James M Samet

Publications of James M Samet

• Omega-3 Fatty Acid Supplementation Appears to Attenuate Particulate Air Pollution Induced Cardiac Effects and Lipid Changes in Healthy Middle-Aged Adults.

  Authors: Haiyan Tong, Ana G Rappold, David Diaz-Sanchez, Susan E Steck, Jon Berntsen, Wayne E Cascio, Robert B Devlin, James M Samet

  Environmental health perspectives. 04/2012;

  Background: Air pollution exposure has been associated with adverse cardiovascular health effects. Findings of a recent epidemiologic study suggested that omega-3 fatty acid (fish oil)Background: Air pollution exposure has been associated with adverse cardiovascular health effects. Findings of a recent epidemiologic study suggested that omega-3 fatty acid (fish oil) supplementation blunted cardiac responses to air pollution exposure. Objectives: We conducted a randomized controlled exposure study to evaluate the efficacy of fish oil supplements in attenuating adverse cardiac effects of exposure to concentrated ambient fine and ultrafine particulate matter (CAP). Methods: Twenty-nine healthy middle-aged participants (mean age 58±1 yr) were supplemented in a randomized, double-blinded manner with 3g/day of either fish oil or olive oil for 4 weeks prior to sequential chamber exposure to filtered air and CAP (mean mass concentration 278±19 µg/m3) for 2 hrs. Cardiac responses were assessed by comparing time- and frequency-domain changes in heart rate variability (HRV) and electrocardiographic repolarization changes measured before, immediately after, and 20 hrs after exposure. Changes in plasma lipids were also evaluated at these time points. Results: Fish oil supplementation appeared to attenuate CAP-induced reductions in high-frequency/low-frequency ratio (HF/LF), as well as elevations in normalized LF (nLF) HRV, and prolongation of the QT interval corrected for heart rate (QTc). Very low-density lipoprotein and triglyceride concentrations increased significantly immediately after exposure to CAP in participants supplemented with olive oil, but not in those supplemented with fish oil. Conclusions: Exposure of healthy middle-aged adults to CAP for 2 hr induced acute cardiac and lipids changes following supplementation with olive oil, but not fish oil. Our findings suggest that omega-3 fatty acid supplements offer protection against the adverse cardiac and lipid effects associated with air pollution exposure.

• Linking oxidative events to inflammatory and adaptive gene expression induced by exposure to an organic particulate matter component.

  Authors: Wan-Yun Cheng, Jenna Currier, Philip A Bromberg, Robert Silbajoris, Steven O Simmons, James M Samet

  Environmental health perspectives. 02/2012; 120(2):267-74.

  Toxicological studies have correlated inflammatory effects of diesel exhaust particles (DEP) with its organic constituents, such as the organic electrophile 1,2-naphthoquinone (1,2-NQ). To elucidateToxicological studies have correlated inflammatory effects of diesel exhaust particles (DEP) with its organic constituents, such as the organic electrophile 1,2-naphthoquinone (1,2-NQ). To elucidate the mechanisms involved in 1,2-NQ-induced inflammatory responses, we examined the role of oxidant stress in 1,2-NQ-induced expression of inflammatory and adaptive genes in a human airway epithelial cell line. We measured cytosolic redox status and hydrogen peroxide (H2O2) in living cells using the genetically encoded green fluorescent protein (GFP)-based fluorescent indicators roGFP2 and HyPer, respectively. Expression of interleukin-8 (IL-8), cyclooxygenase-2 (COX-2), and heme oxygenase-1 (HO-1) mRNA was measured in BEAS-2B cells exposed to 1,2-NQ for 1-4 hr. Catalase overexpression and metabolic inhibitors were used to determine the role of redox changes and H2O2 in 1,2-NQ-induced gene expression. Cells expressing roGFP2 and HyPer showed a rapid loss of redox potential and an increase in H2O2 of mitochondrial origin following exposure to 1,2-NQ. Overexpression of catalase diminished the H2O2-dependent signal but not the 1,2-NQ-induced loss of reducing potential. Catalase overexpression and inhibitors of mitochondrial respiration diminished elevations in IL-8 and COX-2 induced by exposure to 1,2-NQ, but potentiated HO-1 mRNA levels in BEAS cells. These data show that 1,2-NQ exposure induces mitochondrial production of H2O2 that mediates the expression of inflammatory genes, but not the concurrent loss of reducing redox potential in BEAS cells. 1,2-NQ exposure also causes marked expression of HO-1 that appears to be enhanced by suppression of H2O2. These findings shed light into the oxidant-dependent events that underlie cellular responses to environmental electrophiles.

• GSTM1 modulation of IL-8 expression in human bronchial epithelial cells exposed to ozone.

  Authors: Weidong Wu, Vinod Doreswamy, David Diaz-Sanchez, James M Samet, Matt Kesic, Lisa Dailey, Wenli Zhang, Ilona Jaspers, David B Peden

  Free radical biology & medicine. 07/2011; 51(2):522-9.

