Hexavalent chromium exposure activates the non-canonical nuclear factor kappa B pathway to promote immune checkpoint protein programmed death-ligand 1 expression and lung carcinogenesis.

Lung cancer is the leading cause of cancer-related death worldwide; however, the mechanism of lung carcinogenesis has not been clearly defined. Chronic exposure to hexavalent chromium [Cr(VI)], a common environmental and occupational pollutant, causes lung cancer, representing an important lung cancer etiology factor. The mechanism of how chronic Cr(VI) exposure causes lung cancer remains largely unknown. By using cell culture and mouse models and bioinformatics analyses of human lung cancer gene expression profiles, this study investigated the mechanism of Cr(VI)-induced lung carcinogenesis. A new mouse model of Cr(VI)-induced lung carcinogenesis was developed as evidenced by the findings showing that a 16-week Cr(VI) exposure (CaCrO4, 100 µg per mouse once per week) via oropharyngeal aspiration induced lung adenocarcinomas in male and female A/J mice, whereas none of the sham-exposed control mice had lung tumors. Mechanistic studies revealed that chronic Cr(VI) exposure activated the non-canonical NFκB pathway through the long non-coding RNA (lncRNA) ABHD11-AS1/deubiquitinase USP15-mediated tumor necrosis factor receptor-associated factor 3 (TRAF3) down-regulation. The non-canonical NFκB pathway activation increased the interleukin 6 (IL-6)/Janus kinase (Jak)/signal transducer and activator of transcription 3 (Stat3) signaling. The activation of the IL-6/Jak signaling axis by Cr(VI) exposure not only promoted inflammation but also stabilized the immune checkpoint molecule programmed death-ligand 1 (PD-L1) protein in the lungs, reducing T lymphocyte infiltration to the lungs. Given the well-recognized critical role of PD-L1 in inhibiting anti-tumor immunity, these findings suggested that the lncRNA ABHD11-AS1-mediated non-canonical NFκB pathway activation and PD-L1 up-regulation may play important roles in Cr(VI)-induced lung carcinogenesis.

Transcriptome Analysis Reveals Anti-Cancer Effects of Isorhapontigenin (ISO) on Highly Invasive Human T24 Bladder Cancer Cells.

Bladder cancer, the most common malignancy of the urinary tract, has a poor overall survival rate when the tumor becomes muscle invasive. The discovery and evaluation of new alternative medications targeting high-grade muscle invasive bladder cancer (MIBC) are of tremendous importance in reducing bladder cancer mortality. Isorhapontigenin (ISO), a stilbene derivative from the Chinese herb Gnetum cleistostachyum, exhibits a strong anti-cancer effect on MIBCs. Here, we report the whole transcriptome profiling of ISO-treated human bladder cancer T24 cells. A total of 1047 differentially expressed genes (DEGs) were identified, including 596 downregulated and 451 upregulated genes. Functional annotation and pathway analysis revealed that ISO treatment induced massive changes in gene expression associated with cell movement, migration, invasion, metabolism, proliferation, and angiogenesis. Additionally, ISO treatment-activated genes involved in the inflammatory response but repressed genes involved in hypoxia signaling, glycolysis, the actin cytoskeleton, and the tumor microenvironment. In summary, our whole transcriptome analysis demonstrated a shift in metabolism and altered actin cytoskeleton in ISO-treated T24 cells, which subsequently contribute to tumor microenvironment remodeling that suppresses tumor growth and progression.

A combination of three antioxidants decreases the impact of rural particulate pollution in Normal human keratinocytes.

