James F. Scabilloni

Distribution and persistence of pleural penetrations by multi-walled carbon nanotubes

BackgroundMulti-walled carbon nanotubes (MWCNT) are new manufactured nanomaterials with a wide spectrum of commercial applications. The durability and fiber-like dimensions (mean length 3.9 μm long × 49 nm diameter) of MWCNT suggest that these fibers may migrate to and have toxicity within the pleural region. To address whether the pleura received a significant and persistent exposure, C57BL/6J mice were exposed by pharyngeal aspiration to 10, 20, 40 and 80 μg MWCNT or vehicle and the distribution of MWCNT penetrations determined at 1, 7, 28 and 56 days after exposure. Following lung fixation and sectioning, morphometric methods were used to determine the distribution of MWCNT and the number of MWCNT fiber penetrations of three barriers: alveolar epithelium (alveolar penetrations), the alveolar epithelium immediately adjacent to the pleura (subpleural tissue), and visceral pleural surface (intrapleural space).ResultsAt 1 day 18%, 81.6% and 0.6% of the MWCNT lung burden was in the airway, the alveolar, and the subpleural regions, respectively. There was an initial, high density of penetrations into the subpleural tissue and the intrapleural space one day following aspiration which appeared to decrease due to clearance by alveolar macrophages and/or lymphatics by day 7. However, the density of penetrations increased to steady state levels in the subpleural tissue and intrapleural from day 28 - 56. At day 56 approximately 1 in every 400 fiber penetrations was in either the subpleural tissue or intrapleural space. Numerous penetrations into macrophages in the alveolar airspaces throughout the lungs were demonstrated at all times but are not included in the counts presented.ConclusionsThe results document that MWCNT penetrations of alveolar macrophages, the alveolar wall, and visceral pleura are both frequent and sustained. In addition, the findings demonstrate the need to investigate the chronic toxicity of MWCNT at these sites.

Pulmonary fibrotic response to aspiration of multi-walled carbon nanotubes

BackgroundMulti-walled carbon nanotubes (MWCNTs) are new manufactured nanomaterials with a wide spectrum of commercial applications. To address the hypothesis that MWCNTs cause persistent pulmonary pathology, C57BL/6J mice were exposed by pharyngeal aspiration to 10, 20, 40 or 80 μg of MWCNTs (mean dimensions of 3.9 μm × 49 nm) or vehicle. Lungs were preserved at 1, 7, 28 and 56 days post- exposure to determine the potential regions and target cells for impact by MWCNT lung burden. Morphometric measurement of Sirius Red staining was used to assess the connective tissue response.ResultsAt 56 days post-exposure, 68.7 ± 3.9, 7.5 ± 1.9 and 22.0 ± 5.1 percent (mean ± SE, N = 8) of the MWCNT lung burden were in alveolar macrophages, alveolar tissue and granulomatous lesions, respectively. The subpleural tissues contained 1.6% of the MWCNT lung burden. No MWCNTs were found in the airways at 7, 28 or 56 days after aspiration The connective tissue in the alveolar interstitium demonstrated a progressive increase in thickness over time in the 80 μg exposure group (0.12 ± 0.01, 0.12 ± 0.01, 0.16 ± 0.01 and 0.19 ± 0.01 μm for 1, 7, 28 and 56 days post-exposure (mean ± SE, N = 8)). Dose-response determined at 56 days post-exposure for the average thickness of connective tissue in alveolar septa was 0.11 ± 0.01, 0.14 ± .02, 0.14 ± 0.01, 0.16 ± 0.01 and 0.19 ± 0.01 μm (mean ± SE, N = 8) for vehicle, 10, 20, 40 and 80 μg dose groups, respectively.ConclusionsThe distribution of lung burden was predominately within alveolar macrophages with approximately 8% delivery to the alveolar septa, and a smaller but potentially significant burden to the subpleural tissues. Despite the relatively low fraction of the lung burden being delivered to the alveolar tissue, the average thickness of connective tissue in the alveolar septa was increased over vehicle control by 45% in the 40 μg and 73% in the 80 μg exposure groups. The results demonstrate that MWCNTs have the potential to produce a progressive, fibrotic response in the alveolar tissues of the lungs. However, the increases in connective tissue per μg dose of MWCNTs to the interstitium are significantly less than those previously found for single-walled carbon nanotubes (SWCNTs).

