A human relevant mixture of persistent organic pollutants (POPs) and perfluorooctane sulfonic acid (PFOS) enhance nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells.

Disruption of neurite outgrowth is a marker for neurotoxicity. Persistent organic pollutants (POPs) are potential developmental neurotoxicants. We investigated their effect on neurite outgrowth in PC12 rat pheochromocytoma cells, in absence or presence of nerve growth factor (NGF), an inducer of neuronal differentiation. Cells were exposed for 72 h to a defined mixture of POPs with chemical composition and concentrations based on blood levels in the Scandinavian population. We also evaluated perfluorooctane sulfonic acid (PFOS) alone, the most abundant compound in the POP mixture. Only higher concentrations of POP mixture reduced tetrazolium salt (MTT) conversion. High-content analysis showed a decrease in cell number, but no changes for nuclear and mitochondrial cellular health parameters. Robust glutathione levels were observed in NGF-differentiated cells. Live imaging, using the IncuCyte ZOOM platform indicated ongoing cell proliferation over time, but slower in presence of NGF. The pollutants did not inhibit neuritogenesis, but rather increased NGF-induced neurite length. PFOS induced neurite outgrowth to about 50 % of the level seen with the POP mixture. Neither the POP mixture nor PFOS affected neurite length in the absence of NGF. Our observations indicate that realistic complex mixtures of environmental pollutants can affect neuronal connectivity via NGF-induced neurite outgrowth.

Effects of mild steel welding fume particles on pulmonary epithelial inflammation and endothelial activation.

Welders have an increased risk for cardiovascular disease (CVD) following exposure to welding fumes. The underlying mechanisms are largely unknown; however, oxidative stress, systemic inflammation, and endothelial dysfunction have been suggested as contributing factors to particle-induced CVD. We investigated effects of mild steel welding fume (MSWF) on three target cell types: macrophages, pulmonary epithelial, and vascular endothelial cells. Cells were exposed to MSWF at nontoxic doses for 6 h/day, for five consecutive days. The expression of 40 genes involved in inflammation, fibrosis, and endothelial activation was analyzed. Moreover, changes in the reactive oxygen species production and migration capacity of cells were assessed. The expression of matrix metallopeptidase 1 (MMP1) was induced in both epithelial and endothelial cells following repeated exposure to MSWF. Although MMP1 is important in inflammatory responses in vivo, this effect was not concurrent with changes in the inflammatory status, cell proliferation, and migration capacities, nor did it induce oxidative stress in the cells. Thus, repeated exposure with low doses of MSWF was sufficient neither for inducing inflammatory stress in epithelial cells and macrophages nor for endothelial activation, and higher concentrations of MSWF or the nonparticle fraction of MSWF may be critical in causing the increased risk of CVD observed among welders.

Cellulose nanocrystals modulate alveolar macrophage phenotype and phagocytic function.

Nanocellulose is a promising bio-nanomaterial with attractive properties suitable for multiple industrial applications. The increased use of nanocellulose may lead to occupational exposure and negative health outcomes. However, knowledge on its health effects is limited, and while nanocellulose exposure may induce acute inflammatory responses in the lung, the underlying mechanisms are unknown. Alveolar macrophages are key cells in alveolar particle clearance. Their activation and function may be affected by various particles. Here, we investigated the uptake of pristine cellulose nanocrystals (CNC), and their effects on alveolar macrophage polarization and biological function. CNC uptake enhanced the secretion of several cytokines but did not on its own induce a complete macrophage polarization. In presence of macrophage activators, such as LPS/IFNG and IL4/IL13, CNC exposure enhanced the expression of M1 phenotype markers and the secretion of pro-inflammatory cytokines and chemokines, while decreasing M2 markers. CNC exposure also affected the function of activated alveolar macrophages resulting in a prominent cytokine burst and altered phagocytic activity. In conclusion, CNC exposure may result in dysregulation of macrophage activation and function that are critical in inflammatory responses in the lung.

Mechanisms of Toxicity of Industrially Relevant Silicomanganese Dust on Human 1321N1 Astrocytoma Cells: An In Vitro Study.

Tremendous efforts are applied in the ferroalloy industry to control and reduce exposure to dust generated during the production process, as inhalable Mn-containing particulate matter has been linked to neurodegenerative diseases. This study aimed to investigate the toxicity and biological effects of dust particles from laboratory-scale processes where molten silicomanganese (SiMn) was exposed to air, using a human astrocytoma cell line, 1321N1, as model system. Characterization of the dust indicated presence of both nano-sized and larger particles averaging between 100 and 300 nm. The dust consisted mainly of Si, Mn and O. Investigation of cellular mechanisms showed a dose- and time-dependent effect on cell viability, with only minor changes in the expression of proteins involved in apoptosis. Moreover, gene expression of the neurotoxic biomarker amyloid precursor protein (APP) increased, whereas APP protein expression decreased. Finally, induction of gap junctional intercellular communication (GJIC) increased with higher doses and correlated with the other endpoints. Thus, the effects of SiMn dust on 1321N1 cells are highly dependent on the dose of exposure and involves changes in APP, apoptosis-related proteins and intercellular communication.

