Understanding the Early Biological Effects of Isoprene-Derived Particulate Matter Enhanced by Anthropogenic Pollutants.

INTRODUCTION: Airborne fine particulate matter (PM2.5; particulate matter ≤ 2.5 µm in aerodynamic diameter) plays a key role in air quality, climate, and public health. Globally, the largest mass fraction of PM2.5 is organic, dominated by secondary organic aerosol (SOA) formed from atmospheric oxidation of volatile organic compounds (VOCs). Isoprene from vegetation is the most abundant nonmethane VOC emitted into Earth's atmosphere. Isoprene has been recently recognized as one of the major sources of global SOA production that is enhanced by the presence of anthropogenic pollutants, such as acidic sulfate derived from sulfur dioxide (SO2), through multiphase chemistry of its oxidation products. Considering the abundance of isoprene-derived SOA in the atmosphere, understanding mechanisms of adverse health effects through inhalation exposure is critical to mitigating its potential impact on public health. Although previous studies have examined the toxicological effects of certain isoprene-derived gas-phase oxidation products, to date, no systematic studies have examined the potential toxicological effects of isoprene-derived SOA, its constituents, or its SOA precursors on human lung cells. SPECIFIC AIMS: The overall objective of this study was to investigate the early biological effects of isoprene-derived SOA and its subtypes on BEAS-2B cells (a human bronchial epithelial cell line), with a particular focus on the alteration of oxidative stress- and inflammation-related genes. To achieve this objective, there were two specific aims.1. Examine toxicity and early biological effects of SOA derived from the photochemical oxidation of isoprene, considering both urban and downwind-urban types of chemistry.2. Examine toxicity and early biological effects of SOA derived directly from downstream oxidation products of isoprene (i.e., epoxides and hydroperoxides). METHODS: Isoprene-derived SOA was first generated by photooxidation of isoprene under natural sunlight in the presence of nitric oxide (NO) and acidified sulfate aerosols. Experiments were conducted in a 120-m3 outdoor Teflon-film chamber located on the roof of the Gillings School of Global Public Health, University of North Carolina at Chapel Hill (UNC-Chapel Hill). BEAS-2B cells were exposed to chamber- generated isoprene-derived SOA using the Electrostatic Aerosol in Vitro Exposure System (EAVES). This approach allowed us to generate atmospherically relevant compositions of isoprene-derived SOA and to examine its toxicity through in vitro exposures at an air-liquid interface, providing a more biologically relevant exposure model. Isoprene-derived SOA samples were also collected, concurrently with EAVES sampling, onto Teflon membrane filters for in vitro resuspension exposures and for analysis of aerosol chemical composition by gas chromatography/electron ionization-quadrupole mass spectrometry (GC/EI-MS) with prior trimethylsilylation and ultra-performance liquid-chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry equipped with electrospray ionization (UPLC/ESI-HR-QTOFMS). Isoprene-derived SOA samples were also analyzed by the dithiothreitol (DTT) assay in order to characterize their reactive oxygen species (ROS)-generation potential.Organic synthesis of known isoprene-derived SOA precursors, which included isoprene epoxydiols (IEPOX), methacrylic acid epoxide (MAE), and isoprene-derived hydroxyhydroperoxides (ISOPOOH), was conducted in order to isolate major isoprene-derived SOA formation pathways from each other and to determine which of these pathways (or SOA types) is potentially more toxic. Since IEPOX and MAE produce SOA through multiphase chemistry onto acidic sulfate aerosol, dark reactive uptake experiments of IEPOX and MAE in the presence of acidic sulfate aerosol were performed in a 10-m3 flexible Teflon indoor chamber at UNC-Chapel Hill. Since the generation of SOA from ISOPOOH (through a non-IEPOX route) requires a hydroxyl radical (•OH)-initiated oxidation, ozonolysis of tetramethylethylene (TME) was used to form the needed •OH radicals in the indoor chamber. The resultant low-volatility multifunctional hydroperoxides condensed onto nonacidified sulfate aerosol, yielding the ISOPOOH-derived SOA needed for exposures. Similar to the outdoor chamber SOAs, IEPOX, MAE- and ISOPOOH-derived SOAs were collected onto Teflon membrane filters and were subsequently chemically characterized by GC/EI-MS and UPLC/ESI-HR-QTOFMS as well as for ROS-generation potential using the DTT assay. These filters were also used for resuspension in vitro exposures.By conducting gene expression profiling, we provided mechanistic insights into the potential health effects of isoprene-derived SOA. First, gene expression profiling of 84 oxidative stress- and 249 inflammation-associated human genes was performed for cells exposed to isoprene-derived SOA generated in our outdoor chamber experiments in EAVES or by resuspension. Two pathway-focused panels were utilized for this purpose: (1) nCounter GX Human Inflammation Kit comprised of 249 human genes (NanoString), and (2) Human Oxidative Stress Plus RT2 Profiler PCR Array (Qiagen) comprised of 84 oxidative stress-associated genes. We compared the gene expression levels in cells exposed to SOA generated in an outdoor chamber from photochemical oxidation of isoprene in the presence of NO and acidified sulfate seed aerosol to cells exposed to a dark control mixture of isoprene, NO, and acidified sulfate seed aerosol to isolate the effects of the isoprene-derived SOA on the cells using the EAVES and resuspension