Radiation Type- and Dose-Specific Transcriptional Responses across Healthy and Diseased Mammalian Tissues

Ionizing radiation (IR) is a genuine genotoxic agent and a major modality in cancer treatment. IR disrupts DNA sequences and exerts mutagenic and/or cytotoxic properties that not only alter critical cellular functions but also impact tissues proximal and distal to the irradiated site. Unveiling the molecular events governing the diverse effects of IR at the cellular and organismal levels is relevant for both radiotherapy and radiation protection. Herein, we address changes in the expression of mammalian genes induced after the exposure of a wide range of tissues to various radiation types with distinct biophysical characteristics. First, we constructed a publicly available database, termed RadBioBase, which will be updated at regular intervals. RadBioBase includes comprehensive transcriptomes of mammalian cells across healthy and diseased tissues that respond to a range of radiation types and doses. Pertinent information was derived from a hybrid analysis based on stringent literature mining and transcriptomic studies. An integrative bioinformatics methodology, including functional enrichment analysis and machine learning techniques, was employed to unveil the characteristic biological pathways related to specific radiation types and their association with various diseases. We found that the effects of high linear energy transfer (LET) radiation on cell transcriptomes significantly differ from those caused by low LET and are consistent with immunomodulation, inflammation, oxidative stress responses and cell death. The transcriptome changes also depend on the dose since low doses up to 0.5 Gy are related with cytokine cascades, while higher doses with ROS metabolism. We additionally identified distinct gene signatures for different types of radiation. Overall, our data suggest that different radiation types and doses can trigger distinct trajectories of cell-intrinsic and cell-extrinsic pathways that hold promise to be manipulated toward improving radiotherapy efficiency and reducing systemic radiotoxicities

Study of Photodynamic therapy combined with antioxidants in prostate cancer cells

