Heterogeneous ozone effects on the DNA methylome of bronchial cells observed in a crossover study.

We used a randomized crossover experiment to estimate the effects of ozone (vs. clean air) exposure on genome-wide DNA methylation of target bronchial epithelial cells, using 17 volunteers, each randomly exposed on two separated occasions to clean air or 0.3-ppm ozone for two hours. Twenty-four hours after exposure, participants underwent bronchoscopy to collect epithelial cells whose DNA methylation was measured using the Illumina 450 K platform. We performed global and regional tests examining the ozone versus clean air effect on the DNA methylome and calculated Fisher-exact p-values for a series of univariate tests. We found little evidence of an overall effect of ozone on the DNA methylome but some suggestive changes in PLSCR1, HCAR1, and LINC00336 DNA methylation after ozone exposure relative to clean air. We observed some participant-to-participant heterogeneity in ozone responses.

Antioxidant supplementation and nasal inflammatory responses among young asthmatics exposed to high levels of ozone.

The inflammatory response to ozone in atopic asthma suggests that soluble mediators of inflammation are released in response to oxidant stress. Antioxidants may alleviate additional oxidative stress associated with photochemical oxidant pollution. This study investigates the impact of antioxidant supplementation on the nasal inflammatory response to ozone exposure in atopic asthmatic children. We conducted a randomized trial using a double-blinded design. Children with asthma (n = 117), residents of Mexico City, were given randomly a daily supplement of vitamins (50 mg/day of vitamin E and 250 mg/day of vitamin C) or placebo. Nasal lavages were performed three times during the 4-month follow-up and analysed for content of interleukin-6 (IL-6), IL-8, uric acid and glutathione (GSx). IL-6 levels in the nasal lavage were increased significantly in the placebo group after ozone exposure while no increase was observed in the supplement group. The difference in response to ozone exposure between the two groups was significant (P = 0.02). Results were similar for IL-8, but with no significant difference between the groups (P = 0.12). GSx decreased significantly in both groups. Uric acid decreased slightly in the placebo group. Our data suggest that vitamin C and E supplementation above the minimum dietary requirement in asthmatic children with a low intake of vitamin E might provide some protection against the nasal acute inflammatory response to ozone.

Elderly humans exposed to concentrated air pollution particles have decreased heart rate variability.

Air pollution particles are thought to kill > 500,000 people worldwide each year. The population most at risk appears to be elderly people with respiratory and cardiovascular disease. As yet, no commonly accepted mechanism has been proposed which can explain the cause of these deaths. Heart rate variability (HRV) was assessed in healthy elderly adults between the ages of 60 and 80 who were exposed twice for 2 h: once to clean air and once to concentrated ambient air pollution particles (CAPS). Changes in HRV were measured immediately before, immediately following, and 24 h after exposure. Elderly subjects experienced significant decreases in HRV in both time and frequency domains immediately following exposure. Some of these changes persisted for at least 24 h. These data were compared with HRV data collected from young healthy volunteers exposed to CAPS in a previous study, in which no CAPS-induced changes in HRV were found. These concentrated ambient air pollution particle-induced changes in heart rate variability in a controlled human exposure study extend similar findings reported in recent panel studies and suggest potential mechanisms by which particulate matter may induce adverse cardiovascular events.

Attenuation and recovery of pulmonary injury in rats following short-term, repeated daily exposure to ozone.

