ROS/mtROS promotes TNTs formation via the PI3K/AKT/mTOR pathway to protect against mitochondrial damages in glial cells induced by engineered nanomaterials.

BACKGROUND: As the demand and application of engineered nanomaterials have increased, their potential toxicity to the central nervous system has drawn increasing attention. Tunneling nanotubes (TNTs) are novel cell-cell communication that plays a crucial role in pathology and physiology. However, the relationship between TNTs and nanomaterials neurotoxicity remains unclear. Here, three types of commonly used engineered nanomaterials, namely cobalt nanoparticles (CoNPs), titanium dioxide nanoparticles (TiO2NPs), and multi-walled carbon nanotubes (MWCNTs), were selected to address this limitation. RESULTS: After the complete characterization of the nanomaterials, the induction of TNTs formation with all of the nanomaterials was observed using high-content screening system and confocal microscopy in both primary astrocytes and U251 cells. It was further revealed that TNT formation protected against nanomaterial-induced neurotoxicity due to cell apoptosis and disrupted ATP production. We then determined the mechanism underlying the protective role of TNTs. Since oxidative stress is a common mechanism in nanotoxicity, we first observed a significant increase in total and mitochondrial reactive oxygen species (namely ROS, mtROS), causing mitochondrial damage. Moreover, pretreatment of U251 cells with either the ROS scavenger N-acetylcysteine or the mtROS scavenger mitoquinone attenuated nanomaterial-induced neurotoxicity and TNTs generation, suggesting a central role of ROS in nanomaterials-induced TNTs formation. Furthermore, a vigorous downstream pathway of ROS, the PI3K/AKT/mTOR pathway, was found to be actively involved in nanomaterials-promoted TNTs development, which was abolished by LY294002, Perifosine and Rapamycin, inhibitors of PI3K, AKT, and mTOR, respectively. Finally, western blot analysis demonstrated that ROS and mtROS scavengers suppressed the PI3K/AKT/mTOR pathway, which abrogated TNTs formation. CONCLUSION: Despite their biophysical properties, various types of nanomaterials promote TNTs formation and mitochondrial transfer, preventing cell apoptosis and disrupting ATP production induced by nanomaterials. ROS/mtROS and the activation of the downstream PI3K/AKT/mTOR pathway are common mechanisms to regulate TNTs formation and mitochondrial transfer. Our study reveals that engineered nanomaterials share the same molecular mechanism of TNTs formation and intercellular mitochondrial transfer, and the proposed adverse outcome pathway contributes to a better understanding of the intercellular protection mechanism against nanomaterials-induced neurotoxicity.

MMP-3 mediates copper oxide nanoparticle-induced pulmonary inflammation and fibrosis.

BACKGROUND: The increasing production and usage of copper oxide nanoparticles (Nano-CuO) raise human health concerns. Previous studies have demonstrated that exposure to Nano-CuO could induce lung inflammation, injury, and fibrosis. However, the potential underlying mechanisms are still unclear. Here, we proposed that matrix metalloproteinase-3 (MMP-3) might play an important role in Nano-CuO-induced lung inflammation, injury, and fibrosis. RESULTS: Exposure of mice to Nano-CuO caused acute lung inflammation and injury in a dose-dependent manner, which was reflected by increased total cell number, neutrophil count, macrophage count, lactate dehydrogenase (LDH) activity, and CXCL1/KC level in bronchoalveolar lavage fluid (BALF) obtained on day 3 post-exposure. The time-response study showed that Nano-CuO-induced acute lung inflammation and injury appeared as early as day 1 after exposure, peaked on day 3, and ameliorated over time. However, even on day 42 post-exposure, the LDH activity and macrophage count were still higher than those in the control group, suggesting that Nano-CuO caused chronic lung inflammation. The Nano-CuO-induced pulmonary inflammation was further confirmed by H&E staining of lung sections. Trichrome staining showed that Nano-CuO exposure caused pulmonary fibrosis from day 14 to day 42 post-exposure with an increasing tendency over time. Increased hydroxyproline content and expression levels of fibrosis-associated proteins in mouse lungs were also observed. In addition, Nano-CuO exposure induced MMP-3 overexpression and increased MMP-3 secretion in mouse lungs. Knocking down MMP-3 in mouse lungs significantly attenuated Nano-CuO-induced acute and chronic lung inflammation and fibrosis. Moreover, Nano-CuO exposure caused sustained production of cleaved osteopontin (OPN) in mouse lungs, which was also significantly decreased by knocking down MMP-3. CONCLUSIONS: Our results demonstrated that short-term Nano-CuO exposure caused acute lung inflammation and injury, while long-term exposure induced chronic pulmonary inflammation and fibrosis. Knocking down MMP-3 significantly ameliorated Nano-CuO-induced pulmonary inflammation, injury, and fibrosis, and also attenuated Nano-CuO-induced cleaved OPN level. Our study suggests that MMP-3 may play important roles in Nano-CuO-induced pulmonary inflammation and fibrosis via cleavage of OPN and may provide a further understanding of the mechanisms underlying Nano-CuO-induced pulmonary toxicity.

High glucose enhances the activation of NLRP3 inflammasome by ambient fine particulate matter in alveolar macrophages.

