Cell to Cell Variability of Radiation-Induced Foci: Relation between Observed Damage and Energy Deposition

Abstract

Most studies that aim to understand the interactions between different types of photon radiation and cellular DNA assume homogeneous cell irradiation, with all cells receiving the same amount of energy. The level of DNA damage is therefore generally determined by averaging it over the entire population of exposed cells. However, evaluating the molecular consequences of a stochastic phenomenon such as energy deposition of ionizing radiation by measuring only an average effect may not be sufficient for understanding some aspects of the cellular response to this radiation. The variance among the cells associated with this average effect may also be important for the behaviour of irradiated tissue. In this study, we accurately estimated the distribution of the number of radiation-induced γH2AX foci (RIF) per cell nucleus in a large population of endothelial cells exposed to 3 macroscopic doses of gamma rays from 60Co. The number of RIF varied significantly and reproducibly from cell to cell, with its relative standard deviation ranging from 36% to 18% depending on the macroscopic dose delivered. Interestingly, this relative cell-to-cell variability increased as the dose decreased, contrary to the mean RIF count per cell. This result shows that the dose effect, in terms of the number of DNA lesions indicated by RIF is not as simple as a purely proportional relation in which relative SD is constant with dose. To analyse the origins of this observed variability, we calculated the spread of the specific energy distribution for the different target volumes and subvolumes in which RIF can be generated. Variances, standard deviations and relative standard deviations all changed similarly from dose to dose for biological and calculated microdosimetric values. This similarity is an important argument that supports the hypothesis of the conservation of the association between the number of RIF per nucleus and the specific energy per DNA molecule. This comparison allowed us to calculate a volume of 1.6 μm3 for which the spread of the specific energy distribution could explain the entire variability of RIF counts per cell in an exposed cell population. The definition of this volume may allow to use a microdosimetric quantity to predict heterogeneity in DNA damage. Moreover, this value is consistent with the order of magnitude of the volume occupied by the hydrated sugar-phosphate backbone of the DNA molecule, which is the part of the DNA molecule responsible for strand breaks.

Fractal analysis of chromatin as a potential indicator of human exposures to ionizing radiation

“…neutrons, alpha particles), and the biological damage is overdispersed. But at the present work, we obtained This evidence is supported by Gruel et al (2016) [15], who demonstrated that the variance is not constant with the increase of absorbed dose, even for low LET radiations. There are some hypotheses to explain the heterogeneity of damage among irradiated cells, such as:…”

“…Besides the hypothesis presented, one should considerer the volume of the target, in which the molecular changes induced by radiation will cause the global modification of chromatin architecture [5]. Recent studies have demonstrated that the volume of 1.6 μm 3 correspond exactly to the volume filled by the ligation of sugar phosphate of the nucleotides of DNA, which is precisely the region broken by the interaction with ionizing radiation directly or by free radicals, ions and free electrons produced by the radiolysis of water [15].…”

“…Such result may better explain the variability of damage among cells and the quadratic behavior of the dose-effect curve, which depend on the energy of photons/electrons hitting the cells, what have the probability of hitting the tiny region of the target and effectively producing the damage [13,19,15].…”

“…Based on this discussion and on the design of the experiments of the present study, where the effects of irradiation were not studied in a time sufficient for DNA repair, the late hypothesis is well supported by our results. Even the dose being uniform, there is heterogeneity of FDs probably due to the heterogeneity of hits, energy and fluency of electrons that traverses the region of the target [15].…”

“…that even for low LET radiation (such as gamma rays), but at high dose (3 Gy), the variation cell-to-cell is also overdispersed[13,15].…”