Environmental pollutants are considered as a cause of tumorigenesis, but approaches to assess their risk of causing tumors remain insufficient. As an alternative approach, the adverse outcome pathway (AOP) framework is used to assess the risk of tumors caused by environmental pollutants. Arsenic is a pollutant associated with lung cancer, but early assessment of lung cancer risk is lacking. Therefore, we applied the AOP framework to arsenic-induced lung cancer. A systematic review revealed increased risks of lung cancer following exposure to a range of arsenic concentrations in drinking water (OR = 1.83, 95 % CI = 1.46-2.30). We obtained, from public databases, genes related to risk of arsenic-induced lung cancer. Then, Cox and LASSO regressions were used to screen target genes from the risk genes. Subsequently, target genes, phenotypes, and pathways were used to construct the computational AOP network, which was determined by Cytoscape to have 156 edges and 45 nodes. Further, target genes, phenotypes, and pathways were used as molecular initiating events and key events to construct the AOP framework depending on upstream and downstream relationships. In the AOP framework, by Weight of Evidence, arsenic exposure increased levels of EGFR, activated the PI3K/AKT pathway, regulated cell proliferation by promoting the G1/S phase transition, and caused generation of lung cancers. External validation was achieved through arsenite-induced, malignant transformed human bronchial epithelial (HBE) cells. Overall, these results, by integration into existing data to construct an AOP framework, provide insights into the assessment of lung cancer risk for arsenic exposure. Special attention needs to be focused on populations with low-dose arsenic exposure.

Arsenic is a toxic agent that causes respiratory damage. Long non-coding RNAs (lncRNAs) are non-coding transcripts that adsorb specific miRNAs and regulate biological processes of human diseases. N6-Methyladenosine (m6A) is an internal modification of RNAs. However, there are few reports about lncRNAs and m6A modifications as co-regulators of pulmonary fibrosis. For 6 months, C57BL/6 mice were given water containing 0, 10, or 20 ppm arsenite. meRIP-seq and lncRNA-seq analyses showed that the m6A levels of the lncRNA E230001N04Rik were higher, and the levels of E230001N04Rik itself were lower in the high-dose arsenite group than in the controls. Murine lung epithelial 12 (MLE12) cells, exposed to 8 μM arsenite for 8 passages, had elevated METTL3 and miR-20b-3p and low E230001N04Rik. Arsenite induced cellular senescence, as demonstrated by secretion of factors related to the senescence-associated secretory phenotype (SASP). Arsenite-treated MLE12 cells co-cultured with primary lung fibroblasts (PLFs) caused myofibroblast differentiation. These data show that METTL3 reduces E230001N04Rik expression via controlling its m6A levels, which regulate miR-20b-3p and mediate the senescence of alveolar epithelial cells (AECs). Thereby, E230001N04Rik is involved in the arsenite-induced myofibroblast differentiation and in pulmonary fibrosis. These observations provide a prospective mechanism for chronic pulmonary disease caused by arsenite.

Arsenic exposure is connected with lung toxicity and is related to lung fibrotic changes. Idiopathic pulmonary fibrosis (IPF) is characterized by extracellular matrix (ECM) deposition. Various genetic mechanisms and environmental factors induce or exacerbate pulmonary fibrosis. Collagen synthesis induced by sodium arsenite (NaAsO2) is closely associated with IPF. Fibroblasts tend to fine-tune their metabolic networks to support their synthetic requirements in response to environmental stimuli. Alterations in metabolism have an influential role in the pathogenesis of IPF. However, it is unclear how arsenic affects the metabolism in IPF. The urea cycle (UC) is needed for collagen formation, which provides adequate levels of proline (Pro) for biosynthesis of collagen. Carbamoyl phosphate synthetase 1 (CPS1) converts the ammonia to carbamoyl phosphate, which controls the first reaction of the UC. We show that, in arsenite-exposed mice, high amounts of ammonia in the lung microenvironment promotes the expression levels of CPS1 and the Pro metabolism. Reduction of ammonia and CPS1 ablation inhibit collagen synthesis and ameliorate IPF phenotypes induced by arsenite. This work takes advantage of multi-omics data to enhance understanding of the underlying pathogenic mechanisms, the key molecules and the complicated cellular responses to this pollutant, which provide a target for the prevention of pulmonary fibrosis caused by arsenic.