exposure methods. Pathway-based analysis was performed for significantly altered genes using the ConsensusPathDB database, which is a database system for the integration of human gene functional interactions to provide biological pathway information for a gene set of interest. Pathway annotation was performed to provide biological pathway information for each gene set. The gene-gene interaction networks were constructed and visualized using the GeneMANIA Cytoscape app (version 3.4.1) to predict the putative function of altered genes. Lastly, isoprene-derived SOA collected onto filters was used in resuspension exposures to measure select inflammatory biomarkers, including interleukin 8 (IL-8) and prostaglandin-endoperoxide synthase 2 (PTGS2) genes, in BEAS-2B cells to ensure that effects observed from EAVES exposures were attributable to particle-phase organic products. Since EAVES and resuspension exposures compared well, gene expression profiling for IEPOX-, MAE- and ISOPOOH-derived SOA were conducted using only resuspension exposures. RESULTS AND CONCLUSIONS: Chemical characterization coupled with biological analyses show that atmospherically relevant compositions of isoprene-derived SOA alter the levels of 41 oxidative stress-related genes. Of the different composition types of isoprene-derived SOA, MAE- and ISOPOOH-derived SOA altered the greatest number of genes, suggesting that carbonyl and hydroperoxide functional groups are oxidative stress promoters. Taken together, the different composition types accounted for 34 of the genes altered by the total isoprene-derived SOA mixture, while 7 remained unique to the total mixture exposures, indicating that there is either a synergistic effect of the different isoprene-derived SOA components or an unaccounted component in the mixture.The high-oxides of nitrogen (NOx) regime, which yielded MAE- and methacrolein (MACR)-derived SOA, had a higher ROS-generation potential (as measured by the DTT assay) than the low- NOx regime, which included IEPOX- and isoprene-derived SOA. However, ISOPOOH-derived SOA, which also formed in the low- NOx regime, had the highest ROS-generation potential, similar to 1,4-naphthoquinone (1,4-NQ). This suggests that aerosol-phase organic peroxides contribute significantly to particulate matter (PM) oxidative potential. MAE- and MACR- derived SOA showed equal or greater ROS-generation potential than was reported in prior UNC-Chapel Hill studies on diesel exhaust PM, highlighting the importance of a comprehensive investigation of the toxicity of isoprene-derived SOA. Notably, ISOPOOH-derived SOA was one order of magnitude higher in ROS-generation potential than diesel exhaust particles previously examined at UNC-Chapel Hill. As an acellular assay, the DTT assay may not be predictive of oxidative stress; therefore, we also focused on the gene expression results from the cellular exposures.We have demonstrated that the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and the redox-sensitive activation protein-1 (AP-1) transcription factor networks have been significantly altered upon exposure to isoprene-derived SOA. The identification of Nrf2 pathway in cells exposed to isoprene-derived SOA is in accordance with our findings using the DTT assay, which measures the thiol reactivity of PM samples as a surrogate for their ROS-generation potential. Specifically, our results point to the cysteine-thiol modifications within cells that lead to activation of Nrf2-related gene expression.However, based on our gene expression results showing no clear relationship between DTT activity and the number of altered oxidative stress-related genes, the DTT activity of isoprene-derived SOA may not be directly indicative of toxicity relative to other SOA types. While activation of Nrf2-associated genes has been identified with responses to oxidative stress and linked to traffic related air pollution exposure in both toxicological and epidemiological studies, their implicit involvement in this study suggests that activation of Nrf2-related gene expression may occur with exposures to all sorts of PM types.By controlling the exposure time, method, and dose we demonstrated that among the SOA derived from previously identified individual precursors of isoprene-derived SOA, ISOPOOH-derived SOA alters more oxidative stress related genes than does IEPOX-derived SOA, but fewer than MAE-derived SOA. This suggests that the composition of MAE-derived SOA may be the greatest contributor to alterations of oxidative stress-related gene expression observed due to isoprene-derived SOA exposure. Further study on induced levels of protein expression and specific toxicological endpoints is necessary to determine if the observed gene expression changes lead to adverse health effects. In addition, such studies have implications for pollution-control strategies because NOx and SO2 are controllable pollutants that can alter the composition of SOA, and in turn alter its effects on gene expression. The mass fraction of different components of atmospheric isoprene derived SOA should be considered, but altering the fraction of high- NOx isoprene-derived SOA (e.g., MAE derived SOA) may yield greater changes in gene expression than altering the fraction of low- NOx isoprene derived SOA types (ISOPOOH- or IEPOX-derived SOA). Finally, this study confirms that total isoprene-derived SOA alters the expression of a greater number of genes than does SOA derived from the tested precursors. This warrants further work to determine the underlying explanation for this observation, which may be uncharacterized components of isoprene-derived SOA or the potential for synergism between the studied components.