BACKGROUND: Epidemiological studies have demonstrated that individuals with preexisting conditions, including diabetes mellitus (DM), are more susceptible to air pollution. However, the underlying mechanisms remain unclear. In this study, we proposed that a high glucose setting enhances ambient fine particulate matter (PM2.5)-induced macrophage activation and secretion of the proinflammatory cytokine, IL-1ß, through activation of the NLRP3 inflammasome, altering the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). RESULTS: Exposure of mouse alveolar macrophages to non-cytotoxic doses of PM2.5 led to upregulation of IL-1ß, activation of the NLRP3 inflammasome, increased nuclear translocation of the transcription factor NF-κB, increased generation of reactive oxygen species (ROS), and increased expression and enzymatic activity of MMP-9; these effects were enhanced when cells were pretreated with high glucose. However, pretreatment in a high glucose setting alone did not induce significant changes. ROS generation following PM2.5 exposure was abolished when cells were pretreated with ROS scavengers such as Trolox and superoxide dismutase (SOD), or with an NADPH oxidase inhibitor, DPI. Pretreatment of cells with DPI attenuated the effects of a high glucose setting on PM2.5-induced upregulation of IL-1ß, activation of the NLRP3 inflammasome, and nuclear translocation of NF-κB. In addition, enhancement of PM2.5-induced expression and enzymatic activity of MMP-9 following high glucose pretreatment was not observed in primary alveolar macrophages obtained from NLRP3 or IL-1R1 knockout (KO) mice, where pro-IL-1ß cannot be cleaved to IL-1ß or cells are insensitive to IL-1ß, respectively. CONCLUSIONS: This study demonstrated that exposure of mouse alveolar macrophages to PM2.5 in a high glucose setting enhanced PM2.5-induced production of IL-1ß through activation of the NLRP3 inflammasome and nuclear translocation of NF-κB due to PM2.5-induced oxidative stress, leading to MMP-9 upregulation. The key role of NADPH oxidase in PM2.5-induced ROS generation and activation of the IL-1ß secretion pathway and the importance of IL-1ß secretion and signaling in PM2.5-induced increases in MMP-9 enzymatic activity were also demonstrated. This study provides a further understanding of the potential mechanisms underlying the susceptibility of individuals with DM to air pollution and suggests potential therapeutic targets.

MMP-3-mediated cleavage of OPN is involved in copper oxide nanoparticle-induced activation of fibroblasts.

BACKGROUND: Copper oxide nanoparticles (Nano-CuO) are one of the most produced and used nanomaterials. Previous studies have shown that exposure to Nano-CuO caused acute lung injury, inflammation, and fibrosis. However, the mechanisms underlying Nano-CuO-induced lung fibrosis are still unclear. Here, we hypothesized that exposure of human lung epithelial cells and macrophages to Nano-CuO would upregulate MMP-3, which cleaved osteopontin (OPN), resulting in fibroblast activation and lung fibrosis. METHODS: A triple co-culture model was established to explore the mechanisms underlying Nano-CuO-induced fibroblast activation. Cytotoxicity of Nano-CuO on BEAS-2B, U937* macrophages, and MRC-5 fibroblasts were determined by alamarBlue and MTS assays. The expression or activity of MMP-3, OPN, and fibrosis-associated proteins was determined by Western blot or zymography assay. Migration of MRC-5 fibroblasts was evaluated by wound healing assay. MMP-3 siRNA and an RGD-containing peptide, GRGDSP, were used to explore the role of MMP-3 and cleaved OPN in fibroblast activation. RESULTS: Exposure to non-cytotoxic doses of Nano-CuO (0.5 and 1 µg/mL) caused increased expression and activity of MMP-3 in the conditioned media of BEAS-2B and U937* cells, but not MRC-5 fibroblasts. Nano-CuO exposure also caused increased production of cleaved OPN fragments, which was abolished by MMP-3 siRNA transfection. Conditioned media from Nano-CuO-exposed BEAS-2B, U937*, or the co-culture of BEAS-2B and U937* caused activation of unexposed MRC-5 fibroblasts. However, direct exposure of MRC-5 fibroblasts to Nano-CuO did not induce their activation. In a triple co-culture system, exposure of BEAS-2B and U937* cells to Nano-CuO caused activation of unexposed MRC-5 fibroblasts, while transfection of MMP-3 siRNA in BEAS-2B and U937* cells significantly inhibited the activation and migration of MRC-5 fibroblasts. In addition, pretreatment with GRGDSP peptide inhibited Nano-CuO-induced activation and migration of MRC-5 fibroblasts in the triple co-culture system. CONCLUSIONS: Our results demonstrated that Nano-CuO exposure caused increased production of MMP-3 from lung epithelial BEAS-2B cells and U937* macrophages, which cleaved OPN, resulting in the activation of lung fibroblasts MRC-5. These results suggest that MMP-3-cleaved OPN may play a key role in Nano-CuO-induced activation of lung fibroblasts. More investigations are needed to confirm whether these effects are due to the nanoparticles themselves and/or Cu ions.

Prenatal benzo[a]pyrene exposure impairs hippocampal synaptic plasticity and cognitive function in SD rat offspring during adolescence and adulthood via HDAC2-mediated histone deacetylation.

