Medical/biological study (experimental study)

Inhibition of gap junction intercellular communication by extremely low-frequency electromagnetic fields in osteoblast-like models is dependent on cell differentiation med./bio.

By: Yamaguchi DT, Huang J, Ma D, Wang PK

Published in: J Cell Physiol 2002; 190 (2): 180-188

Aim of study (acc. to author)

To study whether extremely low frequency magnetic fields alter gap junction intercellular communication in a pre-osteoblastic model (MC3T3-E1) and a well-differentiated osteoblastic model (ROS 17/2.8).

Background/further details

Gap junctions occur among bone forming cells (osteoblasts) and have been hypothesized to play a role in bone formation.

Endpoint

cell function: gap junction intercellular communication

Exposure

- 30–120 Hz
- 50/60 Hz
- magnetic field
- low frequency
- therapeutic/medical device

Exposure	Parameters
Exposure 1: 30–120 Hz Exposure duration: 1 h before injection; 1.5 h during injection injection method (ROS 17/2.8 cells and MC3T3-E1 cells)	magnetic flux density: 1.25 mT maximum (application of different magnetic flux densities: 0.01, 0.1, 0.3, 0.6, 1.25 mT)
Exposure 2: 60 Hz Exposure duration: experiment 1 (3 days old MC3T3-E1 cells): continuous for 4 h; experiment 2 (4, 8, 15, 23 days old MC3T3-E1 cells): 1 h pre-exposure of parachuting cells + 16 h exposure of all cells parachute method	magnetic flux density: 1.25 mT
Exposure 3: 60 Hz Exposure duration: continuous for 3 h distribution of connexin (MC3T3-E1 cells)	magnetic flux density: 1.25 mT
Exposure 4: 30–120 Hz Exposure duration: continuous for 1 h assessment of calcium (MC3T3-E1 cells)	magnetic flux density: 0.6 mT (application of different frequencies (30 Hz and 120 Hz) at the same magnetic flux density)

General information

Three different, not always for certain assignable exposure setups: i) setup according to Wang 1997: two 1.8 cm long solenoids with a radius of 3.8 cm and a double-layer of 100 turns of wire; for cancellation of the horizontal component of the geomagnetic field: pair of 0.7 cm long coils with a radius of 3.8 cm and 71.4 turns of wire, separated by 7.2 cm ii) setup according to Merrit 1983: four square coils with a sidelenght of 25 cm; upper and lower coils: 40-turn copper magnet wire at a distance of 25 - 37 cm; two middle section coils: 17-turn copper magnet wire at a distance of 6.4 cm iii) Helmholtz coil: exposure system built into a CO₂ tissue culture incubator

Exposure 1

Main characteristics

Frequency	30–120 Hz
Type	magnetic field
Waveform	sinusoidal
Exposure duration	1 h before injection; 1.5 h during injection
Additional info	injection method (ROS 17/2.8 cells and MC3T3-E1 cells)
Additional info	30, 60, 120 Hz

Exposure setup

Exposure source	solenoid setup according to i)
Setup	cells grown on coverslips mounted in a round coverslip holder; in most coverslips 4-5 cells in the central portions of the coverslip were injected
Sham exposure	A sham exposure was conducted.

Parameters

Measurand	Value	Type	Method	Mass	Remarks
magnetic flux density	1.25 mT	maximum	measured	-	application of different magnetic flux densities: 0.01, 0.1, 0.3, 0.6, 1.25 mT

Exposure 2

Main characteristics

Frequency	60 Hz
Type	magnetic field
Waveform	sinusoidal
Exposure duration	experiment 1 (3 days old MC3T3-E1 cells): continuous for 4 h; experiment 2 (4, 8, 15, 23 days old MC3T3-E1 cells): 1 h pre-exposure of parachuting cells + 16 h exposure of all cells
Additional info	parachute method

Exposure setup

Exposure source	Helmholtz coils setup according to ii) or iii) (not clearly assignable)
Setup	cells were plated in plates with 12 wells
Sham exposure	A sham exposure was conducted.

Parameters

Measurand	Value	Type	Method	Mass	Remarks
magnetic flux density	1.25 mT	-	measured	-	-

Exposure 3

Main characteristics

Frequency	60 Hz
Type	magnetic field
Waveform	sinusoidal
Exposure duration	continuous for 3 h
Additional info	distribution of connexin (MC3T3-E1 cells)

Exposure setup

Exposure source	Helmholtz coils setup according to iii)
Sham exposure	A sham exposure was conducted.

Parameters

Measurand	Value	Type	Method	Mass	Remarks
magnetic flux density	1.25 mT	-	measured	-	-

Exposure 4

Main characteristics

Frequency	30–120 Hz
Type	magnetic field
Waveform	sinusoidal
Exposure duration	continuous for 1 h
Additional info	assessment of calcium (MC3T3-E1 cells)

Exposure setup

Exposure source	same setup as exposure 1
Setup	cells grown on coverslips mounted in a round coverslip holder
Sham exposure	A sham exposure was conducted.

