Medical/biological study (experimental study)

Role of NADPH oxidase in MAPK signaling activation by a 50 Hz magnetic field in human neuroblastoma cells med./bio.

By: Martínez MA, Úbeda A, Trillo MÁ

Published in: Electromagn Biol Med 2021; 40 (1): 103-116

Aim of study (acc. to author)

The effects of exposure of NB69 cells to a 50 Hz magnetic field on effects on ROS production and the potential involvement of NADPH oxidase in activation of MAPK pathways should be investigated.

Background/further details

In previous studies by the authors (Martinez et al. 2012, Martinez et al. 2016 and Martinez et al. 2019), it was found that proliferation of NB69 cells was increased by magnetic field exposure and that it was mediated by activation of signal pathways MAPK-ERK1/2 and p38. In the present study, it should be investigated whether NADPH could be involved in the activation of the aforementioned signal pathways. As NADPH oxidase is the main source of ROS in cells, tests were conducted with and without Diphenyleneiodonium (DPI), used to inhibit NADPH oxidase, and N-acetyl-L-cysteine, used as a ROS scavenger.
Cells were exposed for 5-30 minutes. For each exposure group, a respective sham exposure was conducted.

Endpoint

molecular biosynthesis: ROS production and activation of signal pathways

Exposure

- 50/60 Hz
- magnetic field

Exposure	Parameters
Exposure 1: 50 Hz Exposure duration: 5-30 minutes	magnetic flux density: 100 µT effective value

Exposure 1

Main characteristics

Frequency	50 Hz
Type	magnetic field
Waveform	sinusoidal
Polarization	linear
Exposure duration	5-30 minutes

Exposure setup

Exposure source	Helmholtz coils
Setup	one Helmholtz pair was placed inside each of two magnetically shielded chambers located within two identical CO₂ incubators; in each experimental run Petri dishes or well plates, containing the cell samples, were stacked in the central region of the Helmholtz coil gap to ensure uniformity of MF exposure; in each experimental run only one set of coils was energized, the samples in the unenergized set being considered sham-exposed controls; following a random sequence, both coil sets and incubators were alternatively used for MF- or sham-exposure
Sham exposure	A sham exposure was conducted.

Parameters

Measurand	Value	Type	Method	Mass	Remarks
magnetic flux density	100 µT	effective value	measured	-	-

Additional parameter details

the background MF inside the shielded chambers was 0.05 ± 0.02 μT (rms) for AC magnetic fields and 0.04 ± 0.04 μT (rms) for static magnetic fields

Reference articles

Trillo MA et al. (2012): Influence of a 50 Hz magnetic field and of all-transretinol on the proliferation of human cancer cell lines

Exposed system:

intact cell/cell culture
NB69 cells (human neuroblastoma cells)

Methods Endpoint/measurement parameters/methodology

molecular biosynthesis: ROS production (fluorophotometry with dichlorofluorescein) and activation of signal pathways (activation of ERK1/2, p38 und MAPK-JNK and p67phox (component of NADPH oxidase) protein expression; Western blot and immunohistochemical stain, fluorescence microscopy)

Investigated system:

isolated bio./chem. substance
intact cell/cell culture
cell lysates

Time of investigation:

after exposure

Main outcome of study (acc. to author)

Exposure to the magnetic field for 10 minutes significantly increased the level of ROS in NB69 cells compared to the sham exposure and induced transient significanty increased expression of p67phox after 10-15 minutes of exposure. The MF-induced activation of the MAPK-JNK pathway, but not that of -ERK1/2 or -p38 pathways, was prevented in the presence of DPI.
The authors concluded that exposure of NB69 cells to a 50 Hz magnetic field might increase ROS production via NADPH oxidase activity. However, taken together with the results of the previous studies, there seem to be further mechanisms of MF-induced proliferative response mediated by MAPK signaling activation.

Study character:

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

Study funded by

European Defence Agency (EDA)
European Union (EU)/European Commission
Ministerio de Defensa de Espana (Ministry of Defence, Spain)

Martínez MA et al. (2019): Involvement of the EGF Receptor in MAPK Signaling Activation by a 50 Hz Magnetic Field in Human Neuroblastoma Cells
Qiu L et al. (2019): S1P mediates human amniotic cells proliferation induced by a 50-Hz magnetic field exposure via ERK1/2 signaling pathway
Luukkonen J et al. (2017): Modification of p21 level and cell cycle distribution by 50 Hz magnetic fields in human SH-SY5Y neuroblastoma cells
Martinez MA et al. (2016): Power Frequency Magnetic Fields Affect the p38 MAPK-Mediated Regulation of NB69 Cell Proliferation Implication of Free Radicals
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