ABSTRACT. Safety and health hazards associated with workers’ exposure to static magnetic field (SMF) belong to priority bioelectromagnetic research. The characteristics of hazards experienced by workers near various types of MRI scanners (such as vertigo or “flying objects”) - studied experimentally in the context of development of MRI medical diagnostics and scanners design – will be summarized [1]. During approximately 30 years of using MRI diagnostics in Poland, due to changes in a design of scanners (0.1T-7T) and accessories used in MRI units, and in a performance of medical diagnostics – it has been observed the trend of a significant (10-fold) increase in the level of actual and cumulative (daily/annual) SMF exposure. Significant dependence of these parameters from the design (ergonomy) of scanners, organization of radiographers work and the understanding of the nature of SMF hazards by the managers of MRI unit was observed. The exposimetric studies showed that the cumulative exposure of radiographers during 10-20 years reaches the level at which published epidemiological studies have found an increased risk of developing arterial hypertension in people with long-term exposure to SMF. The SMF which impact may be sufficient to cause the noticeable biophysical effects induced in the body of the worker moving next to MRI magnet (such as vertigo) have been found near 1.5-7T scanners. In epidemiological studies focused on the long-term exposure to SMF, the exposure evaluation needs attention for the construction of scanners, work organization and type of executed diagnostics (because the significant fraction of radiographers’ work is executed with attention to patients needs, near any type of MRI scanner - it was found that radiographers’ exposure to SMF near medium, high and very high-field scanners (1.5T-7T) may have similar parameters, despite differences in levels of diagnostic SMF applied to patients).

[1] Selected co-authored articles: Int. J Occupational Safety and Ergonomics 2006; Environmentalist 2007; Bioelectromagnetics 2013; Pol J Radiology 2012; Electromagnetic Biol. Med. 2013; Magnetic Resonance Materials in Physics, Biol. Med., 2017; Int. J. Env. Res. and Public Health 2022.

Funding: the National Programme “Improvement of safety and working conditions” (the state services for the ministry of labour in Poland – task 2.SP.10).

ABSTRACT. People respond differently to environmental factors that induce biological effects. Some people tan easily, some get skin burns and, for the rest of people, the tan/burn susceptibility lies somewhere in-between tan and burn. The same is e.g. with pollens. Some suffer, some don’t, even when pollens are very abundant. Such examples could be given very, very many. To every environmental factor, or pollutant, natural or man-made, there is always a group of more sensitive individuals. Currently, one of the hotly debated man-made environmental factors are electromagnetic fields. Part of the population considers themselves as sensitive to the man-made electromagnetic radiation (EMF) – electromagnetic sensitive (EHS). Sensitivity is characterized by a broad variety of non-specific symptoms that the sensitive people claim to experience when exposed to EMF. While the experienced symptoms are currently considered as a real life impairment, the factor causing these symptoms remains unclear. It is logical to consider that the EHS exists. However, the research was unable to reliably establish causality link between EMF exposures and EHS because the psychology-driven provocation studies, asking about subjective feelings, are too crude and prone to bias. In order to gain objective insight into how EMF exposures affect human body it is necessary to perform studies that examine biochemical effects of EMF exposures in human volunteers. Surprisingly, such research is missing. Most of biochemical effects are known from animal and in vitro studies that not necessarily apply to humans in real life exposures. Human studies are urgently needed because the problem of individual sensitivity to EMF might be much larger than the EHS alone. The International Agency for Research on Cancer (IARC) has classified EMF as possible human carcinogen. When the classification of the RF-EMF was performed, important part of the evidence was provided by studies where animals were co-exposed to RF-EMF and some other known carcinogens. Could EMF/RF-EMF exposures potentiate harmful effects of chemicals that humans are exposed to? It is a possibility but we know nothing in this matter because research has not been done.

