1,800 MHz RF Electromagnetic Irradiation Impairs Neurite Outgrowth With a Decrease in Rap1-GTP in Primary Mouse Hippocampal Neurons and Neuro2a Cells

Abstract

1,800 MHz RF Electromagnetic Irradiation Impairs Neurite Outgrowth With a Decrease in Rap1-GTP in Primary Mouse Hippocampal Neurons and Neuro2a Cells Li Yanqi, Deng Ping, Chen Chunhai, Ma Qinlong, Pi Huifeng, He Mindi, Lu Yonghui, Gao Peng, Zhou Chao, He Zhixin, Zhang Yanwen, Yu Zhengping, Zhang Lei. 1,800 MHz Radiofrequency Electromagnetic Irradiation Impairs Neurite Outgrowth With a Decrease in Rap1-GTP in Primary Mouse Hippocampal Neurons and Neuro2a Cells. Frontiers in Public Health. Vol. 9:1854. 2021. doi: 10.3389/fpubh.2021.771508 Abstract Background: With the global popularity of communication devices such as mobile phones, there are increasing concerns regarding the effect of radiofrequency electromagnetic radiation (RF-EMR) on the brain, one of the most important organs sensitive to RF-EMR exposure at 1,800 MHz. However, the effects of RF-EMR exposure on neuronal cells are unclear. Neurite outgrowth plays a critical role in brain development, therefore, determining the effects of 1,800 MHz RF-EMR exposure on neurite outgrowth is important for exploring its effects on brain development. Objectives: We aimed to investigate the effects of 1,800 MHz RF-EMR exposure for 48 h on neurite outgrowth in neuronal cells and to explore the associated role of the Rap1 signaling pathway. Material and Methods: Primary hippocampal neurons from C57BL/6 mice and Neuro2a cells were exposed to 1,800 MHz RF-EMR at a specific absorption rate (SAR) value of 4 W/kg for 48 h. CCK-8 assays were used to determine the cell viability after 24, 48, and 72 h of irradiation. Neurite outgrowth of primary hippocampal neurons (DIV 2) and Neuro2a cells was observed with a 20 × optical microscope and recognized by ImageJ software. Rap1a and Rap1b gene expressions were detected by real-time quantitative PCR. Rap1, Rap1a, Rap1b, Rap1GAP, and p-MEK1/2 protein expressions were detected by western blot. Rap1-GTP expression was detected by immunoprecipitation. The role of Rap1-GTP was assessed by transfecting a constitutively active mutant plasmid (Rap1-Gly_Val-GFP) into Neuro2a cells. Results: Exposure to 1,800 MHz RF-EMR for 24, 48, and 72 h at 4 W/kg did not influence cell viability. The neurite length, primary and secondary neurite numbers, and branch points of primary mouse hippocampal neurons were significantly impaired by 48-h RF-EMR exposure. The neurite-bearing cell percentage and neurite length of Neuro2a cells were also inhibited by 48-h RF-EMR exposure. Rap1 activity was inhibited by 48-h RF- EMR with no detectable alteration in either gene or protein expression of Rap1. The protein expression of Rap1GAP increased after 48-h RF-EMR exposure, while the expression of p-MEK1/2 protein decreased. Overexpression of constitutively active Rap1 reversed the decrease in Rap1-GTP and the neurite outgrowth impairment in Neuro2a cells induced by 1,800 MHz RF-EMR exposure for 48 h. Conclusion: Rap1 activity and related signaling pathways are involved in the disturbance of neurite outgrowth induced by 48-h 1,800 MHz RF-EMR exposure. The effects of RF-EMR exposure on neuronal development in infants and children deserve greater focus. Open access paper: frontiersin.org

AI evidence extraction

Main findings

Exposure to 1,800 MHz RF-EMR at SAR 4 W/kg did not affect cell viability at 24, 48, or 72 h. After 48 h exposure, neurite outgrowth measures were significantly impaired in primary mouse hippocampal neurons and inhibited in Neuro2a cells. Rap1 activity (Rap1-GTP) decreased without detectable changes in Rap1 gene/protein expression; Rap1GAP increased and p-MEK1/2 decreased, and constitutively active Rap1 reversed the Rap1-GTP decrease and neurite outgrowth impairment in Neuro2a cells.

