A mechanistically approached review upon assorted cell lines stimulated by athermal electromagnetic irradiation

Figures & data

Figure 1. Non-ionizing electromagnetic field spectrum of alternating current ranged between 0–300 GHz frequencies along with several artificial sources.

Figure 2. Common frequencies applied to investigate assorted cell lines through different endpoints.

Table 1. EMF characteristics in the extremely low-frequency range (0–300 Hz) utilized to expose assorted cell lines, along with subsequent biological effects.

Signal (Reference)	EMF emitter (frequency/form/duration/continuity/modulation/direction*)| EMF meter| Dosimeter	Cell line (organism/tissue/morphology)| Temperature	Endpoint** (duration of exposure or postexposure; biomarker; assay; Kit)	Results
Sequential 50/385-Hz pulsed MF [6]	Solenoid magnet (50 Hz and 385 Hz; each for 5 min; perpendicular)| Gaussmeter (20 mT).	A549 (human/lung carcinoma); hLEC (human/lung lymphatic microvascular endothelial); LLC (mouse/lung/epidermoid carcinoma); MDA-MB-231 (human/breast carcinoma)| Room temperature.	Membrane integrity (cytolytic assays, luminescence-based kit, protease release); HS binding capacity (fluorescent FGF-2 binding, flow cytometry) for glycocalyx HS assessment; Sialic acid digestion (AUS sialidase, lectin markers of MAL-II or SNL, flow cytometry); Viability (4-h postexposure, luminescence-based ATP assay); Proliferation (cell count, hemocytometer); Scanning electron microscopy.	A549$\to$Decreased viability; Induced unique membrane rippling and nanoscale pores; Altered membrane integrity due to induced glycocalyx HS charge; Pretreatment with heparin lyase III eliminated this effect by digesting anionic sulfated glycan polymers.| Similar effects occurred in LLC cells. MDA-MB-231$\to$Altered membrane integrity due to induced sialic-acid rich glycocalyx; Pretreatment with AUS sialidase partially abrogated this effect by removing anionic sialic acid.
				hLEC$\to$Unaffected membrane integrity.
50-Hz environmental exposure [10]	Helmholtz coils inside an incubator (50 Hz; sinusoidal; 200 mA; 5 days; continuous; perpendicular)| Tesla meter TM-701 (1 mT RMS)| Simulation by COMSOL 3.4 (11 mT for 50-Hz AC).	CD44^+/CD24^– of MDA-MB-231 cells (human/breast carcinoma)| ${37}^{\circ }\mathrm{C}$.	Cell proliferation or viability (cell counting Kit-8, OD450 nm); RNA expression (RT-PCR, spectrophotometer, OD260 and 280 nm); Cell cycle (PI/RNase A staining, ELITE software); Protein level (western blot); Sphere Formation (21-day follow-up); Colony Formation (Soft agar formation assay); Invasion Assay (microscope).	Decreased mRNA expression of the stemness markers, Bmi-1 and Nanog; Decreased proliferation; Induced G0/G1 cell-cycle arrest; Increased PPAR$\gamma$ mRNA and protein expression; Increased p21 and p27 levels; Decreased cyclin D1, cyclin E, CDK4, and CDK2; Decreased colony formation, CSC sphere number, invasion and CCL2 secretion (ELISA kit). PPAR$\gamma$ inhibitor, GW9662, inverted the EMF effects.
75-Hz EMF therapy for hypoxia or LPS [12]	Rectangular coils inside an incubator (75 Hz; 1.3-ms pulse duration; 2, 4, 6, 12, 24, or 48 h; perpendicular)| Hall gaussmeter (1.5$\pm$0.2 mT); standard coil probe (2.0$\pm$0.5 mV); digital oscilloscope.	Neuron cells of SH-SY5Y (human/bone marrow metastasis/neuroblastoma) and PC12 (rat/adrenal medulla/pheochromocytoma); N9 (mouse/microglia (brain))| ${37}^{\circ }\mathrm{C}$.	Hypoxia-stimulated viability (live/dead cell assay, Nikon fluorescent microscope); Apoptosis (active caspase-3 levels, OD450 nm); HIF-1$\alpha$ activity (Abcam’s kit for HIF-1$\alpha$ Transcription Factor Assay, spectrophotometry at wavelength 450 nm)| ROS production (Abcam’s kit for DCFDA cellular ROS detection assay, Fluorescence)$|$ concentrations of cytokines IL-1$\beta$, TNF-$\alpha$, IL-6, and IL-8 (in the LPS presence; ELISA kit, OD450nm).	SH-SY5Y and PC12$\to$Reduced hypoxia-induced cell death and apoptosis after 12-h, 24-h, or 48-h exposure; Decreased HIF-1$\alpha$ activity after 2-h, 4-h, or 6-h exposure and decreased ROS generation after 24-h or 48-h exposure. N9$\to$Decreased hypoxia-induced ROS generation after 24-h or 48-h exposure; Reduced LPS-induced TNF-$\alpha$, IL-1$\beta$, IL-6, and IL-8 releases.