ABSTRACT. The "non-targeted effects" of ionizing radiation including bystander effects and genomic instability predominate after low dose exposures and dominate response outcomes. These effects are unique in that no classic mutagenic event occurs in the cell showing the effect. In the case of bystander effects, cells which were not in the field affected by the radiation show high levels of mutations, chromosome aberrations, ROS and membrane signaling changes (horizontal transmission of mutations and information which may be damaging) while in the case of genomic instability, generations of cells derived from an irradiated progenitor appear normal but then lethal and non-lethal mutations appear in distant progeny (vertical transmission). The phenomena are characterized by high yields of mutations and distant occurrence of events both in space and time. This precludes a mutator phenotype or other conventional explanation and appears to indicate a generalized form of ROS mediated stress induced mutatgenesis which is well documented in bacteria. The nature of the signal travelling between irradiated and unirradiated cells and organisms is currently unknown but our recent experiments suggest that there may be a physical component such as a vibration wave involved. UV photon mediated transmission has also been documented and the latter mechanisms can induce the release of exosomes which by themselves can induce bystander effects when added to recipient cells . This presentation will discuss the phenomenology of non-targeted effects both in vitro and in vivo, including recent data suggesting that excitation decay-induced photons in the UVA range lead to exosome release and consequent mitochondrial malfunction and elevated ROS in recipient cells. Photons, calcium, and neurochemicals are important in signal production while the exosome cargo, and cytokine mediated pathways especially TGFb determine response to the signal. By highlighting some key challenges and controversies, concerning the mechanisms and more importantly, the reason these effects exist, current ideas about the wider implications of non-targeted effects in radiation protection, evolution and biology in general will be discussed.

ABSTRACT. The continuous increase of the use and diffusion of technologies making use of radiofrequency electromagnetic field (RF-EMF) is generating a continuous growing concern in both policy authorities and general public, about possible effects and risks on health and environment (1). Although several researchers have been performed to identify non-thermal effects by in vitro studies using various assays on different cells and tissues, the reported results are contradictory (2). This is probably due to high experimental variability, linked to the quality of the EMF exposure systems, the different type of cells, and the not optimized and robust experimental design (3). Moreover, the biological effects related to different modulation (continuous and pulsed waves) of the RF signal is a not yet sufficiently investigated topic (4). In light of these observations, the purpose of the present research project is to investigate non-thermal biological effects in human dermal fibroblasts (HDF) exposed to 1.6 GHz using a multi-methodological approach. The HDF will be exposed to continuous (CW) and pulsed (PW) wave for 2 h at the specific absorption rate (SAR) of 0.4 W/kg. The different assays performed in this study will include: γ-H2AX, CREST-micronuclei, chromosome mis-segregation, mitotic spindle, cell cycle, ultrastructural and protein expression analysis. In addition, wide genome expression profile will be evaluated through the RNA sequencing approach by high-throughput NGS. Preliminary results will be presented and discussed.

Acknowledgement: This work is performed in the framework of the European Defence Agency (EDA) project Nº. B 0987 IAP2 GP “Biological Effects of Radiofrequency Electromagnetic Fields (RFBIO)” funded by the Italian MoD.

ABSTRACT. FLASH proton therapy (FLASH-PT) aims to deliver beams of ultra-high dose rates (≥ 40 Gy/s) to induce a normal tissue sparing (FLASH) effect whilst maintaining the anti-tumour effectiveness of conventional dose rates. Compared to conventional radiotherapy techniques, FLASH-PT differ in the beam delivery, dose rate, and fractionation schemes used. Hence, the logistics of the clinical implementation of FLASH-PT, including the treatment planning process, may need to be redefined. This study aims to compare the in silico results produced by a novel FLASH-PT treatment planning system to the results produced for conventional dose rate techniques such as 3DCRT, VMAT, and IMPT. In this study, nine patient cases of bone (3), brain (3), and lung (3) metastases were considered, all previously clinically treated with 3DCRT/VMAT. Treatment plans for FLASH-PT and IMPT were created using a research version of the MIROpt TPS, developed by Ion Beam Applications SA from the open source version of UCLouvain. Conformal FLASH proton beams are produced using monoenergetic spot scanned protons traversing through a conformal energy filter, range shifter, and an aperture. A FLASH dose rate constraint of ≥ 40 Gy/s was included in the plan optimisation. Comparisons of the conformal FLASH-PT plans to the 3DCRT/VMAT and optimised IMPT plans were made using dose volume histograms, boxplots, dose maps, and the Wilcoxon Rank Sum Test (p < 0.05). Conformal FLASH-PT treatment plans, satisfying the FLASH dose rate constraint, exhibited no significant differences when compared to the 3DCRT/VMAT and optimised IMPT plans. This study demonstrates that conformal FLASH-PT treatment plans, showing equivalence to 3DCRT/VMAT and optimised IMPT plans, can be produced. Future work involves the physical verification of the calculated versus delivered doses, to confirm the safety and accuracy needed for the clinical implementation of conformal FLASH-PT.

