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Environmental and occupational exposures to respirable ultrafine fractions of particulate matter (PM) have been implicated in the initiation and exacerbation of lung diseases. However, the precise mechanisms underlying production of cell damage and death attributed to nanoparticles (NP) on human airway epithelium are not fully understood. This study examined the role of neurotrophic pathways in NP-induced airway toxicity. Size and agglomeration of TiO 2 nanoparticles (TiO 2 -NP) and fine (TiO 2 -FP) particles were measured by dynamic light scattering. Expression and signaling of key neurotrophic factors and receptors were assessed by real-time polymerase chain reaction, flow cytometry, immunostaining, and Western blot in various respiratory epithelial cells after exposure to TiO 2 -NP or TiO 2 -FP. Particle-induced cell death was measured by flow cytometry after annexin V/propidium iodide staining. The role of neurotrophin-dependent apoptotic pathways was analyzed with specific blocking antibodies or siRNAs. Exposure of human epithelial cells to TiO 2 -NP enhanced interleukin (IL)-1α synthesis, as well as nerve growth factor (NGF) gene expression and protein levels, specifically the precursor form (proNGF). TiO 2 -NP exposure also increased expression of p75 NRF receptor genes. These neurotropic factor and receptor responses were stimulated by IL-1α and abolished by its specific receptor antagonist (IL-1-ra). TiO 2 -NP also increased JNK phosphorylation and apoptosis, which was prevented by anti-p75 NRF or NGFsiRNA. Data demonstrated that TiO 2 -NP exerted adverse effects in the respiratory tract by inducing unbalanced overexpression of immature neurotrophins, which led to apoptotic death of epithelial cells signaled through the death receptor p75 NTR . This may result in airway inflammation and hyperreactivity after exposure to TiO 2 -NP.

Pulmonary exposure to multiwalled carbon nanotubes (MWCNT) induces an inflammatory and rapid fibrotic response, although the long-term signaling mechanisms are unknown. The aim of this study was to examine the effects of 1, 10, 40, or 80 μg MWCNT administered by pharyngeal aspiration on bronchoalveolar lavage (BAL) fluid for polymorphonuclear cell (PMN) infiltration, lactate dehydrogenase (LDH) activity, and lung histopathology for inflammatory and fibrotic responses in mouse lungs 1 mo, 6 mo, and 1 yr postexposure. Further, a 120-μg crocidolite asbestos group was incorporated as a positive control for comparative purposes. Results showed that MWCNT increased BAL fluid LDH activity and PMN infiltration in a dose-dependent manner at all three postexposure times. Asbestos exposure elevated LDH activity at all 3 postexposure times and PMN infiltration at 1 mo and 6 mo postexposure. Pathological changes in the lung, the presence of MWCNT or asbestos, and fibrosis were noted at 40 and 80 μg MWCNT and in asbestos-exposed mice at 1 yr postexposure. To determine potential signaling pathways involved with MWCNT-associated pathological changes in comparison to asbestos, up- and down-regulated gene expression was determined in lung tissue at 1 yr postexposure. Exposure to MWCNT tended to favor those pathways involved in immune responses, specifically T-cell responses, whereas exposure to asbestos tended to favor pathways involved in oxygen species production, electron transport, and cancer. Data indicate that MWCNT are biopersistent in the lung and induce inflammatory and fibrotic pathological alterations similar to those of crocidolite asbestos, but may reach these endpoints by different mechanisms.

Anecdotal reports in the press and epidemiological studies suggest that deployment to Iraq and Afghanistan may be associated with respiratory diseases and symptoms in U.S. military personnel and veterans. Exposures during military operations were complex, but virtually all service members were exposed to high levels of respirable, geogenic dust. Inhalation of other dusts has been shown to be associated with adverse health effects, but the pulmonary toxicity of ambient dust from Iraq has not been previously studied. The relative toxicity of Camp Victory dust was evaluated by comparing it to particulate matter from northern Kuwait, a standard U.S. urban dust, and crystalline silica using a single intratracheal instillation in rats. Lung histology, protein levels, and cell counts were evaluated in the bronchoalveolar lavage fluid 1-150 d later. The Iraq dust provoked an early significant, acute inflammatory response. However, the level of inflammation in response to the Iraq dust, U.S. urban dust, and Kuwait dust rapidly declined and was nearly at control levels by the end of the study At later times, animals exposed to the Iraq, U.S. urban, or Kuwait dusts showed increased small airway remodeling and emphysema compared to silica-exposed and control animals without evidence of fibrosis or premalignant changes. The severity and persistence of pulmonary toxicity of these three dusts from the Middle East resemble those of a U.S. urban dust and are less than those of silica. Therefore, Iraq dust exposure is not highly toxic, but similar to other poorly soluble low-toxicity dusts.

Welding fume is composed of a complex of different metal particulates. Pulmonary exposure to different welding fumes may exert a negative impact on cardiac function, although the underlying mechanisms remain unclear. To explore the effect of welding fumes on cardiac function, Sprague-Dawley rats were exposed by intratracheal instillation to 2 mg/rat of manual metal arc hard surfacing welding fume (MMA-HS) once per week for 7 wk. Control rats received saline. Cardiomyocytes were isolated enzymatically at d 1 and 7 postexposure. Intracellular calcium ([Ca(2+)]i) transients (fluorescence ratio) were measured on the stage of an inverted phase-contrast microscope using a myocyte calcium imaging/cell length system. Phosphorylation levels of cardiac troponin I (cTnI) were determined by Western blot. The levels of nonspecific inflammatory marker C-reactive protein (CRP) and proinflammatory cytokine interleukin-6 (IL-6) in serum were measured by enzyme-linked immunosorbent assay (ELISA). Contraction of isolated cardiomyocytes was significantly reduced at d 1 and d 7 postexposure. Intracellular calcium levels were decreased in response to extracellular calcium stimulation at d 7 postexposure. Changes of intracellular calcium levels after isoprenaline hydrochloride (ISO) stimulation were not markedly different between groups at either time point. Phosphorylation levels of cTnI in the left ventricle were significantly lower at d 1 postexposure. The serum levels of CRP were not markedly different between groups at either time point. Serum levels of IL-6 were not detectable in both groups. Cardiomyocyte alterations observed after welding fume treatment were mainly due to alterations in intracellular calcium handling and phosphorylation levels of cTnI.

Double-walled carbon nanotubes (DWCNT) are a rather new and unexplored variety of carbon nanotubes. Previously conducted studies established that exposure to a variety of carbon nanotubes produced lung inflammation and fibrosis in mice after pharyngeal aspiration. However, the bioactivity of double-walled carbon nanotubes (DWCNT) has not been determined. In this study, the hypothesis that DWCNT would induce pulmonary toxicity was explored by analyzing the pulmonary bioactivity of DWCNT. To test this hypothesis, C57Bl/6 mice were exposed to DWCNT by pharyngeal aspiration. Mice underwent whole-lung lavage (WLL) to assess pulmonary inflammation and injury, and lung tissue was examined histologically for development of pulmonary disease as a function of dose and time. The results showed that DWCNT exposure produced a dose-dependent increase in WLL polymorphonuclear leukocytes (PMN), indicating that DWCNT exposure initiated pulmonary inflammation. DWCNT exposure also produced a dose-dependent rise in lactate dehydrogenase (LDH) activity, as well as albumin levels, in WLL fluid, indicating that DWCNT exposure promoted cytotoxicity as well as decreases in the integrity of the blood-gas barrier in the lung, respectively. In addition, at 7 and 56 d postexposure, the presence of significant alveolitis and fibrosis was noted in mice exposed to 40 μg/mouse DWCNT. In conclusion, this study provides insight into previously uninvestigated pulmonary bioactivity of DWCNT exposure. Data indicate that DWCNT exposure promotes inflammation, injury, and fibrosis in the lung.

