Chunhai Chen

Exposure to 1800 MHz radiofrequency radiation induces oxidative damage to mitochondrial DNA in primary cultured neurons

Increasing evidence indicates that oxidative stress may be involved in the adverse effects of radiofrequency (RF) radiation on the brain. Because mitochondrial DNA (mtDNA) defects are closely associated with various nervous system diseases and mtDNA is particularly susceptible to oxidative stress, the purpose of this study was to determine whether radiofrequency radiation can cause oxidative damage to mtDNA. In this study, we exposed primary cultured cortical neurons to pulsed RF electromagnetic fields at a frequency of 1800 MHz modulated by 217 Hz at an average special absorption rate (SAR) of 2 W/kg. At 24 h after exposure, we found that RF radiation induced a significant increase in the levels of 8-hydroxyguanine (8-OHdG), a common biomarker of DNA oxidative damage, in the mitochondria of neurons. Concomitant with this finding, the copy number of mtDNA and the levels of mitochondrial RNA (mtRNA) transcripts showed an obvious reduction after RF exposure. Each of these mtDNA disturbances could be reversed by pretreatment with melatonin, which is known to be an efficient antioxidant in the brain. Together, these results suggested that 1800 MHz RF radiation could cause oxidative damage to mtDNA in primary cultured neurons. Oxidative damage to mtDNA may account for the neurotoxicity of RF radiation in the brain.

Hypoxia enhances CXCR4 expression favoring microglia migration via HIF-1α activation

Migration toward pathological area is the first critical step in microglia engagement during the central nervous system (CNS) injury, although the molecular mechanisms underlying microglia mobilization have not been fully understood. Here, we report that hypoxia promotes stromal cell-derived factor-1alpha (SDF-1alpha) induced microglia migration by inducing the CXC chemokine receptor 4 (CXCR4) expression. Exposure to hypoxia significantly enhanced CXCR4 expression levels in N9 microglia cell. Then, cell migration induced by SDF-1, a CXCR4-specific ligand, was observed accelerated. Blockade of hypoxia inducible factor-1alpha (HIF-1alpha) activation by inhibitors of phosphoinositide-3-kinase (PI3K)/Akt signaling pathway abrogated both of hypoxia-induced CXCR4 up-regulation and cell-migration acceleration. These results point to a crucial role of Hypoxia-HIF-1alpha-CXCR4 pathway during microglia migration.

Melatonin ameliorates bisphenol A-induced DNA damage in the germ cells of adult male rats.

Bisphenol A (BPA) is a well-known endocrine-disrupting chemical (EDC) that has received particular attention because of its widespread distribution in humans. Due to its chemical similarity to diethylstilbestrol, which is carcinogenic to mammals, the possible genotoxicity of BPA has already largely been evaluated. However, the results are still inconclusive and controversial. To investigate the genotoxic effects of BPA in rat germ cells and the potential protective action of melatonin against these effects, adult male Sprague-Dawley rats were orally administered BPA at a dose of 200mg/kg body weight per day for ten consecutive days with or without melatonin pretreatment. The thiobarbituric acid reactive substances (TBARS) level and superoxide dismutase (SOD) activity in the testes were evaluated. Subsequently, their spermatocytes were isolated, and DNA damage was assessed using an alkaline comet assay and the meiotic spread method. BPA administration did not significantly affect the weights of rats and their reproductive organs, and no alteration in sperm count was found. However, we demonstrated that BPA administration induced a significant increase in TBARS levels and a decrease in SOD activity that were concomitant with an increase in DNA migration within male germ cells and γH2AX foci formation on the autosomes of pachytene spermatocytes. Furthermore, a decrease in the proportion of 4C-cells was observed. These BPA effects were significantly alleviated by melatonin pretreatment. Nevertheless, the genotoxic effects of BPA were not accompanied by apoptosis in germ cells and morphological changes in the testes. These results indicate that BPA exposure may induce DNA damage accumulation in germ cells via oxidative stress. Moreover, melatonin may be a promising pharmacological candidate for preventing the potential genotoxicity of BPA following occupational or environmental exposure.

