A Systematic Review on the In Vivo Studies on Radiofrequency (100 kHz-300 GHz) Electromagnetic Field Exposure and Co-Carcinogenesis.

In this systematic review, the potential role of in vivo RF-EMF exposure combined with the administration of well-known carcinogens in tumor promotion/progression is assessed. A total of 25 papers were included in the review. Each paper was assessed for Risk of Bias and for the attribution of the quality category. A meta-analysis was conducted on 18 studies, analyzing data for nine different organs/tumors to assess the potential increased risk for the onset of tumors as well as the effects on survival. A descriptive review was performed for the remaining seven eligible papers. In most cases, the results of the meta-analysis did not reveal a statistically significant difference in tumor onset between the sham and co-exposed samples. There was a numerically small increase in the risk of malignant tumors observed in the kidney and liver, as well as benign lung tumors. The level of evidence for health effects indicated "inadequate" evidence for an association between in vivo co-exposure to RF-EMF and known carcinogens and the onset of malignant or benign tumors in most of the analyzed tissues. Nevertheless, the limited number of eligible papers/studies for most of the analyzed tissues suggests that these results cannot be considered definitively conclusive.

Comparing the effects of irradiation with protons or photons on neonatal mouse brain: Apoptosis, oncogenesis and hippocampal alterations.

BACKGROUND AND PURPOSE: Medulloblastoma (MB) is a common primary brain cancer in children. Proton therapy in pediatric MB is intensively studied and widely adopted. Compared to photon, proton radiations offer potential for reduced toxicity due to the characteristic Bragg Peak at the end of their path in tissue. The aim of this study was to compare the effects of irradiation with the same dose of protons or photons in Patched1 heterozygous knockout mice, a murine model predisposed to cancer and non-cancer radiogenic pathologies, including MB and lens opacity. MATERIALS AND METHODS: TOP-IMPLART is a pulsed linear proton accelerator for proton therapy applications. We compared the long-term health effects of 3 Gy of protons or photons in neonatal mice exposed at postnatal day 2, during a peculiarly susceptible developmental phase of the cerebellum, lens, and hippocampus, to genotoxic stress. RESULTS: Experimental testing of the 5 mm Spread-Out Bragg Peak (SOBP) proton beam, through evaluation of apoptotic response, confirmed that both cerebellum and hippocampus were within the SOBP irradiation field. While no differences in MB induction were observed after irradiation with protons or photons, lens opacity examination confirmed sparing of the lens after proton exposure. Marked differences in expression of neurogenesis-related genes and in neuroinflammation, but not in hippocampal neurogenesis, were observed after irradiation of wild-type mice with both radiation types. CONCLUSION: In-vivo experiments with radiosensitive mouse models improve our mechanistic understanding of the dependence of brain damage on radiation quality, thus having important implications in translational research.

Nanodiamond Effects on Cancer Cell Radiosensitivity: The Interplay between Their Chemical/Physical Characteristics and the Irradiation Energy.

Nanoparticles are being increasingly studied to enhance radiation effects. Among them, nanodiamonds (NDs) are taken into great consideration due to their low toxicity, inertness, chemical stability, and the possibility of surface functionalization. The objective of this study is to explore the influence of the chemical/physical properties of NDs on cellular radiosensitivity to combined treatments with radiation beams of different energies. DAOY, a human radioresistant medulloblastoma cell line was treated with NDs-differing for surface modifications [hydrogenated (H-NDs) and oxidized (OX-NDs)], size, and concentration-and analysed for (i) ND internalization and intracellular localization, (ii) clonogenic survival after combined treatment with different radiation beam energies and (iii) DNA damage and apoptosis, to explore the nature of ND-radiation biological interactions. Results show that chemical/physical characteristics of NDs are crucial in determining cell toxicity, with hydrogenated NDs (H-NDs) decreasing either cellular viability when administered alone, or cell survival when combined with radiation, depending on ND size and concentration, while OX-NDs do not. Also, irradiation at high energy (γ-rays at 1.25 MeV), in combination with H-NDs, is more efficient in eliciting radiosensitisation when compared to irradiation at lower energy (X-rays at 250 kVp). Finally, the molecular mechanisms of ND radiosensitisation was addressed, demonstrating that cell killing is mediated by the induction of Caspase-3-dependent apoptosis that is independent to DNA damage. Identifying the optimal combination of ND characteristics and radiation energy has the potential to offer a promising therapeutic strategy for tackling radioresistant cancers using H-NDs in conjunction with high-energy radiation.

Effects of Radiofrequency Electromagnetic Field (RF-EMF) exposure on pregnancy and birth outcomes: A systematic review of experimental studies on non-human mammals.

