Sputum Microbiota in Coal Workers Diagnosed with Pneumoconiosis as Revealed by 16S rRNA Gene Sequencing.

Coal worker's pneumoconiosis (CWP) is an occupationally induced progressive fibrotic lung disease. This irreversible but preventable disease currently affects millions across the world, mainly in countries with developed coal mining industries. Here, we report a pilot study that explores the sputum microbiome as a potential non-invasive bacterial biomarker of CWP status. Sputum samples were collected from 35 former and active coal miners diagnosed with CWP and 35 healthy controls. Sequencing of bacterial 16S rRNA genes was used to study the taxonomic composition of the respiratory microbiome. There was no difference in alpha diversity between CWP and controls. The structure of bacterial communities in sputum samples (ß diversity) differed significantly between cases and controls (pseudo-F = 3.61; p = 0.004). A significant increase in the abundance of Streptococcus (25.12 ± 11.37 vs. 16.85 ± 11.35%; p = 0.0003) was detected in samples from CWP subjects as compared to controls. The increased representation of Streptococcus in sputum from CWP patients was associated only with the presence of occupational pulmonary fibrosis, but did not depend on age, and did not differ between former and current miners. The study shows, for the first time, that the sputum microbiota of CWP subjects differs from that of controls. The results of our present exploratory study warrant further investigations on a larger cohort.

Genomic instability in chronic obstructive pulmonary disease and lung cancer: A systematic review and meta-analysis of studies using the micronucleus assay.

Respiratory tissues are highly susceptible to diseases due to the constant exposure to physical and chemical airborne pollutants. Chronic obstructive pulmonary disease (COPD) and lung cancer are among the most common causes of serious illness and death worldwide. The inflammatory environment associated with these respiratory diseases has long been accepted as the major player in the development of airway abnormalities. The presence and relevance of DNA damage and genomic instability makes the micronucleus assay a suitable candidate to quantitatively estimate these early pathogenetic events. A systematic review and meta-analysis were planned to determine underlying common mechanisms that can explain the relationships between COPD and lung cancer. A total of 17 studies from Jan 1999 to Dec 2019 comparing micronucleus frequency in patients affected by respiratory diseases vs healthy controls were analysed. Our results confirmed the presence of significant association between MN frequency and the diseases investigated, and suggested a circle of events linking inflammation induced oxidative stress to the risk of disease through genomic instability and hypoxia. Therefore, using non-invasive, robust and cost effective genomic instability assays such as the micronucleus assay, would allow us to capture unique phenotypic and biological changes that would allow the identification of subjects at high risk of developing lung diseases and improve early detection strategies.

Mitomycin C induced genotoxic stress in endothelial cells is associated with differential expression of proinflammatory cytokines.

Mitomycin C (MMC) is an alkylating chemotherapy drug that causes DNA crosslinking resulting in transcription arrest and apoptosis. DNA crosslinking is a critical damage to DNA that can be caused not only by MMC and other antitumor drugs, but also by various environmental and anthropogenic endo- and exogenous agents. Mammalian cells exposed to alkylating mutagens are characterized by severe genotoxic stress. Somatic mutations and genotoxic stress may lead to endothelial dysfunction, which is the initial stage of atherosclerosis, a leading cause of morbidity and mortality worldwide. Here we studied DNA damage, protein secretion and gene expression of IL6 and IL8 in primary human coronary artery endothelial cells (HCAEC) and human internal thoracic artery endothelial cells (HITAEC) in vitro exposed to 500 ng/mL MMC. We observed an increase in levels of cytogenetic damage (micronuclei, nucleoplasmic bridges and nuclear buds) in MMC-treated cells compared to control cells. After 6 h incubation with MMC, both HCAEC and HITAEC displayed a decrease in IL8 concentration and the mRNA level of IL6 and IL8 compared to control cells. Removal of MMC from cultures after 6 h followed by 24 h incubation of cells in complete growth media led to a sharp increase in secretion and gene expression of the studied cytokines in both HCAEC and HITAEC. Moreover, HCAEC were more susceptible to mutagenic exposure compared to HITAEC. These findings suggest that the MMC-induced genotoxic stress in endothelial cells derived from different arteries is associated with differential secretion and gene expression of proinflammatory cytokines IL6 and IL8.

Polymorphisms in DNA repair genes in lung cancer patients living in a coal-mining region.

