Medical/biological study (experimental study)

Increased levels of inducible HSP70 in cells exposed to electromagnetic fields med./bio.

By: Alfieri RR, Bonelli MA, Pedrazzi G, Desenzani S, Ghillani M, Fumarola C, Ghibelli L, Borghetti AF, Petronini PG

Published in: Radiat Res 2006; 165 (1): 95-104

Aim of study (acc. to author)

To study the effect of exposure to 50 Hz electromagnetic fields in the range of 50-680 µT on the level of HSP70 in several cell models.

Background/further details

In endothelial cells, the effect of electromagnetic fields on several steps of HSP70 gene expression process (i.e. heat-shock transcription factor 1 (HSF1) activation, gene transcription, mRNA accumulation and HSP70 neosynthesis) and the effect of combined exposure to heat shock (for 30 min at 44°C) and electromagnetic fields were also analyzed. Endothelial cells were transfected by electroporation (with phsplacZ and pEGFP-C1 vectors) to evaluate the HSP70 promotor activity of the transfected gene by an assay of beta-galactosidase.
Since HSP70 is a long-lived protein, and since cytoplasmic proteins are mostly degraded through the ubiquitin/proteasome pathway, its potential stabilization was indirectly measured by proteasome avtivity in cell extracts.

Endpoint

molecular biosynthesis: level of heat-shock protein HSP70

Exposure

- 50/60 Hz
- magnetic field

Exposure	Parameters
Exposure 1: 50 Hz Exposure duration: 20 min, 1 h, 2 h, 4 h, 8 h, 16 h, 18 h, 24 h, 48 h	magnetic flux density: 55 µT minimum magnetic flux density: 150 µT magnetic flux density: 300 µT magnetic flux density: 680 µT maximum

Exposure 1

Main characteristics

Frequency	50 Hz
Type	magnetic field
Waveform	sinusoidal
Exposure duration	20 min, 1 h, 2 h, 4 h, 8 h, 16 h, 18 h, 24 h, 48 h

Exposure setup

Exposure source	solenoid
Setup	Four petri dishes were exposed simultaneously by inserting them in the solenoid. The solenoid and the sample holder resulted in a 38 cm high x 10 cm wide plexiglass chamber. The whole setup was placed in an incubator maintained at 37°C. Control samples were not exposed to MF and were incubated in the same incubator in a position where the environmental magnetic field was minimum (2 µT).
Additional info	In some experiments, cells were exposed to heat stress for 20 or 30 min at 44°C in a water bath.

Parameters

Measurand	Value	Type	Method	Mass	Remarks
magnetic flux density	55 µT	minimum	measured	-	-
magnetic flux density	150 µT	-	measured	-	-
magnetic flux density	300 µT	-	measured	-	-
magnetic flux density	680 µT	maximum	measured	-	-

Measurement and calculation details

Magnetic field within the solenoid was measured both radially and longitudinally with a Hall effect probe. Longitudinal measurements in the center of the solenoid showed nearly constant MF over the region.

Exposed system:

intact cell/cell culture
human leukemia & lymphoma cells (CEM lymphoblastoic leukemia CD4+ cells; U937 (histiocytic lymphoma cells); HL60 (promyelocytic leukemia cells)); endothelial cells (of pig pulmonary arteries; HUVEC cultures); human fibroblasts (WI38 & HuDe (from derma))

Methods Endpoint/measurement parameters/methodology

molecular biosynthesis: levels of heat-shock protein HSP70, Myc and actin (Western blot; densitometry); time course of HSP70 induction; HSP70 mRNA level (Northern blot)
cell function: beta-galactosidase activity in transfected and non-transfected cells (histochemical staining); proteasome activities (chymotrypsin-like, trypsin-like and caspase-like activities of the proteasome; fluorimetry)
HSF1 binding activity (HSF1-HSE (³²P-labeled oligonucleotide) binding (electrophoretic mobility shift assay))

Investigated system:

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

Time of investigation:

during exposure
after exposure

Main outcome of study (acc. to author)

Some cell types (endothelial cells and lymphoma/leukemia cells) showed increased levels of HSP70 protein when exposed for 24 h to 50 Hz, 680 µT electromagnetic fields.
In endothelial cells, electromagnetic fields alone induced only a poor and transient activation of HSF1; however, neither the level of HSP70 mRNA nor the synthesis of HSP70 appeared to be altered significantly.
Transfection experiments involving HSP70 promoter showed that gene transcription was not affected. A marked decrease in proteasome activities in exposed cell extracts was also noted.
The heat-shock-induced levels of HSP70 mRNA and protein were increased by a concomitant weak stressor like electromagnetic fields.
In conslusion the data indicate that in electromagnetic field-exposed endothelial cells, HSP70 gene transcription and translation are unaffected; however, electromagnetic fields alone promoted accumulation of the inducible HSP70 protein, probably by increasing its stability, and it enhanced accumulation and translation of the heat-induced HSP70 mRNA when applied in concert with heat shock.

Study character:

medical/biological study
experimental study
full/main study

Study funded by

Ministero dell' Università e della Ricerca (MIUR (formerly MURST (Ministero dell' Università e della Ricerca Scientifica e Tecnologica); Ministry of University and Research), Italy
University of Parma (UNIPR; Universita degli Studi die Parma), Italy

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