BACKGROUND: The World Health Organization is coordinating an international project aimed at systematically reviewing the evidence regarding the association between radiofrequency electromagnetic field (RF-EMF) exposure and adverse health effects. Within the project, 6 topics have been prioritized by an expert group, which include reproductive health outcomes. OBJECTIVES: According to the protocol published in 2021, a systematic review and meta-analyses on the adverse effects of RF-EMF exposure during pregnancy in offspring of experimental animals were conducted. METHODS: Three electronic databases (PubMed, Scopus and EMF Portal) were last searched on September 8 or 17, 2022. Based on predefined selection criteria, the obtained references were screened by two independent reviewers. Studies were included if they met the following criteria: 1) original, sham controlled experimental study on non-human mammals exposed in utero, published in peer-reviewed journals, 2) the experimental RF-EMF exposure was within the frequency range 100 kHz-300 GHz, 3) the effects of RF-EMF exposure on fecundity (litter size, embryonic/fetal losses), on the offspring health at birth (decrease of weight or length, congenital malformations, changes of sex ratio) or on delayed effects (neurocognitive alterations, female infertility or early-onset cancer) were studied. Study characteristics and outcome data were extracted by two reviewers. Risk of bias (RoB) was assessed using the Office of Health Assessment and Translation (OHAT) guidelines. Study results were pooled in a random effects meta-analysis comparing average exposure to no-exposure and in a dose-response meta-analysis using all exposure doses, after exclusion of studies that were rated at "high concern" for RoB. Subgroup analyses were conducted for species, Specific Absorption Rate (SAR) and temperature increase. The certainty of the evidence was assessed using the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) approach. RESULTS: Eighty-eight papers could be included in this review. Effects on fecundity. The meta-analysis of studies on litter size, conducted at a whole-body average SAR of 4.92 W/kg, did not show an effect of RF-EMF exposure (MD 0.05; 95% CI -0.21 to 0.30). The meta-analysis of studies on resorbed and dead fetuses, conducted at a whole-body average SAR of 20.26 W/kg, showed a significant increase of the incidence in RF-EMF exposed animals (OR 1.84; 95% CI 1.27 to 2.66). The results were similar in the dose-response analysis. Effects on the offspring health at birth. The meta-analysis of studies on fetal weight, conducted at a whole-body average SAR of 9.83 W/kg, showed a small decrease in RF-EMF exposed animals (SMD 0.31; 95% CI 0.15 to 0.48). The meta-analysis of studies on fetal length, conducted at a whole-body average SAR of 4.55 W/kg, showed a moderate decrease in length at birth (SMD 0.45; 95% CI 0.07 to 0.83). The meta-analysis of studies on the percentage of fetuses with malformations, conducted at a whole-body average SAR of 6.75 W/kg, showed a moderate increase in RF-EMF exposed animals (SMD -0.45; 95% CI -0.68 to -0.23). The meta-analysis of studies on the incidence of litters with malformed fetuses, conducted at a whole-body average SAR of 16.63 W/kg, showed a statistically significant detrimental RF-EMF effect (OR 3.22; 95% CI 1.9 to 5.46). The results were similar in the dose-response analyses. Delayed effects on the offspring health. RF-EMF exposure was not associated with detrimental effects on brain weight (SMD 0.10; 95% CI -0.09 to 0.29) and on learning and memory functions (SMD -0.54; 95% CI -1.24 to 0.17). RF-EMF exposure was associated with a large detrimental effect on motor activity functions (SMD 0.79; 95% CI 0.21 to 1.38) and a moderate detrimental effect on motor and sensory functions (SMD -0.66; 95% CI -1.18 to -0.14). RF-EMF exposure was not associated with a decrease of the size of litters conceived by F2 female offspring (SMD 0.08; 95% CI -0.39 to 0.55). Notably, meta-analyses of neurobehavioural effects were based on few studies, which suffered of lack of independent replication deriving from only few laboratories. DISCUSSION: There was high certainty in the evidence for a lack of association of RF-EMF exposure with litter size. We attributed a moderate certainty to the evidence of a small detrimental effect on fetal weight. We also attributed a moderate certainty to the evidence of a lack of delayed effects on the offspring brain weight. For most of the other endpoints assessed by the meta-analyses, detrimental RF-EMF effects were shown, however the evidence was attributed a low or very low certainty. The body of evidence had limitations that did not allow an assessment of whether RF-EMF may affect pregnancy outcomes at exposure levels below those eliciting a well-known adverse heating impact. In conclusion, in utero RF-EMF exposure does not have a detrimental effect on fecundity and likely affects offspring health at birth, based on the meta-analysis of studies in experimental mammals on litter size and fetal weight, respectively. Regarding possible delayed effects of in utero exposure, RF-EMF probably does not affect offspring brain weight and may not decrease female offspring fertility; on the other hand, RF-EMF may have a detrimental impact on neurobehavioural functions, varying in magnitude for different endpoints, but these last findings are very uncertain. Further research is needed on the effects at birth and delayed effects with sample sizes adequate for detecting a small effect. Future studies should use standardized endpoints for testing prenatal developmental toxicity and developmental neurotoxicity (OECD TG 414 and 426), improve the description of the exposure system design and exposure conditions, conduct appropriate dosimetry characterization, blind endpoint analysis and include several exposure levels to better enable the assessment of a dose-response relationship. PROTOCOL REGISTRATION AND PUBLICATION: The protocol was published in Pacchierotti et al., 2021 and registered in PROSPERO CRD42021227746 (https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=227746).