Parameters

Measurand	Value	Type	Method	Mass	Remarks
magnetic flux density	0.6 mT	-	measured	-	application of different frequencies (30 Hz and 120 Hz) at the same magnetic flux density

Reference articles

Wang PK (1997): ELF magnetic field exposure system with feedback-controlled disturbance rejection

Exposed system:

intact cell/cell culture
ROS 17/2.8 (osteoblast-like cells) and MC3T3-E1 cells (mouse osteoblast-like cells)

Methods Endpoint/measurement parameters/methodology

cell function: gap junction intercellular communication (investigated via 1.) injection of Lucifer yellow and monitoring of the dye spread to surrounding cells and 2.) non-invasive dye transfer: exposed cells (4 h) were loaded with BCECF, labeled with PKH26 and released and parachuted onto 1 h pre-exposed cell layers; BCECF transfer from donor cells to surrounding unlabeld cells was assessed); connexin 43 cellular distribution (immunoprecipitation of soluble (cytosolic) and insoluble (membrane) fractions; autoradiography)
alkaline phosphatase activity (as marker for cell differentiation; spectrophotometry); cytosolic calcium concentration (via Fura-2; fluorescence microscopy)

Investigated system:

intact cell/cell culture
cell lysates and supernatants

Time of investigation:

after exposure

Main outcome of study (acc. to author)

The data showed that magnetic fields over a frequency range from 30 to 120 Hz and magnetic flux densities up to 1.25 mT dose dependently decreased gap junction intercellular communication in MC3T3-E1 cells during their proliferative phase of development. The total amount of connexin 43 and the distribution of connexin 43 gap junction protein between cytoplasmic and plasma membrane fractions were unaltered by magnetic field exposure. Cytosolic calcium which can inhibit gap junction communication, was not altered by magnetic field exposure.
Identical exposure conditions did not affect gap junction communication in the ROS 17/2.8 cell line and when MC3T3-E1 cells were more differentiated. Thus, extremely low frequency magnetic fields may affect only less differentiated or pre-osteoblasts and not fully differentiated osteoblasts. Consequently, electromagnetic fields may aid in the repair of bone by effects exerted only on osteo-progenitor cells or pre-osteoblasts.

Study character:

medical/biological study
experimental study
full/main study

Study funded by

Electric Power Research Institute (EPRI), USA
Medical Research Funds of the Veterans Administration, USA

Percherancier Y et al. (2015): Effects of 50 Hz magnetic fields on gap junctional intercellular communication in NIH3T3 cells
Cervellati F et al. (2013): 17-β-Estradiol counteracts the effects of high frequency electromagnetic fields on trophoblastic connexins and integrins
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
Cervellati F et al. (2009): Effect of high-frequency electromagnetic fields on trophoblastic connexins
Soda A et al. (2008): Effect of exposure to an extremely low frequency-electromagnetic field on the cellular collagen with respect to signaling pathways in osteoblast-like cells
Sul AR et al. (2006): Effects of sinusoidal electromagnetic field on structure and function of different kinds of cell lines
Zeng Q et al. (2006): Noise magnetic fields abolish the gap junction intercellular communication suppression induced by 50 Hz magnetic fields
Ramundo-Orlando A et al. (2005): Permeability changes of connexin32 hemi channels reconstituted in liposomes induced by extremely low frequency, low amplitude magnetic fields
Lohmann CH et al. (2003): Pulsed electromagnetic fields affect phenotype and connexin 43 protein expression in MLO-Y4 osteocyte-like cells and ROS 17/2.8 osteoblast-like cells
Marino AA et al. (2003): Extracellular currents alter gap junction intercellular communication in synovial fibroblasts
Zeng QL et al. (2003): ELF magnetic fields induce internalization of gap junction protein connexin 43 in Chinese hamster lung cells
Hu GL et al. (2001): ELF magnetic field inhibits gap junctional intercellular communication and induces hyperphosphorylation of connexin43 in NIH3T3 cells
Griffin GD et al. (2000): Power frequency magnetic field exposure and gap junctional communication in Clone 9 cells
Mullins RD et al. (2000): Mechanisms underlying spontaneous calcium spiking in aequorin-loaded ROS 17/2.8 cells
Li CM et al. (1999): Exposure to 50-Hz electromagnetic fields: Effects of time and field strength on gap junctional intercellular communication
Fear EC et al. (1998): Biological cells with gap junctions in low-frequency electric fields
Ubeda A et al. (1995): A 50 Hz magnetic field blocks melatonin-induced enhancement of junctional transfer in normal C3H/10T1/2 cells
Fitzsimmons RJ et al. (1994): Combined magnetic fields increased net calcium flux in bone cells
McLeod KJ et al. (1993): Electric fields modulate bone cell function in a density-dependent manner
Walleczek J (1992): Electromagnetic field effects on cells of the immune system: the role of calcium signaling
Ozawa H et al. (1989): Electric fields stimulate DNA synthesis of mouse osteoblast-like cells (MC3T3-E1) by a mechanism involving calcium ions
Luben RA et al. (1982): Effects of electromagnetic stimuli on bone and bone cells in vitro: inhibition of responses to parathyroid hormone by low-energy low-frequency fields

Inhibition of gap junction intercellular communication by extremely low-frequency electromagnetic fields in osteoblast-like models is dependent on cell differentiation 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

Reference articles

Methods Endpoint/measurement parameters/methodology

Main outcome of study (acc. to author)

Study funded by

Related articles