ritorno a parlare di danni da campi elettromagnetici – Dr.ssa Loretta Bolgan – NCBI.NLM.NIH.GOV Untargeted metabolomics unveil alterations of biomembranes permeability in human HaCaT keratinocytes upon 60 GHz millimeter-wave exposure A joint metabolomic and lipidomic workflow is used to account for a potential effect of millimeter waves (MMW) around 60 GHz on biological tissues. For this purpose, HaCaT human keratinocytes were exposed at 60.4 GHz with an incident power …

 Loretta Bolgan

ritorno a parlare di danni da campi elettromagnetici.
Qui sotto una lista di link e studi recenti utili per sostenere il principio di precauzione:

MODELLI DA UTILIZZARE:
https://www.alleanzaitalianastop5g.it/442963891
Modello di istanza del Sindaco da inoltrare al Prefetto
Modello di Mozione Stop 5G per il Consiglio Comunale
Modello di Delibera Stop5G di Giunta
Modello di Ordinanza urgente e contingibile Stop 5G che il Sindaco può emanare
Modello di istanza dei cittadini da inoltrare al Sindaco per sospendere il 5G sul territorio
Modello di istanza dei cittadini per la richiesta di accesso agli atti da presentare in Comune e/o Ente pubblico sull’installazione di antenna 5G – Stazione Radio Base
Modello di istanza dei cittadini da presentare al Prefetto per il Ministero dell’Ambiente
Modello di diffida dei cittadini da inoltrare al Sindaco per chiedere di fermare il 5G sul proprio territorio comunale

https://www.alleanzaitalianastop5g.it/442963896
Ricerche, studi, pareri, inchieste

Classificazione 2001 in 2B possibili cancerogeni secondo l’Agenzia Internazionale per la Ricerca sul Cancro
2020-2024, l’Agenzia Internazionale per la Ricerca sul Canmcro rivaluterà la classificazione della cancerogenesi delle radiofrequenze, onde non ionizzanti
Comitato Scientifico sui rischi sanitari ambientali ed emergenti (SCHEER) della Comunità Europea
Direzione generale per le politiche europee del Dipartimento tematico per le politiche economiche, scientifiche e di qualità della vita, incaricato dalla Commissione Industria, Ricerca ed Energia del Parlamento Europeo a svolgere un’approfondita analisi sullo sviluppo del 5G
Consiglio d’Europa, Risoluzione 1815 del 2011
Appello di Friburgo del 2012
Appello di Parigi del 2015
Appello del Prof. Franco Berrino del 2008
Comitato Europeo sui Rischi per le Radiazioni (ECCR) del 2019
L’Alleanza contro il Cancro studia gli effetti delle radiofrequenze
Sentenza TAR Lazio del 2019
Commenti dei medici di ISDE Italia al Rappoorto ISTASAN 2019 dell’Istituto Superiore di Sanità
Corte d’Appello, Tribunale di Brescia, 2009
Prof. Martin Pall: prevedo grandi catastrofi e incendi per il 5G
Prof. Olle Johannson: 5G privo di studi
Prof. Angelo Gino Levis: troppi conflitti d’intertessi
Prof. Ernesto Burgio sulla moratoria del 5G
Studio del Prof. Lennart Hardell
Studi del Prof. Angelo Gino Levis, aggiornamento 2019
Studio del Dott. Agostino Di Ciaula (ISDE Italia)
Studio del Prof. Martin Pall sul 5G
Studio dell’Istituto Ramazzini
Studio dell’Istituto Ramazzini
Studio del National Toxicology Program (USA)
Studio sui ratti esposti al microonde
Studio sugli effetti per la salute dall’uso di tefeonia mobile
Iperglicemia nei ratti esposti al Wi-Fi
5G e taglio degli alberi
Investigate Europe: i conflitti d’interessi sul 5G
Un’altra versione dell’Istituto Superiore di Sanità
I Sindaci possono fermare il 5G
5G, inquietanti retroscena dal Ministero della Salute

VIDEO Youtube:

https://youtu.be/oyt7zm2TDjQ
Di Ciaula ISDE 5G

https://youtu.be/HtCoDWN_nWg
Stop 5G – (9/10) “Onde elettromagnetiche: i risultati scientifici della ricerca” – Fiorella Belpoggi

https://youtu.be/8JyJl6D_U6Y
5G e rischi per la salute secondo l’Istituto Ramazzini

https://youtu.be/rhI63jMKVtM
Roma – Conferenza stampa di Sara Cunial (27.06.19)

https://www.isde.it/speciale-isde-su-5g-materialedocumenti…/

DOCUMENTI, COMUNICATI STAMPA E INTERVISTE A CURA DI ISDE ITALIA
• Comunicato stampa del 2017 che chiede la moratoria sul 5G
• Richiesta di moratoria del 2018 ( per documento in inglese, clicca QUA)
• Lettera del Presidente del comitato scientifico ISDE al Ministro Di Maio
• 5G, l’Italia nella nuova rete. Ma i rischi per la salute sono ignorati, Intervista al Dott. Agostino Di Ciaula
• Rete 5G: gli effetti negativi sono già certificati, servono regolamentazioni governative. Intervista al Dott. Agostino Di Ciaula
• Sperimentazione 5G. La nuova tecnologia ad alto rischio per la salute? A cura di Dott.ssa Patrizia Gentilini, ISDE Italia
• Campi elettromagnetici e rischi per la salute. A cura di Dott.ssa Patrizia Gentilini, ISDE Italia

INIZIATIVE E DOCUMENTI DI ALTRE ASSOCIAZIONI\SOCIETA’ SCIENTIFICHE
• E’ ONLINE IL SITO WEB DELL’ALLEANZA ITALIANA STOP 5G, CLICCA QUA
• Firma l’appello nazionale per richiedere la moratoria sul 5G
• Ecco tutte le azioni promosse dall’Associazione Malattie da Intossicazione Cronica e Ambientale (AMICA)
• 5G: l’appello al Parlamento dell’Istituto Ramazzini
• 5G, diritto alla salute e tutela dei consumatori: una sintesi del quadro europeo e internazionale. A cura di Avv. Sergio Diana, esperto in Diritto Europeo e Relazioni Internazionali
• Il CODACONS invia un esposto a 104 Procure sulla questione 5G

NOTIZIE, DOCUMENTI E ATTIVITA DAI TERRITORI ITALIANI
• Roma, il 5G e i (fastidiosi) alberi monumentali, luglio 2019
• Verso il 5G, possibili rischi sanitari. A cura di Dott. Domenico Scanu, Presidente ISDE Sardegna
• La prima forma di prevenzione è la conoscenza. A cura di C.Zuncheddu, ISDE Sardegna
• 5G:Strumento di comunicazione o strumento di controllo di massa? A cura di Dott. Correddu ISDE Sardegna
• Lettera dei medici sardi al Sindaco di Cagliari

PUBBLICAZIONI SCIENTIFICHE

Towards 5G communication systems: Are there health implications? di A. Di Ciaula
https://www.sciencedirect.com/…/arti…/pii/S1438463917308143…
Il Dr. Agostino Di Ciaula, Presidente del Comitato Scientifico ISDE, in questo articolo (disponibile nel link l’abstract) fa il punto della situazione sulle evidenze scientifiche disponibili sui rischi biologici da esposizione all’elettromagnetismo ad alta frequenza in generale e alle onde millimetriche (quelle che saranno utilizzate dal 5G) in particolare. L’analisi si conclude affermando che “esistono sufficienti evidenze per dimostrare la presenza di effetti biomedici, per invocare il principio di precauzione, per definire i soggetti esposti come potenzialmente vulnerabili e per rivedere i limiti di legge vigenti”

Untargeted metabolomics unveil alterations of biomembranes permeability in human HaCaT keratinocytes upon 60 GHz millimeter-wave exposure di AAVV
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597695/
Il 5G userà onde millimetriche ad elevata frequenza che si sommeranno all’esistente, aggiungendo specificità proprie in termini di effetti biologici. Saranno usate frequenze sino a 26 GHz (“bande pioniere”) “prima di pervenire all’impiego di frequenze più alte… nella gamma tra 24.25 e 86 GHz” (Fonte: AGCOM). Questo articolo, pubblicato su Nature – Scientific Reports da autorevoli ricercatori francesi, è una delle tante spie di pericolo accese e ignorate dalla politica. Si occupa degli effetti biologici (non termici) delle onde millimetriche, quelli completamente ignorati dalla normativa vigente, probabilmente prossima persino ad un aumento dei limiti. Le onde millimetriche possono penetrare nella cute fino a 10mm, una profondità tale da generare effetti sulle cellule cutanee, sulle terminazioni nervose e sul microcircolo, con potenziali effetti locali e sistemici da rilascio di mediatori. L’articolo dimostra, in vitro, una disregolazione del profilo metabolomico dei cheratinociti umani (cellule della cute) attribuibili ad un danno delle membrane cellulari indotto da onde millimetriche a 60GHz. Non si possono certo estrapolare i risultati ottenuti “in vitro” a livello sistemico e clinico ma secondo gli Autori sono “necessari ulteriori studi per valutare gli effetti biologici delle onde millimetriche …prima dell’impiego su larga scala di tecnologie basate su queste specifiche frequenze”. Esattamente il contrario di ciò che sta accadendo.

Systematic Derivation of Safety Limits for Time-Varying 5G Radiofrequency Exposure Based on Analytical Models and Thermal Dose di Esra Neufeld e Niels Kuster
https://insights.ovid.com/pubmed?pmid=30247338
Il Prof. Esra Neufeld lavora a Zurigo. Ha pubblicato oltre 70 lavori su riviste internazionali sugli effetti delle radiofrequenze e le sue pubblicazioni sono state citate oltre 1900 volte da altri Autori. Dunque, una riconosciuta autorità nel suo settore di ricerca. Con questa recente pubblicazione (disponibile nel link l’abstract) ha dimostrato l’inadeguatezza dei limiti ICNIRP (sui quali si basano i limiti vigenti) e che l’esposizione alle frequenze del 5G (oltre 10GHz) “tollerate dall’ICNIRP” (e quindi dai limiti utilizzati a livello internazionale) “possono indurre danno tissutale permanente anche dopo brevi esposizioni”. Secondo i suoi risultati i limiti vigenti “richiedono un’urgente revisione”. Questi risultati si riferiscono ai soli effetti termici (innalzamento della temperatura cutanea), gli unici considerati dagli attuali limiti di legge, che sono inadeguati anche perché ignorano completamente gli effetti biologici delle esposizioni croniche (danni all’espressione genica, stress ossidativo, danno di membrana etc.). Il Dr. Di Ciaula, Presidente del Comitato Scientifico ISDE, ha scritto al Prof. Neufeld chiedendogli se, secondo lui, un adeguamento dei limiti ICNIRP che tenesse conto dei suoi risultati fosse comunque sicuro proprio alla luce dei possibili effetti biologici. Nella sua risposta il Prof. Neufeld confermava i timori dichiarando che sono necessari ulteriori studi e che il suo gruppo sta seguendo questa strada con sperimentazioni in corso e altre ancora da pianificare.