Controlled human and epidemiology studies have demonstrated that during repeated exposure to ozone (O(3)) attenuation of lung function responses may occur. It is yet unknown whether inflammatory and biochemical effects in lower airways of humans, as observed upon single O(3) exposure, also show a diminutive response following repeated exposure to O(3). The aim of this study was to investigate inflammatory, permeability, and histopathological responses in lungs of rats following repeated daily O(3) exposure and to study the time course of attenuation and recovery of these effects using single O(3) challenges at various postexposure times. To aid in animal-to-human extrapolation, this study and a previously reported human study (Devlin et al., 1997) were designed with similar protocols. Wistar rats were exposed for 5 consecutive days to 0.4 ppm O(3) for 12 h/night. Subsequently, the time course of postexposure recovery was determined by a single challenge of 12 h to 0.4 ppm O(3) after a 5-, 10-, 15-, or 20-day recovery period. Bronchoalveolar lavage (BAL) examination and histopathology were performed 12 h after this O(3) challenge. To quantify the magnitude of the O(3) response, results were compared with a group exposed only once for 12 h to 0.4 ppm O(3) and sacrificed simultaneously. The results demonstrate that a single exposure of 0.4 ppm O(3) causes marked permeability and inflammatory responses in lower airways of rats, as evidenced by enhanced BAL fluid levels of proteins, fibronectin, interleukin (IL)-6, and inflammatory cells. However, 5 days of exposure to 0.4 ppm O(3) for 12 h/night resulted in a complete disappearance of these responses, resulting in BAL fluid values that were not different from those observed in unexposed controls. Postexposure analyses of pulmonary response to O(3) challenges demonstrated that these attenuated responses show a gradual recovery. The data indicate that with respect to BAL fluid levels of albumin, IL-6, and number of macrophages and neutrophils, the period for lung tissue to regain its full susceptibility and responsiveness to O(3) following a 5-day preexposure period is approximately 15-20 days. Remarkably, the total protein and fibronectin responses in BAL fluid still exhibited an attenuated response to an O(3) challenge at 20 days postexposure. Morphometry (number of BrdU-labeled cells in terminal bronchiolar epithelium, and number of alveolar macrophages) showed that after a recovery of 5-10 days following a 5-day preexposure the response to a challenge was identical to that after a single exposure. These results suggest that complete repair from lower airway inflammation caused by short-term, repeated exposure to O(3) may take longer than previously assumed.

Nasal biopsies of children exposed to air pollutants.

Southwest Metropolitan Mexico City (SWMMC) atmosphere is a complex mixture of air pollutants, including ozone, particulate matter, and aldehydes. Children in SWMMC are exposed chronically and sequentially to numerous toxicants, and they exhibit significant nasal damage. The objective of this study was to assess p53 accumulation by immunohistochemistry in nasal biopsies of SWMMC children. We evaluated 111 biopsies from 107 children (83 exposed SWMMC children and 24 control children residents in a pollutant-compliant Caribbean island). Complete clinical histories and physical examinations, including an ear-nose-throat (ENT) exam were done. There was a significant statistical difference in the upper and lower respiratory symptomatology and ENT findings between control and exposed children (p < 0.001). Control children gave no respiratory symptomatology in the 3 months prior to the study; their biopsies exhibited normal ciliated respiratory epithelium and were p53-negative. SWMMC children complained of epistaxis, nasal obstruction. and crusting. Irregular areas of whitish-gray recessed mucosa over the inferior and middle turbinates were seen in 25% of SWMMC children, and their nasal biopsies displayed basal cell hyperplasia, decreased numbers of ciliated and goblet cells, neutrophilic epithelial infiltrates, squamous metaplasia. and mild dysplasia. Four of 21 SWMMC children with grossly abnormal mucosal changes exhibited strong transmural nuclear p53 staining in their nasal biopsies (p 0.005, odds ratio 26). In the context of lifetime exposures to toxic and potentially carcinogenic air pollutants, p53 nasal induction in children could potentially represent. a) a checkpoint response to toxic exposures, setting up a selective condition for p53 mutation, or b) a p53 mutation has already occurred as a result of such selection. Because the biological significance of p53 nuclear accumulation in the nasal biopsies of these children is not clear at this point, we strongly suggest that children with macroscopic nasal mucosal abnormalities should be closely monitored by the ENT physician. Parents should be advised to decrease the children's number of outdoor exposure hours and encourage a balanced diet with an important component of fresh fruits and vegetables.

Diffuse alveolar damage after exposure to an oil fly ash.

Epidemiological investigation has established an association between exposure to particulate matter (PM) and both human mortality and diverse indices of human morbidity. However, attributing adverse health effects of specific individuals to PM exposure in these studies is not possible. Consequently, their clinical presentation remains ill-defined. We describe a 42-yr-old male with both respiratory damage, abnormal blood end points, and cardiac effects following an exposure to an emission source air pollution particle aerosolized during the cleaning of his domestic oil-burning stove. Early symptoms of shortness of breath and wheezing progressed over 2 wk to hypoxic respiratory failure necessitating mechanical ventilation. Blood indices were abnormal. Thoracoscopic biopsy demonstrated particle-laden macrophages and diffuse alveolar damage. Symptomatic and objective improvement rapidly followed initiation of corticosteroids. He developed typical anginal symptoms within 2 wk of discharge; however, coronary angiography did not identify any significant narrowing of the epicardial coronary arteries. This patient presents with the aggregate of potential injuries described by epidemiological methods to be associated with air pollution particle exposure.

