Jacqueline D. Carter

METALS ASSOCIATED WITH BOTH THE WATER-SOLUBLE AND INSOLUBLE FRACTIONS OF AN AMBIENT AIR POLLUTION PARTICLE CATALYZE AN OXIDATIVE STRESS

One potential mechanism of injury mediated by air pollution particles is through metal-catalyzed oxidant generation. In one emission source particle, soluble metals have been associated with biological effect and toxicity. However, a majority of metals in ambient air pollution particles can be associated with insoluble components. We tested the hypothesis that concentrations of catalytically active metal in ambient air pollution particles are not equivalent to the concentrations of water-soluble metal. Twelve filters collected from the North Provo, UT, monitoring station were agitated in deionized water. Both the aqueous extract and pellet were isolated, lyophilized, and defined as the water-soluble and insoluble fractions, respectively. The fractions were chemically characterized and ionizable concentrations of metals were measured using inductively coupled plasma emission spectroscopy. While the water-soluble fraction had significantly greater concentrations of ionizable metals per unit mass, the insoluble fraction also had measurable quantities. In vitro oxidant generation by the two fractions, measured as thiobarbituric acid-reactive-products of deoxyribose, corresponded to the concentrations of ionizable rather than total metals. The release of interleukin-8 by cultured respiratory epithelial cells after incubation with the two fractions also coincided with the ionizable metal concentrations. Finally, neutrophil influx and lavage protein levels 24 h after instillation of the two fractions in rats reflected the ionizable metal concentrations, in vitro oxidative stress, and mediator release. We conclude that catalytically active metals can be measured in both the soluble and insoluble fractions of an ambient air pollution particle. These metals corresponded to the biological activity of the two fractions. While in greater concentration in the water-soluble fraction, larger total quantities of catalytically and biologically active metals are likely to be associated with the insoluble fraction as a result of the abundance of the latter.

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.

Neuropeptides and capsaicin stimulate the release of inflammatory cytokines in a human bronchial epithelial cell line.

The role of neuropeptides in initiating and modulating airway inflammation was examined in a human bronchial epithelial cell line (i.e. BEAS-2B). At a range of concentrations, exposure of BEAS-2B cells to Substance P (SP) or calcitonin gene related protein resulted in immediate increases in intracellular calcium ([Ca(2+)](i)), the synthesis of the transcripts for the inflammatory cytokines, IL-6, IL-8 and TNFalpha after 2 h exposure, and the release of their proteins after 6 h exposure. Addition of thiorphan (100 nM), an inhibitor of neutral endopeptidase, enhanced the levels of SP-stimulated cytokine release. Stimulation of IL-6 by SP occurred in a conventional receptor-mediated manner as demonstrated by its differential release by fragments SP 4-11 and SP 1-4 and by the blockage of IL-6 release with the non-peptide, NK-1 receptor antagonist, CP-99 994. In addition to the direct stimulation of inflammatory cytokines, SP (0.5 microM), in combination with TNFalpha (25 units/ml), synergistically stimulated IL-6 release. BEAS-2B cells also responded to the botanical irritant, capsaicin (10 microM) with increases in [Ca(2+)](i) and IL-8 cytokine release after 4 h exposure. The IL-8 release was dependent on the presence of extracellular calcium. Capsaicin-stimulated increases of [Ca(2+)](i) and cytokine release could be reduced to control levels by pre-exposure to capsazepine, an antagonist of capsaicin (i.e. vanilloid) receptor(s) or by deletion of extracellular calcium from the exposure media. The present data indicate that the BEAS-2B human epithelial cell line expresses neuropeptide and capsaicin-sensitive pathways, whose activation results in immediate increases of [Ca(2+)](i) stimulation of inflammatory cytokine transcripts and the release of their cytokine proteins.

Particulate matter in cigarette smoke alters iron homeostasis to produce a biological effect.

RATIONALE Lung injury after cigarette smoking is related to particle retention. Iron accumulates with the deposition of these particles. OBJECTIVES We tested the postulate that (1) injury after smoking correlates with exposure to the particulate fraction of cigarette smoke, (2) these particles alter iron homeostasis, triggering metal accumulation, and (3) this alteration in iron homeostasis affects oxidative stress and inflammation. METHODS Rats and human respiratory epithelial cells were exposed to cigarette smoke, filtered cigarette smoke, and cigarette smoke condensate (the particulate fraction of smoke), and indices of iron homeostasis, oxidative stress, and inflammatory injury were determined. Comparable measures were also evaluated in nonsmokers and smokers. MEASUREMENTS AND MAIN RESULTS After exposure of rats to cigarette smoke, increased lavage concentrations of iron and ferritin, serum ferritin levels, and nonheme iron concentrations in the lung and liver tissue all increased. Lavage ascorbate concentrations were decreased, supporting an oxidative stress. After filtering of the cigarette smoke to remove particles, most of these changes were reversed. Exposure of cultured respiratory epithelial cells to cigarette smoke condensate caused a similar accumulation of iron, metal-dependent oxidative stress, and increased IL-8 release. Lavage samples in healthy smokers and smoking patients with chronic obstructive pulmonary disease revealed elevated concentrations of both iron and ferritin relative to healthy nonsmokers. Lavage ascorbate decreased with cigarette smoking. Serum iron and ferritin levels among smokers were increased, supporting systemic accumulation of this metal after cigarette smoke exposure. CONCLUSIONS We conclude that cigarette smoke particles alter iron homeostasis, both in the lung and systemically.