In Vivo Radiobiological Investigations with the TOP-IMPLART Proton Beam on a Medulloblastoma Mouse Model.

Protons are now increasingly used to treat pediatric medulloblastoma (MB) patients. We designed and characterized a setup to deliver proton beams for in vivo radiobiology experiments at a TOP-IMPLART facility, a prototype of a proton-therapy linear accelerator developed at the ENEA Frascati Research Center, with the goal of assessing the feasibility of TOP-IMPLART for small animal proton therapy research. Mice bearing Sonic-Hedgehog (Shh)-dependent MB in the flank were irradiated with protons to test whether irradiation could be restricted to a specific depth in the tumor tissue and to compare apoptosis induced by the same dose of protons or photons. In addition, the brains of neonatal mice at postnatal day 5 (P5), representing a very small target, were irradiated with 6 Gy of protons with two different collimated Spread-Out Bragg Peaks (SOBPs). Apoptosis was visualized by immunohistochemistry for the apoptotic marker caspase-3-activated, and quantified by Western blot. Our findings proved that protons could be delivered to the upper part while sparing the deepest part of MB. In addition, a comparison of the effectiveness of protons and photons revealed a very similar increase in the expression of cleaved caspase-3. Finally, by using a very small target, the brain of P5-neonatal mice, we demonstrated that the proton irradiation field reached the desired depth in brain tissue. Using the TOP-IMPLART accelerator we established setup and procedures for proton irradiation, suitable for translational preclinical studies. This is the first example of in vivo experiments performed with a "full-linac" proton-therapy accelerator.

In Vivo Studies on Radiofrequency (100 kHz-300 GHz) Electromagnetic Field Exposure and Cancer: A Systematic Review.

The increasing exposure of the human population to radiofrequency electromagnetic fields has increased concern about its possible health effects. The aim of this systematic review is to provide an update of the state of the research on this topic, through a quantitative analysis, to assess the increased risk of tumor incidence in laboratory animals (rodents) without limitations of species, strain, sex or genotype. The review was conducted according to the PRISMA guideline and individual studies were assessed by referring to the OHAT Risk of Bias Rating Tool for Human and Animal Studies. A total of 27 studies were considered eligible for the evaluation of tumor incidence; a meta-analysis was carried out on 23 studies to assess the possible increased risk of both malignant and benign tumors onset at the systemic level or in different organs/tissues. A significant association between exposure to RF and the increased/decreased risk of cancer does not result from the meta-analysis in most of considered tissues. A significant increased/decreased risk can be numerically observed only in heart, CNS/brain, and intestine for malignant tumors. Nevertheless, the assessment of the body of evidence attributes low or inadequate evidence for an association between RF exposure and the onset of neoplasm in all tissues.

Effects of Ultra-Short Pulsed Electric Field Exposure on Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most common brain cancer in adults. GBM starts from a small fraction of poorly differentiated and aggressive cancer stem cells (CSCs) responsible for aberrant proliferation and invasion. Due to extreme tumor heterogeneity, actual therapies provide poor positive outcomes, and cancers usually recur. Therefore, alternative approaches, possibly targeting CSCs, are necessary against GBM. Among emerging therapies, high intensity ultra-short pulsed electric fields (PEFs) are considered extremely promising and our previous results demonstrated the ability of a specific electric pulse protocol to selectively affect medulloblastoma CSCs preserving normal cells. Here, we tested the same exposure protocol to investigate the response of U87 GBM cells and U87-derived neurospheres. By analyzing different in vitro biological endpoints and taking advantage of transcriptomic and bioinformatics analyses, we found that, independent of CSC content, PEF exposure affected cell proliferation and differentially regulated hypoxia, inflammation and P53/cell cycle checkpoints. PEF exposure also significantly reduced the ability to form new neurospheres and inhibited the invasion potential. Importantly, exclusively in U87 neurospheres, PEF exposure changed the expression of stem-ness/differentiation genes. Our results confirm this physical stimulus as a promising treatment to destabilize GBM, opening up the possibility of developing effective PEF-mediated therapies.