Outcomes measured

• Cell viability (CCK-8)
• Neurite outgrowth metrics (neurite length; primary/secondary neurite numbers; branch points) in primary mouse hippocampal neurons
• Neurite-bearing cell percentage and neurite length in Neuro2a cells
• Rap1 activity (Rap1-GTP)
• Rap1a/Rap1b gene expression (qPCR)
• Rap1/Rap1a/Rap1b/Rap1GAP/p-MEK1/2 protein expression (western blot)
• Effect of constitutively active Rap1 (Rap1-Gly_Val-GFP) on RF-EMR-induced changes in Neuro2a cells

Limitations

• In vitro study (primary mouse hippocampal neurons and Neuro2a cells); generalizability to humans not addressed in abstract
• Sample size not reported in abstract
• Exposure conditions limited to 1,800 MHz, SAR 4 W/kg, and primarily 48 h for neurite outcomes

Suggested hubs

• mobile-phones (0.55)
  Background frames exposure as relevant to communication devices such as mobile phones at 1,800 MHz.

{
    "study_type": "in_vitro",
    "exposure": {
        "band": "RF",
        "source": null,
        "frequency_mhz": 1800,
        "sar_wkg": 4,
        "duration": "48 h (also assessed at 24, 48, 72 h for viability)"
    },
    "population": null,
    "sample_size": null,
    "outcomes": [
        "Cell viability (CCK-8)",
        "Neurite outgrowth metrics (neurite length; primary/secondary neurite numbers; branch points) in primary mouse hippocampal neurons",
        "Neurite-bearing cell percentage and neurite length in Neuro2a cells",
        "Rap1 activity (Rap1-GTP)",
        "Rap1a/Rap1b gene expression (qPCR)",
        "Rap1/Rap1a/Rap1b/Rap1GAP/p-MEK1/2 protein expression (western blot)",
        "Effect of constitutively active Rap1 (Rap1-Gly_Val-GFP) on RF-EMR-induced changes in Neuro2a cells"
    ],
    "main_findings": "Exposure to 1,800 MHz RF-EMR at SAR 4 W/kg did not affect cell viability at 24, 48, or 72 h. After 48 h exposure, neurite outgrowth measures were significantly impaired in primary mouse hippocampal neurons and inhibited in Neuro2a cells. Rap1 activity (Rap1-GTP) decreased without detectable changes in Rap1 gene/protein expression; Rap1GAP increased and p-MEK1/2 decreased, and constitutively active Rap1 reversed the Rap1-GTP decrease and neurite outgrowth impairment in Neuro2a cells.",
    "effect_direction": "harm",
    "limitations": [
        "In vitro study (primary mouse hippocampal neurons and Neuro2a cells); generalizability to humans not addressed in abstract",
        "Sample size not reported in abstract",
        "Exposure conditions limited to 1,800 MHz, SAR 4 W/kg, and primarily 48 h for neurite outcomes"
    ],
    "evidence_strength": "low",
    "confidence": 0.7800000000000000266453525910037569701671600341796875,
    "peer_reviewed_likely": "yes",
    "keywords": [
        "1,800 MHz",
        "RF-EMR",
        "SAR 4 W/kg",
        "neurite outgrowth",
        "primary mouse hippocampal neurons",
        "Neuro2a",
        "Rap1-GTP",
        "Rap1GAP",
        "p-MEK1/2"
    ],
    "suggested_hubs": [
        {
            "slug": "mobile-phones",
            "weight": 0.5500000000000000444089209850062616169452667236328125,
            "reason": "Background frames exposure as relevant to communication devices such as mobile phones at 1,800 MHz."
        }
    ]
}

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