Medical/biological study (experimental study)

Calcium signalling in human neutrophil cell lines is not affected by low-frequency electromagnetic fields med./bio.

By: Golbach LA, Philippi JG, Cuppen JJ, Savelkoul HF, Verburg-van Kemenade BM

Published in: Bioelectromagnetics 2015; 36 (6): 430-443

Aim of study (acc. to author)

The adverse effects of exposure of human neutrophil granulocyte-like cells to a 50 Hz magnetic field or to a combination of several extremely low frequency and intermediate frequency fields on calcium signal pathways should be investigated.

Background/further details

In two different cell lines, HL-60 and PLB-985 cells, a differentiation into neutrophil-like cells was induced by the addition of DMSO. The combination of different low-frequency and intermediate frequency fields was called "Immunent field".
Both cell lines were exposed to two different types of fields (50 Hz and Immunent field) and 4 different magnetic flux densities (2,5 mT, 500 µT, 300 µT, and 5 µT) for 3 minutes, 30 minutes, 4 days or 5 days.

Endpoint

cell function: calcium signal pathways in human acute myeloid leukaemia cell lines

Exposure

- 50–2,600 Hz
- 50/60 Hz
- magnetic field
- low frequency
- intermediate frequency
- co-exposure

Exposure	Parameters
Exposure 1: 50 Hz Exposure duration: continuous for 30 minutes	magnetic flux density: 5 µT electric field strength: 1.57 mV/m maximum
Exposure 2: 50 Hz–2.6 kHz Exposure duration: continuous for 30 minutes Immunent field	magnetic flux density: 5 µT electric field strength: 0.3 V/m maximum
Exposure 3: 50 Hz Exposure duration: continuous for 30 minutes, 4 days or 5 days	magnetic flux density: 500 µT electric field strength: 1.57 mV/m maximum
Exposure 4: 50 Hz–2.6 kHz Exposure duration: continuous for 30 minutes, 4 days or 5 days Immunent field	magnetic flux density: 300 µT electric field strength: 0.3 V/m maximum
Exposure 5: 50 Hz Exposure duration: intermittent for a total of up to 3 minutes for flow cytometry	magnetic flux density: 2.5 mT electric field strength: 1.57 mV/m maximum

General information

background alternating current magnetic flux densities at the place of exposure and sham exposure were less than 0.2 µT and ambient direct current static magnetic fields were 10 µT (except for exposure 5)

Exposure 1

Main characteristics

Frequency	50 Hz
Type	magnetic field
Waveform	sinusoidal
Exposure duration	continuous for 30 minutes

Exposure setup

Exposure source	coil(s)
Chamber	cell culture flasks
Setup	coil was placed inside a standard cell culture incubator with 37°C ± 0.2°C and 5% CO₂; cell culture flasks were placed in the central region of the coils, perpendicular to vertical magnetic field lines; copper wire coil was wound on a double co-axial cylinder with an inner (65 turns; Ø 184 mm) and outer cylinder (2 times 8 turns; Ø 400 mm) made of polymethyl methacrylate; inhomogeneity within exposure area was < 0.4%
Sham exposure	A sham exposure was conducted.
Additional info	two identical cell culture incubators with identical exposure coil inside were used and randomly assigned to be active or sham for each experiment

Parameters

Measurand	Value	Type	Method	Mass	Remarks
magnetic flux density	5 µT	-	measured	-	-
electric field strength	1.57 mV/m	maximum	-	-	-

Exposure 2

Main characteristics

Frequency	50 Hz–2.6 kHz
Type	electromagnetic field
Exposure duration	continuous for 30 minutes
Additional info	Immunent field
Additional info	combination of four block waves at frequencies of 320, 730, 880 and 2600 Hz

Exposure setup

Exposure source	same setup as exposure 1
Sham exposure	A sham exposure was conducted.

Parameters

Measurand	Value	Type	Method	Mass	Remarks
magnetic flux density	5 µT	-	measured	-	-
electric field strength	0.3 V/m	maximum	-	-	-

Exposure 3

Main characteristics

Frequency	50 Hz
Type	magnetic field
Waveform	sinusoidal
Exposure duration	continuous for 30 minutes, 4 days or 5 days

Exposure setup

Exposure source	same setup as exposure 1
Sham exposure	A sham exposure was conducted.

