Multiwalled Carbon Nanotubes Induce Fibrosis and Telomere Length Alterations.

Telomere shortening can result in cellular senescence and in increased level of genome instability, which are key events in numerous of cancer types. Despite this, few studies have focused on the effect of nanomaterial exposure on telomere length as a possible mechanism involved in nanomaterial-induced carcinogenesis. In this study, effects of exposure to multiwalled carbon nanotubes (MWCNT) on telomere length were investigated in mice exposed by intrapleural injection, as well as in human lung epithelial and mesothelial cell lines. In addition, cell cycle, apoptosis, and regulation of genes involved in DNA damage repair were assessed. Exposure to MWCNT led to severe fibrosis, infiltration of inflammatory cells in pleura, and mesothelial cell hyperplasia. These histological alterations were accompanied by deregulation of genes involved in fibrosis and immune cell recruitment, as well as a significant shortening of telomeres in the pleura and the lung. Assessment of key carcinogenic mechanisms in vitro confirmed that long-term exposure to the long MWCNT led to a prominent telomere shortening in epithelial cells, which coincided with G1-phase arrest and enhanced apoptosis. Altogether, our data show that telomere shortening resulting in cell cycle arrest and apoptosis may be an important mechanism in long MWCNT-induced inflammation and fibrosis.

Genetic interaction analysis among oncogenesis-related genes revealed novel genes and networks in lung cancer development.

The development of cancer is driven by the accumulation of many oncogenesis-related genetic alterations and tumorigenesis is triggered by complex networks of involved genes rather than independent actions. To explore the epistasis existing among oncogenesis-related genes in lung cancer development, we conducted pairwise genetic interaction analyses among 35,031 SNPs from 2027 oncogenesis-related genes. The genotypes from three independent genome-wide association studies including a total of 24,037 lung cancer patients and 20,401 healthy controls with Caucasian ancestry were analyzed in the study. Using a two-stage study design including discovery and replication studies, and stringent Bonferroni correction for multiple statistical analysis, we identified significant genetic interactions between SNPs in RGL1:RAD51B (OR=0.44, p value=3.27x10-11 in overall lung cancer and OR=0.41, p value=9.71x10-11 in non-small cell lung cancer), SYNE1:RNF43 (OR=0.73, p value=1.01x10-12 in adenocarcinoma) and FHIT:TSPAN8 (OR=1.82, p value=7.62x10-11 in squamous cell carcinoma) in our analysis. None of these genes have been identified from previous main effect association studies in lung cancer. Further eQTL gene expression analysis in lung tissues provided information supporting the functional role of the identified epistasis in lung tumorigenesis. Gene set enrichment analysis revealed potential pathways and gene networks underlying molecular mechanisms in overall lung cancer as well as histology subtypes development. Our results provide evidence that genetic interactions between oncogenesis-related genes play an important role in lung tumorigenesis and epistasis analysis, combined with functional annotation, provides a valuable tool for uncovering functional novel susceptibility genes that contribute to lung cancer development by interacting with other modifier genes.

Lung Cancer Risk in Never-Smokers of European Descent is Associated With Genetic Variation in the 5p15.33 TERT-CLPTM1Ll Region.

INTRODUCTION: Inherited susceptibility to lung cancer risk in never-smokers is poorly understood. The major reason for this gap in knowledge is that this disease is relatively uncommon (except in Asians), making it difficult to assemble an adequate study sample. In this study we conducted a genome-wide association study on the largest, to date, set of European-descent never-smokers with lung cancer. METHODS: We conducted a two-phase (discovery and replication) genome-wide association study in never-smokers of European descent. We further augmented the sample by performing a meta-analysis with never-smokers from the recent OncoArray study, which resulted in a total of 3636 cases and 6295 controls. We also compare our findings with those in smokers with lung cancer. RESULTS: We detected three genome-wide statistically significant single nucleotide polymorphisms rs31490 (odds ratio [OR]: 0.769, 95% confidence interval [CI]: 0.722-0.820; p value 5.31 × 10-16), rs380286 (OR: 0.770, 95% CI: 0.723-0.820; p value 4.32 × 10-16), and rs4975616 (OR: 0.778, 95% CI: 0.730-0.829; p value 1.04 × 10-14). All three mapped to Chromosome 5 CLPTM1L-TERT region, previously shown to be associated with lung cancer risk in smokers and in never-smoker Asian women, and risk of other cancers including breast, ovarian, colorectal, and prostate. CONCLUSIONS: We found that genetic susceptibility to lung cancer in never-smokers is associated to genetic variants with pan-cancer risk effects. The comparison with smokers shows that top variants previously shown to be associated with lung cancer risk only confer risk in the presence of tobacco exposure, underscoring the importance of gene-environment interactions in the etiology of this disease.