ABSTRACT. Cancer cells acquired radioresistance after radiotherapy cause recurrence and metastasis. We previously found that 4-methylumbelliferone (4-MU), which is a hyaluronan (HA) synthesis inhibitor can enhance the radiosensitivity of the radioresistant cancer cells (RR cells). In this study, to elucidate the relationship between HA inhibition and radioresistance, we used the RR cell lines (HSC2-R cells and HSC3-R cells), which were established by long term fractionated X-ray irradiation to the oral squamous cell carcinoma (OSCC) cell lines (HSC2 cells and HSC3 cells). In the RR cells, the mRNA expression of HA synthase (HAS) 3 was significantly higher than that of the parental cells, whereas 4-MU significantly suppressed its expression and HA concentration of the culture medium. The intracellular reactive oxygen species (ROS) level of the RR cells, which is an DNA damage inducer, was lower than that of the parental cells, and significantly enhanced by 4-MU compared with non-treatment control cells. The wound healing ability of the RR cells, which represents cell migration ability was significantly suppressed by 4-MU treatment compared with non-treatment control cells. The same effect was confirmed in the induction of epithelial mesenchymal transition by epithelial growth factor. To confirmed that the extracellular HA involved in the radioresistance and cell migration, it was depleted by using streptomyces-hyaluronidase (St-Hyal), which is a HA degradation enzyme. Although St-Hyal administration completely depleted HA in the culture medium, the cell survival and migration ability were not change. Based on these results, the radiosensitization and anti-migration effects of 4-MU may be caused by suppressing the oncogenic and DNA damage protection signaling via HAS3 expression rather than depletion of extracellular HA. Therefore, we investigated whether the HAS3 knockdown mediated by siRNA sensitize the RR cells. The cell survival of HAS3 knockdown cells treated with X-ray irradiation was significantly suppressed compared with their control cells treated with X-ray, suggesting that HAS3 can be a target for radiosensitization in RR cells. In conclusion, our study suggested that HA synthesis inhibition, especially HAS3 inhibition caused by 4-MU treatment enhances the radiosensitization of RR cells.

ABSTRACT. Introduction: Breast cancer Radiotherapy (RT) has significantly improved patient treatment outcomes, however, it also increased cardiovascular mortality due to radiation-induced cardiovascular disease (RICVD). DNA methylation is an epigenetic mechanism which can regulate gene expression that was found to be dysregulated in cardiovascular diseases. We therefore investigated RT-induced DNA methylation alterations in cardiac-relevant genes and the subsequent effects on gene expression. Methods: Female wistar rats received whole heart fractionated X-irradiation (0, 0.04, 0.3 and 1.2 Gy) for 23 daily fractions. Blood was collected at 1.5, 3, 7 and 12 months after irradiation. SureSelect MethylSeq was used to identify differentially methylated genes (DMG) at 1.5 and 7 months and expression of the top cardiac-relevant DMG was evaluated by RT-qPCR. In addition, methylation and expression levels of these selected DMGs was explored in blood of breast cancer patients receiving RT and sampled at diagnosis, after RT and 6 months after RT using Illumina EPIC beadchip array and RT-qPCR, respectively. Results: Overall, radiation induced global hypomethylation. SLMAP showed hypomethylation at 1.5 months which correlated with significantly increased gene regulation in rats. For the other selected genes, (ITPR2, E2F6 and PTPN2), the correlation between methylation status and gene expression was less clear. In breast cancer patients, ITPR2 and E2F6 exhibited differential methylation which was associated with differential expression. Conclusion: Local rat heart irradiation induces alterations in methylation and expression of SLMAP, ITPR2, E2F6 and PTPN2. ITPR2 and E2F6 were differentially expressed in irradiated rats and in breast cancer patients received radiotherapy, therefore present promising candidates as biomarkers for RICVD risk assessment and warrants further research in a bigger cohort.