Benzo[a]pyrene (B[a]P) is a widespread carcinogenic pollutant in the environment. Although previous studies have demonstrated the neurodevelopmental toxicity of B[a]P, the precise mechanisms underlying the neurotoxic effects induced by prenatal B[a]P exposure remain largely unknown. In the present study, pregnant Sprague-Dawley (SD) rats were injected intraperitoneally with 0, 10, 20, or 40 mg/kg-bw of B[a]P for three consecutive days on embryonic days 17-19. The learning and memory abilities of offspring were determined by Morris Water Maze (MWM) test, while the number of dendritic branches and the density of dendritic spines in hippocampal CA1 and DG regions were evaluated by Golgi-Cox staining at PND 45 and PND 75. The mRNA expression of BDNF, PSD-95, and SYP in offspring hippocampus were detected by qRT-PCR, and the protein expression of BDNF, PSD-95, SYP, HDAC2, acH3K9, and acH3K14 were measured by Western blotting or immunohistochemistry. CHIP-PCR was performed to further detect the levels of acH3K9 and acH3K14 in the promoter regions of BDNF and PSD-95 genes. Our results showed that rats prenatally exposed to B[a]P exhibited impaired spatial learning and memory abilities and the number of dendritic branches and the density of dendritic spines in the hippocampal CA1 and DG regions were significantly reduced during adolescence and adulthood. The expression of HDAC2 protein was significantly upregulated, while acH3K9, acH3K14, BDNF, PSD-95, and SYP protein levels were significantly downregulated in the hippocampus of B[a]P- exposed rats. In addition, CHIP results showed that prenatal B[a]P exposure markedly decreased the level of acH3K9 and acH3K14 in the promoter region of BDNF and PSD-95 gene in the hippocampus of PND 45 and PND 75 offspring. All of the results suggest that prenatal B[a]P exposure impairs cognitive function and hippocampal synaptic plasticity of offspring in adolescence and adulthood, and HDAC2-mediated histone deacetylation plays a crucial role in these deficits.

Transcriptome-wide association study identifies PSMB9 as a susceptibility gene for coal workers' pneumoconiosis.

Coal workers' pneumoconiosis (CWP) is a type of typical occupational lung disease caused by prolonged inhalation of coal mine dust. The individuals' different genetic background may underlie their different susceptibility to develop pneumoconiosis, even under the same exposure level. This study aimed to identify susceptibility genes associated with CWP. Based on our previous genome-wide association study (GWAS, 202 CWP cases vs. 198 controls) and gene expression data obtained by analyzing human lungs and whole blood from the Genotype-Tissue Expression (GTEx) Portal, a transcriptome-wide association study (TWAS) was applied to identify CWP risk-related genes. Luciferase report gene assay, qRT-PCR, Western blot, immunofluorescence assay, and TUNEL assay were conducted to explore the potential role of the candidate gene in CWP. Proteasome 20S subunit beta 9 (PSMB9) was identified as a strong risk-related gene of CWP in both lungs and whole blood (Lungs: PTWAS  = 4.22 × 10-4 ; Whole blood: PTWAS  = 2.11 × 10-4 ). Single nucleotide polymorphisms (SNPs) rs2071480 and rs1351383, which locate in the promoter region and the first intron of the PSMB9 gene, were in high linkage disequilibrium (LD, r2  = 0.98) with the best GWAS SNP rs4713600 (G>T, OR = 0.55, 95% CI: 0.42-0.74, P = 6.86 × 10-5 ). Both rs2071480 and rs1351383 significantly enhanced the transcriptional activity of PSMB9. Functional experiments revealed that silica exposure remarkably reduced the PSMB9 expression and caused cell apoptosis, while overexpression of PSMB9 markedly abolished silica-induced cell apoptosis. We here identified PSMB9 as a novel susceptibility gene for CWP and provided important insights into the further exploration of the CWP pathogenesis.

Phosphorylation of p53 by Cdk5 contributes to benzo[a]pyrene-induced neuronal apoptosis.

Benzo[a]pyrene (B[a]P) is a ubiquitous carcinogenic pollutant in the environment, however, the potential neurotoxic effects of B[a]P has not been elucidated clearly. In the present study, we explored the potential involvement of p53 phosphorylation by Cdk5 in B[a]P-induced neuronal apoptosis at both in vitro and in vivo settings. For in vitro studies, primary cortical neurons isolated from the brains of Sprague Dawley (SD) rat pup were exposed to 0, 10, 20, and 40 µM of B[a]P for 12, 24, or 48 h. For in vivo studies, SD rats were injected intraperitoneally with 0, 1.0, 2.5, and 6.25 mg/kg of B[a]P every other day for 1, 2, or 3 months. Our results demonstrated that exposure to B[a]P caused a dose- and a time-dependent increase in neuronal apoptotic ratio in both in vitro and in vivo studies. There was also a dose- and a time-dependent upregulation of p35, p25, Cdk5, and phosphorylated p53 at Ser15 after B[a]P exposure. In order to explore whether B[a]P-induced increased neuronal apoptosis was through Cdk5/p53 pathway, roscovitine, a specific Cdk5 inhibitor, was applied to pretreat neurons prior to B[a]P exposure. The results showed that pretreatment of neurons with roscovitine partially rescued cells from B[a]P-induced apoptosis, and alleviated B[a]P-induced upregulation of phosphorylated p53 at Ser15. Our results suggest that Cdk5/p53 signaling pathway may be involved in B[a]P-induced neuronal apoptosis, which will provide information to further elucidate the molecular mechanisms of B[a]P-induced neurotoxicity.

Effects of metal nanoparticles on tight junction-associated proteins via HIF-1α/miR-29b/MMPs pathway in human epidermal keratinocytes.

