Ionizing Radiation and Complex DNA Damage: From Prediction to Detection Challenges and Biological Significance

Biological responses to ionizing radiation (IR) have been studied for many years, generally showing the dependence of these responses on the quality of radiation, i.e., the radiation particle type and energy, types of DNA damage, dose and dose rate, type of cells, etc. There is accumulating evidence on the pivotal role of complex (clustered) DNA damage towards the determination of the final biological or even clinical outcome after exposure to IR. In this review, we provide literature evidence about the significant role of damage clustering and advancements that have been made through the years in its detection and prediction using Monte Carlo (MC) simulations. We conclude that in the future, emphasis should be given to a better understanding of the mechanistic links between the induction of complex DNA damage, its processing, and systemic effects at the organism level, like genomic instability and immune responses

Detection of clustred DNA damage in human cells after exposure to ionizing radiation

Η έκθεση σε ιοντίζουσα ακτινοβολία (Ι.Α.) επάγει βλάβες στο κυτταρικό DNA, οι οποίες παρουσιάζουν ποικιλία και αρκετές φορές αυξημένη πολυπλοκότητα. Οι ομαδοποιημένες (σύνθετες) βλάβες του DNA και κυρίως οι δίκλωνες θραύσεις θεωρούνται ως οι περισσότερο επιβλαβείς, καθώς αν δεν επιδιορθωθούν μπορεί να οδηγήσουν σε κρίσιμες συνέπειες για την κυτταρική επιβίωση και αυξημένο κίνδυνο μεταλλάξεων και καρκινογένεσης. Η απόκριση των κυττάρων στις επαγόμενες βλάβες πραγματοποιείται με ενεργοποίηση ενός πολύπλοκου μηχανισμού που τις ανιχνεύει και τις επιδιορθώνει (μονοπάτι απόκρισης σε βλάβη του DNΑ). Στα σημεία των δίκλωνων θραύσεων η ιστόνη Η2ΑΧ φωσφορυλιώνεται άμεσα. Η φωσφορυλιωμένη ιστόνη γ-Η2ΑΧ, αποτελεί καίριας σημασίας παράγοντα στην απόκριση σε βλάβη του DNA και η οπτικοποίησή της με ανοσοφθορισμό (εστίες γ-Η2ΑΧ) επιτρέπει την εκτίμηση της βλάβης, μιας και υπάρχει σχέση αναλογίας μεταξύ του αριθμού των εστιών και των δίκλωνων θραύσεων. Ως εκ τούτου, οι εστίες γ-Η2ΑΧ μπορούν να χρησιμοποιηθούν ως βιοδείκτες της βλάβης, ενώ η σταδιακή μείωσή τους με το πέρας του χρόνου θεωρείται ένδειξη της επιδιόρθωσής της. Σε αυτή την εργασία μελετήθηκε η παρουσία των εστιών γ-Η2ΑΧ σε ανθρώπινα λεμφοκύτταρα περιφερικού αίματος, εκτεθειμένα ex vivo σε δόσεις από 0.2 έως 2 Gy ακτινοβολίας-γ και σε καρκινικά κύτταρα MCF-7 εκτεθειμένα σε δόσεις από 0.5 έως 4 Gy. Τα αποτελέσματα των πειραμάτων υπέδειξαν μια γραμμική συσχέτιση μεταξύ του αριθμού των επαγόμενων εστιών και της δόσης. Στην περίπτωση των λεμφοκυττάρων κρίθηκε σκόπιμη και η μελέτη της επιδιόρθωσης των βλαβών, η οποία αποκάλυψε μια εκθετική μείωση του αριθμού των επαγόμενων εστιών συναρτήσει του χρόνου μετά την ακτινοβόληση και πλήρη επιδιόρθωση των βλαβών λίγες ώρες αργότερα. Τέλος, έγινε προσπάθεια αυτόματης καταμέτρησης των εστιών με το λογισμικό Jcount και σύγκρισης των αποτελεσμάτων με την προηγούμενη μέθοδο. Τα αποτελέσματα της διπλωματικής εργασίας ενισχύουν την αντίληψη πως η γ-Η2ΑΧ μπορεί να χρησιμοποιηθεί ως βιοδοσίμετρο, αλλά και ως βιοδείκτης ακτινοευαισθησίας.Ionizing radiation (IR) - induced DNA damage is diverse and sometimes considerably complex. Clustered (complex) DNA damages, especially double strand breaks (DSBs), are considered to be the most deleterious DNA lesions, which, if left unrepaired, may lead to serious consequences for cell survival, potential mutations and carcinogenesis through chromosomal instability. Cellular response to DNA damage starts with the activation of a complex mechanism, developed to detect and repair such lesions (DNA damage response pathway - DDR). Upon DNA DSB induction, the histone H2AX becomes rapidly phosphorylated. This modified form, γ-Η2ΑΧ, is a key factor for DDR (DSB repair) and its visualization by immunofluorescence (γ-H2AX foci) allows the assessment of DNA damage, as there is a direct relationship between foci number and DSB. Thus, γ-H2AX foci serve as a sensitive marker of DNA DSB induction, whilst reduction in foci number hours after exposure to IR is an evidence of DNA damage repair. In this study the presence of γ-H2AX foci is investigated in human peripheral blood lymphocytes exposed ex vivo to γ-rays, in a dose range of 0.2 to 2 Gy, as well as in MCF-7 cells exposed in a dose range of 0.5 to 4 Gy. Experimental results show that γ-H2AX foci induce linearly with radiation dose. In the case of lymphocytes, the investigation of DNA damage repair was deemed useful and therefore the analysis of the loss of γ-H2AX foci at various times after γ-ray exposure, reveals that the level of γ-H2AX foci reduces with time after irradiation and eventually a few hours is the time needed for full recovery of foci induction. Lastly, there was an effort for automatic focus counting with Jcount software. Results from this study amplify evidence that γ-H2AX may be useful for biodosimetry, as well as for the assessment of individual radiosensitivity.Ιφιγένεια Β. Μαυραγάν