Mechanisms of Breast Cancer in Shift Workers: DNA Methylation in Five Core Circadian Genes in Nurses Working Night Shifts.

Shift work has been suggested to be associated with breast cancer risk, and circadian disruption in shift workers is hypothesized as one of the mechanisms of increased cancer risk. There is, however, insufficient molecular evidence supporting this hypothesis. Using the quantitative methodology of pyrosequencing, epigenetic changes in 5-methyl cytosine (5mC) in five circadian genes CLOCK, BMAL1, CRY1, PER1 and PER2 in female nurses working night shift work (278 breast cancer cases, 280 controls) were analyzed. In breast cancer cases, a medium exposure to night work was associated with increased methylation levels of the CLOCK (p=0.050), BMAL1 (p=0.001) and CRY1 (p=0.040) genes, compared with controls. Within the cases, analysis of the effects of shift work on the methylation patterns showed that methylation of CRY1 was lower in those who had worked night shift and had a high exposure (p=0.006) compared with cases that had worked only days. For cases with a medium exposure to night work, an increase in BMAL1 (p=0.003) and PER1 (p=0.035) methylation was observed compared with day working (unexposed) cases. The methylation levels of the five core circadian genes were also analyzed in relation to the estrogen and progesterone receptors status of the tumors in the cases, and no correlations were observed. Furthermore, nineteen polymorphisms in the five circadian genes were assessed for their effects on the methylation levels of the respective genes, but no associations were found. In summary, our data suggest that epigenetic regulation of CLOCK, BMAL1, CRY1 and PER1 may contribute to breast cancer in shift workers.

Mechanisms of breast cancer risk in shift workers: association of telomere shortening with the duration and intensity of night work.

Occupational factors such as shiftwork and especially night work that involves disruption of the circadian rhythm may contribute to increased breast cancer risk. Circadian disruption may also affect telomere length (TL). While short TL generally is associated with increased cancer risk, its association with breast cancer risk is inconclusive. We suggest that working schedules might be an important factor in assessment of effects of TL on breast cancer risk. Moreover, telomere shortening might be a potential mechanism for night work-related breast cancer. In this study, effects of shift work on TL and its association with breast cancer risk were investigated in a nested breast cancer case-control study of Norwegian nurses. TL was assessed by qPCR in DNA from 563 breast cancer patients and 619 controls. Here, we demonstrate that TL is affected by intensive night work schedules, as work with six consecutive night for a period of more than 5 years was associated with decreased telomere lengths (-3.18, 95% CI: -6.46 to -0.58, P = 0.016). Furthermore, telomere shortening is associated with increased breast cancer risk in workers with long periods of consecutive night shifts. Thus, nurses with longer telomere lengths had a lower risk for breast cancer if they had worked more than four (OR: 0.37, 95% CI: 0.16-0.79, P = 0.014) or five (OR: 0.31, 95% CI: 0.10-0.83, P = 0.029) consecutive night shifts for a period of 5 years or more. These data suggest that telomere shortening is associated with the duration and intensity of night work and may be a contributing factor for breast cancer risk among female shift workers.

Copy number variation, increased gene expression, and molecular mechanisms of neurofascin in lung cancer.

Metastasis and cell adhesion are key aspects of cancer progression. Neurofascin (NFASC) is a member of the immunoglobulin superfamily of adhesion molecules and, while studies on NFASC are inadequate, other members have been indicated pivotal roles in cancer progression and metastasis. This study aimed at increasing the knowledge on the involvement of adhesion molecules in lung cancer progression by studying the regulation and role of NFASC in non-small cell lung cancer (NSCLC). Here, copy number variations in the NFASC gene were analyzed in tumor and non-tumorous lung tissues of 204 NSCLC patients. Frequent gene amplifications (OR = 4.50, 95%CI: 2.27-8.92, P ≤ 0.001) and increased expression of NFASC (P = 0.034) were identified in tumors of NSCLC patients. Furthermore, molecular mechanisms of NFASC in lung cancer progression were evaluated by investigating the effects of NFASC silencing on cell proliferation, viability, migration, and invasion using siRNA technology in four NSCLC cell lines. Silencing of NFASC did not affect cell proliferation or viability but rather decreased NSCLC cell migration (P ≤ 0.001) and led to morphological changes, rearrangements in the actin cytoskeleton and changes in F-actin networks in migrating NSCLC cell lines. This study is the first to report frequent copy number gain and increased expression of NFASC in NSCLC. Moreover, these data suggest that NFASC is a novel regulator of NSCLC cell motility and support a role of NFASC in the regulation of NSCLC progression.

Loss of MKK3 and MK2 Copy Numbers in Non-Small Cell Lung Cancer.

