NAF1 rs4691896 Is Significantly Associated with Coal Workers' Pneumoconiosis in a Chinese Han Population: A Case-Control Study.

BACKGROUND Previous studies have demonstrated the important role of genetic predisposition in coal workers' pneumoconiosis (CWP) in addition to environmental factors. The pathogenesis of pulmonary fibrosis disease is related to telomere activity. We performed this study to assess the association between genetic variants of telomere-related genes and the risk of CWP. MATERIAL AND METHODS We enrolled 652 CWP Chinese Han patients and 648 dust-exposed controls in this case-control design study, genotyping 8 single-nucleotide polymorphisms (SNPs) including TERT (rs2736100), TERC (rs10936599 and rs12696304), and NAF1 (rs7675998, rs3822304, rs12331717, rs936562 and rs4691896) using the Sequenom MassARRAY system. RESULTS We identified a significant allele association between NAF1 rs4691896 and CWP by comparing patients with controls (22.0% vs. 13.0%, odds ratio [OR]: 1.89, 95% confidence interval [CI]: 1.54-2.33, Pc=1.14×10⁻8). The genotype frequency of rs4691896 differed significantly between the patients and controls (Pc=1.49×10⁻8). In addition, rs4691896 was correlated with CWP in an additive genetic model (OR: 1.96, 95% CI: 1.58-2.44, Pc=8.96×10⁻9) and a dominant model (OR: 2.15, 95% CI: 1.70-2.73, Pc=2.39×10⁻9). CONCLUSIONS Our study for the first time demonstrates an association between a telomere-related gene (NAF1) and CWP in a Chinese Han population, and provides valuable insight to further understand the possible pathogenetic mechanism of fibrosis in CWP.

TNF-α-TNFR signal pathway inhibits autophagy and promotes apoptosis of alveolar macrophages in coal worker's pneumoconiosis.

OBJECTIVE: Exposure to coal dust causes the development of coal worker's pneumoconiosis (CWP), which is associated with accumulating macrophages in the lower respiratory tract. This study was performed to investigate the effect of tumor necrosis factor-α (TNF-α)-tumor necrosis factor receptor (TNFR) signal pathway on autophagy and apoptosis of alveolar macrophages (AMs) in CWP. METHODS: AMs from controls exposed to coal dust and CWP patients were collected, in which expressions of TNF-α and TNFR1 were determined. Autophagy was observed by transmission electron microscopy, and apoptosis by light microscope and using terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. AMs in CWP patients were treated with TNF-α or anti-TNF-α antibody. Besides, expressions of autophagy marker proteins, apoptosis-related factors, FAS, caspase-8, and receptor-interacting serine-threonine-protein kinase 3 (RIPK3) were determined by western Blot. Activities of caspase-3 and caspase-8 were determined by a fluorescence kit. Flow cytometry was applied to measure the expression of TNFR1 on the surface of the AM. RESULTS: TNF-α expression and TNFR1 expression on the surface of AM, as well as autophagy and apoptotic index were significantly increased in AMs of CWP patients. In response to the treatment of TNF-α, TNF-α expression and TNFR1 expression on the surface of AM as well as LC3I expression were increased, autophagy was decreased, and LC3, LC3II, Beclin1 and B-cell lymphoma 2 expressions decreased, whereas FAS expression and activity and expression of caspase-3 and caspase-8 increased, and apoptotic index increased. Moreover, the situations were reversed with the treatment of anti-TNF-α antibody. CONCLUSION: TNF-α-TNFR signal pathway was involved in the occurrence and development of CWP by activating FAS-caspase-8 and thus inhibiting autophagy while promoting apoptosis of AM.

Dust induces lung fibrosis through dysregulated DNA methylation.

Pneumoconiosis is a serious occupational disease that often occurs to coal workers with no early diagnosis and effective treatment at present. Diffuse pulmonary fibrosis is the major pathological change of pneumoconiosis, and its mechanism is still unclear. Epigenetics is involved in the development of many diseases, and it is closely associated with fibrosis. In this study, we investigated whether DNA methylation contributes to the pathogenesis of pulmonary fibrosis in pneumoconiosis. By exposure to coal dust or silica dust, we established the models of coal worker's pneumoconiosis (CWP), which showed an increased expression of COL-I, COL-III. We further found that DNMT1, DNMT3a, DNMT3b, MBD2, MeCP2 protein expression changed. Pretreatment with DNMT inhibitor 5-aza-dC reduced expression of COL-I, COL-III, and reduced pulmonary fibrosis. In summary, our results showed that DNA methylation contributes to dust-induced pulmonary fibrosis and that it may serve as a theoretical basis for testing DNA methyltransferase inhibitors in the treatment of CWP.