OBJECTIVE: It is well established that exposure of human skin to airborne pollution, particularly in the form of particulate matter sized 2.5 µm (PM2.5 ), is associated with oxidative stress, DNA damage and inflammation, leading to premature signs of skin aging. Because much of the damage results from oxidative stress, we examined the effects of a topical composition containing three antioxidants in an in vitro model system to assess the potential for amelioration of premature aging. The use of multiple antioxidants was of interest based on the typical composition of therapeutic skincare products. It is important to determine the efficacy of multiple antioxidants together and develop a short-term assay for larger scale efficacy testing. METHODS: Normal human epidermal keratinocytes were exposed to a rural-derived source of PM2.5 in the presence and absence of an antioxidant mixture of resveratrol, niacinamide and GHK peptide. Endpoints related to inflammation, premature aging and carcinogenicity were monitored after 5 h of exposure and included IL-6, CXCL10, MMP-1 and NRF2. Differentially expressed genes were monitored by RNA-seq. RESULTS: Pre-treatment of keratinocytes with the antioxidant preparation in the absence of PM2.5 reduced baseline levels of MMP-1, IL-6 and CYP1A1 and reduced PM2.5 -induced increases in all four endpoints, MMP-1, IL-6, CXCL10 and CYP1A1. Antioxidants significantly increased NRF2 protein in the presence of PM2.5 , indicating a protective response. RNA-seq interrogation of antioxidant-treated cells further showed increased expression of NRF2 inducible genes. The expression of CYP1A1 and genes related to aryl hydrocarbon activation were induced by PM2.5 and suppressed by antioxidants. CONCLUSIONS: Specific signalling pathways known to be correlated with skin inflammation and aging were examined based on their suitability for use in efficacy testing for the prevention of skin damage due to ambient hydrocarbon pollution. Endpoints examined after only 5 h of exposure provide a useful method amenable to high through-put screening. The results obtained reinforce the concept that a multiple antioxidant preparation, topically applied, may reduce pro-inflammatory signalling and cellular damage and thereby reduce premature skin aging due to exposure to rural-derived airborne pollution.

OBJECTIF: Il est bien établi que l'exposition de la peau humaine à la pollution atmosphérique, en particulier sous forme de particules d'une taille de 2,5 µm (PM2,5 ), est associée à un stress oxydatif, à des dommages à l'ADN et à une inflammation entraînant des signes prématurés de vieillissement cutané. Étant donné que la plupart des dommages résultent du stress oxydatif, nous avons examiné les effets d'une composition topique contenant trois antioxydants dans un système de modèle in vitro afin d'évaluer le potentiel d'amélioration du vieillissement prématuré. L'utilisation de plusieurs antioxydants a été intéressante en raison de la composition typique des produits thérapeutiques de soin de la peau. Il est important de déterminer l'efficacité de plusieurs antioxydants combinés et de développer un test à court terme pour des tests d'efficacité à plus grande échelle. MÉTHODES: Des kératinocytes épidermiques humains normaux ont été exposés à une source de PM2,5 rurale en présence et en l'absence d'un mélange antioxydant de resvératrol, de niacinamide et de peptide GHK. Les critères d'évaluation liés à l'inflammation, au vieillissement prématuré et à la carcinogénicité ont été surveillés après 5 heures d'exposition et comprenaient l'IL-6, CXCL10, MMP-1 et le NRF2. Les gènes exprimés de manière différentielle ont été surveillés par séquençage de l'ARN. RÉSULTATS: Le prétraitement des kératinocytes par la préparation antioxydante en l'absence de PM2,5 a réduit les taux initiaux de MMP-1, IL-6 et de CYP1A1 et a réduit les augmentations induites par les PM2,5 dans les quatre critères d'évaluation, MMP-1, IL-6, CXCL10 et CYP1A1. Les antioxydants ont significativement augmenté la protéine NRF2 en présence de PM2,5 , ce qui indique une réponse protectrice. L'interrogation des séquences d'ARN des cellules traitées par antioxydants a également montré une expression accrue des gènes inductibles par NRF2. L'expression du CYP1A1 et des gènes liés à l'activation des hydrocarbures aryles a été induite par les PM2,5 et supprimée par les antioxydants. CONCLUSIONS: Les voies de signalisation spécifiques connues pour être corrélées à l'inflammation cutanée et au vieillissement ont été examinées en fonction de leur adéquation à l'utilisation dans les tests d'efficacité pour la prévention des lésions cutanées dues à la pollution des hydrocarbures ambiants. Les critères d'évaluation examinés après seulement 5 heures d'exposition fournissent une méthode utile pouvant être utilisée pour un dépistage à haut débit. Les résultats obtenus renforcent le principe selon lequel une préparation antioxydante multiple, appliquée par voie topique, peut réduire la signalisation pro-inflammatoire et les dommages cellulaires et ainsi réduire le vieillissement prématuré de la peau résultant de l'exposition à la pollution atmosphérique d'origine rurale.