Direct Fibrogenic Effects of Dispersed Single-Walled Carbon Nanotubes on Human Lung Fibroblasts

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.

Induction of pulmonary fibrosis by cerium oxide nanoparticles

Cerium compounds have been used as a diesel engine catalyst to lower the mass of diesel exhaust particles, but are emitted as cerium oxide (CeO(2)) nanoparticles in the diesel exhaust. In a previous study, we have demonstrated a wide range of CeO(2)-induced lung responses including sustained pulmonary inflammation and cellular signaling that could lead to pulmonary fibrosis. In this study, we investigated the fibrogenic responses induced by CeO(2) in a rat model at various time points up to 84 days post-exposure. Male Sprague Dawley rats were exposed to CeO(2) by a single intratracheal instillation. Alveolar macrophages (AM) were isolated by bronchial alveolar lavage (BAL). AM-mediated cellular responses, osteopontin (OPN) and transform growth factor (TGF)-β1 in the fibrotic process were investigated. The results showed that CeO(2) exposure significantly increased fibrotic cytokine TGF-β1 and OPN production by AM above controls. The collagen degradation enzymes, matrix metalloproteinase (MMP)-2 and -9 and the tissue inhibitor of MMP were markedly increased in the BAL fluid at 1 day- and subsequently declined at 28 days after exposure, but remained much higher than the controls. CeO(2) induced elevated phospholipids in BAL fluid and increased hydroxyproline content in lung tissue in a dose- and time-dependent manner. Immunohistochemical analysis showed MMP-2, MMP-9 and MMP-10 expressions in fibrotic regions. Morphological analysis noted increased collagen fibers in the lungs exposed to a single dose of 3.5mg/kg CeO(2) and euthanized at 28 days post-exposure. Collectively, our studies show that CeO(2) induced fibrotic lung injury in rats, suggesting it may cause potential health effects.

Impairment of Coronary Arteriolar Endothelium-Dependent Dilation after Multi-Walled Carbon Nanotube Inhalation: A Time-Course Study

Engineered nanomaterials have been developed for widespread applications due to many highly unique and desirable characteristics. The purpose of this study was to assess pulmonary inflammation and subepicardial arteriolar reactivity in response to multi-walled carbon nanotube (MWCNT) inhalation and evaluate the time course of vascular alterations. Rats were exposed to MWCNT aerosols producing pulmonary deposition. Pulmonary inflammation via bronchoalveolar lavage and MWCNT translocation from the lungs to systemic organs was evident 24 h post-inhalation. Coronary arterioles were evaluated 24–168 h post-exposure to determine microvascular response to changes in transmural pressure, endothelium-dependent and -independent reactivity. Myogenic responsiveness, vascular smooth muscle reactivity to nitric oxide, and α-adrenergic responses all remained intact. However, a severe impact on endothelium-dependent dilation was observed within 24 h after MWCNT inhalation, a condition which improved, but did not fully return to control after 168 h. In conclusion, results indicate that MWCNT inhalation not only leads to pulmonary inflammation and cytotoxicity at low lung burdens, but also a low level of particle translocation to systemic organs. MWCNT inhalation also leads to impairments of endothelium-dependent dilation in the coronary microcirculation within 24 h, a condition which does not fully dissipate within 168 h. The innovations within the field of nanotechnology, while exciting and novel, can only reach their full potential if toxicity is first properly assessed.

Potential role of apoptotic macrophages in pulmonary inflammation and fibrosis.