Inhalation of PM2.5 does not modulate host defense or immune parameters in blood or lung of normal human subjects.

We tested the hypothesis that exposure of healthy volunteers to concentrated ambient air particles (CAPS) between 0.1 and 2.5 microm in diameter is associated with modulation of human alveolar macrophage (AM) function, cytokine production, and immune phenotype in both blood and lung. Thirty-eight volunteers were exposed to either filtered air or CAPS from the immediate environment of the U.S. Environmental Protection Agency human studies facility in Chapel Hill, North Carolina, USA. Particle concentrations in the chamber during the exposures ranged from 23.1 to 311.1 microg/m3. No symptoms were noted by volunteers after the exposure. Eighteen hours after exposure, analysis of cells obtained by bronchoalveolar lavage (BAL) showed a mild increase in neutrophils in both the bronchial (8.4 +/- 2%) and alveolar fractions (4.2 +/- 1.7%) in subjects exposed to the highest concentration of CAPS compared to neutrophils in the fluids of those exposed to filtered air (bronchial fraction 2.7 +/- 0.6%; alveolar fraction 0.8 +/- 0.3%). There was no change in the percentage of lymphocytes or AMs recovered in the lavage after inhalation of the highest particle levels (mean 207 microg/m3). There was also no change in the proportion of lymphocytes in the BAL expressing CD3, CD4, CD8, CD19, nor activation markers CD25 or CD69. Particle inhalation did not affect the expression of CD11b, CD64, CD16, CD14, CD71 on AM, nor was there an effect on phagocytosis or oxidant generation following stimulation with zymosan A. IL-6 and IL-8 levels detected by enzyme-linked immunoabsorbent assay in the BAL were unrelated to inhaled particle levels. The distribution of lymphocyte subsets in blood obtained 18 hr after exposure to CAPS did not differ from that found before exposure. We conclude that ambient air particles are capable of inducing a mild inflammation in the lower respiratory tract but have no effect on immune phenotype or macrophage function under the conditions tested.

Effect of antioxidant supplementation on ozone-induced lung injury in human subjects.

To determine whether antioxidants can influence human susceptibility to ozone (O(3))-induced changes in lung function and airway inflammation, we placed 31 healthy nonsmoking adults (18 to 35 yr old) on a diet low in ascorbate for 3 wk. At 1 wk, subjects were exposed to filtered air for 2 h while exercising (20 L/min/m(2)), and then underwent bronchoalveolar lavage (BAL) and were randomly assigned to receive either a placebo or 250 mg of vitamin C, 50 IU of alpha-tocopherol, and 12 oz of vegetable cocktail daily for 2 wk. Subjects were then exposed to 0.4 ppm O(3) for 2 h and underwent a second BAL. On the day of the O(3) exposure, supplemented subjects were found to have significantly increased levels of plasma ascorbate, tocopherols, and carotenoids as compared with those of the placebo group. Pulmonary function testing showed that O(3)-induced reductions in FEV(1) and FVC were 30% and 24% smaller, respectively, in the supplemented cohort. In contrast, the inflammatory response to O(3) inhalation, as represented by the percent neutrophils and the concentration of interleukin-6 recovered in the BAL fluid at 1 h after O(3) exposure was not different for the two groups. These data suggest that dietary antioxidants protect against O(3)-induced pulmonary function decrements in humans.

Inflammatory lung injury after bronchial instillation of air pollution particles.

Epidemiologic investigation has established an association between exposure to particulate matter (PM) and human health in the Utah Valley. Reduction of particle mass during the temporary closure of a local steel mill was associated with diminished morbidity and mortality. We tested the hypothesis that the biologic effect of PM would reflect findings of epidemiology with a greater injury after exposure to an equal mass of particles from those years in which the mill was in operation. Filters containing PM were collected prior to closure of the steel mill, during the closure, and after its reopening. Aqueous extracts of the filters were prepared. One of three extracts (500 microg) was instilled through the bronchoscope into the lungs of nonsmoking volunteers. Twenty-four hours later, the same subsegment was lavaged. Exposure to aqueous extracts of PM collected before closure and after reopening of the steel mill provoked a greater inflammatory response relative to PM extract acquired during the plant shutdown. This is the first demonstration that pulmonary effects after experimental exposure of humans to PM can correlate with health outcomes observed in epidemiologic studies of the same material under normal exposure conditions. Findings suggest that mass may not be the most appropriate metric to use in assessing health effects after PM exposure but rather specific components must be identified and assessed.

