Visualisation of γH2AX Foci Caused by Heavy Ion Particle Traversal; Distinction between Core Track versus Non-Track Damage

<div><p>Heavy particle irradiation produces complex DNA double strand breaks (DSBs) which can arise from primary ionisation events within the particle trajectory. Additionally, secondary electrons, termed delta-electrons, which have a range of distributions can create low linear energy transfer (LET) damage within but also distant from the track. DNA damage by delta-electrons distant from the track has not previously been carefully characterised. Using imaging with deconvolution, we show that at 8 hours after exposure to Fe (∼200 keV/µm) ions, γH2AX foci forming at DSBs within the particle track are large and encompass multiple smaller and closely localised foci, which we designate as clustered γH2AX foci. These foci are repaired with slow kinetics by DNA non-homologous end-joining (NHEJ) in G1 phase with the magnitude of complexity diminishing with time. These clustered foci (containing 10 or more individual foci) represent a signature of DSBs caused by high LET heavy particle radiation. We also identified simple γH2AX foci distant from the track, which resemble those arising after X-ray exposure, which we attribute to low LET delta-electron induced DSBs. They are rapidly repaired by NHEJ. Clustered γH2AX foci induced by heavy particle radiation cause prolonged checkpoint arrest compared to simple γH2AX foci following X-irradiation. However, mitotic entry was observed when ∼10 clustered foci remain. Thus, cells can progress into mitosis with multiple clusters of DSBs following the traversal of a heavy particle.</p></div

SCE induction from radioactive solutions.

<p>Radioavtivities (CPM) are at the time of solution transferred. Pooled SCE data induced by water and PBS activated by different hadron radiation types were plotted. Data were fit with semi-log line (R square = 0.722). Observed SCE = 0.017 x log(CPM) + background SCE.</p

Radioactivation after 100 Gy of proton exposure to PBS measured by NaI scintillation detector.

<p>(A) Energy spectrum of NaI detector. A clear peak of annihilation gamma-rays at 0.511 MeV is shown. (B) Time course change of measured count rates of 511 keV gamma-rays. Data was fitted with three-phase exponential decay model with half-lives of 2 min (93%), 10 min (3.5%), and 20 min (4.5%).</p

Radioactivation of the solution after different types of hadron radiation exposure.

<p>Radioactivities were measured as counts per minute (CPM) by GM counter. <b>(</b>A) Comparison of activation of H₂O and PBS. (B) Initial radioactivation per exposed doses. The * indicates that more than 10 Gy of proton exposure induced higher than GM detector’s upper limit. (C-E) Time course of radioactivities after (C) proton, (D) carbon-ion, and (E) iron-ion exposures. Lines were fitted with one-phase exponential decay curves with half-lives of approximately 20 minutes. Experiments were carried out at least three times, and error bars indicate SEM.</p

SCE induction from gamma-ray exposed solution.

<p>No significant difference was observed in the mean SCE frequencies among the samples by one-way ANOVA (p = 0.799). Experiments were carried out at least three times and error bars indicate SEM.</p

Variation in the length and number of tracks between individual flat fibroblast cells.

<p>(A) γH2AX foci in 48BR (WT) primary and 2BN (XLF) hTERT cells were enumerated from 0.5–24 h post 1 Gy X-rays. Foci were scored in 2D using a Zeiss Axioplan microscope. (B) 48BR (WT) cells were fixed at 30 min following 1 Gy Fe ions and stained with γH2AX and DAPI. Asterisks represent non-track cells. (C) Distribution in the percentage of γH2AX tracks following Fe irradiation in B. (D) The average number of tracks per cell following 1 Gy Fe is shown. The predicted fluence of particles traversing the nuclease after 1 Gy horizontal Fe irradiation was estimated to be ∼1.1 with a Poisson distribution predicting and ∼70% cells receiving a particle track (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070107#s2" target="_blank">Materials and Methods</a>). (E) Scatter plots of track length per cell following 1 Gy Fe irradiation are shown in 48BR (WT) and 2BN (XLF) G0/G1 cells. Tracks whose length are <0.025 were excluded from the data, since they were indistinguishable from delta-electron induced foci. ∼250 cells were examined in each analysis (C–E). (F) Clustered γH2AX foci within the tracks is ATM/DNA-PK dependent, indicating that they are DSBs. 48BR (WT) cells were fixed at 30 min post Fe irradiation with/without ATM plus DNA-PK inhibitor. To examine the percentage of track positive cells, >200 cells were scored. Error bars represent the standard deviations (SD) from 2 experiments. The analysis was performed by DeltaVision microscope without deconvolution (B–F). Note that although the cells were exposed to 1 Gy Fe ions, the dose to individual cells can differ due to differing number of particles traversing the cell. In the ensuing analysis, we examine cells which have a single particle traversal.</p

Track structure simulation and spatial distribution of DSBs arising from an Fe ion track.