Frequency-varying (25–6 Hz) Thomas-EMF [7]	Three pairs of solenoids inside an incubator (Thomas-EMF 25–6 Hz; 5 V; frequency-modulated; 1 h/day, for 5 days)| Vector magnetometer (peak intensity 2.5–3.5 $\mu$T); Background 60-Hz EMF inside incubator $\prec 1\mu T$ and static geomagnetic field $50\mu T$; Temperature probe.	B16-BL6 (mouse/melanoma); MDA-MB-231 and MDA-MB-468 (human/pleural effusion metastasis/breast carcinoma); BT-20 (human/breast epithelium/carcinoma); MCF-7 (human/breast/invasive breast ductal carcinoma); HeLa (cervical cancer); normal cells of HEK293 (human/embryonic kidney/epithelium), HSG (human/submandibular gland), and HBL-100 (breast)| $37\pm {0.1}^{\circ }\mathrm{C}$.	Cell proliferation via cAMP or ERK signaling pathway (cell numbers measured at days 1, 2, 3, 4, and 5; trypan blue staining, hemocytometer, MTT assay, OD540 nm); cAMP level (cAMP ELISA, Tropix cAMP Screen System, luminescence, plate reader); Expression of phosphorylated ERK, ERK1/ERK2, and GPF (immunoblot analysis, Densitometric analysis).	Decreased proliferation via cAMP pathway in malignant cells but not in normal cells; Pretreatment with forskolin or SQ22536 eliminated EMF-dependent proliferation inhibition in malignant cells; Transiently-altered level of cAMP under EMF/forskolin co-treatment, which was restored to normal within 6–12 h postexposure| Pretreatment with PD98059 decreased EMF-dependent proliferation inhibition in malignant cells; Promoted phosphorylation level of ERK under EMF in malignant cells and under EGF in all cells; Increase in ERK phosphorylation of B16-BL6 cells transfected with PKA-DN and PKC-Reg, which was correlated with decreased proliferation. No changes in cAMP, ERK phosphorylation, or growth of non-malignant cells.
Co-exposure of X-ray and 30-Hz BEMER EMF therapy [11]	BEMER device (30 Hz; 30-ms pulse duration within a period of 18–22 s; for 8 min, 1 h, or 24 h)| 3D teslameter (0–35 $\mu$T).	UTSCC15 (human/head and neck/squamous carcinoma); A549 (human/lung carcinoma); DLD1 (human/colorectal carcinoma); MiaPaca2 (human/pancreatic ductal adenocarcinoma)| ${37}^{\circ }\mathrm{C}$.	Cell metabolism was investigated only in A549; Investigating Basal tumor cell survival; Clonogical cell survival; DNA damage; and ROS scavenger analysis in all cells.	Alterations in the glycolysis and TCA cycle pathways, such as downregulation in pyruvate, succinate, aspartate, and ADP metabolites, and upregulation in serine| No change in basal cell survival, but 8-min or 1-h BEMER exposure and X-ray co-treatment radiosensitized tumor cells (DLD1 showed no sensitization); Induced radiosensitization dependent on EMF/X-ray treatment intervals and EMF frequency (times); Signal intensity-dependent radiosensitization in all cells except DLD1| Increased DNA double-strand break (DSB) in A549 and UTSCC15 cells; Elevated ROS levels led to increased DSB.
10-Hz, 50-Hz, and 100-Hz ELF-PEMFs [5]	Copper coils inside an incubator (10, 50, or 100 Hz; square wave; 2, 4, or 24 h; continuous).	U87 (human/glioblastoma-astrocytoma/epithelial-like)| ${37}^{\circ }\mathrm{C}$.	Expression of cell-cycle proteins cyclin-D1 and P53, and apoptosis proteins Caspase-3 (Western blots); Viability (MTT assay); Morphology.	24-h, 50-Hz, 100-G exposure$\to$Increased viability and cyclin-D1 expression| 24-h, 100-Hz, 100-G exposure$\to$Decreased viability and Cyclin-D1 expression (This also occurred at 10-Hz, 50-G exposure); Elevated P53 and Caspase-3.
				Cell shape and morphology perturbations.
Cells transfected with miR-26b-5p and exposed to the 50-Hz ELF-EMF [9]	Coils inside a chamber (50 Hz; 1, 2, or 3 mT; 5 min on/10 min off, for 72 h; pulsed exposure).	GC-2 (mouse/spermatocyte-derived)| $37\pm {0.3}^{\circ }\mathrm{C}$.	MiRNA and mRNA expressions (quantitative RT-PCR); Viability (Cell counting kit-8, OD450 nm); Apoptosis and cell cycle (flow cytometry, Annexin V-FITC and PI double staining; PtdIns staining); Protein concentration (Western blot).	50-Hz ELF-EMF$\to$ MiR-26b-5p expression did not change at 1 mT, increased at 2 mT, and decreased at 3 mT; Unmethylation status in CTDSD1; Cyclin D2 did not change at 1 mT, decreased at 2 mT, and increased at 3 mT, at both the mRNA and protein levels.
				Transfected miR-26b-5p + 50 Hz EMF caused no effect on viability, apoptosis or cell cycle but decreased the G0/G1-phase cells and slightly increased the S-phase cells.
50-Hz ELF-EMF [8]	Coils inside an incubator (AC 50 Hz, 12 $\mu$T RMS; square wave; DC 45 $\mu$T; 7 days exposure, 3 days exposure, 6 days exposure + 24 h without EMF, or 3 days exposure + 4 days without EMF)$|$ BGF 1–2 $\mu$T.	SKBR3 (human/breast carcinoma); HT29 (human/colon epithelium/adenocarcinoma)| ${37}^{\circ }\mathrm{C}$.	Mitochondrial activity (JC-1 cytofluorimetric evaluation of membrane potential) and mitochondrial mRNA transcription (RT-PCR of COX II and COX IV); ATP (ATP Bioluminescent Assay Kit: FL-AA, Sigma); Expression of mitochondrial proteins (Western blot, DC protein assay); Intracellular LDH activity (OD340 nm).	Increased mitochondrial membrane potential, but unchanged ATP synthesis| Unchanged cytosolic LDH activity| Unaffected mitochondrial mRNA transcription in COX II and IV; Unaffected total lysates and nuclear levels of proteins| Downregulated expression of mitochondrial proteins phospho-ERK, p53, and cytochrome c.

