James M. Samet

Activation of MAPKs in human bronchial epithelial cells exposed to metals

We have previously shown that in vitro exposure to metallic compounds enhances expression of interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha in human bronchial epithelial cells. To characterize signaling pathways involved in metal-induced expression of inflammatory mediators and to identify metals that activate them, we studied the effects of As, Cr, Cu, Fe, Ni, V, and Zn on the mitogen-activated protein kinases (MAPK) extracellular receptor kinase (ERK), c-Jun NH2-terminal kinase (JNK), and P38 in BEAS cells. Noncytotoxic concentrations of As, V, and Zn induced a rapid phosphorylation of MAPK in BEAS cells. Activity assays confirmed marked activation of ERK, JNK, and P38 in BEAS cells exposed to As, V, and Zn. Cr and Cu exposure resulted in a relatively small activation of MAPK, whereas Fe and Ni did not activate MAPK under these conditions. Similarly, the transcription factors c-Jun and ATF-2, substrates of JNK and P38, respectively, were markedly phosphorylated in BEAS cells treated with As, Cr, Cu, V, and Zn. The same acute exposure to As, V, or Zn that activated MAPK was sufficient to induce a subsequent increase in IL-8 protein expression in BEAS cells. These data suggest that MAPK may mediate metal-induced expression of inflammatory proteins in human bronchial epithelial cells.

Effects of aqueous extracts of PM10 filters from the Utah Valley on human airway epithelial cells

We hypothesized that the reduction in hospital respiratory admissions in the Utah Valley during closure of a local steel mill in 1986-1987 was attributable in part to decreased toxicity of ambient air particles. Sampling filters for particulate matter < 10 μm (PM10) were obtained from a Utah Valley monitoring station for the year before ( year 1), during ( year 2), and after ( year 3) the steel mill closure. Aqueous extracts of the filters were analyzed for metal content and oxidant production and added to cultures of human respiratory epithelial (BEAS-2B) cells for 2 or 24 h. Year 2 dust contained the lowest concentrations of soluble iron, copper, and zinc and showed the least oxidant generation. Only dust from year 3 caused cytotoxicity (by microscopy and lactate dehydrogenase release) at 500 μg/ml. Year 1 and year 3, but not year 2, dust induced expression of interleukin-6 and -8 in a dose-response fashion. The effects of ambient air particles on human respiratory epithelial cells vary significantly with time and metal concentrations.We hypothesized that the reduction in hospital respiratory admissions in the Utah Valley during closure of a local steel mill in 1986-1987 was attributable in part to decreased toxicity of ambient air particles. Sampling filters for particulate matter < 10 micrometer (PM(10)) were obtained from a Utah Valley monitoring station for the year before (year 1), during (year 2), and after (year 3) the steel mill closure. Aqueous extracts of the filters were analyzed for metal content and oxidant production and added to cultures of human respiratory epithelial (BEAS-2B) cells for 2 or 24 h. Year 2 dust contained the lowest concentrations of soluble iron, copper, and zinc and showed the least oxidant generation. Only dust from year 3 caused cytotoxicity (by microscopy and lactate dehydrogenase release) at 500 microgram/ml. Year 1 and year 3, but not year 2, dust induced expression of interleukin-6 and -8 in a dose-response fashion. The effects of ambient air particles on human respiratory epithelial cells vary significantly with time and metal concentrations.

Activation of the EGF receptor signaling pathway in human airway epithelial cells exposed to metals.