<p>A Simulation of the structure of damage arising from a single Fe ion particle traversal. The black marks and the red marks represent the tracks of Fe ions and the position of DSBs, respectively. The blue ellipsoid shape shows the size of the nucleus. The slighter wider band of the damage within the tracks observed in these experiments is likely explained by DNA movement after radiation exposure. The number of electrons generated decreases as the electron energy decreases. For 416 MeV/n Fe ions, ∼90% of electrons generated are estimated to have energies less than 100 eV. The range of such low-energy electrons is less than a few nm. The majority of other electrons have a range from a nm to 5 µm. The highest energy electrons with a range of a few hundred µm can also arise but at a low frequency (<0.1%).</p

γH2AX foci at sites distinct to the tracks showing less clustered damage.

<p>(A) Typical image of DSB formation outside of the particle track following Fe ion irradiation. 48BR (WT) primary G0/G1 cells were irradiated with 1 Gy Fe irradiation, fixed at 30 min, and stained with γH2AX and DAPI. (B) Percentage of individual foci within a cluster at non-track regions was analysed from >100 individual foci. 48BR (WT) cells were irradiated and fixed at 30 min post 1 Gy Fe irradiation. (C) The width of clustered γH2AX foci post Fe irradiation within the particles tracks, at non-track regions following Fe irradiation or following X-irradiation. >50 foci were analysed in each sample. The diameter of clustered γH2AX foci after Fe ions is ∼3–4 fold greater than the width of γH2AX foci arising after X-rays, whilst the foci diameter of non-track γH2AX foci is similar to that arising following X-rays. The width of foci is measured by IMARIS in 3D using images taken by the DeltaVision microscope. (D) Foci complexity is negatively correlated with distance from the particle track. γH2AX foci at non-track regions in 48BR cells were analysed following 1 Gy Fe irradiation. Cluster analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070107#pone-0070107-g003" target="_blank">Figure 3</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070107#pone-0070107-g004" target="_blank">4</a>. To allow the analysis of γH2AX foci close to the particle track, we used a distance of 1 µm to distinguish non-track from track damage and carried out the analysis at 30 min exposure to minimise the movement of γH2AX foci within tracks. Images were taken by DeltaVision microscope followed by deconvolution and analysed (A–D). Cells forming a single γH2AX track of length >8 µm and width >1 µm were analysed in B and C.</p

Clustered γH2AX foci arising within the particle tracks represent a signature of high LET particle radiation and are repaired slowly by NHEJ in G1.

<p>(A) 48BR (WT) primary and 2BN (XLF) hTERT G0/G1 cells were irradiated in a horizontal direction with 1 Gy Fe irradiation. Images are taken using the DeltaVision microscope followed by deconvolution. Representative images at 8 and 24 h post Fe irradiation are shown. The nucleus outline is drawn with a dashed line from the DAPI staining. (B, C) Percentage of individual foci within a cluster was analysed from >100 individual clusters at each time point. Similar results were obtained in two independent experiments. Cells forming a single γH2AX track of length >8 µm and width >1 µm were analysed.</p

Generation of “simple” γH2AX foci at distances away from the particle track and they are repaired rapidly by NHEJ.

<p>(A) Representative image of γH2AX foci formation at non-track regions. 48BR (WT) primary and 2BN (XLF) hTERT G0/G1 cells were irradiated with 1 Gy pencil Fe ions and stained with γH2AX and DAPI. (B) γH2AX foci at non-track regions in cells which have a single track per nucleus were enumerated following 1 Gy Fe ion irradiation. (C) To investigate whether non-track induced γH2AX foci formation is due to DSBs, the number of γH2AX foci at non-track regions was enumerated with/without ATM plus DNA-PK inhibitor. Since ATM/DNA-PK inhibitor treated cells do not form γH2AX tracks, foci number was enumerated without any bias in these cells. (D) Distribution in the percentage of γH2AX tracks following 0.1 Gy Fe irradiation. (E, F) γH2AX foci at non-track regions with a single track per nucleus was examined following 0.1 Gy Fe irradiation. A region which is located greater than 2 µm from the track was excluded from the analysis (B and F). Images were taken by Olympus BX51 microscope without deconvolution (A and E). γH2AX foci were analysed using Olympus BX51 or Zeiss Axioplan microscope by 2D (B, C, D and F). Cells forming a single γH2AX track of length >8 µm and width >1 µm were analysed in B and F.</p