  Exposure to the major air pollutant ozone can aggravate asthma and other lung diseases. Our recent study in human volunteers has shown that the glutathione S-transferase Mu 1 (GSTM1)-null genotype isExposure to the major air pollutant ozone can aggravate asthma and other lung diseases. Our recent study in human volunteers has shown that the glutathione S-transferase Mu 1 (GSTM1)-null genotype is associated with increased airway neutrophilic inflammation induced by inhaled ozone. The aim of this study was to examine the effect of GSTM1 modulation on interleukin 8 (IL-8) production in ozone-exposed human bronchial epithelial cells (BEAS-2B) and the underlying mechanisms. Exposure of BEAS-2B cells to 0.4 ppm ozone for 4 h significantly increased IL-8 release, with a modest reduction in intracellular reduced glutathione (GSH). Ozone exposure induced reactive oxygen species (ROS) production and NF-κB activation. Pharmacological inhibition of NF-κB activation or mutation of the IL-8 promoter at the κB-binding site significantly blocked ozone-induced IL-8 production or IL-8 transcriptional activity, respectively. Knockdown of GSTM1 in BEAS-2B cells enhanced ozone-induced NF-κB activation and IL-8 production. Consistently, an ozone-induced overt increase in IL-8 production was detected in GSTM1-null primary human bronchial epithelial cells. In addition, supplementation with reduced GSH inhibited ozone-induced ROS production, NF-κB activation, and IL-8 production. Taken together, GSTM1 deficiency enhances ozone-induced IL-8 production, which is mediated by generated ROS and subsequent NF-κB activation in human bronchial epithelial cells.

• Ambient particulate matter induces interleukin-8 expression through an alternative NF-κB (nuclear factor-kappa B) mechanism in human airway epithelial cells.

  Authors: Robert Silbajoris, Alvaro R Osornio-Vargas, Steven O Simmons, William Reed, Philip A Bromberg, Lisa A Dailey, James M Samet

  Environmental health perspectives. 06/2011; 119(10):1379-83.

  Exposure to ambient air particulate matter (PM) has been shown to increase rates of cardiopulmonary morbidity and mortality, but the underlying mechanisms are still not well understood.Objective: WeExposure to ambient air particulate matter (PM) has been shown to increase rates of cardiopulmonary morbidity and mortality, but the underlying mechanisms are still not well understood.Objective: We examined signaling events involved in the expression of the inflammatory gene interleukin-8 (IL-8) in human airway epithelial cells (HAECs) exposed to ambient PM collected in an urban area of Mexicali, Mexico. We studied IL-8 expression and regulatory signaling pathways in cultured HAECs exposed to Mexicali PM suspended in media for 0-4 hr. Exposure resulted in a dose-dependent, 2- to 8-fold increase in IL-8 mRNA expression relative to controls. PM exposure induced IL-8 transcriptional activity in BEAS-2B cells that was dependent on the nuclear factor-kappa B (NF-κB) response element in the IL-8 promoter. Chromatin immunoprecipitation (ChIP) assays showed a 3-fold increase in binding of the p65 (RelA) NF-κB isoform to the IL-8 promoter sequence in HAECs exposed to PM. Western blot analyses showed elevated levels of phosphorylation of p65 but no changes in IκBα phosphorylation or degradation. IL-8 expression was blunted in a dose-dependent manner in BEAS-2B cells transduced with a lentivirus encoding a dominant negative p65 mutant in which phosphorylation sites were inactivated. Taken together, these findings show that the increase in IL-8 mRNA expression in HAECs exposed to PM10 (PM ≤ 10 μm in aerodynamic diameter) is mediated through an NF-κB-dependent signaling mechanism that occurs through a pathway involving direct phosphorylation of the transcription factor p65 in the absence of IκBα degradation. These data show that exposure to PM10 in ambient air can induce inflammatory responses by activating specific signaling mechanisms in HAECs.

• Darkfield-confocal microscopy detection of nanoscale particle internalization by human lung cells.

  Authors: Eugene A Gibbs-Flournoy, Philip A Bromberg, Thomas P J Hofer, James M Samet, Robert M Zucker

  Particle and fibre toxicology. 01/2011; 8(1):2.