Air pollutants and ionizing radiation are well-known carcinogens involved in the pathogenesis of lung cancer, and residents of coal-mining regions are exposed routinely to these agents. Polymorphisms in DNA repair genes may be associated with an increased risk of malignant transformation. We investigated associations between the risk of lung cancer in residents of the coal-mining region and polymorphisms in the genes APEX1 (rs1130409), hOGG1 (rs1052133), XRCC1 (rs25489, rs25487), XRCC2 (rs3218536), XRCC3 (rs861539), ADPRT/PARP1 (rs1136410), XPD/ERCC2 (rs13181), XPG/ERCC5 (rs17655), XPC (rs2228001), ATM (rs1801516), and NBS1 (rs1805794). Three hundred and forty residents of the Kemerovo Region (a coal-mining region of western Siberia) were lung cancer patients exposed to air pollutants and ionizing radiation (case) and 335 were healthy donors (control). Genotyping was performed by real-time PCR and allele-specific PCR. We discovered that polymorphisms in the XPD gene in men [log-additive model: odds ratio (OR) = 1.64, 95% confidence interval (CI): 1.17-2.31], the ATM gene in women and nonsmokers (codominant model: OR = 0.11, 95% CI: 0.02-0.49 and OR = 0.25, 95% CI: 0.08-0.72, respectively), the APEX1 gene for smokers (recessive model: OR = 2.55, 95% CI: 1.34-4.85), and the NBS1 gene for those who work in the coal industry (overdominant model: OR = 0.40, 95% CI: 0.21-0.75) are associated with an increased risk of lung cancer. Using the multifactor dimensionality reduction method, we found a model of gene-gene interactions associated with the risk of lung cancer: NBS1 (rs1805794)-XRCC1 (rs25487)-hOGG1 (rs1052133)-XPG (rs17655). These results indicate an association between combinations of polymorphisms in the studied genes and the risk of lung cancer in residents of a coal-mining region.

Associations of polymorphisms in the cytokine genes IL1ß (rs16944), IL6 (rs1800795), IL12b (rs3212227) and growth factor VEGFA (rs2010963) with anthracosilicosis in coal miners in Russia and related genotoxic effects.

Anthracosilicosis (AS), a prevalent form of pneumoconiosis among coal miners, results from the accumulation of carbon and silica in the lungs from inhaled coal dust. This study investigated genotoxic effects and certain cytokine genes polymorphic variants in Russian coal miners with АS. Peripheral leukocytes were sampled from 129 patients with AS confirmed by X-ray and tissue biopsy and from 164 asymptomatic coal miners. Four single-nucleotide polymorphisms were genotyped in the extracted DNA samples: IL1ß T-511C (rs16944), IL6 C-174G (rs1800795), IL12b A1188C (rs3212227) and VEGFA C634G (rs2010963). Genotoxic effects were assessed by the analysis of chromosome aberrations in cultured peripheral lymphocytes. The mean frequency of chromatid-type aberrations and chromosome-type aberrations, namely, chromatid-type breaks and dicentric chromosomes, was found to be higher in AS patients [3.70 (95% confidence interval {CI}, 3.29-4.10) and 0.28 (95% CI, 0.17-0.38)] compared to the control group [2.41 (95% CI, 2.00-2.82) and 0.09 (95% CI, 0.03-0.15)], respectively. IL1ß gene T/T genotype (rs16944) was associated with AS [17.83% in AS patients against 4.35% in healthy donors, odds ratio = 4.77 (1.88-12.15), P < 0.01]. A significant increase in the level of certain chromosome interchanges among AS donors is of interest because such effects are typical for radiation damage and caused by acute oxidative stress. IL1ß T allele probably may be considered as an AS susceptibility factor among coal miners.

Chromosome aberrations in peripheral blood lymphocytes of lung cancer patients exposed to radon and air pollution.

Lung cancer is one of the most common forms of cancer. The aim of this study was to validate chromosome aberrations in peripheral blood lymphocytes of lung cancer patients living in a region with high air pollution and increased background radon levels as a biomarker of cancer risk. A total of 417 lung cancer patients and 468 control participants were analysed using a chromosome aberration assay in peripheral blood lymphocytes. The results showed that chromatid-type aberrations (2.26±1.58 vs. 1.60±1.58) and chromosome-type aberrations (CSAs) (0.96±1.36 vs. 0.42±0.70) in lung cancer patients were increased significantly in comparison with the controls. The most significant two-fold increase was detected for CSAs (nonsmoking patients: 0.84±1.54 vs. 0.41±0.73%, smoking patients: 0.99±1.31 vs. 0.44±0.67%). The frequency of dicentric and ring chromosomes, double minutes and rogue cells was significantly higher (P=0.002, 0.00002, 0.01, 0.0007) in the lung cancer patients. As both analysed groups lived in the same environment, our results show that increased radon levels were not the only source for the detected genome damage. Using binomial logistic regression, the estimated odds ratios and 95% confidence intervals adjusted for the main confounders (smoking, occupational exposure, age) were 1.31 (1.20-1.40) for chromatid-type aberrations, 1.28 (1.17-1.33), and 1.68 (1.49-1.88) for CSAs. It may be suggested that lung cancer patients show a significant increase in genome damage that may be caused by an interplay between exposure and individual low capacity of DNA repair, leading to genome instability.

