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The mechanisms of action of 50 Hz magnetic field-induced of epidermal growth factor receptor clustering in human amniotic epithelial cells should be investigated.
In previous studies (Sun et al. 2004, Sun et al. 2008), the authors found that exposure to a magnetic field could induce epidermal growth factor receptor (EGFR) clustering in cultured cells. These data suggested that EGFR is one of the likely targets of magnetic fields on cell membrane. In a further study, Park et al. (2013) found that clustering might be mediated by reactive oxygen species. However, the source of reactive oxygen species production remained unclear. Cells were divided into the following groups: exposure to a 0.4 mT magnetic field for 1) 5 minutes, 2) 15 minutes, 3) 30 minutes or 4) 60 minutes and exposure to a magnetic field of 5) 0.1 mT or 6) 0.2 mT for 15 minutes and 7) sham exposure. Positive control was conducted.
pathway for epidermal growth factor receptor clustering
two exposure chambers in CO2 incubator (95% humidity and 5% CO2)
each chamber contained a set of square Helmholtz coils (20 cm x 20 cm), which were double-wrapped with two lines of copper wire, and was encased by mu-metal; a fan on the wall ventilated and maintained air and temperature uniformity between chamber and incubator; temperature in the chambers was maintained at 37.0 ± 0.1°C throughout the entire exposure period; cell dishes were put in the center of the coils; the magnetic field was perpendicular to the dishes
A sham exposure was conducted.
current was fed into the coils with the same direction in the exposure chamber, whereas opposite direction currents were fed into the coils in the sham exposure chamber; difference in temperature between exposure and sham exposure conditions did not exceed 0.1°C
there was almost no 50 HzMF at the center of the sham exposure coils, when current was fed into coils in the exposure chamber; the mu-metal enclosure attenuated the geomagnetic field, as well as magnetic fields from the fans, by more than 30 dB to a level of less than 1 µT and the AC magnetic field background in the chamber was also less than 0.1 µT; the heterogeneity of field distribution within the 12 x 12 x 20 cm3 space in the exposure chamber was less than 1%; the sham isolation rate was more than 43 dB and electric fields were less than 1 V/m
分子生合成: EGFR-clustering (only with group 2; immunohistochemical EGFR stain, nuclear DAPI stain, confocal microscopy)
oxidative stress: total production of reactive oxygen species (dichlorofluorescein diacetate stain), cytoplasmic (dihydroethidium stain) and mitochondrial superoxide production (MitoSOX red stain) (fluorophotometry)
The total reactive oxygen species production was significantly increased in groups 1, 2, 3 and 6 compared to the sham exposure group, with the most distinct increase in group 2 (0.4 mT). Groups 5 and 6 were not regarded in further tests. Cytoplasmic reactive oxygen species production was significantly increased in groups 1-3 compared to the sham exposure group. To examine the NADPH oxidase as a potential source of reactive oxygen species, these groups were also tested with addition of the NADPH oxidase inhibitor diphenylene iodonium. These co-exposed cells showed a significantly reduced cytoplasmic reactive oxygen species production compared to cells exposed to the magnetic field alone, comparable to sham exposure level. Mitochondrial reactive oxygen species production was significantly increased in groups 2 and 3 compared to sham exposed cells, however, treatment with the NADPH oxidase inhibitor had no significant effect. Confocal microscopy showed EGFR clustering was significantly higher in group 2 compared to the sham exposure group. However, pretreatment with the NADPH oxidase inhibitor completely inhibited MF-induced EGFR clustering. The authors conclude that the NADPH oxidase might mediate the 50 Hz magnetic field-induced epidermal growth factor receptor clustering in human amniotic epithelial cells via superoxide production.