Oral Health Effects of Combusted and Smokeless Tobacco Products.

The oral cavity is usually the first part of a consumer's body exposed to the constituents of tobacco products or their emissions. Consequently, the oral cavity is a frequent site for carcinogenic, microbial, immunologic, and clinical effects of tobacco use. This article summarizes 5 presentations on various aspects of oral health affected by combusted or noncombusted tobacco products from a recent conference, "Oral Health Effects of Tobacco Products: Science and Regulatory Policy," sponsored by the American Association for Dental Research and the Food and Drug Administration.

Design and testing of Electrostatic Aerosol in Vitro Exposure System (EAVES): an alternative exposure system for particles.

Conventional in vitro exposure methods for cultured human lung cells rely on prior suspension of particles in a liquid medium; these have limitations for exposure intensity and may modify the particle composition. Here electrostatic precipitation was used as an effective method for such in vitro exposures. An obsolete electrostatic aerosol sampler was modified to provide a viable environment within the deposition field for human lung cells grown on membranous support. Particle deposition and particle-induced toxicological effects for a variety of particles including standardized polystyrene latex spheres (PSL) and diesel exhaust emission particle mixtures are reported. The Electrostatic Aerosol in Vitro Exposure System (EAVES) efficiently deposited particles from an air stream directly onto cells. Cells exposed to the electric field of the EAVES in clean air or in the presence of charged PSL spheres exhibited minimal cytotoxicity, and their release of inflammatory cytokines was indistinguishable from that of the controls. For the responses tested here, there are no significant adverse effects caused neither by the electric field alone nor by the mildly charged particles. Exposure to diesel exhaust emissions using the EAVES system induced a threefold increase in cytokines and cytotoxicity as compared to the control. Taken together, these data show that the EAVES can be used to expose human lung cells directly to particles without prior collection in media, thereby providing an efficient and effective alternative to the more conventional particle in vitro exposure methods.

Selenium deficiency alters epithelial cell morphology and responses to influenza.

