Radiofrequency radiation reshapes tumor immune microenvironment into antitumor phenotype in pulmonary metastatic melanoma by inducing active transformation of tumor-infiltrating CD8+ T and NK cells.

Immunosuppression by the tumor microenvironment is a pivotal factor contributing to tumor progression and immunotherapy resistance. Priming the tumor immune microenvironment (TIME) has emerged as a promising strategy for improving the efficacy of cancer immunotherapy. In this study we investigated the effects of noninvasive radiofrequency radiation (RFR) exposure on tumor progression and TIME phenotype, as well as the antitumor potential of PD-1 blockage in a model of pulmonary metastatic melanoma (PMM). Mouse model of PMM was established by tail vein injection of B16F10 cells. From day 3 after injection, the mice were exposed to RFR at an average specific absorption rate of 9.7 W/kg for 1 h per day for 14 days. After RFR exposure, lung tissues were harvested and RNAs were extracted for transcriptome sequencing; PMM-infiltrating immune cells were isolated for single-cell RNA-seq analysis. We showed that RFR exposure significantly impeded PMM progression accompanied by remodeled TIME of PMM via altering the proportion and transcription profile of tumor-infiltrating immune cells. RFR exposure increased the activation and cytotoxicity signatures of tumor-infiltrating CD8+ T cells, particularly in the early activation subset with upregulated genes associated with T cell cytotoxicity. The PD-1 checkpoint pathway was upregulated by RFR exposure in CD8+ T cells. RFR exposure also augmented NK cell subsets with increased cytotoxic characteristics in PMM. RFR exposure enhanced the effector function of tumor-infiltrating CD8+ T cells and NK cells, evidenced by increased expression of cytotoxic molecules. RFR-induced inhibition of PMM growth was mediated by RFR-activated CD8+ T cells and NK cells. We conclude that noninvasive RFR exposure induces antitumor remodeling of the TIME, leading to inhibition of tumor progression, which provides a promising novel strategy for TIME priming and potential combination with cancer immunotherapy.

Cadmium Exposure Enhances Bisphenol A-Induced Genotoxicity through 8-Oxoguanine-DNA Glycosylase-1 OGG1 Inhibition in NIH3T3 Fibroblast Cells.

BACKGROUND: Both cadmium (Cd) and bisphenol A (BPA) are commonly encountered in humans' daily activities, but their combined genotoxic effects remain unclear. METHODS: In the present study, we exposed a mouse embryonic fibroblast cell line (NIH3T3) to Cd for 24 h, followed by a 24 h BPA exposure to evaluate toxicity. The cytotoxicity was evaluated by viability with CCK-8 assay and lactate dehydrogenase (LDH) release. Reactive oxygen species (ROS) production was measured by 2',7'-dichlorofluorescein diacetate (DCFH-DA). And DNA damage was measured by 8-hydroxydeoxyguanosine (8-OHdG), phosphorylated H2AX (γH2AX) and the comet assay. The flow cytometry was used to detect cell cycle distribution, and apoptosis was determined by TUNEL assay and western blot against poly-ADP-ribose polymerase (PARP). RESULTS: The results showed that Cd or BPA treatments alone (with the exception of BPA exposure at 50 µM) did not alter cell viability. However, pre-treatment with Cd aggravated the BPA-induced reduction in cell viability; increased BPA-induced LDH release, ROS production, DNA damage and G2 phase arrest; and elevated BPA-induced TUNEL-positive cells and the expression levels of cleaved PARP. Cd exposure concurrently decreased the expression of 8-oxoguanine-DNA glycosylase-1 (OGG1), whereas OGG1 over-expression abolished the enhancement of Cd on BPA-induced genotoxicity and cytotoxicity. CONCLUSION: These findings indicate that Cd exposure aggravates BPA-induced genotoxicity and cytotoxicity through OGG1 inhibition.

NiO nanoparticles induce apoptosis through repressing SIRT1 in human bronchial epithelial cells.