Please note that we use symbols forward slash (/) or vertical bar (|) as separators in Tables to present a more readable text.

*Field direction$\to$Perpendicular, inclined, or parallel means that the electromagnetic field is perpendicular, inclined, or parallel to the culture dish.

**Endpoint$\to$A conclusion that marks a biological effect of irradiation.

**Biological marker or biomarker$\to$A measurable factor that indicates alterations in biological processes under irradiation (endpoint).

**Assay$\to$A procedure to investigate the quantity of a component (biomarker).

**Kit$\to$A set of equipment used to carry out an investigation (assay).

AC$\to$alternating current; ADP$\to$adenosindiphosphate; ATP$\to$Adenosine triphosphate; BEMER$\to$Bio-Electro-Magnetic-Energy-Regulation; BGF$\to$background field; CAMP$\to$cyclic adenosine monophosphate; CDK4$\to$cyclin dependent kinase 4; COX II and IV$\to$cytochrome c oxidase subunits 2 and 4; DSB$\to$double strand break; EGF$\to$epidermal growth factor; ELF$\to$extremely low frequency; ELISA$\to$enzyme-linked immunosorbent assay ERK$\to$extracellular-signal-regulated kinase; FGF-2$\to$fibroblast growth factor-2; forskolin$\to$cAMP level activator; HIF-1$\alpha \to$hypoxia-inducible factor 1$\alpha$; HS$\to$heparan sulfate; IL-1$\beta \to$interleukin-1$\beta$; JC-1$\to$5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethyl-benzimidazolyl carbocyanine iodide LDH$\to$lactate dehydrogenase; LPS$\to$lipopolysaccaaride; MAL-II$\to$Maackia Amurensis Lectin-II; MF$\to$Magnetic field; MTT$\to$3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OD450 nm$\to$Optical density at 450 nm; PEMF$\to$pulsed electromagnetic field; PI$\to$propidium iodide; PKA$\to$protein kinase A; PPAR-$\gamma \to$Peroxisome proliferator-activated receptor $\gamma$; RMS$\to$root mean square; RNA$\to$Ribonucleic acid; ROS$\to$reactive oxygen species; RT-PCR$\to$Reverse transcription-polymerase chain reaction; SNL$\to$Sambucus Nigra Lectin; SQ22536$\to$cAMP level inhibitor; TCA$\to$tricarboxylic acid; TNF-$\alpha \to$tumor necrosis factor–$\alpha$.