135 σ.Εθνικό Μετσόβιο Πολυτεχνείο--Μεταπτυχιακή Εργασία. Διεπιστημονικό-Διατμηματικό Πρόγραμμα Μεταπτυχιακών Σπουδών (Δ.Π.Μ.Σ.) “Μικροσυστήματα και Νανοδιατάξεις”Η Φωτοδυναμική Θεραπεία(ΦΔΘ) είναι μια πολλά υποσχόμενη μέθοδος αντιμετώπισης καλοήθων και μη ασθενειών, οι οποίες εμφανίζουν υπερανάπτυξη ανεπιθύμητων ή μη φυσιολογικών κυττάρων. Η βασική ιδέα στηρίζεται στη χορήγηση μιας μη τοξικής ουσίας, του φωτοευαισθητοποιητή (ΦΕ), ο οποίος συσσωρεύεται επιλεκτικά στα κύτταρα του όγκου, καθιστώντας τα ευάλωτα στην οπτική ακτινοβολία. Όταν ολοκληρωθεί η επωαστική περίοδος με τον ΦΕ, ακολουθεί ακτινοβόληση του όγκου με ορατό φως. Παρουσία οξυγόνου, η ενεργοποίηση του ΦΕ από την ακτινοβολία προκαλεί την παραγωγή ελευθέρων ριζών, οι οποίες είναι κυτταροτοξικές. Αποτέλεσμα της επαγόμενης κυτταροτοξικότητας είναι ο κυτταρικός θάνατος και επομένως η αποδόμηση του πάσχοντος ιστού. Η χρήση της ΦΔΘ στην αντιμετώπιση του καρκίνου θεωρείται ιδιαιτέρως ελκυστική, δεδομένης της θεμελιώδους εξειδίκευσης και επιλεκτικότητας που παρέχει. Στην παρούσα εργασία ερευνάται η συνδυαστική δράση Φωτοδυναμικής Θεραπείας και αντιοξειδωτικών σε ανθρώπινα καρκινικά κύτταρα προστάτη. Ειδικότερα μελετήθηκε η Φωτοδυναμική δράση με δύο σκευάσματα του ΦΕ mTHPC (απλή μορφή-Foscan& λιποσωμική μορφή-Fospeg) και εκχύλισμα προερχόμενο από τον φλοιό του πεύκουPinushalepensis,πλούσιο σε πολυφαινόλες. Η αντιοξειδωτική ή/και προοξειδωτική δράση του εκχυλίσματος μελετήθηκαν συστηματικά. Καθώς παρόμοιες μελέτες οι οποίες συνδυάζουν τη ΦΔΘ με αντιοξειδωτικά σε προοξειδωτικές δόσεις έχουν δείξει συνεργειακή δράση, μελετάται εδώ το συγκριμένο εκχύλισμα. Το κείμενο είναι χωρισμένο σε δύο μέρη, στο θεωρητικό και το πειραματικό.Το θεωρητικό μέρος ξεκινά με την εισαγωγή στη Φωτοδυναμική Θεραπεία όπου παρουσιάζονται αναλυτικά οι δύο κύριες συνιστώσες της, οι φωτοευαισθητοποιητές και τα lasers. Στη συνέχεια εξηγούνται οι μηχανισμοί επίδρασης σε ατομικό, μοριακό, χημικό, υποκυττάριο και κυτταρικό επίπεδο. Ακολούθως αναφέρονται οι μηχανισμοί καταστροφής των όγκων από κυτταρικό επίπεδο μέχρι την ενεργοποίηση του ανοσοποιητικού συστήματος. Τα τελευταία δύο κεφάλαια του πρώτου μέρους αφιερώνονται στη συμβολή της Νανοτεχνολογίας στη ΦΔΘ και τους τρόπους ενίσχυσης της Φωτοδυναμικής δράσης. Στο πειραματικό μέρος περιγράφονται τα πρωτόκολλα που αναπτύχθηκαν, αναφέρονται τα υλικά, οι διατάξεις και οι μέθοδοι που χρησιμοποιήθηκαν και παρουσιάζονται τα αποτελέσματα που εξήχθησαν. Στο τέλος γίνεται συζήτηση για τα συμπεράσματα και τις μελλοντικές προοπτικές. H μελέτη της επίδρασης του εκχυλίσματος του φλοιού του πεύκου Pinus halepensis, πραγματοποιήθηκε με πειράματα βιωσιμότητας, στα ακόλουθα στάδια: Έλεγχος Ι: τοξικότητα σκότους των δύο σκευασμάτων του ΦΕ mTHPC, Foscan και Fospeg. Έλεγχος ΙΙ: επίδραση μόνο της ακτινοβόλησης. Έλεγχος ΙΙΙ: κλασική ΦΔΘ με τα δύο προαναφερθέντα σκευάσματα. Έλεγχος ΙV: προσδιορισμός συγκέντρωσης αντιοξειδωτικού στην οποία ξεκινά η προοξειδωτική δράση. Έλεγχος V: τοξικότητα σκότους αντιοξειδωτικού και Foscan, τοξικότητα σκότους αντιοξειδωτικού και Fospeg. Έλεγχος VI: τοξικότητα αντιοξειδωτικού και ακτινοβόλησης. Έλεγχος VII: πιθανή συνεργειακή δράση ΦΔΘ και αντιοξειδωτικού.Photodynamic therapy is considered to be a promising method for the treatmentof malignant and non-malignant diseases that are generally characterized by overgrowth of unwanted or abnormal cells. The basic concept for this method consists of administering a non-toxic drug, known as a photosensitizer, which selectively accumulates by the tumor cells. After the incubation time, irradiation of the tumor follows, with visible light. In presence of oxygen, the activation of the photosensitizer through irradiation, leads to the generation of cytotoxic species and consequently to cell death and tissue devastation. The use of Photodynamic Therapy as a cancer therapy is considered particularly attractive, due to its fundamental specificity and selectivity. In the present study is investigated the combination of Photodynamic Therapy and the use of antioxidants on human prostate cancer cells. Speciffically the photodynamic action of two formulations of PS mTHPC (simple form – Foscan & liposomal form – Fospeg) and the extract from the pine Pinus halepensis bark, rich in polyphenols, is investigated. The antioxidant and/or prooxidant properties of this extract were studied in depth. Since similar studies compining PDT with antioxidants in prooxidant concentrations have demonstrated synergetic action, this particular extract is investigated. This thesis is divided in two parts, the theoretical and the experimental. The theoretical part includes firstly an introduction to Photodynamic Therapy which details its two main components, the photosensitizers and lasers. Secondly, themechanisms of the PD effect inatomic, molecular, chemical, subcellular and cellular level are explainedfollowed by the mechanisms of tumor destruction at a cellular level leading to the activation of the immune system. The last two chapters of the first part are devoted to the contribution of Nanotechnology in PDT and other ways of enhancing the photodynamic action. The experimental part details the protocols developed during the experiments, the materials, devices and methods that were used. Finally the results are presented followed by the conclusions and a discussion about future prospects. Control I: Dark toxicity of the two formulations of PS mTHPC, Foscαn and Fospeg. Control II: The effects of illumination Control III: Classic PDT using the two previously mentioned formulatios Control IV: Determination of the antioxidants concentration where its prooxidant action begins Control V: Dark toxicity of antioxidant and Foscan, dark toxicity of antioxidant ant Fospeg. Control VI: Toxicity of antioxidant under illumination Control VII: Possible synergetic action of PDT with the antioxidant.Ζαχαρένια Γ. Νικητάκ