Exposure to 1800 MHz radiofrequency electromagnetic radiation induces oxidative DNA base damage in a mouse spermatocyte-derived cell line

Whether exposure to radiofrequency electromagnetic radiation (RF-EMR) emitted from mobile phones can induce DNA damage in male germ cells remains unclear. In this study, we conducted a 24h intermittent exposure (5 min on and 10 min off) of a mouse spermatocyte-derived GC-2 cell line to 1800 MHz Global System for Mobile Communication (GSM) signals in GSM-Talk mode at specific absorption rates (SAR) of 1 W/kg, 2 W/kg or 4 W/kg. Subsequently, through the use of formamidopyrimidine DNA glycosylase (FPG) in a modified comet assay, we determined that the extent of DNA migration was significantly increased at a SAR of 4 W/kg. Flow cytometry analysis demonstrated that levels of the DNA adduct 8-oxoguanine (8-oxoG) were also increased at a SAR of 4 W/kg. These increases were concomitant with similar increases in the generation of reactive oxygen species (ROS); these phenomena were mitigated by co-treatment with the antioxidant α-tocopherol. However, no detectable DNA strand breakage was observed by the alkaline comet assay. Taking together, these findings may imply the novel possibility that RF-EMR with insufficient energy for the direct induction of DNA strand breaks may produce genotoxicity through oxidative DNA base damage in male germ cells.

A TRPC1-mediated increase in store-operated Ca2+ entry is required for the proliferation of adult hippocampal neural progenitor cells

Adult hippocampal neurogenesis plays an important role in brain function and neurological diseases. Adult neural progenitor cell (aNPC) proliferation is a critical first step in hippocampal neurogenesis. However, the mechanisms that modulate aNPC proliferation have not been fully identified. Ample evidence has demonstrated that cell proliferation is dependent on the intracellular Ca(2+) concentration. We hypothesized that store-operated Ca(2+) channels (SOCs), which are ubiquitously expressed in all cell types, participate in aNPC proliferation. We found that store-operated Ca(2+) entry (SOCE) was involved in the proliferation of aNPCs and that 2-APB, Gd(3+) and SKF96365, antagonists of SOCE and canonical transient receptor potential (TRPC), respectively, inhibited the increase in SOCE and aNPC proliferation. We therefore analyzed the expression of TRPCs in aNPCs and showed that TRPC1 is the most significantly upregulated member under proliferative conditions. Interestingly, knockdown of TRPC1 and using an antibody against TRPC1 markedly reduced the degree of SOCE and aNPC proliferation. In parallel, we observed the suppression of aNPC proliferation was found to be associated with cell cycle arrest in G0/G1 phase. Furthermore, gene expression microarray analysis revealed a selective up- or downregulation of 10 genes in aNPCs following TRPC1 silencing. Knockdown of Orai1 or STIM1 also induced a significant inhibition of SOCE and proliferation in aNPCs, and all three proteins were colocalized in the plasma membrane region of cells. Together, these results indicate that SOCE represents a principal mechanism regulating the proliferation of aNPCs and that TRPC1 is an essential component of this pathway. This discovery may be important in improving adult hippocampal neurogenesis and treating cognitive deficits.

Protective effects of lycopene against amyloid β-induced neurotoxicity in cultured rat cortical neurons.