Identification of genetic alterations in members of the p38 mitogen-activated protein kinase (MAPK) pathway is important as these proteins have dynamic roles in tumor progression and may serve as potential therapeutic targets in cancer. We analyzed tumor and non-tumorous lung tissue of 233 non-small cell lung cancer (NSCLC) patients for the presence of copy number alterations (CNAs) in the MAPK kinase 3 (MKK3) and MAPK-activated kinase 2 (MK2) genes. We report frequent CNAs in MKK3 and MK2 genes in NSCLC. Copy number losses were detected in 31% of NSCLC tumors (odds ratio: 7.08, 95% confidence interval: 3.2-15.6, P<0.001) for the MKK3 gene and in 28% of tumors for the MK2 gene (odds ratio: 3.68, 95% confidence interval: 1.9-7.2, P<0.001). Several of the non-tumorous tissues showed an elevated MKK3 copy number, with a concurrent loss of this in 89% of the paired tumors. MKK3 gene deletions were significantly more frequent in squamous and large cell carcinoma than in adenocarcinoma. These data demonstrate a novel loss of MKK3 and MK2 genomic copy numbers in NSCLC tumors, and suggest these genes as interesting therapeutic candidates in NSCLC.

Mutations in TP53 increase the risk of SOX2 copy number alterations and silencing of TP53 reduces SOX2 expression in non-small cell lung cancer.

BACKGROUND: Amplifications of the transcription factor, SRY (sex determining region Y)-box 2 (SOX2), are common in non-small cell lung cancer (NSCLC). SOX2 signaling is important in maintaining the stem cell-like phenotype of cancer cells and contributes to the pathogenesis of lung cancer. TP53 is known to inhibit gene amplifications and to repress many stem cell-associated genes following DNA damage. The aim of this study was to investigate if TP53 mutational status affected SOX2 copy number variation and gene expression in early-stage NSCLC patients; moreover, to assess if TP53 regulates SOX2 expression in human lung cancer cells. METHODS: 258 early-stage lung cancer patients were included in the study. Exons 4-9 in the TP53 gene were sequenced for mutations in tumor tissues. SOX2 copy number as well as TP53 and SOX2 gene expression were analyzed in tumor and in adjacent non-tumorous tissues by qPCR. TP53 and SOX2 were silenced using gene-specific siRNAs in human lung adenocarcinoma A427 cells, and the expression of TP53, SOX2 and subset of selected miRNAs was analyzed by qPCR. The odds ratios (ORs) for associations between copy number variation and lung cancer were estimated by conditional logistic regression, and the correlation between gene status and clinicopathological characteristics was assessed by Chi-square or Fisher's exact test. Gene expression data was analyzed using nonparametric Mann-Whitney test. RESULTS: TP53 mutations were associated with an increased risk of acquiring a SOX2 copy number alteration (OR = 2.08, 95% CI: 1.14-3.79, p = 0.017), which was more frequently occurring in tumor tissues (34%) than in adjacent non-tumorous tissues (3%). Moreover, SOX2 and TP53 expression levels were strongly correlated in tumor tissues. In vitro studies showed that a reduction in TP53 was associated with decreased SOX2 expression in A427 cells. Furthermore, TP53 knockdown reduced the miRNA hsa-miR-145, which has previously been shown to regulate SOX2 expression. CONCLUSIONS: TP53 signaling may be important in the regulation of SOX2 copy number and expression in NSCLC tumors, and the miRNA hsa-miR-145-5p may be one potential driver. This prompts for further studies on the mechanisms behind the TP53-induced regulation of SOX2 expression and the possible importance of hsa-miR-145 in lung cancer.

The matricellular "cysteine-rich protein 61" is released from activated platelets and increased in the circulation during experimentally induced sepsis.

Sepsis and sepsis-induced organ dysfunction remain lethal and common conditions among intensive care patients. Accumulating evidence suggests that the matricellular Cyr61/CCN1 (cysteine-rich, angiogenic-inducer, 61) protein is involved in the regulation of inflammatory responses and possesses organ-protective capabilities in diseases of an inflammatory etiology. However, its regulation in sepsis remains largely unexplored. The present study provides a comprehensive description of CCN1 regulation in the circulation and vital organs during experimentally induced sepsis with developing organ dysfunction. Female CD-1 mice served as baseline controls or were subjected to cecal ligation and puncture (CLP) for 18 to 96 h, and CCN1 regulation was analyzed in selected organs and in the circulation. A 5-, 5-, and 3-fold increases in circulating CCN1 protein were observed at 18, 48, and 96 h after CLP, respectively. Hepatic and pulmonary CCN1 mRNA expression was down-regulated by 80%, 60%, and 55% and 85%, 80%, and 65% at 18, 48, and 96 h after CLP and undetectable in circulating white blood cells. To identify a potential source for the circulating protein, mouse and human platelets were explored and revealed to contain CCN1. Human platelets were stimulated by thrombin and a specific PAR1 agonist (SFLLRN) in vitro. Both agonists induced an instant CCN1 release, and the effect of SFLLRN was blocked by the specific antagonist RWJ56110. The current study demonstrates that experimental sepsis is associated with a robust increase in circulating CCN1 protein levels and a paradoxical downregulation of CCN1 mRNA expression in vital organs. It provides evidence that CCN1 is released from activated platelets, suggesting that platelets constitute a novel source for CCN1 release to the circulation during sepsis.