It is unknown whether nutritional deficiencies affect the morphology and function of structural cells, such as epithelial cells, and modify the susceptibility to viral infections. We developed an in vitro system of differentiated human bronchial epithelial cells (BEC) grown either under selenium-adequate (Se+) or selenium-deficient (Se-) conditions, to determine whether selenium deficiency impairs host defense responses at the level of the epithelium. Se- BECs had normal SOD activity, but decreased activity of the selenium-dependent enzyme GPX1. Interestingly, catalase activity was also decreased in Se- BECs. Both Se- and Se+ BECs differentiated into a mucociliary epithelium; however, Se- BEC demonstrated increased mucus production and increased Muc5AC mRNA levels. This effect was also seen in Se+ BEC treated with 3-aminotriazole, an inhibitor of catalase activity, suggesting an association between catalase activity and mucus production. Both Se- and Se+ were infected with influenza A/Bangkok/1/79 and examined 24 h postinfection. Influenza-induced IL-6 production was greater while influenza-induced IP-10 production was lower in Se- BECs. In addition, influenza-induced apoptosis was greater in Se- BEC as compared to the Se+ BECs. These data demonstrate that selenium deficiency has a significant impact on the morphology and influenza-induced host defense responses in human airway epithelial cells.

Aspirin enhances multidrug resistance gene 1 expression in human Molt-4 T lymphoma cells.

BACKGROUND: We recently found that aspirin induces the expression of P-glycoprotein (P-gp), a protein mediating drug resistance, in human prostate cancer cells. The purpose of this study was to evaluate the effect of aspirin on the expression of P-gp in a different human cancer type, i.e., T lymphoma. Furthermore, we analyzed this effect at the level of the gene encoding P-gp, MDR1, and of the transcription factor (NF-IL6), regulating this gene. MATERIALS AND METHODS: NF-IL6 was assayed by the electrophoretic mobility shift assay, MDR1 mRNA was assayed by the reverse transcriptase polymerase chain reaction (RT-PCR), and P-gp was assayed by Western blotting. RESULTS: aspirin, at plasma attainable levels, induced NF-IL6 DNA-binding activity, and increased MDR1 mRNA expression (by up to 140%), as well as the expression of P-gp, in Molt-4 cells. CONCLUSIONS: This study suggests that treatment with aspirin induces a cellular signal culminating in the enhancement of P-gp expression in T lymphoma Molt-4 cells.

Gaseous VOCs rapidly modify particulate matter and its biological effects - Part 1: Simple VOCs and model PM.

This is the first of a three-part study designed to demonstrate dynamic entanglements among gaseous organic compounds (VOC), particulate matter (PM), and their subsequent potential biological effects. We study these entanglements in increasingly complex VOC and PM mixtures in urban-like conditions in a large outdoor chamber. To the traditional chemical and physical characterizations of gas and PM, we added new measurements of gas-only- and PM-only-biological effects, using cultured human lung cells as model indicators. These biological effects are assessed here as increases in cellular damage or expressed irritation (i.e., cellular toxic effects) from cells exposed to chamber air relative to cells exposed to clean air. The exposure systems permit gas-only- or PM-only-exposures from the same air stream containing both gases and PM in equilibria, i.e., there are no extractive operations prior to cell exposure.Our simple experiments in this part of the study were designed to eliminate many competing atmospheric processes to reduce ambiguity in our results. Simple volatile and semi-volatile organic gases that have inherent cellular toxic properties were tested individually for biological effect in the dark (at constant humidity). Airborne mixtures were then created with each compound and PM that has no inherent cellular toxic properties for another cellular exposure. Acrolein and p-tolualdehyde were used as model VOCs and mineral oil aerosol (MOA) was selected as a surrogate for organic-containing PM. MOA is appropriately complex in composition to represent ambient PM, and it exhibits no inherent cellular toxic effects and thus did not contribute any biological detrimental effects on its own.Chemical measurements, combined with the responses of our biological exposures, clearly demonstrate that gas-phase pollutants can modify the composition of PM (and its resulting detrimental effects on lung cells) - even if the gas-phase pollutants are not considered likely to partition to the condensed phase: the VOC-modified-PM showed significantly more damage and inflammation to lung cells than did the original PM. Because gases and PM are transported and deposited differently within the atmosphere and the lungs, these results have significant consequences. For example, current US policies for research and regulation of PM do not recognize this "effect modification" phenomena (NAS, 2004).These results present an unambiguous demonstration that - even in these simple mixtures - physical and thermal interactions alone can cause a modification of the distribution of species among the phases of airborne pollution mixtures and can result in a non-toxic phase becoming toxic due to atmospheric thermal processes only. Subsequent work extends the simple results reported here to systems with photochemical transformations of complex urban mixtures and to systems with diesel exhaust produced by different fuels.