  Concerns over the health effects of nanomaterials in the environment have created a need for microscopy methods capable of examining the biological interactions of nanoparticles (NP). Unfortunately,Concerns over the health effects of nanomaterials in the environment have created a need for microscopy methods capable of examining the biological interactions of nanoparticles (NP). Unfortunately, NP are beyond the diffraction limit of resolution for conventional light microscopy (~200 nm). Fluorescence and electron microscopy techniques commonly used to examine NP interactions with biological substrates have drawbacks that limit their usefulness in toxicological investigation of NP. EM is labor intensive and slow, while fluorescence carries the risk of photobleaching the sample and has size resolution limits. In addition, many relevant particles lack intrinsic fluorescence and therefore can not be detected in this manner. To surmount these limitations, we evaluated the potential of a novel combination of darkfield and confocal laser scanning microscopy (DF-CLSM) for the efficient 3D detection of NP in human lung cells. The DF-CLSM approach utilizes the contrast enhancements of darkfield microscopy to detect objects below the diffraction limit of 200 nm based on their light scattering properties and interfaces it with the power of confocal microscopy to resolve objects in the z-plane. Validation of the DF-CLSM method using fluorescent polystyrene beads demonstrated spatial colocalization of particle fluorescence (Confocal) and scattered transmitted light (Darkfield) along the X, Y, and Z axes. DF-CLSM imaging was able to detect and provide reasonable spatial locations of 27 nm TiO2 particles in relation to the stained nuclei of exposed BEAS 2B cells. Statistical analysis of particle proximity to cellular nuclei determined a significant difference between 5 min and 2 hr particle exposures suggesting a time-dependent internalization process. DF-CLSM microscopy is an alternative to current conventional light and electron microscopy methods that does not rely on particle fluorescence or contrast in electron density. DF-CLSM is especially well suited to the task of establishing the spatial localization of nanoparticles within cells, a critical topic in nanotoxicology. This technique has advantages to 2D darkfield microscopy as it visualizes nanoparticles in 3D using confocal microscopy. Use of this technique should aid toxicological studies related to observation of NP interactions with biological endpoints at cellular and subcellular levels.

• Differential cardiopulmonary effects of size-fractionated ambient particulate matter in mice.

  Authors: Haiyan Tong, Wan-Yun Cheng, James M Samet, M Ian Gilmour, Robert B Devlin

  Cardiovascular toxicology. 12/2010; 10(4):259-67.

  A growing body of evidence from epidemiological and toxicological studies provides a strong link between exposure to ambient particulate matter (PM) of varying size and increased cardiovascular andA growing body of evidence from epidemiological and toxicological studies provides a strong link between exposure to ambient particulate matter (PM) of varying size and increased cardiovascular and respiratory morbidity and mortality. This study was designed to evaluate the cardiopulmonary effects of ambient coarse, fine, and ultrafine particles collected in Chapel Hill, NC. Mice were exposed to each size fraction by oropharyngeal instillation. Twenty-four hours later, pulmonary inflammation was assessed by bronchoalveolar lavage and cardiac injury was measured using a Langendorff cardiac perfusion preparation. Recovery of post-ischemic left ventricular developed pressure and infarct size were measured as indeces of cardiac ischemia/reperfusion injury. Coronary flow rate was measured before, during, and after ischemia. We demonstrate that coarse PM caused the most significant pulmonary inflammatory responses. In contrast, hearts from ultrafine-exposed mice had significantly lower post-ischemic functional recovery and greater infarct size, while hearts from coarse and fine PM-exposed mice had no significant responses to ischemia/reperfusion. The coronary flow rate was significantly reduced in the ultrafine PM group. This study shows that exposure of mice to coarse PM results in significant pulmonary toxicity while ultrafine PM appears to enhance cardiac ischemia/reperfusion injury.

• An integrated imaging approach to the study of oxidative stress generation by mitochondrial dysfunction in living cells.

  Authors: Wan-Yun Cheng, Haiyan Tong, Evan W Miller, Christopher J Chang, James Remington, Robert M Zucker, Philip A Bromberg, James M Samet, Thomas P J Hofer

  Environmental health perspectives. 04/2010; 118(7):902-8.

  The mechanisms of action of many environmental agents commonly involve oxidative stress resulting from mitochondrial dysfunction. Zinc is a common environmental metallic contaminant that has beenThe mechanisms of action of many environmental agents commonly involve oxidative stress resulting from mitochondrial dysfunction. Zinc is a common environmental metallic contaminant that has been implicated in a variety of oxidant-dependent toxicological responses. Unlike ions of other transition metals such as iron, copper, and vanadium, Zn(2+) does not generate reactive oxygen species (ROS) through redox cycling. To characterize the role of oxidative stress in zinc-induced toxicity. We used an integrated imaging approach that employs the hydrogen peroxide (H2O2)-specific fluorophore Peroxy Green 1 (PG1), the mitochondrial potential sensor 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolylcarbocyanine iodide (JC-1), and the mitochondria-targeted form of the redox-sensitive genetically encoded fluorophore MTroGFP1 in living cells. Zinc treatment in the presence of the Zn(2+) ionophore pyrithione of A431 skin carcinoma cells preloaded with the H(2)O(2)-specific indicator PG1 resulted in a significant increase in H(2)O(2) production that could be significantly inhibited with the mitochondrial inhibitor carbonyl cyanide 3-chlorophenylhydrazone. Mitochondria were further implicated as the source of zinc-induced H(2)O(2) formation by the observation that exposure to zinc caused a loss of mitochondrial membrane potential. Using MTroGFP1, we showed that zinc exposure of A431 cells induces a rapid loss of reducing redox potential in mitochondria. We also demonstrated that zinc exposure results in rapid swelling of mitochondria isolated from mouse hearts. Taken together, these findings show a disruption of mitochondrial integrity, H(2)O(2) formation, and a shift toward positive redox potential in cells exposed to zinc. These data demonstrate the utility of real-time, live-cell imaging to study the role of oxidative stress in toxicological responses.

