Autophagy (also referred to as auto-phagocytosis) means the process in which cells digest cellular components. In the present paper, only macroautophagy was examined. Here, cell components are enclosed in a so called autophagosome in a first step. In a second step, the fusion with a lysosome starts the degradation. LC3 is a protein that is distributed ubiquitously in the cell. During autophagic processes, it concentrates in autophagosomes as LC3-II and gets degraded there. Both forms are common markers for autophagy. To ensure that an increase of LC3-II represents an actual increase of the autophagic flux and not a block of the fusion of autophagosome and lysosome for instance, tests with chloroquine were conducted. Chloroquine blocks the fusion and hence induces an increase of LC3-II without an increase of the autophagic flux.
Rapamycin was used as positive control for autophagy and the activity of the mTOR (mammalian target of rapamycin) pathway. Phosphorylated p70S6 kinase is a direct substrate of mTOR activity and a decrease is used as a common marker for mTOR activity. Positive controls were performed for flow cytometry and the TUNEL assay.
Exposure duration: continuous for 0.5, 2, 6, 12 and 24 h
|Exposure duration||continuous for 0.5, 2, 6, 12 and 24 h|
|Chamber||2 chambers were placed in one incubator with constant environmental conditions (37 °C, 5% CO2); each chamber was composed of a series of Helmholtz coils|
|Setup||the connection of the coils in one chamber was in-phase to generate a magnetic field for exposure, whereas an opposite phase connection in the other chamber generated an offset magnetic field for sham exposure|
|Sham exposure||A sham exposure was conducted.|
|magnetic flux density||2 mT||-||-||-||-|
Although an increase of autophagic markers could be observed at all times, only the 6-hour exposure showed a significant and consistent increase in autophagy in Western Blot, confocal laser scanning microscopy and transmission electron microscopy compared to sham exposure. The tests with chloroquine confirmed that this was the result of an actual increase of the autophagic flux. The mTOR pathway was inactive during these processes. Moreover, a significant higher level of reactive oxygen species could be found in the 6-hour exposure compared to sham exposure. The tests on apoptosis did not show any differences.
The authors conclude that an in vitro exposure of mouse embryonic fibroblasts to a 50 Hz magnetic field for 6 h causes a significant increase in autophagy, which might be related to a simultaneous increase in reactive oxygen species and works without an activation of the common autophagy signaling pathway involving mTOR.