Quantification of damage to plasmid DNA from 35 MeV electrons, 228 MeV protons and 300 kVp X-rays in varying hydroxyl radical scavenging environments.

The pBR322 plasmid DNA was irradiated with 35 MeV electrons, 228 MeV protons and 300 kVp X-rays to quantify DNA damage and make comparisons of DNA damage between radiation modalities. Plasmid was irradiated in a medium containing hydroxyl radical scavengers in varying concentrations. This altered the amount of indirect hydroxyl-mediated DNA damage, to create an environment that is more closely associated with a biological cell. We show that increasing hydroxyl scavenger concentration significantly reduced post-irradiation DNA damage to pBR322 plasmid DNA consistently and equally with three radiation modalities. At low scavenging capacities, irradiation with both 35 MeV electrons and 228 MeV protons resulted in increased DNA damage per dose compared with 300 kVp X-rays. We quantify both single-strand break (SSB) and double-strand break (DSB) induction between the modalities as a ratio of yields relative to X-rays, referred to as relative biological effectiveness (RBE). RBESSB values of 1.16 ± 0.15 and 1.18 ± 0.08 were calculated for protons and electrons, respectively, in a low hydroxyl scavenging environment containing 1 mM Tris-HCl for SSB induction. In higher hydroxyl scavenging capacity environments (above 1.1 × 106 s-1), no significant differences in DNA damage induction were found between radiation modalities when using SSB induction as a measure of RBE. Considering DSB induction, significant differences were only found between X-rays and 35 MeV electrons, with an RBEDSB of 1.72 ± 0.91 for 35 MeV electrons, indicating that electrons result in significantly more SSBs and DSBs per unit of dose than 300 kVp X-rays.

A computational approach to quantifying miscounting of radiation-induced double-strand break immunofluorescent foci.

Immunofluorescent tagging of DNA double-strand break (DSB) markers, such as γ-H2AX and other DSB repair proteins, are powerful tools in understanding biological consequences following irradiation. However, whilst the technique is widespread, there are many uncertainties related to its ability to resolve and reliably deduce the number of foci when counting using microscopy. We present a new tool for simulating radiation-induced foci in order to evaluate microscope performance within in silico immunofluorescent images. Simulations of the DSB distributions were generated using Monte Carlo track-structure simulation. For each DSB distribution, a corresponding DNA repair process was modelled and the un-repaired DSBs were recorded at several time points. Corresponding microscopy images for both a DSB and (γ-H2AX) fluorescent marker were generated and compared for different microscopes, radiation types and doses. Statistically significant differences in miscounting were found across most of the tested scenarios. These inconsistencies were propagated through to repair kinetics where there was a perceived change between radiation-types. These changes did not reflect the underlying repair rate and were caused by inconsistencies in foci counting. We conclude that these underlying uncertainties must be considered when analysing images of DNA damage markers to ensure differences observed are real and are not caused by non-systematic miscounting.

Impact of DNA Geometry and Scoring on Monte Carlo Track-Structure Simulations of Initial Radiation-Induced Damage.

Track structure Monte Carlo simulations are a useful tool to investigate the damage induced to DNA by ionizing radiation. These simulations usually rely on simplified geometrical representations of the DNA subcomponents. DNA damage is determined by the physical and physicochemical processes occurring within these volumes. In particular, damage to the DNA backbone is generally assumed to result in strand breaks. DNA damage can be categorized as direct (ionization of an atom part of the DNA molecule) or indirect (damage from reactive chemical species following water radiolysis). We also consider quasi-direct effects, i.e., damage originated by charge transfers after ionization of the hydration shell surrounding the DNA. DNA geometries are needed to account for the damage induced by ionizing radiation, and different geometry models can be used for speed or accuracy reasons. In this work, we use the Monte Carlo track structure tool TOPAS-nBio, built on top of Geant4-DNA, for simulation at the nanometer scale to evaluate differences among three DNA geometrical models in an entire cell nucleus, including a sphere/spheroid model specifically designed for this work. In addition to strand breaks, we explicitly consider the direct, quasi-direct, and indirect damage induced to DNA base moieties. We use results from the literature to determine the best values for the relevant parameters. For example, the proportion of hydroxyl radical reactions between base moieties was 80%, and between backbone, moieties was 20%, the proportion of radical attacks leading to a strand break was 11%, and the expected ratio of base damages and strand breaks was 2.5-3. Our results show that failure to update parameters for new geometric models can lead to significant differences in predicted damage yields.

The suitability of micronuclei as markers of relative biological effect.

Micronucleus (MN) formation is routinely used as a biodosimeter for radiation exposures and has historically been used as a measure of DNA damage in cells. Strongly correlating with dose, MN are also suggested to indicate radiation quality, differentiating between particle and photon irradiation. The "gold standard" for measuring MN formation is Fenech's cytokinesis-block micronucleus (CBMN) cytome assay, which uses the cytokinesis blocking agent cytochalasin-B. Here, we present a comprehensive analysis of the literature investigating MN induction trends in vitro, collating 193 publications, with 2476 data points. Data were collected from original studies that used the CBMN assay to quantify MN in response to ionizing radiation in vitro. Overall, the meta-analysis showed that individual studies mostly have a linear increase of MN with dose [85% of MN per cell (MNPC) datasets and 89% of percentage containing MN (PCMN) datasets had an R2 greater than 0.90]. However, there is high variation between studies, resulting in a low R2 when data are combined (0.47 for MNPC datasets and 0.60 for PCMN datasets). Particle type, species, cell type, and cytochalasin-B concentration were suggested to influence MN frequency. However, variation in the data meant that the effects could not be strongly correlated with the experimental parameters investigated. There is less variation between studies when comparing the PCMN rather than the number of MNPC. Deviation from CBMN protocol specified timings did not have a large effect on MN induction. However, further analysis showed less variation between studies following Fenech's protocol closely, which provided more reliable results. By limiting the cell type and species as well as only selecting studies following the Fenech protocol, R2 was increased to 0.64 for both measures. We therefore determine that due to variation between studies, MN are currently a poor predictor of radiation-induced DNA damage and make recommendations for futures studies assessing MN to improve consistency between datasets.

Mechanistic Modelling of Slow and Fast NHEJ DNA Repair Pathways Following Radiation for G0/G1 Normal Tissue Cells.

Mechanistic in silico models can provide insight into biological mechanisms and highlight uncertainties for experimental investigation. Radiation-induced double-strand breaks (DSBs) are known to be toxic lesions if not repaired correctly. Non-homologous end joining (NHEJ) is the major DSB-repair pathway available throughout the cell cycle and, recently, has been hypothesised to consist of a fast and slow component in G0/G1. The slow component has been shown to be resection-dependent, requiring the nuclease Artemis to function. However, the pathway is not yet fully understood. This study compares two hypothesised models, simulating the action of individual repair proteins on DSB ends in a step-by-step manner, enabling the modelling of both wild-type and protein-deficient cell systems. Performance is benchmarked against experimental data from 21 cell lines and 18 radiation qualities. A model where resection-dependent and independent pathways are entirely separated can only reproduce experimental repair kinetics with additional restraints on end motion and protein recruitment. However, a model where the pathways are entwined was found to effectively fit without needing additional mechanisms. It has been shown that DaMaRiS is a useful tool when analysing the connections between resection-dependent and independent NHEJ repair pathways and robustly matches with experimental results from several sources.