Genotoxic Effects in Human Fibroblasts Exposed to Microwave Radiation di AAVV
https://insights.ovid.com/pubmed?pmid=29787439
L’action plan per la diffusione del 5G nel nostro Paese prevede, nell’ultima fase, l’utilizzo di frequenze superiori a 24.2 GHz. E’ stato dimostrato con questo articolo (disponibile abstract nel link) che esporre cellule umane a 25 GHz per 20 minuti genera alterazioni cromosomiali predisponenti al cancro.

The effect of the built and natural environment on millimetric radio waves di Department of Digital, Culture, Media and Sport ( UK)
https://insights.ovid.com/pubmed?pmid=29787439
Questo documento contiene la ricerca inglese realizzata per comprendere l’impatto del 5G.
Nello specifico, questo documento:
• fornisce i dettagli delle ricerche svolte da Ordnance Survey, 5GIC e Met Office
• definisce uno strumento di pianificazione della rete basato sulla ricerca
• trae conclusioni sull’impatto dei fattori geospaziali e meteorologici nell’introduzione del 5G nelle aree geografiche ad alto valore nel Regno Unito.

5G Planning – geospatial considerations. A guide for planners and local authorities di Department of Digital, Culture, Media and Sport (UK)
https://assets.publishing.service.gov.uk/…/OS_Final_report_…

Exposure of Insects to Radio-Frequency Electromagnetic Fields from 2 to 120 GHz
https://assets.publishing.service.gov.uk/…/OS_Final_report_…
La magnetite contenuta nel corpo delle api ha un ruolo importante per le loro funzioni neurologiche e contribuisce al senso di orientamento (ad es. rende possibile il ritorno alle colonie di appartenenza) ma le rende anche sensibili ai campi elettromagnetici. Per questo l’esposizione alle frequenze proprie della telefonia mobile può avere su questi insetti effetti negativi (Sharma e Kumar, Current Science 2010, El Halabi et al, IEEE 2013). Le api sono per fortuna oggi poco esposte alle frequenze utilizzate dalla telefonia mobile ma la situazione potrebbe cambiare con il 5G. Questo articolo, pubblicato su Nature – Scientific Reports, è stato il primo studio che ha valutato direttamente l’assorbimento delle radiofrequenze da parte degli insetti, dimostrando che dipende dalla frequenza utilizzata. Nel caso delle api, a 1V/m (il limite attuale è 6 V/m) l’assorbimento è massimo con onde millimetriche oltre i 6 GHz, frequenze che saranno usate su scala globale per il 5G. Gli Autori dello studio prevedono un incremento significativo delle radiofrequenze che saranno assorbite dagli insetti in seguito al passaggio dal 3G-4G al 5G. Questo, secondo gli Autori, potrà comportare “con il tempo modificazioni del comportamento, della fisiologia e della morfologia degli insetti”.

Global 5G wireless networks threaten weather forecasts
https://www.nature.com/articles/d41586-019-01305-4
La prestigiosa rivista Nature ha pubblicato un articolo che spiega come il 5G interferirà con le possibilità di prevedere gli eventi meteorologici a causa di interferenze con le trasmissioni satellitari. Proprio quando, a causa delle modificazioni climatiche causate da insostenibili attività umane, eventi meteorici intensi, tempeste e tornado stanno diventando più frequenti e stanno conquistando nuove aree geografiche, i meteorologi potrebbero avere gravi difficoltà a prevedere questi eventi in tempo utile negli Stati Uniti e in Europa. Il meteorologo Tony McNail, dell’European Centre for Medium-Range Weather Forecast esprime in una intervista rilasciata al “Guardian” la sua forte preoccupazione: “Il modo in cui il 5G si sta introducendo compromette gravemente la nostra capacità di prevedere le tempeste più violente. Alla fine potrebbe fare la differenza tra la vita e la morte. Siamo molto preoccupati per questo”
(https://www.theguardian.com/…/5g-mobile-networks-threat-to-…).

Approfondimenti scientifici

La banda 2.6 GHz è una di quelle vendute all’asta dal nostro Governo per il 5G. L’ICNIRP, l’ONG privata che ha formulato i limiti di legge validi a livello internazionale, ritiene che la popolazione esposta sia al sicuro al di sotto di una SAR (l’energia elettromagnetica assorbita dal corpo quando questo viene esposto a una radiofrequenza) di 0.08 W/Kg. L’ICNIRP, tuttavia, ha calcolato i suoi limiti considerando i soli effetti termici acuti delle esposizioni e ignorando completamente ciò che invece più ci interessa dal punto di vista clinico: gli effetti biologici derivanti da esposizioni croniche, quelle che concretamente si verificano nella realtà quotidiana. Numerosi studi hanno dimostrato che l’irradiazione con radiofrequenze è una sorgente ambientale di stress ossidativo, un processo fisiopatologico alla base di numerose patologie. Tra gli organi più vulnerabili c’è sicuramente il cervello, nel quale si genera lipoperossidazione lipidica che può promuovere danni neuronali. Lo studio sintetizzato nell’immagine, recente, ha dimostrato che topi esposti solo per due ore al giorno ad una frequenza di 2.45 GHz, con SAR di 0.014 W/Kg (circa 6 volte più bassa del limite ICNIRP) sviluppano, indipendentemente dagli effetti termici, alterazioni della memoria spaziale e di apprendimento, aumento dei livelli sierici di corticosterone proporzionali al tempo di esposizione, stress ossidativo e danni, a livello dell’ippocampo, sia dei neuroni cerebrali che di cellule non neuronali. Certo, non siamo topi. Con il 5G, tuttavia, saremo esposti h24 (dunque più di quei topi) a 2.6 GHz, a tutte le altre frequenze che il 5G utilizzerà (onde millimetriche) ed a tutte le frequenze sino ad ora utilizzate (3G, 4G), alcune delle quali dotate di effetti biologici molto specifici e completamente ignorati dai limiti vigenti. Anche se il nostro cervello non è molto diverso, funzionalmente, da quello dei topi, non siamo al momento autorizzati a trarre conclusioni sulla possibile presenza di effetti fisiopatologici o clinici sull’uomo, simili a quelli ben documentati sugli animali. (Agostino Di Ciaula)

Environ Toxicol. 2018 Sep;33(9):931-945. doi: 10.1002/tox.22578. Epub 2018 Jul 3.
2.45 GHz microwave radiation induced oxidative and nitrosative stress mediated testicular apoptosis: Involvement of a p53 dependent bax-caspase-3 mediated pathway.
Shahin S1, Singh SP2, Chaturvedi CM1.
https://www.ncbi.nlm.nih.gov/pubmed/29968967
Abstract
Deleterious effects of MW radiation on the male reproduction are well studied. Previous reports although suggest that 2.45 GHz MW irradiation induced oxidative and nitrosative stress adversely affects the male reproductive function but the detailed molecular mechanism occurring behind it has yet to be elucidated. The aim of present study was to investigate the underlying detailed pathway of the testicular apoptosis induced by free radical load and redox imbalance due to 2.45 GHz MW radiation exposure and the degree of severity along with the increased exposure duration. Twelve-week old male mice were exposed to 2.45 GHz MW radiation [continuous-wave (CW) with overall average Power density of 0.0248 mW/cm2 and overall average whole body SAR value of 0.0146 W/kg] for 2 hr/day over a period of 15, 30, and 60 days. Testicular histology, serum testosterone, ROS, NO, MDA level, activity of antioxidant enzymes, expression of pro-apoptotic proteins (p53 and Bax), anti-apoptotic proteins (Bcl-2 and Bcl-xL ), cytochrome-c, inactive/active caspase-3, and uncleaved PARP-1 were evaluated.
Findings suggest that 2.45 GHz MW radiationexposure induced testicular redox imbalance not only leads to enhanced testicular apoptosis via p53 dependent Bax-caspase-3 mediated pathway, but also increases the degree of apoptotic severity in a duration dependent manner.

Toxicol Sci. 2015 Dec;148(2):380-99. doi: 10.1093/toxsci/kfv205. Epub 2015 Sep 22.
2.45 GHz Microwave Radiation Impairs Learning and Spatial Memory via Oxidative/Nitrosative Stress Induced p53-Dependent/Independent HippocampalApoptosis: Molecular Basis and Underlying Mechanism.
Shahin S1, Banerjee S1, Singh SP2, Chaturvedi CM3.
https://www.ncbi.nlm.nih.gov/pubmed/26396154
Abstract
A close association between microwave (MW) radiation exposure and neurobehavioral disorders has been postulated but the direct effects of MW radiation on central nervous system still remains contradictory. This study was performed to understand the effect of short (15 days) and long-term (30 and 60 days) low-level MW radiation exposure on hippocampus with special reference to spatial learning and memory and its underlying mechanism in Swiss strain male mice, Mus musculus. Twelve-weeks old mice were exposed to 2.45 GHz MW radiation (continuous-wave [CW] with overall average power density of 0.0248 mW/cm(2) and overall average whole body specific absorption rate value of 0.0146 W/Kg) for 2 h/day over a period of 15, 30, and 60 days). Spatial learning and memory was monitored by Morris Water Maze. We have checked the alterations in hippocampal oxidative/nitrosative stress, neuronal morphology, and expression of pro-apoptotic proteins (p53 and Bax), inactive executioner Caspase- (pro-Caspase-3), and uncleaved Poly (ADP-ribose) polymerase-1 in the hippocampal subfield neuronal and nonneuronal cells (DG, CA1, CA2, and CA3). We observed that, short-term as well as long-term 2.45 GHz MW radiation exposure increases the oxidative/nitrosative stress leading to enhanced apoptosis in hippocampal subfield neuronal and nonneuronal cells. Present findings also suggest that learning and spatial memory deficit which increases with the increased duration of MW exposure (15 < 30 < 60 days) is correlated with a decrease in hippocampal subfield neuronal arborization and dendritic spines. These findings led us to conclude that exposure to CW MW radiation leads to oxidative/nitrosative stress induced p53-dependent/independent activation of hippocampal neuronal and nonneuronal apoptosis associated with spatial memory loss.