The neurotoxicity of amyloid β (Aβ) has been implicated as a critical cause in the pathogenesis of Alzheimers disease (AD). Among antioxidant phytochemicals derived from fruit and vegetables, lycopene has recently received considerable attention for its potent protective properties already demonstrated in several models of oxidative damage. The present study aims to investigate whether lycopene could provide protective effects against Aβ-induced neurotoxicity in primary cultured rat cortical neurons. The cultured cortical neurons were pretreated with different dose of lycopene for 4h, followed by the challenge with 25 μM Aβ(25-35) for 24h. The results showed that pretreatment with lycopene efficiently attenuated Aβ(25-35)-induced neurotoxicity, as evidenced by the improved cell viability and the decreased apoptotic rate. In addition, lycopene inhibited the reactive oxygen species generation and mitochondrial membrane potential depolarization caused by Aβ(25-35). Lycopene also restored the levels of proapoptotic Bax, antiapoptotic Bcl-2, and inhibited caspase-3 activation. These beneficial effects may contribute to the protection against Aβ-induced neurotoxicity. Together, our results suggest that the natural antioxidant lycopene has potential for neuroprotection and therefore, may be a promising candidate for AD treatment.

The role of the JAK2-STAT3 pathway in pro-inflammatory responses of EMF-stimulated N9 microglial cells

BackgroundIn several neuropathological conditions, microglia can become overactivated and cause neurotoxicity by initiating neuronal damage in response to pro-inflammatory stimuli. Our previous studies have shown that exposure to electromagnetic fields (EMF) activates cultured microglia to produce tumor necrosis factor (TNF)-α and nitric oxide (NO) through signal transduction involving the activator of transcription STAT3. Here, we investigated the role of STAT3 signaling in EMF-induced microglial activation and pro-inflammatory responses in more detail than the previous study.MethodsN9 microglial cells were treated with EMF exposure or a sham treatment, with or without pretreatment with an inhibitor (Pyridone 6, P6) of the Janus family of tyrosine kinases (JAK). The activation state of microglia was assessed via immunoreaction using the microglial marker CD11b. Levels of inducible nitric oxide synthase (iNOS), TNF-α and NO were measured using real-time reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and the nitrate reductase method. Activation of JAKs and STAT3 proteins was evaluated by western blotting for specific tyrosine phosphorylation. The ability of STAT3 to bind to DNA was detected with an electrophoresis mobility shift assay (EMSA).ResultsEMF was found to significantly induce phosphorylation of JAK2 and STAT3, and DNA-binding ability of STAT3 in N9 microglia. In addition, EMF dramatically increased the expression of CD11b, TNF-α and iNOS, and the production of NO. P6 strongly suppressed the phosphorylation of JAK2 and STAT3 and diminished STAT3 activity in EMF-stimulated microglia. Interestingly, expression of CD11b as well as gene expression and production of TNF-α and iNOS were suppressed by P6 at 12 h, but not at 3 h, after EMF exposure.ConclusionsEMF exposure directly triggers initial activation of microglia and produces a significant pro-inflammatory response. Our findings confirm that the JAK2-STAT3 pathway may not mediate this initial microglial activation but does promote pro-inflammatory responses in EMF-stimulated microglial cells. Thus, the JAK2-STAT3 pathway might be a therapeutic target for reducing pro-inflammatory responses in EMF-activated microglia.

Mortalin overexpression attenuates beta-amyloid-induced neurotoxicity in SH-SY5Y cells

Amyloid-beta peptide (Aβ) is shown to be toxic to the mitochondria and implicates this organelle in the pathogenesis of Alzheimers disease. Previous studies suggest that targeting mitochondria for protection may be a useful strategy to reduce Aβ-induced neurotoxicity. Mortalin is the mitochondrial located member of the heat shock protein 70 family, which serves as a major mitochondrial molecular chaperone and plays a key role in mitochondrial import of proteins. Several studies have demonstrated the protective potential of Hsp75 overexpression against apoptosis induced by various forms of stresses. To investigate whether mortalin overexpression could provide protective effects on Aβ toxicity, SH-SY5Y cells were used to transfect human mortalin gene and then treated with Aβ(1-42) for 24h. It is found that overexpression of mortalin efficiently attenuated Aβ(1-42)-induced cell viability damage and apoptosis. Additionally, inhibition of mortalin expression by mortalin-specific siRNA oligonucleotides sensitized SH-SY5Y cells to Aβ(1-42)-induced neurotoxicity. Furthermore, mortalin overexpression significantly inhibited the Aβ(1-42)-induced depolarization of mitochondrial membrane potential, reversed the Aβ(1-42)-induced reduction in cytochrome c oxidase activity and ATP generation, and suppressed the Aβ(1-42)-induced reactive oxygen species accumulation and lipid peroxidation. Together, our results suggest that mortalin can afford protection against Aβ(1-42)-induced neurotoxicity in SH-SY5Y cells. These beneficial effects of mortalin overexpression may be attributable to its roles in maintaining mitochondrial function and reducing oxidative stress.

