MODELLING Γ-H2ax FOCI INDUCTION TO MIMIC LIMITATIONS IN THE SCORING TECHNIQUE

Barbieri, Sofia; Baiocco, G.; Babini, Gabriele; Morini, Jacopo; Friedland, W.; Grilj, Veljko; Brenner, David J.; Ottolenghi, A.

doi:10.1093/rpd/ncy217

Cited by 8 publications

(10 citation statements)

References 10 publications

(13 reference statements)

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“…Besides, from our simulation results, it can be seen that about 55% of the DSBs occurred in the euchromatin regions and 45% in the heterochromatin ones for the three X-rays energies considered in the present work. It should also be noted that overlapping phenomena can occur between different foci during microscopy detection [52]. In the end, it appears that the number of foci is experimentally underestimated [53].…”

Section: Discussionmentioning

confidence: 98%

Assessment of Radio-Induced Damage in Endothelial Cells Irradiated with 40 kVp, 220 kVp, and 4 MV X-rays by Means of Micro and Nanodosimetric Calculations

Tang

Bueno

Meylan³

et al. 2019

IJMS

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The objective of this work was to study the differences in terms of early biological effects that might exist between different X-rays energies by using a mechanistic approach. To this end, radiobiological experiments exposing cell monolayers to three X-ray energies were performed in order to assess the yields of early DNA damage, in particular of double-strand breaks (DSBs). The simulation of these irradiations was set in order to understand the differences in the obtained experimental results. Hence, simulated results in terms of microdosimetric spectra and early DSB induction were analyzed and compared to the experimental data. Human umbilical vein endothelial cells (HUVECs) were irradiated with 40, 220 kVp, and 4 MV X-rays. The Geant4 Monte Carlo simulation toolkit and its extension Geant4-DNA were used for the simulations. Microdosimetric calculations aiming to determine possible differences in the variability of the energy absorbed by the irradiated cell population for those photon spectra were performed on 10,000 endothelial cell nuclei representing a cell monolayer. Nanodosimetric simulations were also carried out using a computation chain that allowed the simulation of physical, physico-chemical, and chemical stages on a single realistic endothelial cell nucleus model including both heterochromatin and euchromatin. DNA damage was scored in terms of yields of prompt DSBs per Gray (Gy) and per giga (109) base pair (Gbp) and DSB complexity was derived in order to be compared to experimental data expressed as numbers of histone variant H2AX (γ-H2AX) foci per cell. The calculated microdosimetric spread in the irradiated cell population was similar when comparing between 40 and 220 kVp X-rays and higher when comparing with 4 MV X-rays. Simulated yields of induced DSB/Gy/Gbp were found to be equivalent to those for 40 and 220 kVp but larger than those for 4 MV, resulting in a relative biological effectiveness (RBE) of 1.3. Additionally, DSB complexity was similar between the considered photon spectra. Simulated results were in good agreement with experimental data obtained by IRSN (Institut de radioprotection et de sûreté nucléaire) radiobiologists. Despite differences in photon energy, few differences were observed when comparing between 40 and 220 kVp X-rays in microdosimetric and nanodosimetric calculations. Nevertheless, variations were observed when comparing between 40/220 kVp and 4 MV X-rays. Thanks to the simulation results, these variations were able to be explained by the differences in the production of secondary electrons with energies below 10 keV.

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Section: Discussionmentioning

confidence: 98%

Assessment of Radio-Induced Damage in Endothelial Cells Irradiated with 40 kVp, 220 kVp, and 4 MV X-rays by Means of Micro and Nanodosimetric Calculations

Tang

Bueno

Meylan³

et al. 2019

IJMS

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“…PARTRAC allows simulations with photons and charged particles in a wide energy range: as reference low-LET radiation, we implemented as source the spectrum of X-rays generated by a 220 kVp machine (2 mm Cu filter). Photons were generated randomly from the bottom surface of the cytoplasm, emitted parallel to the z axis, and calculations were stopped when the dose to the nuclear compartment was reached (1, 2 and 5 Gy), as previously done in a pilot study from our group 51 .…”

Section: Methodsmentioning

confidence: 99%

“…The algorithm to reproduce the read-out of γ-H2AX foci in ICC images taken with 2D microscopy (introduced in 51 , referred to as 2D algorithm in the following) proceeds through the following steps:a slice of thickness Δz centered in the middle of the nucleus is selected, and only DSBs within this slice are kept for further analysis;selected DSBs are projected onto the x-y plane, their planar coordinates are stored;a recursive algorithm searches for DSBs falling within a planar distance r , referred to as clustering radius , and it merges them in a single focus;…”