Table 2. EMF characteristics in the intermediate frequency range (300 Hz-300 kHz) utilized to expose assorted cell lines, along with subsequent biological effects.

Signal (Reference)	EMF Characteristics	Cell line Features	Endpoint	Results
100-kHz asymmetrically induced electric fields [14]	Helmholtz coil equipped with a microscope (100 kHz; asymmetric sawtooth waveform; continuous; three-time cycle of 10 $\mu$s, 30 $\mu$s)| LCR meter, fluxgate sensor and oscilloscope (100 $\mu$V/cm).	MDA-MB-231 (human/breast carcinoma); MCF10CA1a (human/breast invasive ductal carcinoma); MCF10A (human/breast epithelium)| ${37}^{\circ }\mathrm{C}$.	Motility or directed migration (microfluidic bi-directional microtrack assay, modified transwell migration assay); EGFR expression and Akt signaling (western blot, immunofluorescence staining); F-actin distribution (polarization ratio, Immunofluorescence, phalloidin and DAPI staining).	Increased migration speed and cell persistence under antiparallel iEFs (to the direction of cell migration) without EGF gradients| Decreased cancer cell motility under iEF/EGF gradients (chemokine) and the opposite effect in normal cells| Promoted EGFR aggregation in MDA-MB-231 but not in the other two cells; Downregulated EGFR phosphorylation (at the Tyr-1068 site) and inhibited EGF-promoted F-actin aggregation in the cancerous cells|
				iEF/MK2206 co-treatment$\to$Nullified cell sensitivity to iEF directionality and inhibited EGF-promoted motility.
TTFields at 100 and 200 kHz [13]	Four stainless steel electrode plates dipped into the culture medium (100 or 200 kHz; 72 h; voltages 5–12 Vpp; continuous)| Impedance Analyser and Dielectric Probe Kit; LCR Meter; Oscilloscope DS1102E; Digital temperature sensor.	BT4Ca, C6, F98, and RG-2 cell lines (rat, glioma)| Room temperature$\pm$1.1 K.	Cell proliferation under frequencies 100–1000 kHz at different intensities and times during the Telophase/cytokinesis stage (CASY TT cell counter, laser scanning microscope, EM solver of Sim4Life software for electric field and SAR simulations).	Decreased cell proliferation dependent on commutation time and intensity at frequencies 100 or 200 kHz out of 100, 200, 500, and 1000 kHz, without completely being stopped; Induced excessive electric fields or SAR in the cleavage furrow region for frequencies around 100 kHz with parallel field orientation; Decreased power absorption when the furrow is not parallel to the applied field.
200-kHz TTFields combined with Ca^2+ antagonists [16]	Single-cell TTField applicator (200 kHz; 0, 0.25, 1.25, or 2.5 V/cm; sine waves; 5–7 days; perpendicular).	U251 and T98G (human/glioblastoma/epithelium)| ${37}^{\circ }\mathrm{C}$. ${ }_{free}[C{a}^{2+}{]}_i$ was recorded at Ca^2+-containing NaCl solution, Ca^2+-free NaCl solution, or Ca^2+-containing NaCl solution + L-, N-, T-type Ca^2+ channel blocker, benidipine, or the L-type inhibitor, nifedipine.	Calcium signaling (Fura-2 Fluorescence Imaging of ${ }_{free}[C{a}^{2+}{]}_i$); Ion channel activity in the plasma membrane (patch-clamp recordings); Voltage-gated Ca^2+ ($\mathrm{C}{\mathrm{a}}_v$) channel expressions (quantitative RT-PCR); Cell cycle, cell death (sub-G1 population), cell division (hyper-G population), and clonogenic survival (DNA content, Mitochondrial Membrane Potential, TMRE staining, flow cytometry, and pre-plated colony formation assay).	