Exposure to wet aerosols generated during use of spray products containing silver (Ag) has not been evaluated. The goal was to assess the potential for cardiopulmonary toxicity following an acute inhalation of wet silver colloid. Rats were exposed by inhalation to a low concentration (100 μg/m(3) ) using an undiluted commercial antimicrobial product (20 mg/L total silver; approximately 33 nm mean aerodynamic diameter [MAD]) or to a higher concentration (1000 μg/m(3)) using a suspension (200 mg/L total silver; approximately 39 nm MAD) synthesized to possess a similar size distribution of Ag nanoparticles for 5 h. Estimated lung burdens from deposition models were 0, 1.4, or 14 μg Ag/rat after exposure to control aerosol, low, and high doses, respectively. At 1 and 7 d postexposure, the following parameters were monitored: pulmonary inflammation, lung cell toxicity, alveolar air/blood barrier damage, alveolar macrophage activity, blood cell differentials, responsiveness of tail artery to vasoconstrictor or vasodilatory agents, and heart rate and blood pressure in response to isoproterenol or norepinephrine, respectively. Changes in pulmonary or cardiovascular parameters were absent or nonsignificant at 1 or 7 d postexposure with the exceptions of increased blood monocytes 1 d after high-dose Ag exposure and decreased dilation of tail artery after stimulation, as well as elevated heart rate in response to isoproterenol 1 d after low-dose Ag exposure, possibly due to bioavailable ionic Ag in the commercial product. In summary, short-term inhalation of nano-Ag did not produce apparent marked acute toxicity in this animal model.

Concerns over the potential for multiwalled carbon nanotubes (MWCNT) to induce lung carcinogenesis have emerged. This study sought to (1) identify gene expression signatures in the mouse lungs following pharyngeal aspiration of well-dispersed MWCNT and (2) determine if these genes were associated with human lung cancer risk and progression. Genome-wide mRNA expression profiles were analyzed in mouse lungs (n = 160) exposed to 0, 10, 20, 40, or 80 μg of MWCNT by pharyngeal aspiration at 1, 7, 28, and 56 d postexposure. By using pairwise statistical analysis of microarray (SAM) and linear modeling, 24 genes were selected, which have significant changes in at least two time points, have a more than 1.5-fold change at all doses, and are significant in the linear model for the dose or the interaction of time and dose. Additionally, a 38-gene set was identified as related to cancer from 330 genes differentially expressed at d 56 postexposure in functional pathway analysis. Using the expression profiles of the cancer-related gene set in 8 mice at d 56 postexposure to 10 μg of MWCNT, a nearest centroid classification accurately predicts human lung cancer survival with a significant hazard ratio in training set (n = 256) and test set (n = 186). Furthermore, both gene signatures were associated with human lung cancer risk (n = 164) with significant odds ratios. These results may lead to development of a surveillance approach for early detection of lung cancer and prognosis associated with MWCNT in the workplace.

Multiwalled carbon nanotubes (MWCNT) have elicited great interest in biomedical applications due to their extraordinary physical, chemical, and optical properties. Intravenous administration of MWCNT-based medical imaging agents and drugs in animal models was utilized. However, the potential harmful health effects of MWCNT administration in humans have not yet been elucidated. Furthermore, to date, there are no apparent reports regarding the precise mechanisms of translocation of MWCNT into target tissues and organs from blood circulation. This study demonstrates that exposure to MWCNT leads to an increase in cell permeability in human microvascular endothelial cells (HMVEC). The results obtained from this study also showed that the MWCNT-induced rise in endothelial permeability is mediated by reactive oxygen species (ROS) production and actin filament remodeling. In addition, it was found that MWCNT promoted cell migration in HMVEC. Mechanistically, MWCNT exposure elevated the levels of monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule 1 (ICAM-1) in HMVEC. Taken together, these results provide new insights into the bioreactivity of MWCNT, which may have implications in the biomedical application of MWCNT in vascular targeting, imaging, and drug delivery. The results generated from this study also elucidate the potential adverse effects of MWCNT exposure on humans at the cellular level.

Consequent to the 2010 Deepwater Horizon oil spill in the Gulf of Mexico, there is an emergent concern about the short- and long-term adverse health effects of exposure to crude oil, weathered-oil products, and oil dispersants among the workforce employed to contain and clean up the spill. Oil dispersants typically comprise of a mixture of solvents and surfactants that break down floating oil to micrometer-sized droplets within the water column, thus preventing it from reaching the shorelines. As dispersants are generally sprayed from the air, workers are at risk for exposure primarily via inhalation. Such inhaled fractions might potentially permeate or translocate to the brain via olfactory or systemic circulation, producing central nervous system (CNS) abnormalities. To determine whether oil dispersants pose a neurological risk, male Sprague-Dawley rats were exposed by whole-body inhalation exposure to a model oil dispersant, COREXIT EC9500A (CE; approximately 27 mg/m(3) × 5 h/d × 1 d), and various molecular indices of neural dysfunction were evaluated in discrete brain areas, at 1 or 7 d postexposure. Exposure to CE produced partial loss of olfactory marker protein in the olfactory bulb. CE also reduced tyrosine hydroxylase protein content in the striatum. Further, CE altered the levels of various synaptic and neuronal intermediate filament proteins in specific brain areas. Reactive astrogliosis, as evidenced by increased expression of glial fibrillary acidic protein, was observed in the hippocampus and frontal cortex following exposure to CE. Collectively, these findings are suggestive of disruptions in olfactory signal transduction, axonal function, and synaptic vesicle fusion, events that potentially result in an imbalance in neurotransmitter signaling. Whether such acute molecular aberrations might persist and produce chronic neurological deficits remains to be ascertained.

These studies characterized cardiovascular responses after an acute inhalation exposure to COREXIT EC9500A, the oil dispersant used in the Deepwater Horizon oil spill. Male Sprague-Dawley rats underwent a single 5-h inhalation exposure to COREXIT EC9500A (average exposure level 27.12 mg/m(3)) or air. On d 1 and 7 following the exposure, rats were implanted with indwelling catheters and changes in heart rate and blood pressure were assessed in response to increasing levels of adrenoreceptor agonists. A separate group of rats was euthanized at the same time points, ventral tail arteries were dissected, and vascular tone along with dose-dependent responses to vasoconstricting and dilating factors were assessed in vitro. Agonist-induced dose-dependent increases in heart rate and blood pressure were greater in COREXIT EC9500A-exposed than in air-exposed rats at 1 d but not 7 d after the exposure. COREXIT EC9500A exposure also induced a rise in basal tone and reduced responsiveness of tail arteries to acetylcholine-induced vasodilation at 1 d but not 7 d following the exposure. These findings demonstrate that an acute exposure to COREXIT EC9500A exerts transient effects on cardiovascular and peripheral vascular functions.

COREXIT EC9500A (COREXIT) was used to disperse crude oil during the 2010 Deepwater Horizon oil spill. While the environmental impact of COREXIT has been examined, the pulmonary effects are unknown. Investigations were undertaken to determine whether inhaled COREXIT elicits airway inflammation, alters pulmonary function or airway reactivity, or exerts pharmacological effects. Male rats were exposed to COREXIT (mean 27 mg/m(3), 5 h). Bronchoalveolar lavage was performed on d 1 and 7 postexposure. Lactate dehydrogenase (LDH) and albumin were measured as indices of lung injury; macrophages, neutrophils, lymphocytes, and eosinophils were quantified to evaluate inflammation; and oxidant production by macrophages and neutrophils was measured. There were no significant effects of COREXIT on LDH, albumin, inflammatory cell levels or oxidant production at either time point. In conscious animals, neither breathing frequency nor specific airway resistance were altered at 1 hr, 1 d and 7 d postexposure. Airway resistance responses to methacholine (MCh) aerosol in anesthetized animals were unaffected at 1 and 7 d postexposure, while dynamic compliance responses were decreased after 1 d but not 7 d. In tracheal strips, in the presence or absence of MCh, low concentrations of COREXIT (0.001% v/v) elicited relaxation; contraction occurred at 0.003-0.1% v/v. In isolated, perfused trachea, intraluminally applied COREXIT produced similar effects but at higher concentrations. COREXIT inhibited neurogenic contractile responses of strips to electrical field stimulation. Our findings suggest that COREXIT inhalation did not initiate lung inflammation, but may transiently increase the difficulty of breathing.

