Shielding of the geomagnetic field reduces hydrogen peroxide production in human neuroblastoma cell and inhibits the activity of CuZn superoxide dismutase

Accumulative evidence has shown the adverse effects of a geomagnetic field shielded condition, so called a hypomagnetic field (HMF), on the metabolic processes and oxidative stress in animals and cells. However, the underlying mechanism remains unclear. In this study, we evaluate the role of HMF on the regulation of cellular reactive oxygen species (ROS) in human neuroblastoma SH-SY5Y cells. We found that HMF exposure led to ROS decrease, and that restoring the decrease by additional H2O2 rescued the HMF-enhanced cell proliferation. The measurements on ROS related indexes, including total anti-oxidant capacity, H2O2 and superoxide anion levels, and superoxide dismutase (SOD) activity and expression, indicated that the HMF reduced H2O2 production and inhibited the activity of CuZn-SOD. Moreover, the HMF accelerated the denaturation of CuZn-SOD as well as enhanced aggregation of CuZn-SOD protein, in vitro. Our findings indicate that CuZn-SOD is able to response to the HMF stress and suggest it a mediator of the HMF effect. Electronic supplementary material The online version of this article (doi:10.1007/s13238-017-0403-9) contains supplementary material, which is available to authorized users.


Methane dicarboxylic aldehyde (MDA) assay
MDA is the end product of lipid peroxidation and commonly considered as an indicator of oxidative stress. Cellular MDA levels were measured with the lipid peroxidation MDA assay Kit (Beyotime, Jiangsu, China) by the thiobarbituric acid (TBA) method. Cells were seeded into 60 mm petri dishes at the density of 3.0 × 10 4 cells/cm 2 . Cell lysates and protein concentration were prepared as described in the material and methods for TAC assay. Absorbance at 532 nm was measured by using a microplate reader (Bio-Rad, USA). The final data of MDA levels were normalized by protein concentration. Each measurement was performed in triplets.

Lactate dehydrogenase (LDH) assay
The activity of LDH released in the supernatant medium was measured with a LDH cytotoxicity assay kit (Cayman Chemical Company, Ann Arbor, MI, USA).
The activity of LDH was determined by the conversion of tetrazolium salt into a red formazon product. Cells were seeded in 60 mm petri dishes as described above. In brief, 100 μL medium was transferred to a new 96-well plate and mixed with 100 μL reaction solution. After incubating the plate with gentle shaking for 30 minutes at RT, absorbance at 490 nm was measured using a microplate reader (Biorad, USA). The concentration of the released LDH was calculated by the prepared LDH standard curve. Each measurement was performed in triplets.

Catalase (CAT) assay
The activity of cellular CAT was measured with a catalase assay kit (Beyotime, China). Cells were seeded in 60 mm petri dishes as described above. Cell lysates and protein concentration were prepared as described above. Cell ℃ for 30 min. The absorbance at 520 nm was measured using a microplate reader (Biorad, USA). The activity of CAT was calculated by a prepared standard curve. The final data of CAT activity was normalized by protein concentration. Each measurement was performed in triplets.

Glutathione peroxidase (GPx) assays
The cellular GPx activity was measured with a cellular GPx assay kit (Beyotime, China). Cells were seeded in 60 mm petri dishes as described above. Cell lysates and protein concentration were prepared as described above. Mix 10 μL GPx working solution, 176 μL assay buffer and 4 μL H 2 O 2 (15 mM) with 10 μL cell lysate. The absorbance at 340 nm (A 340 ) of the mixture was measure every 30 s for 3 min at 25 ℃ by a microplate reader (Biorad, USA). The GPx activity was determined by the rate of A 340 change (∆A340/min). The final data of GSH reductase activity were normalized by protein concentration. Each measurement was performed in triplets.

Circular dichroism (CD) spectrum assay
CD measurements were performed using a Chirascan plus (Applied Photophysics, Leatherhead, Surrey, UK) at RT. The CD scans were recorded within a wavelength range of 180 to 260 nm. All measurements were performed in a cuvette with a volume of 200 μL in ddH 2 O. Individual titrations were performed with 0.176mg/ml CuZn-SOD in a ddH 2 O incubated in GMF and HMF at 37°C for 0.5 h. ddH 2 O was used as the blank. The spectra were measured based on an average of three runs.

Fluorescence spectra assay
CuZn-SOD (sigma, USA) solution (0.88 μg/ml) were prepared by diluting the stock solution with ddH 2 O. The solution was incubated in the GMF and HMF condition at 37℃ for 30 min. The intrinsic fluorescence spectra of the solutions