U251 and T98G$\to$ Increased ${ }_{free}[C{a}^{2+}{]}_i$; Ca^2+-activated BK K${ }^{+}$ channels; ${ }_{free}[C{a}^{2+}{]}_i$ rise was abolished by decrease in extracellular Ca^2+; Contribution of $\mathrm{C}{\mathrm{a}}_v$1.2 channels in the induced dihydropyridine-sensitive Ca^2+ entry. T98G$\to$Decreased G1 population and increased S and G2 populations, augmented by Benidipine; Induced cell death and increased hyper-G population by TTField/Benidipine; Induced $\mathrm{\Delta }{\psi }_m$ dissipation by TTField but not by Benidipine; Decline of clonogenic survival.
				U251$\to$Increased G1 population and decreased G2 population unaffected by Benidipine; Hyper-G population decreased by TTField and unaffected by Benidipine; No cell death occurred; TTField and Benidipine induced $\mathrm{\Delta }{\psi }_m$ dissipation and apoptosis.
300-kHz transient EMF exposure [17]	TEM Cell in an incubator, ${37}^{\circ }\mathrm{C}$, or an open area, ${25}^{\circ }\mathrm{C}$ (300 kHz; 30 kV/m (peak); 25 pulse/min for 1.5 h).	PC3 (human/bone metastasis/prostate carcinoma); MIN6 (mouse/pancreatic $\beta$).	Evaluating necessity of integrated incubator system; Viability (Cell Counting Kit-8, spectrophotometry, OD450 nm); ROS level (DCFH-DA, fluorescence microplate reader); mitochondrial membrane potential (JC-1 staining, fluorescence spectrophotometry).	A significant difference between cell processes in two environments (incubator or open area); Unchanged viability in the incubator but decreased viability in the open area (more decrease in MIN6 cell viability); Higher ROS increase in the incubator than that in open area; Cellular MMP increased in the incubator but decreased in the open area (PC3 was more sensitive).
Amplitude modulated EMF at 500 Hz-22 kHz [15]	Spoon-shaped applicator (carrier frequency 27.12 MHz; Frequency range 500 Hz-22 kHz; 3h/day, for 7 days; Amplitude Modulated)$|$ Dosimeter: EX3DV3 probe S/N 3515 + DASY; dosimetry simulator: SEMCAD X (SARs 30 and 400 mW/kg).	Human HCC Xenograft in mice (in-vivo); MHCC97-L, HCCLM3, Huh7, Hep3B, and HepG2 (human/liver/hepatoblastoma); THLE2 (human/liver/epithelial)$|$ $36.4\pm {0.17}^{\circ }\mathrm{C}$.	Proliferation via protein antigens (tritiated thymidine incorporation assay); Differentiation via Ca^2+ (Fluo-4 Calcium Imaging Kit, Fluostar fluorescent plate reader, flow cytometry; addition of BAPTA/Ethosuximide); shRNA knockdown (CACNA1G, CACNA1H, and CACNA1I shRNA Plasmid Kits); IP3/DAG signalling pathway via microRNAs gene expression (quantitative RT-PCR).	Huh7 in vivo (SAR 67 mW/kg)$\to$Decreased tumor volume, tumor ki-67, and cyclin D1; increased p21; and unchanged BrdU staining, which show proliferation changes and tumor shrinkage. Absence of necrosis; Fibroblast-like cells intertwined with Huh7 cells$|$ Intracellular Ca^2+ increased through CACNA1H in Huh7 for 30-min, 1-h, 3-h, or 6-h exposure, and Hep3B for 3-h exposure; Ethosuximide/BAPTA abrogated HCCMF’s Ca^2+ influx and inhibition of Huh7 cell proliferation$|$ CACNA1H knockdown abrogated downregulation of CSCs and decreased sphere formation in Huh7 and Hep3B.