• Phosphorylation of p65 is required for zinc oxide nanoparticle-induced interleukin 8 expression in human bronchial epithelial cells.

  Authors: Weidong Wu, James M Samet, David B Peden, Philip A Bromberg

  Environmental health perspectives. 03/2010; 118(7):982-7.

  Exposure to zinc oxide (ZnO) in environmental and occupational settings causes acute pulmonary responses through the induction of proinflammatory mediators such as interleukin-8 (IL-8). WeExposure to zinc oxide (ZnO) in environmental and occupational settings causes acute pulmonary responses through the induction of proinflammatory mediators such as interleukin-8 (IL-8). We investigated the effect of ZnO nanoparticles on IL-8 expression and the underlying mechanisms in human bronchial epithelial cells. We determined IL-8 mRNA and protein expression in primary human bronchial epithelial cells and the BEAS-2B human bronchial epithelial cell line using reverse-transcriptase polymerase chain reaction and the enzyme-linked immunosorbent assay, respectively. Transcriptional activity of IL-8 promoter and nuclear factor kappa B (NFkappaB) in ZnO-treated BEAS-2B cells was measured using transient gene transfection of the luciferase reporter construct with or without p65 constructs. Phosphorylation and degradation of IkappaBalpha, an inhibitor of NF-kappaB, and phosphorylation of p65 were detected using immunoblotting. Binding of p65 to the IL-8 promoter was examined using the chromatin immunoprecipitation assay. ZnO exposure (2-8 microg/mL) increased IL-8 mRNA and protein expression. Inhibition of transcription with actinomycin D blocked ZnO-induced IL-8 expression, which was consistent with the observation that ZnO exposure increased IL-8 promoter reporter activity. Further study demonstrated that the kappaB-binding site in the IL-8 promoter was required for ZnO-induced IL-8 transcriptional activation. ZnO stimulation modestly elevated IkappaBalpha phosphorylation and degradation. Moreover, ZnO exposure also increased the binding of p65 to the IL-8 promoter and p65 phosphorylation at serines 276 and 536. Overexpression of p65 constructs mutated at serines 276 or 536 significantly reduced ZnO-induced increase in IL-8 promoter reporter activity. p65 phosphorylation and IkappaBalpha phosphorylation and degradation are the primary mechanisms involved in ZnO nanoparticle-induced IL-8 expression in human bronchial epithelial cells.

• Toxicological disruption of signaling homeostasis: tyrosine phosphatases as targets.

  Authors: James M Samet, Tamara L Tal

  Annual review of pharmacology and toxicology. 01/2010; 50:215-35.

  The protein tyrosine phosphatases (PTPs) consist of a diverse group of enzymes whose activity opposes that of the tyrosine kinases. As such, the PTPs have critical roles in maintaining signalingThe protein tyrosine phosphatases (PTPs) consist of a diverse group of enzymes whose activity opposes that of the tyrosine kinases. As such, the PTPs have critical roles in maintaining signaling quiescence in resting cells and in restoring homeostasis by effecting signal termination. Interest in these enzymes has increased in recent years following the discovery that the activity of PTPs is modulated through redox mechanisms during signaling. The molecular features that enable redox regulation of PTPs during physiological signaling also render them highly susceptible to oxidative and electrophilic inactivation by a broad spectrum of structurally disparate xenobiotic compounds. The loss of PTP activity results in a profound disregulation of protein phosphotyrosine metabolism, leading to widespread and persistent activation of signaling cascades in the cell.

• Diesel Particle-induced Transcription Expression of P21 Involves Activation of EGFR, SRC and STAT3.

  Authors: Dongsun Cao, Philip A Bromberg, James M Samet

  American journal of respiratory cell and molecular biology. 04/2009;

  Exposure to diesel exhaust particle has been associated with adverse health outcomes, such as inflammation, adjuvancy and mutagenesis. However, the molecular mechanisms by which DEP inhalation exertsExposure to diesel exhaust particle has been associated with adverse health outcomes, such as inflammation, adjuvancy and mutagenesis. However, the molecular mechanisms by which DEP inhalation exerts these effects are still largely unknown. We previously reported that exposure to DEP activates the transcription factor Stat3 in airway epithelial cells, a primary target cell of inhaled DEP. To elucidate the functional role of Stat3 activation in these cells, we investigated the function of Stat3 in DEPâinduced expression of the p21 gene in the human bronchial epithelial cell line BEAS-2B. We report that DEP exposure induces increased levels of p21 mRNA and protein in a manner that is independent of p53 and Sp1 expression or DNA binding to the p21 gene. Using Chromatin Immuoprecipitation assays and expression of a dominant negative Stat3 mutant, we show that activation of Stat3 and its binding to the p21 promoter are required for DEP-induced expression of p21, suggesting that Stat3 plays an essential role in the induction of p21 by DEP. Additional experiments demonstrated that activation of p21 gene expression is dependent on the activation of EGFR and Src kinase activities. Lastly, we provide evidence suggesting that DEP exposure can inhibit the proliferation of human bronchial epithelial cells, suggesting a functional role of p21 activation airway epithelial cells exposed to DEP.

