Fundamental Interaction Mechanisms of Engineered Nanomaterials with DNA

Summary

We utilized isotope-dilution liquid chromatography/mass spectrometry (LC/MS) to determine the levels of the oxidatively damaged DNA bases, 8-hydroxydeoxyguanosine (8-OH-dG) and 8-hydroxydeoxyadenosine (8-OH-dA), in simple bicomponent solutions consisting of calf-thymus DNA and 10, 30, or 60 nm gold nanoparticles (Au-NPs). No consistent changes in the levels of 8-OH-dA were observed; however, analysis of 8-OH-dG demonstrated a size-dependent interaction of the Au-NPs with DNA. The 10 nm Au-NPs produced a significant and consistent reduction, versus control samples, in the measured level of 8-OH-dG that was not observable with either the 30 or 60 nm Au-NPs.

Description

Intended impact

Nanotechnology research has resulted in the rapid creation of engineered nanomaterials with many foreseeable applications in medical imaging/diagnosis and in drug delivery. However, there is a notable scarcity of both acute and chronic human toxicity data for these new materials. Exposure to nanomaterials, whether during manufacturing/research procedures (inhalation/skin absorption) or during therapeutic/diagnostic procedures (ingestion/injection), is an immediate and significant concern for human health and safety. This concern derives from the observation that certain nanomaterial characteristics (size, surface coating, charge, shape, aspect ratio, etc.) have the capability to generate reactive oxygen species (ROS), reactive nitrogen species (RNS) or other free radicals in biological systems. Free radicals are naturally present in the body; however, an overabundance of free radicals can overwhelm the body's natural antioxidant defense system and lead to oxidative stress. Oxidative damage to the body's genomic DNA is one of the consequences of oxidative stress. It is not known at a fundamental molecular level if engineered nanomaterials promote or hinder the formation of free radicals in the body or how these nanomaterials might interact with specific molecular targets such as DNA. By using simple solutions of DNA and Au-NPs as a preliminary model system, the present work aims to generate fundamental in vitro data that will enable continued and deeper research into the potential mechanisms of NP interactions (if any) with the DNA in mammalian cells.

Objective

Develop methods and conduct research to understand the potential physicochemical interactions of engineered NPs with DNA that could potentially result in cytotoxicity and/or genotoxicity.

Goals

• Measure oxidatively-induced DNA damage resulting from the interaction of Au-NPs with DNA in simple bicomponent solutions.
• Determine the mechanism of interaction of Au-NPs with DNA in simple bicomponent solutions and develop a working hypothesis about potential toxic implications.
• Measure oxidatively-induced DNA damage resulting from the interaction of Au-NPs and other biomedically-relevant engineered NPs with mammalian cell lines and develop a standardized platform for characterizing NP genotoxicity/cytotoxicity based on application of targeted metabolomics.

Research activities and technical approach

Single-stage LC/MS has been utilized to quantitatively determine the level of oxidatively-induced DNA damage in DNA that has been extracted from incubated bicomponent solutions of Au-NP/DNA. Samples of the Au-NP/DNA solutions, with NP concentrations of 1, 10, and 90 nmol/L were incubated at 37 °C for 1 h and the DNA was precipitated, washed and digested into its component bases. Digests were analyzed by LC/MS for the quantitative determination of 8-OH-dG.

Major Accomplishments

• Measured the presence of oxidatively-induced DNA lesions in simple bicomponent solutions of Au-NP/DNA.
• Determined that Au-NPs in the presence of DNA solutions are not strictly "inert". Au-NPs appear to have a significant size-dependent interaction with DNA that has implications for Au-NP interactions with nuclear DNA.