Analysis of 8-oxo-7, 8-dihydroGuanine formation and oxidation mediated by Fenton reaction induced DNA oxidative stress

DNA undergoes an estimated 10,000 oxidative hits per day. Oxidative DNA damage caused by reactive oxygen species (ROS) can result in multiple base modifications, which have been implicated in mutagenesis, disease and aging. The primary product of G oxidation is 8-oxo-7,8-dihydroguanine (8- oxoG), which is considered by many as a biomarker for oxidative DNA damage. 8-oxoG has an oxidation potential about 0.5 V lower than G, and so can be accurately quantified using electrochemical (EC) detection. EC detection coupled to HPLC resulted in a sensitive and accurate mode of detection for 8- oxoG without requiring any preconcentration or removal of undamaged G, which was simultaneously detected by UV detection. The aim of this investigation was to measure the rate of 8-oxoG formation in DNA subjected to continuous oxidative attack. The hydroxyl radical, the most aggressive ROS, was generated via the iron-mediated Fenton reaction, and used to generate 8-oxoG in both free G and double stranded DNA. HPLC-UV-EC was utilised for the quantitative analysis of both G and 8-oxoG concentrations with respect to incubation time with the hydroxyl radical. The concentration of 8-oxoG was observed to oscillate with respect to time. After approximately 18 min incubation with Fenton reagents, a maximum 8-oxoG concentration of 0.68 uM was detected in DNA. Thereafter, however, there was an overall decrease in 8-oxoG concentration over time. 8-oxoG concentration was not found to be proportional to the level of oxidative damage which occurred. The concentration of G was also observed to decrease with increasing DNA oxidation, so that as oxidation continued, both G and 8-oxoG were oxidised. Copper, another important biological metal ion, binds tightly to DNA, inducing significant oxidative DNA damage. It was also investigated as a metal catalyst for the Fenton reaction-mediated DNA oxidation. Again, 8-oxoG concentration was found to oscillate with increasing oxidation of DNA. There were significant differences between the iron- and copper-mediated oxidation of DNA. Oscillation periods for copper-mediated oxidation were shorter, with greater concentration amplitudes. A maximum 8-oxoG concentration of 4.2 fiM was detected after 35 min oxidation. Overall, however, the trend was again towards oxidation of both G and 8-oxoG with increasing oxidation of DNA. 8-oxoG is a hotspot for further oxidation. It was observed in both studies outlined above to be further oxidised during DNA oxidation by the Fenton reaction. The final products of 8-oxoG oxidation were determined using HPLCMS/ MS. Oxidised guanidinohydantoin (oxGh) was identified as the primary product of 8-oxoG oxidation, when both iron and copper catalysts were used. A mechanism for the formation of oxGh by hydroxyl radical attack of 8-oxoG was also proposed

Separation of oxidatively damaged DNA Nucleobases and Nucleosides on Packed and Monolith C18 Columns by HPLC-UV-EC

This study involves the incorporation of a commercially available Phenomenex Onyx C18 monolith column into the separation and detection of oxidative DNA damage. It includes thorough investigation of monolith performance and a comparison of the performance of monolith columns with a commercially available packed Restek reverse phase Ultra C18 column for the separation of DNA bases and nucleosides. The performance of the monolith was examined using efficiency, resolution, plate height, asymmetry and retention times, and in each case showed improved or at least comparable results in the separation of a mix of DNA bases and nucleosides. A 90% reduction, from just under 40 min. to just under 4 min., was obtained in the elution time of this separation. To the best of our knowledge, this is the first report of a fast monolith column separation successfully coupled to both a UV-vis and EC detector, which is especially useful for analysis of oxidative DNA damage. The determination of 8-oxoG and 8-OH-dG, oxidation products of guanine and 2’-deoxyguanosine, respectively, may be compromised by their ease of oxidation and therefore the fast separation, selective and sensitive detection, with no artifactual oxidation, detailed in this report, is ideal

Inverse-opal conducting polymer monoliths in microfluidic channels

Inverse opal monolithic flow-through structures of polyaniline (PANI) were achieved in microfluidic channels for lab-on-a-chip (LOC) applications. In order to achieve the uniformly porous monolith, polystyrene (PS) colloidal crystal (CC) templates were fabricated in channel. An inverse opal PANI structure was achieved on-chip, through a two-step process involving the electrochemical growth of PANI and subsequent removal of the template. The effect of electropolymerisation on these structures is discussed. It was found that growth time is critical in achieving an ordered structure with well-defined flow-through pores. This is significant in order to fabricate optimal porous PANI structures that maximise surface area of the monolith and also provide well-defined flow profiles through the micro-channel

Nickel(II)-catalysed oxidative guanine and DNA damage beyond 8-oxoguanine

Oxidative DNA damage is one of the most important and most studied mechanisms of disease. It has been associated with a range of terminal diseases such as cancer, heart disease, hepatitis, and HIV, as well as with a variety of everyday ailments. There are various mechanisms by which this type of DNA damage can be initiated, through radiation and chemical oxidation, among others; however, these mechanisms have yet to be fully elucidated. A HPLC-UV-EC study of the oxidation of DNA mediated by nickel(II) obtained results that show an erratic, almost oscillatory formation of 8-oxoguanine (8-oxoG) from free guanine and from guanine in DNA. Sporadic 8-oxoG concentrations were also observed when 8-oxoG alone was subjected to these conditions. A HPLC-MS/MS study showed the formation of oxidised-guanidinohydantoin (oxGH) from free guanine at pH 11, and the formation of guanidinohydantoin (GH) from DNA at pH 5.5

Development and characterisation of switchable polyaniline-functionalised flow-through capillary monoliths

Polymer monoliths were prepared in capillary format (250 mm i.d.) and used as solid supports for the immobilisation of the conducting polymer polyaniline (PANI). The immobilisation of PANI was confirmed on the large macro-porous structure of a polystyrene–divinylbenzene (PS-co-DVB) monolith. The surface coverage of polyaniline was characterised by field emission scanning electron microscopy (FESEM) and by capacitively coupled contactless conductivity detection (C4D), which was operated in scanning mode to non-invasively visualise the axial distribution of the immobilised PANI and to provide information on its doping state. To further demonstrate the successful functionalisation of the monoliths, the PANI-functionalised monoliths were demonstrated as switchable, weak anion-exchange stationary phases as confirmed by studying the retention of iodide using a perchlorate eluent

Inverse-opal conducting polymer monoliths in micro-fluidic channels.