Activation of the EGF receptor signaling pathway in airway epithelial cells exposed to Utah Valley PM.

Exposure to ambient particulate matter (PM) in the Utah Valley has previously been associated with a variety of adverse health effects. To investigate intracellular signaling mechanisms for pulmonary responses to Utah Valley PM inhalation, human primary airway epithelial cells were exposed to aqueous extracts of PM collected from the year before (Y1), during (Y2), and after (Y3) the closure of a local steel mill located in the Utah Valley in this study. Transfection with kinase-deficient extracellular signal-regulated kinase (ERK) 1 constructs partially blocked Utah Valley PM-induced interleukin (IL)-8 promoter reporter activity. The mitogen-activated protein kinase/ERK kinase (MEK) activity inhibitor PD-98059 significantly abolished IL-8 released in response to Utah Valley PM, as did the epidermal growth factor (EGF) receptor kinase inhibitor AG-1478. Western blotting showed that Utah Valley PM induced phosphorylation of EGF receptor tyrosine, MEK1/2, and ERK1/2, which could be ablated with AG-1478 or PD-98059. For all findings, the potency of Utah Valley PM collected during Y2 was found to be lower relative to that of Y1 and Y3. These data demonstrate that Utah Valley PM can induce IL-8 expression partially through the activation of the EGF receptor signaling.

Canines as sentinel species for assessing chronic exposures to air pollutants: part 1. Respiratory pathology.

A complex mixture of air pollutants is present in the ambient air in urban areas. People, animals, and vegetation are chronically and sequentially exposed to outdoor pollutants. The objective of this first of 2 studies is to evaluate by light and electron microscopy the lungs of Mexico City dogs and compare the results to those of 3 less polluted cities in MEXICO: One hundred fifty-two clinically healthy stray mongrel dogs (91 males/61 females), including 43 dogs from 3 less polluted cities, and 109 from southwest and northeast metropolitian Mexico City (SWMMC, NEMMC) were studied. Lungs of dogs living in Mexico City and Cuernavaca exhibited patchy chronic mononuclear cell infiltrates along with macrophages loaded with particulate matter (PM) surrounding the bronchiolar walls and extending into adjacent vascular structures; bronchiolar epithelial and smooth muscle hyperplasia, peribronchiolar fibrosis, microthrombi, and capillary and venule polymorphonuclear leukocytes (PMN) margination. Ultrafine PM was seen in alveolar type I and II cells, endothelial cells, interstitial macrophages (Mtheta), and intravascular Mtheta-like cells. Bronchoalveolar lavage showed significant numbers of alveolar macrophages undergoing proliferation. Exposure to complex mixtures of pollutants-predominantly particulate matter and ozone-is causing lung structural changes induced by the sustained inflammatory process and resulting in airway and vascular remodeling and altered repair. Cytokines released from both, circulating inflammatory and resident lung cells in response to endothelial and epithelial injury may be playing a role in the pathology described here. Deep concern exists for the potential of an increasing rise in lung diseases in child populations exposed to Mexico City's environment.

Canines as sentinel species for assessing chronic exposures to air pollutants: part 2. Cardiac pathology.