Toxicol Sci. 2018 Feb 1;161(2):349-374. doi: 10.1093/toxsci/kfx221.
From the Cover: 2.45-GHz Microwave Radiation Impairs Hippocampal Learning and Spatial Memory: Involvement of Local Stress Mechanism-Induced Suppression of iGluR/ERK/CREB Signaling.
Shahin S1, Banerjee S1, Swarup V2, Singh SP3, Chaturvedi CM1.
https://www.ncbi.nlm.nih.gov/pubmed/…
Abstract
Microwave (MW) radiation induced oxidative stress reduces dendritic arborization, spine density and number of hippocampal pyramidal neurons and hence, impair learning and spatial memory through p53-dependent/independent apoptosis of hippocampal neuronal and nonneuronal cells. However, the mechanisms responsible for MW radiation induced impairment in memory formation remains still unknown. This study elucidates the effect of short (15 days) and long-term (30 and 60 days) low level 2.45 GHz MW radiation-induced local stress on the hippocampal spatial memory formation pathway in adult male mice. Twelve-weeks old mice were exposed to 2.45 GHz MW radiation (continuous-wave with overall average Power density of 0.0248 mW/cm2 and overall average whole body SAR value of 0.0146 W/Kg) @ 2 h/d for 15, 30, and 60 days. Learning and spatial memory was assessed by 8-arm radial maze. We have investigated the alterations in serum corticosterone level and the expression of glucocorticoid receptor, corticotropin-releasing hormone (CRH), inducible nitric oxide synthase (i-NOS), iGluRs, PSD-95-neuronal NOS (n-NOS) system, protein kinase A, protein kinase Cε-ERK1/2-pERK1/2 in all the hippocampal subregions, viz. CA1, CA2, CA3, and DG through immunohistochemistry/immunofluorescence and alterations in the expression of hippocampal glucocorticoid receptor, CRH-receptor 1 (CRH-R1), cAMP-response element-binding (CREB), and phosphorylated-CREB (p-CREB) through western blot analysis. We observed that 2.45 GHz MW irradiated mice showed slow learning and significantly increased number of working and reference memory errors in radial maze task. Further, 2.45 GHz MW radiationexposure increases serum corticosterone level and the expression of CRH, CRH-R1, and i-NOS, while the expression of iGluRs, n-NOS, PSD-95, protein kinase Cε, protein kinase A, ERK-p-ERK, CREB, and p-CREB decreases in above mentioned hippocampal subregions in a duration dependent manner. Our findings led us to conclude that 2.45 GHz MW radiation exposure induced local stress suppresses signaling mechanism(s) of hippocampal memory formation.

Biomed Environ Sci. 2019 Mar;32(3):189-198. doi: 10.3967/bes2019.026.
Behavioral Abnormality along with NMDAR-related CREB Suppression in Rat Hippocampus after Shortwave Exposure.
Yu C1, Bai YX1, Xu XP1, Gao YB1, Hao YH1, Wang H1, Tan SZ1, Li WC1, Zhang J1, Yao BW1, Dong J1, Zhao L1, Peng RY1.
http://www.besjournal.com/…/No3/201904/t20190411_200982.html
Abstract
OBJECTIVE:
To estimate the detrimental effects of shortwave exposure on rat hippocampal structure and function and explore the underlying mechanisms.
METHODS:
One hundred Wistar rats were randomly divided into four groups (25 rats per group) and exposed to 27 MHz continuous shortwave at a power density of 5, 10, or 30 mW/cm2 for 6 min once only or underwent sham exposure for the control. The spatial learning and memory, electroencephalogram (EEG), hippocampal structure and Nissl bodies were analysed. Furthermore, the expressions of N-methyl-D-aspartate receptor (NMDAR) subunits (NR1, NR2A, and NR2B), cAMP responsive element-binding protein (CREB) and phosphorylated CREB (p-CREB) in hippocampal tissue were analysed on 1, 7, and 14 days after exposure.
RESULTS:
The rats in the 10 and 30 mW/cm2 groups had poor learning and memory, disrupted EEG oscillations, and injured hippocampal structures, including hippocampal neurons degeneration, mitochondria cavitation and blood capillaries swelling. The Nissl body content was also reduced in the exposure groups. Moreover, the hippocampaltissue in the 30 mW/cm2 group had increased expressions of NR2A and NR2B and decreased levels of CREB and p-CREB.
CONCLUSION:
Shortwave exposure (27 MHz, with an average power density of 10 and 30 mW/cm2) impaired rats’ spatial learning and memory and caused a series of dose-dependent pathophysiological changes. Moreover, NMDAR-related CREB pathway suppression might be involved in shortwave-induced structural and functional impairments in the rat hippocampus.

Biomol Ther (Seoul). 2019 May 1;27(3):265-275. doi: 10.4062/biomolther.2018.152.
Possible Effects of Radiofrequency Electromagnetic Field Exposure on Central Nerve System.
Kim JH1, Lee JK1, Kim HG1, Kim KB2, Kim HR1.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6513191/
Abstract
Technological advances of mankind, through the development of electrical and communication technologies, have resulted in the exposure to artificial electromagnetic fields (EMF). Technological growth is expected to continue; as such, the amount of EMF exposure will continue to increase steadily. In particular, the use-time of smart phones, that have become a necessity for modern people, is steadily increasing. Social concerns and interest in the impact on the cranial nervous system are increased when considering the area where the mobile phone is used. However, before discussing possible effects of radiofrequency-electromagnetic field (RF-EMF) on the human body, several factors must be investigated about the influence of EMFs at the level of research using in vitro or animal models. Scientific studies on the mechanism of biological effects are also required. It has been found that RF-EMF can induce changes in central nervous system nerve cells, including neuronal cell apoptosis, changes in the function of the nerve myelin and ion channels; furthermore, RF-EMF act as a stress source in living creatures. The possible biological effects of RF-EMF exposure have not yet been proven, and there are insufficient data on biological hazards to provide a clear answer to possible health risks. Therefore, it is necessary to study the biological response to RF-EMF in consideration of the comprehensive exposure with regard to the use of various devices by individuals. In this review, we summarize the possible biological effects of RF-EMF exposure.

I dubbi sui possibili effetti biologici delle onde millimetriche del 5G non riguardano solo le conseguenze dirette sulla salute umana ma anche quelle potenzialmente legate all’azione sui microbi (compresi quelli “buoni”) e sugli insetti. La magnetite contenuta nel corpo delle api ha un ruolo importante per le loro funzioni neurologiche e contribuisce al senso di orientamento (ad es. rende possibile il ritorno alle colonie di appartenenza) ma le rende anche sensibili ai campi elettromagnetici. Per questo l’esposizione alle frequenze proprie della telefonia mobile può avere su questi insetti effetti negativi (Sharma e Kumar, Current Science 2010, El Halabi et al, IEEE 2013). Le api sono per fortuna oggi poco esposte alle frequenze utilizzate dalla telefonia mobile ma la situazione potrebbe cambiare con il 5G. Quello citato nell’immagine, pubblicato nel 2018 su Nature – Scientific Reports, è stato il primo studio che ha valutato direttamente l’assorbimento delle radiofrequenze da parte degli insetti, dimostrando che dipende dalla frequenza utilizzata. Nel caso delle api, a 1V/m (il limite attuale è 6 V/m) l’assorbimento è massimo con onde millimetriche oltre i 6 GHz, frequenze che saranno usate su scala globale per il 5G. Gli Autori dello studio prevedono un incremento significativo delle radiofrequenze che saranno assorbite dagli insetti in seguito al passaggio dal 3G-4G al 5G. Questo, secondo gli Autori, potrà comportare “con il tempo modificazioni del comportamento, della fisiologia e della morfologia degli insetti”.
L’inquinamento atmosferico e l’uso massivo di pesticidi stanno già compromettendo in maniera significativa gli ecosistemi. L’insieme degli inquinanti chimici e fisici (come le radiofrequenze) è potenzialmente in grado di amplificare i danni soprattutto a carico degli organismi più vulnerabili.

Sci Rep. 2018 Mar 2;8(1):3924. doi: 10.1038/s41598-018-22271-3.
Exposure of Insects to Radio-Frequency Electromagnetic Fields from 2 to 120 GHz.
Thielens A1,2, Bell D3, Mortimore DB4, Greco MK5, Martens L6, Joseph W6.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834628/
Insects are continually exposed to Radio-Frequency (RF) electromagnetic fields at different frequencies. The range of frequencies used for wireless telecommunication systems will increase in the near future from below 6 GHz (2 G, 3 G, 4 G, and WiFi) to frequencies up to 120 GHz (5 G). This paper is the first to report the absorbed RF electromagnetic power in four different types of insects as a function of frequency from 2 GHz to 120 GHz. A set of insect models was obtained using novel Micro-CT (computer tomography) imaging. These models were used for the first time in finite-difference time-domain electromagnetic simulations. All insects showed a dependence of the absorbed power on the frequency. All insects showed a general increase in absorbed RF power at and above 6 GHz, in comparison to the absorbed RF power below 6 GHz. Our simulations showed that a shift of 10% of the incident power density to frequencies above 6 GHz would lead to an increase in absorbed power between 3-370%.

Bioinformation. 2018 Dec 21;14(9):421-424. doi: 10.6026/97320630014521. eCollection 2018.
Colony Collapse Disorder (CCD) in Honey Bees Caused by EMF Radiation.
Santhosh Kumar S1.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563664/
Abstract
Honey bees are one of the treasures in the world. An increase of waveform communication leads to good information exchange of mankind. In the biological view, it causes a lot of side effects and lifestyle changes in other living organisms. The drastic changes are causing the natural imbalance in the ecosystem and become a global issue. There are significant reasons for bee colony collapse disorder (CCD) like pesticides, disease and climate change. Recent studies reveal that a cell phone tower and mobile phone handset are also causing side effects to honey bees due to radiation emission. Most of the researchers concentrated on biological and behavioral changes in a honey bee due to radiation effects. For that, the real-time radiation levels have experimented but the different technical perspectives such as radiation emission levels, handset radiation emission measures and multi-sources of radiation are needed to be considered during research. This study aimed to provide possible research extensions of colony collapse disordercaused by cell tower and mobile handsets.
KEYWORDS:
honey bees; Electromagnetic radiation colony collapse disorder.; cell towers; colony collapse disorder.