The role of soluble components in ambient fine particles-induced changes in human lungs and blood.

Normal individuals developed pulmonary neutrophilic inflammation and increased blood fibrinogen following inhalation of concentrated ambient particles (CAPS). In this study, we sought to determine how soluble components in CAPS contributed to these changes. We expanded and reanalyzed data from 37 young healthy volunteers from a previous study (Ghio et al., 2000) who were exposed to either filtered air or CAPS. Postexposure bronchoalveolar lavage (BAL) as well as pre- and postexposure venous blood samples was analyzed for cellular and acute inflammatory endpoints. Nine most abundant components in the water-soluble fraction of CAPS were correlated with these endpoints using principal component analysis. We found that a sulfate/Fe/Se factor was associated with increased BAL percentage of neutrophils and a Cu/Zn/V factor with increased blood fibrinogen. The concentrations of sulfate, Fe, and Se correlated highly with PM mass (R > 0.75) while the correlations between PM and Cu/Zn/V were modest (R = 0.2-0.6). These results from controlled human exposure linked specific PM components to pulmonary neutrolphil influx and blood fibrinogen increase, and indicated the soluble components of pollutant particles may differentially affect pulmonary and hematological systems in humans exposed to PM.

Pollutant Particles Produce Vasoconstriction and Enhance MAPK Signaling via Angiotensin Type I Receptor

Exposure to particulate matter (PM) is associated with acute cardiovascular mortality and morbidity, but the mechanisms are not entirely clear. In this study, we hypothesized that PM may activate the angiotensin type 1 receptor (AT1R), a G protein-coupled receptor that regulates inflammation and vascular function. We investigated the acute effects of St. Louis, Missouri, urban particles (UPs; Standard Reference Material 1648) on the constriction of isolated rat pulmonary artery rings and the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinases (MAPKs) in human pulmonary artery endothelial cells with or without losartan, an antagonist of AT1R. UPs at 1–100 μg/mL induced acute vaso-constriction in pulmonary artery. UPs also produced a time- and dose-dependent increase in phosphorylation of ERK1/2 and p38 MAPK. Losartan pretreatment inhibited both the vasoconstriction and the activation of ERK1/2 and p38. The water-soluble fraction of UPs was sufficient for inducing ERK1/2 and p38 phosphorylation, which was also losartan inhibitable. Copper and vanadium, two soluble transition metals contained in UPs, induced pulmonary vasoconstriction and phosphorylation of ERK1/2 and p38, but only the phosphorylation of p38 was inhibited by losartan. The UP-induced activation of ERK1/2 and p38 was attenuated by captopril, an angiotensin-converting enzyme inhibitor. These results indicate that activation of the local renin–angiotensin system may play an important role in cardiovascular effects induced by PM.

Mitochondrial Oxidative Stress in Aortic Stiffening With Age: The Role of Smooth Muscle Cell Function

Objective—Age-related aortic stiffness is an independent risk factor for cardiovascular diseases. Although oxidative stress is implicated in aortic stiffness, the underlying molecular mechanisms remain unelucidated. Here, we examined the source of oxidative stress in aging and its effect on smooth muscle cell (SMC) function and aortic compliance using mutant mouse models. Methods and Results—Pulse wave velocity, determined using Doppler, increased with age in superoxide dismutase 2 (SOD2)+/− but not in wild-type, p47phox−/− and SOD1+/− mice. Echocardiography showed impaired cardiac function in these mice. Increased collagen I expression, impaired elastic lamellae integrity, and increased medial SMC apoptosis were observed in the aortic wall of aged SOD2+/− versus wild-type (16-month-old) mice. Aortic SMCs from aged SOD2+/− mice showed increased collagen I and decreased elastin expression, increased matrix metalloproteinase-2 expression and activity, and increased sensitivity to staurosporine-induced apoptosis versus aged wild-type and young (4-month-old) SOD2+/− mice. Smooth muscle &agr;-actin levels were increased with age in SOD2+/− versus wild-type SMCs. Aged SOD2+/− SMCs had attenuated insulin-like growth factor-1-induced Akt and Forkhead box O3a phosphorylation and prolonged tumor necrosis factor-&agr;–induced Jun N-terminal kinase 1 activation. Aged SOD2+/− SMCs had increased mitochondrial superoxide but decreased hydrogen peroxide levels. Finally, dominant-negative Forkhead box O3a overexpression attenuated staurosporine-induced apoptosis in aged SOD2+/− SMCs. Conclusion—Mitochondrial oxidative stress over a lifetime causes aortic stiffening, in part by inducing vascular wall remodeling, intrinsic changes in SMC stiffness, and aortic SMC apoptosis.