• Concentrated Ambient Ultrafine Particle Exposure Induces Cardiac Changes in Young Healthy Volunteers.

  Authors: James M Samet, Ana Rappold, Donald Graff, Wayne E Cascio, Jon H Berntsen, Yuh-Chin T Huang, Margaret Herbst, Maryann Bassett, Tracey Montilla, Milan J Hazucha, Philip A Bromberg, Robert B Devlin

  American journal of respiratory and critical care medicine. 03/2009;

  RATIONALE: Exposure to ambient ultrafine particles has been associated with cardiopulmonary toxicity and mortality. Adverse effects specifically linked to ultrafine particles include loss ofRATIONALE: Exposure to ambient ultrafine particles has been associated with cardiopulmonary toxicity and mortality. Adverse effects specifically linked to ultrafine particles include loss of sympathovagal balance and altered hemostasis. OBJECTIVE: To characterize the effects of acute exposure to ambient ultrafine particle in young healthy humans METHODS: Twenty healthy non-smoking male and female subjects between the ages of 18 and 35 were exposed to filtered air or to an atmosphere in which captured ultrafine (<0.16 um) particles were concentrated by a factor of up to 20 fold over ambient levels with the use of particle concentrators fitted with size-selective outlets (UFCAP). Subjects underwent bronchoalveolar lavage 18 hrs following each exposure. Cardiovascular endpoints measured included pulmonary function, clinical chemistry and hematological parameters as well as heart rate variability and repolarization indices. MEASUREMENTS AND MAIN RESULTS: Exposure to UFCAP was statistically associated with an increase in frequency domain markers of heart rate variability, specifically indicative of elevated vagal input to the heart. Consistent with this finding were increases in the variance associated with the duration of the QT interval. In addition, UFCAP exposure resulted in a significant increase in blood levels of the fibrin degradation product D-dimer as well as a modest elevation in the inflammatory chemokine IL-8 recovered in the lavage fluid. CONCLUSIONS: These findings show mild inflammatory and pro-thrombic responses, and are suggestive of alterations in cardiac repolarization induced by UFCAP inhalation.

• Epidermal growth factor receptor activation by diesel particles is mediated by tyrosine phosphatase inhibition.

  Authors: Tamara L Tal, Philip A Bromberg, Yumee Kim, James M Samet

  Toxicology and applied pharmacology. 10/2008;

  Exposure to particulate matter (PM) is associated with increased cardiopulmonary morbidity and mortality. Diesel exhaust particles (DEP) are a major component of ambient PM and may contribute toExposure to particulate matter (PM) is associated with increased cardiopulmonary morbidity and mortality. Diesel exhaust particles (DEP) are a major component of ambient PM and may contribute to PM-induced pulmonary inflammation. Proinflammatory signaling is mediated by phosphorylation-dependent signaling pathways whose activation is opposed by the activity of protein tyrosine phosphatases (PTPases) which thereby function to maintain signaling quiescence. PTPases contain an invariant catalytic cysteine that is susceptible to electrophilic attack. DEP contain electrophilic oxy-organic compounds that may contribute to the oxidant effects of PM. Therefore, we hypothesized that exposure to DEP impairs PTPase activity allowing for unopposed basal kinase activity. Here we report that exposure to 30 mug/cm(2) DEP for 4 h induces differential activation of signaling in primary cultures of human airway epithelial cells (HAEC), a primary target cell in PM inhalation. In-gel kinase activity assay of HAEC exposed to DEPs of low (L-DEP), intermediate (I-DEP) or high (H-DEP) organic content showed differential activation of intracellular kinases. Exposure to these DEP also induced varying levels of phosphorylation of the receptor tyrosine kinase EGFR in a manner that requires EGFR kinase activity but does not involve receptor dimerization. We demonstrate that treatment with DEP results in an impairment of total and EGFR-directed PTPase activity in HAEC with a potency that correlates with the organic content of these particles. These data show that DEP-induced EGFR-phosphorylation in HAEC is the result of a loss of PTPase activities which normally function to dephosphorylate EGFR in opposition to baseline EGFR kinase activity.

• Regulation of cyclooxygenase-2 expression by cAMP response element and mRNA stability in a human airway epithelial cell line exposed to zinc.