Ozone-induced IL-8 expression and transcription factor binding in respiratory epithelial cells.

Ozone, one of the most reactive oxidant gases to which humans are routinely exposed, induces inflammation in the lower airways. The airway epithelium is one of the first targets that inhaled ozone will encounter, but its role in airway inflammation is not well understood. Expression of inducible genes involved in the inflammatory response, such as interleukin (IL)-8, is controlled by transcription factors. Expression of the IL-8 gene is regulated by the transcription factors nuclear factor (NF)-kappaB, NF-IL-6, and possibly activator protein-1 (AP-1). Type II-like epithelial cells (A549) were grown on a collagen-coated membrane and exposed in vitro to 0.1 ppm ozone or air. Exposure to ozone induced DNA-binding activity of NF-kappaB, NF-IL-6, and AP-1. IL-8 mRNA and IL-8 protein levels were also increased after ozone exposure. These results link ozone-induced DNA-binding activity of transcription factors and the production of IL-8 by epithelial cells thus demonstrating a potential cellular cascade resulting in the recruitment of inflammatory cells into the airway lumen.

Changes in the glucose-6-phosphatase complex in hepatomas.

Hepatomas tend to have a decreased glucose-6-phosphatase activity. We have observed phenotypic stability for this change in Morris hepatomas transplanted in rats. To determine if this decrease is selective for translocase functions or the hydrolase activity associated with glucose-6-phosphatase, we have compared activities in liver and hepatomas with glucose-6-phosphate or mannose-6-phosphate as substrates and with intact or histone-disrupted microsomes. In five out of seven subcutaneously transplanted rat hepatoma lines, the microsomal mannose-6-phosphatase activity was lower than in preparations from liver of normal or tumor-bearing rats. With liver microsomes and with most hepatoma microsomes, preincubation with calf thymus histones caused a greater increase in mannose-6-phosphatase than in glucose-6-phosphatase activity. In studies with liver and hepatoma microsomes there were similar increases in mannose-6-phosphatase activity with total calf thymus histones and arginine-rich histones. A smaller increase was seen with lysine-rich histones. The effect of polylysine was similar to the action of lysine-rich histones. There was only a small effect with protamine at the same concentration (1 mg/ml). Rat liver or hepatoma H1 histones gave only about half the activation seen with core nucleosomal histones. Our data suggested that microsomes of rat hepatomas tend to have decreased translocase and hydrolase functions of glucose-6-phosphatase relative to activities in untransformed liver.

Induction of interleukin-8 by ozone is mediated by tyrosine kinase and protein kinase A, but not by protein kinase C.

Ozone is one of the most common air pollutants humans routinely inhale. We have previously shown that in vitro ozone exposure induces the DNA-binding activities of NF-kappaB and NF-IL6 as well as the expression of interleukin 8 in respiratory epithelial cells. In this study, we investigated intracellular signaling steps mediating ozone-induced inflammatory mediator release. A549 cells, a type II like alveolar epithelial cell line, were exposed in vitro to air or 0.1 ppm of ozone in the presence of several kinase inhibitors. Exposure to ozone increased interleukin 8 expression and transcription factor activities in a protein tyrosine kinase (PTK)-dependent and protein kinase A (PKA)-dependent, yet protein kinase C (PKC)-independent, manner. Furthermore, ozone-induced PTK and PKA activities but failed to induce PKC activity. In addition, our results suggest that ozone-induced PTK and PKA activities were reactive oxygen intermediate dependent and occurred in parallel, because specific inhibitors for PTK and PKA failed to block the other kinase's activity. These results indicate that PTK and PKA activities are early events in the signal transduction cascade mediating the ozone-induced activation of NF-kappaB and NF-IL6 as well as the release of interleukin 8.

Vanadium-induced kappaB-dependent transcription depends upon peroxide-induced activation of the p38 mitogen-activated protein kinase.