Sci Total Environ. 2019 Apr 15;661:553-562. doi: 10.1016/j.scitotenv.2019.01.154. Epub 2019 Jan 15.
Effects of radiofrequency electromagnetic radiation (RF-EMF) on honey bee queen development and mating success.
Odemer R1, Odemer F2.
https://www.ncbi.nlm.nih.gov/pubmed/30682608
Abstract
Mobile phones can be found almost everywhere across the globe, upholding a direct point-to-point connection between the device and the broadcast tower. The emission of radiofrequency electromagnetic fields (RF-EMF) puts the surrounding environment inevitably into contact with this radiation. We have therefore exposed honey bee queen larvae to the radiation of a common mobile phone device (GSM band at 900 MHz) during all stages of their pre-adult development including pupation. After 14 days of exposure, hatching of adult queens was assessed and mating success after further 11 days, respectively. Moreover, full colonies were established of five of the untreated and four of the treated queens to contrast population dynamics. We found that mobile phone radiation had significantly reduced the hatching ratio but not the mating success. If treated queens had successfully mated, colony development was not adversely affected. We provide evidence that mobile phone radiation may alter pupal development, once succeeded this point, no further impairment has manifested in adulthood. Our results are discussed against the background of long-lasting consequences for colony performance and the possible implication on periodic colony losses.

Il 5G userà onde millimetriche ad elevata frequenza che si sommeranno all’esistente, aggiungendo specificità proprie in termini di effetti biologici. Saranno usate frequenze sino a 26 GHz (“bande pioniere”) “prima di pervenire all’impiego.. di frequenze più alte… nella gamma tra 24.25 e 86 GHz” (Fonte: AGCOM). Questo articolo, pubblicato su Nature – Scientific Reports da autorevoli ricercatori francesi, è uno dei tanti che, secondo alcuni, “non esistono”. Una delle tante spie di pericolo accese e ignorate dalla politica. Si occupa degli effetti biologici (non termici) delle onde millimetriche, quelli COMPLETAMENTE IGNORATI dalla normativa vigente, probabilmente prossima persino ad un aumento dei limiti. Le onde millimetriche possono penetrare nella cute fino a 10mm, una profondità tale da generare effetti sulle cellule cutanee, sulle terminazioni nervose e sul microcircolo, con potenziali effetti locali e sistemici da rilascio di mediatori. L’articolo dimostra, in vitro, una disregolazione del profilo metabolomico dei cheratinociti umani (cellule della cute) attribuibili ad un danno delle membrane cellulari indotto da onde millimetriche a 60GHz. Non si possono certo estrapolare i risultati ottenuti “in vitro” a livello sistemico e clinico ma secondo gli Autori sono “necessari ulteriori studi per valutare gli effetti biologici delle onde millimetriche … PRIMA DELL’IMPIEGO SU LARGA SCALA DI TECNOLOGIE BASATE SU QUESTE SPECIFICHE FREQUENZE”. Esattamente il contrario di ciò che sta accadendo: la politica ci sta imponendo rapidamente un “impiego su larga scala” delle onde millimetriche ignorando tutte le evidenze disponibili sugli effetti biologici generali delle radiofrequenze e ignorando le moltissime raccomandazioni del mondo scientifico sugli effetti biologici SPECIFICI delle onde millimetriche, ancora in fase di studio e con risultati preliminari preoccupanti.

Sci Rep. 2019 Jun 27;9(1):9343. doi: 10.1038/s41598-019-45662-6.
Untargeted metabolomics unveil alterations of biomembranes permeability in human HaCaT keratinocytes upon 60 GHz millimeter-wave exposure.
Le Pogam P1, Le Page Y2, Habauzit D2, Doué M1, Zhadobov M1, Sauleau R1, Le Dréan Y2, Rondeau D3,4.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597695/
Abstract
A joint metabolomic and lipidomic workflow is used to account for a potential effect of millimeter waves (MMW) around 60 GHz on biological tissues. For this purpose, HaCaT human keratinocytes were exposed at 60.4 GHz with an incident power density of 20 mW/cm², this value corresponding to the upper local exposure limit for general public in the context of a wide scale deployment of MMW technologies and devices. After a 24h-exposure, endo- and extracellular extracts were recovered to be submitted to an integrative UPLC-Q-Exactive metabolomic and lipidomic workflow. R-XCMS data processing and subsequent statistical treatment led to emphasize a limited number of altered features in lipidomic sequences and in intracellular metabolomic analyses, whatever the ionization mode (i.e 0 to 6 dysregulated features). Conversely, important dysregulations could be reported in extracellular metabolomic profiles with 111 and 99 frames being altered upon MMW exposure in positive and negative polarities, respectively. This unexpected extent of modifications can hardly stem from the mild changes that could be reported throughout transcriptomics studies, leading us to hypothesize that MMW might alter the permeability of cell membranes, as reported elsewhere.

È in corso una corposa campagna propagandistica pro-5G. Come ampiamente previsto, questa si accompagna alla ripetizione ossessiva della “necessità” di aumentare il limite di legge italiano (6V/m), portandolo ai livelli della molto più alta media europea (circa 50 V/m). Mantenere i limiti vigenti ostacolerebbe la strada del 5G e, ovviamente, gli affari. La normativa vigente si basa sulle determinazioni della ICNIRP (una ONG privata), che valuta le soglie per la sola insorgenza di EFFETTI TERMICI (riscaldamento cutaneo) in seguito ad ESPOSIZIONI ACUTE, ignorando completamente i numerosi effetti BIOLOGICI (non-termici: soprattutto riproduttivi, neurologici, metabolici) successivi ad esposizioni CRONICHE, anche di bassa intensità. Un gruppo internazionale di scienziati indipendenti (il BioInitiative report) ha, al contrario, considerato anche gli effetti biologici descritti da numerosi studi, indicando come soglia di sicurezza 0.04 V/m (di molto inferiore ai 6V/m della normativa italiana). Mostro a titolo di esempio uno studio condotto su un gruppo di studenti di età compresa tra 12 e 17 anni, che frequentavano due diverse scuole esposte a intensità molto inferiori ai 6V/m (prossimità a ripetitori di radiotelefonia). Secondo gli Autori, l’esposizione per 6 ore al giorno a 0.2 V/m (valore 30 volte più basso del limite italiano) aumenta il rischio di diabete. I risultati di questo studio, pur essendo in linea con evidenze precedenti su modelli animali, hanno bisogno di ulteriori conferme nell’essere umano. Indipendentemente da questo, comunque, le evidenze disponibili certamente NON ESCLUDONO la possibilità di effetti biologici per esposizioni croniche inferiori ai limiti italiani, come la IARC (OMS) NON ESCLUDE la possibilità di cancro da esposizione a radiofrequenze. Andare incontro ad un aumento delle esposizioni (h24 per TUTTI), all’utilizzo ampio, specifico e inedito delle onde millimetriche e proporre non una riduzione (come si dovrebbe) ma addirittura un aumento dei limiti vigenti, ignorando completamente le evidenze sull’inadeguatezza dei limiti ICNIRP, è da irresponsabili. Abbiamo bisogno di maggiore prudenza, non di cellulari più costosi o di lavatrici collegate al 5G.

Int J Environ Res Public Health. 2015 Nov 13;12(11):14519-28. doi: 10.3390/ijerph121114519.
Association of Exposure to Radio-Frequency Electromagnetic Field Radiation (RF-EMFR) Generated by Mobile Phone Base Stations with Glycated Hemoglobin(HbA1c) and Risk of Type 2 Diabetes Mellitus.
Meo SA1, Alsubaie Y2, Almubarak Z3, Almutawa H4, AlQasem Y2, Hasanato RM5.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4661664/
Installation of mobile phone base stations in residential areas has initiated public debate about possible adverse effects on human health. This study aimed to determine the association of exposure to radio frequency electromagnetic fieldradiation (RF-EMFR) generated by mobile phone base stations with glycated hemoglobin (HbA1c) and occurrence of type 2 diabetes mellitus. For this study, two different elementary schools (school-1 and school-2) were selected. We recruited 159 students in total; 96 male students from school-1, with age range 12-16 years, and 63 male students with age range 12-17 years from school-2. Mobile phone base stations with towers existed about 200 m away from the school buildings. RF-EMFR was measured inside both schools. In school-1, RF-EMFR was 9.601 nW/cm² at frequency of 925 MHz, and students had been exposed to RF-EMFR for a duration of 6 h daily, five days in a week. In school-2, RF-EMFR was 1.909 nW/cm² at frequency of 925 MHz and students had been exposed for 6 h daily, five days in a week. 5-6 mL blood was collected from all the students and HbA1c was measured by using a Dimension Xpand Plus Integrated Chemistry System, Siemens. The mean HbA1c for the students who were exposed to high RF-EMFR was significantly higher (5.44 ± 0.22) than the mean HbA1c for the students who were exposed to low RF-EMFR (5.32 ± 0.34) (p = 0.007). Moreover, students who were exposed to high RF-EMFR generated by MPBS had a significantly higher risk of type 2 diabetes mellitus (p = 0.016) relative to their counterparts who were exposed to low RF-EMFR. It is concluded that exposure to high RF-EMFR generated by MPBS is associated with elevated levels of HbA1c and risk oftype 2 diabetes mellitus.
Comment in
Comments on Meo et al. Association of Exposure to Radio-Frequency Electromagnetic Field Radiation (RF-EMFR) Generated by Mobile Phone Base Stations with Glycated Hemoglobin (HbA1c) and Risk of Type 2 Diabetes Mellitus. Int. J. Environ. Res. Public Health, 2015, 12, 14519-14528. [Int J Environ Res Public Health. 2016]
Response to Comments on Meo et al. Association of Exposure to Radio-Frequency Electromagnetic Field Radiation (RF-EMFR) Generated by Mobile Phone Base Stations with Glycated Hemoglobin (HbA1c) and Risk of Type 2 Diabetes Mellitus. Int. J. Environ. Res. Public Health, 2015, 12, 14519-14528. [Int J Environ Res Public Health. 2016]

Il Prof. Esra Neufeld lavora a Zurigo. Ha pubblicato oltre 70 lavori su riviste internazionali sugli effetti delle radiofrequenze e le sue pubblicazioni sono state citate oltre 1900 volte da altri Autori. Dunque, una riconosciuta autorità nel suo settore di ricerca. Con questa recente pubblicazione (un’altra delle tante evidenze che secondo alcuni NON ESISTONO) ha dimostrato l’inadeguatezza dei limiti vigenti e che l’esposizione alle frequenze del 5G (oltre 10GHz) “tollerate dall’ICNIRP” (e quindi dai limiti utilizzati a livello internazionale) “possono indurre danno tissutale permanente anche dopo brevi esposizioni”. Secondo i suoi risultati i limiti vigenti “richiedono un’urgente revisione”. Questi risultati si riferiscono ai soli effetti termici (innalzamento della temperatura cutanea), gli unici considerati dagli attuali limiti di legge, che sono inadeguati anche perché ignorano completamente gli effetti biologici delle esposizioni croniche (danni all’espressione genica, stress ossidativo, danno di membrana etc.). Ho scritto al Prof. Neufeld chiedendogli se, secondo lui, un adeguamento dei limiti ICNIRP che tenesse conto dei suoi risultati fosse comunque sicuro proprio alla luce dei possibili effetti biologici. Nella sua risposta confermava i miei timori dichiarando che sono necessari ulteriori studi e che il suo gruppo sta seguendo questa strada con sperimentazioni in corso e altre ancora da pianificare. Un’ulteriore dimostrazione del salto nel buio che il nostro Paese insieme ad altri sta compiendo, procedendo con decisione ma con incoscienza assoluta verso un incremento esponenziale delle esposizioni e con limiti di legge palesemente inadeguati, che non ci tutelano.