Protocol for a systematic review of the in vivo studies on radiofrequency (100 kHz-300 GHz) electromagnetic field exposure and cancer.

BACKGROUND: An Italian project aims to review the scientific literature on the possible carcinogenicity of radiofrequency (100 kHz-300 GHz) electromagnetic field (RF-EMF) exposure. The ENEA team has to carry out a systematic review of the in vivo studies on this topic. OBJECTIVES: Development of a protocol for a systematic review (meta-analysis included) to investigate the potential carcinogenic risk following RF-EMF in vivo exposure to doses above or within legal limits. The aims of this review are (1) to provide a descriptive and, if possible, a quantitative summary of the results of the examined RF-EMF in vivo studies, together with an assessment of the consistency of observations and of the causes of heterogeneity, and (2) to assess the weight of evidence to support or refute the hypothesis of carcinogenic effects caused by RF-EMF exposure and to draw conclusions about the potential for carcinogenicity of RF-EMF exposure. METHODS: We will search for relevant studies in electronic academic databases and in the reference list of selected papers and reviews on the topic, including the descriptive reviews on RF-EMF carcinogenic effect carried out by international panels of experts since 2011. The following elements of the PECO question were defined: experimental studies on rodents of both sexes, all ages and species, all genetic backgrounds (Population) exposed to RF-EMF alone, or in combination with other physical or chemical agents (Exposure); only studies reporting outcome data in exposed and sham control groups (Comparison); and all types of cancer with all tumor-related outcome measures (Outcome) will be included. Only peer-reviewed articles written in English will be considered without limit in the publication date. Eligibility criteria were defined for papers to be included. A risk of bias assessment will be performed using a tool specifically developed for animal studies. A meta-analysis will be performed, if feasible, for all outcome measures; for subgroup analysis, a minimum of 3 studies per subgroup will be required. If meta-analysis will not be possible, a narrative synthesis of the results will be reported. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42020191105 HIGHLIGHTS: An Italian collaborative research agreement aims to review the scientific literature on the possible carcinogenicity of RF-EMF (100 kHz - 300 GHz). The ENEA team will systematically review and, if possible, meta-analyse estimates the effects of in vivo exposure to RF-EMF exposure on cancer. The ENEA group is a multidisciplinary team of researchers with a consolidated experience both in carcinogenicity experiments and radiofrequency dosimetric assessment. The proposed protocol uses the NTP OHAT Approach for Systematic Review as an organizing framework. The proposed protocol aims to lead to the first systematic review providing a strength of evidence assessment on this topic.

Biological effects of ultrashort electric pulses in a neuroblastoma cell line: the energy density role.

BACKGROUND: Despite the numerous literature results about biological effects of electromagnetic field (EMF) exposure, the interaction mechanisms of these fields with organisms are still a matter of debate. Extremely low frequency (ELF) MFs can modulate redox homeostasis and we showed that 24 h exposure to 50 Hz-1 mT has a pro-oxidant effect and effects on the epigenome of SH-SY5Y cells, decreasing miR-34b/c expression through the hypermethylation of their promoter. METHODS: Here, we investigated the role of the electromagnetic deposited energy density (ED) during exposures lasting 24 h to 1 mT amplitude MFs at a frequency of 50 Hz in inducing the above mentioned effects. To this end, we delivered ultrashort electric pulses, in the range of microsecond and nanosecond duration, with the same ED of the previously performed magnetic exposure to SH-SY5Y cells. Furthermore, we explored the effect of higher deposited energy densities. Analysis of i) gene and microRNA expression, ii) cell morphology, iii) reactive oxygen species (ROS) generation, and iv) apoptosis were carried out. RESULTS: We observed significant changes in egr-1 and c-fos expression at very low deposited ED levels, but no change of the ROS production, miR-34b/c expression, nor the appearance of indicators of apoptosis. We thus sought investigating changes in egr-1 and c-fos expression caused by ultrashort electric pulses at increasing deposited ED levels. The pulses with the higher deposited ED caused cell electroporation and even other morphological changes such as cell fusion. The changes in egr-1 and c-fos expression were more intense, but, again, no change of the ROS production, miR-34b/c expression, nor apoptosis induction was observed. CONCLUSIONS: These results, showing that extremely low levels of electric stimulation (never investigated until now) can cause transcriptional changes, also reveal the safety of the electroporating pulses used in biomedical applications and open up the possibility to further therapeutic applications of this technology.