Toxic element contaminations of prenatal vitamins.

The detrimental effects of gestational and lactational exposure to adverse chemical agents are gathering increasing attention. In our study, the presence of toxic heavy metals in several prenatal vitamins from six brands available in supermarkets and pharmacies was measured using ICP mass spectrometry. Several toxic heavy metals were detected, some at levels that could have potential toxicity to the fetus and the mother as well. Previous studies have also detected toxic heavy metals in prenatal and other vitamins. One of the reasons for toxic heavy metals in "natural vitamins" sold to consumers is that they are produced from naturally grown material and not synthesized. They are likely exposed to the heavy metals from the ground that they are grown in and there has not been any significant attempt to get rid of them before the vitamin pill was sold to consumers. Thus, this problem is not an isolated issue and regulatory agencies should be dealing more aggressively than they have been doing. In fact, several papers have already been published showing similar findings as we are reporting here. The vitamin pills we analyzed have elevated levels of boron, aluminum, molybdenum, barium, lead, titanium, nickel, arsenic, strontium, and cadmium. The levels of total chromium were also elevated but we did not separately determine Cr(III) and the much more hazardous Cr(VI), because of the tedious procedure required to separate these two forms of Cr.

Longitudinal Impact of WTC Dust Inhalation on Rat Cardiac Tissue Transcriptomic Profiles.

First responders (FR) exposed to the World Trade Center (WTC) Ground Zero air over the first week after the 9/11 disaster have an increased heart disease incidence compared to unexposed FR and the general population. To test if WTC dusts were causative agents, rats were exposed to WTC dusts (under isoflurane [ISO] anesthesia) 2 h/day on 2 consecutive days; controls received air/ISO or air only. Hearts were collected 1, 30, 240, and 360 d post-exposure, left ventricle total RNA was extracted, and transcription profiles were obtained. The data showed that differentially expressed genes (DEG) for WTC vs. ISO rats did not reach any significance with a false discovery rate (FDR) < 0.05 at days 1, 30, and 240, indicating that the dusts did not impart effects beyond any from ISO. However, at day 360, 14 DEG with a low FDR were identified, reflecting potential long-term effects from WTC dust alone, and the majority of these DEG have been implicated as having an impact on heart functions. Furthermore, the functional gene set enrichment analysis (GSEA) data at day 360 showed that WTC dust could potentially impact the myocardial energy metabolism via PPAR signaling and heart valve development. This is the first study showing that WTC dust could significantly affect some genes that are associated with the heart/CV system, in the long term. Even > 20 years after the 9/11 disaster, this has potentially important implications for those FR exposed repeatedly at Ground Zero over the first week after the buildings collapsed.

Longitudinal impact on rat cardiac tissue transcriptomic profiles due to acute intratracheal inhalation exposures to isoflurane.

Isoflurane (ISO) is a widely used inhalation anesthetic in experiments with rodents and humans during surgery. Though ISO has not been reported to impart long-lasting side effects, it is unknown if ISO can influence gene regulation in certain tissues, including the heart. Such changes could have important implications for use of this anesthetic in patients susceptible to heart failure/other cardiac abnormalities. To test if ISO could alter gene regulation/expression in heart tissues, and if such changes were reversible, prolonged, or late onset with time, SHR (spontaneously hypertensive) rats were exposed by intratracheal inhalation to a 97.5% air/2.5% ISO mixture on two consecutive days (2 hr/d). Control rats breathed filtered air only. On Days 1, 30, 240, and 360 post-exposure, rat hearts were collected and total RNA was extracted from the left ventricle for global gene expression analysis. The data revealed differentially-expressed genes (DEG) in response to ISO (compared to naïve control) at all post-exposure timepoints. The data showed acute ISO exposures led to DEG associated with wounding, local immune function, inflammation, and circadian rhythm regulation at Days 1 and 30; these effects dissipated by Day 240. There were other significantly-increased DEG induced by ISO at Day 360; these included changes in expression of genes associated with cell signaling, differentiation, and migration, extracellular matrix organization, cell-substrate adhesion, heart development, and blood pressure regulation. Examination of consistent DEG at Days 240 and 360 indicated late onset DEG reflecting potential long-lasting effects from ISO; these included DEG associated with oxidative phosphorylation, ribosome, angiogenesis, mitochondrial translation elongation, and focal adhesion. Together, the data show acute repeated ISO exposures could impart variable effects on gene expression/regulation in the heart. While some alterations self-resolved, others appeared to be long-lasting or late onset. Whether such changes occur in all rat models or in humans remains to be investigated.