Health Phys. 2018 Sep 21. doi: 10.1097/HP.0000000000000930. [Epub ahead of print]
Systematic Derivation of Safety Limits for Time-Varying 5G Radiofrequency Exposure Based on Analytical Models and Thermal Dose.
Neufeld E1, Kuster N1,2.
https://www.ncbi.nlm.nih.gov/pubmed/30247338
Abstract
Extreme broadband wireless devices operating above 10 GHz may transmit data in bursts of a few milliseconds to seconds. Even though the time- and area-averaged power density values remain within the acceptable safety limits for continuous exposure, these bursts may lead to short temperature spikes in the skin of exposed people. In this paper, a novel analytical approach to pulsed heating is developed and applied to assess the peak-to-average temperature ratio as a function of the pulse fraction α (relative to the averaging time [INCREMENT]T; it corresponds to the inverse of the peak-to-average ratio). This has been analyzed for two different perfusion-related thermal time constants (τ1 = 100 s and 500 s) corresponding to plane-wave and localized exposures. To allow for peak temperatures that considerably exceed the 1 K increase, the CEM43 tissue damage model, with an experimental-data-based damage threshold for human skin of 600 min, is used to allow large temperature oscillations that remain below the level at which tissue damage occurs. To stay consistent with the current safety guidelines, safety factors of 10 for occupational exposure and 50 for the general public were applied. The model assumptions and limitations (e.g., employed thermal and tissue damage models, homogeneous skin, consideration of localized exposure by a modified time constant) are discussed in detail. The results demonstrate that the maximum averaging time, based on the assumption of a thermal time constant of 100 s, is 240 s if the maximum local temperature increase for continuous-wave exposure is limited to 1 K and α ≥ 0.1. For a very low peak-to-average ratio of 100 (α ≥ 0.01), it decreases to only 30 s. The results also show that the peak-to-average ratio of 1,000 tolerated by the International Council on Non-Ionizing Radiation Protection guidelines may lead to permanent tissue damage after even short exposures, highlighting the importance of revisiting existing exposure guidelines.
Health Phys. 2019 Jul;117(1):67-69. doi: 10.1097/HP.0000000000001090.
Comments on Neufeld and Kuster, “Systematic Derivation of Safety Limits for Time-varying 5G Radiofrequency Exposure Based on Analytical Models and Thermal Dose”.
Foster KR1.
https://www.ncbi.nlm.nih.gov/pubmed/31135642

Spesso il dibattito sul 5G è volutamente distorto dal “cancrocentrismo” e questo può essere fuorviante, soprattutto se si riflette sui rischi in età pediatrica. Dal punto di vista epidemiologico il cancro è forse il minore dei problemi, seppur grave. Molto più frequente potrebbe essere la possibilità di conseguenze più subdole ma meglio documentate, come quelle neurologiche e riproduttive. Queste non portano alla morte ma hanno un peso sanitario e sociale elevatissimo, specie se iniziano a manifestarsi in età pediatrica. Nella slide sono mostrati gli effetti dell’esposizione di ratti a 2.45 GHz per 2 ore al giorno per 40 giorni. La fase ora in corso dell’implementazione del 5G prevede, prima di arrivare all’utilizzo di frequenze superiori a 20GHz (probabilmente entro il prossimo anno), l’utilizzo nell’infrastruttura 5G delle frequenze 2.6 GHz e 3.7 GHz. Non siamo ratti ma il nostro sistema nervoso è funzionalmente molto simile al loro, pur essendo molto più complesso. L’area encefalica sulla quale sono stati dimostrati gli effetti mostrati nella slide è la stessa sulla quale agiscono, soprattutto nei bambini, molti pesticidi e, in parte, l’inquinamento atmosferico, generando una potenziale sinergia negativa e estremamente preoccupante. Il rischio è quello di ridurre le capacità intellettive che consentirebbero alle future generazioni di risolvere i problemi che NOI gli stiamo causando. Come i ratti di questo studio non potremo scappare dalle nostre gabbie per evitare l’esposizione, considerata la capillare proliferazione di microripetitori e l’obiettivo del milione di devices connessi al 5G per ogni Km2. C’è la possibilità che queste siano solo inutili e orrende fantasie allarmistiche anche se, purtroppo, basate su solide evidenze scientifiche. Il problema è che non ci sarà la possibilità di verificarle o smentirle perché l’implementazione è già in atto e la gestione monopolistica del MISE non ci ha concesso gli strumenti adeguati per valutare questi aspetti.

Toxicol Ind Health. 2018 Oct 21:748233718798976. doi: 10.1177/0748233718798976. [Epub ahead of print]
2.45 GHz microwave radiation impairs learning, memory, and hippocampal synaptic plasticity in the rat.
Karimi N1, Bayat M2, Haghani M1,3, Saadi HF4, Ghazipour GR5.
https://www.ncbi.nlm.nih.gov/pubmed/…
Abstract
Microwave (MW) radiation has a close relationship with neurobehavioral disorders. Due to the widespread usage of MW radiation, especially in our homes, it is essential to investigate the direct effect of MW radiation on the central nervous system.
Therefore, this study was carried out to determine the effect of MW radiation on memory and hippocampal synaptic plasticity. The rats were exposed to 2.45 GHz MW radiation (continuous wave with overall average power density of 0.016 mW/cm2 and overall average whole-body specific absorption rate value of 0.017 W/kg) for 2 h/day over a period of 40 days. Spatial learning and memory were tested by radial maze and passive avoidance tests. We evaluated the synaptic plasticity and hippocampal neuronal cells number by field potential recording and Giemsa staining, respectively. Our results showed that MW radiation exposure decreased the learning and memoryperformance that was associated with decrement of long-term potentiation induction and excitability of CA1 neurons. However, MW radiation did not have any effects on short-term plasticity and paired-pulse ratio as a good indirect index for measurement of glutamate release probability. The evaluation of hippocampal morphology indicated that the neuronal density in the hippocampal CA1 area was significantly decreased by MW.

L’action plan per la diffusione del 5G nel nostro Paese prevede, nell’ultima fase, l’utilizzo di frequenze superiori a 24.2 GHz. E’ stato dimostrato che esporre cellule umane a 25 GHz per soli 20 minuti genera alterazioni cromosomiali predisponenti al cancro. Questo è solo uno degli studi che, secondo alcuni, NON esistono e NON sono mai stati condotti, pur essendo pubblicati su riviste medico-scientifiche internazionali. Non so se sia ignoranza o malafede ma è certamente inaccettabile fare finta di nulla e continuare a puntare sull’obiettivo dichiarato del milione di dispositivi per Km2 connessi al 5G senza assumere nessuna precauzione. Non so quanti italiani non vedano l’ora di collegare il proprio frigo o la propria auto a internet ma so che prima di farlo dovrebbero pretendere maggiori garanzie di sicurezza per sé e per i propri figli da chi deve tutelare la salute pubblica, che sembra invece al momento unicamente impegnato a soddisfare le esigenze degli operatori telefonici. Non solo queste garanzie sono al momento inesistenti ma chi gioisce per l’avanzata del 5G nel nostro Paese sta già invocando l’innalzamento di limiti di legge che andrebbero invece ulteriormente abbassati. E’ davvero questo ciò che gli italiani vogliono?

Health Phys. 2018 Jul;115(1):126-139. doi: 10.1097/HP.0000000000000871.
Genotoxic Effects in Human Fibroblasts Exposed to Microwave Radiation.
Franchini V1,2, Regalbuto E1,2, De Amicis A1, De Sanctis S1, Di Cristofaro S1, Coluzzi E2, Marinaccio J2, Sgura A2, Ceccuzzi S3, Doria A3, Gallerano GP3, Giovenale E3, Ravera GL3, Bei R4, Benvenuto M4, Modesti A4, Masuelli L5, Lista F1.
https://www.ncbi.nlm.nih.gov/pubmed/29787439
Abstract
In the last decades, technological development has led to an increasing use of devices and systems based on microwave radiation. The increased employment of these devices has elicited questions about the potential long-term health consequences associated with microwave radiation exposure. From this perspective, biological effects of microwave radiation have been the focus of many studies, but the reported scientific data are unclear and contradictory. The aim of this study is to evaluate the potential genotoxic and cellular effects associated with in vitro exposure of human fetal and adult fibroblasts to microwave radiation at the frequency of 25 GHz. For this purpose, several genetic and biological end points were evaluated. Results obtained from comet assay, phosphorylation of H2AX histone, and antikinetochore antibody (CREST)-negative micronuclei frequency excluded direct DNA damage to human fetal and adult fibroblasts exposed to microwaves. No induction of apoptosis or changes in prosurvival signalling proteins were detected. Moreover, CREST analysis showed for both the cell lines an increase in the total number of micronuclei and centromere positive micronuclei in exposed samples, indicating aneuploidy induction due to chromosome loss.