Nanomaterials, including single-walled carbon nanotubes (SWCNT), are being developed for a variety of commercial products. However, adverse health effects attributed to these new materials are not well understood. Recent reports showed that exposure of mice to dispersed SWCNT (DSWCNT) produced a rapid and progressive interstitial lung fibrosis without persistent inflammation. To understand the mechanism underlying this unusual fibrogenicity of DSWCNT, the present investigation focused on the direct bioactivity of DSWCNT using a cell culture of lung fibroblasts that represent a major cell type resident in the lung interstitium and responsible for the production of collagen matrix. At concentrations relevant to those used in vivo, in vitro exposure of lung fibroblasts to DSWCNT stimulated cell proliferation and induced collagen production without producing cell damage. One of the major matrix metalloproteinases (MMP), MMP-9, which is known to be involved in lung fibrosis, was also elevated by DSWCNT treatment both in vitro and in vivo. Taken together, these results suggest that direct stimulation of fibroblasts by DSWCNT translocated into the interstitium may play a significant role in DSWCNT-induced lung fibrosis. Our data also suggest that the dispersion status and/or size of the SWCNT structures is a critical factor in determining nanoparticle fibrogenicity and that MMP-9 may be involved in the fibrogenic process. The results obtained may aid in the development of in vitro models for rapid screening of the potential fibrogenicity of carbon nanotubes, which are lacking and urgently needed.

Nanoparticles are widely used in nanomedicines, including for targeted delivery of pharmacological, therapeutic, and diagnostic agents. Since nanoparticles might translocate across cellular barriers from the circulation into targeted organs, it is important to obtain information concerning the pathophysiologic effects of these particles through systemic migration. In the present study, acute pulmonary responses were examined after intraperitoneal (ip) administration of ultrafine titanium dioxide (TiO(2), 40 mg/kg) in mice at rest or in lungs primed with lipopolysaccharide (LPS, ip, 5 mg/kg). Ultrafine TiO(2) exposure increased neutrophil influx, protein levels in bronchoalveolar lavage (BAL) fluid, and reactive oxygen species (ROS) activity of BAL cells 4 h after exposure. Concomitantly, the levels of proinflammatory mediators, such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and macrophage inflammatory protein (MIP)-2 in BAL fluid and mRNA expression of TNF-alpha and IL-1beta in lung tissue were elevated post ultrafine TiO(2) exposure. Ultrafine TiO(2) exposure resulted in significant activation of inflammatory signaling molecules, such as c-Src and p38 MAP kinase, in lung tissue and alveolar macrophages, and the nuclear factor (NF)-kappaB pathway in pulmonary tissue. Furthermore, ultrafine TiO(2) additively enhanced these inflammatory parameters and this signaling pathway in lungs primed with lipopolysaccharide (LPS). Contrary to this trend, a synergistic effect was found for TNF-alpha at the level of protein and mRNA expression. These results suggest that ultrafine TiO(2) (P25) induces acute lung inflammation after ip administration, and exhibits additive or synergistic effects with LPS, at least partly, via activation of oxidant-dependent inflammatory signaling and the NF-kappaB pathway, leading to increased production of proinflammatory mediators.

Exposure to ambient nanoparticles (defined as particulate matter [PM] having one dimension <100 nm) is associated with increased risk of childhood and adult asthma. Nanomaterials feature a smaller aerodynamic diameter and a higher surface area per unit mass ratio compared to fine or coarse-sized particles, resulting in greater lung deposition efficiency and an increased potential for biological interaction. The neurotrophins nerve growth factor and brain-derived neurotrophic factor are key regulatory elements of neuronal development and responsiveness of airway sensory neurons. Changes in their expression are associated with bronchoconstriction, airway hyperresponsiveness, and airway inflammation. The neurogenic-mediated control of airway responses is a key pathophysiological mechanism of childhood asthma. However, the effects of nanoparticle exposure on neurotrophin-driven airway responses and their potential role as a predisposing factor for developing asthma have not been clearly elucidated. In this study, in vivo inhalation exposure to titanium dioxide nanoparticles (12 mg/m(3); 5.6 h/d for 3 d) produced upregulation of lung neurotrophins in weanling (2-wk-old) and newborn (2-d-old) rats but not in adult (12-wk-old) animals compared to controls. This effect was associated with increased airway responsiveness and upregulation of growth-related oncogene/keratine-derived chemokine (GRO/KC; CXCL1, rat equivalent of human interleukin [IL]-8) in bronchoalveolar lavage fluid. These data show for the first time that exposure to nanoparticulate upregulates the expression of lung neurotrophins in an age-dependent fashion and that this effect is associated with airway hyperresponsiveness and inflammation. These results suggest the presence of a critical window of vulnerability in earlier stages of lung development, which may lead to a higher risk of developing asthma.

Engineered nanoparticles (NP) are being developed and incorporated in a number of commercial products, raising the potential of human exposure during manufacture, use, and disposal. Although data concerning the potential toxicity of some NP have been reported, validated simple assays are lacking for predicting their in vivo toxicity. The aim of this study was to evaluate new response metrics based on chemical and biological activity of NP for screening assays that can be used to predict NP toxicity in vivo. Two cell-free and two cell-based assays were evaluated for their power in predicting in vivo toxicity of eight distinct particle types with widely differing physicochemical characteristics. The cell-free systems comprised fluorescence- and electron spin resonance-based assays of oxidant activity. The cell-based systems also used electron spin resonance (ESR) as well as luciferase reporter activity to rank the different particle types in comparison to benchmark particles of low and high activity. In vivo experiments evaluated acute pulmonary inflammatory responses in rats. Endpoints in all assays were related to oxidative stress and responses were expressed per unit NP surface area to compare the results of different assays. Results indicated that NP are capable of producing reactive species, which in biological systems lead to oxidative stress. Copper NP had the greatest activity in all assays, while TiO(2) and gold NP generally were the least reactive. Differences in the ranking of NP activity among the assays were found when comparisons were based on measured responses. However, expressing the chemical (cell-free) and biological (cells; in vivo) activity per unit particle surface area showed that all in vitro assays correlated significantly with in vivo results, with the cellular assays correlating the best. Data from this study indicate that it is possible to predict acute in vivo inflammatory potential of NP with cell-free and cellular assays by using NP surface area-based dose and response metrics, but that a cellular component is required to achieve a higher degree of predictive power.

Carbon nanotubes (CNT), since their discovery, have become one of the most promising nanomaterials in many industrial and biomedical applications. Due to their unique physicochemical properties, interest is growing in the manufacture of CNT-based products and their subsequent marketing. Since their discovery, the prospect of possible undesirable human health effects has been a focus of many scientific studies. Although CNT possess unique physical properties that include (1) nanoscale diameter, (2) a wide length distribution ranging from tens of nanometers to several micrometers, and (3) high aspect ratio, the fibrous-like shape and durability suggest that their toxic properties may be analogous to those observed with other fibrous particles, such as asbestos. The present study provides a summary of published findings on CNT bioactivity, such as the potential of CNT, especially of multi-wall carbon nanotubes (MWCNT), to activate signaling pathways modulating transcription factor activity, induce apoptosis, induce DNA damage, and initiate biological responses. Assessment of risks to human health and adoption of appropriate exposure controls is critical for the safe and successful introduction of CNT -based products for future applications.

Perfluorooctane sulfonate (PFOS) is a member of the perfluoroalkyl acids (PFAA) containing an eight-carbon backbone. PFOS is a man-made chemical with carbon-fluorine bonds that are among the strongest in organic chemistry, and PFOS is widely used in industry. Human occupational and environmental exposure to PFOS occurs globally. PFOS is non-biodegradable and is persistent in the human body and environment. In this study, data demonstrated that exposure of human microvascular endothelial cells (HMVEC) to PFOS induced the production of reactive oxygen species (ROS) at both high and low concentrations. Morphologically, it was found that exposure to PFOS induced actin filament remodeling and endothelial permeability changes in HMVEC. Furthermore, data demonstrated that the production of ROS plays a regulatory role in PFOS-induced actin filament remodeling and the increase in endothelial permeability. Our results indicate that the generation of ROS may play a role in PFOS-induced aberrations of the endothelial permeability barrier. The results generated from this study may provide a new insight into the potential adverse effects of PFOS exposure on humans at the cellular level.

