RadPhysBio: A Radiobiological Database for the Prediction of Cell Survival upon Exposure to Ionizing Radiation.

Based on the need for radiobiological databases, in this work, we mined experimental ionizing radiation data of human cells treated with X-rays, γ-rays, carbon ions, protons and α-particles, by manually searching the relevant literature in PubMed from 1980 until 2024. In order to calculate normal and tumor cell survival α and ß coefficients of the linear quadratic (LQ) established model, as well as the initial values of the double-strand breaks (DSBs) in DNA, we used WebPlotDigitizer and Python programming language. We also produced complex DNA damage results through the fast Monte Carlo code MCDS in order to complete any missing data. The calculated α/ß values are in good agreement with those valued reported in the literature, where α shows a relatively good association with linear energy transfer (LET), but not ß. In general, a positive correlation between DSBs and LET was observed as far as the experimental values are concerned. Furthermore, we developed a biophysical prediction model by using machine learning, which showed a good performance for α, while it underscored LET as the most important feature for its prediction. In this study, we designed and developed the novel radiobiological 'RadPhysBio' database for the prediction of irradiated cell survival (α and ß coefficients of the LQ model). The incorporation of machine learning and repair models increases the applicability of our results and the spectrum of potential users.

Vibrational spectroscopies for biochemical investigation of X-ray exposure effects on SH-SY5Y human neuroblastoma cells.

Neuroblastoma is the most recurring cancer in childhood and adolescence. The SH-SY5Y neuroblastoma cell line is generally adopted for elaborating new therapeutical approaches and/or elaborating strategies for the prevention of central nervous system disturbances. In fact, it represents a valid model system for investigating in vitro the effects on the brain of X-ray exposure using vibrational spectroscopies that can detect early radiation-induced molecular alterations of potential clinical usefulness. In recent years, we dedicated significant efforts in the use of Fourier-transform and Raman microspectroscopy techniques for characterizing such radiation-induced effects on SH-SY5Y cells by examining the contributions from different cell components (DNA, proteins, lipids, and carbohydrates) to the vibrational spectra. In this review, we aim at revising and comparing the main results of our studies to provide a wide outlook of the latest outcomes and a framework for future radiobiology research using vibrational spectroscopies. A short description of our experimental approaches and data analysis procedures is also reported.

A Sphingolipidomic Profiling Approach for Comparing X-ray-Exposed and Unexposed HepG2 Cells.

An analytical method based on tandem mass spectrometry-shotgun is presently proposed to obtain sphingolipidomic profiles useful for the characterization of lipid extract from X-ray-exposed and unexposed hepatocellular carcinoma cells (HepG2). To obtain a targeted lipidic profile from a specific biological system, the best extraction method must be identified before instrumental analysis. Accordingly, four different classic lipid extraction protocols were compared in terms of efficiency, specificity, and reproducibility. The performance of each procedure was evaluated using the Fourier-transform infrared spectroscopic technique; subsequently, the quality of extracts was estimated using electrospray ionization tandem mass spectrometry. The selected procedure based on chloroform/methanol/water was successfully used in mass spectrometry-based shotgun sphingolipidomics, allowing for evaluation of the response of cells to X-ray irradiation, the most common anticancer therapy. Using a relative quantitative approach, the changes in the sphingolipid profiles of irradiated cell extracts were demonstrated, confirming that lipidomic technologies are also useful tools for studying the key sphingolipid role in regulating cancer growth during radiotherapy.

Identification of a BRCA1-mRNA splicing complex required for efficient DNA repair and maintenance of genomic stability.

Mutations within BRCA1 predispose carriers to a high risk of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through the assembly of multiple protein complexes involved in DNA repair, cell-cycle arrest, and transcriptional regulation. Here, we report the identification of a DNA damage-induced BRCA1 protein complex containing BCLAF1 and other key components of the mRNA-splicing machinery. In response to DNA damage, this complex regulates pre-mRNA splicing of a number of genes involved in DNA damage signaling and repair, thereby promoting the stability of these transcripts/proteins. Further, we show that abrogation of this complex results in sensitivity to DNA damage, defective DNA repair, and genomic instability. Interestingly, mutations in a number of proteins found within this complex have been identified in numerous cancer types. These data suggest that regulation of splicing by the BRCA1-mRNA splicing complex plays an important role in the cellular response to DNA damage.