Complex DNA Damage: A Route to Radiation-Induced Genomic Instability and Carcinogenesis

Cellular effects of ionizing radiation (IR) are of great variety and level, but they are mainly damaging since radiation can perturb all important components of the cell, from the membrane to the nucleus, due to alteration of different biological molecules ranging from lipids to proteins or DNA. Regarding DNA damage, which is the main focus of this review, as well as its repair, all current knowledge indicates that IR-induced DNA damage is always more complex than the corresponding endogenous damage resulting from endogenous oxidative stress. Specifically, it is expected that IR will create clusters of damage comprised of a diversity of DNA lesions like double strand breaks (DSBs), single strand breaks (SSBs) and base lesions within a short DNA region of up to 15–20 bp. Recent data from our groups and others support two main notions, that these damaged clusters are: (1) repair resistant, increasing genomic instability (GI) and malignant transformation and (2) can be considered as persistent “danger” signals promoting chronic inflammation and immune response, causing detrimental effects to the organism (like radiation toxicity). Last but not least, the paradigm shift for the role of radiation-induced systemic effects is also incorporated in this picture of IR-effects and consequences of complex DNA damage induction and its erroneous repair

Key mechanisms involved in ionizing radiation-induced systemic effects. A current review

International audienceOrganisms respond to physical, chemical and biological threats by a potent inflammatory response, aimed at preserving tissue integrity and restoring tissue homeostasis and function. Systemic effects in an organism refer to an effect or phenomenon which originates at a specific point and can spread throughout the body affecting a group of organs or tissues. Ionizing radiation (IR)-induced systemic effects arise usually from a local exposure of an organ or part of the body. This stress induces a variety of responses in the irradiated cells/tissues, initiated by the DNA damage response and DNA repair (DDR/R), apoptosis or immune response, including inflammation. Activation of this IR-response (IRR) system, especially at the organism level, consists of several subsystems and exerts a variety of targeted and non-targeted effects. Based on the above, we believe that in order to understand this complex response system better one should follow a 'holistic' approach including all possible mechanisms and at all organization levels. In this review, we describe the current status of knowledge on the topic, as well as the key molecules and main mechanisms involved in the 'spreading' of the message throughout the body or cells. Last but not least, we discuss the danger-signal mediated systemic immune effects of radiotherapy for the clinical setup

Complex DNA Damage: A Route to Radiation-Induced Genomic Instability and Carcinogenesis

Cellular effects of ionizing radiation (IR) are of great variety and level, but they are mainly damaging since radiation can perturb all important components of the cell, from the membrane to the nucleus, due to alteration of different biological molecules ranging from lipids to proteins or DNA. Regarding DNA damage, which is the main focus of this review, as well as its repair, all current knowledge indicates that IR-induced DNA damage is always more complex than the corresponding endogenous damage resulting from endogenous oxidative stress. Specifically, it is expected that IR will create clusters of damage comprised of a diversity of DNA lesions like double strand breaks (DSBs), single strand breaks (SSBs) and base lesions within a short DNA region of up to 15-20 bp. Recent data from our groups and others support two main notions, that these damaged clusters are: (1) repair resistant, increasing genomic instability (GI) and malignant transformation and (2) can be considered as persistent “danger” signals promoting chronic inflammation and immune response, causing detrimental effects to the organism (like radiation toxicity). Last but not least, the paradigm shift for the role of radiation-induced systemic effects is also incorporated in this picture of IR-effects and consequences of complex DNA damage induction and its erroneous repair

Investigation of the Biological Implications of Complex DNA Damage with Emphasis in Cancer Radiotherapy through a Systems Biology Approach.

Different types of DNA lesions forming in close vicinity, create clusters of damaged sites termed as "clustered/complex DNA damage" and they are considered to be a major challenge for DNA repair mechanisms resulting in significant repair delays and induction of genomic instability. Upon detection of DNA damage, the corresponding DNA damage response and repair (DDR/R) mechanisms are activated. The inability of cells to process clustered DNA lesions efficiently has a great impact on the normal function and survival of cells. If complex lesions are left unrepaired or misrepaired, they can lead to mutations and if persistent, they may lead to apoptotic cell death. In this in silico study, and through rigorous data mining, we have identified human genes that are activated upon complex DNA damage induction like in the case of ionizing radiation (IR) and beyond the standard DNA repair pathways, and are also involved in cancer pathways, by employing stringent bioinformatics and systems biology methodologies. Given that IR can cause repair resistant lesions within a short DNA segment (a few nm), thereby augmenting the hazardous and toxic effects of radiation, we also investigated the possible implication of the most biologically important of those genes in comorbid non-neoplastic diseases through network integration, as well as their potential for predicting survival in cancer patients

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

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

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

<p>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 <i>in situ</i> 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.</p