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:

• Nanoparticles
• nano-surfaces
• nanotoxicology
• DNA damage

Acknowledgements

We thank the National Institutes of Health (NIH-NIBIB 1R15EB014560) for financial support. Electron microscopy characterization was supported The South Carolina Bioengineering Center of Regeneration and Formation of Tissues (BioCRAFT) center funded under NIGMS of the National Institutes of Health, award number 5P20GM103444-07. C. A. M and K. V. T. N. thank the Clemson University Chemistry Department for graduate fellowships. C. A. M. thanks the Department of Science of the Government of Costa Rica for a graduate fellowship. K. V. T. N. received support from the Vietnam Education Foundation fellowship.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.

Additional information

Funding

We thank the National Institutes of Health (NIH-NIBIB 1R15EB014560) for financial support. Electron microscopy characterization was supported The South Carolina Bioengineering Center of Regeneration and Formation of Tissues (BioCRAFT) center funded under NIGMS of the National Institutes of Health, award number 5P20GM103444-07.