Could Polyphenols Really Be a Good Radioprotective Strategy?

Currently, radiotherapy is one of the most effective strategies to treat cancer. However, deleterious toxicity against normal cells indicate for the need to selectively protect them. Reactive oxygen and nitrogen species reinforce ionizing radiation cytotoxicity, and compounds able to scavenge these species or enhance antioxidant enzymes (e.g., superoxide dismutase, catalase, and glutathione peroxidase) should be properly investigated. Antioxidant plant-derived compounds, such as phenols and polyphenols, could represent a valuable alternative to synthetic compounds to be used as radio-protective agents. In fact, their dose-dependent antioxidant/pro-oxidant efficacy could provide a high degree of protection to normal tissues, with little or no protection to tumor cells. The present review provides an update of the current scientific knowledge of polyphenols in pure forms or in plant extracts with good evidence concerning their possible radiomodulating action. Indeed, with few exceptions, to date, the fragmentary data available mostly derive from in vitro studies, which do not find comfort in preclinical and/or clinical studies. On the contrary, when preclinical studies are reported, especially regarding the bioactivity of a plant extract, its chemical composition is not taken into account, avoiding any standardization and compromising data reproducibility.

The RNA processing factors THRAP3 and BCLAF1 promote the DNA damage response through selective mRNA splicing and nuclear export.

mRNA splicing and export plays a key role in the regulation of gene expression, with recent evidence suggesting an additional layer of regulation of gene expression and cellular function through the selective splicing and export of genes within specific pathways. Here we describe a role for the RNA processing factors THRAP3 and BCLAF1 in the regulation of the cellular DNA damage response (DDR) pathway, a key pathway involved in the maintenance of genomic stability and the prevention of oncogenic transformation. We show that loss of THRAP3 and/or BCLAF1 leads to sensitivity to DNA damaging agents, defective DNA repair and genomic instability. Additionally, we demonstrate that this phenotype can be at least partially explained by the role of THRAP3 and BCLAF1 in the selective mRNA splicing and export of transcripts encoding key DDR proteins, including the ATM kinase. Moreover, we show that cancer associated mutations within THRAP3 result in deregulated processing of THRAP3/BCLAF1-regulated transcripts and consequently defective DNA repair. Taken together, these results suggest that THRAP3 and BCLAF1 mutant tumors may be promising targets for DNA damaging chemotherapy.

Multivariate Analysis of Difference Raman Spectra of the Irradiated Nucleus and Cytoplasm Region of SH-SY5Y Human Neuroblastoma Cells.

Previous works showed that spatially resolved Raman spectra of cytoplasm and nucleus region of single cells exposed to X-rays evidence different features. The present work aims to introduce a new approach to profit from these differences to deeper investigate X-ray irradiation effects on single SH-SY5Y human neuroblastoma cells. For this aim, Raman micro-spectroscopy was performed in vitro on single cells after irradiation by graded X-ray doses (2, 4, 6, 8 Gy). Spectra from nucleus and cytoplasm regions were selectively acquired. The examination by interval Principal Component Analysis (i-PCA) of the difference spectra obtained by subtracting each cytoplasm-related spectrum from the corresponding one detected at the nucleus enabled us to reveal the subtle modifications of Raman features specific of different spatial cell regions. They were discussed in terms of effects induced by X-ray irradiation on DNA/RNA, lipids, and proteins. The proposed approach enabled us to evidence some features not outlined in previous investigations.

Graphene-Based Raman Spectroscopy for pH Sensing of X-rays Exposed and Unexposed Culture Media and Cells.