With application of nano-sized nickel-containing particles (Nano-Ni) expanding, the health concerns about their adverse effects on the pulmonary system are increasing. However, the mechanisms for the pulmonary toxicity of these materials remain unclear. In the present study, we focused on the impacts of NiO nanoparticles (NiONPs) on sirtuin1 (SIRT1), a NAD-dependent deacetylase, and investigated whether SIRT1 was involved in NiONPs-induced apoptosis. Although the NiONPs tended to agglomerate in fluid medium, they still entered into the human bronchial epithelial cells (BEAS-2B) and released Ni(2+) inside the cells. NiONPs at doses of 5, 10, and 20µg/cm(2) inhibited the cell viability. NiONPs' produced cytotoxicity was demonstrated through an apoptotic process, indicated by increased numbers of Annexin V positive cells and caspase-3 activation. The expression of SIRT1 was markedly down-regulated by the NiONPs, accompanied by the hyperacetylation of p53 (tumor protein 53) and overexpression of Bax (Bcl-2-associated X protein). However, overexpression of SIRT1 through resveratrol treatment or transfection clearly attenuated the NiONPs-induced apoptosis and activation of p53 and Bax. Our results suggest that the repression of SIRT1 may underlie the NiONPs-induced apoptosis via p53 hyperacetylation and subsequent Bax activation. Because SIRT1 participates in multiple biologic processes by deacetylation of dozens of substrates, this knowledge of the impact of NiONPs on SIRT1 may lead to an improved understanding of the toxic mechanisms of Nano-Ni and provide a molecular target to antagonize Nano-Ni toxicity.

The amelioration of phagocytic ability in microglial cells by curcumin through the inhibition of EMF-induced pro-inflammatory responses.

BACKGROUND: Insufficient clearance by microglial cells, prevalent in several neurological conditions and diseases, is intricately intertwined with MFG-E8 expression and inflammatory responses. Electromagnetic field (EMF) exposure can elicit the pro-inflammatory activation and may also trigger an alteration of the clearance function in microglial cells. Curcumin has important roles in the anti-inflammatory and phagocytic process. Here, we evaluated the ability of curcumin to ameliorate the phagocytic ability of EMF-exposed microglial cells (N9 cells) and documented relative pathways. METHODS: N9 cells were pretreated with or without recombinant murine MFG-E8 (rmMFG-E8), curcumin and an antibody of toll-like receptor 4 (anti-TLR4), and subsequently treated with EMF or a sham exposure. Their phagocytic ability was evaluated using phosphatidylserine-containing fluorescent bioparticles. The pro-inflammatory activation of microglia was assessed via CD11b immunoreactivity and the production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and nitric oxide (NO) via the enzyme-linked immunosorbent assay or the Griess test. We evaluated the ability of curcumin to ameliorate the phagocytic ability of EMF-exposed N9 cells, including checking the expression of MFG-E8, αvß3 integrin, TLR4, nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) using Western blotting. RESULTS: EMF exposure dramatically enhanced the expression of CD11b and depressed the phagocytic ability of N9 cells. rmMFG-E8 could clearly ameliorate the phagocytic ability of N9 cells after EMF exposure. We also found that EMF exposure significantly increased the secretion of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß) and the production of NO; however, these increases were efficiently chilled by the addition of curcumin to the culture medium. This reduction led to the amelioration of the phagocytic ability of EMF-exposed N9 cells. Western blot analysis revealed that curcumin and naloxone restored the expression of MFG-E8 but had no effect on TLR4 and cytosolic STAT3. Moreover, curcumin significantly reduced the expression of NF-κB p65 in nuclei and phospho-STAT3 (p-STAT3) in cytosols and nuclei. CONCLUSIONS: This study indicates that curcumin ameliorates the depressed MFG-E8 expression and the attenuated phagocytic ability of EMF-exposed N9 cells, which is attributable to the inhibition of the pro-inflammatory response through the NF-κB and STAT3 pathways.

Mobile phone radiation induces mode-dependent DNA damage in a mouse spermatocyte-derived cell line: a protective role of melatonin.

PURPOSE: To evaluate whether exposure to mobile phone radiation (MPR) can induce DNA damage in male germ cells. MATERIALS AND METHODS: A mouse spermatocyte-derived GC-2 cell line was exposed to a commercial mobile phone handset once every 20 min in standby, listen, dialed or dialing modes for 24 h. DNA damage was determined using an alkaline comet assay. RESULTS: The levels of DNA damage were significantly increased following exposure to MPR in the listen, dialed and dialing modes. Moreover, there were significantly higher increases in the dialed and dialing modes than in the listen mode. Interestingly, these results were consistent with the radiation intensities of these modes. However, the DNA damage effects of MPR in the dialing mode were efficiently attenuated by melatonin pretreatment. CONCLUSIONS: These results regarding mode-dependent DNA damage have important implications for the safety of inappropriate mobile phone use by males of reproductive age and also suggest a simple preventive measure: Keeping mobile phones as far away from our body as possible, not only during conversations but during 'dialed' and 'dialing' operation modes. Since the 'dialed' mode is actually part of the standby mode, mobile phones should be kept at a safe distance from our body even during standby operation. Furthermore, the protective role of melatonin suggests that it may be a promising pharmacological candidate for preventing mobile phone use-related reproductive impairments.