Association of DNA repair gene polymorphisms with genotoxic stress in underground coal miners.

In underground coal mining, numerous harmful substances and ionising radiation pose a major threat to the occupational safety and health of workers. Because cell DNA repair machinery eliminates genotoxic stress conferred by these agents, we examined whether single nucleotide polymorphisms in hOGG1 (rs1052133), XRCC1 (rs25487), ADPRT (rs1136410), XRCC4 (rs6869366) and LIG4 (rs1805388) genes modulate the genotoxic damage assessed by the cytokinesis-block micronucleus assay in lymphocytes from 143 underground coal miners and 127 healthy non-exposed males. We also analyzed models of gene-gene interactions associated with increased cytogenetic damage in coal miners and determined 'protective' and 'risk' combinations of alleles. We showed that miners with the G/G genotype of the hOGG1 (rs1052133) gene had a significantly increased frequency of binucleated lymphocytes with micronuclei (13.17‰, 95% CI = 10.78-15.56) compared to the C/C genotype carriers (10.35‰, 95% CI = 9.59-11.18). In addition, in the exposed group this indicator was significantly increased in carriers of the T/T genotype of the LIG4 (rs1805388) gene compared to miners harbouring the C/T genotype (13.00‰, 95% CI = 10.96-15.04 and 9.69‰, 95% CI = 8.32-11.06, respectively). Using the multifactor dimensionality reduction method, we found the three-locus model of gene-gene interactions hOGG1 (rs1052133) × ADPRT (rs1136410) × XRCC4 (rs6869366) associated with high genotoxic risk in coal miners. These results indicate that the studied polymorphisms and their combinations are associated with cytogenetic status in miners and may be used as molecular predictors of occupational risks in underground coal mines.

Polymorphisms of GSTM1, GSTT1, GSTP1 genes and chromosomal aberrations in lung cancer patients.

PURPOSE: To study the potential links between genetic polymorphisms in the GSTT1, GSTM1, GSTP1 genes and the frequency of chromosomal aberrations (CAs) in lung cancer patients and healthy residents in Russian Federation. METHODS: 200 cells in well-spread metaphase with 46 chromosomes were examined for 353 newly diagnosed lung cancer patients (males) who received medical treatment in the Kemerovo Regional Oncology Center (Kemerovo, Russian Federation), and 300 healthy males from Kemerovo, Russian Federation. The polymorphisms of the GSTM1 del and GSTT1 del genes were analysed by multiplex PCR. Genotyping of the polymorphic variants in the GSTP1 (A313G, T341C) gene was performed using Real-time PCR with competing TaqMan probes complementary to the polymorphic DNA sites. The data analysis was performed using software STATISTICA 8.0 (StatSoft Inc., USA). RESULTS: We discovered that a GSTM1 del polymorphism increases the frequency of chromosomal damage in smoking patients with lung cancer, a general group of lung cancer patients, donors with non-small cell lung cancer and patients in the latest stages of the malignant process. The synergetic effects of occupational exposure and the malignant process can induce some modifications in the cytogenetic status in lung cancer patients harbouring the GSTM1 del polymorphism. CONCLUSIONS: CAs in peripheral blood lymphocytes can be used as biomarkers of the early biological effects of exposure to genotoxic carcinogens and may predict future cancer incidence in several epidemiologic studies. Genetic changes in genes encoding phase II detoxification enzymes are linked to decreases in the metabolic detoxification of environmentally derived genotoxic carcinogens.

Assessment of DNA damage in underground coal miners using the cytokinesis-block micronucleus assay in peripheral blood lymphocytes.