Lycopene protects against trimethyltin-induced neurotoxicity in primary cultured rat hippocampal neurons by inhibiting the mitochondrial apoptotic pathway

Lycopene is a potent free radicals scavenger with demonstrated protective efficacy in several experimental models of oxidative damage. Trimethyltin (TMT) is an organotin compound with neurotoxic effects on the hippocampus and other limbic structures and is used to model neurodegenerative diseases targeting these brain areas. Oxidative stress is widely accepted as a central pathogenic mechanism of TMT-mediated neurotoxicity. The present study investigated whether the plant carotene lycopene protects against TMT-induced neurotoxicity in primary cultured rat hippocampal neurons. Lycopene pretreatment improved cell viability in TMT-treated hippocampal neurons and inhibited neuronal apoptosis. Microfluorometric imaging revealed that lycopene inhibited the accumulation of mitochondria-derived reactive oxygen species (ROS) during TMT exposure. Moreover, lycopene ameliorated TMT-induced activation of the mitochondrial permeability transition pore (mPTP) and the concomitant depolarization of the mitochondrial membrane potential (ΔΨ(m)). Consequently, cytochrome c release from the mitochondria and ensuing caspase-3 activation were markedly reduced. These findings reveal that lycopene protects against TMT-induced neurotoxicity by inhibiting the mitochondrial apoptotic pathway. The anti-apoptotic effect of lycopene on hippocampal neurons highlights the therapeutic potential of plant-derived antioxidants against neurodegenerative diseases.

Extremely Low-Frequency Electromagnetic Fields Affect Transcript Levels of Neuronal Differentiation-Related Genes in Embryonic Neural Stem Cells

Previous studies have reported that extremely low-frequency electromagnetic fields (ELF-EMF) can affect the processes of brain development, but the underlying mechanism is largely unknown. The proliferation and differentiation of embryonic neural stem cells (eNSCs) is essential for brain development during the gestation period. To date, there is no report about the effects of ELF-EMF on eNSCs. In this paper, we studied the effects of ELF-EMF on the proliferation and differentiation of eNSCs. Primary cultured eNSCs were treated with 50 Hz ELF-EMF; various magnetic intensities and exposure times were applied. Our data showed that there was no significant change in cell proliferation, which was evaluated by cell viability (CCK-8 assay), DNA synthesis (Edu incorporation), average diameter of neurospheres, cell cycle distribution (flow cytometry) and transcript levels of cell cycle related genes (P53, P21 and GADD45 detected by real-time PCR). When eNSCs were induced to differentiation, real-time PCR results showed a down-regulation of Sox2 and up-regulation of Math1, Math3, Ngn1 and Tuj1 mRNA levels after 50 Hz ELF-EMF exposure (2 mT for 3 days), but the percentages of neurons (Tuj1 positive cells) and astrocytes (GFAP positive cells) were not altered when detected by immunofluorescence assay. Although cell proliferation and the percentages of neurons and astrocytes differentiated from eNSCs were not affected by 50 Hz ELF-EMF, the expression of genes regulating neuronal differentiation was altered. In conclusion, our results support that 50 Hz ELF-EMF induce molecular changes during eNSCs differentiation, which might be compensated by post-transcriptional mechanisms to support cellular homeostasis.