  Authors: Weidong Wu, Robert A Silbajoris, Dongsun Cao, Philip A Bromberg, Qiao Zhang, David B Peden, James M Samet

  Toxicology and applied pharmacology. 05/2008;

  Exposure to zinc-laden particulate matter in ambient and occupational settings has been associated with proinflammatory responses in the lung. Cyclooxygenase 2-derived eicosanoids are importantExposure to zinc-laden particulate matter in ambient and occupational settings has been associated with proinflammatory responses in the lung. Cyclooxygenase 2-derived eicosanoids are important modulators of airway inflammation. In this study, we characterized the transcriptional and posttranscriptional events that regulate COX-2 expression in a human bronchial epithelial cell line BEAS-2B exposed to Zn(2+). Zn(2+) exposure resulted in pronounced increases in COX-2 mRNA and protein expression, which were prevented by pretreatment with the transcription inhibitor actinomycin D, implying the involvement of transcriptional regulation. This was supported by the observation of increased COX-2 promoter activity in Zn(2+)-treated BEAS-2B cells. Mutation of the cAMP response element (CRE), but not the kappaB-binding sites in the COX-2 promoter markedly reduced COX-2 promoter activity induced by Zn(2+). Inhibition of NFkappaB activation did not block Zn(2+)-induced COX-2 expression. Measurement of mRNA stability demonstrated that Zn(2+) exposure impaired the degradation of COX-2 mRNA in BEAS-2B cells. This message stabilization effect of Zn(2+) exposure was shown to be dependent on the integrity of the 3'-untranslated region found in the COX-2 transcript. Taken together, these data demonstrate that the CRE and mRNA stability regulates COX-2 expression induced in BEAS-2B cells exposed to extracellular Zn(2+).

• Capacitative calcium entry contributes to the differential transactivation of the epidermal growth factor receptor in response to thiazolidinediones.

  Authors: Brian J Dewar, Olivia S Gardner, Ching-Shih Chen, H Shelton Earp, James M Samet, Lee M Graves

  Molecular pharmacology. 12/2007; 72(5):1146-56.

  Thiazolidinediones (TZDs) are synthetic ligands for the peroxisome proliferator-activated receptor gamma (PPARgamma) but also elicit PPARgamma-independent effects, most notably activation ofThiazolidinediones (TZDs) are synthetic ligands for the peroxisome proliferator-activated receptor gamma (PPARgamma) but also elicit PPARgamma-independent effects, most notably activation of mitogen-activated protein kinases (MAPKs). Ciglitazone rapidly activates extracellular signal-regulated kinase (Erk) MAPK, an event requiring c-Src kinase-dependent epidermal growth factor receptor (EGFR) transactivation, whereas troglitazone only weakly activates Erk and does not induce EGFR transactivation; the mechanism underlying this difference remains unclear. In this study, both ciglitazone and troglitazone increased Src activation. Similar effects were observed with Delta2-derivatives of each TZD, compounds that bind PPARgamma but do not lead to its activation, further indicating a PPARgamma-independent mechanism. Neither EGFR kinase nor Pyk2 inhibition prevented Src activation; however, inhibition of Src kinase activity prevented Pyk2 activation. Intracellular calcium chelation blocks TZD-induced Pyk2 activation; here, Src activation by both TZDs and ciglitazone-induced EGFR transactivation were prevented by calcium chelation. Accordingly, both TZDs increased calcium concentrations from intracellular stores; however, only ciglitazone produced a secondary calcium influx in the presence of extracellular calcium. Removal of extracellular calcium or inhibition of capacitative calcium entry by 2-APB prevented ciglitazone-induced EGFR transactivation and Erk activation but did not affect upstream kinase signaling pathways. These results demonstrate that upstream kinases (i.e., Src and Pyk2) are required but not sufficient for EGFR transactivation by TZDs. Moreover, influx of extracellular calcium through capacitative calcium entry may be an unrecognized component that provides a mechanism for the differential induction of EGFR transactivation by these compounds.

• COX-2 expression induced by diesel particles involves chromatin modification and degradation of HDAC1.

  Authors: Dongsun Cao, Philip A Bromberg, James M Samet

  American journal of respiratory cell and molecular biology. 08/2007; 37(2):232-9.

  Cyclooxygenase-2 (COX-2) plays an important role in the inflammatory response induced by physiologic and stress stimuli. Exposure to diesel exhaust particulate matter (DEP) has been shown to induceCyclooxygenase-2 (COX-2) plays an important role in the inflammatory response induced by physiologic and stress stimuli. Exposure to diesel exhaust particulate matter (DEP) has been shown to induce pulmonary inflammation and exacerbate asthma and chronic obstructive pulmonary disease. DEP is a potent inducer of inflammatory reponses in human airway epithelial cells. The mechanism through which DEP inhalation induces inflammatory mediator expression is not understood. In this report, we demonstrate that DEP can induce the expression of COX-2 gene in a human bronchial epithelial cell line (BEAS-2B) at both transcriptional and protein levels. The induction of COX-2 gene expression involves chromatin modification, in particular acetylation and deacetylation of histones. We show that exposure to DEP increases the acetylation of histone H4 associated with the COX-2 promoter and causes degradation of histone deacetylase 1 (HDAC1). Further, we establish that HDAC1 plays a pivotal role in mediating the transcriptional activation of the COX-2 gene in BEAS-2B cells exposed to DEP, supported by evidence that the down-regulation of HDAC1 using siRNA leads to activation of COX-2 gene expression, whereas overexpression of HDAC1 results in its repression. Finally, DEP exposure induced recruitment of histone acetyltransferase (HAT) p300 to the promoter of the COX-2 gene, suggesting that acetylation is also important in regulating its expression in response to DEP exposure. These results show for the first time acetylation via selective degradation of HDAC1, and that recruitment of HAT plays an important role in DEP-induced expression of the COX-2 gene.