Graphene provides a unique way of sensing the local pH level of substances on the micrometric scale, with important implications for the monitoring of cellular metabolic activities where proton excretion could occur. Accordingly, an innovative biosensing approach for the quantification of the pH value of biological fluids, to be used also with small amounts of fluids, was realized and tested. It is based on the use of micro-Raman spectroscopy to detect the modifications of the graphene doping level induced by the contact of the graphene with the selected fluids. The approach was preliminarily tested on aqueous solutions of known pH values. It was then used to quantify the pH values of cell culture media directly exposed to different doses of X-ray radiation and to media exposed to X-ray-irradiated cells. The Raman response of cells placed on graphene layers was also examined.

Seed Priming by Low-Dose Radiation Improves Growth of Lactuca sativa and Valerianella locusta.

Valerian salad and lettuce are edible species that are easy to grow rapidly, and have traits useful for commercial purposes. The consumption of these species is increasing worldwide for their nutritional properties. Seed germination and seedling development are critical stages in the life cycle of plants. Seed priming, including the use of high-energy radiation, is a set of techniques based on the idea that low stress levels stimulate plant responses, thereby improving seed germination and plant growth. In this study, we evaluated in hydroponic culture (i) the germination performance; (ii) morphological traits; and (iii) antioxidant and phenol contents at different endpoints in Lactuca sativa and Valerianella locusta that were developed from seeds exposed to X-rays (1 Gy and 10 Gy doses). Under radiation, biomass production increased in both species, especially in lettuce, where also a reduction in the mean germination time occurred. Radiation increased the level of phenols during the first growth weeks, under both doses for lettuce, and only 1 Gy was required for valerian salad. The species-specific responses observed in this research suggest that the use of radiations in seed priming needs to be customized to the species.

Impact on the Transcriptome of Proton Beam Irradiation Targeted at Healthy Cardiac Tissue of Mice.

Proton beam therapy is considered a step forward with respect to electromagnetic radiation, thanks to the reduction in the dose delivered. Among unwanted effects to healthy tissue, cardiovascular complications are a known long-term radiotherapy complication. The transcriptional response of cardiac tissue from xenografted BALB/c nude mice obtained at 3 and 10 days after proton irradiation covering both the tumor region and the underlying healthy tissue was analyzed as a function of dose and time. Three doses were used: 2 Gy, 6 Gy, and 9 Gy. The intermediate dose had caused the greatest impact at 3 days after irradiation: at 2 Gy, 219 genes were differently expressed, many of them represented by zinc finger proteins; at 6 Gy, there were 1109, with a predominance of genes involved in energy metabolism and responses to stimuli; and at 9 Gy, there were 105, mainly represented by zinc finger proteins and molecules involved in the regulation of cardiac function. After 10 days, no significant effects were detected, suggesting that cellular repair mechanisms had defused the potential alterations in gene expression. The nonlinear dose-response curve indicates a need to update the models built on photons to improve accuracy in health risk prediction. Our data also suggest a possible role for zinc finger protein genes as markers of proton therapy efficacy.

Evaluation of morpho-physiological responses and genotoxicity in Eruca sativa (Mill.) grown in hydroponics from seeds exposed to X-rays.

Due to its potential applications in cultivated plants, ionizing radiation (IR) and its effect on organisms is increasingly studied. Here we measured the effects of ionizing radiation on Eruca sativa by analyzing plants from irradiated seeds (1 and 10 Gy) grown in hydroponics. We measured several morpho-physiological traits and genotoxicity. Radiation stress induced a noticeable variability of the morpho-physiological traits highlighting decreased plant vigor. Shoot length and leaf number were significantly higher in 1 Gy-treated samples, whereas root length was significantly higher in 10 Gy treated plants. Stomata number significantly increased with IR dose, whereas both pigment and Rubisco content decreased under radiation stress. Phenol content significantly increased in 1 Gy treated samples, otherwise from total antioxidants, which were not different from control. Most results could find a feasible explanation in a hormesis-like pattern and in a decreased plant vigor under radiation stress. IR induced genotoxic damage, evaluated by ISSR markers, in 15 day old leaves; specifically, a severe decrease in the genome template stability was observed. However, a partial recovery occurred after 2 weeks, especially under the lowest dose (i.e., 1 Gy), suggesting that DNA damage detection and repair mechanisms are active. Pigment content and genotoxic damage may serve as proxies for evaluating plant responses to IR stress, since they show univocal dose-dependent trends. The use of more checkpoints for analyses and more doses over a wider range, as well as the focus on different metabolites, could help elucidate plant response in terms of morpho-physiological changes.