BAPTA$\to$a chelator of Ca²⁺; BK$\to$big potassium; CACNA1H$\to$$\mathrm{C}{\mathrm{a}}_v$3.2 T-type voltage-gated calcium channels; CFDA-SE or briefly CFSE$\to$5,6-carboxyfluorescein diacetate succinimidyl ester; CSCs$\to$cancer stem cells; $\mathrm{\Delta }{\psi }_m\to$mitochondrial membrane potential; DCFH-DA$\to$$2^{\prime }$,7${ }^{\prime }$dichlorofluorescein diacetate method; EGFR$\to$epidermal growth factor receptor; Ethosuximide$\to$an inhibitor of all three T-type VGCCs ($\mathrm{C}{\mathrm{a}}_v$3.1, 3.2, 3.3); HCC$\to$hepatocellular carcinoma; iEF$\to$induced electric fields; MK2206$\to$an inhibitor of phosphorylation of Akt-1, Akt-2, and Akt-3; MMP$\to$mitochondrial membrane potential; NO$\to$nitric oxide; TMRE$\to$tetramethylrhodamine ethyl ester perchlorate; TTFields$\to$tumor treating fields; TUNEL$\to$terminal deoxynucleotidyl transferase-mediated Nick-End Labeling VGCCs$\to$voltage gated Ca²⁺ channels; Vpp$\to$peak to peak voltage.

Table 3. EMF characteristics in the radio frequency range (300 kHz-300 GHz) utilized to expose assorted cell lines, along with subsequent biological effects.

Signal (Reference)	EMF Characteristics	Cell line Features	Endpoint	Results
900-MHz GSM mobile phone [19]	900-MHz GSM mobile phone connected to a cavity inside an incubator (900 MHz; 1.96 W; 12 or 24 h; continuous)$|$ HFSS/CST microwave software (SAR 0.2–0.4 w/kg); Thermocouple temperature device.	CHO (hamster/ovary/epithelium)$|$ $37.1\pm {0.1}^{\circ }\mathrm{C}$.	Apoptosis (AO/EB staining using flow cytometry); Autophagy (acidic vesicular organelles by flow cytometry); DNA damage (olive moment, comet assay, CBMN assay); Membrane permeability (Trypan blue dye exclusion assay); Redox activity (MTT assay); ROS evaluation (A fluorescent probe, DCFH-DA); Bystander effect (Medium transfer procedure).	No induced apoptosis or autophagy; Decreased colony formation continued with time; Occurrence of Bystander effect and colony reduction due to conditional culture medium; Increased DNA damage after 24-h direct or indirect exposure; Increased extracellular and intracellular ROS; No increase in cell membrane permeability or redox activity.
1.7-GHz LTE RF-EMF [18]	Radial transmission line system using conical antenna inside an incubator (1.7 GHz; SAR 1 or 2 W/kg; 60 W; 72 h; continuous)$|$ SAR measured using fiber optic thermometer.	Human adipose stem cells (ASCs); Huh7 and Hep3B (liver/CSCs); HeLa and SH-SY5Y cancer cells; normal fibroblast IMR-90 cells$|$ $35.5\pm {0.5}^{\circ }\mathrm{C}$.	Proliferation (cell counting using Nexcelom Bioscience); DNA DSB ($\gamma$-H2AX expression, flow cytometry); Apoptosis (cleaved PARP, western blot); Cell cycle (PI staining, DNA content, flow cytometry, cell senescence markers: p21 expression, phosphorylation of p53 at serine 15 and phosphorylation of retinoblastoma (Rb) at serine 780, western blot, $\beta$-galactosidase staining, Nikon microscope); ROS generation (carboxy-H2DCFDA staining, MitoSOX Red staining, fluorescence microscopy).	Decreased proliferation in all cells with the least effect in ASCs. ASCs, Huh7$\to$No DNA DSBs or apoptotic cell death; Induced cell senescence; Expressed molecular markers for cellular senescence including upregulated p21 and p53, downregulated phosphorylation of Rb; Slight delay in the G1-S cell cycle transition; Increased intracellular Reactive Oxygen Species (specific detection of $O_2^{\circ -}$, superoxide anion radical).
Black carbon (BC) and 2.45-GHz EMF co-treatment [21]	GTEM radiation chamber (2.45 GHz; continuous; Sine; 24 or 72 h)$|$ SAR measurement via SEMCAD X (FDTD)-based software (0.4 W/Kg); Isotropic probe (102.8 V/m).	RAW 264.7 macrophage cell line (mouse)$|$ ${37}^{\circ }\mathrm{C}$.	Viability or cytotoxicity (MTT assay, OD540 nm, microplate reader; and Trypan Blue assay, cell counts, Neubauer haemocytometer under a microscope); phagocytic activity (light microscopy); Oxidative stress or NO production (NO assay, Griess reaction); Activity of pro-inflammatory cytokines TNF-$\alpha$ and IL-1$\beta$, and pre-apoptotic activity via caspase-3 expression (quantitative RT-PCR).	Inefficient MTT assay; Under co-treatment, decreased viability assayed via Trypan Blue (BC concentration- and exposure time-dependent); Prolonged phagocytosis decreased with time; The highest nitrite levels obtained in cells stimulated by LPS endotoxin under 24-h exposure; Prolongation of innate and inflammatory immune responses due to increased TNF-$\alpha$, IL-1$\beta$, and caspase-3 levels.
3.5-GHz microwave [20]	Axial vircator (Chundoong) in an open area (3.5 GHz; 260 kV; 10 kA; 20 MW; 50 ns; 5 or 45 shots, each shot after one min, 0.6 J/shot)$|$ Thermal imager FLUKE Ti90.	G-361 and SKMel-31 (human/malignant melanoma); Normal human dermal fibroblast (NHDF) cell line$|$ ${25}^{\circ }\mathrm{C}$.	Viability evaluated with a 5-h, 24-h, 48-h, or 72-h follow-up (Alamar blue (AB) dye, plate-reading spectrometer, fluorescence measurement)$|$ Cell death and Apoptosis (PI uptake, 24-h exposure, flow cytometry; luminescence-based detection of phosphatidyl-serine); ATP generation (Cell Titer-Glo Luminescent Cell Viability Assay, luminescence, microplate reader, and Mitochondrial ToxGlo Assay, luminometer); Proliferation or cell growth (OD490 nm); Superoxide dismutase (SOD) activity (Enzychrome assay kit, OD 440nm); mRNA expression (spectrophotometer, RT-PCRs).	Increased viability at 45 shots continued for 24-h after exposure and disappeared afterward (G361, the most affected cell, and NHDF, the least affected); Unaffected morphology and no cell death occurrence. G-361$\to$Increased ATP level, mitochondrial activity, and proliferation; Unaffected SOD activity; Altered gene expression related to proliferation (increased Ki67 and c-Myc under 45 shots), apoptosis (decreased CASP3 and increased CASP9 under 5 shots), or cell division and mitochondrion (unaffected CDC2, increased CENPF, increased ATP5A1, and decreased ATP2B1 under 5 shots).

