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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.

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.

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.

Highlights • Male infertility and adverse pregnancy outcomes are relevant human health problems. • Radiofrequency electromagnetic fields are widespread in the human environment. • A link between radiofrequency and adverse reproductive outcomes is controversial. • This is the protocol of WHO-funded systematic review and meta-analysis on this issue., Background Radiofrequency Electromagnetic Fields (RF-EMF) at environmental level have been reported to induce adverse effects on the male reproductive system and developing embryos. However, despite the number of experiments conducted since the 1970s, the diversity of testing approaches and exposure conditions, inconsistencies among results, and dosimetric flaws have not yet permitted a solid assessment of the relationship between RF-EMF exposure and such effects, warranting a more systematic and methodologically rigorous approach to the evaluation of available data. Objectives This study aims at evaluating the effects of RF-EMF exposure on male fertility and pregnancy outcomes by a systematic review (SR) of experimental studies, conducted in compliance with international guidelines. The evidence will be organized into three streams: 1) Studies evaluating the impact of RF-EMF on the male reproductive system of experimental mammals; 2) studies evaluating the impact of RF-EMF on human sperm exposed in vitro; 3) studies evaluating the impact of RF-EMF on adverse pregnancy, birth outcomes and delayed effects in experimental mammals exposed in utero. Study eligibility and criteria Eligible studies will include peer-reviewed articles reporting of original results about effects of controlled exposures to RF-EMF in the frequency range 100 kHz–300 GHz on the selected outcomes without any language or year-of-publication restrictions. Eligible studies will be retrieved by calibrated search strings applied to three electronic databases, PubMed, Scopus and EMF Portal and by manual search of the list of references of included papers and published reviews. Study appraisal and synthesis method The internal validity of the studies will be evaluated using the Risk of Bias (RoB) Rating Tool developed by National Toxicology Program/Office of Health Assessment and Translation (NTP/OHAT) integrated with input from the SYRCLE RoB tool. Given sufficient commensurate data, meta-analyses will be performed, otherwise narrative syntheses will be produced. Finally, the certainty of the effects of RF-EMF exposure on male fertility and pregnancy and birth outcomes will be established following GRADE. Funding The study is financially supported by the World Health Organization. Registration OSF Registration DOI https://doi.org/10.17605/OSF.IO/7MUS3; PROSPERO CRD42021227729, CRD42021227746.

The denitrosylase S‐nitrosoglutathione reductase (GSNOR) has been suggested to sustain mitochondrial removal by autophagy (mitophagy), functionally linking S‐nitrosylation to cell senescence and aging. In this study, we provide evidence that GSNOR is induced at the translational level in response to hydrogen peroxide and mitochondrial ROS. The use of selective pharmacological inhibitors and siRNA demonstrates that GSNOR induction is an event downstream of the redox‐mediated activation of ATM, which in turn phosphorylates and activates CHK2 and p53 as intermediate players of this signaling cascade. The modulation of ATM/GSNOR axis, or the expression of a redox‐insensitive ATM mutant influences cell sensitivity to nitrosative and oxidative stress, impairs mitophagy and affects cell survival. Remarkably, this interplay modulates T‐cell activation, supporting the conclusion that GSNOR is a key molecular effector of the antioxidant function of ATM and providing new clues to comprehend the pleiotropic effects of ATM in the context of immune function., Hydrogen peroxide and pro‐oxidant conditions activate ATM via oxidation of Cys2991. The resulting phospho‐signal activates CHK2 and p53 and culminates in enhanced translation of the denitrosylase GSNOR to sustain mitophagy and protect the cells against oxidative stress.