Differential Radiomodulating Action of Olea europaea L. cv. Caiazzana Leaf Extract on Human Normal and Cancer Cells: A Joint Chemical and Radiobiological Approach.

The identification of a natural compound with selectively differential radiomodulating activity would arguably represent a valuable asset in the striving quest for widening the therapeutic window in cancer radiotherapy (RT). To this end, we fully characterized the chemical profile of olive tree leaf polyphenols from the Caiazzana cultivar (OLC), autochthonous to the Campania region (Italy), by ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HR-MS). Oleacein was the most abundant molecule in the OLC. Two normal and two cancer cells lines were X-ray-irradiated following 24-h treatment with the same concentration of the obtained crude extract and were assessed for their radioresponse in terms of micronucleus (MN) induction and, for one of the normal cell lines, of premature senescence (PS). Irradiation of pre-treated normal cells in the presence of the OLC reduced the frequency of radiation-induced MN and the onset of PS. Conversely, the genotoxic action of ionising radiation was exacerbated in cancer cells under the same experimental conditions. To our knowledge, this is the first report on the dual action of a polyphenol-rich olive leaf extract on radiation-induced damage. If further confirmed, these findings may be pre-clinically relevant and point to a substance that may potentially counteract cancer radioresistance while reducing RT-associated normal tissue toxicity.

New Discoveries in Radiation Science.

This series of 16 articles (8 original articles and 8 reviews) was written by internationally recognized scientists attending the 44th Congress of the European Radiation Research Society (Pécs, Hungary) [...].

The Proton-Boron Reaction Increases the Radiobiological Effectiveness of Clinical Low- and High-Energy Proton Beams: Novel Experimental Evidence and Perspectives.

Protontherapy is a rapidly expanding radiotherapy modality where accelerated proton beams are used to precisely deliver the dose to the tumor target but is generally considered ineffective against radioresistant tumors. Proton-Boron Capture Therapy (PBCT) is a novel approach aimed at enhancing proton biological effectiveness. PBCT exploits a nuclear fusion reaction between low-energy protons and 11B atoms, i.e. p+11B→ 3α (p-B), which is supposed to produce highly-DNA damaging α-particles exclusively across the tumor-conformed Spread-Out Bragg Peak (SOBP), without harming healthy tissues in the beam entrance channel. To confirm previous work on PBCT, here we report new in-vitro data obtained at the 62-MeV ocular melanoma-dedicated proton beamline of the INFN-Laboratori Nazionali del Sud (LNS), Catania, Italy. For the first time, we also tested PBCT at the 250-MeV proton beamline used for deep-seated cancers at the Centro Nazionale di Adroterapia Oncologica (CNAO), Pavia, Italy. We used Sodium Mercaptododecaborate (BSH) as 11B carrier, DU145 prostate cancer cells to assess cell killing and non-cancer epithelial breast MCF-10A cells for quantifying chromosome aberrations (CAs) by FISH painting and DNA repair pathway protein expression by western blotting. Cells were exposed at various depths along the two clinical SOBPs. Compared to exposure in the absence of boron, proton irradiation in the presence of BSH significantly reduced DU145 clonogenic survival and increased both frequency and complexity of CAs in MCF-10A cells at the mid- and distal SOBP positions, but not at the beam entrance. BSH-mediated enhancement of DNA damage response was also found at mid-SOBP. These results corroborate PBCT as a strategy to render protontherapy amenable towards radiotherapy-resilient tumor. If coupled with emerging proton FLASH radiotherapy modalities, PBCT could thus widen the protontherapy therapeutic index.

Cooperative biological effects between ionizing radiation and other physical and chemical agents.