AO/EB$\to$acridine orange/ethidium bromide; carboxy-H2DCFDA$\to$5-(and-6) carboxy-2${ }^{\prime }$,7${ }^{\prime }$-dichlorodihydrofuorescein diacetate; CBMN$\to$cytokinesis-block micronucleus; CDC2$\to$cell division control 2; CENPF$\to$centromere protein F; comet assay$\to$alkaline single-cell gel electrophoresis; c-Myc$\to$cellular myelocytomatosis; GSM$\to$global system for mobile communications; GTEM$\to$gigahertz transverse electromagnetic; $\gamma$-H2AX$\to$Phospho-histone 2AX; LTE$\to$long-term evolution; PARP$\to$poly ADP-ribose polymerase (marker of apoptosis); RF$\to$radio frequency.

Figure 3. Various factors influencing the suspected cause-effect linkage between EMF irradiation exposure and biological effects in an in vitro setup.

Figure 4. Correlation of cellular processes stimulated with electromagnetic field.

Figure 5. Activation of a likely cascade of cell compositions within the cell cycle by the electromagnetic field.

Figure 6. A likely cascade activation of cell compositions associated with mitochondria stimulated by the electromagnetic field.

Figure 7. Plausible impact of the electromagnetic field in the regulation of ion channels.

Ashdown CP, Johns SC, Aminov E, et al. Pulsed low-frequency magnetic fields induce tumor membrane disruption and altered cell viability. Biophys J. 2020;118(7):1552–1563.

 PubMed Web of Science ®Google Scholar

Oh IR, Raymundo B, Jung SA, et al. Extremely low-frequency electromagnetic field altered PPARγ and CCL2 levels and suppressed CD44+/CD24- breast cancer cells characteristics. Bull Korean Chem Soc. 2020;41(8):812–823.