BACKGROUND: The increasing use of metal nanoparticles in industry and biomedicine raises the risk for unintentional exposure. The ability of metal nanoparticles to penetrate the skin ranges from stopping at the stratum corneum to passing below the dermis and entering the systemic circulation. Despite the potential health risks associated with skin exposure to metal nanoparticles, the mechanisms underlying the toxicity of metal nanoparticles on skin keratinocytes remain unclear. In this study, we proposed that exposure of human epidermal keratinocytes (HaCaT) to metal nanoparticles, such as nickel nanoparticles, dysregulates tight-junction associated proteins by interacting with the HIF-1α/miR-29b/MMPs axis. METHODS: We performed dose-response and time-response studies in HaCaT cells to observe the effects of Nano-Ni or Nano-TiO2 on the expression and activity of MMP-2 and MMP-9, and on the expression of tight junction-associated proteins, TIMP-1, TIMP-2, miR-29b, and HIF-1α. In the dose-response studies, cells were exposed to 0, 10, or 20 µg/mL of Nano-Ni or Nano-TiO2 for 24 h. In the time-response studies, cells were exposed to 20 µg/mL of Nano-Ni for 12, 24, 48, or 72 h. After treatment, cells were collected to either assess the expression of mRNAs and miR-29b by real-time PCR or to determine the expression of tight junction-associated proteins and HIF-1α nuclear accumulation by Western blot and/or immunofluorescent staining; the conditioned media were collected to evaluate the MMP-2 and MMP-9 activities by gelatin zymography assay. To further investigate the mechanisms underlying Nano-Ni-induced dysregulation of tight junction-associated proteins, we employed a HIF-1α inhibitor, CAY10585, to perturb HIF-1α accumulation in one experiment, and transfected a miR-29b-3p mimic into the HaCaT cells before Nano-Ni exposure in another experiment. Cells and conditioned media were collected, and the expression and activities of MMPs and the expression of tight junction-associated proteins were determined as described above. RESULTS: Exposure of HaCaT cells to Nano-Ni resulted in a dose-dependent increase in the expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 and the activities of MMP-2 and MMP-9. However, exposure of cells to Nano-TiO2 did not cause these effects. Nano-Ni caused a dose-dependent decrease in the expression of miR-29b and tight junction-associated proteins, such as ZO-1, occludin, and claudin-1, while Nano-TiO2 did not. Nano-Ni also caused a dose-dependent increase in HIF-1α nuclear accumulation. The time-response studies showed that Nano-Ni caused significantly increased expressions of MMP-2 at 24 h, MMP-9 at 12, 24, and 48 h, TIMP-1 from 24 to 72 h, and TIMP-2 from 12 to 72 h post-exposure. The expression of miR-29b and tight junction-associated proteins such as ZO-1, occludin, and claudin-1 decreased as early as 12 h post-exposure, and their levels declined gradually over time. Pretreatment of cells with a HIF-1α inhibitor, CAY10585, abolished Nano-Ni-induced miR-29b down-regulation and MMP-2/9 up-regulation. Introduction of a miR-29b-3p mimic into HaCaT cells by transfection before Nano-Ni exposure ameliorated Nano-Ni-induced increased expression and activity of MMP-2 and MMP-9 and restored Nano-Ni-induced down-regulation of tight junction-associated proteins. CONCLUSION: Our study herein demonstrated that exposure of human epidermal keratinocytes to Nano-Ni caused increased HIF-1α nuclear accumulation and increased transcription and activity of MMP-2 and MMP-9 and down-regulation of miR-29b and tight junction-associated proteins. Nano-Ni-induced miR-29b down-regulation was through Nano-Ni-induced HIF-1α nuclear accumulation. Restoration of miR-29b level by miR-29b-3p mimic transfection abolished Nano-Ni-induced MMP-2 and MMP-9 activation and down-regulation of tight junction-associated proteins. In summary, our results demonstrated that Nano-Ni-induced dysregulation of tight junction-associated proteins in skin keratinocytes was via HIF-1α/miR-29b/MMPs pathway.

Nickel nanoparticle-induced cell transformation: involvement of DNA damage and DNA repair defect through HIF-1α/miR-210/Rad52 pathway.