Exposure to ionizing radiation (IR), at environmentally and therapeutically relevant doses or as a result of diagnostics or accidents, causes cyto- and genotoxic damage. However, exposure to IR alone is a rare event as it occurs in spatial and temporal combination with several physico-chemical agents. Some of these are of known noxiousness, as is the case with chemical compounds at high dose, hence additive/synergistic effects can be expected or have been demonstrated. Conversely, the cellular toxicity of other agents, such as non-ionizing electromagnetic fields (EMFs), is only presumed and their short- and long-term cooperation on IR-induced damage remains undetermined. In this review, we shall examine evidence in support of the interplay between spatially and/or temporally related environmentally relevant stressors. In vitro or animal-based studies as well as epidemiological surveys have generally examined the combined action of no more than a couple of known or potentially DNA-damaging agents. Moreover, most existing research mainly focused on short-term effects of combined exposures. Hence, it is important that quantitative research addresses the issue of the possible cooperation between chronic exposure to environmental trace contaminants and exposure to EMFs, examining not only the modulation of damage acutely induced by IR but also long-term genome stability.

Raman spectroscopy monitoring of MCF10A cells irradiated by protons at clinical doses.

PURPOSE: Proton therapy has been recently proposed as a radiotherapy form for breast cancer treatment in view of its potentially decreased normal-tissue toxicity compared with conventional photon-based radiotherapy. However, the risks for the healthy tissue cannot be completely eliminated. In the present study, the suitability of Raman spectroscopy to monitor the radiosensitivity of normal cells exposed to clinical proton beam was investigated. MATERIALS AND METHODS: MCF10A normal human breast cells were irradiated at two different proton doses: 0.5 Gy and 4 Gy. They were fixed immediately after irradiation and measured by means of Raman spectroscopy technique. The obtained data were analyzed both by evaluating the intensity ratio of specific Raman spectral peaks and through Multivariate Distance Matrix Regression technique. RESULTS: Certain Raman peaks associated with DNA showed a systematic suppression at both dose levels. In particular, the intensity of a Raman peak at 784 cm-1, related to a stretching mode inside the phosphate group of DNA, is very sensitive to the proton beam exposure, even at the lowest investigated dose. Therefore, it could be considered as a spectral marker of cytogenetic damage. CONCLUSIONS: The obtained results are encouraging for the future of Raman spectroscopy in radiobiology research, particularly for improving risk assessment in the field of proton radiotherapy. Specifically, these findings validate Raman spectroscopy to measure biological response in human breast cells exposed to standard proton therapy doses used in clinical setting.

Proton-irradiated breast cells: molecular points of view.

Breast cancer (BC) is the most common cancer in women, highly heterogeneous at both the clinical and molecular level. Radiation therapy (RT) represents an efficient modality to treat localized tumor in BC care, although the choice of a unique treatment plan for all BC patients, including RT, may not be the best option. Technological advances in RT are evolving with the use of charged particle beams (i.e. protons) which, due to a more localized delivery of the radiation dose, reduce the dose administered to the heart compared with conventional RT. However, few data regarding proton-induced molecular changes are currently available. The aim of this study was to investigate and describe the production of immunological molecules and gene expression profiles induced by proton irradiation. We performed Luminex assay and cDNA microarray analyses to study the biological processes activated following irradiation with proton beams, both in the non-tumorigenic MCF10A cell line and in two tumorigenic BC cell lines, MCF7 and MDA-MB-231. The immunological signatures were dose dependent in MCF10A and MCF7 cell lines, whereas MDA-MB-231 cells show a strong pro-inflammatory profile regardless of the dose delivered. Clonogenic assay revealed different surviving fractions according to the breast cell lines analyzed. We found the involvement of genes related to cell response to proton irradiation and reported specific cell line- and dose-dependent gene signatures, able to drive cell fate after radiation exposure. Our data could represent a useful tool to better understand the molecular mechanisms elicited by proton irradiation and to predict treatment outcome.

Effectiveness of monoenergetic and spread-out bragg peak carbon-ions for inactivation of various normal and tumour human cell lines.