 Web of Science ®Google Scholar

Vincenzi F, Ravani A, Pasquini S, et al. Pulsed electromagnetic field exposure reduces hypoxia and inflammation damage in neuron-like and microglial cells. J Cell Physiol. 2017;232(5):1200–1208.

 PubMed Web of Science ®Google Scholar

Buckner CA, Buckner AL, Koren SA, et al. Exposure to a specific time-varying electromagnetic field inhibits cell proliferation via cAMP and ERK signaling in cancer cells. Bioelectromagnetics. 2018;39(3):217–230.

 PubMed Web of Science ®Google Scholar

Storch K, Dickreuter E, Artati A, et al. Bemer electromagnetic field therapy reduces cancer cell radioresistance by enhanced ros formation and induced DNA damage. PLoS ONE. 2016;11(12):e0167931.

 PubMed Web of Science ®Google Scholar

Akbarnejad Z, Eskandary H, Vergallo C, et al. Effects of extremely low-frequency pulsed electromagnetic fields (ELF-PEMFs) on glioblastoma cells (U87. Electromagn Biol Med. 2017;36(3):238–247.

 PubMed Web of Science ®Google Scholar

Liu Y, Liu W, Liu K, et al. Overexpression of miR-26b-5p regulates the cell cycle by targeting CCND2 in GC-2 cells under exposure to extremely low frequency electromagnetic fields. Cell Cycle. 2016;15(3):357–367.

 PubMed Web of Science ®Google Scholar

Destefanis M, Viano M, Leo C, et al. Extremely low frequency electromagnetic fields affect proliferation and mitochondrial activity of human cancer cell lines. Int J Radiat Biol. 2015;91(12):964–972.

 PubMed Web of Science ®Google Scholar

Garg AA, Jones TH, Moss SM, et al. Electromagnetic fields alter the motility of metastatic breast cancer cells. Commun Biol. 2019;2(1):1–16.

 PubMedGoogle Scholar

Berkelmann L, Bader A, Meshksar S, et al. Tumour-treating fields (TTFields): investigations on the mechanism of action by electromagnetic exposure of cells in telophase/cytokinesis. Sci Rep. 2019;9(1):1–11.

 PubMedGoogle Scholar

Neuhaus E, Zirjacks L, Ganser K, et al. Alternating electric fields (TTFields) activate Cav1.2 channels in human glioblastoma cells. Cancers (Basel). 2019;11(1):110.

 PubMed Web of Science ®Google Scholar

Peng WY, Li KJ, Xie YZ, et al. Development of a TEM-cell-integrated CO2 incubator for cell-based transient electromagnetic field bioeffect study. Electromagn Biol Med. 2020;39(4):290–297.

 PubMed Web of Science ®Google Scholar

Jimenez H, Wang M, Zimmerman JW, et al. Tumour-specific amplitude-modulated radiofrequency electromagnetic fields induce differentiation of hepatocellular carcinoma via targeting Cav3.2 T-type voltage-gated calcium channels and Ca2+ influx. EBioMedicine. 2019;44:209–224.

 PubMed Web of Science ®Google Scholar

Jooyan N, Goliaei B, Bigdeli B, et al. Direct and indirect effects of exposure to 900 MHz GSM radiofrequency electromagnetic fields on CHO cell line: evidence of bystander effect by non-ionizing radiation. Environ Res. 2019;174:176–187.

 PubMed Web of Science ®Google Scholar

Choi J, Min K, Jeon S, et al. Continuous exposure to 1.7 GHz LTE electromagnetic fields increases intracellular reactive oxygen species to decrease human cell proliferation and induce senescence. Sci Rep. 2020;10(1):1–15.

 PubMed Web of Science ®Google Scholar

Sueiro-Benavides RA, Leiro-Vidal JM, Salas-Sánchez AÁ, et al. Radiofrequency at 2.45 ghzGhz increases toxicity, pro-inflammatory and pre-apoptotic activity caused by black carbon in the RAW 264.7 macrophage cell line. Sci Total Environ. 2021;765:142681.

 PubMed Web of Science ®Google Scholar

Mumtaz S, Bhartiya P, Kaushik N, et al. Pulsed high-power microwaves do not impair the functions of skin normal and cancer cells in vitro: a short-term biological evaluation. J Adv Res. 2020;22:47–55.

 PubMed Web of Science ®Google Scholar

Data availability statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.