Activation of nuclear factor (NF)-kappaB and subsequent proinflammatory gene expression in human airway epithelial cells can be evoked by oxidative stress. In this study we examined signal transduction pathways activated by vanadyl sulfate (V(IV))-induced oxidative stress in normal human bronchial epithelial cells. Both nuclear translocation of NF-kappaB and enhanced kappaB-dependent transcription induced by V(IV) were inhibited by overexpression of catalase, but not Cu,Zn superoxide dismutase (Cu,Zn-SOD), indicating that peroxides rather than superoxides initiated signaling. Catalase selectively blocked the response to V(IV) because it inhibited neither NF-kappaB translocation nor kappaB-dependent transcription evoked by the proinflammatory cytokine tumor necrosis factor (TNF)-alpha. The V(IV)-induced kappaB-dependent transcription was dependent upon activation of the p38 mitogen-activated protein kinase because overexpression of dominant-negative mutants of the p38 MAPK pathway inhibited V(IV)-induced kappaB-dependent transcription. This inhibition was not due to suppression of NF-kappaB nuclear translocation because NF-kappaB DNA binding was unaffected by the inhibition of p38 activity. Overexpression of catalase, but not Cu,Zn-SOD, inhibited p38 activation, indicating that peroxides activated p38. Catalase failed to block V(IV)- induced increases in phosphotyrosine levels, suggesting that the catalase-sensitive signaling components were independent of V(IV)-induced tyrosine phosphorylation. The data demonstrate that V(IV)-induced oxidative stress activates at least two distinct pathways, NF-kappaB nuclear translocation and p38-dependent transactivation of NF-kappaB, both of which are required to fully activate kappaB-dependent transcription. Moreover, V(IV)-induced oxidative stress activated these pathways in bronchial epithelial cells by upstream signaling cascades that were distinct at some level from those used by the proinflammatory cytokine TNF-alpha.

Arsenite exposure of cultured airway epithelial cells activates kappaB-dependent interleukin-8 gene expression in the absence of nuclear factor-kappaB 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)-kappaB. We compared intracellular signaling mediating IL-8 gene expression in bronchial epithelial cells cultured in vitro and exposed to two inducers of cellular stress, sodium arsenite (As(III)), and vanadyl sulfate (V(IV)). 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-kappaB depressed both basal and metal-induced IL-8 expression. Low levels of nuclear NF-kappaB were constitutively present in unstimulated cultures. These levels were augmented by exposure to V(IV), but not As(III). Accordingly, V(IV) induced IkappaBalpha breakdown and NF-kappaB nuclear translocation, whereas As(III) did not. However, both As(III) and V(IV) enhanced kappaB-dependent transcription. In addition, As(III) activation of an IL-8 promoter-reporter construct was partially kappaB-dependent. These data suggested that As(III) enhanced IL-8 gene transcription independently of IkappaB breakdown and nuclear translocation of NF-kappaB in part by enhancing transcription mediated by low levels of constitutive nuclear NF-kappaB.

Ozone inactivates cyclooxygenase in human tracheal epithelial cells without altering PGHS-2 mRNA or protein.

Exposure of human tracheal epithelial (TE) cells to ozone (0.1-0.5 ppm) leads to a transient increase followed by decreased production of prostaglandin (PG) E2 concomitant with dose-dependent loss and delayed recovery of cyclooxygenase (CO) activity [S.E. Alpert and R.W. Walenga. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L734-L743, 1995]. Formation of reactive oxygen species (ROS) in cultured tracheobronchial epithelial cells during ozone exposure was recently demonstrated (L.C. Chen and Q.Qu. Toxicol. Appl. Pharmacol. 143: 96-101, 1997). In the present study, we investigated if ROS generated by ozone-exposed human TE cells contribute to PGE2 production and/or CO inactivation and whether the delay in recovery of CO activity after ozone reflects impaired gene transcription and/or protein synthesis. Rapid, dose-dependent ROS generation, assessed by fluorescence of dihydrorhodamine 123, was detected in human TE monolayers exposed to 0.21-0.63 ppm ozone. In a different system, TE cells were exposed to air or 0.5 ppm ozone for 1 h by serial renewal/collection of an adherent film of media. Ozone-induced ROS formation, the transient increase and decline in PGE2, and CO inactivation were attenuated by an intracellular hydroxyl radical scavenger, 1,3-dimethyl-2-thiourea. Ibuprofen, a reversible CO inhibitor, prevented PGE2 release during ozone exposure (and hence autocatalytic CO inactivation) but not loss of CO activity. Although CO activity remained depressed for hours after ozone exposure, compared with air-exposed cultures, no differences were detected in mRNA and protein levels of prostaglandin endoperoxide G/H synthase 2 (PGHS-2), the only CO isoform present in human TE cells, or in the rate of de novo PGHS-2 synthesis. Our findings suggest that ozone-induced PGE2 production and CO inactivation are primarily the result of formation of intracellular oxidant molecules and that delayed recovery of CO activity in human TE cells after short-term ozone exposure is due to persistent inactivation of PGHS-2, rather than to interference with its synthesis.