BACKGROUND: Nickel nanoparticles (Nano-Ni) are increasingly used in industry and biomedicine with the development of nanotechnology. However, the genotoxic and carcinogenic effects of Nano-Ni and the underlying mechanisms are still unclear. METHODS: At first, dose-response (0, 10, 20, and 30 µg/mL) and time-response (0, 3, 6, 12, and 24 h) studies were performed in immortalized normal human bronchial epithelial cells BEAS-2B to observe the effects of Nano-Ni on DNA damage response (DDR)-associated proteins and the HIF-1α/miR-210/Rad52 pathway by real-time PCR or Western blot. Then, a Hsp90 inhibitor (1 µM of 17-AAG, an indirect HIF-1α inhibitor), HIF-1α knock-out (KO) cells, and a miR-210 inhibitor (20 nM) were used to determine whether Nano-Ni-induced Rad52 down-regulation was through HIF-1α nuclear accumulation and miR-210 up-regulation. In the long-term experiments, cells were treated with 0.25 and 0.5 µg/mL of Nano-Ni for 21 cycles (~ 150 days), and the level of anchorage-independent growth was determined by plating the cells in soft agar. Transduction of lentiviral particles containing human Rad52 ORF into BEAS-2B cells was used to observe the role of Rad52 in Nano-Ni-induced cell transformation. Nano-Ni-induced DNA damage and dysregulation of HIF-1α/miR-210/Rad52 pathway were also investigated in vivo by intratracheal instillation of 50 µg per mouse of Nano-Ni. gpt delta transgenic mice were used to analyze mutant frequency and mutation spectrum in mouse lungs after Nano-Ni exposure. RESULTS: Nano-Ni exposure caused DNA damage at both in vitro and in vivo settings, which was reflected by increased phosphorylation of DDR-associated proteins such as ATM at Ser1981, p53 at Ser15, and H2AX. Nano-Ni exposure also induced HIF-1α nuclear accumulation, miR-210 up-regulation, and down-regulation of homologous recombination repair (HRR) gene Rad52. Inhibition of or knocking-out HIF-1α or miR-210 ameliorated Nano-Ni-induced Rad52 down-regulation. Long-term low-dose Nano-Ni exposure led to cell malignant transformation, and augmentation of Rad52 expression significantly reduced Nano-Ni-induced cell transformation. In addition, increased immunostaining of cell proliferation markers, Ki-67 and PCNA, was observed in bronchiolar epithelial cells and hyperplastic pneumocytes in mouse lungs at day 7 and day 42 after Nano-Ni exposure. Finally, using gpt delta transgenic mice revealed that Nano-Ni exposure did not cause increased gpt mutant frequency and certain DNA mutations, such as base substitution and small base insertions/deletions, are not the main types of Nano-Ni-induced DNA damage. CONCLUSIONS: This study unraveled the mechanisms underlying Nano-Ni-induced cell malignant transformation; the combined effects of Nano-Ni-induced DNA damage and DNA repair defects through HIF-1α/miR-210/Rad52 pathway likely contribute to Nano-Ni-induced genomic instability and ultimately cell transformation. Our findings will provide information to further elucidate the molecular mechanisms of Nano-Ni-induced genotoxicity and carcinogenicity.

miR-770-5p inhibits the activation of pulmonary fibroblasts and silica-induced pulmonary fibrosis through targeting TGFBR1.

Silicosis is a devastating interstitial lung disease arising from long-term exposure to inhalable silica. Regrettably, no therapy currently can effectively reverse the silica-induced fibrotic lesion. Emerging evidence has indicated that the dysregulation of microRNAs is involved in silica-induced pulmonary fibrosis. The aim of this study is to explore the expression pattern and underlying mechanisms of miR-770-5p in silica-induced pulmonary fibrosis. Consistent with our previous miRNA microarray analysis, the results of qRT-PCR showed that miR-770-5p expression was downregulated in silica-induced pulmonary fibrosis in humans and animal models. Administration of miR-770-5p agomir significantly reduced the fibrotic lesions in the lungs of mice exposed to silica dust. MiR-770-5p also exhibited a dramatic reduction in TGF-ß1-activated human pulmonary fibroblasts (MRC-5). Transfection of miR-770-5p mimics significantly decreased the viability, migration ability, and S/G0 phase distribution, as well as the expression of fibronectin, collagen I, and α-SMA in TGF-ß1-treated MRC-5 cells. Transforming growth factor-ß receptor 1 (TGFBR1) was confirmed as a direct target of regulation by miR-770-5p. The expression of TGFBR1 was significantly increased in pulmonary fibrosis. Knockdown of TGFBR1 blocked the transduction of the TGF-ß1 signaling pathway and attenuated the activation of MRC-5 cells, while overexpression of TGFBR1 effectively restored the activation of MRC-5 cells inhibited by miR-770-5p. Together, our results demonstrated that miR-770-5p exerted an anti-fibrotic effect in silica-induced pulmonary fibrosis by targeting TGFBR1. Targeting miR-770-5p might provide a new therapeutic strategy to prevent the abnormal activation of pulmonary fibroblasts in silicosis.

Comparison of the neurotoxicity associated with cobalt nanoparticles and cobalt chloride in Wistar rats.

Cobalt nanoparticles (CoNPs) have been widely used in industry given their physical, chemical and magnetic properties; however, CoNPs may cause neurological symptoms and diseases in human, yet their mechanisms of toxicity remain unknown. Here, we used male Wistar rats to investigate differences in the toxic effects associated with CoNPs and CoCl2. Upon exposure to CoCl2, and 96 nm or 123 nm CoNPs at the same concentration, the Co2+ content in CoCl2 group was significantly higher than that in either the CoNPs groups in brain tissues and blood, but lower in liver. Significant neural damage was observed in both hippocampus and cortex of the temporal lobe. Increase malondialdehyde (MDA) content and CASPASE 9 protein level were associated both with CoCl2 and CoNPs treatments, consistent with lipid perioxidation and apoptosis. Heme oxygenase-1 and (NF-E2) p45-related factor-2 protein levels were elevated in response to 96 nm CoNPs exposure. In PC12 cells, NRF2 downregulation led to reduced cell viability and increased apoptotic rate. In conclusion, both CoNPs and CoCl2 cause adverse neural effects, with nanoparticles showing greater neurotoxic potency. In addition, NRF2 protects neural cells from damage induced by CoCl2 and CoNPs by activating downstream antioxidant responses.

Comparative mouse lung injury by nickel nanoparticles with differential surface modification.