During hematopoiesis it is clearly emerging that microRNAs are integrated with target genes in regulatory circuitries involved in the decisions regarding the ability to self-renew and to generate a differentiated progeny in hematopoietic cells including myeloid cells (1). microRNAs are able to modulate genes expression mainly by tuning the rate of proteins’ synthesis inhibiting the initiation or later stages of translation. A change in the association of an mRNA with polysomes is indicative of changes in its translation state. For instance, a block in translational initiation would result in reduced ribo- some density on the affected mRNA and a shift toward the lower-density fractions of the gradient (2). Prediction algorithms usually provide hundreds of target genes for each microRNA and the identification of reliable target genes is feasible only through single-gene approaches. To identify microRNA-mRNA networks relevant for the transition from myeloblasts to monocytes, we here evaluated the localization of microRNAs and mRNAs in ribosomal/polysomal cell fractions obtained by sucrose density gradient centrifugation from the myeloblastic cell line HL60 induced or not to differentiate by 1,25-dihydroxyvitamin D3 treatment to induce monocyte/macrophage differentiation. The co-localization of miRNAs and predicted target mRNAs in low-density riboso- mal fractions is strongly indicative of their functional interaction. Intersection between mRNAs shifted across the fractions after treatment with putative target genes of modulated microRNAs showed a series of molecular net- works relevant for the monocyte cell fate determination, as for example the post-tran- scriptional regulation of the Polo-like kinase 1 (PLK1) by miR-22-3p and let-7e-5p. The disclosing of new molecular players involved in myeloid cell fate determina- tion paves the way for the identification of new potentially interesting molecular tar- gets for the treatment of acute myeloid leukemia cells., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Background Numerous health benefits have been attributed to the Ginkgo biloba leaf extract (GBLE), one of the most extensively used phytopharmaceutical drugs worldwide. Recently, concerns of the safety of the extract have been raised after a report from US National Toxicology Program (NTP) claimed high doses of GBLE increased liver and thyroid cancer incidence in mice and rats. A safety study has been designed to assess, in a population of elderly residents in nursing homes, clinical and genomic risks associated to GBLE treatment. Methods GiBiEx is a multicentre randomized clinical trial, placebo controlled, double blinded, which compared subjects randomized to twice-daily doses of either 120-mg of IDN 5933 (also known as Ginkgoselect®Plus) or to placebo for a 6-months period. IDN 5933 is extracted from dried leaves and contains 24.3% flavone glycosides and 6.1% of terpene lactones (2.9% bilobalide, 1.38% ginkgolide A, 0.66% ginkgolide B, 1.12% ginkgolide C) as determined by HPLC. The study was completed by 47 subjects, 20 in the placebo group and 27 in the treatment group. Clinical (adverse clinical effect and liver injury) and genomic (micronucleus frequency, comet assay, c-myc, p53, and ctnnb1 expression profile in lymphocytes) endpoints were assessed at the start and at the end of the study. Results No adverse clinical effects or increase of liver injury markers were reported in the treatment group. The frequency of micronuclei [Mean Ratio (MR) = 1.01, 95% Confidence Intervals (95% CI) 0.86–1.18), and DNA breaks (comet assay) (MR = 0.91; 95% CI 0.58–1.43), did not differ in the two study groups. No significant difference was found in the expression profile of the three genes investigated. Conclusions None of the markers investigated revealed a higher risk in the treatment group, supporting the safety of IDN 5933 at doses prescribed and for duration of six months. Trial registration ClinicalTrials.gov Identifier: NCT03004508, December 20, 2016. Trial retrospectively registered. Electronic supplementary material The online version of this article (10.1186/s12906-018-2080-5) contains supplementary material, which is available to authorized users.

Paired cultures of early-passage melanoma cells and melanocytes were established from metastatic lesions and the uninvolved skin of five patients. In this stringent autologous setting, cDNA profiling was used to analyze a subset of 1477 genes selected by the Gene Ontology term 'immune response'. Human Leukocyte Antigen E (HLA-E) was ranked 19th among melanoma-overexpressed genes and was embedded in a transformation signature including its preferred peptide ligand donors HLA-A, HLA-B, HLA-C, and HLA-G. Mostly undetectable in normal skin and 39 nevi (including rare and atypical lesions), HLA-E was detected by immunohistochemistry in 17/30 (57%) and 32/48 (67%) primary and metastatic lesions, respectively. Accordingly, surface HLA-E was higher on melanoma cells than on melanocytes and protected the former (6/6 cell lines) from lysis by natural killer (NK) cells, functionally counteracting co-expressed triggering ligands. Although lacking HLA-E, melanocytes (4/4 cultures) were nevertheless (and surprisingly) fully protected from NK cell lysis. © 2013 John Wiley & Sons A/S.