It is well known that 5-methylcytosine (m5C) is involved in variety of crucial biological processes in cancers. However, its biological roles in lung adenocarcinoma (LAUD) remain to be determined. The LUAD samples were used to assess the clinical value of NOP2/Sun RNA Methyltransferase 2 (NSUN2). Dot blot was used to determine global m5C levels. ChIP and dual-luciferase assays were performed to investigate the MYC-associated zinc finger protein (MAZ)-binding sites in NSUN2 promoter. RNA-seq was used to explore the downstream molecular mechanisms of NSUN2. Dual luciferase reporter assay, m5C-RIP-qPCR, and mRNA stability assay were conducted to explore the effect of NSUN2-depletion on target genes. Cell viability, transwell, and xenograft mouse model were designed to demonstrate the characteristic of NSUN2 in promoting LUAD progression. The m5C methyltransferase NSUN2 was highly expressed and caused elevated m5C methylation in LUAD samples. Mechanistically, MAZ positively regulated the transcription of NSUN2 and was related to poor survival of LUAD patients. Silencing NSUN2 decreased the global m5C levels, suppressed proliferation, migration and invasion, and inhibited activation of PI3K-AKT signaling in A549 and SPAC-1 cells. Phosphoinositide-3-Kinase Regulatory Subunit 2 (PIK3R2) was upregulated by NSUN2-mediated m5C methylation by enhancing its mRNA stabilization and activated the phosphorylation of the PI3K-AKT signaling. The present study explored the underlying mechanism and biological function of NSUN2-meditated m5C RNA methylation in LUAD. NSUN2 was discovered to facilitate the malignancy progression of LUAD through regulating m5C modifications to stabilize PIK3R2 activating the PI3K-AKT signaling, suggesting that NSUN2 could be a novel biomarker and promising therapeutic target for LUAD patients.

Lung cancer is a commonly diagnosed disease worldwide, with non-small cell lung cancers (NSCLCs) accounting for ≈ 85% of cases. Cigarette smoke is an environmental exposure promoting progression of NSCLC, but its role is poorly understood. This study reports that smoking-induced accumulation of M2-type tumor-associated macrophages (M2-TAMs) surrounding NSCLC tissues promotes malignancy. Specifically, extracellular vesicles (EVs) from cigarette smoke extract (CSE)-induced M2 macrophages promoted malignancy of NSCLC cells in vitro and in vivo. circEML4 in EVs from CSE-induced M2 macrophages is transported to NSCLC cells, where it reduced the distribution of ALKBH5 in the nucleus by interacting with Human AlkB homolog H5 (ALKBH5), resulting in elevated N6-methyladenosine (m6A) modifications. m6A-seq and RNA-seq revealed suppressor of cytokine signaling 2 (SOCS2)-mediated activation of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway by regulating m6A modification of SOCS2 via ALKBH5. Down-regulation of circEML4 in EVs from CSE-induced M2 macrophages reversed EVs-enhanced tumorigenicity and metastasis in NSCLC cells. Furthermore, this study found that smoking patients showed an increase in circEML4-positive M2-TAMs. These results indicate that smoking-induced M2-TAMs via circEML4 in EVs promote the NSCLC progression through ALKBH5-regulated m6A modification of SOCS2. This study also reveals that circEML4 in EVs from TAMs acts as a diagnostic biomarker for NSCLC, especially for patients with smoking history.

Arsenic is a recognized environmental pollutant naturally occurring in aquifers through geological processes. Toxicological studies have revealed that liver is the main target organ harmed by arsenic exposure. However, systematic studies of non-alcoholic fatty liver disease (NAFLD) are not comprehensive, and information regarding threats and risk assessment remains insufficient. This research aimed to examine the association between arsenic exposure and NAFLD and uncover the role of molecular initiating events and key events in disease development using the Adverse Outcome Pathway (AOP). Data from 8,104 adults in the National Health and Nutrition Examination Survey were used to explore the relationship between urinary arsenic and NAFLD. In a logistic regression model, urinary inorganic arsenic levels positively correlated with NAFLD (odds ratio = 1.12, 95 % confidence interval = 1.07-1.16). Subsequently, to gain a deeper understanding of arsenic-induced NAFLD, an AOP framework was constructed, revealing that arsenic exposure led to elevate levels of TNF-α, which regulated the NF-κB pathway and led to hepatic lipid deposition, causing NAFLD. This AOP was assessed as "high" according to the Organization for Economic Co-operation and Development users' handbook, and in vitro and in vivo models validated the AOP framework. In summary, this study highlights the potential mechanisms of arsenic-induced NAFLD. We combined the AOP with classical toxicological approaches with a view of establishing, rapidly and accurately, the lowest level at which environmental arsenic exposure can have adverse effects on the body, thereby contributing to risk assessment strategies for arsenic exposure through iterative and animal modeling at the population level.