The principal objective of this study is to evaluate by light and electron microscopy (LM, EM) the heart tissues in stray southwest and northeast metropolitan Mexico City (SWMMC, NEMMC) dogs and compare their findings to those from 3 less polluted cities (Cuernavaca, Tlaxcala, and Tuxpam). Clinically healthy mongrel dogs, including 109 from highly polluted SWMMC and NEMMC, and 43 dogs from less polluted cities were studied. Dogs residing in cities with lower levels of pollutants showed little or no cardiac abnormalities. Mexico City and Cuernavaca dogs exhibited LM myocardial alterations including apoptotic myocytes, endothelial and immune effector cells, degranulated mast cells associated with scattered foci of mononuclear cells in left and right ventricles and interventricular septum, and clusters of adipocytes interspersed with mononuclear cells. Vascular changes included scattered polymorphonuclear leukocytes (PMN) margination and microthrombi in capillaries, and small venous and arteriolar blood vessels. Small veins exhibited smooth muscle cell hyperplasia, and arteriolar blood vessels showed deposition of particulate matter (PM) in the media and adventitia. Unmyelinated nerve fibers showed endoneural and epineural degranulated mast cells. EM examination of myocardial mast cells showed distended and abundant rough endoplasmic reticulum with few secretory granules. Myocardial capillaries exhibited fibrin deposition and their endothelial cells displayed increased luminal and abluminal pinocytic activity and the formation of anemone-like protrusions of the endothelium into the lumen. A close association between myocardial findings, lung epithelial and endothelial pathology, and chronic inflammatory lung changes was noted. The myocardial changes described in dogs exposed to ambient air pollutants may form the basis for developing hypothesis-driven mechanistic studies that might explain the epidemiological data of increased cardiovascular morbidity and mortality in people exposed to air pollutants.

Ultrastructural nasal pathology in children chronically and sequentially exposed to air pollutants.

Southwest Metropolitan Mexico City (SWMMC) children are repeatedly exposed to a complex mixture of air pollutants, including ozone, particulate matter, and aldehydes. Nasal biopsies taken from these children exhibit a wide range of histopathologic alterations: marked changes in ciliated and goblet cell populations, basal cell hyperplasia, squamous metaplasia, and mild dysplasias. We studied the ultrastructural features of 15 nasal biopsies obtained from clinically healthy children 4 to 15 yr of age, growing up in SWMMC. The results were compared with nasal biopsies from 11 children growing up in Veracruz and exposed to low pollutant levels. Ultrathin sections of nasal biopsies revealed an unremarkable mucociliary epithelium in control children, whereas SWMMC children showed an epithelium comprised of variable numbers of basal, ciliated, goblet, and squamous metaplastic as well as intermediate cells. Nascent ciliated cells, as evidenced by the presence of migratory kinetosomes, were common, as were ciliary abnormalities, including absent central microtubules, supernumerary central and peripheral tubules, ciliary microtubular discontinuities, and compound cilia. Dyskinesia associated with these abnormal cilia was suggested by the altered orientation of the central microtubules in closely adjacent cilia. A transudate was evident between epithelial cells, suggesting potential deficiencies in epithelial junction integrity. Particulate matter was present in heterolysosomal bodies in epithelial cells and it was also deposited in intercellular spaces. The severe structural alteration of the nasal epithelium together with the prominent acquired ciliary defects are likely the result of chronic airway injury in which ozone, particulate matter, and aldehydes are thought to play a crucial role. The nasal epithelium in SWMMC children is fundamentally disordered, and their mucociliary defense mechanisms are no longer intact. A compromised nasal epithelium has less ability to protect the lower respiratory tract and may potentially leave the distal acinar airways more vulnerable to reactive gases. Impairment of mucociliary clearance has the potential to increase the contact time between deposited mutagenic particulate matter and the epithelial surface, thus increasing the risk for nasal carcinogenesis. Chronic exposures to air pollutants affect the whole respiratory tract; the nasal epithelium is an accessible and valuable sentinel to monitor exposures to toxic or carcinogenic substances.

Concentrated ambient air particles induce mild pulmonary inflammation in healthy human volunteers.

We tested the hypothesis that exposure of healthy volunteers to concentrated ambient particles (CAPS) is associated with an influx of inflammatory cells into the lower respiratory tract. Thirty-eight volunteers were exposed to either filtered air or particles concentrated from the immediate environment of the Environmental Protection Agency (EPA) Human Studies Facility in Chapel Hill, North Carolina. Particle concentrations in the chamber during the exposures ranged from 23.1 to 311.1 microgram/m(3). While in the exposure chamber, volunteers alternated between moderate exercise (15 min) and rest (15 min) for a total exposure time of 2 h. There were no symptoms noted by volunteers after the exposure. Similarly, there were no decrements in pulmonary function. Eighteen hours after exposure, analysis of cells and fluid obtained by bronchoalveolar lavage showed a mild increase in neutrophils in both the bronchial and alveolar fractions in those individuals exposed to CAPS (8.44 +/- 1.99 and 4.20 +/- 1.69%, respectively, in those with the greatest exposure) relative to filtered air (2.69 +/- 0.55 and 0.75 +/- 0.28%, respectively). Blood obtained 18 h after exposure to CAPS contained significantly more fibrinogen relative to samples obtained before exposure. We conclude that ambient air particles are capable of inducing a mild inflammation in the lower respiratory tract, as well as an increased concentration of blood fibrinogen.