Parameters

Measurand	Value	Type	Method	Mass	Remarks
magnetic flux density	500 µT	-	measured	-	-
electric field strength	1.57 mV/m	maximum	-	-	-

Exposure 4

Main characteristics

Frequency	50 Hz–2.6 kHz
Type	electromagnetic field
Exposure duration	continuous for 30 minutes, 4 days or 5 days
Additional info	Immunent field
Additional info	combination of four block waves at frequencies of 320, 730, 880 and 2600 Hz

Exposure setup

Exposure source	same setup as exposure 1
Sham exposure	A sham exposure was conducted.

Parameters

Measurand	Value	Type	Method	Mass	Remarks
magnetic flux density	300 µT	-	measured	-	-
electric field strength	0.3 V/m	maximum	-	-	-

Exposure 5

Main characteristics

Frequency	50 Hz
Type	magnetic field
Waveform	sinusoidal
Exposure duration	intermittent for a total of up to 3 minutes
Additional info	for flow cytometry

Exposure setup

Exposure source	coil(s)
Chamber	test tubes
Setup	custom-made exposure system fitting around the test tube inside the flow cytometer; the circular coil consisted of 0.5 mm tightly wound copper wire around a polyether ether ketone body with an inner diameter of 38 mm (100 turns); the direction of the field was parallel to the height of the test tube
Sham exposure	A sham exposure was conducted.

Parameters

Measurand	Value	Type	Method	Mass	Remarks
magnetic flux density	2.5 mT	-	measured	-	-
electric field strength	1.57 mV/m	maximum	-	-	-

Additional parameter details

background alternating current magnetic flux densities at place of exposure and sham exposure were less than 0.3 µT and ambient direct current static magnetic fields were 47 µT

Reference articles

Bouwens M et al. (2012): Low-frequency electromagnetic fields do not alter responses of inflammatory genes and proteins in human monocytes and immune cell lines
de Kleijn S et al. (2011): Extremely low frequency electromagnetic field exposure does not modulate toll-like receptor signaling in human peripheral blood mononuclear cells

Exposed system:

intact cell/cell culture
HL-60 and PLB-985 cells (human acute myeloid leukaemia cell lines)

Methods Endpoint/measurement parameters/methodology

molecular biosynthesis: gene expression of calcium signal pathway genes (during differentiation and after exposure and differentiation; gene expression of differentiation marker CD11b, calcium channel proteins STIM1, STIM2, Orai1, TRPC6, calcium signal pathway proteins calmodulin, IP3 receptor type 1 and cytoskeletal components Arp2/3, beta-actin; quantitative real-time RT-PCR)
cell function: intracellular calcium concentration (after exposure and differentiation; fluorescence spectrophotometry; before, during and after exposure and differentiation; flow cytometry) (stimulation with platelet-activating factor, staining with calcium indicator Fluo-4)
morphol./histopathol. changes: phenotype of differentiated cells according to amount and shape of microvilli on cell surface and cell polarity (after exposure and differentiation; scanning electron microscopy)

Investigated system:

isolated bio./chem. substance
DNA/RNA
intact cell/cell culture
calcium

Time of investigation:

before exposure
during exposure
after exposure

Main outcome of study (acc. to author)

No significant differences could be found between the exposure groups and their corresponding sham exposure groups.
The authors conclude that exposure of human neutrophil granulocyte-like cells to a 50 Hz magnetic field or to a combination of several extremely low frequency and intermediate frequency fields has no effect on calcium signal pathways.