Element-based prognostics of occupational pneumoconiosis using micro-proton-induced X-ray emission analysis.

Pneumoconiosis is an occupational disease accompanied by long-term lung impairment, for which prediction of prognosis is poorly understood because of the complexity of the inhaled particles. Micro-proton-induced X-ray emission (micro-PIXE) analysis, which is advantageous for high-sensitivity, two-dimensional element mapping of lung tissues, was used to investigate element-based predictive factors of prognosis in Chinese patients with welder's and coal miner's pneumoconiosis. Chest radiographs and lung function tests showed that most of the coal miners deteriorated, whereas symptoms in some welders were alleviated after 5 yr, as determined by comparing percent vital capacity (%VC) and forced expiratory volume in the 1st second over forced vital capacity (FEV1.0/FVC) to values taken at the initial diagnosis. Micro-PIXE analysis suggested that the most abundant particulates in welder's pneumoconiosis were Fe, Mn, and Ti (metallic oxide),which were accompanied by particulates containing Si, Al, and Ca (aluminum silicate) or only Si (SiO2); the most abundant particulates in coal miner's pneumoconiosis were composed of C, Si, Al, K, and Ti, which were accompanied by particulates containing Ca or Fe. Particulates containing Al, Si, S, K, Ca, and Ti (orthoclase and anorthite) were correlated with severity of fibrosis. Multivariable linear regression suggested that long-term FEV1.0/FVC decrease was independently associated with Si and smoking index, whereas %VC decrease was associated with Si and Ti. A risk index comprised of these factors was developed to predict the prognosis of pneumoconiosis. Micro-PIXE analysis is feasible for the evaluation of elemental composition and dust exposure, especially for patients whose exposure is mixed or uncertain.

Polymorphisms in interleukin 17A gene and coal workers' pneumoconiosis risk in a Chinese population.

BACKGROUND: The interleukin 17A (IL-17A) which is located on chromosome 6p and has been linked to chronic inflammation, is an important candidate gene conferring coal workers' pneumoconiosis (CWP). The purpose of this study was to investigate the genetic association between single nucleotide polymorphisms (SNPs) of IL-17A and CWP in a Chinese population. METHODS: We conducted a case-control study to investigate the role of four common SNPs in the IL-17A gene, and evaluated the relationship between these four SNPs and dust-exposure year, tobacco smoking and stages of CWP. A total of 1391 subjects was enrolled in this study, including 694 subjects in control group and 697 in case group. TaqMan based qRT-PCRs were taken to genotype rs2275913, rs3748067, rs4711998, and rs8193036 within the IL-17A gene. Luciferase assays were used to determine the effects of rs8193036 C > T alleles on the expression of IL-17A. RESULTS: Unconditional logistic regression analysis showed that the genotypes of rs3748067 AA (adjusted OR = 0.43, 95 % CI = 0.23-0.83) and rs8193036 TT (adjusted OR = 0.59, 95 % CI = 0.40-0.86) were associated with a decreased risk of CWP, particularly among subgroups of smokers (adjusted OR =0.34, 95 % CI = 0.13-0.86 for rs3748076; adjusted OR = 0.41, 95 % CI = 0.23-0.71 for 8193036) and CWP cases with stage I (adjusted OR = 0.45, 95 % CI = 0.21-0.98 for rs3748076; adjusted OR = 0.46, 95 % CI = 0.28-0.74 for 8193036). Furthermore, the polymorphism of rs3748067 significantly reduced the CWP risk among cases with over 27 years of dust exposure (adjusted OR = 0.42, 95 % CI = 0.18-0.97). The luciferase assays in two cell lines showed that the rs8193036 C > T substitution could reduce the expression of IL-17A, which was consistent with the findings of our association study. CONCLUSIONS: The rs3748067 G > A and rs8193036 C > T polymorphisms decrease CWP risk. These findings could be helpful in identifying individuals at decreased risk for CWP and further studies are warranted to validate them.

ERCC1 which affects lipids metabolism and actin dynamics in coal workers' pneumoconiosis is a candidate biomarker for early warning and diagnosis.

