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The effects of exposure of mouse and human adrenal cortex-derived cells to a 60 Hz magnetic field on steroid hormone production and the underlying mechanisms of action should be investigated.
Except for one test, all experiments, a magnetic flux density of 1.5 mT was used. Only for the quantification of corticosterone, mouse adrenal cortex-derived cells were exposed to 0.25 mT, 0.5 mT, 1.5 mT and 3 mT.
内分泌の変化: steroid hormone production in adrenal cells
the exposure apparatus consisted of a cylindrical plastic (polyvinyl chloride) container (37 cm outer diameter, 34.5 cm inner diameter, 33 cm length) placed vertically with a coil inside; the coil consisted of one primary coil and two secondary coils that were placed on the upper and lower ends of the primary coil; coils were made of enameled wires (2.12 mm diameter); for the primary coil, the wire was wound 150 times around the plastic container; for the two secondary coils, the wire was wound 33 times around every layer to correct the uneven distribution of the magnetic flux density of the primary coil; a plastic incubator (25 cm length, 20 cm width and 10 cm height) containing the culture dishes was placed in the middle of each plastic container; to maintain the temperature of 37 ± 0.5°C and 5% CO2 in the incubator, warm water (40 ± 0.5°C) and 5% CO2 air were supplied in the container; the temperature difference between ELF-MF and sham exposure apparatuses was maintained within ± 0.5°C
A sham exposure was conducted.
sham exposure was performed simultaneously with exposure using a sham apparatus, which was made using the same materials with the same size, except for the coil
mouse adrenal cortex-derived cells (Y-1) and human adrenal cortex-derived cells (H295R)
分子生合成: gene expression of adrenal steroid hormone synthesis related genes after 24 h: mouse steroidogenic acute regulatory protein (StAR), mouse cytochrome P450 (Cyp)11a1, Cyp11b1, Cyp11b2 (only in Y-1 cells) and human StAR, human cytochrome P450 (Cyp)11a1, Cyp11b1, Cyp11b2, Cyp17a1 (only in H295R cells) (real-time RT-PCR); protein expression after 6 h and 24 h of StAR, CYP11B2, CYP17A1, CREB and phosphorylated CREB (Western blot); cyclic adenosine monophosphate (cAMP) quantification after 24 h (only with Y-1 cells with and without addition of 10 µM or 30 µM NF449 (inhibitor of G protein), ELISA); total protein concentration (BCA protein assay)
細胞機能: phosphodiesterase (for degradation of cAMP) enzyme activity after 1 h, 6 h and 24 h (commercial kit), intracellular calcium concentration after 24 h (FURA-2 stain, fluorescence analysis) (only with Y-1 cells)
内分泌の変化: quantification of adrenal steroid hormones (corticosterone (only in Y-1 cells), aldosterone and cortisol (only in H295R cells) after 1 h, 6 h, 24 h and 48 h of exposure (ELISA))
Exposure to the magnetic field was found to significantly increase the secretion of corticosterone and aldosterone and the gene expression and/or protein expression of Cyp11a1 and Cyp11b2 in mouse Y-1 cells compared to the control group. It was also found that only a magnetic flux density of 0.5 mT or more could induce a significant increase in corticosterone secretion in exposed Y-1 cells compared to the control group. Exposed Y-1 cells showed significant decreases in phosphodiesterase enzyme activity and intracellular calcium concentration and significant increases in cAMP concentration and CREB phosphorylation compared to cells of the control group. The increase in cAMP concentration was not inhibited by treatment with NF449, suggesting that it did not result from an activation of G protein-coupled receptors. No effects were found in human adrenal cortex-derived cells. The authors conclude that exposure of mouse adrenal cortex-derived cells to a 60 Hz magnetic field might stimulate the steroid hormone production via an increase in intracellular cAMP caused by the inhibition of phosphodiesterase enzyme activity.