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.

Cellular Responses of Industrially Relevant Silica Dust on Human Glial Cells In Vitro.

Despite the rigorous emission control measures in the ferroalloy industry, there are still emissions of dust during the production of various alloys. Dust particles were collected from laboratory scale processes where oxide particulate matter was formed from liquid silicon (metallurgical grade). The dust was produced in a dry air atmosphere to mimic industrial conditions. To investigate possible effects of ultrafine dust on the central nervous system, a human astrocytic cell line was employed to investigate inflammatory effects of particles as astrocytes play a number of active and neuron supporting roles in the brain. Toxicity on the astrocytes by amorphous silica generated in laboratory scale was compared to crystalline macro-sized silica using several doses to determine toxicological dose response curves. The cell viability experiments indicated that low particle doses of amorphous silica induced a small nonsignificant reduction in cell viability compared to crystalline silica which led to increased levels of toxicity. The gene expression of amyloid precursor protein (APP), a biomarker of neurodegenerative disease, was affected by particle exposure. Furthermore, particle exposure, in a dose-and time-dependent manner, affected the ability of the cells to communicate through gap junction channels. In conclusion, in vitro studies using low doses of particles are important to understand mechanisms of toxicity of occupational exposure to silica particles. However, these studies cannot be extrapolated to real exposure scenarios at work place, therefore, controlling and keeping the particle exposure levels low at the work place, would prevent potential negative health effects.

Physicochemical predictors of Multi-Walled Carbon Nanotube-induced pulmonary histopathology and toxicity one year after pulmonary deposition of 11 different Multi-Walled Carbon Nanotubes in mice.

Multi-walled carbon nanotubes (MWCNT) are widely used nanomaterials that cause pulmonary toxicity upon inhalation. The physicochemical properties of MWCNT vary greatly, which makes general safety evaluation challenging to conduct. Identification of the toxicity-inducing physicochemical properties of MWCNT is therefore of great importance. We have evaluated histological changes in lung tissue 1 year after a single intratracheal instillation of 11 well-characterized MWCNT in female C57BL/6N BomTac mice. Genotoxicity in liver and spleen was evaluated by the comet assay. The dose of 54 µg MWCNT corresponds to three times the estimated dose accumulated during a work life at a NIOSH recommended exposure limit (0.001 mg/m3 ). Short and thin MWCNT were observed as agglomerates in lung tissue 1 year after exposure, whereas thicker and longer MWCNT were detected as single fibres, suggesting biopersistence of both types of MWCNT. The thin and entangled MWCNT induced varying degree of pulmonary inflammation, in terms of lymphocytic aggregates, granulomas and macrophage infiltration, whereas two thick and straight MWCNT did not. By multiple regression analysis, larger diameter and higher content of iron predicted less histopathological changes, whereas higher cobalt content significantly predicted more histopathological changes. No MWCNT-related fibrosis or tumours in the lungs or pleura was found. One thin and entangled MWCNT induced increased levels of DNA strand breaks in liver; however, no physicochemical properties could be related to genotoxicity. This study reveals physicochemical-dependent difference in MWCNT-induced long-term, pulmonary histopathological changes. Identification of diameter size and cobalt content as important for MWCNT toxicity provides clues for designing MWCNT, which cause reduced human health effects following pulmonary exposure.

Mendelian Randomization and mediation analysis of leukocyte telomere length and risk of lung and head and neck cancers.