As epigenetic modifications, lactylation and N6-methyladenosine (m6A) have attracted wide attention. Arsenite is an environmental pollutant that has been proven to induce idiopathic pulmonary fibrosis (IPF). However, the molecular mechanisms of lactylation and m6A methylation are unclear in arsenite-related IPF (As-IPF). In view of the limited understanding of molecular mechanism of m6A and lactylation in As-IPF, MeRIP-seq, RNA-seq and ChIP-seq were analyzed to verify the target gene regulated by m6A and H3K18 lactylation (H3K18la). We found that, for As-IPF, the global levels of m6A, levels of YTHDF1 and m6A-modified neuronal protein 3.1 (NREP) were elevated in alveolar epithelial cells (AECs). The secretion levels of TGF-β1 were increased via YTHDF1/m6A/NREP, which promoted the fibroblast-to-myofibroblast transition (FMT). Further, extracellular lactate from myofibroblasts elevated levels of the global lactylation (Kla) and H3K18la via the lactate monocarboxylate transporter 1 (MCT1), and, in AECs, H3K18la facilitated the transcription of Ythdf1. This report highlights the role of crosstalk between AECs and myofibroblasts via lactylation and m6A and the significance of H3K18la regulation of YTHDF1 in the progression of As-IPF, which may be useful for finding effective therapeutic targets.

Cigarette smoking killed about 8 million people every year and promoted non-small cell lung cancer (NSCLC). We investigated the molecular mechanism of smoking-promoted NSCLC progression. Relative to non-smokers, NSCLC patients who were smokers had a higher tumor malignancy. For NSCLC cells, cigarette smoke extract (CSE) increased levels of HIF-1α, METTL3, Cyclin E1, and CDK2 and promoted the G1/S transition, which promoted cell proliferation. Down-regulation HIF-1α or METTL3 reversed these effects. meRIP-seq and RNA-seq revealed the m6A modification in Cyclin Dependent Kinase 2 Associated Protein 2 (CDK2AP2) mRNA as the key downstream target. Further, for NSCLC cells exposed to CSE, HIF-1α activated METTL3 transcription. Xenografts in nude mice demonstrated that HIF-1α via METTL3 participated in tumor growth. In NSCLC tissues of smokers, protein levels of HIF-1α and METTL3 were higher, and levels of CDK2AP2 were lower. In conclusion, HIF-1α via METTL3 regulation of the m6A modification of CDK2AP2 mRNA drives smoking-induced progression of NSCLC through promoting cell proliferation. This is a previously unknown molecular mechanism for smoking-induced NSCLC progression. The results have potential value for treatment of NSCLC, especially for patients who smoke.

Arsenic compounds are toxins that are widely distributed in the environment. Chronic exposure to low levels of these compounds can cause hepatic fibrosis and other damage. Th17 differentiation of CD4+ T cells and the secretion of IL-17 activates hepatic stellate cells (HSCs), which are involved in hepatic fibrosis, but their mechanisms in arsenic-induced hepatic fibrosis are unclear. We found, in arsenite-induced fibrotic livers of mice, increases of CD4+ T cell infiltration, Th17 cell nuclear receptor retinoic acid receptor-related orphan receptor γt (RORγt), and secretion of the pro-inflammatory cytokine IL-17. There were also elevated levels of the lncRNA, HOTAIR. For Jurkat cells, arsenite elevated levels of HOTAIR and protein levels of RORγt and IL-17A, decreased miR‐17‐5p, promoted Th17 cell differentiation, and released IL-17. The culture medium of arsenite-treated Jurkat cells activated LX-2 cells. Down-regulation of HOTAIR or up-regulation of miR‐17‐5p blocked arsenite-induced Th17 cell differentiation, which inhibited the LX-2 cell activation. However, down-regulation of HOTAIR and miR‐17‐5p reversed this inhibitory effect. For mice, silencing of HOTAIR diminished the hepatic levels of RORγt and IL-17A and alleviated arsenite-induced hepatic fibrosis. These results demonstrate that, for CD4+ T cells, arsenite promotes RORγt-mediated Th17 cell differentiation through HOTAIR down-regulation of miR‐17‐5p, and increases the secretion of cytokine IL-17A, which activates HSCs; the activated HSCs facilitate hepatic fibrosis. The findings reveal a new mechanism and a potential therapeutic target for arsenite-induced hepatic fibrosis.