Long-Term Effects of Ionizing Radiation on the Hippocampus: Linking Effects of the Sonic Hedgehog Pathway Activation with Radiation Response.

Radiation therapy represents one of the primary treatment modalities for primary and metastatic brain tumors. Although recent advances in radiation techniques, that allow the delivery of higher radiation doses to the target volume, reduce the toxicity to normal tissues, long-term neurocognitive decline is still a detrimental factor significantly affecting quality of life, particularly in pediatric patients. This imposes the need for the development of prevention strategies. Based on recent evidence, showing that manipulation of the Shh pathway carries therapeutic potential for brain repair and functional recovery after injury, here we evaluate how radiation-induced hippocampal alterations are modulated by the constitutive activation of the Shh signaling pathway in Patched 1 heterozygous mice (Ptch1+/-). Our results show, for the first time, an overall protective effect of constitutive Shh pathway activation on hippocampal radiation injury. This activation, through modulation of the proneural gene network, leads to a long-term reduction of hippocampal deficits in the stem cell and new neuron compartments and to the mitigation of radio-induced astrogliosis, despite some behavioral alterations still being detected in Ptch1+/- mice. A better understanding of the pathogenic mechanisms responsible for the neural decline following irradiation is essential for identifying prevention measures to contain the harmful consequences of irradiation. Our data have important translational implications as they suggest a role for Shh pathway manipulation to provide the therapeutic possibility of improving brain repair and functional recovery after radio-induced injury.

Radiation dose from medical imaging in end stage renal disease patients: a Nationwide Italian Survey.

BACKGROUND AND OBJECTIVES: End stage renal disease (ESRD) patients are exposed to the risk of ionizing radiation during repeated imaging studies. The variability in diagnostic imaging policies and the accompanying radiation doses across various renal units is still unknown. We studied this variability at the centre level and quantified the associated radiation doses at the patient level. METHODS: Fourteen Italian nephrology departments enrolled 739 patients on haemodialysis and 486 kidney transplant patients. The details of the radiological procedures performed over one year were recorded. The effective doses and organ doses of radiation were estimated for each patient using standardized methods to convert exposure parameters into effective and organ doses RESULTS: Computed tomography (CT) was the major contributor (> 77%) to ionizing radiation exposure. Among the haemodialysis and kidney transplant patients, 15% and 6% were in the high (≥ 20 mSv per year) radiation dose groups, respectively. In haemodialysis patients, the most exposed organs were the liver (16 mSv), the kidney (15 mSv) and the stomach (14 mSv), while the uterus (6.2 mSv), the lung (5.7 mSv) and the liver (5.5 mSv) were the most exposed in kidney transplant patients. The average cumulative effective dose (CED) of ionizing radiation among centres in this study was highly variable both in haemodialysis (from 6.4 to 18.8 mSv per patient-year; p = 0.018) and even more so in kidney transplant (from 0.6 to 13.7 mSv per patient-year; p = 0.002) patients. CONCLUSIONS: Radiation exposure attributable to medical imaging is high in distinct subgroups of haemodialysis and transplant patients. Furthermore, there is high inter-centre variability in radiation exposure, suggesting that nephrology units have substantially different clinical policies for the application of diagnostic imaging studies.

Microsecond Pulsed Electric Fields: An Effective Way to Selectively Target and Radiosensitize Medulloblastoma Cancer Stem Cells.

PURPOSE: Cancer stem cells constitute an endless reserve for the maintenance and progression of tumors, and they could be the reason for conventional therapy failure. New therapeutic strategies are necessary to specifically target them. In this context, microsecond pulsed electric fields have been selected to expose D283Med cells, a human medulloblastoma cell line resulted to be rich in cancer stem cells, and normal human astrocytes. METHODS: We analyzed in vitro different endpoints at different times after microsecond pulsed electric field exposure, such as permeabilization, reactive oxygen species generation, cell viability/proliferation, cell cycle, and clonogenicity, as well as the expression of different genes involved in cell cycle, apoptosis, and senescence. Furthermore, the response of D283Med cells exposed to microsecond pulsed electric fields was validated in vivo in a heterotopic mouse xenograft model. RESULTS: Our in vitro results showed that a specific pulse protocol (ie, 0.3 MV/m, 40 µs, 5 pulses) was able to induce irreversible membrane permeabilization and apoptosis exclusively in medulloblastoma cancer stem cells. In the surviving cells, reactive oxygen species generation was observed, together with a transitory G2/M cell-cycle arrest with a senescence-associated phenotype via the upregulation of GADD45A. In vivo results, after pulsed electric field exposure, demonstrated a significant tumor volume reduction with no eradication of tumor mass. In conjunction, we verified the efficacy of electric pulse pre-exposure followed by ionizing irradiation in vivo to enable complete inhibition of tumor growth. CONCLUSIONS: Our data reveal novel therapeutic options for the targeting of medulloblastoma cancer stem cells, indicating nonionizing pulsed electric field pre-exposure as an effective means to overcome the radioresistance of cancer stem cells.