BACKGROUND: Evidence from observational studies of telomere length (TL) has been conflicting regarding its direction of association with cancer risk. We investigated the causal relevance of TL for lung and head and neck cancers using Mendelian Randomization (MR) and mediation analyses. METHODS: We developed a novel genetic instrument for TL in chromosome 5p15.33, using variants identified through deep-sequencing, that were genotyped in 2051 cancer-free subjects. Next, we conducted an MR analysis of lung (16 396 cases, 13 013 controls) and head and neck cancer (4415 cases, 5013 controls) using eight genetic instruments for TL. Lastly, the 5p15.33 instrument and distinct 5p15.33 lung cancer risk loci were evaluated using two-sample mediation analysis, to quantify their direct and indirect, telomere-mediated, effects. RESULTS: The multi-allelic 5p15.33 instrument explained 1.49-2.00% of TL variation in our data (p = 2.6 × 10-9). The MR analysis estimated that a 1000 base-pair increase in TL increases risk of lung cancer [odds ratio (OR) = 1.41, 95% confidence interval (CI): 1.20-1.65] and lung adenocarcinoma (OR = 1.92, 95% CI: 1.51-2.22), but not squamous lung carcinoma (OR = 1.04, 95% CI: 0.83-1.29) or head and neck cancers (OR = 0.90, 95% CI: 0.70-1.05). Mediation analysis of the 5p15.33 instrument indicated an absence of direct effects on lung cancer risk (OR = 1.00, 95% CI: 0.95-1.04). Analysis of distinct 5p15.33 susceptibility variants estimated that TL mediates up to 40% of the observed associations with lung cancer risk. CONCLUSIONS: Our findings support a causal role for long telomeres in lung cancer aetiology, particularly for adenocarcinoma, and demonstrate that telomere maintenance partially mediates the lung cancer susceptibility conferred by 5p15.33 loci.

Kinetics and tissue distribution of bismuth, tin and lead after implantation of miniature shotgun alloy pellets in rats.

INTRODUCTION: Shotgun pellets containing bismuth (Bi) as substitute for lead (Pb) are increasingly being used due to environmental concerns. Information on toxicokinetics of Bi is lacking for the assessment of humans accidentally shot by Bi-containing shotgun alloy pellets. METHODS: Male Wistar rats were exposed to miniature alloy pellets containing Bi, tin (Sn) and minor amounts of Pb by implantation in muscle tissues of the hind legs. RESULTS: The concentrations of Bi in whole blood and urine increased up to 53 weeks after implantation. The highest concentrations of Sn in whole blood were observed three weeks after implantation, then declining to background levels 53 weeks after implantation. Lead in whole blood increased up to 13 weeks of exposure, and declined for the remaining observation period. Bismuth and Sn accumulated mainly in kidney, but also in liver, testicle and brain. Analytical field emission scanning electron microscopy of post-implant pellets showed depletion of Pb towards the pellet surface. Oxygen and chlorine accumulated in Sn rich lamellas in areas next to the pellet surface. The distribution of Bi remained visually unaffected as compared to pre-implant pellets. CONCLUSION: The concentration of Bi increased during the whole observation period in blood, urine, kidney, brain, testicle and liver. The decline in the concentrations of Pb and Sn in blood and urine after reaching the peak concentration may be related to alterations in the chemical composition and element distribution of the implanted alloy pellets.

Inflammation in the pleural cavity following injection of multi-walled carbon nanotubes is dependent on their characteristics and the presence of IL-1 genes.