Hydrogen peroxide has opposing effects on IKK activity and IkappaBalpha breakdown in airway epithelial cells.

Recent studies have advanced our knowledge about the signal transduction cascade involved in the activation of nuclear factor (NF) kappaB, including the identification and characterization of IkappaB kinases (IKKs). Although exposure to hydrogen peroxide (H2O2) in vitro can activate NF-kappaB, this response is not universal and depends on the cell type and transformation state. In this study, we examined the effects of H2O2 on IKKs and activation of NF-kappaB in primary normal human bronchial epithelial (NHBE) cells. Our results demonstrate that treatment with H2O2 increased IKK activity, phosphorylation, and ubiquitination of IkappaBalpha in NHBE cells. However, there was no significant proteolytic degradation of IkappaBalpha, nuclear translocation of p65, or NF-kappaB DNA binding activity in cells treated with H2O2. Treatment with H2O2 also inhibited tumor necrosis factor (TNF)-alpha-induced IkappaBalpha breakdown, NF-kappaB DNA binding activity, and NF-kappaB-dependent transcription but had no effect on TNF-alpha-induced IkappaBalpha phosphorylation or ubiquitination. Furthermore, treatment with H2O2 alone or in combination with TNF-alpha increased the levels of other ubiquitinated proteins in NHBE cells, suggesting general inhibition of proteasomal activity by H2O2. Taken together, these results demonstrate that in airway epithelial cells treatment with H2O2 has opposing effects on IKK activity and proteasomal degradation of IkappaBalpha, and suggest that H2O2 may suppress TNF-alpha-induced NF-kappaB- dependent gene expression.

Tumor necrosis factor-alpha-induced activating protein-1 activity is modulated by nitric oxide-mediated protein kinase G activation.

We tested the hypothesis that protein kinase (PK)G activation in response to nitric oxide ((*)NO) mediates tumor necrosis factor (TNF)-alpha-induced activation of the transcription factor activating protein-1 (AP-1) in pulmonary microvessel endothelial monolayers (PEM). The DNA-binding activity of AP-1 was assessed using the electrophoretic mobility shift assay. TNF treatment (1,000 U/ml) for 4 h induced a significant increase in DNA binding of AP-1. The effects of TNF were prevented by the superoxide radical scavenger superoxide dismutase (SOD) (100 U/ml), the (*)NO synthase inhibitor aminoguanidine (100 microM), the guanylate cyclase inhibitor ODQ (100 microM), and the PKG inhibitors KT5823 (1 microM) and 8-bromo-cyclic guanosine monophosphate (cGMP)-thioate (100 microM). Spermine-NO (1 microM) and L-arginine (400 microM) prevented the aminoguanidine-induced ablation of AP-1 activation in response to TNF. Phosphorylation of H-Arg-Lys-Ile-Ser-Ala-Ser-Glu-Phe-Asp-Arg-Pro-Leu-Arg-OH (BPDEtide), a specific substrate for PKG, measured the activity of cGMP-dependent protein kinase (PKG). TNF for 0.5 h induced an increase in PKG activity that was prevented by aminoguanidine, ODQ, KT5823, and 8-bromo-cGMP-thioate; however, SOD had no effect. The PKG agonist 8-bromo-cGMP (100 microM), when given alone, increased PKG activity but induced significant DNA-binding activity of AP-1 only when given in the ODQ + TNF Group. SIN-1 (1 mM, a peroxynitrite agonist) increased DNA-binding activity of AP-1. SOD prevented SIN-1-induced AP-1 activation, a response similar to that of the SOD + TNF Group. PEM were transfected with the chloramphenicol acetyltransferase (CAT) reporter plasmid pBLCAT2, which contains a regulation sequence responsive to AP-1. The pharmacologic profile of TNF-induced CAT activity was identical to TNF-induced DNA binding by AP-1. Thus, TNF-induced AP-1-dependent gene transcription is modulated by (*)NO-dependent mediated activation of PKG.