Exposure of the SH-SY5Y Human Neuroblastoma Cells to 50-Hz Magnetic Field: Comparison Between Two-Dimensional (2D) and Three-Dimensional (3D) In Vitro Cultures.

We here characterize the response to the extremely low-frequency (ELF) magnetic field (MF, 50 Hz, 1 mT) of SH-SY5Y human neuroblastoma cells, cultured in a three-dimensional (3D) Alvetex® scaffold compared to conventional two-dimensional (2D) monolayers. We proved that the growing phenotype of proliferating SH-SY5Y cells is not affected by the culturing conditions, as morphology, cell cycle distribution, proliferation/differentiation gene expression of 3D-cultures overlap what reported in 2D plates. In response to 72-h exposure to 50-Hz MF, we demonstrated that no proliferation change and apoptosis activation occur in both 2D and 3D cultures. Consistently, no modulation of Ki67, MYCN, CCDN1, and Nestin, of invasiveness and neo-angiogenesis-controlling genes (HIF-1α, VEGF, and PDGF) and of microRNA epigenetic signature (miR-21-5p, miR-222-3p and miR-133b) is driven by ELF exposure. Conversely, intracellular glutathione content and SOD1 expression are exclusively impaired in 3D-culture cells in response to the MF, whereas no change of such redox modulators is observed in SH-SY5Y cells if grown on 2D monolayers. Moreover, ELF-MF synergizes with the differentiating agents to stimulate neuroblastoma differentiation into a dopaminergic (DA) phenotype in the 3D-scaffold culture only, as growth arrest and induction of p21, TH, DAT, and GAP43 are reported in ELF-exposed SH-SY5Y cells exclusively if grown on 3D scaffolds. As overall, our findings prove that 3D culture is a more reliable experimental model for studying SH-SY5Y response to ELF-MF if compared to 2D conventional monolayer, and put the bases for promoting 3D systems in future studies addressing the interaction between electromagnetic fields and biological systems.

Expression of a Functional Recombinant Human Glycogen Debranching Enzyme (hGDE) in N. benthamiana Plants and in Hairy Root Cultures.

BACKGROUND: Glycogen storage disease type III (GSDIII, Cori/Forbes disease) is a metabolic disorder due to the deficiency of the Glycogen Debranching Enzyme (GDE), a large monomeric protein (about 176 kDa) with two distinct enzymatic activities: 4-α-glucantransferase and amylo-α-1,6-glucosidase. Several mutations along the amylo-alpha-1,6-glucosidase,4-alphaglucanotransferase (Agl) gene are associated with loss of enzymatic activity. The unique treatment for GSDIII, at the moment, is based on diet. The potential of plants to manufacture exogenous engineered compounds for pharmaceutical purposes, from small to complex protein molecules such as vaccines, antibodies and other therapeutic/prophylactic entities, was shown by modern biotechnology through "Plant Molecular Farming". OBJECTIVE AND METHODS: In an attempt to develop novel protein-based therapeutics for GSDIII, the Agl gene, encoding for the human GDE (hGDE) was engineered for expression as a histidinetagged GDE protein both in Nicotiana benthamiana plants by a transient expression approach, and in axenic hairy root in vitro cultures (HR) from Lycopersicum esculentum and Beta vulgaris. RESULTS: In both plant-based expression formats, the hGDE protein accumulated in the soluble fraction of extracts. The plant-derived protein was purified by affinity chromatography in native conditions showing glycogen debranching activity. CONCLUSION: These investigations will be useful for the design of a new generation of biopharmaceuticals based on recombinant GDE protein that might represent, in the future, a possible therapeutic option for GSDIII.

A combined ANXA2-NDRG1-STAT1 gene signature predicts response to chemoradiotherapy in cervical cancer.