Polyadenylation of Histone H3.1 mRNA Promotes Cell Transformation by Displacing H3.3 from Gene Regulatory Elements.

Replication-dependent canonical histone messenger RNAs (mRNAs) do not terminate with a poly(A) tail at the 3' end. We previously demonstrated that exposure to arsenic, an environmental carcinogen, induces polyadenylation of canonical histone H3.1 mRNA, causing transformation of human cells in vitro. Here we report that polyadenylation of H3.1 mRNA increases H3.1 protein, resulting in displacement of histone variant H3.3 at active promoters, enhancers, and insulator regions, leading to transcriptional deregulation, G2/M cell-cycle arrest, chromosome aneuploidy, and aberrations. In support of these observations, knocking down the expression of H3.3 induced cell transformation, whereas ectopic expression of H3.3 attenuated arsenic-induced cell transformation. Notably, arsenic exposure also resulted in displacement of H3.3 from active promoters, enhancers, and insulator regions. These data suggest that H3.3 displacement might be central to carcinogenesis caused by polyadenylation of H3.1 mRNA upon arsenic exposure. Our findings illustrate the importance of proper histone stoichiometry in maintaining genome integrity.

PBMC gene expression profiles of female Bangladeshi adults chronically exposed to arsenic-contaminated drinking water.

Arsenic, a class I human carcinogen, is ubiquitously found throughout the environment and around the globe, posing a great public health concern. Notably, Bangladesh and regions of West Bengal have been found to have high levels (0.5-4600 µg/L) of arsenic drinking water contamination, and approximately 50 million of the world's 200 million people chronically exposed to arsenic in Bangladesh alone. This study was carried out to examine genome-wide gene expression changes in individuals chronically exposed to arsenic-contaminated drinking water. Our study population includes twenty-nine Bangladeshi female participants with urinary arsenic levels ranging from 22.32 to 1828.12 µg/g creatinine. RNA extracted from peripheral blood mononuclear cells (PBMCs) were evaluated using RNA-Sequencing analysis. Our results indicate that a total of 1,054 genes were significantly associated with increasing urinary arsenic levels (FDR p < 0.05), which include 418 down-regulated and 636 up-regulated genes. Further Ingenuity Pathway Analysis revealed potential target genes (DAPK1, EGR2, APP), microRNAs (miR-155, -338, -210) and pathways (NOTCH signaling pathway) related to arsenic carcinogenesis. The selection of female-only participants provides a homogenous study population since arsenic has significant sex dependent effects, and the wide exposure range provides new insight for key gene expression changes that correlate with increasing urinary arsenic levels.

Role of miR-31 and SATB2 in arsenic-induced malignant BEAS-2B cell transformation.

