Reactive oxygen species generation by copper(II) oxide nanoparticles determined by DNA damage assays and EPR spectroscopy

Abstract

Copper(II) oxide nanoparticles (^NPCuO) have many industrial applications, but are highly cytotoxic because they generate reactive oxygen species (ROS). It is unknown whether the damaging ROS are generated primarily from copper leached from the nanoparticles, or whether the nanoparticle surface plays a significant role. To address this question, we separated nanoparticles from the supernatant containing dissolved copper, and measured their ability to damage plasmid DNA with addition of hydrogen peroxide, ascorbate, or both. While DNA damage from the supernatant (measured using an electrophoresis assay) can be explained solely by dissolved copper ions, damage by the nanoparticles in the presence of ascorbate is an order of magnitude higher than can be explained by dissolved copper and must, therefore, depend primarily upon the nanoparticle surface. DNA damage is time-dependent, with shorter incubation times resulting in higher EC₅₀ values. Hydroxyl radical (^•OH) is the main ROS generated by ^NPCuO/hydrogen peroxide as determined by EPR measurements; ^NPCuO/hydrogen peroxide/ascorbate conditions generate ascorbyl, hydroxyl, and superoxide radicals. Thus, ^NPCuO generate ROS through several mechanisms, likely including Fenton-like and Haber-Weiss reactions from the surface or dissolved copper ions. The same radical species were observed when ^NPCuO suspensions were replaced with the supernatant containing leached copper, washed ^NPCuO, or dissolved copper solutions. Overall, ^NPCuO generate significantly more ROS and DNA damage in the presence of ascorbate than can be explained simply from dissolved copper, and the ^NPCuO surface must play a large role.

Keywords: DNA damage; Nanoparticles; nano-surfaces; nanotoxicology.

Figure 1

Flowchart illustrating separation of ^NPCuO components to evaluate DNA damage. ^NPCuO: whole suspension of CuO nanoparticles, ^wCuO: washed CuO nanoparticles, ^lCuO: leachate of CuO nanoparticles.

Figure 2

A) Gel electrophoresis image of plasmid DNA (p) treated with ^NPCuO (1–1000 μM) and H₂O₂ (50 μM) for 150 min at pH 7 (MOPS, 10 mM). Lane 0: 1 kb molecular weight ladder; 1: p; 2: p + H₂O₂ (50 μM), 3: p + ^NPCuO (1000 μM); 4: p + Cu²⁺ (6 μM) + ascorbate (7.5 μM) + H₂O₂ (50 μM); lanes 5–13: p + H₂O₂ (50 μM) + increasing concentrations of ^NPCuO (1, 5, 10, 25, 50, 100, 250, 500, and 1000 μM, respectively). B) Dose-response curve fitting for the gel data in A to obtain an EC₅₀ value.

Figure 3

Comparative scheme of DNA damage (shown in gel images) caused by ^NPCuO, ^wCuO, and ^lCuO fractions (50 μM) with ascorbate and H₂O₂ for 150 min.

Figure 4

Comparison of the EC₅₀ plots for DNA damage caused by ^NPCuO, ascorbate (1.25 equiv; 1.25 – 1250 μM), and H₂O₂ (50 μM) for 30 min (open circles) and 150 min (filled circles).

Figure 5

EPR spectra of ^wCuO (300 μM) with A) H₂O₂ (22.5 mM), B) ascorbate (375 μM), and C) H₂O₂ (22.5 mM) and ascorbate (375 μM). All samples in buffer at pH 7 (MOPS, 10 mM) with DMPO (30 mM) as a spin trap. Asterisks indicate DMPO-OOH resonances. A₁ and g₁; A₂ and g₂; and g₃ and A₃ correspond to DMPO-OH, AscH^•, and DMPO-OOH resonances, respectively. A₄ is the second hyperfine coupling constant for the DMPO-OOH resonance.

Figure 6

Comparison of EPR spectra with CuO fractions (^NPCuO, ^wCuO, or ^lCuO; 300 μM) and A) H₂O₂ (22.5 mM), B) ascorbate (375 μM), or C) H₂O₂ (22.5 mM) and ascorbate (375 μM). All samples in buffer at pH 7 (MOPS, 10 mM) with DMPO (30 mM) as a spin trap.

Figure 7

EPR spectra of CuSO₄ (300 μM),H₂O₂ (22.5 mM), and ascorbate using A) DMPO (30 mM) and B) TEMP (30 mM) as a spin trap.