Exposure to fine particulate matter (PM, mean aerodynamic diameter

Titanium dioxide (TiO(2)), a commercially important material, is used in a wide variety of products. Although TiO(2) is generally regarded as nontoxic, the cytotoxicity, pathogenicity, and carcinogenicity of TiO(2) nanoparticles have been recently recognized. The present study investigated TiO(2) nanoparticle-induced cell apoptosis and molecular mechanisms involved in this process in a mouse epidermal (JB6) cell line. Using the 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay, TiO(2) nanoparticles were found to exhibit higher cytotoxicity than fine particles. YO-PRO-1 iodide (YP) staining demonstrated that both TiO(2) nanoparticles and fine particles induced cell death through apoptosis. The signaling pathways involved in TiO(2) particle-induced apoptosis were investigated. Western-blot analysis showed an activation of caspase-8, Bid, BAX, and caspase-3 and a decrease of Bcl-2 in JB6 cells treated with TiO(2) particles. Time-dependent poly(ADP)ribose polymerase (PARP) cleavage induced by TiO(2) nanoparticles was observed. TiO(2) particles also induced cytochrome c release from mitochondria to cytosol. Further studies demonstrated that TiO(2) nanoparticles induced significant changes in mitochondrial membrane permeability, suggesting the involvement of mitochondria in the apoptotic process. In conclusion, evidence indicated that TiO(2) nanoparticles exhibit higher cytotoxicity and apoptotic induction compared to fine particles in JB6 cells. Caspase-8/Bid and mitochondrial signaling may play a major role in TiO(2) nanoparticle-induced apoptosis involving the intrinsic mitochondrial pathway. Unraveling the complex mechanisms associated with these events may provide further insights into TiO(2) nanoparticle-induced pathogenicity and potential to induce carcinogenicity.

Pulmonary responses to diesel exhaust particles (DEP) exposure are mediated through enhanced production of reactive oxygen species (ROS) and nitric oxide (NO) by alveolar macrophages (AM). The current study examined the differential roles of ROS and NO in DEP-induced lung injury using C57B/6J wild-type (WT) and inducible NO synthase knockout (iNOS KO) mice. Mice exposed by pharyngeal aspiration to DEP or carbon black particles (CB) (35 mg/kg) showed an inflammatory profile that included neutrophil infiltration, increased lactate dehydrogenase (LDH) activity, and elevated albumin content in bronchoalveolar lavage fluid (BALF) at 1, 3, and 7 d postexposure. The organic extract of DEP (DEPE) did not induce an inflammatory response. Comparing WT to iNOS KO mice, the results show that NO enhanced DEP-induced neutrophils infiltration and plasma albumin content in BALF and upregulated the production of the pro-inflammatory cytokine interleukin 12 (IL-12) by AM. DEP-exposed AM from iNOS KO mice displayed diminished production of IL-12 and, in response to ex vivo lipopolysaccharide (LPS) challenge, decreased production of IL-12 but increased production of IL-10 when compared to cells from WT mice. DEP, CB, but not DEPE, induced DNA damage and mitochondria dysfunction in AM, however, that is independent of cellular production of NO. These results demonstrate that DEP-induced immune/inflammatory responses in mice are regulated by both ROS- and NO-mediated pathways. NO did not affect ROS-mediated mitochondrial dysfunction and DNA damage but upregulated IL-12 and provided a counterbalance to the ROS-mediated adaptive stress response that downregulates IL-12 and upregulates IL-10.

Crystalline silica (silica), a suspected human carcinogen, produces an increase in reactive oxygen species (ROS) when fractured using mechanical tools used in several occupations. Although ROS has been linked to apoptosis, DNA damage, and carcinogenesis, the role of enhanced ROS production by silica in silica-induced carcinogenesis is not completely understood. The goal of this study was to compare freshly fractured and aged silica-induced molecular alterations in human immortalized/transformed bronchial epithelial cells (BEAS-IIB) and lung cancer cells with altered (H460) or deficient (H1299) p53 expression. Exposure to freshly fractured or aged silica produced divergent cellular responses in certain downstream cellular events, including ROS production, apoptosis, cell cycle and chromosomal changes, and gene expression. ROS production increased significantly following exposure to freshly fractured silica compared to aged silica in BEAS-IIB and H460 cells. Apoptosis showed a comparable enhanced level of induction with freshly fractured or aged silica in both cancer lines with p53 functional changes. p53 protein was present in the BEAS-IIB and was absent in cancer cell lines after silica exposure. Exposure to freshly fractured silica also resulted in a rise in aneuploidy in cancer cells with a significantly greater increase in p53-deficient cells. Cytogenetic analysis demonstrated increased metaphase spreads, chromosome breakage, rearrangements, and endoreduplication in both cancer cells. These results suggest that altered and deficient p53 affects the cellular response to freshly fractured silica exposure, and thereby enhances susceptibility and augments cell proliferation and lung cancer development.

Ultrafine or fine titanium dioxide (TiO(2)) particles are widely used in the production of white pigments, for sunscreens, and in cleanup techniques. However, currently knowledge is deficient concerning cellular responses to these particles. The study evaluated and compared the biological activity of ultrafine and fine TiO(2) particles in RAW 264.7 macrophages according to an oxidative stress paradigm. In vitro exposure of macrophages to ultrafine or fine TiO(2) in the range of 0.5-200 microg/ml did not significantly alter cell viability. However, ultrafine TiO(2) enhanced intracellular generation of reactive oxygen species (ROS) to a greater extent than fine TiO(2) at each exposure concentration. Ultrafine TiO(2) induced ERK1/2 activation in a concentration-dependent manner, while the fine TiO(2)-induced changes were minimal. Phosphorylation of ERK1/2 occurred following 10 min exposure to higher concentrations of ultrafine TiO(2) (> or = 25 microg/ml). Similarly, ultrafine TiO(2) exposure significantly enhanced tumor necrosis factor (TNF)-alpha and macrophage inflammatory protein (MIP)-2 secretion in a concentration-dependent manner, and its potency was higher than fine TiO(2). These findings suggest that when exposure concentration is based upon equivalent mass, ultrafine TiO(2) exerts greater biological activity as measured by ROS generation, ERK 1/2 activation, and proinflammatory mediator secretion in RAW 264.7 macrophages than fine TiO(2).

With the development of nanotechnology, there is a tremendous growth of the application of nanomaterials, which increases the risk of human exposure to these nanomaterials through inhalation, ingestion, and dermal penetration. Among different types of nanoparticles, single-walled carbon nanotubes (SWCNT) with extremely small size (1 nm in diameter) exhibit extraordinary properties and offer possibilities to create materials with astounding features. Since the release of nanoparticles in an enclosed environment is of great concern, a study of possible genotoxic effects is important. Our previous data showed that pharyngeal aspiration of SWCNT elicited pulmonary effects in C57BL/6 mice that was promoted by a robust, acute inflammatory reaction with early onset resulting in progressive interstitial fibrogenic response and the formation of granulomas. In the present study, the genotoxic potential of SWCNT was evaluated in vitro. The genotoxic effects of nanoparticles were examined using three different test systems: the comet assay and micronucleus (MN) test in a lung fibroblast (V79) cell line, and the Salmonella gene mutation assay in strains YG1024/YG1029. Cytotoxicity tests showed loss of viability in a concentration- and time-dependent manner after exposure of cells to SWCNT. Results from the comet assay demonstrated the induction of DNA damage after only 3 h of incubation with 96 microg/cm2 of SWCNT. The MN test indicated some but not significant micronucleus induction by SWCNT in the V79 cell line at the highest concentrations tested. With two different strains of Salmonella typhimurium, no mutations were found following SWCNT exposure.

1-->3-Beta-glucan has been associated with pulmonary inflammation induced by exposure to fungal or yeast cell wall dust. 1-->3-Beta-glucan is the major cell wall component of yeast or fungi. However, the yeast cell wall contains several other components besides 1-->3-beta-glucans, such as mannan and chitin. Few studies evaluated the contribution of these other cell wall components to pulmonary inflammation. The present study compares a crude particulate yeast cell wall preparation (zymosan A) to purified yeast glucan, purified yeast glucan mannan, or purified yeast glucan chitin particles for their potency to induce mouse pulmonary inflammation after in vivo exposure. Mannan is the second most abundant polysaccharide in the yeast cell wall, whereas chitin content is a minor component. The results show that pulmonary injury is mediated by both chitin and 1-->3-beta-glucan and to a lesser degree by mannan. There is also evidence that zymosan is more potent than purified 1-->3-beta-glucan alone. Evidence indicates that 1-->3-beta-glucan is the major inflammatory component in yeast and fungal cell walls.