BACKGROUND: A better understanding of locally advanced cervical cancer (LACC) is mandatory for further improving the rates of disease control, since a significant proportion of patients still fail to respond or undergo relapse after concurrent chemoradiation treatment (CRT), and survival for these patients has generally remained poor. METHODS: To identify specific markers of CRT response, we compared pretreatment biopsies from LACC patients with pathological complete response (sensitive) with those from patients showing macroscopic residual tumor (resistant) after neoadjuvant CRT, using a proteomic approach integrated with gene expression profiling. The study of the underpinning mechanisms of chemoradiation response was carried out through in vitro models of cervical cancer. RESULTS: We identified annexin A2 (ANXA2), N-myc downstream regulated gene 1 (NDRG1) and signal transducer and activator of transcription 1 (STAT1) as biomarkers of LACC patients' responsiveness to CRT. The dataset collected through qPCR on these genes was used as training dataset to implement a Random Forest algorithm able to predict the response of new patients to this treatment. Mechanistic investigations demonstrated the key role of the identified genes in the balance between death and survival of tumor cells. CONCLUSIONS: Our results define a predictive gene signature that can help in cervical cancer patient stratification, thus providing a useful tool towards more personalized treatment modalities.

Evidences of plasma membrane-mediated ROS generation upon ELF exposure in neuroblastoma cells supported by a computational multiscale approach.

BACKGROUND: Molecular mechanisms of interaction between cells and extremely low frequency magnetic fields (ELF-MFs) still represent a matter of scientific debate. In this paper, to identify the possible primary source of oxidative stress induced by ELF-MF in SH-SY5Y human neuroblastoma cells, we estimated the induced electric field and current density at the cell level. METHODS: We followed a computational multiscale approach, estimating the local electric field and current density from the whole sample down to the single cell level. The procedure takes into account morphological modeling of SH-SY5Y cells, arranged in different topologies. Experimental validation has been carried out: neuroblastoma cells have been treated with Diphenyleneiodonium (DPI) -an inhibitor of the plasma membrane enzyme NADPH oxidase (Nox)- administered 24 h before exposure to 50 Hz (1 mT) MF. RESULTS: Macroscopic and microscopic dosimetric evaluations suggest that increased current densities are induced at the plasma membrane/extra-cellular medium interface; identifying the plasma membrane as the main site of the ELF-neuroblastoma cell interaction. The in vitro results provide an experimental proof that plasma membrane Nox exerts a key role in the redox imbalance elicited by ELF, as DPI treatment reverts the generation of reactive oxygen species induced by ELF exposure. GENERAL SIGNIFICANCE: Microscopic current densities induced at the plasma membrane are likely to play an active physical role in eliciting ELF effects related to redox imbalance. Multiscale computational dosimetry, supported by an in vitro approach for validation, is proposed as the innovative and rigorous paradigm to unveil mechanisms underlying the complex ELF-MF interactions.

Bioproduction of a Therapeutic Vaccine Against Human Papillomavirus in Tomato Hairy Root Cultures.

Human papillomavirus (HPV) tumor disease is a critical public health problem worldwide, especially in the developing countries. The recognized pathogenic function of E5, E6, and E7 oncoproteins offers the opportunity to devise therapeutic vaccines based on engineered recombinant proteins. The potential of plants to manufacture engineered compounds for pharmaceutical purposes, from small to complex protein molecules, allows the expression of HPV antigens and, possibly, the regulation of immune functions to develop very specific therapies as a reinforcement to available nonspecific therapies and preventive vaccination also in developed countries. Among plant-based expression formats, hairy root cultures are a robust platform combining the benefits of eukaryotic plant-based bioreactors, with those typical of cell cultures. In this work, to devise an experimental therapeutic vaccine against HPV, hairy root cultures were used to express a harmless form of the HPV type 16 E7 protein (E7*) fused to SAPKQ, a noncytotoxic form of the saporin protein from Saponaria officinalis, that we had shown to improve E7-specific cell-mediated responses as a fusion E7*-SAPKQ DNA vaccine. Hairy root clones expressing the E7*-SAPKQ candidate vaccine were obtained upon infection of leaf explants of Solanum lycopersicum using a recombinant plant expression vector. Yield was approximately 35.5 µg/g of fresh weight. Mouse immunization with vaccine-containing crude extracts was performed together with immunological and biological tests to investigate immune responses and anticancer activity, respectively. Animals were primed with either E7*-SAPKQ DNA-based vaccine or E7*-SAPKQ root extract-based vaccine and boosted with the same (homologous schedule) or with the other vaccine preparation (heterologous schedule) in the context of TC-1 experimental mouse model of HPV-associated tumor. All the formulations exhibited an immunological response associated to anticancer activity. In particular, DNA as prime and hairy root extract as boost demonstrated the highest efficacy. This work, based on the development of low-cost technologies, highlights the suitability of hairy root cultures as possible biofactories of therapeutic HPV vaccines and underlines the importance of the synergic combination of treatment modalities for future developments in this field.

