この研究は、50Hz磁界（MF；0.4mT）にさまざまなばく露時間（5-60分間）のばく露を与えたヒト羊膜（FL）細胞でのセラミドレベル（高性能液晶クロマトグラフィ・タンデム質量分析法）、カテプシンD活性（蛍光アッセイ）、タンパク質脱リン酸化酵素2A（PP2A、ウエスタンブロット法）を分析した。60分ばく露群については、ばく露後に0、3、6、12、36時間培養後のアポトーシス発生率をフローサイトメトリで評価した。その結果、さまざまなばく露時間のMFばく露は、スフィンゴミエリンの新たな合成および加水分解を介したセラミド生成の有意な上昇を引き起こし、その影響はばく露時間により差が見られた；しかし、60分ばく露後に36時間培養した細胞でのアポトーシスに有意な変化は見られなかった；また、セラミドの上昇はその下流の信号分子であるカテプシンDおよびPP2Aを活性化しなかった、と報告している。
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The effects of exposure of human amniotic cells to a 50 Hz magnetic field on the ceramide metabolism and associated signaling pathways for apoptosis should be investigated.
Previous studies by the authors (Ke et al. 2008, Sun et al. 2008) have shown that exposure to a 50 Hz magnetic field could induce epidermal growth factor receptor clustering, which is related to ceramide. The two major direct targets for ceramide in cells are cathepsin D and PP2Ac (a protein phosphatase 2A subunit), which usually transduce cellular apoptotic signals.
For ceramide metabolism experiments, cells were tested with or without pretreatment with imipramine (50 µM, 4 h). Imipramine inhibits the hydrolysis of sphingomyelin, a main ceramide production way beside the de novo synthesis during cellular stress response. Additionally, sphinganine was investigated as it is the crucial intermediate product of the ceramide de novo synthesis.
A positive control was conducted.
| 周波数 | 50 Hz |
|---|---|
| タイプ |
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| 波形 |
|
| ばく露時間 | continuous for 5, 15, 30 or 60 min |
| ばく露の発生源／構造 | |
|---|---|
| チャンバの詳細 | two exposure chambers placed inside a CO2 incubator; a fan in the chamber wall ventilated and maintained air and temperature uniformity between chamber and incubator |
| ばく露装置の詳細 | each chamber was composed of a set of square Helmholtz coils (20 x 20 cm2) which were double-wrapped with two lines of copper wire and were encased by mu-metal; culture dishes were put in the center of the coils; the MF was perpendicular to the dishes; the incubator was aired with 95% humidity air and 5% CO2; the temperature in the chambers was recorded real time and kept at 37.0°C ± 0.2°C throughout the entire exposure period; difference in temperature between MF- and sham-exposed conditions did not exceed 0.2°C |
| Sham exposure | A sham exposure was conducted. |
| Additional information | one chamber was used for exposure, the other one for sham exposure; 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; the conditions of the setting (exposure or sham exposure) were blind to the experimenters who did the assays |
| 測定量 | 値 | 種別 | Method | Mass | 備考 |
|---|---|---|---|---|---|
| 磁束密度 | 0.4 mT | - | 測定値 | - | - |
After 5, 15 and 30 minutes, the contents of individual cermids were signficantly increased in MF exposed cells compared to sham exposed cells. After 60 minutes of exposure, the highest increase was found with significantly increased values of all ceramides (C16, C18 and C24) compared to the sham exposure. Sphingomylein (C16, C24:1 and C24) levels were significantly decreased in exposed cells compared to sham exposed cells after 60 Minutes while the sphinganine level was significantly increased at the same time.
Preatreatment with imipramin inhibited the increase of C18 and C24 ceramides and the decrease of sphingomylein levels in exposed cells after 60 minutes.
Exposure to the magnetic field did not affect cell apoptosis and had no significant effects on the cathepsin D enzyme activity and activation of PP2Ac.
The authors conclude that exposure of human amniotic cells to a 50 Hz magnetic field might increase the ceramide production via both hydrolysis of sphingomyelin and de novo synthesis. However, no effects on apoptosis were found.
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