In Situ Detection of Complex DNA Damage Using Microscopy: A Rough Road Ahead

Complexity of DNA damage is considered currently one if not the primary instigator of biological responses and determinant of short and long-term effects in organisms and their offspring. In this review, we focus on the detection of complex (clustered) DNA damage (CDD) induced for example by ionizing radiation (IR) and in some cases by high oxidative stress. We perform a short historical perspective in the field, emphasizing the microscopy-based techniques and methodologies for the detection of CDD at the cellular level. We extend this analysis on the pertaining methodology of surrogate protein markers of CDD (foci) colocalization and provide a unique synthesis of imaging parameters, software, and different types of microscopy used. Last but not least, we critically discuss the main advances and necessary future direction for the better detection of CDD, with important outcomes in biological and clinical setups

Using Machine Learning Techniques for Asserting Cellular Damage Induced by High-LET Particle Radiation

This is a study concerning the use of Machine Learning (ML) techniques to ascertain the impacts of particle ionizing radiation (IR) on cell survival and DNA damage. Current empirical models do not always take into account intrinsic complexities and stochastic effects of the interactions of IR and cell populations. Furthermore, these models often lack in biophysical interpretations of the irradiation outcomes. The linear quadratic (LQ) model is a common way to associate the biological response of a cell population with the radiation dose. The parameters of the LQ model are used to extrapolate the relation between the dosage and the survival fraction of a cell population. The goal was to create a ML-based model that predicts the α and β parameters of the well known and established LQ model, along with the key metrics of DNA damage induction. The main target of this effort was, on the one hand, the development of a computational framework that will be able to assess key radiobiophysical quantities, and on the other hand, to provide meaningful interpretations of the outputs. Based on our results, as some metrics of the adaptability and training efficiency, our ML models exhibited 0.18 median error (relative root mean squared error (RRMSE)) in the prediction of the α parameter and errors of less than 0.01 for various DNA damage quantities; the prediction for β exhibited a rather large error of 0.75. Our study is based on experimental data from a publicly available dataset of irradiation studies. All types of complex DNA damage (all clusters), and the number of double-stranded breaks (DSBs), which are widely accepted to be closely related to cell survival and the detrimental biological effects of IR, were calculated using the fast Monte Carlo Damage Simulation software (MCDS). We critically discussed the varying importance of physical parameters such as charge and linear energy transfer (LET); we also discussed the uncertainties of our predictions and future directions, and the dynamics of our approach

A Mathematical Radiobiological Model (MRM) to Predict Complex DNA Damage and Cell Survival for Ionizing Particle Radiations of Varying Quality

Predicting radiobiological effects is important in different areas of basic or clinical applications using ionizing radiation (IR); for example, towards optimizing radiation protection or radiation therapy protocols. In this case, we utilized as a basis the ‘MultiScale Approach (MSA)’ model and developed an integrated mathematical radiobiological model (MRM) with several modifications and improvements. Based on this new adaptation of the MSA model, we have predicted cell-specific levels of initial complex DNA damage and cell survival for irradiation with 11Β, 12C, 14Ν, 16Ο, 20Νe, 40Αr, 28Si and 56Fe ions by using only three input parameters (particle’s LET and two cell-specific parameters: the cross sectional area of each cell nucleus and its genome size). The model-predicted survival curves are in good agreement with the experimental ones. The particle Relative Biological Effectiveness (RBE) and Oxygen Enhancement Ratio (OER) are also calculated in a very satisfactory way. The proposed integrated MRM model (within current limitations) can be a useful tool for the assessment of radiation biological damage for ions used in hadron-beam radiation therapy or radiation protection purposes

GENE EXPRESSION COLLECTIVE DATA ANALYSIS FOR STUDYING THE EFFECTS OF HIGH-LET IONIZING RADIATION: A BIOINFORMATICS APPROACH