A number of toxic heavy metals, such as cadmium (Cd) and mercury (Hg), are widely used in occupational settings, and exposure to these metals is associated with the development of pulmonary diseases. Cytotoxicity, apoptosis, and reactive oxygen species (ROS) generation were tested to compare the biological reactivity of these two heavy metals using a human bronchial epithelial cell line, BEAS-2B. Further, heat-shock protein 70 (Hsp70) expression was observed as a sensitive indicator of cellular stress. Exposure to metals (0-50 microM) for 72 h showed more significant cytotoxicity in Cd-treated than Hg-treated cells. Apoptosis was significantly increased in the cells exposed to 50 microM of Cd (3.5-fold) and Hg (3.6-fold). Cd and Hg produced an induction of Hsp70 protein as assayed by Western blotting and enzyme-linked immunosorbent assay (ELISA). Induction of Hsp70 protein by these metals was inhibited by addition of N-acetylcysteine. However, addition of catalase blocked the synthesis of Hsp70 only in Hg-treated cells. Hsp70B and Hsp70C mRNA expression was induced by both metals, while Hsp70A mRNA expression showed no change. Electron spin resonance (ESR) tests showed that hydroxyl radical generation was greater in the reaction of cells with Hg compared to Cd. Intracellular generation of ROS was detected in the cells exposed to both Cd and Hg. These results suggest that both cytotoxicity and apoptosis were significantly elevated with all metals tested; however, Cd was relatively more toxic. Hsp70 protein and mRNA were sensitive to exposure to these metals. Depletion of sulfhydryl groups of cellular proteins and generation of ROS may be involved in metal-induced lung cell damage.

Arginase induction was reported in several inflammatory lung diseases, suggesting that this may be a common feature underlying the pathophysiology of such diseases. As little is known regarding arginase expression in silicosis, the induction and cellular localization of arginase were elucidated in lungs of Sprague-Dawley rats 24 h following exposure to varying doses of silica by intratracheal instillation. Arginase expression was evaluated by activity assay, quantification of arginase I and arginase II mRNA levels using real-time polymerase chain reaction (PCR), and immunohistochemistry. Analyses of cells and fluid obtained by bronchoalveolar lavage (BAL) showed that markers of pulmonary inflammation, tissue damage, activation of alveolar macrophages (AM) and NO production were significantly increased by all silica doses. Arginase activity was increased also in AMs isolated from BAL fluid of silica-treated rats. Silica produced two- and three-fold increases in arginase activity of whole lung at doses of 1 and 5 mg/100 g body weight, respectively. Levels of arginase I mRNA, but not of arginase II mRNA, were similarly elevated. In control lungs, arginase I immunoreactivity was observed only in AMs sparsely dispersed throughout the lung; no inducible nitric oxide synthase (iNOS) immunoreactivity was detected. In silica-treated lungs, arginase I and iNOS were co-expressed in most AMs that were abundantly clustered at inflammatory foci. The rapid induction of arginase I expression in inflammatory lung cells, similar to induction of arginase in other inflammatory lung diseases, implicates elevated arginase activity as a factor in the development of lung damage following exposure to silica.

Agricultural workers are exposed to a variety of airborne dusts, including crystalline silica and other inorganic minerals. This study was designed to characterize the organic and inorganic components of agricultural dusts in California grape- and citrus-farm fields and to compare their cytotoxicity using in vitro toxicity bioassays as predictors of pathogenicity. Aerosolized dusts collected from farm fields were characterized by scanning-electron-microscopic energy-dispersive x-ray analysis, x-ray diffraction, trace metal analysis by plasma emission spectroscopy, and surface area measurements. As indicators of cytotoxicity, cell viability, release of alveolar enzymes activities (lactate dehydrogenase, N-acetyl glucosaminidase), production of reactive oxygen species (ROS), such as H2O2 and hydroxyl radical (OH), and lipid peroxidation were monitored after exposure of cells to grape- and citrus-farm dusts or inorganic components of these dusts. In addition, activation of nuclear factor kappa B and activator protein-1 were evaluated at the peak time for response of 36 h postexposure. All toxicity studies were done in comparison with crystalline silica of similar particle size and diameter using the same mass concentrations as farm dusts. The results showed that inorganic minerals in the aerosolized farm dust fractions were mostly composed of aluminum silicates, crystalline silica, and free iron. Crystalline silica used in these studies was more cytotoxic than grape- and citrus-farm dusts. However, in general, citrus farm dust exhibited the greatest ability to generate ROS and induce lipid peroxidation. These results support human epidemiologic studies, reporting an increased incidence of pulmonary fibrosis in farm workers, by documenting the potential of farm dusts to induce oxidative stress and initiate disease development.

It is well known that ozone (O3), a potent reactive oxidant and air pollutant, induces respiratory inflammation and hyperresponsiveness upon inhalation. It was previously shown that O3 exposure (0.6 ppm, 10 h/day for 15 days) not only results in local bronchial inflammation, but also affects the nervous system and thymocyte proliferation, and places mice under oxidative stress. In the present study, data showed that O3 exposure could impair both the natural killer (NK) cell activity and the proliferation potential of spleen T cells to a specific antigen stimulus. Immunological function assays indicated that O3 exposure attenuated the proliferation of spleen mononuclear cells induced by concanavalin A and decreased CD4+ and CD28+ lymphocyte subsets. However, supplementation with natural antioxidants protected mice from O3-induced dysfunction of splenocyte proliferation. Meanwhile, O3 exposure resulted in a decline of mitogen-induced IL-2 production in splenocytes. It was also found that O3 exposure dramatically enhanced the proliferation of CD4-CD8- thymocytes stimulated by recombinant mouse interleukin-7 (rmIL-7), which is usually observed during the mammal aging process. Taken together, data conclude that short-term repetitive O3 exposure damages both innate and acquired immunity via altering the lymphocyte subset and cytokine profile, and via impact on thymocyte early development. O3-induced oxidative damage is one of the key factors leading to immune dysfunction in this mouse model.

Intercellular communications between alveolar macrophages (AM) and alveolar epithelial type II (TII) cells have been suggested to be important in cellular responses. The main objective of this study was to improve our understanding of the interactions between AM and TII cells that might occur in the lung. In the present investigation, this interaction was studied under different interaction conditions (transwell or mixed coculture) and different exposure conditions (basal, lipopolysaccharide [LPS] exposure, or silica exposure). Studies also attempted different approaches to identify specific mediator(s) involved in this interaction. The following findings were made: (1) Surfactant released from TII cells appears to exert an inhibitory effect on AM activity. (2) Basal transwell coculture conditions are better than mixed coculture conditions to study AM/TII cell interactions, since the inhibitory effect of the surfactant in the transwell coculture is minimized. (3) AM/TII cell interaction is dependent on cell culture (transwell vs mixed) and exposure conditions. (4) Oxidants, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, prostaglandins, and leukotrienes probably do not independently affect the AM/TII intercellular interaction; instead, they appear to indirectly modulate the complex pathways of AM/TII communication.

Involvement of protein tyrosine kinases (PTK) in lipopolysaccharide (LPS)-induced nuclear factor-kappa B (NF-kappaB) activation has been demonstrated. Studies investigated the role of PTK and the underlying mechanisms by which PTK play a role in LPS induction of pathways leading to NF-kappaB activation in macrophages. Inhibitors of PTK-genistein, herbimycin A, or AG126-blocked LPS-induced NF-kappaB activation. Genistein also blocked pervanadate-induced NF-kappaB activation. Furthermore, Src TK selective inhibitors-damnacanthal or PP1-blocked LPS-induced NF-kappaB activation over a range of nanomolar concentrations. Genistein, damnacanthal, or PP1 blocked the LPS-induced serine phosphorylation, the degradation of IkappaB-alpha, and the consequent translocation of the p65 subunit of NF-kappaB to the nucleus. In addition to serine phosphorylation of IkappaB-alpha, LPS-induced NF-kappaB activation also required tyrosine phosphorylation of IkappaB-alpha. These TK inhibitors blocked substantially LPS induction of tyrosine phosphorylation of IkappaB-alpha. Furthermore, cSrc and Lck were physically associated with IkappaB-alpha. These results suggest that the LPS-induced NF-kappaB pathways are dependent on both serine and tyrosine phosphorylation of IkappaB-alpha, and that Src TK, such as cSrc and Lck, are key components of the LPS signaling pathway through at least two different mechanisms associated with NF-kappaB activation.