Inverse opal monolithic flow-through structures of polyaniline (PANI) were achieved in microfluidic channels for lab-on-a-chip (LOC) applications. In order to achieve the uniformly porous monolith, polystyrene (PS) colloidal crystal (CC) templates were fabricated in channel. An inverse opal PANI structure was achieved on-chip, through a two-step process involving the electrochemical growth of PANI and subsequent removal of the template. The effect of electropolymerisation on these structures is discussed. It was found that growth time is critical in achieving an ordered structure with well-defined flow-through pores. This is significant in order to fabricate optimal porous PANI structures that maximise surface area of the monolith and also provide well-defined flow profiles through the micro-channel

Presence of pharmaceuticals in Irish surface water

At each stage of a pharmaceutical lifecycle, there is a significant risk of environmental exposure. For this reason, it is imperative to implement both source directed and end of pipe control measures. This will mitigate any potential hazards to the environment or to humans. The ever-increasing use and availability of pharmaceuticals in the last decade have led to the contamination of surface water ecosystems with ng/L to µg/L concentrations. The environmental fate and toxicological implications of many pharmaceuticals and their residues are not fully understood. Additionally, the stability and biological activity of these “micropollutants” can lead to chronic environmental exposure causing behavioural and health-related effects. This research investigates pharmaceuticals chosen from the updated surface water “Watch List” (Decision (EU) 2018 /840), followed by pharmaceuticals which are commonly found in European surface water and pharmaceuticals which have a low removal efficiency in wastewater treatment plants. This project aims to create a comprehensive prioritisation framework and a risk-based assessment by calculating their risk quotient for each of the chosen pharmaceuticals

Electroactive monolith μchips based on nanostructured polyaniline

The extensive application of monolithic columns for HPLC is severely hindered by a lack of column-to-column reproducibility. EMμ(Electroactive Monolithic μChip) is a new concept that solves the significant reproducibility problems, as well as allowing miniaturization and improving overall efficiency through electrochemically controlled dynamic separations. This novel μchip has a micro-structured monolith fabricated from intelligent, electroactive polymer. By application of a specific potential, conducting polymers such as polyaniline (PANI) can be reproducibly grown and readily fine-tuned in terms of porosity, hydrophobicity an d ionic capacity. This unique chip provides for an Electroac tive Monolithic μchip capable of multi-dimensional chromatographic separations. The monolith microstructuring (provided by templating) wi ll provide reproducibility and improve efficiency by decre asing the A-term of the Van Deemter equation. Furthermore, the use of these intelligen t materials will enable gradient control and redox reaction s to be exploited during separations of large biomolecules

Lifecycle assessment of pharmaceuticals in Irish surface waters

At each stage of a pharmaceutical lifecycle, there is a significant risk of environmental exposure. For this reason, it is imperative to implement both source directed and end of pipe control measures. This will mitigate any potential hazards to the environment or to humans. The ever-increasing use and availability of pharmaceuticals in the last decade have led to the contamination of surface water ecosystems with ng/L to µg/L concentrations. The environmental fate and toxicological implications of many pharmaceuticals and their residues are not fully understood. Additionally, the stability and biological activity of these “micropollutants” can lead to chronic environmental exposure causing behavioural and health-related effects. This research investigates pharmaceuticals chosen from the updated surface water “Watch List” (Decision (EU) 2018/840) followed by pharmaceuticals which are commonly found in European surface water and pharmaceuticals which have a low removal efficiency in wastewater treatment plants. This project aims to create a comprehensive prioritisation framework and a risk-based assessment by calculating their risk quotient for each of the chosen pharmaceuticals

Production of polystyrene spheres for use as a templating material for polyaniline monolith structures.

Polystyrene (PS) spheres are potentially useful as a reproducible, sacrificial templating material for monolith columns once they can be utilised to create a uniform microstructured packing which enables a higher monolith batch to batch reproducibility. To achieve PS spheres which can meet these requirements, their synthesis was optimised. Parameters investigated included variation of reactant concentrations, along with optimisation of reaction conditions temperature, agitation speed and nitrogen flow during aeration. Temperature and agitati on played vital roles in the size and homogeneity of the synthesised PS spheres. Temperature affected the equilibrium concentration of monomer in the aqueous phase. When reaction temperature was increased, sphere size reduced and as reaction temperature decreased sphere size increased. A similar trend was seen when agitation speed was varied. At higher agitation speed average PS sphere size decreased as the rate of polymerisation increased. At lower agitation speed the average PS sphere size increased as the rate of polymerisation decreased. Ensuring fluctuations in both temperature and agitation were kept to a minimum was key to maintaining reproducibility. Any fluctuation above ~10% in either temperature or agitation speed affected standard deviation irreversibly. The facile dissolution of the PS spheres was also investigated. If the spheres produced could not be dissolved, their use as a sacrificial templating material would not be possible. By decreasing the original concentration of cross-linker, dissolution increased dramaticall