BACKGROUND: Previous studies have demonstrated that exposure to nickel nanoparticles (Nano-Ni) causes oxidative stress and severe, persistent lung inflammation, which are strongly associated with pulmonary toxicity. However, few studies have investigated whether surface modification of Nano-Ni could alter Nano-Ni-induced lung injury, inflammation, and fibrosis in vivo. Here, we propose that alteration of physicochemical properties of Nano-Ni through modification of Nano-Ni surface may change Nano-Ni-induced lung injury, inflammation, and fibrosis. METHODS: At first, dose-response and time-response studies were performed to observe lung inflammation and injury caused by Nano-Ni. In the dose-response studies, mice were intratracheally instilled with 0, 10, 20, 50, and 100 µg per mouse of Nano-Ni and sacrificed at day 3 post-exposure. In the time-response studies, mice were intratracheally instilled with 50 µg per mouse of Nano-Ni and sacrificed at days 1, 3, 7, 14, 28, and 42 post-instillation. At the end of the experiment, mice were bronchoalveolar lavaged (BAL) and the neutrophil count, CXCL1/KC level, LDH activity, and concentration of total protein in the BAL fluid (BALF) were determined. In the comparative studies, mice were intratracheally instilled with 50 µg per mouse of Nano-Ni or with the same molar concentration of Ni as Nano-Ni of either partially [O]-passivated Nano-Ni (Nano-Ni-P) or carbon-coated Nano-Ni (Nano-Ni-C). At day 3 post-exposure, BAL was performed and the above cellular and biochemical parameters in the BALF were analyzed. The MMP-2/9 protein levels and activities in the BALF and mouse lung tissues were also determined. Mouse lung tissues were also collected for H&E staining, and measurement of thiobarbituric acid reactive substances (TBARS) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the genomic DNA. At day 42 post-exposure, mouse right lung tissues were collected for H&E and Trichrome stainings, and left lung tissues were collected to determine the hydroxyproline content. RESULTS: Exposure of mice to Nano-Ni resulted in a dose-response increase in acute lung inflammation and injury reflected by increased neutrophil count, CXCL1/KC level, LDH activity, and concentration of total protein in the BALF. The time-response study showed that Nano-Ni-induced acute lung inflammation and injury appeared as early as day 1, peaked at day 3, and attenuated at day 7 post-instillation. Although the neutrophil count, CXCL1/KC level, LDH activity, and concentration of total protein in the BALF dramatically decreased over the time, their levels were still higher than those of the controls even at day 42 post-exposure. Based on the results of the dose- and time-response studies, we chose a dose of 50 µg per mouse of Nano-Ni, and day 3 post-exposure as short-term and day 42 post-exposure as long-term to compare the effects of Nano-Ni, Nano-Ni-P, and Nano-Ni-C on mouse lungs. At day 3 post-exposure, 50 µg per mouse of Nano-Ni caused acute lung inflammation and injury that were reflected by increased neutrophil count, CXCL1/KC level, LDH activity, concentration of total protein, and MMP-2/9 protein levels and activities in the BALF. Nano-Ni exposure also caused increased MMP-2/9 activities in the mouse lung tissues. Histologically, infiltration of large numbers of neutrophils and macrophages in the alveolar space and interstitial tissues was observed in mouse lungs exposed to Nano-Ni. Nano-Ni-P exposure caused similar acute lung inflammation and injury as Nano-Ni. However, exposure to Nano-Ni-C only caused mild acute lung inflammation and injury. At day 42 post-exposure, Nano-Ni caused extensive interstitial fibrosis and proliferation of interstitial cells with inflammatory cells infiltrating the alveolar septa and alveolar space. Lung fibrosis was also observed in Nano-Ni-P-exposed lungs, but to a much lesser degree. Only slight or no lung fibrosis was observed in Nano-Ni-C-exposed lungs. Nano-Ni and Nano-Ni-P, but not Nano-Ni-C, caused significantly elevated levels of TBARS in mouse lung tissues and 8-OHdG in mouse lung tissue genomic DNA, suggesting that Nano-Ni and Nano-Ni-P induce lipid peroxidation and oxidative DNA damage in mouse lung tissues, while Nano-Ni-C does not. CONCLUSION: Our results demonstrate that short-term Nano-Ni exposure causes acute lung inflammation and injury, while long-term Nano-Ni exposure causes chronic lung inflammation and fibrosis. Surface modification of Nano-Ni alleviates Nano-Ni-induced pulmonary effects; partially passivated Nano-Ni causes similar effects as Nano-Ni, but the chronic inflammation and fibrosis were at a much lesser degree. Carbon coating significantly alleviates Nano-Ni-induced acute and chronic lung inflammation and injury.

Paraquat and MPTP alter microRNA expression profiles, and downregulated expression of miR-17-5p contributes to PQ-induced dopaminergic neurodegeneration.

Recent evidence indicates that microRNAs (miRNAs) play a key role in neurodegenerative diseases. However, the toxic effects of paraquat (PQ) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on miRNA expression profiles in dopaminergic neurons have not been investigated. In the present study, we used microarray analysis to show that PQ and MPTP induce alterations of miRNA expression in neuro-2a cells. The results reveal that treatment with 300 µm PQ caused miRNA deregulation, such that 60 miRNAs were upregulated and 228 miRNAs were downregulated. Following treatment with 300 µm MPTP, a total of 576 miRNAs were dysregulated, of which 506 were upregulated and 70 were downregulated. Alterations in the expression of miR-17-5p, miR-210-3p, miR-374-5p, miR-378-3p and miR-503-5p were verified by real-time quantitative reverse transcriptase polymerase chain reaction. Moreover, overexpression of miR-17-5p in Neuro-2a cells enhanced cell proliferation, suppressed apoptosis and promoted S phase transition of the cell cycle after PQ treatment. Taken together, our study demonstrates that characteristic changes in miRNA expression profiles occur after PQ and MPTP treatment, which suggests that miRNAs may be involved in the development of PQ- and MPTP-induced neurodegeneration. Downregulated miR-17-5p expression contributes to PQ-induced dopaminergic neurodegeneration.