Elevated levels of arsenic may be present in groundwater, and long-term exposure to arsenic increases hepatic fibrosis. T helper 2 (Th2) cells are involved in the fibrotic cascade, and cell metabolism is a regulatory factor participating in CD4+ T cell differentiation and function. However, the mechanism for Th2 cell regulation of arsenite-induced hepatic fibrosis is not fully understood. In present study, for arsenite-fed mice, activated hepatic stellate cells may be involved in the infiltration of CD4+ T cells, accompanied by up-regulation of GATA3, a transcription factor, and IL-13, the major Th2 cytokine. Exposed to arsenite, Jurkat cells had increased aerobic glycolysis to promote the cell cycle and cell proliferation. Further, this process elevated levels of marker molecules, including those of the Th2 paradigm characterized by GATA3, IL-4, and IL-13. LX-2 cells were activated when treated with culture medium from Jurkat cells exposed to arsenite. miR-21 may be a therapeutic target for arsenite-induced hepatic fibrosis. In vitro, miR-21 knock-down caused inhibition of the PTEN/PI3K/AKT pathway induced by arsenite. It also reversed the elevated glycolysis and the accelerated cell cycle and cell proliferation. Indeed, this alteration led to diminished expression of GATA3, IL-4, and IL-13 in T cells differentiated under Th2 conditions, which inhibits activation of LX-2 cells. Consistent with the results in vitro, miR-21 knock-out in mice reversed hepatic fibrosis and attenuated the levels of GATA3 and IL-13 induced by arsenite. These findings indicate that miR-21 regulates the glycolysis of CD4+ T cells through the PTEN/PI3K/AKT pathway to accelerate the cell cycle, thereby facilitating CD4+ T cell polarization toward Th2 and releasing the fibrogenic factor IL-13, which participates in arsenite-associated hepatic fibrosis. Inhibition of Th2 polarization of CD4+T cells or miR-21 could be a therapeutic strategy to combat hepatic fibrosis caused by exposure to arsenic.

As an environmental toxicant, arsenic causes damage to various organs and systems of the body and has attracted worldwide attention. It is well-known that exposure to arsenic can induce pulmonary fibrosis, but the molecular mechanisms are elusive. Glycolysis is involved in the process of various diseases, including pulmonary fibrosis. Extracellular vehicles (EVs) are mediators of cell communication through transporting miRNAs. The potential of miRNAs in EVs as liquid biopsy biomarkers for various diseases has been reported, and they have been applied in clinical diagnoses. In the present investigation, we focused on the roles and mechanisms of miR-21 in EVs on arsenic-induced glycolysis and pulmonary fibrosis through experiments with human populations, experimental animals, and cells. The results for arsenicosis populations showed that the serum levels of hydroxyproline, lactate, and EVs-miRNAs were elevated and that EVs-miR-21 levels were positively related to the levels of hydroxyproline and lactate. For mice, chronic exposure to arsenite led to high levels of miR-21, AKT activation, elevated glycolysis, and pulmonary fibrosis; however, these effects were blocked by the depletion of miR-21 in miR-21 knockout (miR-21KO) mice. After MRC-5 cells were co-cultured with arsenite-treated HBE cells, the levels of miR-21, AKT activation, glycolysis, and myofibroblast differentiation were enhanced, effects that were blocked by reducing miR-21 and by inhibiting the EVs in HBE cells. The down-regulation of PTEN in MRC-5 cells and primary lung fibroblasts (PLFs) reversed the blocking effect of inhibiting miR-21 in HBE cells. Thus, miR-21 down-regulates PTEN and promotes glycolysis via activating AKT, which is associated with arsenite-induced myofibroblast differentiation and pulmonary fibrosis. Our results provide a new approach for the construction of clinical diagnosis technology based on analysis of the mechanism of arsenite-induced pulmonary fibrosis.