Exposure to air pollution is associated with lung hyperinflation in healthy children and adolescents in Southwest Mexico City: a pilot study.

Air pollution produces adverse health effects. The consequences of lifelong daily exposures to atmospheric pollutants upon the respiratory apparatus of healthy children are of considerable clinical importance. We investigated the association between exposure to a highly polluted urban environment with a complex mixture of air pollutants-ozone and particulate matter the predominant ones-and chest x-ray abnormalities in 59 healthy Mexican children who are lifelong residents of Southwest Metropolitan Mexico City (SWMMC), with a negative history of tobacco exposure and respiratory illnesses. Their clinical results and x-ray findings were compared to those of 19 Mexican control children, residents of a low-pollution area, with a similar negative history of tobacco exposure and respiratory illnesses. Ozone concentrations in SWMMC exceeded the U.S. Environmental Protection Agency (U.S. EPA) National Ambient Air Quality Standard (NAAQS) for O(3): 0.08 ppm as 1-h maximal concentration, not to be exceeded more than 4 times a year, on 71% of days in 1986 and 95% in 1997, with values as high as 0.48 ppm. Ozone maximal peaks are usually recorded between 2 and 5 pm coinciding with children's outdoor physical activities. Children in the control group reported no upper or lower respiratory symptomatology. Every SWMMC child complained of upper and/or lower respiratory symptoms, including epistaxis, nasal dryness and crusting, cough, shortness of breath, and chest discomfort. Children aged 7-13 yr had the most symptomatology, while 5- to 6-year olds and adolescents with the lowest number of statistically significant outdoor exposure hours had less respiratory symptoms. Bilateral symmetric mild lung hyperinflation was significantly associated with exposure to the SWMMC atmosphere (p = .0004). Chronic and sustained inhalation of a complex mixture of air pollutants, including ozone and particulate matter (PM), is associated with lung hyperinflation, suggestive of small airway disease, in a population of clinically healthy children and adolescents. Small airways are a target of air pollutants in SWMMC children, with ozone and PM being most likely responsible, based on experimental animal, controlled-chamber, and epidemiological data available. Our main concern is the potential likelihood for the development of chronic lung disease in this highly exposed population.

Respiratory tract pathology and cytokine imbalance in clinically healthy children chronically and sequentially exposed to air pollutants.

Chronic exposure of children to a complex mixture of air pollutants leads to recurrent episodes of upper and lower respiratory tract injury. An altered nasal mucociliary apparatus leaves the distal acinar airways more vulnerable to reactive gases and particulate matter (PM). The heterogeneity of structure in the human lung can impart significant variability in the distribution of ozone dose and particle deposition; this, in turn, influences the extent of epithelial injury and repair in chronically exposed children. Cytokines are low-molecular-weight proteins that act as intercellular mediators of inflammatory reactions, including lung injury of various etiologies. Cytokines are involved in generating inflammatory responses that contribute to injury at the lung epithelial and endothelial barriers. Mexico City is a 20-million-person megacity with severe air pollution problems. Southwest Metropolitan Mexico City (SWMMC) atmosphere is characterized by a complex mixture of air pollutants, including ozone, PM, and aldehydes. There is radiological evidence that significant lower respiratory tract damage is taking place in clinically healthy children chronically and sequentially exposed to air pollutants while growing up in SWMMC. We hypothesize that there is an imbalanced and dysregulated cytokine network in SWMMC children with overproduction of proinflammatory cytokines and cytokines involved in lung tissue repair and fibrosis. The nature of the sustained imbalance among the different cytokines ultimately determines the final lung histopathology, which would include subchronic inflammation, emphysema, and fibrosis. Cytokines likely would reach the systemic circulation and produce systemic effects. Individuals with an underlying respiratory or cardiovascular disease are less able to maintain equilibrium of the precarious cytokine networks.