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 2.45 GHz electromagnetic fields elicits an HSP-related stress response in rat hippocampus.

The issue of possible neurobiological effects of the electromagnetic field (EMF) exposure is highly controversial. To determine whether electromagnetic field exposure could act as an environmental stimulus capable of producing stress responses, we employed the hippocampus, a sensitive target of electromagnetic radiation, to assess the changes in its stress-related gene and protein expression after EMF exposure. Adult male Sprague-Dawley rats with body restrained were exposed to a 2.45 GHz EMF at a specific absorption rate (SAR) of 6 W/kg or sham conditions. cDNA microarray was performed to examine the changes of gene expression involved in the biological effects of electromagnetic radiation. Of 2048 candidate genes, 23 upregulated and 18 downregulated genes were identified. Of these differential expression genes, two heat shock proteins (HSP), HSP27 and HSP70, are notable because expression levels of both proteins are increased in the rat hippocampus. Result from immunocytochemistry revealed that EMF caused intensive staining for HSP27 and HSP70 in the hippocampus, especially in the pyramidal neurons of cornu ammonis 3 (CA3) and granular cells of dentate gyrus (DG). The gene and protein expression profiles of HSP27 and HSP70 were further confirmed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Our data provide direct evidence that exposure to electromagnetic fields elicits a stress response in the rat hippocampus.

Tau protein is involved in morphological plasticity in hippocampal neurons in response to BDNF.

Tau protein, a microtubule-associated protein involved in a number of neurological disorders such as Alzheimer's disease (AD), may undergo modifications under both physiological and pathological conditions. However, the signaling pathways that couple tau protein to neuronal physiology such as synaptic plasticity have not yet been elucidated. Here we report that tau protein is involved in morphological plasticity in response to brain derived neurotrophic factor (BDNF). Stimulation of the cultured rat hippocampal neurons with BDNF resulted in increased tau protein expression, as detected by Western blotting. Furthermore, tau protein accumulated in the distal region of the neurite when treated with taxol or taxol plus BDNF. The increased tau protein also protected neurons against nocodazole-induced dendrite loss. Moreover, BDNF promoted spine growth as well as tau protein over-expression. Knockdown of tau protein using specific short-hairpin RNA (shRNA) significantly decreased the spine density. And BDNF could not increase the spine density of tau-knockdown neurons. These results highlight a possible role for tau protein in the dynamic rearrangement of cytoskeletal fibers vital for BDNF-induced synaptic plasticity.

[Relationship between activation of microglia and Jaks phosphorylation induced by microwave irradiation].

OBJECTIVE: To explore the relationship between microglial proinflammatory and electromagnetic radiation and unveil the role of microglia in microwave radiation induced central nervous system injury. METHODS: N9 microglia cells cultured in vitro were exposed to microwave at 90 mW/cm2. Cell flow cytometry was used to observe the expression of CD11b at different time points after exposure; ELISA was used to detect the concentration of TNF-alpha in N9 cell culture supernatant; RT-PCR analysis confirmed iNOS mRNA expression in N9 microglia cells; and Nitrate Reductase Method was used to test NO amount in culture supernatant. RESULTS: The CD11b positive microglial cells increased significantly at 3 h after microwave exposure (P < 0.05), continued to increase until 24 h and peaked at 6 h after exposure. The amount of TNF-alpha rose dramatically from 1 h to 24 h after exposure (P < 0.01) and peaked at 3 h [(762.1 +/- 61.5) pg/ml] after exposure (P < 0.01). The level of NO started to increase at 1 h [(4.48-0.59) micromol/L] and lasted for 24 h after exposure. The expression of iNOS mRNA increased significantly at 1 h (P < 0.05), and tripled the original expression at 6 h after exposure, hereafter, it decreased slightly, but all were higher than the control group within 24 h after exposure. CONCLUSION: Microwave radiation could induce the activation of microglia cells. The activated microglia cells could induce microglial proinflammatory by producing large amounts of TNF-alpha, NO, etc.