Altri studi recenti

Environ Res. 2018 Aug;165:484-495. doi: 10.1016/j.envres.2018.01.016. Epub 2018 Apr 11.
5 G wireless telecommunications expansion: Public health and environmental implications.
Russell CL1.
https://www.ncbi.nlm.nih.gov/pubmed/29655646
Abstract
The popularity, widespread use and increasing dependency on wireless technologies has spawned a telecommunications industrial revolution with increasing public exposure to broader and higher frequencies of the electromagnetic spectrum to transmit data through a variety of devices and infrastructure. On the horizon, a new generation of even shorter high frequency 5G wavelengths is being proposed to power the Internet of Things (IoT). The IoT promises us convenient and easy lifestyles with a massive 5G interconnected telecommunications network, however, the expansion of broadband with shorter wavelength radiofrequency radiation highlights the concern that health and safety issues remain unknown. Controversy continues with regards to harm from current 2G, 3G and 4G wireless technologies. 5G technologies are far less studied for human or environmental effects. It is argued that the addition of this added high frequency 5G radiation to an already complex mix of lower frequencies, will contribute to a negative public health outcome both from both physical and mental health perspectives. Radiofrequency radiation (RF) is increasingly being recognized as a new form of environmental pollution. Like other common toxic exposures, the effects of radiofrequency electromagnetic radiation (RF EMR) will be problematic if not impossible to sort out epidemiologically as there no longer remains an unexposed control group. This is especially important considering these effects are likely magnified by synergistic toxic exposures and other common health risk behaviors. Effects can also be non-linear. Because this is the first generation to have cradle-to-grave lifespan exposure to this level of man-made microwave (RF EMR) radiofrequencies, it will be years or decades before the true health consequences are known. Precaution in the roll out of this new technology is strongly indicated. This article will review relevant electromagnetic frequencies, exposure standards and current scientific literature on the health implications of 2G, 3G, 4G exposure, including some of the available literature on 5G frequencies. The question of what constitutes a public health issue will be raised, as well as the need for a precautionary approach in advancing new wireless technologies.

Int J Oncol. 2019 Jan;54(1):111-127. doi: 10.3892/ijo.2018.4606. Epub 2018 Oct 24.
Comments on the US National Toxicology Program technical reports on toxicology and carcinogenesis study in rats exposed to whole-body radiofrequency radiationat 900 MHz and in mice exposed to whole-body radiofrequency radiation at 1,900 MHz.
Hardell L1, Carlberg M1.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6254861/
Abstract
During the use of handheld mobile and cordless phones, the brain is the main target of radiofrequency (RF) radiation. An increased risk of developing glioma and acoustic neuroma has been found in human epidemiological studies. Primarily based on these findings, the International Agency for Research on Cancer (IARC) at the World Health Organization (WHO) classified in May, 2011 RF radiation at the frequency range of 30 kHz 300 GHz as a ‘possible’ human carcinogen, Group 2B. A carcinogenic potential for RF radiation in animal studies was already published in 1982. This has been confirmed over the years, more recently in the Ramazzini Institute rat study. An increased incidence of glioma in the brain and malignant schwannoma in the heart was found in the US National Toxicology Program (NTP) study on rats and mice. The NTP final report is to be published; however, the extended reports are published on the internet for evaluation and are reviewed herein in more detail in relation to human epidemiological studies. Thus, the main aim of this study was to compare earlier human epidemiological studies with NTP findings, including a short review of animal studies. We conclude that there is clear evidence that RF radiation is a human carcinogen, causing glioma and vestibular schwannoma (acoustic neuroma). There is some evidence of an increased risk of developing thyroid cancer, and clear evidence that RF radiation is a multi site carcinogen. Based on the Preamble to the IARC Monographs, RF radiation should be classified as carcinogenic to humans, Group 1.

Int J Oncol. 2017 Aug;51(2):405-413. doi: 10.3892/ijo.2017.4046. Epub 2017 Jun 21.
World Health Organization, radiofrequency radiation and health – a hard nut to crack (Review).
Hardell L1.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5504984/
Abstract
In May 2011 the International Agency for Research on Cancer (IARC) evaluated cancer risks from radiofrequency (RF) radiation. Human epidemiological studies gave evidence of increased risk for glioma and acoustic neuroma. RF radiation was classified as Group 2B, a possible human carcinogen. Further epidemiological, animal and mechanistic studies have strengthened the association. In spite of this, in most countries little or nothing has been done to reduce exposure and educate people on health hazards from RF radiation. On the contrary ambient levels have increased. In 2014 the WHO launched a draft of a Monograph on RF fields and health for public comments. It turned out that five of the six members of the Core Group in charge of the draft are affiliated with International Commission on Non-Ionizing Radiation Protection (ICNIRP), an industry loyal NGO, and thus have a serious conflict of interest. Just as by ICNIRP, evaluation of non-thermal biological effects from RF radiation are dismissed as scientific evidence of adverse health effects in the Monograph. This has provoked many comments sent to the WHO. However, at a meeting on March 3, 2017 at the WHO Geneva office it was stated that the WHO has no intention to change the Core Group.

Bioelectromagnetics. 2017 Jul;38(5):329-338. doi: 10.1002/bem.22052. Epub 2017 Apr 20.
Acute effects of radiofrequency electromagnetic field emitted by mobile phone on brain function.
Zhang J1, Sumich A2, Wang GY3.
https://www.ncbi.nlm.nih.gov/pubmed/28426166
Due to its attributes, characteristics, and technological resources, the mobile phone (MP) has become one of the most commonly used communication devices. Historically, ample evidence has ruled out the substantial short-term impact of radiofrequency electromagnetic field (RF-EMF) emitted by MP on human cognitive performance. However, more recent evidence suggests potential harmful effects associated with MP EMF exposure. The aim of this review is to readdress the question of whether the effect of MP EMF exposure on brain function should be reopened. We strengthen our argument focusing on recent neuroimaging and electroencephalography studies, in order to present a more specific analysis of effects of MP EMF exposure on neurocognitive function. Several studies indicate an increase in cortical excitability and/or efficiency with EMF exposure, which appears to be more prominent in fronto-temporal regions and has been associated with faster reaction time. Cortical excitability might also underpin disruption to sleep. However, several inconsistent findings exist, and conclusions regarding adverse effects of EMF exposure are currently limited. It also should be noted that the crucial scientific question of the effect of longer-term MP EMF exposure on brain function remains unanswered and essentially unaddressed.

Environ Int. 2015 Dec;85:343-51. doi: 10.1016/j.envint.2015.09.025. Epub 2015 Oct 30.
Memory performance, wireless communication and exposure to radiofrequency electromagnetic fields: A prospective cohort study in adolescents.
Schoeni A1, Roser K1, Röösli M2.
https://www.ncbi.nlm.nih.gov/pubmed/26474271
Abstract
BACKGROUND:
The aim of this study is to investigate whether memory performance in adolescents is affected by radiofrequency electromagnetic fields (RF-EMF) from wireless device use or by the wireless device use itself due to non-radiation related factors in that context.
METHODS:
We conducted a prospective cohort study with 439 adolescents. Verbal and figural memory tasks at baseline and after one year were completed using a standardized, computerized cognitive test battery. Use of wireless devices was inquired by questionnaire and operator recorded mobile phone use data was obtained for a subgroup of 234 adolescents. RF-EMF dose measures considering various factors affecting RF-EMF exposure were computed for the brain and the whole body. Data were analysed using a longitudinal approach, to investigate whether cumulative exposure over one year was related to changes in memory performance. All analyses were adjusted for relevant confounders.
RESULTS:
The kappa coefficients between cumulative mobile phone call duration and RF-EMF brain and whole body dose were 0.62 and 0.67, respectively for the whole sample and 0.48 and 0.28, respectively for the sample with operator data. In linear exposure-response models an interquartile increase in cumulative operator recorded mobile phone call duration was associated with a decrease in figural memory performance score by -0.15 (95% CI: -0.33, 0.03) units. For cumulative RF-EMF brain and whole body dose corresponding decreases in figural memory scores were -0.26 (95% CI: -0.42, -0.10) and -0.40 (95% CI: -0.79, -0.01), respectively. No exposure-response associations were observed for sending text messages and duration of gaming, which produces tiny RF-EMF emissions.
CONCLUSIONS:
A change in memory performance over one year was negatively associated with cumulative duration of wireless phone use and more strongly with RF-EMF dose. This may indicate that RF-EMF exposure affects memory performance.

VARIE

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597695/?fbclid=IwAR17dkSO00DCABJhELOtw-Z7mbnR3-yGodJ8WkursowjT0kUctpG1Ib3D0U

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A joint metabolomic and lipidomic workflow is used to account for a potential effect of millimeter waves (MMW) around 60 GHz on biological tissues. For this purpose, HaCaT human keratinocytes were exposed at 60.4 GHz with an incident power …
Logo of scirep
. 2019; 9: 9343.
Published online 2019 Jun 27. doi: 10.1038/s41598-019-45662-6
PMCID: PMC6597695
PMID: 31249327

Untargeted metabolomics unveil alterations of biomembranes permeability in human HaCaT keratinocytes upon 60 GHz millimeter-wave exposure

Associated Data

Supplementary Materials
Data Availability Statement

Abstract

A joint metabolomic and lipidomic workflow is used to account for a potential effect of millimeter waves (MMW) around 60 GHz on biological tissues. For this purpose, HaCaT human keratinocytes were exposed at 60.4 GHz with an incident power density of 20 mW/cm², this value corresponding to the upper local exposure limit for general public in the context of a wide scale deployment of MMW technologies and devices. After a 24h-exposure, endo- and extracellular extracts were recovered to be submitted to an integrative UPLC-Q-Exactive metabolomic and lipidomic workflow. R-XCMS data processing and subsequent statistical treatment led to emphasize a limited number of altered features in lipidomic sequences and in intracellular metabolomic analyses, whatever the ionization mode (i.e 0 to 6 dysregulated features). Conversely, important dysregulations could be reported in extracellular metabolomic profiles with 111 and 99 frames being altered upon MMW exposure in positive and negative polarities, respectively. This unexpected extent of modifications can hardly stem from the mild changes that could be reported throughout transcriptomics studies, leading us to hypothesize that MMW might alter the permeability of cell membranes, as reported elsewhere.

Subject terms: Metabolomics, Diagnostic markers

Introduction

Owing to the saturation of the lower part of the microwave spectrum and to the growing demand for higher data rates, the millimeter waves (MMW) are increasingly used for wireless communications, i.e. for Wireless Local/Personal/Body Area Networks (WLAN, PAN, BAN) or in the context of the upcoming 5 G mobile standard. These electromagnetic radiations correspond to frequencies ranging from 30 to 300 GHz (free-space wavelengths spanning from 10 to 1 mm). Interestingly, MMW have been used for therapeutic purposes indicating that physiological processes might be altered upon exposure to these radiations. Accordingly, the assessment of the potential effects of MMW on human health is thus of paramount importance prior to the wide scale deployment of technologies exploiting this band. MMW are known for their shallow penetration of human tissues (between a few tenth of millimeter to a millimeter), making skin keratinocytes and free nerve endings the primary targets for MMW. It is assumed that some general effects of MMW may be initiated by the release of skin-secreted factors into the blood stream or through the stimulation of superficial free nerve endings,.