We have previously shown that exposure to combustion-derived metals rapidly (within 20 min) activated mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK), in the human bronchial epithelial cell line BEAS. To study the mechanisms responsible for metal-induced activation of ERK, we examined the effect of noncytotoxic exposures to As, Cu, V, or Zn on the kinases upstream of ERK in the epidermal growth factor (EGF) receptor signaling pathway. Western blotting using phospho-specific ERK1/2 antibody demonstrated the selective MEK1/2 inhibitor PD-98059 blocked metal-induced phosphorylation of ERK1/2. Meanwhile, Western blotting using a phospho-specific MEK1/2 antibody showed that these metals induce a rapid phosphorylation of MEK1/2. Kinase activity assays confirmed the activation of MEK1/2 by metal treatment. Immunoprecipitation studies demonstrated that As, Cu, V, or Zn induces EGF receptor phosphorylation. Furthermore, the EGF receptor-specific tyrosine kinase inhibitor (PD-153035) significantly blocked the phosphorylation of MEK1/2 initiated by metals. Interestingly, we observed low levels of Raf-1 activity that were not increased by metal exposure in these cells through kinase activity assay. Finally, transfection assays showed that MEK1/2 inhibition could inhibit trans-activation of Elk1, a transcription factor in the ERK pathway, in BEAS cells exposed to metals. Together, these data demonstrate that As, Cu, V, and Zn can activate the EGF receptor signaling pathway in BEAS cells and suggest that this mechanism may be involved in pulmonary responses to metal inhalation.We have previously shown that exposure to combustion-derived metals rapidly (within 20 min) activated mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK), in the human bronchial epithelial cell line BEAS. To study the mechanisms responsible for metal-induced activation of ERK, we examined the effect of noncytotoxic exposures to As, Cu, V, or Zn on the kinases upstream of ERK in the epidermal growth factor (EGF) receptor signaling pathway. Western blotting using phospho-specific ERK1/2 antibody demonstrated the selective MEK1/2 inhibitor PD-98059 blocked metal-induced phosphorylation of ERK1/2. Meanwhile, Western blotting using a phospho-specific MEK1/2 antibody showed that these metals induce a rapid phosphorylation of MEK1/2. Kinase activity assays confirmed the activation of MEK1/2 by metal treatment. Immunoprecipitation studies demonstrated that As, Cu, V, or Zn induces EGF receptor phosphorylation. Furthermore, the EGF receptor-specific tyrosine kinase inhibitor (PD-153035) significantly blocked the phosphorylation of MEK1/2 initiated by metals. Interestingly, we observed low levels of Raf-1 activity that were not increased by metal exposure in these cells through kinase activity assay. Finally, transfection assays showed that MEK1/2 inhibition could inhibit trans-activation of Elk1, a transcription factor in the ERK pathway, in BEAS cells exposed to metals. Together, these data demonstrate that As, Cu, V, and Zn can activate the EGF receptor signaling pathway in BEAS cells and suggest that this mechanism may be involved in pulmonary responses to metal inhalation.

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

RATIONALE 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. OBJECTIVES To characterize the effects of acute exposure to ambient ultrafine particles in young healthy humans. METHODS Nineteen healthy nonsmoking 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 microm) 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 (ultrafine concentrated ambient particles [UFCAPs]). Subjects underwent bronchoalveolar lavage 18 hours after 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 UFCAPs 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 prothrombic responses and are suggestive of alterations in cardiac repolarization induced by UFCAP inhalation.

Mechanisms of Zn2+-induced signal initiation through the epidermal growth factor receptor ☆

Zn(2+) is a ubiquitous ambient air contaminant that is found as a constituent of airborne particulate matter (PM). Previous studies have associated Zn(2+) levels in PM with health effects in exposed populations and have shown proinflammatory properties of Zn(2+) exposure in vivo and in vitro. In the present study, we studied the mechanisms of epidermal growth factor receptor (EGFR) dimerization, phosphorylation, and kinase activity in A431 cells treated with Zn(2+). EGF, but not Zn(2+), induced dimerization of EGFR in A431 cells and membrane extracts. Like EGF, Zn(2+) induced phosphorylation of EGFR at tyrosines 845, 1068, and 1173. However, unlike EGF, Zn(2+) failed to induce detectable dimerization of EGFR. The EGFR kinase inhibitor PD153035 ablated all phosphorylation induced by EGF but none caused by Zn(2+). PD153035 abolished EGF-induced phosphorylation of the EGFR substrate Cbl, but had no effect on levels of phospho-Cbl caused by Zn(2+). Inhibition of EGFR kinase activity did, however, blunt Zn(2+)-induced phosphorylation of ERK. Exposure to Zn(2+), but not EGF, induced phosphorylation of the activating site of c-Src (tyrosine 416), and Zn(2+)-induced phosphorylation of EGFR at tyrosines 845 and 1068 was blocked by the c-Src kinase activity inhibitor PP2. In summary, Zn(2+) ions induce EGFR phosphorylation in a manner dependent on c-Src but not on EGFR dimerization or EGFR kinase activation, suggesting that Zn(2+) induces EGFR transactivation by c-Src.

