Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)

Abstract

Detrimental effects of ionizing radiation (IR) are correlated to the varying efficiency of IR to induce complex DNA damage. A double strand break (DSB) can be considered the simpler form of complex DNA damage. These types of damage can consist of DSBs, single strand breaks (SSBs) and/or non-DSB lesions such as base damages and apurinic/apyrimidinic (AP; abasic) sites in different combinations. Enthralling theoretical (Monte Carlo simulations) and experimental evidence suggests an increase in the complexity of DNA damage and therefore repair resistance with linear energy transfer (LET). In this study, we have measured the induction and processing of DSB and non-DSB oxidative clusters using adaptations of immunofluorescence. Specifically, we applied foci colocalization approaches as the most current methodologies for the in situ detection of clustered DNA lesions in a variety of human normal (FEP18-11-T1) and cancerous cell lines of varying repair efficiency (MCF7, HepG2, A549, MO59K/J) and radiation qualities of increasing LET, that is γ-, X-rays 0.3–1 keV/μm, α-particles 116 keV/μm and ³⁶Ar ions 270 keV/μm. Using γ-H2AX or 53BP1 foci staining as DSB probes, we calculated a DSB apparent rate of 5–16 DSBs/cell/Gy decreasing with LET. A similar trend was measured for non-DSB oxidized base lesions detected using antibodies against the human repair enzymes 8-oxoguanine-DNA glycosylase (OGG1) or AP endonuclease (APE1), that is damage foci as probes for oxidized purines or abasic sites, respectively. In addition, using colocalization parameters previously introduced by our groups, we detected an increasing clustering of damage for DSBs and non-DSBs. We also make correlations of damage complexity with the repair efficiency of each cell line and we discuss the biological importance of these new findings with regard to the severity of IR due to the complex nature of its DNA damage.

Keywords:

• Complex DNA damage
• non-DSB clusters
• foci colocalization
• Monte Carlo simulation
• high LET ionizing radiations
• DNA repair

Acknowledgements

The authors acknowledge the valuable help of Dr. Pavel N. Lobachevsky (Sir Peter MacCallum Department of Oncology, The University of Melbourne) for providing the “foci” software analysis JQuantPro and his technical support. The authors would like also to sincerely thank the staff at the heavy ions accelerator facility at the Centre Interdisciplinaire de Recherche Ions Lasers (CIRIL) at GANIL, Caen, France, for dosimetry and support of the heavy ion experiments, especially Dr. Isabelle Testard, Dr. Yannick Satigny, Florent Durantel (GANIL), and all the members of the Cellular Biodiagnostics group at DLR for their help during beam times (Prof. Dr. Christa Baumstark-Khan, Dr. Luis F. Spitta, Sebastian Feles, Claudia Schmitz, Sebastian Diegeler, Bikash Konda, Bernd Henschenmacher). We also sincerely thank Dr. Gabriel Pantelias (NCSR “Demokritos”) for helpful discussions and continuous support. C.E.H. acknowledges the support by the European Union program ENSAR for the travel costs to GANIL.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.