Induction of apoptosis has been associated with a variety of exposures which result in inflammatory and fibrotic lung disorders. Macrophages are key regulatory cells in the lung; however, the role of apoptotic macrophages in those pulmonary disorders is not well characterized. In the present investigation, apoptotic macrophages were instilled into the lungs of rats to study directly the pulmonary responses to apoptotic cells. The effects of apoptotic macrophages on lung inflammation and fibrosis, as well as associated protein expression of TNF‐α, TGF‐β, and matrix metalloproteinases (MMPs) were examined. Induction of macrophage apoptosis was carried out in vitro using a variety of known apoptosis inducers. Intratracheal administration of apoptotic macrophages (5 × 106 cells/rat) into the lung of rats caused an increase in pulmonary infiltration of macrophages and lung cell apoptosis 4 weeks after the treatment as indicated by terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling (TUNEL) assay. In contrast, pulmonary instillation of saline or normal control macrophages had no effect. Histological analysis of lung sections showed collagen deposition and fibrotic lesions after apoptotic cell treatment but not in control groups. Immunohistochemical studies revealed increased expression of TNF‐α, TGF‐β, MMP2, and MMP9 in the treatment group 4 weeks after the treatment. These results suggest a role for macrophage apoptosis in the initiation of these lung disorders. This study provides direct evidence that apoptotic macrophages can induce lung inflammation and fibrosis and that this induction may be associated with increased expression of TNF‐α, TGF‐β, MMP2, and MMP9. Published 2002 Wiley‐Liss, Inc.

ACUTE INFLAMMATORY REACTION IN RATS AFTER INTRATRACHEAL INSTILLATION OF MATERIAL COLLECTED FROM A NYLON FLOCKING PLANT

Several cases of interstitial lung disease have been diagnosed among workers at a nylon flock plant, but the etiologic agent for the disease outbreak was unknown. The results of a medical survey and industrial hygiene study indicated that the dust present in the plant may be responsible. Thus, airborne dust collected at the plant was examined for its inflammatory potential in rat lungs. The endpoints measured were: (1) breathing rates, (2) differential cell counts of bronchoalveolar lavage cells, (3) alveolar macrophage (AM) chemiluminescence, (4) albumin concentration and matrix metalloprotease activities in the acellular fluid from the initial bronchoalveolar lavage, and (5) pulmonary histopathology. In the first study, rats received a single dose of the airborne dust sample (10 mg/kg body weight) by intratracheal (IT) instillation. At 1 d post-IT, all inflammatory endpoints were significantly increased versus controls, but by 29 d post-IT they did not differ significantly from controls. Histopathology demonstrated mild to moderate, multifocal, suppurative pneumonia, usually centered around bronchioles, at 1 d post-IT. At 29 d post-IT, pulmonary inflammation was minimal to mild and characterized by alveolar histocytosis usually restricted to the immediate area of retained bire-fringent fibers. In subsequent experiments, airborne dust was extracted with water and the dust (washed airborne dust) and water extract (soluble fraction) were separated by centrifugation for further study. Nylon tow dust was prepared in the laboratory by milling uncut nylon strands (called tow) that had not been treated with the finish or dyes that are commonly used in the flock plants. Rats were administered a single dose of a dust sample (10 mg/kg body weight) or the soluble fraction (1.3 ml/kg body weight) by IT administration and the same endpoints were measured at 1 d post-IT. The dust samples caused significant increases in all of the inflammatory endpoints; however, the soluble fraction was much less active. Histological analysis of the lungs 1 d post-IT confirmed lung inflammation was occurring and tended to center around bronchioles. The results suggest that: (1) nylon flocking generates particles of respirable size that can interact with AM in the lung and can be detected in the lung 29 d after exposure, (2) the dust samples examined cause an inflammatory response, (3) water-extractable agent(s) from airborne dust contribute only minimally to the inflammatory response, and (4) the acute inflammatory response to these dusts is substantial when compared to other pathologic occupational dusts previously examined in our laboratory.