Phosphorylation of P65 Is Required for Zinc Oxide Nanoparticle-Induced Interleukin 8 Expression in Human Bronchial Epithelial Cells

Background 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). Objective We investigated the effect of ZnO nanoparticles on IL-8 expression and the underlying mechanisms in human bronchial epithelial cells. Methods 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 (NFκB) 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 IκBα, an inhibitor of NF-κB, and phosphorylation of p65 were detected using immunoblotting. Binding of p65 to the IL-8 promoter was examined using the chromatin immunoprecipitation assay. Results ZnO exposure (2–8 μg/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 κB-binding site in the IL-8 promoter was required for ZnO-induced IL-8 transcriptional activation. ZnO stimulation modestly elevated IκBα 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. Conclusion p65 phosphorylation and IκBα phosphorylation and degradation are the primary mechanisms involved in ZnO nanoparticle-induced IL-8 expression in human bronchial epithelial cells.

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

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 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.

A Comparison of Studies on the Effects of Controlled Exposure to Fine, Coarse and Ultrafine Ambient Particulate Matter from a Single Location

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 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.

Differential transcriptional regulation of IL-8 expression by human airway epithelial cells exposed to diesel exhaust particles

Exposure to diesel exhaust particles (DEP) induces inflammatory signaling characterized by MAP kinase-mediated activation of NFkB and AP-1 in vitro and in bronchial biopsies obtained from human subjects exposed to DEP. NFkB and AP-1 activation results in the upregulation of genes involved in promoting inflammation in airway epithelial cells, a principal target of inhaled DEP. IL-8 is a proinflammatory chemokine expressed by the airway epithelium in response to environmental pollutants. The mechanism by which DEP exposure induces IL-8 expression is not well understood. In the current study, we sought to determine whether DEP with varying organic content induces IL-8 expression in lung epithelial cells, as well as, to develop a method to rapidly evaluate the upstream mechanism(s) by which DEP induces IL-8 expression. Exposure to DEP with varying organic content differentially induced IL-8 expression and IL-8 promoter activity human airway epithelial cells. Mutational analysis of the IL-8 promoter was also performed using recombinant human cell lines expressing reporters linked to the mutated promoters. Treatment with a low organic-containing DEP stimulated IL-8 expression by a mechanism that is predominantly NFkB-dependent. In contrast, exposure to high organic-containing DEP induced IL-8 expression independently of NFkB through a mechanism that requires AP-1 activity. Our study reveals that exposure to DEP of varying organic content induces proinflammatory gene expression through multiple specific mechanisms in human airway epithelial cells. The approaches used in the present study demonstrate the utility of a promoter-reporter assay ensemble for identifying transcriptional pathways activated by pollutant exposure.

Arsenite Exposure of Cultured Airway Epithelial Cells Activates κB-dependent Interleukin-8 Gene Expression in the Absence of Nuclear Factor-κB Nuclear Translocation

Airway epithelial cells respond to certain environmental stresses by mounting a proinflammatory response, which is characterized by enhanced synthesis and release of the neutrophil chemotactic and activating factor interleukin-8 (IL-8). IL-8 expression is regulated at the transcriptional level in part by the transcription factor nuclear factor (NF)-κB. We compared intracellular signaling mediating IL-8 gene expression in bronchial epithelial cells culturedin vitro and exposed to two inducers of cellular stress, sodium arsenite (AsIII), and vanadyl sulfate (VIV). Unstimulated bronchial epithelial cells expressed IL-8, and exposure to both metal compounds significantly enhanced IL-8 expression. Overexpression of a dominant negative inhibitor of NF-κB depressed both basal and metal-induced IL-8 expression. Low levels of nuclear NF-κB were constitutively present in unstimulated cultures. These levels were augmented by exposure to VIV, but not AsIII. Accordingly, VIV induced IκBα breakdown and NF-κB nuclear translocation, whereas AsIIIdid not. However, both AsIII and VIV enhanced κB-dependent transcription. In addition, AsIII activation of an IL-8 promoter-reporter construct was partially κB-dependent. These data suggested that AsIII enhanced IL-8 gene transcription independently of IκB breakdown and nuclear translocation of NF-κB in part by enhancing transcription mediated by low levels of constitutive nuclear NF-κB.