// Lorena Paris 1, 2, * , Paola Giardullo 3, 4, * , Simona Leonardi 1, * , Barbara Tanno 1 , Roberta Meschini 2 , Eugenia Cordelli 1 , Barbara Benassi 1 , Maria Grazia Longobardi 5 , Alberto Izzotti 5, 6 , Alessandra Pulliero 5 , Mariateresa Mancuso 1 , Francesca Pacchierotti 1 1 Division of Health Protection Technologies, Agenzia Nazionale per le Nuove Tecnologie, l’Energia e lo Sviluppo Economico Sostenibile (ENEA), Rome, Italy 2 Department of Ecological and Biological Sciences, Tuscia University, Viterbo, Italy 3 Department of Radiation Physics, Guglielmo Marconi University, Rome, Italy 4 Department of Sciences, University of Roma Tre, Rome, Italy 5 Department of Health Sciences, University of Genoa, Genoa, Italy 6 IRCCS AOU San Martino IST Genoa, Italy * These authors have contributed equally to this work Correspondence to: Mariateresa Mancuso, e-mail: mariateresa.mancuso@enea.it Francesca Pacchierotti, e-mail: francesca.pacchierotti@enea.it Keywords: transgenerational carcinogenesis, epigenetic inheritance, medulloblastoma, patched1 knockout mice, microRNA Received: July 30, 2015 Accepted: September 21, 2015 Published: October 03, 2015 ABSTRACT The hypothesis of transgenerational induction of increased cancer susceptibility after paternal radiation exposure has long been controversial because of inconsistent results and the lack of a mechanistic interpretation. Here, exploiting Ptch1 heterozygous knockout mice, susceptible to spontaneous and radiation-induced medulloblastoma, we show that exposure of paternal germ cells to 1 Gy X-rays, at the spermatogonial stage, increased by a considerable 1.4-fold the offspring susceptibility to medulloblastoma induced by neonatal irradiation. This effect gained further biological significance thanks to a number of supporting data on the immunohistochemical characterization of the target tissue and preneoplastic lesions (PNLs). These results altogether pointed to increased proliferation of cerebellar granule cell precursors and PNLs cells, which favoured the development of frank tumours. The LOH analysis of tumor DNA showed Ptch1 biallelic loss in all tumor samples, suggesting that mechanisms other than interstitial deletions, typical of radiation-induced medulloblastoma, did not account for the observed increased cancer risk. This data was supported by comet analysis showing no differences in DNA damage induction and repair in cerebellar cells as a function of paternal irradiation. Finally, we provide biological plausibility to our results offering evidence of a possible epigenetic mechanism of inheritance based on radiation-induced changes of the microRNA profile of paternal sperm.

Low-dose exposure of ionizing radiation triggers cell-to-cell communications and tissue interplay alterations. These alterations may play a fundamental role in non-cancer effects, overwhelming the theory of the DNA centric approach. Neither the mechanisms of these effects are fully understood nor is it possible to dissect the real incidence of quality and quantity of incident radiation during in vivo exposure, overall for particulate high-linear energy transfer (LET) radiation. Moreover, the knowledge of particulate high-LET radiation is mandatory for the human deep space exploration and to gain efficiency in the dose/effect ratio for radiotherapy. The aim of this minireview was to describe an integrated system approach to the radiation biology of cosmic interest which could be set up in the framework of a future Sino-Italy cooperation among participating laboratories. We propose, in particular, to deliver X-rays and neutron irradiation at ENEAFNG (Frascati, Italy) and heavy ion irradiation at IMPCAS (Lanzhou, China) to in vivo models. The integrated system approach will focus on the correlation between the quality and quantity of radiation exposure and its in vivo biological effects. Wide range molecular profiling will analyze mainly cell and DNA damages and cell-to-cell and tissues interplay, meanwhile biochemical and chemical specific composition will be detected by infrared spectroscopy. The recently characterized alteration of leptin metabolism is discussed in more detail to present a successful example of systemic approach to cosmic radiation biology. ﾩ Accademia Nazionale dei Lincei 2013.

Cancer cells are characterized by altered ubiquitination of many proteins. The ubiquitin-specific protease 2a (USP2a) is a deubiquitinating enzyme overexpressed in prostate adenocarcinomas, where it exhibits oncogenic behavior in a variety of ways including targeting c-Myc via the miR-34b/c cluster. Here we demonstrate that USP2a induces drug resistance in both immortalized and transformed prostate cells. Specifically, it confers resistance to typically pro-oxidant agents, such as cisplatin (CDDP) and doxorubicin (Doxo), and to taxanes. USP2a overexpression protects from drug-induced oxidative stress by reducing reactive oxygen species (ROS) production and stabilizing the mitochondrial membrane potential (Δ Ψ), thus impairing downstream p38 activation and triggering of apoptosis. The molecular mediator of the USP2a protective function is the glutathione (GSH). Through miR-34b/c-driven c-Myc regulation, USP2a increases intracellular GSH content, thus interfering with the oxidative cascade triggered by chemotherapeutic agents. In light of these findings, targeting Myc and/or miR-34b/c might revert chemo-resistance. © 2013 Macmillan Publishers Limited. All rights reserved.