A human and mouse homolog of the Schizosaccharomyces pombe rad1+ cell cycle checkpoint control gene.

The Schizosaccharomyces pombe rad1+ cell cycle checkpoint control gene is required for S-phase and G2/M arrest in response to both DNA damage and incomplete DNA replication. We isolated and characterized the putative human RAD1 (hRAD1) and mouse RAD1 (mRAD1) homologs of the S. pombe Rad1 (Rad1) protein. The human RAD1 open reading frame (ORF) encodes a protein of 282 amino acids; the mRAD1 ORF codes for a protein of 280 amino acids. The human RAD1 and mRAD1 messengers are highly expressed in the testis as different mRNA species (varying from 1.0, 1.4, 1.5, to 3.0 kb). The hRAD1 and mRAD1 proteins are 30% identical and 56% similar to the S. pombe Rad1 protein. Sequence homology was also noted with the Saccharomyces cerevisiae Rad17p, the putative 3'-5' exonuclease Rec1 from Ustilago maydis, and the structurally related polypeptides from Arabidopsis thaliana and Caenorhabditis elegans. The degree of conservation between the mammalian RAD1 proteins and those of the other species is consistent with the evolutionary distance between the species, implicating that these proteins are most likely true counterparts. Together, this suggests that the structure and function of the checkpoint "rad" genes in the G2/M checkpoint pathway are evolutionarily conserved between yeasts and higher eukaryotes. The human RAD1 gene could be localized on human chromosome 5p13, a region that has been implicated in the etiology of small cell lung carcinomas, squamous cell carcinomas, adenocarcinomas, and bladder cancer.

Human and mouse homologs of the Schizosaccharomyces pombe rad17+ cell cycle checkpoint control gene.

The Schizosaccharomyces pombe rad17+ cell cycle checkpoint control gene is required for S-phase and G2/M arrest in response to both DNA damage and incomplete DNA replication. We isolated and characterized the putative human (RAD17Sp) and mouse (mRAD17Sp) homologs of the S. pombe Rad17 (Rad17Sp) protein. The human RAD17Sp open reading frame (ORF) encodes a protein of 681 amino acids; the mRAD17Sp ORF codes for a protein of 688 amino acids. The mRAD17Sp messenger is highly expressed in the testis as a single 3-kb mRNA species. The human RAD17Sp and mRAD17Sp proteins are 24% identical and 46% similar to the S.pombe Rad17Sp protein. Sequence homology was also noted with the Saccharomyces cerevisiae Rad24Sc (which is the structural counterpart of S.pombe Rad17Sp) and structurally related polypeptides from Caenorhabditis elegans, Arabidopsis thaliana, Pyrococcus horikoshii, and Drosophila melanogaster. The degree of conservation between the mammalian RAD17Sp proteins and those of the other species is consistent with the evolutionary distance between the species, indicating that these proteins are most likely true counterparts. In addition, homology was found between the Rad17Sp homologs and proteins identified as components of mammalian replication factor C (RF-C)/activator 1, especially in several highly conserved RF-C-like domains including a "Walker A" motif. Using FISH and analysis of a panel of rodent-human cell hybrids, the human RAD17Sp gene (HGMW-approved symbol RAD17 could be localized on human chromosome 5q13-q14, a region implicated in the etiology of small cell lung carcinoma, non-small-cell lung carcinoma, duodenal adenocarcinoma, and head and neck squamous cell carcinoma. Our results suggest that the structure and function of the checkpoint "rad" genes in the G2/M checkpoint pathway are evolutionary conserved between yeast and higher eukaryotes.

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.