Upon inhalation, multi-walled carbon nanotubes (MWCNTs) may reach the subpleura and pleural spaces, and induce pleural inflammation and/or mesothelioma in humans. However, the mechanisms of MWCNT-induced pathology after direct intrapleural injections are still only partly elucidated. In particular, a role of the proinflammatory interleukin-1 (IL-1) cytokines in pleural inflammation has so far not been published. We examined the MWCNT-induced pleural inflammation, gene expression abnormalities, and the modifying role of IL-1α and ß cytokines following intrapleural injection of two types of MWCNTs (CNT-1 and CNT-2) compared with crocidolite asbestos in IL-1 wild-type (WT) and IL-1α/ß KO (IL1-KO) mice. Histopathological examination of the pleura 28 days post-exposure revealed mesothelial cell hyperplasia, leukocyte infiltration, and fibrosis occurring in the CNT-1 (Mitsui-7)-exposed group. The pleura of these mice also showed the greatest changes in mRNA and miRNA expression levels, closely followed by CNT-2. In addition, the CNT-1-exposed group also presented the greatest infiltrations of leukocytes and proliferation of fibrous tissue. WT mice were more prone to development of sustained inflammation and fibrosis than IL1-KO mice. Prominent differences in genetic and epigenetic changes were also observed between the two genotypes. In conclusion, the fibrotic response to MWCNTs in the pleura depends on the particles' physico-chemical properties and on the presence or absence of the IL-1 genes. Furthermore, we found that CNT-1 was the most potent inducer of inflammatory responses, followed by CNT-2 and crocidolite asbestos.

Criteria for grouping of manufactured nanomaterials to facilitate hazard and risk assessment, a systematic review of expert opinions.

With the emergence of nanotechnology the number of manufactured nanomaterials (MNM) in production and use is constantly increasing. Exposure of workers to MNM is of concern, because still much is unknown about health effects. MNM may have different properties, testing of each material is time consuming and costly. Experts have proposed various approaches to categorize MNM to facilitate risk assessment of human health effects based on shared properties of various materials. A systematic literature survey was undertaken to identify expert opinions on grouping of MNM published between the years 2000 and 2015. We summarized and synthesized the opinions according to a systematic review of text and opinion. We identified 22 articles that fulfilled our inclusion criteria reporting 17 proposals with three proposals for groups and 14 proposals for criteria for grouping. Five proposals suggested one or more of the following groups of concern: fibrous, biopersistent, high solubility with high toxicity, chemically active. Criteria proposed in multiple studies were: viable testing options, mode of action, physicochemical properties predicting toxicity. We conclude that a limited number of groups have been proposed to categorize MNM according to human health concern. Further research should be conducted to underpin the proposed groups with empirical evidence.

Characterization of the proteome and lipidome profiles of human lung cells after low dose and chronic exposure to multiwalled carbon nanotubes.

The effects of long-term chronic exposure of human lung cells to multi-walled carbon nanotubes (MWCNT) and their impact upon cellular proteins and lipids were investigated. Since the lung is the major target organ, an in vitro normal bronchial epithelial cell line model was used. Additionally, to better mimic exposure to manufactured nanomaterials at occupational settings, cells were continuously exposed to two non-toxic and low doses of a MWCNT for 13-weeks. MWCNT-treatment increased ROS levels in cells without increasing oxidative DNA damage and resulted in differential expression of multiple anti- and pro-apoptotic proteins. The proteomic analysis of the MWCNT-exposed cells showed that among more than 5000 identified proteins; more than 200 were differentially expressed in the treated cells. Functional analyses revealed association of these differentially regulated proteins to cellular processes such as cell death and survival, cellular assembly, and organization. Similarly, shotgun lipidomic profiling revealed accumulation of multiple lipid classes. Our results indicate that long-term MWCNT-exposure of human normal lung cells at occupationally relevant low-doses may alter both the proteome and the lipidome profiles of the target epithelial cells in the lung.

Genome-wide interaction study of smoking behavior and non-small cell lung cancer risk in Caucasian population.

