Medical/biological study (experimental study)

Extremely Low-Frequency Electromagnetic Fields Cause G1 Phase Arrest through the Activation of the ATM-Chk2-p21 Pathway med./bio.

By: Huang CY, Chang CW, Chen CR, Chuang CY, Chiang CS, Shu WY, Fan TC, Hsu IC

Published in: PLoS One 2014; 9 (8): e104732

Aim of study (acc. to author)

To investigate the biological effects of exposure to extremely low frequency magnetic fields in HaCaT cells at the transcriptional, protein and cellular level.

Background/further details

Ionizing radiation (UV-B, 233 J/m²) was performed as a positive control.
Further controls were performed using siRNA (small interfering RNA), which is a class of double-stranded RNA molecules, 20-25 base pairs in length and do not code for proteins. It is most notable in the interference of the expression of specific genes with complementary nucleotide sequences resulting in a prevention of translation.

Endpoint

molecular biosynthesis: gene expression, protein expression
cell viability/cell division/proliferation: cell proliferation, cell viability, cell cycle distribution

Exposure

- 50/60 Hz
- magnetic field
- shielding/field deprivation

Exposure	Parameters
Exposure 1: 60 Hz Exposure duration: continuous up to 144 hours (24, 48, 72, 96, 120, 144 hours)	magnetic flux density: 1.5 mT (± 0.03 µT, in the central area of the coils)

Exposure 1

Main characteristics

Frequency	60 Hz
Type	magnetic field
Exposure duration	continuous up to 144 hours (24, 48, 72, 96, 120, 144 hours)

Exposure setup

Exposure source	Helmholtz coils
Setup	pair of coil apparatuses each had a diameter of 34 cm and were 17 cm apart. AC power to the Helmholtz coil system was supplied by a step-down transformer connected to a 60-Hz, 110 V AC source
Sham exposure	A sham exposure was conducted.

Parameters

Measurand	Value	Type	Method	Mass	Remarks
magnetic flux density	1.5 mT	-	measured	-	± 0.03 µT, in the central area of the coils

Additional parameter details

background magnetic fields in the incubator were 0.47 ± 0.03 mT (upper part), 0.25 ± 0.01 mT (middle part), and 1.15 ± 0.47 mT (lower part)

Exposed system:

intact cell/cell culture
HaCaT cells (human keratinocytes) partially transfected with CHK2 siRNA

Methods Endpoint/measurement parameters/methodology

molecular biosynthesis: general gene expression (microarray), gene expression of the cell cycle-related genes CDKN1A (encodes cyclin-dependent kinase inhibitor 1, also called p21), CDC25B, CDC20, CDC2, CCNA2, CCNB1 to confirm the results of the microarray (quantitative real-time PCR); protein expression of ATM, phosphorylated ATM, Chk2, phosphorylated Chk2, p21 (Western blot)
cell viability/cell division/proliferation: cell proliferation (hemocytometer, microscopy); cell viability (colony forming units); cell cycle distribution (propidium iodide staining, flow cytometry)

Investigated system:

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

Time of investigation:

after exposure

Main outcome of study (acc. to author)

In 144 hours-exposed cell cultures, the number of cells in the G1 phase was significant increased ("G1 phase cell cycle arrest") compared to the sham exposure, while the cell proliferation and colony forming units were significantly decreased. The protein expression of phosphorylated ATM, phosphorylated Chk2 and p21 was higher in the exposed cell cultures (after 96, 120 and 144 hours) than in the sham exposed ones. The magnetic field-induced G1 phase cell cycle arrest was diminished when the gene expression of CHK2 was prevented by transfection with the siRNA.
The authors conclude that extremely low frequency magnetic fields can trigger cell cycle arrest in the G1 phase in HaCaT cells. Furthermore, they conclude that the results indicate an involvement of an ATM-Chk2-p21 pathway.

Study character:

medical/biological study
experimental study
full/main study

Study funded by

National Science Council, Taiwan

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