Cobalt nanoparticles induce lung injury, DNA damage and mutations in mice.

BACKGROUND: We and other groups have demonstrated that exposure to cobalt nanoparticles (Nano-Co) caused oxidative stress and inflammation, which have been shown to be strongly associated with genotoxic and carcinogenic effects. However, few studies have reported Nano-Co-induced genotoxic effects in vivo. Here, we propose that Nano-Co may have high genotoxic effects due to their small size and high surface area, which have high capacity for causing oxidative stress and inflammation. METHODS: gpt delta transgenic mice were used as our in vivo study model. They were intratracheally instilled with 50 µg per mouse of Nano-Co. At day 1, 3, 7 and 28 after exposure, bronchoalveolar lavage (BAL) was performed and the number of neutrophils, CXCL1/KC level, LDH activity and concentration of total protein in the BAL fluid (BALF) were determined. Mouse lung tissues were collected for H&E staining, and Ki-67, PCNA and γ-H2AX immunohistochemical staining. 8-OHdG level in the genomic DNA of mouse lungs was determined by an OxiSelect™ Oxidative DNA Damage ELISA Kit, and mutant frequency and mutation spectrum in the gpt gene were also determined in mouse lungs at four months after Nano-Co exposure by 6-TG selection, colony PCR, and DNA sequencing. RESULTS: Exposure of mice to Nano-Co (50 µg per mouse) resulted in extensive acute lung inflammation and lung injury which were reflected by increased number of neutrophils, CXCL1/KC level, LDH activity and concentration of total protein in the BALF, and infiltration of large amount of neutrophils and macrophages in the alveolar space and interstitial tissues. Increased immunostaining of cell proliferation markers, Ki-67 and PCNA, and the DNA damage marker, γ-H2AX, was also observed in bronchiolar epithelial cells and hyperplastic type II pneumocytes in mouse lungs at day 7 after Nano-Co exposure. At four months after exposure, extensive interstitial fibrosis and proliferation of interstitial cells with inflammatory cells infiltrating the alveolar septa were observed. Moreover, Nano-Co caused increased level of 8-OHdG in genomic DNA of mouse lung tissues. Nano-Co also induced a much higher mutant frequency as compared to controls, and the most common mutation was G:C to T:A transversion, which may be explained by Nano-Co-induced increased formation of 8-OHdG. CONCLUSION: Our study demonstrated that exposure to Nano-Co caused oxidative stress, lung inflammation and injury, and cell proliferation, which further resulted in DNA damage and DNA mutation. These findings have important implications for understanding the potential health effects of nanoparticle exposure.

Effects of urban particulate matter with high glucose on human monocytes U937.

Epidemiological studies and animal experiments have shown that individuals with preexisting diseases, such as diabetes mellitus (DM), are more susceptible to particulate matter (PM)-related cardiovascular diseases. However, the underlying mechanisms are still unclear. We hypothesized that PM and high glucose combined would cause enhanced effects on activation of monocytes and p38 mitogen-activated protein kinase (MAPK) by inducing oxidative stress, which would further activate matrix metalloproteinases (MMPs). Human monocytes U937 were used to test the effects of urban particulate matter (U-PM) and high glucose. The results showed that exposure of monocytes to non-toxic doses of U-PM alone caused generation of reactive oxygen species (ROS), increased phosphorylation of p38, and activation of monocytes which was reflected by up-regulation of MMP-2, MMP-9 and proinflammatory cytokines IL-1ß and IL-8 expression and increased activity of pro-MMP-2 and pro-MMP-9. These effects were enhanced significantly when cells were exposed to U-PM in a high-glucose environment. Our results also showed that pre-treatment of cells with ROS scavengers or inhibitors abolished U-PM and high glucose-induced increased phosphorylation of p38. Up-regulation of pro-MMP-2 and pro-MMP-9 activity by U-PM in the setting of high glucose level was dramatically attenuated by treatment of cells with the p38-specific inhibitor, SB203580. These results suggest that activation of MMPs by U-PM with high glucose is partly through p38 phosphorylation that is induced by oxidative stress. Our findings may have important implications in understanding the potential health effects of PM on susceptible populations such as those with DM.

Urinary enterolactone associated with liver enzyme levels in US adults: National Health and Nutrition Examination Survey (NHANES).

Phyto-oestrogens are a family of plant-derived xeno-oestrogens that appear to have beneficial effects on human health. To date, no data are available about phyto-oestrogen consumption affecting liver health in a population. The present study aimed to explore the relationship of urinary phyto-oestrogen metabolites with serum liver enzymes in US adults. A nationally representative sample of US adults in the National Health and Nutrition Examination Survey (NHANES) 2003-10 was analysed. The cross-sectional study sample consisted of 6438 adults with data on urinary phyto-oestrogen levels, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and γ-glutamyl transaminase (GGT) concentrations and data on other potential confounders. Multivariate logistic regression and linear regression were applied to assess associations between urinary phyto-oestrogen levels and ALT, AST, ALP and GGT concentrations. We found a remarkable association between urinary enterolactone and GGT in both adult males (OR 0.37, 95 % CI 0.22, 0.61; P= 0.003) and females (OR 0.37, 95 % CI 0.26, 0.54; P= 0.009). Moreover, elevated enterolactone levels were inversely associated with ALT and AST levels in adult males. However, no association was present between levels of urinary daidzein, O-desmethylangolensin, equol, enterodiol or genistein with liver enzyme levels in this population. The present study results provide epidemiological evidence that urinary enterolactone levels are associated with liver GGT levels in humans. This suggests a potential protective effect of enterolactone on human liver function. However, the underlying mechanisms still need further investigation.