Section: Methodsmentioning

confidence: 99%

Predicting DNA damage foci and their experimental readout with 2D microscopy: a unified approach applied to photon and neutron exposures

Barbieri

Babini

Morini

et al. 2019

Sci Rep

Self Cite

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The consideration of how a given technique affects results of experimental measurements is a must to achieve correct data interpretation. This might be challenging when it comes to measurements on biological systems, where it is unrealistic to have full control (e.g. through a software replica) of all steps in the measurement chain. In this work we address how the effectiveness of different radiation qualities in inducing biological damage can be assessed measuring DNA damage foci yields, only provided that artefacts related to the scoring technique are adequately considered. To this aim, we developed a unified stochastic modelling approach that, starting from radiation tracks, predicts both the induction, spatial distribution and complexity of DNA damage, and the experimental readout of foci when immunocytochemistry coupled to 2D fluorescence microscopy is used. The approach is used to interpret γ-H2AX data for photon and neutron exposures. When foci are reconstructed in the whole cell nucleus, we obtain information on damage characteristics “behind” experimental observations, as the average damage content of a focus. We reproduce how the detection technique affects experimental findings, e.g. contributing to the saturation of foci yields scored at 30 minutes after exposure with increasing dose and to the lack of dose dependence for yields at 24 hours.

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“…Track-structure approaches coupled to a realistic description of the DNA target can be used for the investigation of damage characteristics behind radiation-induced foci (17) . Notably, a saturation of the signal of γ-H2AX foci as a function of increasing dose is observed experimentally, also for low LET reference radiation, occurring at a dose dependent on the readout technique (18) . This might render the RBE concept intrinsically not applicable to this endpoint.…”

Section: Track-structure and Microdosimetry Approachesmentioning

confidence: 91%

“…What is measured in standard immunocytochemistry protocols with 2D microscopy is the yield of foci that can be counted in a projected image of a cell nucleus slice, selected as a result of optical focusing, with a resolution limit dictated by the size of the smallest detectable focus. Experimental results can then be compared to what is obtained starting from a track-structure based prediction of the 3D spatial distribution of damages in the whole cell nucleus, when the readout is also simulated (18) , thus obtaining a quantification of the actual damage behind observations (also carefully considering the time dynamics of foci induction and disappearance).…”

Section: Track-structure and Microdosimetry Approachesmentioning

confidence: 99%

WHAT ROLES FOR TRACK-STRUCTURE AND MICRODOSIMETRY IN THE ERA OF -omics AND SYSTEMS BIOLOGY?

Baiocco

Babini

Barbieri

et al. 2018

Radiation Protection Dosimetry

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Ionizing radiation is a peculiar perturbation when it comes to damage to biological systems: it proceeds through discrete energy depositions, over a short temporal scale and a spatial scale critical for subcellular targets as DNA, whose damage complexity determines the outcome of the exposure. This lies at the basis of the success of track structure (and nanodosimetry) and microdosimetry in radiation biology. However, such reductionist approaches cannot account for the complex network of interactions regulating the overall response of the system to radiation, particularly when effects are manifest at the supracellular level and involve long times. Systems radiation biology is increasingly gaining ground, but the gap between reductionist and holistic approaches is becoming larger. This paper presents considerations on what roles track structure and microdosimetry can have in the attempt to fill this gap, and on how they can be further exploited to interpret radiobiological data and inform systemic approaches.

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MODELLING Γ-H2ax FOCI INDUCTION TO MIMIC LIMITATIONS IN THE SCORING TECHNIQUE

Cited by 8 publications

References 10 publications

Assessment of Radio-Induced Damage in Endothelial Cells Irradiated with 40 kVp, 220 kVp, and 4 MV X-rays by Means of Micro and Nanodosimetric Calculations

Assessment of Radio-Induced Damage in Endothelial Cells Irradiated with 40 kVp, 220 kVp, and 4 MV X-rays by Means of Micro and Nanodosimetric Calculations

Predicting DNA damage foci and their experimental readout with 2D microscopy: a unified approach applied to photon and neutron exposures

WHAT ROLES FOR TRACK-STRUCTURE AND MICRODOSIMETRY IN THE ERA OF -omics AND SYSTEMS BIOLOGY?

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