Arsenic is a naturally occurring and highly potent metalloid known to elicit serious public health concerns. Today, approximately 200 million people around the globe are exposed to arsenic-contaminated drinking water at levels greater than the World Health Organization's recommended limit of 10 parts per billion. As a class I human carcinogen, arsenic exposure is known to elicit various cancers, including lung, skin, liver, and kidney. Current evidence suggests that arsenic is capable of inducing both genotoxic and cytotoxic injury, as well as activating epigenetic pathways to induce carcinogenesis. Our study identifies a novel pathway that is implicated in arsenic-induced carcinogenesis. Arsenic down-regulated miRNA-31 and the release of this inhibition caused overexpression of special AT-rich sequence-binding protein 2 (SATB2). Arsenic is known to disrupt miRNA expression, and here we report for the first time that arsenic is capable of inhibiting miR-31 expression. As a direct downstream target of miR-31, SATB2 is a prominent transcription factor, and nuclear matrix binding protein implicated in many types of human diseases including lung cancer. Results from this study show that arsenic induces the overexpressing SATB2 by inhibiting miR-31 expression, which blocks the translation of SATB2 mRNA, since levels of SATB2 mRNA remain the same but protein levels decrease. Overexpression of SATB2 induces malignant transformation of human bronchial epithelial (BEAS-2B) cells indicating the importance of the expression of miR-31 in preventing carcinogenesis by suppressing SATB2 protein levels.

Transcription factors and stress response gene alterations in human keratinocytes following Solar Simulated Ultra Violet Radiation.

Ultraviolet radiation (UVR) from sunlight is the major effector for skin aging and carcinogenesis. However, genes and pathways altered by solar-simulated UVR (ssUVR), a mixture of UVA and UVB, are not well characterized. Here we report global changes in gene expression as well as associated pathways and upstream transcription factors in human keratinocytes exposed to ssUVR. Human HaCaT keratinocytes were exposed to either a single dose or 5 repetitive doses of ssUVR. Comprehensive analyses of gene expression profiles as well as functional annotation were performed at 24 hours post irradiation. Our results revealed that ssUVR modulated genes with diverse cellular functions changed in a dose-dependent manner. Gene expression in cells exposed to a single dose of ssUVR differed significantly from those that underwent repetitive exposures. While single ssUVR caused a significant inhibition in genes involved in cell cycle progression, especially G2/M checkpoint and mitotic regulation, repetitive ssUVR led to extensive changes in genes related to cell signaling and metabolism. We have also identified a panel of ssUVR target genes that exhibited persistent changes in gene expression even at 1 week after irradiation. These results revealed a complex network of transcriptional regulators and pathways that orchestrate the cellular response to ssUVR.

Tungsten exposure causes a selective loss of histone demethylase protein.

In the course of our investigations into the toxicity of tungstate, we discovered that cellular exposure resulted in the loss of the histone demethylase protein. We specifically investigated the loss of two histone demethylase dioxygenases, JARID1A and JMJD1A. Both of these proteins were degraded in the presence of tungstate and this resulted in increased global levels of H3K4me3 and H3K9me2, the substrates of JARID1A and JMJD1A, respectively. Treatment with MG132 completely inhibited the loss of the demethylase proteins induced by tungstate treatment, suggesting that tungstate activated the proteasomal degradation of these proteins. The changes in global histone marks and loss of histone demethylase protein persisted for at least 48 h after removing sodium tungstate from the culture. The increase in global histone methylation remained when cells were cultured in methionine-free media, indicating that the increased histone methylation did not depend upon any de novo methylation process, but rather was due to the loss of the demethylase protein. Similar increases of H3K4me3 and H3K9me2 were observed in the livers of the mice that were acutely exposed to tungstate via their drinking water. Taken together, our results indicated that tungstate exposure specifically reduced histone demethylase JARID1A and JMJD1A via proteasomal degradation, leading to increased histone methylation.

Hexavalent Chromium (Cr(VI)) Down-Regulates Acetylation of Histone H4 at Lysine 16 through Induction of Stressor Protein Nupr1.