Coal miners are exposed to coal dust, containing mineral particles, inorganic compounds and polycyclic aromatic hydrocarbons, and to ionizing radiation. These factors can induce oxidative stress and promote inflammation that leads to DNA damage. The aim of this investigation is to analyse the degree of DNA damage in miners working in underground coal mines in Kemerovo Region (Russian Federation) using the cytokinesis-block micronucleus assay (CBMN) in peripheral blood lymphocytes. The exposed group included 143 coal miners (mean age = 50.11±7.36 years; mean length of service in coal mining conditions = 23.26±9.66 years). As a control group, we have used venous blood extracted from 127 healthy non-exposed men. The mean age in this group was 47.67±8.45 years. We have discovered that coal miners are characterized by a significant increase in the frequency of binucleated lymphocytes with micronuclei (MN), nucleoplasmic bridges (NPBs) and protrusions (NBUDs) compared to non-exposed donors. In addition, we report, for the first time, a reduction of cell proliferation in a cohort of coal miners. These data are evidence of the genotoxic and cytostatic effects of occupational harmful factors of the coal mining industry. No correlation between the level of chromosome damage and age, smoking status or length of service in coal mining conditions were discovered. We suggest that the CBMN assay would be useful in biomonitoring studies to monitor hygiene and prevention strategies in occupational settings in coal mining countries.

DNA excision repair and double-strand break repair gene polymorphisms and the level of chromosome aberration in children with long-term exposure to radon.

PURPOSE: To study polymorphic variants of repair genes in people affected by long-term exposure to radon. The chromosome aberration frequency in peripheral blood lymphocytes was used as the biological marker of genotoxicity. MATERIALS AND METHODS: Genotyping of 12 single nucleotide polymorphisms in DNA repair genes (APE, XRCC1, OGG1, ADPRT, XpC, XpD, XpG, Lig4 and NBS1) was performed in children with long-term resident exposure to radon. Quantification of the aberrations was performed using light microscopy. RESULTS: The total frequency of aberrations was increased in carriers of the G/G genotype for the XpD gene (rs13181) polymorphism in recessive model confirmed by the results of ROC-analysis ('satisfactory predictor', AUC = 0.609). Single chromosome fragments frequency was increased in carriers of the G/G genotype in comparison with the T/T genotype. In respect to the total frequency of aberrations, the G/G genotype for the XpG gene (rs17655) polymorphism was also identified as a 'satisfactory predictor' (AUC = 0.605). Carriers of the T/C genotype for the ADPRT gene (rs1136410) polymorphism were characterized by an increased level of single fragments relative to the T/T genotype. CONCLUSION: The relationships with several types of cytogenetic damage suggest these three SNP (rs13181, rs17655 and rs1136410) may be considered radiosensitivity markers.

Modifying influence of occupational inflammatory diseases on the level of chromosome aberrations in coal miners.

Coal miners are exposed to a wide range of genotoxic agents that can induce genome damage. In addition, miners are characterised by a high risk of the initiation of different occupational inflammatory as well as non-inflammatory diseases. The aim of this investigation is to analyse the modifying influence of occupational pulmonary inflammatory diseases on the level of chromosome aberrations (CAs) in miners working in underground coal mines in Kemerovo Region (Russian Federation). The study group included 90 coal miners with the following pulmonary diseases: chronic dust-induced bronchitis (CDB) and coal-workers' pneumoconiosis (CWP) (mean age = 53.52±2.95 years; mean work experience in coal-mining conditions = 27.70±3.61 years). As a population control (control 1), we have used venous blood extracted from 124 healthy unexposed men. The mean age in this group was 50.92±4.56 years. Control 2 was the venous blood extracted from 42 healthy coal miners (mean age = 51.56±6.38 years; mean work experience in coal-mining conditions = 25.43±8.14 years). We have discovered that coal miners are characterised by an increased general level of CAs as well as an increased frequency of several types of CAs. The significant increase in the frequency of aberration per 100 cells and aberration of chromosome type was discovered in the group of pulmonary disease patients (study group). No correlations of the level of chromosome damage with age, smoking status and work experience in coal-mining conditions were discovered.

Assessing the level of chromosome aberrations in peripheral blood lymphocytes in long-term resident children under conditions of high exposure to radon and its decay products.

In this study, the frequency and spectrum of chromosomal aberrations were analysed in samples of peripheral blood from 372 (mean age = 12.24 ± 2.60 years old) long-term resident children in a boarding school (Tashtagol city, Kemerovo Region, Russian Federation) under conditions of high exposure to radon and its decay products. As a control group, we used blood samples from people living in Zarubino village (Kemerovo Region, Russian Federation). We discovered that the average frequencies of single and double fragments, chromosomal exchanges, total number of aberrations, chromatid type, chromosome type and all types of aberrations were significantly increased in the exposed group. This is evidence of considerable genotoxicity to children living under conditions of high exposure to radon compared to children living under ecological conditions without increased radon radiation.