Phosphorylated histone-H2AX (gamma-H2AX) foci formation is a sensitive indicator to detect DNA damage, especially DNA double-strand breaks. Further investigations were focused on interferences between gamma-H2AX foci formation and wortmannin (a phosphatidylinositol 3-kinase (PI3K) family inhibitor), Rad50 (a DNA damage-repair factor) and noise magnetic field.
The mouse embryos were examined in two groups: 2-cell (n=40) and 8-cell (n=30) embryos. In each experiment, 80 embryos in 2-cell group or 60 embryos in 8-cell group were used. All the experiments were repeated at least 3 times.
Positive controls were methyl-nitro-nitrosoguanidine (MNNG) treated embryos.
The experiments were conducted in 9 treatment groups with 4 dishes in each group: 1) sham exposure, 2) 0.3 mT EMF for 24 h, 3) 0.3 mT EMF for 48 h, 4) 0.5 mT EMF for 24 h, 5) 0.5 mT EMF for 48 h, 6) pretreated with 200 µM wortmannin for 30 min and 0.5 mT EMF for 24 h; 7) 0.5 mT EMF for 24 h and no exposure for another 24 h; 8) 0.5 mT noise MF alone for 24 h, 9) 0.5 mT EMF and 0.5 mT noise MF for 24 h.
In each experiment, 80 embryos in 2-cell group or 60 embryos in 8-cell group were used.
|Setup||The coil system generated highly uniform magnetic fields in the center of a 10 X 10 X 10 cm cube where well plates containing 30 embryos (8-cell embryos groups) or 40 embryos (2-cell embryos groups) were located. The plates were placed coaxially with the centerline in the central area of the coils, and the EMF was perpendicular to the plates. The sham exposure system was the same as exposure system but without current or signal input.|
|Additional info||To generate a noise MF, the above system was double-wrapped with two lines of copper wire. One of the double wires was provided with a 30-90 Hz white noise signal while the other one was excited in the same way as in the experiment with MF alone to get a combined MF and noise exposure. For positive control, embryos were treated with MNNG (N-methyl-N'-nitro-N-nitrosoguanidine).|
The data demonstrated a magnetic field dependent decrease of the cleavage rate of preimplantation mouse embryos. Gamma-H2AX foci formation was significantly increased in exposed embryos. The inducing effects of electromagnetic fields on gamma-H2AX foci formation could be inhibited by the treatment of noise magnetic fields or wortmannin. Furthermore, the data also showed that electromagnetic fields could activate the DNA repair mechanism by recruiting repair factor Rad50.
These findings suggest that electromagnetic fields could cause DNA damage in preimplantation embryos in vitro and that the adverse effects of electromagnetic fields on embryo development might at least partly act through DNA damage.