Study character:

medical/biological study
experimental study
full/main study
partially blind study

Study funded by

ZonMw, The Netherlands

Yang X et al. (2019): Involvement of calcium in 50-Hz magnetic field-induced activation of sphingosine kinase 1 signaling pathway
Wei J et al. (2015): Effects of extremely low frequency electromagnetic fields on intracellular calcium transients in cardiomyocytes
Luo FL et al. (2014): Exposure to extremely low frequency electromagnetic fields alters the calcium dynamics of cultured entorhinal cortex neurons
Cui Y et al. (2014): Exposure to extremely low-frequency electromagnetic fields inhibits T-type calcium channels via AA/LTE signaling pathway
Bouwens M et al. (2012): Low-frequency electromagnetic fields do not alter responses of inflammatory genes and proteins in human monocytes and immune cell lines
Hwang YH et al. (2011): Intracellular Ca Mobilization and Beta-hexosaminidase Release Are Not Influenced by 60 Hz-electromagnetic Fields (EMF) in RBL 2H3 Cells
de Kleijn S et al. (2011): Extremely low frequency electromagnetic field exposure does not modulate toll-like receptor signaling in human peripheral blood mononuclear cells
Zhang X et al. (2010): Magnetic Fields at Extremely Low-Frequency (50 Hz, 0.8 mT) Can Induce the Uptake of Intracellular Calcium Levels in Osteoblasts
McCreary CR et al. (2006): Real-time measurement of cytosolic free calcium concentration in Jurkat cells during ELF magnetic field exposure and evaluation of the role of cell cycle
Ikehara T et al. (2005): Effects of a time-varying strong magnetic field on transient increase in Ca(2+) release induced by cytosolic Ca(2+) in cultured pheochromocytoma cells
Gobba F et al. (2003): Effects of 50 Hz magnetic fields on fMLP-induced shape changes in invertebrate immunocytes: The role of calcium ion channels
Craviso GL et al. (2002): Intracellular calcium activity in isolated bovine adrenal chromaffin cells in the presence and absence of 60 Hz magnetic fields
Ikehara T et al. (2002): Effects of a time varying strong magnetic field on release of cytosolic free Ca2+ from intracellular stores in cultured bovine adrenal chromaffin cells
McCreary CR et al. (2002): Factors confounding cytosolic calcium measurements in Jurkat E6.1 cells during exposure to ELF magnetic fields
Mattsson MO et al. (2001): [Ca2+](i) rise in Jurkat E6-1 cell lines from different sources as a response to 50 Hz magnetic field exposure as a reproducible effect and independent of poly-L-lysine treatment
Shahidain R et al. (2001): Calcium spiking activity and baseline calcium levels in ROS 17/2.8 cells exposed to extremely low frequency electromagnetic fields (ELF EMF)
Nishimura I et al. (1999): Linearly and circularly polarized, 50 Hz magnetic fields did not alter intracellular calcium in rat immune cells
Galvanovskis J et al. (1999): Cytoplasmic Ca2+ oscillations in human leukemia T-cells are reduced by 50 Hz magnetic fields
Sontag W (1998): Action of extremely low frequency electric fields on the cytosolic calcium concentration of differentiated HL-60 cells: nonactivated cells
Lyle DB et al. (1997): Intracellular calcium signaling by Jurkat T-lymphocytes exposed to a 60 Hz magnetic field
Galvanovskis J et al. (1996): The influence of 50-Hz magnetic fields on cytoplasmic Ca2+ oscillations in human leukemia T-cells
Lindstrom E et al. (1995): Intracellular calcium oscillations in a T-cell line after exposure to extremely-low-frequency magnetic fields with variable frequencies and flux densities
Lindstrom E et al. (1993): Intracellular calcium oscillations induced in a T-cell line by a weak 50 Hz magnetic field
Lyle DB et al. (1991): Calcium uptake by leukemic and normal T-lymphocytes exposed to low frequency magnetic fields

Calcium signalling in human neutrophil cell lines is not affected by low-frequency electromagnetic fields med./bio.

Aim of study (acc. to author)

Background/further details

Endpoint

Exposure

General information

Exposure 1

Main characteristics

Exposure setup

Parameters

Exposure 2

Main characteristics

Exposure setup

Parameters

Exposure 3

Main characteristics

Exposure setup

Parameters

Exposure 4

Main characteristics

Exposure setup

Parameters

Exposure 5

Main characteristics

Exposure setup

Parameters

Additional parameter details

Reference articles

Methods Endpoint/measurement parameters/methodology

Main outcome of study (acc. to author)

Study funded by

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