Urinary enterolactone is associated with obesity and metabolic alteration in men in the US National Health and Nutrition Examination Survey 2001-10.

Phyto-oestrogens are a family of plant-derived xeno-oestrogens that have been shown to prevent cancer in some studies. Whether phyto-oestrogen intake affects obesity status in a population is still unclear. In the present cross-sectional study, we examined the association of urinary phyto-oestrogen metabolites with obesity and metabolic parameters in children and adults. Data from 1294 children (age 6-19 years) and from 3661 adults (age ≥ 20 years) who participated in the US National Health and Nutrition Examination Survey 2001-10 were analysed. Multivariate logistic regression was applied to investigate the associations of BMI, waist circumference, serum metabolites (total cholesterol, HDL-cholesterol, LDL-cholesterol, TAG, fasting glucose and fasting insulin) and the metabolic syndrome with urinary phyto-oestrogen levels. When stratified by age and sex, we found a stronger association (OR 0·30, 95 % CI 0·17, 0·54; P< 0·001) between urinary enterolactone levels and obesity in adult males (age 20-60 years) than in children (age 12-19 years) or the elderly (age >60 years) in the same survey. However, no associations with urinary daidzein, O-desmethylangolensin, equol, enterodiol or genistein were found in the overall population. We also found that the elevation of enterolactone levels was inversely associated with TAG levels, fasting glucose levels, fasting insulin levels and the metabolic syndrome in males aged 20-60 years, but positively associated with HDL-cholesterol levels. The present results provide epidemiological evidence that urinary enterolactone is inversely associated with obesity in adult males.

miR-486 regulates metastasis and chemosensitivity in hepatocellular carcinoma by targeting CLDN10 and CITRON.

AIM: miRNA-486 (miR-486) was first identified from the human fetal liver cDNA library and considered to be associated with hepatocellular carcinoma (HCC) development. Its roles in regulation of HCC metastasis and chemosensitivity have not been explored yet. METHODS: miR-486 expression in HCC tissues, cell lines and serum was evaluated by real-time polymerase chain reaction. miR-486 overexpression or downregulation in the cell lines SMMC-7721/LM3 was conducted by lentivirus transfection. Cell proliferation, migration and apoptosis were quantitated using commercial assays. Matrix metalloproteinase activity was quantitated by gelatin zymography. The target genes of miR-486 were screened by 3'-untranslated region luciferase report assays and their function was validated by small RNA interference. RESULTS: We show here that miR-486 is frequently down-expressed in HCC tissues and cell lines. Lentivirus-mediated restoration of miR-486 in HCC cell lines resulted in significant reduction in the ability of cell growth, colony formation and migration. However, miR-486 inhibition enhances proliferation and invasion of HCC cells. Two genes, CITRON and CLDN10 which regulate cell proliferation and invasion, respectively, were identified as the direct targets of miR-486 in HCC cells. CITRON and CLDN10 knockdown by siRNA results in similar phenotypes of miR-486 restoration in HCC cell lines. In addition, miR-486 enhances the chemosensitivity of HCC cells to sorafenib. CONCLUSION: Our data indicated that miR-486 may function as a novel tumor suppressor in HCC.

Up-regulation of Gadd45α after exposure to metal nanoparticles: the role of hypoxia inducible factor 1α.

The increased development and use of nanoparticles in various fields may lead to increased exposure, directly affecting human health. Our current knowledge of the health effects of metal nanoparticles such as cobalt and titanium dioxide (Nano-Co and Nano-TiO2 ) is limited but suggests that some metal nanoparticles may cause genotoxic effects including cell cycle arrest, DNA damage, and apoptosis. The growth arrest and DNA damage-inducible 45α protein (Gadd45α) has been characterized as one of the key players in the cellular responses to a variety of DNA damaging agents. The aim of this study was to investigate the alteration of Gadd45α expression in mouse embryo fibroblasts (PW) exposed to metal nanoparticles and the possible mechanisms. Non-toxic doses of Nano-Co and Nano-TiO2 were selected to treat cells. Our results showed that Nano-Co caused a dose- and time-dependent increase in Gadd45α expression, but Nano-TiO2 did not. To investigate the potential pathways involved in Nano-Co-induced Gadd45α up-regulation, we measured the expression of hypoxia inducible factor 1α (HIF-1α) in PW cells exposed to Nano-Co and Nano-TiO2 . Our results showed that exposure to Nano-Co caused HIF-1α accumulation in the nucleus. In addition, hypoxia inducible factor 1α knock-out cells [HIF-1α (-/-)] and its wild-type cells [HIF-1α (+/+)] were used. Our results demonstrated that Nano-Co caused a dose- and time-dependent increase in Gadd45α expression in wild-type HIF-1α (+/+) cells, but only a slight increase in HIF-1α (-/-) cells. Pre-treatment of PW cells with heat shock protein 90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), prior to exposure to Nano-Co significantly abolished Nano-Co-induced Gadd45α expression. These results suggest that HIF-1α accumulation may be partially involved in the increased Gadd45α expression in cells exposed to Nano-Co. These findings may have important implications for understanding the potential health effects of metal nanoparticle exposure.