Chronic obstructive pulmonary disease (COPD) affects the health of more than 300 million people worldwide; at present, there is no effective drug to treat COPD. Smoking is the most important risk factor, but the molecular mechanism by which smoking causes the disease is unclear. The senescence of lung epithelial cells is related to development of COPD. Regulation of miRNAs is the main epigenetic mechanism related to aging. β-Galactose staining showed that the lung tissues of smokers have a higher degree of cellular senescence, and the expression of miR-125a-5p is high. This effect is obvious for smokers with COPD/emphysema, and there is a negative correlation between miR-125a-5p levels and values for forced expiratory volume in one second (FEV1)/forced vital capacity (FVC). After Balb/c mice were chronically exposed to various concentrations of cigarette smoke (CS), plethysmography showed that lung function was impaired, lung tissue senescence was increased, and the senescence-associated secretory phenotype (SASP) in bronchoalveolar lavage fluid was increased. For mouse lung epithelial (MLE)-12 cells treated with cigarette smoke extract (CSE), Sp1 and SIRT1 levels were low, HIF-1α acetylation levels were high, and cell senescence and secretion of SASP factors were elevated. Down-regulation of miR-125a-5p or up-regulation of Sp1 reversed these effects. In addition, compared with mice exposed to CS, knockdown of miR-125a-5p reduced lung epithelial cell senescence and COPD/emphysema. Therefore, in smoking-induced COPD, elevated miR-125a-5p participates in the senescence of lung epithelial cells through Sp1/SIRT1/HIF-1α. These findings provide evidence related to the pathogenesis of COPD/emphysema caused by chronic smoking.

Long-term exposure to arsenic, a widely distributed environmental toxicant, may result in damage to various organs, including the liver. Mice exposed chronically to arsenite developed hepatic damage, inflammation, and fibrosis, as well as increased levels of microRNA-21 (miR-21) and hypoxia-inducible factor (HIF)-1 alpha. The levels of miR-21 and HIF-1 alpha were also enhanced in primary hepatocytes and L-02 cells exposed to arsenite. The culture media from these cells induced the activation of hepatic stellate cells (HSCs), as demonstrated by up-regulation of the protein levels of alpha-smooth muscle actin (alpha-SMA) and collagen1A2 (COL1A2) and by increased activity in gel contractility assays. For L-02 cells, knockdown of miR-21 blocked the arsenite-induced up-regulation of HIF-1 alpha and vascular endothelial growth factor (VEGF), which prevented the activation of LX-2 cells induced by medium from arsenite-exposed L-02 cells. However, these effects were reversed by down-regulation of von Hippel Lindau protein (pVHL). In arsenite-treated L-02 cells, miR-21 knockdown elevated the levels of ubiquitination and accelerated the degradation of HIF-1 alpha via pVHL. In the livers of miR-21(-/-) mice exposed chronically to arsenite, there were less hepatic damage, lower fibrosis, lower levels of HIF-1 alpha and VEGF, and higher levels of pVHL than for wild-type mice. In summary, we propose that miR-21, acting via the HIF-1 alpha/VEGF signaling pathway, is involved in arsenite-induced hepatic fibrosis through mediating aberrant cross-talk of hepatocytes and HSCs. The findings provide evidence relating to the pathogenesis of hepatic fibrosis induced by exposure to arsenic. (C) 2020 Elsevier Ltd. All rights reserved.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Angiogenesis is the major hallmark in NSCLC. So, further elucidation of molecular mechanisms underlying the angiogenesis of NSCLC is urgently needed. Here, we found that microRNA-206 (miR-206) decreased the angiogenic ability in NSCLC via inhibiting the 14-3-3ζ/STAT3/HIF-1α/VEGF pathway. Briefly, 14-3-3ζ bond with phosphorylated-STAT3, and in turn, elevated the expression of HIF-1α. Then, by enhancing the recruitment of HIF-1α to VEGF promoter, 14-3-3ζ increased the angiogenesis. However, miR-206 decreased the angiogenesis by targeting 14-3-3ζ, and inhibiting the STAT3/HIF-1α/VEGF pathway. In NSCLC cell xenograft model, either overexpression of miR-206 or inhibition of 14-3-3ζ inhibited the STAT3/HIF-1α/VEGF pathway and decreased the tumor growth and angiogenesis. Furthermore, there was a negative correlation between miR-206 and 14-3-3ζ in NSCLC specimens. NSCLC patients with low expressions of miR-206 but high expressions of 14-3-3ζ had the worst survival. Collectively, our findings provided the underlying mechanisms of miR-206/14-3-3ζ in tumor growth and angiogenesis, and implicated miR-206 and 14-3-3ζ as potential therapeutic targets for NSCLC.