Evidence suggests that hyperproduction of reactive oxidants and inflammatory mediators plays a critical role in adverse pulmonary responses to silica or lipopolysaccharide (LPS). The objective of this study was to evaluate the role of alveolar macrophages (AM) and alveolar epithelial type II cells (TII) in the induction of pulmonary inflammation and injury in response to these pulmonary toxicants. To support this objective, the release of several inflammatory mediators from primary rat AMs and TII cells was compared under similar culture and exposure conditions. The responsiveness of RLE-6TN, a rat type II cell line, was also compared to primary rat TII cells under the same culture conditions, following exposure to LPS or silica. The following findings were made. (1) Although AMs were generally found to release more inflammatory mediators than TII cells following LPS or silica exposure, primary TII cells clearly produced significant levels of mediators that could be capable of contributing considerably to lung inflammation and injury. (2) Since the responses of the RLE-6TN cell line to LPS or silica exposure were generally considerably less intense and required higher concentrations of stimulant than those measured in primary rat TII cells, RLE-6TN cells may not be an ideal substitute for primary TII cells in studying pulmonary inflammation. (3) LPS was more potent than silica in inducing inflammatory cytokine release from the three cell types. However, compared to LPS, silica exhibited equal or greater potency as an inducer of cellular oxidant generation, especially from primary TII cells.

Tumor necrosis factor-alpha (TNF-a) is produced by alveolar macrophages (AM) in response to bleomycin (BLM) exposure. This cytokine has been linked to BLM-induced pulmonary inflammation, an early drug effect, and to lung fibrosis, the ultimate toxic effect of BLM. The present study was carried out to study the time dependence of apoptotic signaling pathways and the potential roles of TNF receptors in BLM-induced AM apoptosis. Male Sprague-Dawley rats were exposed to saline or BLM (1 mg/kg) by intratracheal instillation. At 1, 3, or 7 d postexposure, AM were isolated by bronchoalveolar (BAL) lavage and evaluated for apoptosis by ELISA. The release of cytochrome c from mitochrondria, the activation of caspase-3, -8, and -9, the cleavage of nuclear poly(ADP-ribose) polymerase (PARP), and the expression of TNF receptors (TNF-R1/p55 and TNF-R2/p75), TNF-R-associated factor 2 (TRAF2), and cellular inhibitor of apoptosis 1 (c-IAP1) were determined by immunoblotting. The results showed that BLM exposure induced AM apoptosis, with the highest apoptotic effect occurring at 1 d after exposure and gradually decreasing at 3 and 7 d postexposure, but still remaining significantly above the control level. The maximal translocation of cytochromec from mitochondria into the cytosol was observed at 1 d postexposure, whereas the activation of caspase-9 and caspase-3 and caspase-3-dependent cleavage of PARP was found to reach a peak level at 3 d postexposure. BLM exposure had no marked effect on AM expression of TNF-R1 or caspase-8 activation, but significantly increased the expression of TNF-R2 that was accompanied by a rise in c-IAP1 and a decrease in TRAF2. This induction of TNF-R2 by BLM was significant on d 1 and increased with greater exposure time. In vitro studies showed that pretreatment of naive AM with a TNF-R2 antibody significantly inhibited BLM-induced caspase-3 activity and apoptosis. These results suggest that BLM-induced apoptosis involves multiple pathways in a time-dependent manner. Since maximal BLM-induced AM apoptosis (1 d postexposure) preceded maximal changes in caspase-9 and -3 (3 d postexposure), it is possible that a caspase-independent mechanism is involved in this initial response. These results indicate that the sustained expression of TNF-R2 in AM by BLM exposure may sensitize these cells to TNF-a-mediated toxicity., (Copyright Taylor & Francis Inc.)

Inhalation of crystalline silica can produce lung inflammation and fibrosis. Inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) is believed to be involved in silica-induced lung disease. To investigate the role of iNOS-derived NO in this disease, the responses of iNOS knockout (KO) versus C57Bl/6J wild-type (WT) mice to silica were compared. Male mice (8-10 wk old, mean body weight 24.0 g) were anesthetized and exposed, by aspiration, to silica (40 mg/kg) or saline. At 24 h and 42 d postexposure, lungs were lavaged with saline. The first bronchoalveolar lavage (BAL) fluid supernatant was analyzed for lactate dehydrogenase (LDH) activity, levels of albumin, tumor necrosis factor-alpha (TNF-alpha), and macrophage inflammatory protein-2 (MIP-2), as well as total antioxidant capacity (TAC). The cellular fraction of the total BAL was used to determine alveolar macrophage (AM) and polymorphonuclear leukocyte (PMN) counts, and zymosanstimulated AM chemiluminescence (AM-CL). In separate mice, lung histopathological changes were evaluated 42 d postexposure. Acute (24-h) silica exposure decreased AMs, increased PMNs, increased LDH activity and levels of albumin, TNF-alpha, and MIP-2 in BAL fluid, and enhanced AM-CL in both iNOS KO and WT mice. However, iNOS KO mice exhibited less AM activation (defined as increased AM-CL and decreased AM yield) than WT. Furthermore, TAC following acute silica decreased in WT but was maintained in iNOS KO mice. Pulmonary reactions to subchronic (42 d) silica exposure were similar to acute. However, histopathological and BAL fluid indices of lung damage and inflammation, AM activation, and lung hydroxyproline levels were significantly less in iNOS KO compared to WT mice. These results suggest that iNOS-derived NO contributes to the pathogenesis of silica-induced lung disease in this mouse model., (Copyright Taylor and Francis Inc.)

Inhalation of residual oil fly ash (ROFA) has been shown to impair lung defense mechanisms in laboratory animals and susceptible populations. Bioavailability of soluble transition metals has been shown to play a key role in lung injury caused by ROFA exposure. The goal of this study was to evaluate the effect of soluble metals on lung defense and injury in animals preexposed to ROFA followed by pulmonary challenge with a bacterial pathogen. ROFA was suspended in saline (ROFA-TOTAL), incubated overnight at 37 degrees C, and separated by centrifugation into soluble (ROFA-SOL) and insoluble (ROFA-INSOL) fractions. A portion of the soluble sample was treated with the metal-binding resin Chelex for 24 h at 37 degrees C. Sprague-Dawley rats were intratracheally dosed at d 0 with ROFA-TOTAL (1.0 mg/100 g body weight), ROFA-INSOL, ROFA-SOL, saline, saline + Chelex, or ROFA-SOL + Chelex. At d 3, 5 x 10(5) Listeria monocytogenes were intratracheally instilled into rats from each treatment group. At d 6, 8, and 10, left lungs were removed, homogenized, and cultured to assess bacterial clearance. Histopathological analysis was performed on the right lungs. Pulmonary exposure of ROFA-TOTAL or ROFA-SOL before infection led to a marked increase in lung injury and inflammation at all three time points after inoculation, and an increase in morbidity in comparison to saline control rats. Treatment with ROFA-INSOL, saline + Chelex, or ROFA-SOL + Chelex caused no significant increases in lung damage and morbidity when compared to control. By d 10, the ROFA-SOL and ROFA-TOTAL groups had approximately 200-fold more bacteria in the lung than saline control, indicating the inability of these groups to effectively respond to the infection. None of the other treatment groups had significant impairments in bacterial clearance when compared to saline. In conclusion, exposure to ROFA-TOTAL and ROFA-SOL significantly suppressed the lung response to infection. These results suggest that soluble metals present in ROFA may play a key role in increased susceptibility to pulmonary infection in exposed populations.