The single-nucleotide polymorphisms of genes related to DNA damage repair and inflammasomes and mutated gene expression in coal workers' pneumoconiosis (CWP) were analysed to identify the risk factors of CWP and potential biomarkers for early warning and diagnosis. Further, mutated gene pathways were analysed based on proteome and metabolome. Han Chinese male subjects were randomly selected and divided into 4 or 5 groups according to the process of CWP. MassARRAY was used to sequence single-nucleotide polymorphism genotypes. Mutated gene expression in plasma was tested using enzyme-linked immunosorbent assay (ELISA). Odds ratios (ORs) and receiver operating characteristic curves (ROC) were calculated. The serum different proteins and metabolites were identified by Ultra Performance Liquid Chromatography Quadrupole time of flight/Mass Spectrum (UPLC-Q-TOF/MS) and analysed using bioinformation software. As CWP progressed, the CC and CA genotypes of ERCC1 rs3212986 decreased and increased significantly, respectively. AA (OR = 3.016) and CA (OR = 2.130) genotypes were identified as risk factors for stage II. ERCC1 significantly decreased in processing of CWP. The cutoff value of ERCC1 was 5.265 pg/ml, with a sensitivity of 90.0% and specificity of 86.7%. ERCC1 had an indirect interaction with activator protein-1 and insulin and its pathways were mainly made with molecules related to lipid metabolism and actin dynamics. ERCC1 is a candidate biomarker for detection and precise intervention in CWP. If it reaches the threshold, workers will change other jobs in time and will not develop and diagnose as pneumoconiosis and will help the employers spend less money. Meanwhile, the signal molecules of ERCC1 pathway could be as a candidate target for drug discovery.

Laboratory studies of the impact of calcite on in vitro and in vivo effects of coal dust: a potential preventive agent for coal workers' pneumoconiosis?

BACKGROUND: Bioavailable iron (BAI) in coal, which may play a key role in causing coal workers' pneumoconiosis (CWP), is present at relatively high levels in Appalachian coals. Calcite decreases BAI and is more plentiful in Western coals than in Appalachian coals, possibly explaining the lower CWP prevalence among Western miners. METHODS: We measured effects of calcite on BAI in non-cellular and cellular systems involving Pennsylvania (PA) coal dust. We also tested in vivo effects of calcite on transferrin receptor and markers of epithelial mesenchymal transition (EMT) and inflammation in mice exposed to PA coal. RESULTS: Calcite rapidly eliminated BAI in an aqueous suspension of PA coal. Ferritin induction in human lung epithelial cells exposed to PA coal was effectively eliminated by calcite. Mouse lung tissue markers indicated increased EMT after exposure to PA coal dust, but not after exposure to PA coal plus calcite. Markers of inflammation increased following exposure to PA coal alone, but not following exposure to PA coal plus calcite. CONCLUSION: Additional research may lead to the use of supplemental calcite in coal mining as a safe and effective way to prevent CWP among Appalachian coal miners.

Pyrite-driven reactive oxygen species formation in simulated lung fluid: implications for coal workers' pneumoconiosis.

The origin of coal worker's pneumoconiosis (CWP) has been long debated. A recent epidemiological study shows a correlation between what is essentially the concentration of pyrite within coal and the prevalence of CWP in miners. Hydrogen peroxide and hydroxyl radical, both reactive oxygen species (ROS), form as byproducts of pyrite oxidative dissolution in air-saturated water. Motivated by the possible importance of ROS in the pathogenesis of CWP, we conducted an experimental study to evaluate if ROS form as byproducts in the oxidative dissolution of pyrite in simulated lung fluid (SLF) under biologically applicable conditions and to determine the persistence of pyrite in SLF. While the rate of pyrite oxidative dissolution in SLF is suppressed by 51% when compared to that in air-saturated water, the initial amount of hydrogen peroxide formed as a byproduct in SLF is nearly doubled. Hydroxyl radical is also formed in the experiments with SLF, but at lower concentrations than in the experiments with water. The formation of these ROS indicates that the reaction mechanism for pyrite oxidative dissolution in SLF is no different from that in water. The elevated hydrogen peroxide concentration in SLF suggests that the decomposition, via the Fenton mechanism to hydroxyl radical or with Fe(III) to form water and molecular oxygen, is initially inhibited by the presence of SLF components. On the basis of the oxidative dissolution rate of pyrite measured in this paper, it is calculated that a respirable two micron pyrite particle will take over 3 years to dissolve completely.

[Bone mineral density changes in coal workers' pneumoconiosis in Two and Triple stages with increasing ages].