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.

Effects of aqueous extracts of PM(10) 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 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.

Ozone effects on airway responsiveness, lung injury, and inflammation. Comparative rat strain and in vivo/in vitro investigations.

Asthmatic individuals appear to be particularly sensitive to the effects of certain air pollutants-including ozone (O(3)), an oxidant ambient air pollutant-for reasons that are poorly understood. The general purpose of these studies, therefore, was to expand and improve upon toxicologic methods for assessing ozone-induced effects on the airways of the rat by (1) developing an in vivo testing procedure that allows detection of airway responsiveness changes in rats exposed to ozone; (2) identifying a strain of rat that may be inherently more sensitive to the effects of ozone; and (3) validation of an in vitro epithelial culture system to more directly assess airway cellular/subcellular effects of ozone. Using methacholine inhalation challenges, we detected increased airway responsiveness in senescent F344 rats acutely after ozone exposure (2 ppm x 2 h). We also determined that acutely after ozone exposure (0.5 ppm x 8 h), Wistar rats developed significantly greater lung injury, neutrophilic inflammation, and bronchoalveolar lavage (BAL) fluid concentrations of IL-6 than either Sprague-Dawley (SD) or F344 rats. SD rats had greater BAL fluid concentrations of prostaglandin E(2) (PGE(2)), while F344 rats consistently exhibited the least effect. Wistar rat-derived tracheal epithelial (RTE) cultures were exposed in vitro to air or ozone (0.1-1.0 ppm x 1 h), and examined for analogous effects. In a concentration-dependent manner, ozone exposure resulted in acute but minor cytotoxicity. RT polymerase chain reaction (PCR) analysis of RNA isolated from ozone-exposed cells demonstrated variable increases in steady-state gene expression of IL-6 at 4 h postexposure, while at 24 h cellular fibronectin expression (EIIIA domain) was decreased. Exposure was without effect on macrophage inflammatory protein 2 (MIP-2) or gamma-glutamyl cysteine synthetase expression. At 6 h postexposure, IL-6 synthesis and apical release appeared increased in ozone-exposed cells (1 ppm x 1 h). MIP-2 release was not significantly increased in ozone-exposed cells. At 2 h postexposure, ozone exposure resulted in minor increases in apical fibronectin, but exposure was without effect on basolateral accumulation of fibronectin. Exposure to 1.0, but not 0.1 ppm (x 1 h), increased production of cyclooxygenase (i.e., PGE(2)) and noncyclooxygenase products of arachidonic acid. Results demonstrate that multiple inflammatory mediator pathways are affected by ozone exposure. Such effects could exacerbate morbidity in individuals with preexisting airway inflammation such as asthmatics.

Inflammatory response in humans exposed to 2.0 ppm nitrogen dioxide.

Nitrogen dioxide (NO2) is a common indoor air pollutant, especially in homes with unvented combustion appliances. Epidemiological studies suggest that children living in homes with unvented heating sources are more prone to respiratory infections than children living in homes with lower levels of NO2. However, experimental studies in which human volunteers were exposed acutely to moderate levels of NO2 (0.5-2.0 ppm) have shown little evidence of lung inflammation or decreased host resistance capacity. In the study reported here, 8 healthy volunteers were exposed to 2.0 ppm NO2 and to filtered air for 4 h while undergoing intermittent moderate exercise. Bronchoalveolar lavage was performed the following morning. The lavage was divided into a predominantly bronchial washing (first 20 ml of lavage; BL) and a predominantly alveolar washing (BAL). In the BL, NO2 exposure caused increases in polymorphonuclear neutrophils (PMNs), interleukin 6 (IL-6), IL-8, alpha1-antitrypsin, and tissue plasminogen activator, and decreases in epithelial cells. In the BAL, there were no NO2-induced changes in either cell numbers or soluble mediators. On the other hand, alveolar macrophages from BAL showed a decrease in the ability to phagocytose unopsonized Candida albicans and a decrease in superoxide production. No difference in susceptibility to virus infection was found between the NO2- and air-exposed macrophages. No changes in lung function were observed, but the aerosol bolus recovery technique revealed a statistically significant (p <.05) decrease in the fraction of aerosol recovered following nitrogen dioxide exposure, which is suggestive of small obstructive changes induced by NO2.