This work aimed at measuring cell-killing effectiveness of monoenergetic and Spread-Out Bragg Peak (SOBP) carbon-ion beams in normal and tumour cells with different radiation sensitivity. Clonogenic survival was assayed in normal and tumour human cell lines exhibiting different radiosensitivity to X- or gamma-rays following exposure to monoenergetic carbon-ion beams (incident LET 13-303 keV/microm) and at various positions along the ionization curve of a therapeutic carbon-ion beam, corresponding to three dose-averaged LET (LET(d)) values (40, 50 and 75 keV/microm). Chinese hamster V79 cells were also used. Carbon-ion effectiveness for cell inactivation generally increased with LET for monoenergetic beams, with the largest gain in cell-killing obtained in the cells most radioresistant to X- or gamma-rays. Such an increased effectiveness in cells less responsive to low LET radiation was found also for SOBP irradiation, but the latter was less effective compared with monoenergetic ion beams of the same LET. Our data show the superior effectiveness for cell-killing exhibited by carbon-ion beams compared to lower LET radiation, particularly in tumour cells radioresistant to X- or gamma-rays, hence the advantage of using such beams in radiotherapy. The observed lower effectiveness of SOBP irradiation compared to monoenergetic carbon beam irradiation argues against the radiobiological equivalence between dose-averaged LET in a point in the SOBP and the corresponding monoenergetic beams.

Ultrasound-assisted aqueous extraction, LC-MS/MS analysis and radiomodulating capability of autochthonous Italian sweet cherry fruits.

The current cancer treatment scenario lacks drugs acting as both radiosensitizer and radioprotector agents. In this context, the radiomodulatory properties exerted by an aqueous extract from the fruits of the Italian Prunus avium cv. Della Recca (PaDRw) were investigated. The extract, obtained through an environmentally-friendly ultrasound-assisted extraction, seemed to act as a radioprotector at lower tested doses (25 and 50 µg mL-1) and a radiosensitizer at 400 and 500 µg mL-1 dose levels towards the neuroblastoma SH-SY5Y cell line, irradiated with four graded X-ray doses (0, 0.5, 2, and 4 Gy). The fractionation of PaDRw by Amberlite XAD-4 non-ionic polymeric resin, coupled to LC-UV-MS/MS techniques, proved to be efficient also in the disclosure of lower constituents. About 63% of the whole PaDRw extract was constituted of hexitol, followed by fructose (∼22.8%) and glucose (∼10.7%). Chlorogenic acids and flavonoids, which accounted only for ∼2.2%, were hypothesized to be the main actors in PaDRw-induced radiomodulation.

Gene expression profiling of breast cancer cell lines treated with proton and electron radiations.

OBJECTIVE: Technological advances in radiation therapy are evolving with the use of hadrons, such as protons, indicated for tumors where conventional radiotherapy does not give significant advantages or for tumors located in sensitive regions, which need the maximum of dose-saving of the surrounding healthy tissues. The genomic response to conventional and non-conventional linear energy transfer exposure is a poor investigated topic and became an issue of radiobiological interest. The aim of this work was to analyze and compare molecular responses in term of gene expression profiles, induced by electron and proton irradiation in breast cancer cell lines. METHODS: We studied the gene expression profiling differences by cDNA microarray activated in response to electron and proton irradiation with different linear energy transfer values, among three breast cell lines (the tumorigenic MCF7 and MDA-MB-231 and the non-tumorigenic MCF10A), exposed to the same sublethal dose of 9 Gy. RESULTS: Gene expression profiling pathway analyses showed the activation of different signaling and molecular networks in a cell line and radiation type-dependent manner. MCF10A and MDA-MB-231 cell lines were found to induce factors and pathways involved in the immunological process control. CONCLUSION: Here, we describe in a detailed way the gene expression profiling and pathways activated after electron and proton irradiation in breast cancer cells. Summarizing, although specific pathways are activated in a radiation type-dependent manner, each cell line activates overall similar molecular networks in response to both these two types of ionizing radiation. Advances in knowledge: In the era of personalized medicine and breast cancer target-directed intervention, we trust that this study could drive radiation therapy towards personalized treatments, evaluating possible combined treatments, based on the molecular characterization.