Non-small cell lung cancer is the most common type of lung cancer. Both environmental and genetic risk factors contribute to lung carcinogenesis. We conducted a genome-wide interaction analysis between single nucleotide polymorphisms (SNPs) and smoking status (never- versus ever-smokers) in a European-descent population. We adopted a two-step analysis strategy in the discovery stage: we first conducted a case-only interaction analysis to assess the relationship between SNPs and smoking behavior using 13336 non-small cell lung cancer cases. Candidate SNPs with P-value <0.001 were further analyzed using a standard case-control interaction analysis including 13970 controls. The significant SNPs with P-value <3.5 × 10-5 (correcting for multiple tests) from the case-control analysis in the discovery stage were further validated using an independent replication dataset comprising 5377 controls and 3054 non-small cell lung cancer cases. We further stratified the analysis by histological subtypes. Two novel SNPs, rs6441286 and rs17723637, were identified for overall lung cancer risk. The interaction odds ratio and meta-analysis P-value for these two SNPs were 1.24 with 6.96 × 10-7 and 1.37 with 3.49 × 10-7, respectively. In addition, interaction of smoking with rs4751674 was identified in squamous cell lung carcinoma with an odds ratio of 0.58 and P-value of 8.12 × 10-7. This study is by far the largest genome-wide SNP-smoking interaction analysis reported for lung cancer. The three identified novel SNPs provide potential candidate biomarkers for lung cancer risk screening and intervention. The results from our study reinforce that gene-smoking interactions play important roles in the etiology of lung cancer and account for part of the missing heritability of this disease.

Effects on human bronchial epithelial cells following low-dose chronic exposure to nanomaterials: A 6-month transformation study.

The most plausible exposure route to manufactured nanomaterials (MNM) remains pulmonary inhalation. Yet, few studies have attempted to assess carcinogenic properties in vitro following long-term exposure of human pulmonary cells to low and occupationally relevant doses. The most advanced in vitro tests for carcinogenicity, the cell transformation assay (CTA), rely mostly on rodent cells and short-term exposure. We hypothesized that long-term exposure of human bronchial epithelial cells with a normal phenotype could be a valuable assay for testing carcinogenicity of nanomaterials. Therefore, this study (performed within the framework of the FP7-NANoREG project) assessed carcinogenic potential of chronic exposure (up to 6months) to low doses of multi-walled carbon nanotubes (MWCNT, NM-400 and NM-401) and TiO2 materials (NM62002 and KC7000). In order to harmonize and standardize the experiments, standard operating protocols of MNM dispersion (NANOGENOTOX) were used by three different NANoREG project partners. All nanomaterials showed low cytotoxicity in short-term tests for the tested doses (0.96 and 1.92µg/cm2). During long-term exposure, however, NM-401 clearly affected cell proliferation. In contrast, no cell transformation was observed for NM-401 by any of the partners. NM-400 and NM62002 formed some colonies after 3months. We conclude that agglomerated NM-401 in low doses affect cell proliferation but do not cause cell transformation in the CTA assay used.

Differences in inflammation and acute phase response but similar genotoxicity in mice following pulmonary exposure to graphene oxide and reduced graphene oxide.

We investigated toxicity of 2-3 layered >1 µm sized graphene oxide (GO) and reduced graphene oxide (rGO) in mice following single intratracheal exposure with respect to pulmonary inflammation, acute phase response (biomarker for risk of cardiovascular disease) and genotoxicity. In addition, we assessed exposure levels of particulate matter emitted during production of graphene in a clean room and in a normal industrial environment using chemical vapour deposition. Toxicity was evaluated at day 1, 3, 28 and 90 days (18, 54 and 162 µg/mouse), except for GO exposed mice at day 28 and 90 where only the lowest dose was evaluated. GO induced a strong acute inflammatory response together with a pulmonary (Serum-Amyloid A, Saa3) and hepatic (Saa1) acute phase response. rGO induced less acute, but a constant and prolonged inflammation up to day 90. Lung histopathology showed particle agglomerates at day 90 without signs of fibrosis. In addition, DNA damage in BAL cells was observed across time points and doses for both GO and rGO. In conclusion, pulmonary exposure to GO and rGO induced inflammation, acute phase response and genotoxicity but no fibrosis.

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.

A Two-Gene Prognostic Classifier for Early-Stage Lung Squamous Cell Carcinoma in Multiple Large-Scale and Geographically Diverse Cohorts.