The environmental and occupational carcinogen Hexavalent Chromium (Cr(VI)) has been shown to cause lung cancer in humans when inhaled. In spite of a considerable research effort, the mechanisms of Cr(VI)-induced carcinogenesis remain largely unknown. Nupr1 (nuclear protein 1) is a small, highly basic, and unfolded protein with molecular weight of 8,800 daltons and is induced by a variety of stressors. Studies in animal models have suggested that Nupr1 is a key factor in the development of lung and pancreatic cancers, with little known about the underlying molecular mechanisms. Here we report that the level of Nupr1 is significantly increased in human bronchial epithelial BEAS2B cells following exposure to Cr(VI) through epigenetic mechanisms. Interestingly, Cr(VI) exposure also results in the loss of acetylation at histone H4K16, which is considered a 'hallmark' of human cancer. Cr(VI)-induced reduction of H4K16 acetylation appears to be caused by the induction of Nupr1, since (a) overexpression of Nupr1 decreased the levels of both H4K16 acetylation and the histone acetyltransferase MOF (male absent on the first; also known as Kat8, Myst 1), which specifically acetylates H4K16; (b) the loss of acetylation of H4K16 upon Cr(VI) exposure is greatly compromised by knockdown of Nupr1. Moreover, Nupr1-induced reduction of H4K16 acetylation correlates with the transcriptional down-regulation at several genomic loci. Notably, overexpression of Nupr1 induces anchorage-independent cell growth and knockdown of Nupr1 expression prevents Cr(VI)-induced cell transformation. We propose that Cr(VI) induces Nupr1 and rapidly perturbs gene expression by downregulating H4K16 acetylation, thereby contributing to Cr(VI)-induced carcinogenesis.

Molecular Mechanisms of Malignant Transformation by Low Dose Cadmium in Normal Human Bronchial Epithelial Cells.

Cadmium is a carcinogenic metal, the mechanisms of which are not fully understood. In this study, human bronchial epithelial cells were transformed with sub-toxic doses of cadmium (0.01, 0.05, and 0.1 µM) and transformed clones were characterized for gene expression changes using RNA-seq, as well as other molecular measurements. 440 genes were upregulated and 47 genes were downregulated in cadmium clones relative to control clones over 1.25-fold. Upregulated genes were associated mostly with gene ontology terms related to embryonic development, immune response, and cell movement, while downregulated genes were associated with RNA metabolism and regulation of transcription. Several embryonic genes were upregulated, including the transcription regulator SATB2. SATB2 is critical for normal skeletal development and has roles in gene expression regulation and chromatin remodeling. Small hairpin RNA knockdown of SATB2 significantly inhibited growth in soft agar, indicating its potential as a driver of metal-induced carcinogenesis. An increase in oxidative stress and autophagy was observed in cadmium clones. In addition, the DNA repair protein O6-methylguanine-DNA-methyltransferase was depleted by transformation with cadmium. MGMT loss caused significant decrease in cell viability after treatment with the alkylating agent temozolomide, demonstrating diminished capacity to repair such damage. Results reveal various mechanisms of cadmium-induced malignant transformation in BEAS-2B cells including upregulation of SATB2, downregulation of MGMT, and increased oxidative stress.

Solar Simulated Ultraviolet Radiation Induces Global Histone Hypoacetylation in Human Keratinocytes.

Ultraviolet radiation (UVR) from sunlight is the primary effector of skin DNA damage. Chromatin remodeling and histone post-translational modification (PTM) are critical factors in repairing DNA damage and maintaining genomic integrity, however, the dynamic changes of histone marks in response to solar UVR are not well characterized. Here we report global changes in histone PTMs induced by solar simulated UVR (ssUVR). A decrease in lysine acetylation of histones H3 and H4, particularly at positions of H3 lysine 9, lysine 56, H4 lysine 5, and lysine 16, was found in human keratinocytes exposed to ssUVR. These acetylation changes were highly associated with ssUVR in a dose-dependent and time-specific manner. Interestingly, H4K16ac, a mark that is crucial for higher order chromatin structure, exhibited a persistent reduction by ssUVR that was transmitted through multiple cell divisions. In addition, the enzymatic activities of histone acetyltransferases were significantly reduced in irradiated cells, which may account for decreased global acetylation. Moreover, depletion of histone deacetylase SIRT1 in keratinocytes rescued ssUVR-induced H4K16 hypoacetylation. These results indicate that ssUVR affects both HDAC and HAT activities, leading to reduced histone acetylation.

SATB2 expression increased anchorage-independent growth and cell migration in human bronchial epithelial cells.