Accumulation of Iron in the Rat Lung after Tracheal Instillation of Diesel Particles

Oxidant generation catalyzed by metals has been postulated to account for a lung injury following exposure to air pollution particles. In particles that are predominantly carbonaceous, it is difficult to implicate such an oxidative stress as the responsible mechanism, since concentrations of metals can be extremely low. Comparable to these air pollution particles, mineral oxide particles can include only minute amounts of metal, but lung injury following their exposure can be associated with an accumulation of endogenous iron from the host and an oxidative stress. We tested the hypothesis that diesel exhaust particulate (DEP) effects an accumulation of biologically active iron in the rat lung, with both oxidative stress and a lung injury resulting. Characterization of the DEP confirmed a high concentration of carbon, whereas metals were low in quantity. The concentration of total lavage iron in animals instilled with saline was low, but this concentration increased with exposure to DEP. Non-heme iron in lung tissue was similarly elevated after instillation of the diesel product. Particle instillation was associated with a decrease in lavage ascorbate concentration supporting an oxidative stress. Relative to saline exposure, DEP resulted in elevated lavage concentrations of the inflammatory mediators macrophage inflammatory protein-2 and tumor necrosis factor. Finally, an injury after particle instillation was evident with increased neutrophils and an elevation of lavage protein and lactic dehydrogenase. We conclude that DEP exposure effected an accumulation of iron in the rat lung. This accrual of iron was associated with an oxidative stress, release of oxidant-sensitive mediators, and a neutrophilic lung injury.

ACTIVATION OF EGF RECEPTORS MEDIATES PULMONARY VASOCONSTRICTION INDUCED BY RESIDUAL OIL FLY ASH

Residual oil fly ash (ROFA)is a constituent of pollutant particles that can producelung injury and activate protein tyrosine phosphorylation cascade. In this study, we determined whether or not protein tyrosine phosphorylation caused lung injury, and if so, identified critical tyrosine phosphorylated proteins that mediated the injury. ROFA was instilled intratracheally into perfused rabbit lungs and injury responses, including increase in pulmonary artery pressure (Ppa), lung weight gain, as well as release of interleukin (IL)-1 β, IL-6, IL-8, and nitrite/nitrate were measured. ROFA increased Ppa and IL-1 β, but inhibited nitrite/nitrate accumulation. Vanadyl sulfate at concentration equivalent to the amount of vanadium detected in the perfusate of ROFA-treated lungs induced similar changes. ROFAenhanced tyrosine phosphorylation of lung proteins, including a 170-kDa protein, likely the epidermal growth factor (EGF)receptor, as shown by immunoprecipitation. Pretreatment with genistein, a tyrosine kinase inhibitor, blocked the increase in Ppa and tyrosine phosphorylation of the 170-kDa protein. Intravascular administration of human EGF increased Ppa, and pretreatment with PD153035, an EGF receptor-specific tyrosine kinase inhibitor, attenuated ROFA-induced pulmonary vasoconstriction. These results indicate that tyrosine phosphorylation of EGF receptors in the lung, possibly as a result of inhibition of protein tyrosine phosphatases, mediates constriction of pulmonary vessels induced by ROFA.

Iron and iron-related proteins in the lower respiratory tract of patients with acute respiratory distress syndrome

ObjectiveAn increased oxidative stress in the lower respiratory tract of individuals with acute respiratory distress syndrome is considered to be one mechanism of lung injury in these patients. Cell and tissue damage resulting from an oxidative stress can ultimately be the consequence of a disruption of normal iron metabolism and an increased availability of catalytically active metal. Using bronchoalveolar lavage fluid, we quantified concentrations of iron and iron-related proteins in the lower respiratory tract in patients with acute respiratory distress syndrome and healthy volunteers. DesignA clinical study to quantify iron and iron-related proteins in the lower respiratory tract in patients with acute respiratory distress syndrome and healthy volunteers. PatientsWe studied 14 patients with acute respiratory distress syndrome and 28 healthy volunteers. Main ResultsComparable to previous investigation, protein, albumin, and cytokine concentrations in the bronchoalveolar lavage fluid were significantly increased in acute respiratory distress syndrome patients. The concentrations of total and nonheme iron were also increased in the lavage fluid of patients. Concentrations of hemoglobin, haptoglobin, transferrin, transferrin receptor, lactoferrin, and ferritin in the bronchoalveolar lavage fluid were all significantly increased in acute respiratory distress syndrome patients. ConclusionsWe conclude that bronchoalveolar lavage fluid indices reflect a disruption of normal iron metabolism in the lungs of acute respiratory distress syndrome patients. Increased concentrations of available iron in acute respiratory distress syndrome may participate in catalyzing oxidant generation destructive to the tissues of the lower respiratory tract. However, increased metal availability is also likely to elicit an increased expression of transferrin receptor, lactoferrin, and ferritin in the lower respiratory tract which will function to diminish this oxidative stress.