Nanomaterials and Oxidative Stress

With the rapid development of new nanomaterials, it was recognized early that together with their beneficial properties, nanomaterials may pose a risk to human health and the environment. Evidence has accumulated over the last twenty years in support of oxidative stress as a broad mechanistic concept to explain the interaction of engineered nanoparticles with biological substances. As oxidative stress as a physiological response was recognized in redox biology, its wide-ranging use in nanotoxicology has exposed new challenges and limitations. In this commentary, we review certain oxidative stress concepts and their relevance to nanotoxicology

Electrochemical Potential Gradient as a Quantitative in Vitro Test Platform for Cellular Oxidative Stress

Oxidative stress in a biological system is often defined as a redox imbalance within cells or groups of cells within an organism. Reductive-oxidative (redox) imbalances in cellular systems have been implicated in several diseases, such as cancer. To better understand the redox environment within cellular systems, it is important to be able to characterize the relationship between the intensity of the oxidative environment, characterized by redox potential, and the biomolecular consequences of oxidative damage. In this study, we show that an in situ electrochemical potential gradient can serve as a tool to simulate exogenous oxidative stress in surface-attached mammalian cells. A culture plate design, which permits direct imaging and analysis of the cell viability, following exposure to a range of solution redox potentials, was developed. The in vitro oxidative stress test vessel consists of a cell growth flask fitted with two platinum electrodes that support a direct current along the flask bottom. The applied potential span and gradient slope can be controlled by adjusting the constant current magnitude across the vessel with spatially localized media potentials measured with a sliding reference electrode. For example, the viability of Chinese Hamster Ovary cells under a gradient of redox potentials indicated that cell death was initiated at approximately 0.4 V vs. standard hydrogen electrode (SHE) media potential and this potential could be modified with antioxidants. This experimental platform may facilitate studies of oxidative stress characteristics on different types of cells by enabling imaging live cell cultures that have been exposed to a gradient of exogenous redox potentials

Conformational analysis of the telomerase RNA pseudoknot hairpin by Raman spectroscopy

We have measured the temperature-dependent Raman spectra of two 30-mer ribonucleotides that represent the wild-type (WT) and dyskeratosis congenita (DKC) mutant (MT) GC (107–108) → AG structures of the pseudoknot hairpin region of human telomerase RNA. We have used these structures, previously characterized by UV-melting and NMR, as a model system for our Raman investigation. We observe that Raman hypochromism of vibrational bands, previously assigned to specific bases or conformational RNA markers, reflect temperature-dependent alterations in the pentaloop and stem structures of these two oligonucleotides. We also observe that the intense ν(s)(O-P-O) band at 812 cm(−1) indicates the presence of A-form backbone structure at relatively low temperatures in both the WT and MT RNA sequences. The mutation induces a decrease in the intensity of the uridine (rU) band at 1244 cm(−1) associated with C2′-endo/anti ribose conformation in the pentaloop. Two transition temperatures (T(m)) were determined from the analysis of Raman difference intensity-temperature profiles of the 1256 cm(−1) band, which is associated with vibrations of cytidine (rC) residues, in particular, the C2′-endo/anti ribose conformation (T(m)1 = 23.6 ± 1.6°C for WT and 19.7 ± 2.8°C for MT; T(m)2 = 68.9 ± 1.8°C for WT and 70.9 ± 1.1°C for MT). From these results we can conclude that the DKC mutant 30-mer exhibits a lower stability in the pentaloop region and a slightly higher stability in the stem region than the WT 30-mer. This demonstrates that Raman bands, previously assigned to specific bases or conformational RNA markers, can be used to probe local structural features of the telomerase pseudoknot hairpin sequence

In situ Electrochemical Small-Angle Neutron Scattering (<i>e</i>SANS) for Quantitative Structure and Redox Properties of Nanoparticles

The rapid growth in nanomaterial applications have revealed limitations in available physicochemical characterization methods. An in situ electrochemical small-angle neutron scattering (eSANS) methodology was devised that enables direct measurements of nanomaterial dispersion structure while undergoing reduction–oxidation (redox) reactions at the vitreous carbon electrode. Furthermore, these porous electrodes are amenable to contrast-variant neutron scattering strategies to measure nanoparticle structure and polymer conformation in multicomponent systems. The eSANS method was tested for feasibility by characterizing ZnO nanoparticles in 50 mmol/L NaCl deuterium oxide solution undergoing bulk electrolysis at negative potentials. Irreversible nanoparticle structural changes are observed during the potential cycle. The complete reduction of Zn²⁺ to Zn⁰ nanoparticles is unlikely, but a peak in the characteristic correlation length occurs during the redox bias with reduced average characteristic size

Cellular Reference Materials for DNA Damage Using Electrochemical Oxidation

Reference materials are needed to quantify the level of DNA damage in cells, to assess sources of measurement variability and to compare results from different laboratories. The comet assay (single cell gel electrophoresis) is a widely used method to determine DNA damage in the form of strand breaks. Here we examine the use of electrochemical oxidation to produce DNA damage in cultured mammalian cells and quantify its percentage using the comet assay. Chinese hamster ovary (CHO) cells were grown on an indium tin oxide electrode surface and exposed 12 h to electrochemical potentials ranging from 0.5 V to 1.5 V (vs Ag/AgCl). The resulting cells were harvested and analyzed by comet and a cell viability assay. We observed a linear increase in the percentage (DNA in tail) of strand breaks along with a loss of cell viability with increasing oxidation potential value. The results indicate that electrochemically induced DNA damage can be produced in mammalian cells under well-controlled conditions and could be considered in making a cellular reference material for the comet assay

Temperature dependence of the formal reduction potential of putidaredoxin

AbstractPutidaredoxin (Pdx), a [2Fe–2S] redox protein of size Mr 11 600, transfers two electrons in two separate steps from the flavin containing putidaredoxin reductase to the heme protein, cytochrome CYP101 in the P450cam catalytic cycle. It has recently come to light, through NMR measurements, that there can be appreciable differences in the Pdx conformational dynamics between its reduced and oxidized states. The redox reaction entropy, ΔSrc0′=(SPdxr0′−SPdxo0′), as determined from measurements of the variation in formal potential with temperature, E0′(T), provides a measure of the strength of this influence on Pdx function. We designed a spectroelectrochemical cell using optically transparent tin oxide electrodes, without fixed or diffusible mediators, to measure E0′(T) over the temperature range 0–40°C. The results indicate that the redox reaction entropy for Pdx is biphasic, decreasing from −213±27 J mol−1 K−1 over 0–27°C, to −582±150 J mol−1 K−1 over 27–40°C. These redox reaction entropy changes are significantly more negative than the changes reported for most cytochromes, although our measurement over the temperature interval 0–27°C is in the range reported for other iron–sulfur proteins. This suggests that Pdx (and other ferredoxins) is a less rigid system than monohemes, and that redox-linked changes in conformation, and/or conformational dynamics, impart to these proteins the ability to interact with a number of redox partners