The effect of diesel exhaust particulate (DEP) exposure on innate, cellular and humoral pulmonary immunity was studied using high-dose, acute-exposure rat, mouse, and cell culture models. DEP consists of a complex mixture of petrochemical-derived organics adsorbed onto elemental carbon particles. DEP is a major component of particulate urban air pollution and a health concern in both urban and occupational environments. The alveolar macrophage is considered a key cellular component in pulmonary innate immunity. DEP and DEP organic extracts have been found to suppress alveolar macrophage function as demonstrated by reduced production of cytokines (interleukin-1 [IL-1], tumor necrosis factor- alpha [TNF- alpha]) and reactive oxygen species (ROS) in response to a variety of agents, including lipopolysaccharide (LPS), interferon- gamma (IFN- gamma), and bacteria. Fractionation of DEP organic extract suggests that this activity was predominately in polyaromatic-containing and more polar (resin) fractions. Organic-stripped DEP did not alter these innate pulmonary immune responses. DEP also depressed pulmonary clearance of Listeria monocytogenes and Bacillus Calmette-Guerin (BCG). The contribution of the organic component of DEP is less well defined with respect to acquired and humoral immunity. Indeed, both DEP and carbon black enhanced humoral immune responses (specific immunoglobulin [Ig] E and IgG) in an ovalbumin-sensitized rat model. It is concluded that both the particulate and adsorbed organics may contribute to DEP-mediated immune alterations.

Carbon nanotubes represent a relatively recently discovered allotrope of carbon that exhibits unique properties. While commercial interest in the material is leading to the development of mass production and handling facilities, little is known of the risk associated with exposure. In a two-part study, preliminary investigations have been carried out into the potential exposure routes and toxicity of single-walled carbon nanotube material (SWCNT)--a specific form of the allotrope. The material is characterized by bundles of fibrous carbon molecules that may be a few nanometers in diameter, but micrometers in length. The two production processes investigated use-transition metal catalysts, leading to the inclusion of nanometer-scale metallic particles within unrefined SWCNT material. A laboratory-based study was undertaken to evaluate the physical nature of the aerosol formed from SWCNT during mechanical agitation. This was complemented by a field study in which airborne and dermal exposure to SWCNT was investigated while handling unrefined material. Although laboratory studies indicated that with sufficient agitation, unrefined SWCNT material can release fine particles into the air, concentrations generated while handling material in the field were very low. Estimates of the airborne concentration of nanotube material generated during handling suggest that concentrations were lower than 53 microg/m(3) in all cases. Glove deposits of SWCNT during handling were estimated at between 0.2 mg and 6 mg per hand.

Carbon nanotubes are new members of carbon allotropes similar to fullerenes and graphite. Because of their unique electrical, mechanical, and thermal properties, carbon nanotubes are important for novel applications in the electronics, aerospace, and computer industries. Exposure to graphite and carbon materials has been associated with increased incidence of skin diseases, such as carbon fiber dermatitis, hyperkeratosis, and naevi. We investigated adverse effects of single-wall carbon nanotubes (SWCNT) using a cell culture of immortalized human epidermal keratinocytes (HaCaT). After 18 h of exposure of HaCaT to SWCNT, oxidative stress and cellular toxicity were indicated by formation of free radicals, accumulation of peroxidative products, antioxidant depletion, and loss of cell viability. Exposure to SWCNT also resulted in ultrastructural and morphological changes in cultured skin cells. These data indicate that dermal exposure to unrefined SWCNT may lead to dermal toxicity due to accelerated oxidative stress in the skin of exposed workers.

The role of nitric oxide (NO) in pulmonary disease has been controversial with both antiinflammatory (scavenging radicals and inhibiting NF-êB activation) and proinflammatory (forming highly reactive peroxynitrite and augmenting NF-êB activation by inflammatory agents) actions reported. Therefore, a study has been initiated to determine whether deletion of the inducible nitric oxide synthase (iNOS) gene in the C57BL/6J mouse alters the pulmonary macrophage response to lipopolysaccharide (LPS) or silica. The objective of the initial phase of this study was to determine the difference in responsiveness of alveolar macrophages (AMs), harvested from naive wild-type (WT) or iNOS knockout (iNOS KO) mice, to an in vitro LPS or silica exposure. Primary AMs were obtained by bronchoalveolar lavage (BAL) from age- and weight-matched iNOS KO and WT mice. The cells were treated with interferon-gamma (IFN-ã) (50 U/ml), IFN-ã (50 U/ml) + LPS (1 microg/ml), LPS (0.01-100 microg/ml), or silica (25-250 microg/ml). The following parameters were measured: nitrate and nitrite (NOx), tumor necrosis factor-á (TNF-á), macrophage inflammatory protein-2 (MIP-2), intracellular generation of the reactive oxygen species (ROS) hydrogen peroxide (H(2)O(2) and superoxide (O(*-2)), and basal (unstimulated) total antioxidant capacity. Data show a significant increase in NOx production upon exposure to IFN-ã +/- LPS in the WT but not iNOS KO AMs. NOx production by iNOS KO or WT AMs was not altered by in vitro exposure to LPS or silica alone. LPS, but not silica, induced TNF-á and MIP-2 production in both iNOS KO and WT AMs. Statistical analysis of concentration response curves found a significant tendency for greater mediator production in the iNOS KO versus WT AMs. Basal intracellular production of H(2)O(2) and O(*- 2) was significantly greater in the iNOS KO compared to WT AMs. In contrast, LPS- (10 microg/ml) or silica- (100 microg/ml) stimulated intracellular oxidant production was lower in iNOS KO AMs, but overall (basal + stimulated) inflammatory capacity was similar between the cell types. The basal total antioxidant production of the iNOS KO AMs was approximately twofold higher than the WT AMs. In conclusion, certain compensatory changes appear to occur in AMs from iNOS KO mice. In response to the inability to induce NO production, iNOS KO AMs exhibit significantly higher basal generation of H(2)O(2) and (O(*- 2)) as well as higher total antioxidant levels. In addition, LPS induced TNF-á and MIP-2 production tend to be higher in AMs from iNOS KO mice. Such compensatory changes in the AM response may affect the response of iNOS KO mice to inflammatory exposures.

Asphalt fumes have been reported to produce nasal irritation in road workers. Since inhaled irritants can increase substance P (SP) production in airway neurons, the effects of asphalt fumes on SP production in trigeminal ganglia (TG) sensory neurons innervating the nasal mucosa were investigated. The effects of asphalt fumes on nasal mucosal innervation were examined by measuring SP and calcitonin-gene-related peptide (CGRP) levels in rat TG neurons projecting to the nasal epithelium. Female Sprague-Dawley rats were exposed to asphalt fumes at 16.0 +/- 8.1mg /m3 for 5 consecutive days, 3.5 h/d. Inflammatory cells were measured in nasal cavity lavage fluid. SP and CGRP immunoreactivity (IR) was measured in the cell bodies of trigeminal ganglion sensory neurons projecting to the nasal cavity. A significant increase in neutrophils and macrophages was observed after asphalt fume exposure indicating an inflammatory response in the nasal cavity. The percentage of SP-IR neurons increased significantly in the asphalt-exposed rats, and the proportion of CGRP-IR neurons was also elevated following asphalt exposure. These results indicate that exposure to asphalt fumes produces inflammation and increases the levels of SP and CGRP in TG neurons projecting to the nasal epithelium. The findings are consistent with asphalt-induced activation of sensory C-fibers in the nasal cavity. Enhanced sensory neuropeptide release from nerve terminals in the nasal cavity may produce neurogenic inflammation associated with nasal irritation following exposure to asphalt fumes.

1-->3-beta-Glucans produce pulmonary inflammation in rats and are commonly found in indoor air dust samples. Conformation is an important factor determining the biological activity of 1-->3-beta-glucans. The partially opened triple-helix conformation induced by NaOH treatment and the annealed triple-helix conformation have been identified by fluorescence resonance energy transfer spectroscopy in our previous study. The objective of this study was to examine the role of these conformations of 1-->3-beta-glucans in the induction of pulmonary inflammation in rats. A partially opened triple-helix conformation of the known inflammatory 1-->3-beta-glucan zymosan was prepared by treating zymosan with NaOH followed by neutralization and dialysis. The annealed triple-helix conformation was prepared by allowing the partially opened triple-helix conformation to anneal for 9 d at room temperature. Rats were exposed to fresh or annealed NaOH-treated zymosan via intratracheal instillation. The results show that the zymosan-induced pulmonary inflammatory responses were significantly reduced after the 9-d annealing period, which suggests that this inflammatory response was dependent on the conformation of zymosan. Freezing NaOH-treated zymosan (for 7 d) inhibited the annealing process. Exposure of rats to thawed preparations of zymosan resulted in the same inflammatory responses as the freshly prepared partially opened triple-helix zymosan. In contrast, the potency of untreated zymosan did not change significantly following a 7-d annealing period, indicating that annealing occurs only after the conformation has been modified by NaOH treatment. This study indicates that the partially opened triple helix of 1-->3-beta-glucans is more active than the closed conformation in inducing pulmonary inflammation in rats.