The use of high linear energy (LET) ionizing radiation (IR) is progressively being incorporated in radiation therapy (RT) due to its precise dose localization and high relative biological effectiveness. At the same time, these benefits of particle radiation become a high risk for astronauts in the case of inevitable long-term cosmic radiation exposure. Nonetheless, DNA Damage Response (DDR) activated via complex DNA damage on healthy tissue, occurring from such types of radiation, may be instrumental in the induction of various chronic and late effects. A method of approach in understanding the possible underlying mechanisms, is studying alterations in gene expression. To this end we identified Differentially Expressed Genes (DEGs) in IR-exposed healthy human tissue, utilizing microarray data available in public repositories. DEG analysis was conducted using R programming language. Consequently, through functional enrichment and biological network analysis, we identified biological pathways and processes implicated in DDR. By comparing low and high-LET radiation effects, our primary results indicate the induction of a differential biological response for high-LET, like an enhanced inflammatory response.In addition, patterns of DNA repair are substantially distinct compared to low-LET. Finally, we expanded our study in search of possible comorbidities for HZE particle exposure. Pathway enrichment analysis suggests the involvement of mechanisms, tightly correlated with neurodegenerative disorders like amyloid fibrils formation. Regarding blood tissue, platelet activation signaling was found, upholding the connection to cardiovascular disease. This holistic bioinformatics approach revealed cellular trends towards inflammation and degeneration which might be central to the development of late effects of high-LET radiation exposure. It can contribute to the identification of molecular targets for effective countermeasures

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

Bridging Plant and Human Radiation Response and DNA Repair through an In Silico Approach

The mechanisms of response to radiation exposure are conserved in plants and animals. The DNA damage response (DDR) pathways are the predominant molecular pathways activated upon exposure to radiation, both in plants and animals. The conserved features of DDR in plants and animals might facilitate interdisciplinary studies that cross traditional boundaries between animal and plant biology in order to expand the collection of biomarkers currently used for radiation exposure monitoring (REM) in environmental and biomedical settings. Genes implicated in trans-kingdom conserved DDR networks often triggered by ionizing radiation (IR) and UV light are deposited into biological databases. In this study, we have applied an innovative approach utilizing data pertinent to plant and human genes from publicly available databases towards the design of a plant radiation biodosimeter', that is, a plant and DDR gene-based platform that could serve as a REM reliable biomarker for assessing environmental radiation exposure and associated risk. From our analysis, in addition to REM biomarkers, a significant number of genes, both in human and Arabidopsis thaliana, not yet characterized as DDR, are suggested as possible DNA repair players. Last but not least, we provide an example on the applicability of an Arabidopsis thalianabased plant system monitoring the role of cancer-related DNA repair genes BRCA1, BARD1 and PARP1 in processing DNA lesions

Monte Carlo Simulation-Based Calculations of Complex DNA Damage for Incidents of Environmental Ionizing Radiation Exposure

In this paper, we present a useful Monte Carlo (MC)-based methodology that can be utilized to calculate the absorbed dose and the initial levels of complex DNA damage (such as double strand breaks-DSBs) in the case of an environmental ionizing radiation (IR) exposure incident (REI) i.e., a nuclear accident. Our objective is to assess the doses and complex DNA damage by isolating only one component of the total radiation released in the environment after a REI that will affect the health of the exposed individual. More specifically, the radiation emitted by radionuclide 137Cs in the ground (under the individual’s feet). We use a merging of the Monte Carlo N-Particle Transport code (MCNP) with the Monte Carlo Damage Simulation (MCDS) code. The DNA lesions have been estimated through simulations for different surface activities of a 137Cs ground-based γ radiation source. The energy spectrum of the emitted secondary electrons and the absorbed dose in typical mammalian cells have been calculated using the MCNP code, and then these data are used as an input in the MCDS code for the estimation of critical DNA damage levels and types. As a realistic application, the calculated dose is also used to assess the Excess Lifetime Cancer Risk (ELCR) for eight hypothetical individuals, living in different zones around the Chernobyl Nuclear Power Plant, exposed to different time periods at the days of the accident in 1986. We conclude that any exposition of an individual in the near zone of Chernobyl increases the risk of cancer at a moderate to high grade, connected also with the induction of complex DNA damage by radiation. Generally, our methodology has proven to be useful for assessing γ rays-induced complex DNA damage levels of the exposed population, in the case of a REI and for better understanding the long-term health effects of exposure of the population to IR