50-Hz MF does not affect global DNA methylation of SH-SY5Y cells treated with the neurotoxin MPP.

Exposure to extremely low frequency magnetic fields (ELF-MFs) has been associated with an increased risk of neurodegenerative disorders. The underlying mechanisms, however, are still debated. Since epigenetics play a key role in the neurodegenerative process, we investigated whether exposure to ELF-MF (50 Hz, 1 mT) might affect global DNA methylation of SH-SY5Y dopaminergic-like neuroblastoma cells. We assessed the percentage of 5-methylcytosine (5-mC) of three repetitive interspersed sequences (ALU, LINE-1, or SATα), through pyrosequencing analysis. We demonstrated that ELF exposure (up to 72 h) does not induce any change in the methylation pattern of ALU, LINE-1, and SATα in both proliferating and differentiated SH-SY5Y cells. Furthermore, when administered in combination with 1-methyl-4-phenylpyridinium (MPP+ ), a neurotoxin mimicking the Parkinson's Disease (PD) phenotype, ELF-MF exposure does not trigger any modulation in the percentage of 5-mC of the repetitive elements. Our findings demonstrate that exposure to 50-Hz MF does not affect global DNA methylation in proliferating and dopaminergic differentiated SH-SY5Y cells, either under basal culture conditions or under neurotoxic stress. Bioelectromagnetics. 40:33-41, 2019. © 2018 Bioelectromagnetics Society.

50-Hz magnetic field impairs the expression of iron-related genes in the in vitro SOD1G93A model of amyotrophic lateral sclerosis.

PURPOSE: We characterized the response to the extremely low frequency magnetic field (ELF-MF) in an in vitro model of familial Amyotrophic Lateral Sclerosis (fALS), carrying two mutant variants of the superoxide dismutase 1 (SOD1) gene. MATERIALS AND METHODS: SH-SY5Y human neuroblastoma cells, stably over-expressing the wild type, the G93A or the H46R mutant SOD1 cDNA, were exposed to either the ELF-MF (50 Hz, 1 mT) or the sham control field, up to 72 h. Analysis of (i) viability, proliferation and apoptosis, (ii) reactive oxygen species generation, and (iii) assessment of the iron metabolism, were carried out in all clones in response to the MF exposure. RESULTS: We report that 50-Hz MF exposure induces: (i) no change in proliferation and viability; (ii) no modulation of the intracellular superoxide and H2O2 levels; (iii) a significant deregulation in the expression of iron-related genes IRP1, MFRN1 and TfR1, this evidence being exclusive for the SOD1G93A clone and associated with a slight (p = .0512) difference in the total iron content. CONCLUSIONS: 50-Hz MF affects iron homeostasis in the in vitro SOD1G93A ALS model.

Fifty-Hertz Magnetic Field Affects the Epigenetic Modulation of the miR-34b/c in Neuronal Cells.

The exposure to extremely low-frequency magnetic fields (ELF-MFs) has been associated to increased risk of neurodegenerative diseases, although the underlying molecular mechanisms are still undefined. Since epigenetic modulation has been recently encountered among the key events leading to neuronal degeneration, we here aimed at assessing if the control of gene expression mediated by miRNAs, namely miRs-34, has any roles in driving neuronal cell response to 50-Hz (1 mT) magnetic field in vitro. We demonstrate that ELF-MFs drive an early reduction of the expression level of miR-34b and miR-34c in SH-SY5Y human neuroblastoma cells, as well as in mouse primary cortical neurons, by affecting the transcription of the common pri-miR-34. This modulation is not p53 dependent, but attributable to the hyper-methylation of the CpG island mapping within the miR-34b/c promoter. Incubation with N-acetyl-l-cysteine or glutathione ethyl-ester fails to restore miR-34b/c expression, suggesting that miRs-34 are not responsive to ELF-MF-induced oxidative stress. By contrast, we show that miRs-34 control reactive oxygen species production and affect mitochondrial oxidative stress triggered by ELF-MFs, likely by modulating mitochondria-related miR-34 targets identified by in silico analysis. We finally demonstrate that ELF-MFs alter the expression of the α-synuclein, which is specifically stimulated upon ELF-MFs exposure via both direct miR-34 targeting and oxidative stress. Altogether, our data highlight the potential of the ELF-MFs to tune redox homeostasis and epigenetic control of gene expression in vitro and shed light on the possible mechanism(s) producing detrimental effects and predisposing neurons to degeneration.