ABSTRACT. Ionizing radiation (IR) kills cells primarily by generating reactive oxygen species (ROS) and cytotoxic double strand breaks (DSBs) in DNA. Cancer stem cells (CSC) have often higher expression of antioxidant and an effective DNA repair systems that protect them from the effect of IR and lead to relapse, consequently. The main objectives of our project are to establish CSC line with combined deficiencies in two pathways namely oxidative stress response and DNA repair, using inhibitors and/or knock down of the involved genes and to analyze the effect of combined inhibitions with IR (x-rays, protons and carbon ions) on the radioresistant of the cells. U87 glioblastoma cancer stem cells were cultured as spheres in medium in present of Parp1, Nrf2 inhibitors. Counting of the sphere numbers and sizes showed that treatment of the cells with inhibitors decreases the sphere formation and the size of sphere. Our preliminary results showed that pre-treatment of cells 76 hours prior exposure to IR with Parp1i and Nrf2i has no effect on the number of formed spheres as compared to control without inhibitors. However, when the inhibitors were present 76 hours before and 7 days after irradiation, the number of spheres decreased, with Nrf2 inhibitor after carbon ions irradiation and with the double inhibition (Parp1 and Nrf2) after X-ray exposure. Further, to compare the effect of radiation quality, we irradiate the cells (+/- inhibitors) with x-rays, protons and carbon ions. Sphere formation assay showed carbon ions irradiation is more effective in killing cancer stem cells, comparing to the protons and x-rays. Preliminary results indicate that treatment with the inhibitors modify the effect of x-ray more than carbon ions and the analysis of proton irradiated cells are ongoing.

ABSTRACT. Mitochondria play important roles in cellular response to various stresses including ionizing radiation (IR). Previously, we reported that mitochondrial ribosome protein death-associated protein 3 (DAP3) regulates radioresistance of human lung adenocarcinoma cell lines A549 and H1299 [1]. However, the underlying mechanisms by which DAP3 regulates the radioresistance remain to be elucidated. Here, we investigated the role of DAP3 in cell cycle regulation after IR to obtain clues for the mechanism of DAP3-mediated radioresistance in A549 and H1299. We prepared DAP3-knockdown A549 and H1299 cells, and analyzed the effect of DAP3 knockdown on cell cycle regulation after IR. Notably, although DAP3 knockdown hardly affected cell cycle distribution of non-irradiated cells, it attenuated the increase in G2/M population by IR. Therefore, we next analyzed the protein expression of G2/M regulators such as cdc2-Tyr15 phosphorylation (pcdc2). The results showed that DAP3 knockdown suppressed IR-increased pcdc2 expression accompanied by G2/M arrest. Furthermore, the analysis of phosphorylated expression of checkpoint kinase 1 (pchk1)-Ser296 and pchk2-Thr68, which coordinate cell cycle progression, revealed that DAP3 knockdown decreased IR-induced pchk1 and pchk2 expression. Intriguingly, chk1, but not chk2, inhibitor could suppress IR-induced G2/M arrest. Collectively, these results suggest that DAP3 regulates IR-induced G2/M arrest through chk1-cdc2 axis in human lung adenocarcinoma cells. 1Sato et al., international journal of molecular sciences, 22(1), 420. 2021.