• Zn2+-induced NF-kappaB-dependent transcriptional activity involves site-specific p65/RelA phosphorylation.

  Authors: Yu-Mee Kim, Dongsun Cao, William Reed, Weidong Wu, Ilona Jaspers, Tamara Tal, Philip A Bromberg, James M Samet

  Cellular signalling. 04/2007; 19(3):538-46.

  Zinc is an essential micronutrient, but is proinflammatory when inhaled into the lung. While it is recognized that zinc exposure of airway epithelial cells activates the transcription factorZinc is an essential micronutrient, but is proinflammatory when inhaled into the lung. While it is recognized that zinc exposure of airway epithelial cells activates the transcription factor NF-kappaB and increases the expression of inflammatory cytokines to mediate this response, the underlying mechanism of NF-kappaB activation remains to be characterized. In this study, we investigated these Zn2+-induced signaling mechanisms in the BEAS-2B human airway epithelial cell line. Fifty micromolars Zn2+ induced NF-kappaB-dependent transcriptional activity. However, this occurred independently of IkappaBalpha degradation, an essential event in activation of the canonical NF-kappaB pathway, which is induced by physiological stimuli such as TNFalpha and IL-1beta. We also observed that 50 microM Zn2+ exposure caused p65/RelA phosphorylation on Ser 276, Ser 529, and Ser 536 in both cytoplasmic and nuclear cell fractions. Mutational analysis pointed to Ser 536 of p65/RelA as the determinant of Zn2+-induced NF-kappaB transactivation in BEAS-2B cells. Pharmacological inhibition of IKKalpha/beta activity reduced both Zn2+-induced p65/RelA phosphorylation at Ser 536 and NF-kappaB-dependent transcriptional activity, suggesting that IKKalpha/beta is necessary for these Zn2+-induced effects. Taken together, these data show that exposure to supraphysiological concentrations of Zn2+ induces NF-kappaB-dependent transcription through an alternate mechanism, suggesting a novel pathway for cellular responses to environmental stress.

• Diesel exhaust particulate-induced activation of Stat3 requires activities of EGFR and Src in airway epithelial cells.

  Authors: Dongsun Cao, Tamara L Tal, Lee M Graves, Ian Gilmour, William Linak, William Reed, Philip A Bromberg, James M Samet

  American journal of physiology. Lung cellular and molecular physiology. 03/2007; 292(2):L422-9.

  In vivo exposure to diesel exhaust particles (DEP) elicits acute inflammatory responses in the lung characterized by inflammatory cell influx and elevated expression of mediators such as cytokinesIn vivo exposure to diesel exhaust particles (DEP) elicits acute inflammatory responses in the lung characterized by inflammatory cell influx and elevated expression of mediators such as cytokines and chemokines. Signal transducers and activators of transcription (STAT) proteins are a family of cytoplasmic transcription factors that are key transducers of signaling in response to cytokine and growth factor stimulation. One member of the STAT family, Stat3, has been implicated as a regulator of inflammation but has not been studied in regard to DEP exposure. The results of this study show that DEP induces Stat3 phosphorylation as early as 1 h following stimulation and that phosphorylated Stat3 translocates into the nucleus. Inhibition of epidermal growth factor receptor (EGFR) and Src activities by the inhibitors PD-153035 and PP2, respectively, abolished the activation of Stat3 by DEP, suggesting that Stat3 activation by DEP requires EGFR and Src kinase activation. The present study suggests that oxidative stress induced by DEP may play a critical role in activating EGFR signaling, as evidenced by the fact that pretreatment with antioxidant prevented the activation of EGFR and Stat3. These findings demonstrate that DEP inhalation can activate proinflammatory Stat3 signaling in vitro.

• A comparison of studies on the effects of controlled exposure to fine, coarse and ultrafine ambient particulate matter from a single location.

  Authors: James M Samet, Donald Graff, Jon Berntsen, Andrew J Ghio, Yuh-Chin T Huang, Robert B Devlin

  Inhalation toxicology. 02/2007; 19 Suppl 1:29-32.