OBJECTIVE: To observe the bone mineral density changes in coal workers' pneumoconiosis in Two and Triple stages with increasing ages. METHODS: Chose 70 cases of coal workers pneumoconiosis in Two and Triple stages in Jincheng Coal Mining Group, all of workers were male, of 55-years old-80 years old, an average of 67 years old. 10 years of ages to grouping, whole body bone mineral densities were measured by body dual-energy X-ray absorptiometry. We analyzed the BMD changes bone loss, osteoporosis occurrence. RESULTS: Chest bone, pelvis, spine bone mineral densities of coal workers pneumoconiosis in Two and Triple stages were significantly decreased. We found that the rate of Pelvic BMD decline of coal workers' pneumoconiosis patients in Two stage was significantly faster after 65 years of age. In Different age groups of coal workers' pneumoconiosis patients in Two and Triple stages, incidence of bone loss and osteoporosis were significantly increased. CONCLUSION: With the age increasing, Coal workers' pneumoconiosis in Two and Triple stages significantly accelerated the speed of the rate of BMD decline. This phenomenon was most obvious in the chest bone, pelvis, and spine.

[Bone mineral density changes in coal workers' pneumoconiosis].

OBJECTIVE: To observe the bone mineral density changes of coal workers' pneumoconiosis. METHODS: We chose 150 cases of One-Triple coal workers pneumoconiosis in Jincheng Coal Mining Group, all of workers were male, of 55-years old-80 years old, an average of 67 years old. 10 years of age to grouping, Whole body bone mineral density and T value were measured by body dual-energy X-ray absorptiometry. We analyzed the BMD changes bone loss, osteoporosis occurrence. RESULTS: The BMD of six parts were not declined obviously in One stage of coal workers' pneumoconiosis; the BMD of Chest bone, pelvis and spine were declined obviously in two stage of coal workers' pneumoconiosis; the BMD of six parts were declined obviously in Triple stage of coal workers' pneumoconiosis; The occurrence rate of bone loss was significantly higher in Two and Triple coal workers pneumoconiosis. The occurrence rate of osteoporosis was significantly higher in Triple coal workers pneumoconiosis. CONCLUSION: With the increase in the severity of coal workers' pneumoconiosis, the BMD of six parts were declined, The occurrence rate of bone loss osteoporosis was significantly higher.

Exploration study on serum metabolic profiles of Chinese male patients with artificial stone silicosis, silicosis, and coal worker's pneumoconiosis.

Long-term exposure to inhaled silica dust induces pneumoconiosis, which remains a heavy burden in developing countries. Modern industry provides new resources of occupational SiO2 leading to artificial stone silicosis especially in developed countries. This study aimed to characterize the serum metabolic profile of pneumoconiosis and artificial stone silicosis patients. Our case-control study recruited 46 pairs of pneumoconiosis patients and dust-exposed workers. Nontargeted metabolomics and lipidomics by ultra-high-performance liquid chromatography-tandem mass spectrometry platform were conducted to characterize serum metabolic profile in propensity score-matched (PSM) pilot study. 54 differential metabolites were screened, 24 of which showed good screening efficiency through receiver operating characteristics (ROC) in pilot study and validation study (both AUC > 0.75). 4 of the 24 metabolites can predict pneumoconiosis stages, which are 1,2-dioctanoylthiophosphatidylcholine, phosphatidylcholine(O-18:1/20:1), indole-3-acetamide and l-homoarginine. Kynurenine, N-tetradecanoylsphingosine 1-phosphate, 5-methoxytryptophol and phosphatidylethanolamine(22:6/18:1) displayed the potential as specific biomarkers for artificial stone silicosis. Taken together, our results confirmed that tryptophan metabolism is closely related to pneumoconiosis and may be related to disease progression. Hopefully, our results could supplement the biomarkers of pneumoconiosis and provide evidence for the discovery of artificial stone silicosis-specific biomarkers.

Targeting the RAS axis alleviates silicotic fibrosis and Ang II-induced myofibroblast differentiation via inhibition of the hedgehog signaling pathway.

The hedgehog (HH) signaling pathway plays an important role in lung development, but its significance in silicosis is unclear. We showed that in human coal pneumoconiosis autopsy specimens, Sonic Hedgehog (SHH) and the Glioma-associated oncogene homolog transcription factors family (GLI) 1 proteins were up-regulated, whereas Patch-1 (PTC) was down-regulated. The protein levels of SHH, smoothened (SMO), GLI1, GLI2, α-smooth muscle actin (α-SMA) and collagen type Ⅰ (Col Ⅰ) were also elevated gradually in the bronchoalveolar lavage fluid (BALF) of different stages of coal pneumoconiosis patients, dynamic silica-inhalation rat lung tissue and MRC-5 cells induced by Ang II at different time points, whereas the PTC and GLI3 levels were diminished gradually. Ac-SDKP, an active peptide of renin-angiotensin system (RAS), is an anti-fibrotic tetrapeptide. Targeting RAS axis also has anti-silicotic fibrosis effects. However, their roles on the HH pathway are still unknown. Here, we reported that Ac-SDKP + Captopril, Ac-SDKP, Captopril, or Ang (1-7) could alleviate silicotic fibrosis and collagen deposition, as well as improve the lung functions of silicotic rat. These treatments decreased the expression of SHH, SMO, GLI1, GLI2, α-SMA, and Col Ⅰ and increased the expression of PTC and GLI3 on both the silicotic rat lung tissue and MRC-5 cells induced by Ang II. We also reported that Ang II may promote myofibroblast differentiation via the GLI1 transcription factor and independently of the SMO receptor.