The shallow penetration depth of MMW in skin, results in elevated levels of Specific Absorption Rates (SAR) compared to lower microwave frequencies with the same Incident Power Density (IPD). As a consequence, this leads to a noticeable local heating for IPD exceeding typically 5 mW/cm2. Accordingly, one of the main safety concerns regarding these frequencies is the local heating of skin caused by the absorption of MMW energy in the human body. Whether electromagnetic-field specific effects occur upon exposure to MMW or not has been a matter of debate for a long period of time. Besides the warm-up effect, the mechanisms accounting for non-thermal effects occurring at low-power exposure have not been fully demonstrated yet so that their understanding remains an open challenge. Consequently, the guidelines established by the International Commission on Non Ionizing Radiation Protection (ICNIRP) aim at circumventing thermal hazards associated with MMW exposure. This led to define two exposure scenarios. For far-field exposure, the IPD is limited to 1 mW/cm². For body-centric wireless networks that are related to very limited exposition area, the IPD limit is set at 20 mW/cm² (averaged over 1 cm²). While such exposure scenarios may trigger a heat-shock response, it is assumed that the thermoregulation due to blood flow effect might be efficient to avoid any thermal damage.

Notwithstanding decades of investigations and a significant number of studies dedicated at unravelling the effects of MMW on biosystems, the number of reliable and reproducible experimental data remains scarce, most likely stemming for difficulties in dissociating thermal and electromagnetically pure effects. Our group aimed at evaluating the biological effects of MMW in the 60 GHz band. Previous in vitro studies revealed that MMW at 57–64 GHz exert no influence on protein homeostasis for IPD low enough to prevent any temperature increase. Moreover, transcriptomic studies highlighted no, or very weak, effect of MMW on keratinocyte gene expression under athermal conditions. By contrast, it must be mentioned that some authors have pointed out the MMW effects on cell membranes, indicating that permeability changes could be induced by a direct effect on membrane proteins or phospholipids domain organization. The possibility of permeation across the lipid bilayer led us to consider the metabolomic profiling of human keratinocytes as a pertinent next step to better understand the interactions between MMW radiation and cell membrane in the context of human body exposure to 60 GHz waves. The goal of the present study is to apply an untargeted metabolomic strategy based on UHPLC-HRMS assessment of metabolic changes appearing upon exposure to MMW in human HaCaT keratinocytes cell lines. Cells were exposed at 60.4 GHz with an IPD of 20 mW/cm² with the temperature being artificially maintained constant between non-exposed and exposed samples. As metabolites represent the sharp end of systems biology, including multiple up-stream steps, the keratinocytes were exposed for 24 hours to enable possible changes. Then, to afford as wide a coverage of HaCaT keratinocytes chemistry as possible, lipidomic and metabolomic profilings were performed in both intra- and extracellular fractions in positive and negative polarities using a metabolomic workflow that we had previously validated.

Results and Discussion

Data quality assurance

The analytical strategy adopted herein was based on two different chromatographic separation methods (HILIC and RPLC, see experimental section) coupled to detection in mass spectrometry involving both positive and negative-ion modes. This strategy was applied by distinguishing the endo- and exocellular fractions as well to deepen the coverage of the metabolome. As a result, this led to analyze each biological sample 8 times, in distinct sessions of analyses.

Prior to any data processing or analysis, several quality checks were adopted (i.e column pressure checking, variations of internal standards in QC/study samples and instrumental stability in terms of retention time and accurate masses) along the whole batch. Accordingly, internal standards were spiked into each sample prior to LC-HRMS analyses to assess retention time stability, the consistency of signal intensities and mass accuracy along the whole batch. No significant drift in retention time could be evidenced. Likewise, standard accurate mass measurements errors always remained below 5 ppm. For metabolomic analyses, the selected external standards were leucine-d3 (RT 5.97 min), tryptophan-d3 (RT 6.10 min), indole acetic acid-d5 (RT, 1.81 min) and tetradecanedioic acid-d24 (RT 1.38 min). Creatine-d3(RT 8.22 min) and L-lysine-d4 (RT 14.31 min) were selected as internal standards. As to lipidomic sequences, phosphatidylcholine (15:0) (RT 8.37 min), lysophosphatidylcholine (15:0) (RT 1.75 min), ceramide (d18:1) (RT 17.95 min), C15:0 (RT 3.19 min) and C23:0 (RT 9.27 min) were used as external standards, whereas C17:0 (RT 4.30 min) served as an internal standard. All these standards revealed a coefficient of variation below 30% except in the case of C17:0 which displayed a slightly higher value in negative-ion mode of the exo-lipidomics sequence (33.7%) (Tables S1 and S2).

The analysis of QC samples served as a further criterion to establish both the quality of the generated data and the stability of the instrumental platform. For this purpose, it is highly recommended that QC peak tables should pass pre-determined criteria, a widely admitted one being that a majority of features (i.e. 70%) shows a coefficient of variation less than 30%. With respect to this parameter, most sequences displayed satisfying values, with only negative-ion mode exo-lipidomics sequence exhibiting a slightly lower value of 56.3% (Table 1). The stability of the analytical instrumentation could also be assessed based on the clustering of the QC samples in a Principal Component Analysis (PCA) score plot of all tested sequences.

Table 1

Validation of the generated data set using R-XCMS data processing.

Metabolome Fraction Ion mode Ions Ions with CV QC < 30% (% of total ions) Ions with both CV QC < 30% and FC > 2
Exo Lipido Positive 3081 2161 (70.82) 289
Negative 1920 1126 (56.25) 126
Metabo Positive 2750 2076 (75.42) 662
Negative 1632 1263 (77.37) 506
Endo Lipido Positive 4295 3920 (91.27) 540
Negative 2468 2164 (87.68) 264
Metabo Positive 1910 1525 (79.84) 210
Negative 1017 843 (83.00) 58

Altogether, these parameters underscore both the robustness of the LC-HRMS system operating conditions and the validity of the retrieved metabolomic data, paving the way for their statistical processing.

Lipidomic RPLC-MS analyses

As to intracellular fractions, the filtered data subsets were represented by 540 and 264 features, for positive and negative ionization polarities respectively. Non-exposed and exposed samples clustered separately, being discriminated along PC1 component in these two sequences (Fig. 1).

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PCA plots generated from endocellular lipidomic sequences from the features displaying both a Fold-Change ≥2 and a p-value < 0.05 upon R-XCMS data processing performed from UHPLC-HRMS sequences recorded in (A) Positive-Ion Mode and (B) Negative-Ion Mode. Note that SHi (with i = 1 to 4) and MMWj (with j = 1 to 4) are related to the non-exposed and exposed samples, respectively; QC represents the overall of the quality control samples.

In positive and negative polarities, univariate analyses respectively identified six and four masses as the main discriminators between the two sample groups (Figs S1 and S2). Regarding positive-ion mode, tentative hits could be retrieved for two out of five species, which might correspond to ceramide derivatives and/or N-palmitoylsphingosine derivatives (Table S3). Putative matches could be proposed for two ions appearing as dysregulated in negative-ion mode, tentatively corresponding to a further ceramide derivative (Cer (d18:0/16:0)) and to a diglyceride derivative DG (36:3) (Table S4). Besides the very limited number of features appearing as dysregulated, one should note that their corresponding Fold-Change indices display modest values remaining below 5 for all features identified in the course of negative-ion mode and below 10 for those pinpointed throughout positive-ion mode analyses. The only exception was M453T283 in this latter group, for which no putative identification could be retrieved.

Filtered RPLC datasets related to extracellular fractions consisted of 289 and 126 features in positive and negative-ion modes, respectively. In both ionization modes, PCA plots accounted for roughly 60% of the total variance between the two groups. However, whatever the considered ionization mode, these plots did not lead to a clear-cut discrimination of the samples according to their exposure status (Fig. 2).

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PCA plots yielded by exocellular lipidomic analyses from the ions having a Fold-Change ≥2 with a p-value < 0.05 upon R-XCMS data processing performed from UHPLC-HRMS sequences recorded in (A) Positive-Ion Mode and (B) Negative-Ion Mode. Note that SHi (with i = 1 to 4) and MMWj (with j = 1 to 4) are related to the non-exposed and exposed samples, respectively; QC represents the overall of the quality control samples.

While no discriminating feature could be highlighted during the course of negative-ion mode data processing, two ions of interest could however be evidenced in positive polarity, i.e. M627T51 and M783T478 (Fig. S3). Putative hits could only be retrieved for this latter feature that displayed an important FC of 764, which corresponds to a phosphatidylcholine derivative with a sum composition of (36:4) (Table S5).

Metabolomic HILIC analyses

Endocellular data subset with features satisfying both FC > 2 and ANOVA p-value < 0.05 were respectively represented by 210 and 58 features in positive and negative-ion modes. Generated PCA plots roughly represented 75% of the total variance and could discriminate between non-exposed and exposed samples along PC2 component in both ion modes (Fig. 3).

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PCA plots obtained from endocellular metabolomic analyses from the features displaying both a Fold-Change ≥2 and a p-value < 0.05 upon R-XCMS data processing performed from UHPLC-HRMS sequences recorded in (A) Positive-Ion Mode and (B) Negative-Ion Mode. Note that SHi (with i = 1 to 4) and MMWj (with j = 1 to 4) are related to the non-exposed and exposed samples, respectively; QC represents the overall of the quality control samples.

Notwithstanding this discrimination, the statistical processing workflow did not lead to emphasize any relevant biomarker in the negative-ion mode data set. Positive-ion mode endometabolomics only resulted in highlighting one biomarker with a moderate FC value of 2.8 for which two putative identifications could be retrieved from HMDB database (i.e. either 2-aminoheptanedioic acid or N-carboxyethyl-γ-aminobutyric acid) (Fig. S4 and Table S6).