The special AT-rich sequence-binding protein 2 (SATB2) is a protein that binds to the nuclear matrix attachment region of the cell and regulates gene expression by altering chromatin structure. In our previous study, we reported that SATB2 gene expression was induced in human bronchial epithelial BEAS-2B cells transformed by arsenic, chromium, nickel and vanadium. In this study, we show that ectopic expression of SATB2 in the normal human bronchial epithelial cell-line BEAS-2B increased anchorage-independent growth and cell migration, meanwhile, shRNA-mediated knockdown of SATB2 significantly decreased anchorage-independent growth in Ni transformed BEAS-2B cells. RNA sequencing analyses of SATB2 regulated genes revealed the enrichment of those involved in cytoskeleton, cell adhesion and cell-movement pathways. Our evidence supports the hypothesis that SATB2 plays an important role in BEAS-2B cell transformation.

Nuclear Factor κB1/RelA Mediates Inflammation in Human Lung Epithelial Cells at Atmospheric Oxygen Levels.

Oxygen levels range from 2% to 9% in vivo. Atmospheric O2 levels (21%) are known to induce cell proliferation defects and cellular senescence in primary cell cultures. However, the mechanistic basis of the deleterious effects of higher O2 levels is not fully understood. On the other hand, immortalized cells including cancer cell lines, which evade cellular senescence are normally cultured at 21% O2 and the effects of higher O2 on these cells are understudied. Here, we addressed this problem by culturing immortalized human bronchial epithelial (BEAS-2B) cells at ambient atmospheric, 21% O2 and lower, 10% O2. Our results show increased inflammatory response at 21% O2 but not at 10% O2. We found higher RelA binding at the NF-κB1/RelA target gene promoters as well as upregulation of several pro-inflammatory cytokines in cells cultured at 21% O2. RelA knockdown prevented the upregulation of the pro-inflammatory cytokines at 21% O2, suggesting NF-κB1/RelA as a major mediator of inflammatory response in cells cultured at 21% O2. Interestingly, unlike the 21% O2 cultured cells, exposure of 10% O2 cultured cells to H2O2 did not elicit inflammatory response, suggesting increased ability to tolerate oxidative stress in cells cultured at lower O2 levels.

In Vivo Exposures to Particulate Matter Collected from Saudi Arabia or Nickel Chloride Display Similar Dysregulation of Metabolic Syndrome Genes.

Particulate matter (PM) exposures have been linked to mortality, low birth weights, hospital admissions, and diseases associated with metabolic syndrome, including diabetes mellitus, cardiovascular disease, and obesity. In a previous in vitro and in vivo study, data demonstrated that PM(10µm) collected from Jeddah, Saudi Arabia (PMSA), altered expression of genes involved in lipid and cholesterol metabolism, as well as many other genes associated with metabolic disorders. PMSA contains a relatively high concentration of nickel (Ni), known to be linked to several metabolic disorders. In order to evaluate whether Ni and PM exposures induce similar gene expression profiles, mice were exposed to 100 µg/50 µl PM(SA) (PM-100), 50 µg/50 µl nickel chloride (Ni-50), or 100 µg/50 µl nickel chloride (Ni-100) twice per week for 4 wk and hepatic gene expression changes were determined. Ultimately, 55 of the same genes were altered in all 3 exposures. However, where the two Ni groups differed markedly was in the regulation (up or down) of these genes. Ni-100 and PM-100 groups displayed similar regulations, whereby 104 of the 107 genes were similarly modulated. Many of the 107 genes are involved in metabolic syndrome and include ALDH4A1, BCO2, CYP1A, CYP2U, TOP2A. In addition, the top affected pathways, such as fatty acid α-oxidation, and lipid and carbohydrate metabolism, are involved in metabolic diseases. Most notably, the top diseased outcome affected by these changes in gene expression was cardiovascular disease. Given these data, it appears that Ni and PM(SA) exposures display similar gene expression profiles, modulating the expression of genes involved in metabolic disorders.

Gene expression and pathway analysis of human hepatocellular carcinoma cells treated with cadmium.