  Particle size has been implicated by epidemiological and toxicological studies as an important determinant of the toxicity of ambient particulate matter (PM). In an effort to characterize theParticle size has been implicated by epidemiological and toxicological studies as an important determinant of the toxicity of ambient particulate matter (PM). In an effort to characterize the cardiovascular, hematological and pulmonary effects of different PM size fractions in humans, we have conducted controlled human exposures of normal volunteers to ultrafine-, fine- and coarse- fraction PM concentrated from ambient air in Chapel Hill, North Carolina. Healthy non-smoking male and female subjects between the ages of 18 and 35 participated in these studies. Exposures were undertaken with the use of particle concentrators fitted with size-selective outlets. These devices are capable of generating concentration factors between 10- and 20-fold over ambient levels. Cardiovascular endpoints measured include heart rate variability and T-wave alternans, as well as pulmonary function parameters. Subjects underwent bronchoscopy and bronchoalveolar lavage 18 hrs following exposure to PM or to clean air. Lavage fluids and blood samples were assayed for a battery of markers of hematological, cytotoxic and inflammatory injury. The design of these studies permits direct comparison of the effects of concentrated ambient PM as a function of particle size. The data to be presented reveal modest size fraction-dependent effects of concentrated PM exposure on cardiovascular, pulmonary and hematological parameters in normal adult human subjects. These findings have relevant implications for the design of future chamber studies and the role of particle size fraction in the adverse health effects of PM exposure in humans.

• [Expression of phosphorylated ERK1/2 induced by crocidolite fibers in BEAS-2B cells]

  Authors: Xin-Chao Wang, Yi-Ming Wu, James M Samet, Adrew J Ghio

  Zhonghua lao dong wei sheng zhi ye bing za zhi = Zhonghua laodong weisheng zhiyebing zazhi = Chinese journal of industrial hygiene and occupational diseases. 11/2006; 24(10):597-600.

  OBJECTIVE: To explore the characteristic of the signal transduction in BEAS cells induced by the crocidolite fibers. METHODS: The human respiratory airway epithelial cells BEAS-2B were cultured inOBJECTIVE: To explore the characteristic of the signal transduction in BEAS cells induced by the crocidolite fibers. METHODS: The human respiratory airway epithelial cells BEAS-2B were cultured in vitro. The final 100 microg/ml crocidolite concentration and lOnM of epidermal growth factor were cocultured with BEAS-2B cells for 30 minutes and 120 minutes. Phosphorylated ERKl/2 and MEKl/2 were detected by Western Blotting using specific antibodies. RESULTS: A rapid phosphorylation expression of ERK1/2 (molecular weight at 44 kD and 42 kD, also called as p44 and p42) was observed by treatment of the BEAS-2B cells with 100 microg/ml crocidolite or 100 ng/ml EGF (the proven activator of the ERK signaling pathway) at 30 minutes. This phosphorylation could be still detected by incubation the cells at 2 hours. However no expression was changed for the total ERKl/2 expression at 30 minutes or 120 minutes. Treatment of BEAS cells with 100 microg/ml crocidolite fiber or 100 ng/ml EGF led to the rapid increased phosphorylation of MEK1/2 at 30 minutes; similarly, the overexpression of MEK1/2 could last 2 hours. CONCLUSION: The crocidolite induces the MAPK (ERK1/2 and MEK1/2) phosphorylation within a shorter time. It indicates that the MAPKs signals are involved in the process of crocidolite induced damage.

• Zn2+-induced IL-8 expression involves AP-1, JNK, and ERK activities in human airway epithelial cells.

  Authors: Yu-Mee Kim, William Reed, Weidong Wu, Philip A Bromberg, Lee M Graves, James M Samet

  American journal of physiology. Lung cellular and molecular physiology. 06/2006; 290(5):L1028-35.

  Exposure to zinc-laden particulate matter in ambient and occupational settings has been associated with proinflammatory responses in the lung. IL-8 is an important proinflammatory cytokine in theExposure to zinc-laden particulate matter in ambient and occupational settings has been associated with proinflammatory responses in the lung. IL-8 is an important proinflammatory cytokine in the human lung and is induced in human airway epithelial cells exposed to zinc. In this study, we examined the cellular mechanisms responsible for Zn(2+)-induced IL-8 expression. Zn(2+) stimulation resulted in pronounced increases in both IL-8 mRNA and protein expression in the human airway epithelial cell line (BEAS-2B). IL-8 promoter activity was significantly increased by Zn(2+) exposure in BEAS-2B cells, indicating that Zn(2+)-induced IL-8 expression is transcriptionally mediated. Mutation of the activating protein (AP)-1 response element in an IL-8 promoter-enhanced green fluorescent protein construct reduced Zn(2+)-induced IL-8 promoter activity. Moreover, Zn(2+) exposure of BEAS-2B cells induced the phosphorylation of the AP-1 proteins c-Fos and c-Jun. We observed that Zn(2+) exposure induced the phosphorylation of ERK, JNK, and p38 MAPKs, whereas inhibition of ERK or JNK activity blocked IL-8 mRNA and protein expression in BEAS-2B cells treated with Zn(2+). In addition, we investigated the role of protein tyrosine phosphatases in the activation of signaling by Zn(2+). Zn(2+) treatment inhibited ERK- and JNK-directed phosphatase activities in BEAS-2B cells. These results suggested that Zn(2+)-induced inhibition of phosphatase activity is an initiating event in MAPK and AP-1 activation that leads to enhanced IL-8 expression by human airway epithelial cells.