PCA plots related to exometabolomic sequences revealed clear-cut discriminations according to millimeter wave exposure status in both polarities. Owing to the large number of features in the filtered data subset in positive-ion mode (662), the frames were split into two halves prior to generating the PCA plots after being ordered by increasing masses. For both these data subsets, PCA plots roughly accounted for 70% of the total variance (ca. 60% for PC1 and ~ 9% for PC2). In these two groups, the samples could be discriminated in a straightforward manner along PC1 component (Fig. 4A,B). Regarding negative-ion mode, the PCA plot (engulfing 506 features) accounted for 74.1% of the total variance among the two groups, where PC1 and PC2 had respective contributions of 62.7 and 8.4%. Control and exposed samples also exhibited an obvious separation along PC1 dimension (Fig. 4C).

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PCA plots of exocellular metabolomics sequences, selecting ions having both a FC > 2 and a p-value < 0.05 upon R-XCMS data processing performed from UHPLC-HRMS sequences recorded in (A,B) Positive-Ion Mode and (C) Negative-Ion Mode. Note that SHi (with i = 1 to 4) and MMWj (with j = 1 to 4) are related to the non-exposed and exposed samples, respectively; QC represents the overall of the quality control samples.

The careful validation of both extracted ion chromatograms and box-and-whisker plot as described in the Data Processing section led applied to retainment of 111 and 99 features as dysregulated in positive and negative polarities, respectively (Figs S5 and S6). The current study was performed using a single HRMS analyser, so that the identification of the dysregulated features could only rely on exact mass data, including any limitation coming along with it in terms of structural resolution. Resultantly, the structural assignments remain tentative and most often do not lead to reach unambiguous metabolites. From a practical viewpoint, handling an array of references covering the structural diversity of candidate biomarkers is not a realistic purpose in the frame of a metabolomic study pinpointing such an elevated number of features of interest. Despite this structural uncertainty, these putative identifications reveal that dysregulated features encompass an array of structurally diverse metabolites (Tables S7 and S8). Emphasizing such a large extent of dysregulations, especially in the exo-metabolomic sequences, appears as an intriguing outcome. Indeed, as upstream transcriptomics analyses did not highlight significant alterations in gene expression, such dramatic changes would not have been expected. This led us to assume that these modifications might not be related to modifications of enzyme expression but rather stem from alterations in membrane permeability. Cell membranes are regarded as major targets for the interactions between millimeter waves and biological systems since a variety of bioeffects were reported upon exposure to these radiofrequencies. As an example, 60 GHz exposure with an incident power density of 0.9 mW/cm² (i.e. in the typical range of values expected from wireless communications) was proved to induce structural modifications of artificial biomembranes. Consequently, MMW exposure was demonstrated to increase the lateral pressure of phospholipid monolayers although not strongly enough to disturb phospholipid microdomain organization in biomembranes. Further biochemical processes could be evidenced such as the externalization of phosphatidylserine, even though the biological relevance of this event remains to be determined. Likewise, 53-GHz radiations and 130-GHz pulse modulated exposures were shown to alter the permeability of phospholipid vesicles. MMW action on the orientation of charged and dipolar molecules in the region located between the aqueous phase and the hydrocarbon interior of the membrane has been hypothesized to represent the driving force to rearrange phospholipids bilayers, resulting in an increase of small molecules permeability across the membrane. This new organization of the bilayer presumably displays a higher curvature which would elicit metabolites leakage. It can also be assumed that electromagnetic radiations of specific frequencies might excite components of cell membrane, depending on their electric dipoles and possibly leading to form Bose-condensed phonons. Such assumptions are consistent with structural changes observed in cells upon MMW exposure, with different deformations being reported in bacteria.

Likewise, it can be assumed that a similar phenomenon might trigger the leakage of intracellular metabolites into the extracellular medium shown in this manuscript. This inference is further strengthened by the huge majority of dysregulated features which are found to be upregulated in the treated group (107/111 and 98/99 in positive and negative-ion modes, respectively. It is interesting to note that under different exposure conditions, membrane permeabilization upon electric pulses of nanosecond duration were previously reported to occur in mammalian cells,.

Conclusion

As far as can be ascertained, this report represents the first metabolomic investigation focusing on the effects of MMW. To get as wide an insight into cellular processes as possible, a joint metabolomic and lipidomic profiling strategy was designed and the extra and intracellular contents were discriminated. It appeared that all lipidomic sequences and intracellular metabolomic profiles were slightly affected by MMW but drastic changes in extracellular metabolomic sequences could be evidenced. During these experiments, we put great emphasis on controlling cell culture parameters (temperature, pH, incubator humidity). Much attention has been paid to temperature control, however, it cannot be ruled out that unexpected changes at subcellular scale have occurred and could be responsible for the differences found. Moreover, by making the choice to put the non-exposed control in the same place in our exposure system, we ensure that these cells are in the same growing conditions that those that are exposed. Nevertheless, it should be noted that this strategy has the disadvantage of inducing a shift of one day of culture between the control and the exposed samples. While the unusually high number of dysregulated features makes of their unambiguous structural assignment an unrealistic purpose, the current study enables drawing significant and unprecedented conclusions as to the effects of MMW exposure on cellular systems, especially when combining them with former studies carried out by our group. Accordingly, as upstream pan-transcriptomic studies in this cellular system did not led to emphasize any significant change upon 60-GHz MMW exposure, it is reasonable to assume that the vast amount of dysregulations reported in these sequences do not stem from alterations of gene expression but rather from alterations in membrane permeability, consistently with previous reports on acellular phospholipidic systems. The tentative metabolites identified throughout the current workflow might serve as a ground to focus on subsets of metabolites through the so-called target-based metabolomics. For this purpose, multiplex LC-MS-MRM pipelines proved useful for the quantitative profiling of some of the hundreds of expected metabolites in complex biological samples with no structural/identity ambiguity. Based on the current findings, such follow-up studies could be limited to the exocellular compartment. Finally, we can conclude that our model, purely in vitro, haven’t to be lead to a direct extrapolation of our results at the organism level. In the future, further studies will be necessary to assess MMW bioeffects on animal models and to investigate potential dysregulations induced by lower IPD values prior to the wide-scale deployment of technologies based on these specific frequencies.

Methods

Chemicals, reagents and materials

LC-MS grade water, methanol (MeOH), methyl tert-butyl ether (MTBE), chloroform (CHCl3), acetonitrile (MeCN), 2-propanol (IPA), ammonium acetate and acetic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). The standard mixtures Calmix-positive (i.e. caffeine, L-methionyl-arginyl-phenylalanylalanine acetate and Ultramark 1621) and Calmix-negative (i.e. acetic acid, sodium dodecyl sulfate, taurocholic acid sodium salt hydrate and Ultramark 1621), were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

1,2-Dipentadecanoyl-sn-glycero-3-phosphocholine (PC(15:0/15:0)), 1-pentadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (Lyso PC(15:0)), 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine (PE(17:0/17:0)) and N-heptadecanoyl-D-erythro-sphingosine (Cer(d18:1/17:0)) were obtained from Coger (Paris, France). L-Tryptophan-2,3,3-d3 (Trypto-d3), indole-2,4,5,6,7-d5-3-acetic acid (Ind-AA-d5), 1,14-tetradecanedioic-d24 acid (Tetra-A-d24) were purchased from Cluzeau Info Labo (Sainte-Foy-La-Grande, France). 1,2,3-triheptadecanoyl-glycerol (TG (17:0)), pentadecanoic acid (C15:0), tricosanoic acid (C23:0), heptadecanoic acid (C17:0), leucine-5,5,5-d3 (Leu-d3), creatine-(methyl-d3) (Crea-d3) and lysine-4,4,5,5-d4 (Lys-d4) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Stock standard solutions (1 mg/L) were prepared in CHCl3 (for lipidic compounds) or in MeOH and kept at −20 °C. The internal standard (IS) solution contains C17:0, Crea-d3 and Lys-d4 at 10 ng/µL in a MeOH/H2O mixture (4/1). Lipidomic external standard (ES) solution containing (PC (15:0/15:0), Lyso PC (15:0), PE (17:0/17:0), TG (17:0), Cer (d18:1/17:0), C15:0 and C23:0 at 0.5 ng/µL and metabolomic (ES) solution containing Leu-d3, Trypto-d3, Ind-AA-d5 and Tetra-A-d24 at 1 ng/µL were subsequently prepared in CHCl3 (lipidomics ES) or in MeOH (metabolomics ES).

Cell culture

A human keratinocyte cell line (HaCaT) was cultured as described elsewhere. In an attempt to circumvent any senescence or drift of the cellular populations, keratinocytes were exposed at early passages (between 10 and 16). To enable proper cross-sample comparison, the quantity of cellular material was estimated through the evaluation of total ERK protein amount (Western Blot using anti ERK1 (K-23) antibody (Santa Cruz Biotechnology, Dallas, Texas, USA)). The exposure medium designed to keep pH buffering in the non-gassed incubator of the exposure system as described elsewhere. Briefly, it consists in powder reconstituted DMEM medium, completed with fetal calf serum, 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES) and antibiotics.

Experimental setup for sample exposition

Sample irradiation was performed as previously reported. A thorough description of the exposure system can be found in Zhadobov et al.. Briefly, a 6-well culture plate was positioned in a MEMMERT UE400 incubator to be exposed from the bottom by a standard pyramidal horn antenna. Cells were then irradiated or not by this antenna with an average incident power density (IPD) of 20 mW/cm² for 24 hours, according to the guidelines established by the International Commission on Non-Ionizing Radiation Protection (ICNIRP) for MMW with limited exposition area. Both MMW-exposures and non-exposed samples were performed inside the same incubator, on different days. To mitigate exposure variability between sample groups, the non-exposed and MMW-Exposed cell samples were located in the same position within the same incubator. To avoid a thermal effect associated with MMW exposure, the temperature increase was counteracted by lowering the incubator set point by the predicted increment. Consistently with literature, MMW exposure at such IPD resulted in an elevation of 8 °C leading to set the temperature of the incubator at 28 °C in the MMW group to reach 36 °C, as in non-exposed cells. Assuming that thermal convection currents occurring in the radiofrequency-exposed groups may lead to a differential condensation of the culture medium, the consistency of the pH value was monitored in both non-exposed and exposed sample groups. An identical pH value of 7.7 could be measured from the different culture media, irrespective of their exposure status. HaCaT keratinocytes cell viability was formerly reported to be of 100% for pH values spanning across the 7.0–8.2 range.

Metabolomics workflow

As MMW exposure might result in evidencing little to no biological effect, it was very important to validate the ability of the retained metabolomic workflow to emphasize the dysregulations triggered by a known interfering agent. This preliminary study, carried out using the cytotoxic drug 2-deoxyglucose, demonstrated the adequacy of the proposed workflow for metabolomics purposes. The whole sample preparation workflow is summarized in Fig. 5.

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