized for amount of enzyme. Cyt P450s used were 1B1(blue), 1A2 (red), cam (green), and 2E1 (purple).

Figure 7

(a) CapLC chromatogram showing elution of reaction media after 10-min exposure of Mb immobilized in a thin film on SiO₂ microspheres to B[a]P/H₂O₂ (red). Controls are B[a]P only (blue) and 100 mM B[a]P reacted with 0.5 mM H₂O₂ for 10 min in the absence of Mb (green). (b) MS for the 11–12-min eluting peaks in (a) along with representative epoxides and diol forms of B[a]P. (c) CapLC chromatogram (select ion mode) measuring total ion current m/z 402 from neutral hydrolysate of DNA/Mb film immobilized on carbon cloth after 10-min exposure to 100 mM B[a]P + 0.5 mM H₂O₂. (d) MS/MS of the 17-min elution peak in (c) with expected fragmentation pattern of parent B[a]P–guanine ion.

Scheme 1. Conceptual Diagram of the ECL Array Instrumentation^a

^a RuPVP polymer, DNA, and enzymes are located in each spot on a (a) pyrolytic graphite block; with (b) Ag/AgCl reference electrode, (c) Pt wire counter electrode, (d) CCD camera, (e) computer, (f) gel-doc dark room, and (g) potentiostat for applied voltage control.

Scheme 3. Summary of Possible Metabolic Routes for B[a]P in Vivo^a

^a Pathway A shows the formation of possible proximal epoxides (1) by cyt P450 followed by direct nucleophilic attack to form guanine adducts at the N2 position (2) or hydrolysis by epoxide hydrolase to form a diol product, and a second cyt P450-mediated epoxidation forming diol epoxides (3), and the final nucleophilic attack forming a guanine adduct at the N² position (4). Pathway B shows the one-electron oxidation by cyt P450 to form the reactive cation radical (5) with subsequent nucleophilic attack on guanine (6) or adenine (7) forming adducts at the N7 position on the nucleobases.