Serum and BAL cytokine and antioxidant enzyme levels at different stages of pneumoconiosis in coal workers.

Coal workers' pneumoconiosis (CWP) is an occupational pulmonary disease that occurs by chronic inhalation of coal dust. CWP is divided into two stages depending on the extent of the disease, as simple pneumoconiosis (SP) and progressive massive fibrosis (PMF). In the present study, serum and bronchoalveolar lavage (BAL) cytokine (interleukin-1beta [IL-1beta], IL-6, tumor necrosis factor-alpha [TNF-alpha], transforming growth factor-beta [TGF-beta]) and antioxidant enzymes levels, their relation with the disease severity, and whether they can be considered as biological markers were investigated. Serum and BAL levels of IL-1beta, IL-6, and TNF-alpha were higher in SP and PMF patient groups compared with that in active and retired miner groups. Serum and BAL IL-1beta, IL-6, and TNF-alpha levels were also found to be higher in patients with PMF compared with the SP group. BAL superoxide dismutase (SOD), glutathione peroxidase, and catalase levels and serum SOD level were increased in both patient groups compared with the control group. In addition, mean serum and BAL TGF-beta levels were found to be increased in patients with SP compared with PMF group. Based on these results, BAL and serum cytokine and antioxidant enzymes levels were evaluated and discussed as potential biomarkers for different stages of CWP.

EGFR overexpression in canine primary lung cancer: pathogenetic implications and impact on survival.

This study reports the main clinicopathological features of primary lung cancer (PLC) in 37 dogs, with special regard to the pathogenetic and prognostic role of epidermal growth factor receptor (EGFR) overexpression. For each case the following characteristics were evaluated: tumour-node-metastasis (TNM) stage, tumour histotype, histological grade, mitotic activity and immunohistochemical expression of EGFR. In samples with available normal lung tissue, the amount of background anthracosis was also measured by image analysis. In 27 tumours (73%) a variable number of cells (20-100%) stained positively for EGFR. The proportion of EGFR-positive tumours was significantly higher in cases with background anthracosis, and the amount of anthracosis was correlated with the percentage of positive tumour cells. Additionally, a trend towards shortened survival for the high EGFR group was observed. These findings suggest an involvement of EGFR signalling pathway in canine PLC, a negative prognostic significance of protein overexpression and its potential implication in air pollution carcinogenesis.

Expression levels of surfactant-associated proteins and inflammation cytokines in serum and bronchoalveolar lavage fluid among coal miners: a case-control study.

OBJECTIVE: To investigate whether the Th1/Th2 balance and expressions of surfactant-associated proteins and cytokines in serum and bronchoalveolar lavage fluid (BALF) are associated with the development of coal workers' pneumoconiosis (CWP). METHODS: A case-control study was conducted among 72 CWP cases and 68 controls. Th1 and Th2 populations were measured by flow cytometry. Expressions of surfactant-associated proteins A and D (SPA and SPD) and cytokines in serum and BALF were detected by enzyme-linked-immunosorbent serologic assay. Data were analyzed by t test and logistic regression. RESULTS: Higher Th2 and lower Th1/Th2 were observed in CWP (P < 0.05). Increased CWP risk was associated with elevated BALF-interleukin-10 (odds ratio [OR]: 25.97; 95% confidence interval [CI]: 2.17 to 49.77), serum-SPA/BALF-SPA (OR: 12.87; 95% CI: 1.11 to 148.72), and serum-SPD/BALF-SPD (OR: 77.91; 95% CI: 5.64 to 161.46), decreased BALF-SPA (OR: 0.06; 95% CI: 0.01 to 0.31) and BALF-SPD (OR: 0.07; 95% CI: 0.02 to 0.36). CONCLUSIONS: The development of CWP might be associated with Th1/Th2 imbalance and increased BALF-interleukin-10, serum-SPA/BALF-SPA, and serum-SPD/BALF-SPD.