Although diesel exhaust particles (DEP) are known to produce pulmonary disorders, the xenobiotic metabolic pathways associated with DEP detoxification and bioactivation remain unclear. In this study, the effect of acute exposure of DEP on phase I and phase II enzymes of rat lung was investigated. Intratracheal administration of DEP produced an induction of cytochrome P-450 (CYP) 1A1 enzyme protein and activity at 1 d postexposure, with the enzyme level returning to control at 5 d postexposure. On the other hand, carbon black (CB), a particle control, did not show any induction of CYP1A1 protein or enzyme activity. However, both DEP and CB significantly decreased CYP2B1 protein and enzyme activity at 1 d postexposure. The decrease in CYP2B1 enzyme protein and activity by DEP or CB treatment was observed up to 7 d postexposure. DEP and CB treatments also significantly attenuated glutathione S-transferase (GST)-pi protein at 1 d postexposure. Both DEP and CB at 35 mg/kg significantly decreased the activities of GST and catalase at 1 and 7 d postexposure. DEP, but not CB, significantly induced quinone reductase (QR) activity at 7 d postexposure. This study suggests that DEP may induce CYP1A1 and QR enzymes via a chemical effect, while the carbonaceous core may be involved in the attenuation of CYP2B1, GST, and catalase proteins and enzyme activities.

1-->3-beta-Glucans, derived from the inner cell wall of yeasts and fungi, are commonly found in indoor air dust samples and have been implicated in organic dust toxic syndrome. In a previous study, it was reported that 1-->3-beta-glucan (zymosan A) induced acute pulmonary inflammation in rats. This study investigates which form of 1-->3-beta-glucans, particulate or soluble, is more potent in inducing pulmonary inflammation. Zymosan A was suspended in 0.25 N NaOH for 30 min, neutralized, dialyzed for 2 d using deionized water, and particulate and soluble fractions were collected. Male Sprague-Dawley rats were exposed via intratracheal instillation to NaOH-soluble or NaOH-insoluble zymosan A. At 18 h postexposure, various indicators of pulmonary response were monitored, including indicators of lung damage, such as serum albumin concentration and lactate dehydrogenase (LDH) activity in acellular bronchoalveolar lavage fluid. Inflammation was characterized by an increase in lavageable polymorphonuclear leukocytes (PMN). Pulmonary irritation (breathing frequency increase) and oxidant production (nitric oxide and chemiluminescence, CL) were also monitored. Exposure to the particulate form of NaOH-treated zymosan produced a significant increase in all these indicators. In contrast, rats exposed to the NaOH-soluble fraction were not markedly affected except for LDH, PMN, and CL. However, these increases were significantly less than with exposure to NaOH-insoluble zymosan. Therefore, results demonstrate that particulate zymosan A is more potent in inducing pulmonary inflammation and damage in rats than the soluble form of this beta-glucan.

Exposure to asphalt fumes is a health concern due to the presence of polycyclic aromatic compounds (PACs) in asphalt. Bioactivation of many PACs requires metabolism by the cytochrome P-450 (P-450) system. The objective of this study was to evaluate the effects of exposure of rats to asphalt fume condensate (AFC), collected at the top of a paving asphalt storage tank, on the pulmonary microsomal P-450 system and to determine the genotoxic effects of such exposure. Male Sprague-Dawley rats were intratracheally instilled with saline or with 0.45, 2.22, or 8.88 mg/kg AFC for 3 consecutive days and sacrificed the following day. Lung microsomes were isolated by differential centrifugation of lung homogenates. Microsomal protein level, NADPH cytochrome c reductase activity, and the activities and protein levels of cytochrome P-450 isozymes CYP1A1 and CYP2B1 were monitored to assess the effects of AFC exposure on pulmonary P-450. The activities of CYP2B1 and CYP1A1 were determined by monitoring xenobiotic metabolism of 7-pentoxyresorufin and 7-ethoxyresorufin, respectively. CYP2B1 and CYP1A1 levels were determined by immunochemical analysis. Micronucleus (MN) formation in bone-marrow polychromatic erythrocytes (PCEs) was determined to assess the genotoxic effects of AFC exposure. The results showed that exposure of rats to AFC did not significantly affect total cytochrome P-450 content or cytochrome c reductase activity in the lung. CYP2B1 levels and enzyme activity were not significantly affected by AFC exposure. In contrast, CYP1A1 levels and activity were significantly increased in microsomes isolated from AFC-exposed lungs. Increased MN formation was observed only in high-dose AFC-exposed bone marrow PCEs. These results demonstrate that AFC exposure induced CYP1A1 activity and increased the enzyme levels of CYP1A1 in lung microsomes, suggesting that AFC exposure may alter metabolism of PACs by the cytochrome P-450 system in the lung. Alteration of cytochrome P-450 metabolism of PACs may contribute to the AFC-induced genotoxic effects demonstrated as MN formation.

Blasting sand is used for abrasive blasting, but its inhalation is associated with pulmonary inflammation and fibrosis. Consequently, safer substitute materials for blasting sand are needed. In a previous study from this laboratory, the comparative pulmonary toxicity of five abrasive blasting substitutes and blasting sand was reported. In this study, the pulmonary toxicity of blasting sand was compared to five additional abrasive blasting substitutes: steel grit, copper slag, nickel slag, crushed glass, and olivine. Exposed rats received by intratracheal instillation 10 mg of respirable-size particles of blasting sand or an abrasive blasting substitute, while controls were instilled with vehicle. Pulmonary inflammation, damage, and fibrosis were examined 28 d postexposure. Pulmonary inflammation was monitored by determining bronchoalveolar lavage polymorphonuclear cell counts and alveolar macrophage activation by chemiluminescence. Pulmonary damage was assessed by acellular bronchoalveolar (BAL) fluid serum albumin concentrations and lactate dehydrogenase activities. Histological examination of lung tissue samples was made to assess the severity and distribution of pulmonary fibrosis, alveolitis, and alveolar epithelial cell hypertrophy and hyperplasia. In comparison to blasting sand, olivine exposed rats had higher levels of pulmonary inflammation and damage with a similar level of fibrosis. Steel grit-exposed rats had lower levels of pulmonary inflammation and damage, and did not develop fibrosis. However, steel grit-exposed rats had a level of epithelial cell hypertrophy and hyperplasia similar to blasting sand. The other abrasive blasting substitutes gave a mixed profile of toxicity. The data demonstrate that steel grit produced less acute pulmonary toxicity than blasting sand or any of the other abrasive blasting substitutes. Notwithstanding, the data also suggest that chronic exposure to steel grit may pose a health risk due to its effects on epithelial cell proliferation in the lung.

Metalloporphyrins have been shown to be protective in oxidative stress models. However, the molecular basis for the antioxidative and antiinflammatory activities of iron tetrakis (N-methyl-4'-pyridyl) porphyrinato (FeTMPyP) is not known. The objective of this study was to determine whether FeTMPyP exhibited the ability to (1) scavenge reactive oxygen species (ROS), (2) inhibit the activation of nuclear factor kappa B (NF-kappaB), or (3) block the production of interleukin 1 (IL-1) in RAW 264.7 cultured macrophages. The results indicate that FeTMPyP is a potent scavenger of hydroxyl radicals and superoxide anion radicals, and an effective inhibitor of stimulant-induced NF-kappaB activation and IL-1 production by RAW 264.7 cells. Therefore, FeTMPyP may be a useful tool to investigate the molecular mechanisms involved in stimulant-induced signal pathways, and may possess therapeutic utility in diseases associated with overproduction of ROS.