Lung toxicity and biodistribution of Cd/Se-ZnS quantum dots with different surface functional groups after pulmonary exposure in rats

BackgroundThe potential use of quantum dots (QD) in biomedical applications, as well as in other systems that take advantage of their unique physiochemical properties, has led to concern regarding their toxicity, potential systemic distribution, and biopersistence. In addition, little is known about workplace exposure to QD in research, manufacturing, or medical settings. The goal of the present study was to assess pulmonary toxicity, clearance, and biodistribution of QD with different functional groups in rats after pulmonary exposure.MethodsQD were composed of a cadmium-selenide (CdSe) core (~5nm) with a zinc sulfide (ZnS) shell functionalized with carboxyl (QD-COOH) or amine (QD-NH2) terminal groups. Male Sprague–Dawley rats were intratracheally-instilled (IT) with saline, QD-COOH, or QD-NH2 (12.5, 5.0, or 1.25 μg/rat). On days 0, 1, 3, 5, 7, 14, and 28 post-IT, the left lung, lung-associated lymph nodes (LALN), heart, kidneys, spleen, liver, brain, and blood were collected for metal analysis of Cd content by neutron activation to evaluate clearance and biodistribution. One right lobe was ligated and fixed for microscopy and histopathological analysis. The remaining right lobes from rats in each group were subjected to bronchoalveolar lavage (BAL) to retrieve BAL fluid and cells for analysis of injury and inflammation.ResultsLung injury and inflammation was found to be dose-dependent and peaked at days 7 and 14 post-exposure for both forms of QD, with slight variations in degree of toxicity at early and later time points. Both QD appeared to lose their fluorescent properties and destabilize after 1 week in the lung. Cd persisted up to 28 days for both forms of QD; however, clearance rate was slightly greater for QD-COOH over time. No Cd was detected in the liver, spleen, heart, brain, or blood at any time point. Cd appeared in the LALN and kidneys beginning at 1–2 weeks post-exposure.ConclusionsQD-COOH and QD-NH2 differed in clearance rate and differed slightly in degree of toxicity at different time points; however, the overall pattern of toxicity and biodistribution was similar between the two particles. Toxicity may be dependent on the dissolution rate and bioavailability of free Cd.

Pulmonary toxicity, distribution, and clearance of intratracheally instilled silicon nanowires in rats

Silicon nanowires (Si NWs) are being manufactured for use as sensors and transistors for circuit applications. The goal was to assess pulmonary toxicity and fate of Si NW using an in vivo experimental model. Male Sprague-Dawley rats were intratracheally instilled with 10, 25, 50, 100, or 250 μg of Si NW (~20–30 nm diameter; ~2–15 μm length). Lung damage and the pulmonary distribution and clearance of Si NW were assessed at 1, 3, 7, 28, and 91 days after-treatment. Si NW treatment resulted in dose-dependent increases in lung injury and inflammation that resolved over time. At day 91 after treatment with the highest doses, lung collagen was increased. Approximately 70% of deposited Si NW was cleared by 28 days with most of the Si NW localized exclusively in macrophages. In conclusion, Si NW induced transient lung toxicity which may be associated with an early rapid particle clearance; however, persistence of Si NW over time related to dose or wire length may lead to increased collagen deposition in the lung.

Role of lung surfactant in phagocytic clearance of apoptotic cells by macrophages in rats.

Two of the common features of inflammatory lung diseases are the increased production of pulmonary surfactant and the induction of lung cell apoptosis. However, the relationship between these two events has not been addressed. To investigate the role of surfactant in pulmonary inflammation and apoptosis, we instilled natural lung surfactant (Survanta) (1.6–12.5 mg) into the rat lungs and determined the number of alveolar macrophages (AMs) and apoptotic lung cells. High-dose treatments of Survanta (>6.25 mg/rat) caused an increase in macrophage cell influx and lung cell apoptosis at 4 weeks post-treatment. In vitro studies using lavaged macrophages showed Survanta did not cause apoptosis. We then examined the role of Survanta on ability of macrophages phagocytizing apoptotic cells. This study demonstrated that macrophages were able to eliminate apoptotic cells more efficiently in the absence of surfactant than in its presence. In vivo, high doses of Survanta decreased the ability to clear exogenously instilled apoptotic cells or bacteria. Taken together, our results suggest that excessive accumulation of lung surfactant by Survanta treatment can impair or overwhelm the phagocytic clearance function of AMs and that this impairment may lead to increased presence of apoptotic cells in the lung and bacterial survival.