INTRODUCTION: There are no validated molecular methods that prospectively identify patients with surgically resected lung squamous cell carcinoma (SCC) at high risk for recurrence. By focusing on the expression of genes with known functions in development of lung SCC and prognosis, we sought to develop a robust prognostic classifier of early-stage lung SCC. METHODS: The expression of 253 genes selected by literature search was evaluated in microarrays from 107 stage I/II tumors. Associations with survival were evaluated by Cox regression and Kaplan-Meier survival analyses in two independent cohorts of 121 and 91 patients with SCC, respectively. A classifier score based on multivariable Cox regression was derived and examined in six additional publicly available data sets of stage I/II lung SCC expression profiles (n = 358). The prognostic value of this classifier was evaluated in meta-analysis of patients with stage I/II (n = 479) and stage I (n = 326) lung SCC. RESULTS: Dual specificity phosphatase 6 gene (DUSP6) and actinin alpha 4 gene (ACTN4) were associated with prognostic outcome in two independent patient cohorts. Their expression values were utilized to develop a classifier that identified patients with stage I/II lung SCC at high risk for recurrence (hazard ratio [HR] = 4.7, p = 0.018) or cancer-specific mortality (HR = 3.5, p = 0.016). This classifier also identified patients at high risk for recurrence (HR = 2.7, p = 0.008) or death (HR = 2.2, p = 0.001) in publicly available data sets of stage I/II and in meta-analysis of stage I patients. CONCLUSIONS: We have established and validated a prognostic classifier to inform clinical management of patients with lung SCC after surgical resection.

Estrogen receptor expression and gene promoter methylation in non-small cell lung cancer - a short report.

PURPOSE: In the past, anomalous estrogen receptor (ER) regulation has been associated with various lung pathologies, but so far its involvement in lung cancer initiation and/or progression has remained unclear. Here, we aimed at assessing in vivo and in vitro ER expression and its possible epigenetic regulation in non-small cell lung cancer (NSCLC) samples and their corresponding normal tissues and cells. METHODS: ERα and ERß gene expression levels were assessed using real time quantitative PCR (RT-qPCR), whereas ERα and ERß gene promoter methylation levels were assessed using DNA bisulfite conversion followed by pyrosequencing. We included NSCLC (n = 87) and adjacent histologically normal lung tissue samples from lung cancer patients (n = 184), primary normal bronchial epithelial-derived cell cultures (n = 11), immortalized bronchial epithelial-derived cell lines (n = 3) and NSCLC derived cell lines (n = 9). RESULTS: Using RT-qPCR we found significantly lower ERα and ERß expression levels in the NSCLC tissue samples compared to their normal adjacent tissue samples. These lower ER expression levels were confirmed in vitro using primary normal bronchial epithelial-derived cell cultures, immortalized bronchial epithelial-derived cell lines and NSCLC-derived cell lines. By using this latter panel of cells, we found that ER gene promoter hypermethylation was associated with decreased ER expression. In addition we found that in tumor and normal lung tissues, smoking was associated with decreased ER expression and that normal lung tissues with a low ERß expression level exhibited increased smoking-related DNA adducts. CONCLUSIONS: Taken together, our results indicate that decreased ER expression mediated by DNA methylation may play a role in NSCLC development.

Genetic Risk Can Be Decreased: Quitting Smoking Decreases and Delays Lung Cancer for Smokers With High and Low CHRNA5 Risk Genotypes - A Meta-Analysis.

BACKGROUND: Recent meta-analyses show that individuals with high risk variants in CHRNA5 on chromosome 15q25 are likely to develop lung cancer earlier than those with low-risk genotypes. The same high-risk genetic variants also predict nicotine dependence and delayed smoking cessation. It is unclear whether smoking cessation confers the same benefits in terms of lung cancer risk reduction for those who possess CHRNA5 risk variants versus those who do not. METHODS: Meta-analyses examined the association between smoking cessation and lung cancer risk in 15 studies of individuals with European ancestry who possessed varying rs16969968 genotypes (N=12,690 ever smokers, including 6988 cases of lung cancer and 5702 controls) in the International Lung Cancer Consortium. RESULTS: Smoking cessation (former vs. current smokers) was associated with a lower likelihood of lung cancer (OR=0.48, 95%CI=0.30-0.75, p=0.0015). Among lung cancer patients, smoking cessation was associated with a 7-year delay in median age of lung cancer diagnosis (HR=0.68, 95%CI=0.61-0.77, p=4.9∗10-10). The CHRNA5 rs16969968 risk genotype (AA) was associated with increased risk and earlier diagnosis for lung cancer, but the beneficial effects of smoking cessation were very similar in those with and without the risk genotype. CONCLUSION: We demonstrate that quitting smoking is highly beneficial in reducing lung cancer risks for smokers regardless of their CHRNA5 rs16969968 genetic risk status. Smokers with high-risk CHRNA5 genotypes, on average, can largely eliminate their elevated genetic risk for lung cancer by quitting smoking- cutting their risk of lung cancer in half and delaying its onset by 7years for those who develop it. These results: 1) underscore the potential value of smoking cessation for all smokers, 2) suggest that CHRNA5 rs16969968 genotype affects lung cancer diagnosis through its effects on smoking, and 3) have potential value for framing preventive interventions for those who smoke.