Cadmium (Cd) is a toxic and carcinogenic metal naturally occurring in the Earth's crust. A common route of human exposure is via diet and cadmium accumulates in the liver. The effects of Cd exposure on gene expression in human hepatocellular carcinoma (HepG2) cells were examined in this study. HepG2 cells were acutely-treated with 0.1, 0.5, or 1.0 µM Cd for 24h; or chronically-treated with 0.01, 0.05, or 0.1 µM Cd for three weeks and gene expression analysis was performed using Affymetrix GeneChip® Human Gene 1.0 ST Arrays. Acute and chronic exposures significantly altered the expression of 333 and 181 genes, respectively. The genes most upregulated by acute exposure included several metallothioneins. Downregulated genes included the monooxygenase CYP3A7, involved in drug and lipid metabolism. In contrast, CYP3A7 was upregulated by chronic Cd exposure, as was DNAJB9, an anti-apoptotic J protein. Genes downregulated following chronic exposure included the transcriptional regulator early growth response protein 1. Ingenuity Pathway Analysis revealed that the top networks altered by acute exposure were lipid metabolism, small molecule biosynthesis, cell morphology, organization, and development; while top networks altered by chronic exposure were organ morphology, cell cycle, cell signaling, and renal and urological diseases/cancer. Many of the dysregulated genes play important roles in cellular growth, proliferation, and apoptosis, and may be involved in carcinogenesis. In addition to gene expression changes, HepG2 cells treated with cadmium for 24h indicated a reduction in global levels of histone methylation and acetylation that persisted 72 h post-treatment.

Malignant human cell transformation of Marcellus Shale gas drilling flow back water.

The rapid development of high-volume horizontal hydraulic fracturing for mining natural gas from shale has posed potential impacts on human health and biodiversity. The produced flow back waters after hydraulic stimulation are known to carry high levels of saline and total dissolved solids. To understand the toxicity and potential carcinogenic effects of these wastewaters, flow back waters from five Marcellus hydraulic fracturing oil and gas wells were analyzed. The physicochemical nature of these samples was analyzed by inductively coupled plasma mass spectrometry and scanning electron microscopy/energy dispersive X-ray spectroscopy. A cytotoxicity study using colony formation as the endpoint was carried out to define the LC50 values of test samples using human bronchial epithelial cells (BEAS-2B). The BEAS-2B cell transformation assay was employed to assess the carcinogenic potential of the samples. Barium and strontium were among the most abundant metals in these samples and the same metals were found to be elevated in BEAS-2B cells after long-term treatment. BEAS-2B cells treated for 6weeks with flow back waters produced colony formation in soft agar that was concentration dependent. In addition, flow back water-transformed BEAS-2B cells show better migration capability when compared to control cells. This study provides information needed to assess the potential health impact of post-hydraulic fracturing flow back waters from Marcellus Shale natural gas mining.

Tungsten-induced carcinogenesis in human bronchial epithelial cells.

Metals such as arsenic, cadmium, beryllium, and nickel are known human carcinogens; however, other transition metals, such as tungsten (W), remain relatively uninvestigated with regard to their potential carcinogenic activity. Tungsten production for industrial and military applications has almost doubled over the past decade and continues to increase. Here, for the first time, we demonstrate tungsten's ability to induce carcinogenic related endpoints including cell transformation, increased migration, xenograft growth in nude mice, and the activation of multiple cancer-related pathways in transformed clones as determined by RNA sequencing. Human bronchial epithelial cell line (Beas-2B) exposed to tungsten developed carcinogenic properties. In a soft agar assay, tungsten-treated cells formed more colonies than controls and the tungsten-transformed clones formed tumors in nude mice. RNA-sequencing data revealed that the tungsten-transformed clones altered the expression of many cancer-associated genes when compared to control clones. Genes involved in lung cancer, leukemia, and general cancer genes were deregulated by tungsten. Taken together, our data show the carcinogenic potential of tungsten. Further tests are needed, including in vivo and human studies, in order to validate tungsten as a carcinogen to humans.