Multi-walled carbon nanotube physicochemical properties predict pulmonary inflammation and genotoxicity.

Lung deposition of multi-walled carbon nanotubes (MWCNT) induces pulmonary toxicity. Commercial MWCNT vary greatly in physicochemical properties and consequently in biological effects. To identify determinants of MWCNT-induced toxicity, we analyzed the effects of pulmonary exposure to 10 commercial MWCNT (supplied in three groups of different dimensions, with one pristine and two/three surface modified in each group). We characterized morphology, chemical composition, surface area and functionalization levels. MWCNT were deposited in lungs of female C57BL/6J mice by intratracheal instillation of 0, 6, 18 or 54 µg/mouse. Pulmonary inflammation (neutrophil influx in bronchoalveolar lavage (BAL)) and genotoxicity were determined on day 1, 28 or 92. Histopathology of the lungs was performed on day 28 and 92. All MWCNT induced similar histological changes. Lymphocytic aggregates were detected for all MWCNT on day 28 and 92. Using adjusted, multiple regression analyses, inflammation and genotoxicity were related to dose, time and physicochemical properties. The specific surface area (BET) was identified as a positive predictor of pulmonary inflammation on all post-exposure days. In addition, length significantly predicted pulmonary inflammation, whereas surface oxidation (-OH and -COOH) was predictor of lowered inflammation on day 28. BET surface area, and therefore diameter, significantly predicted genotoxicity in BAL fluid cells and lung tissue such that lower BET surface area or correspondingly larger diameter was associated with increased genotoxicity. This study provides information on possible toxicity-driving physicochemical properties of MWCNT. The results may contribute to safe-by-design manufacturing of MWCNT, thereby minimizing adverse effects.

Effects of carbon nanotubes on intercellular communication and involvement of IL-1 genes.

An increasing amount of products containing engineered nanoparticles is emerging. Among these particles are carbon nanotubes (CNTs) which are of interest for a wide range of industrial and biomedical applications. There have been raised concerns over the effects of CNTs on human health. Some types of CNTs are classified as group 2B carcinogens by the International Agency for Research on Cancer. CNTs may also induce pulmonary inflammatory and fibrotic effects. By utilizing CNTs of different lengths, we investigated the role of the proinflammatory cytokine, interleukin-1 (IL-1) on gap junctional intercellular communication (GJIC) by using IL-1 wild-type (IL1-WT) and IL-1 knock-out (IL1-KO) cells. GJIC decreased equally in both cell types after CNT exposure. Immunofluorescence staining showed Gja1 and Gjb2 in gap junctions and hemichannels for both cell types. Gjb1 and Gjb2 expression was low in IL1-KO cells, which was confirmed by protein analysis. Gja1 was upregulated with both CNTs, whereas Gjb1 was down-regulated by CNT-2 in IL1-WT cells. Connexin mRNA expression was regulated differently by the CNTs. CNT-1 affected Gja1 and Gjb2, whereas CNT-2 had an effect on Gjb1. CNTs negatively affect GJIC through gap junctions independently of the length of CNT and IL-1 status. Furthermore, connexin gene expression was affected by IL-1 at transcriptional and translational levels. As both CNTs used in this study are cytotoxic to the cells and reduce cell survival, we suggest that CNT-induced reduction in GJIC may be important for inhibiting transfer of cell survival signals between cells.