| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1Pathology and Physiology Research Branch, Health Effect Laboratory Division, National Institute for Occupational Safety and Health, Morgantown; and 2Department of Physiology and Pharmacology, West Virginia University, Morgantown, West Virginia
Submitted 10 May 2007 ; accepted in final form 11 November 2007
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
toxicology; nanoparticles; particulates; emerging technologies; lung injury
There are relatively few toxicity studies of SWCNT. Lam et al. (10) were the first to report the granulomatous lesions in the mouse 7 and 90 days after intratracheal instillation of SWCNT. This research demonstrated that, on an equal-weight basis, SWCNT were more toxic than carbon black or quartz. Warheit et al. (26) conducted higher dose intratracheal instillation studies in the rat, which demonstrated granulomatous lesions but also reported that the granulomas were not dose dependent and that exposure to large agglomerates obstructed conducting airways, causing asphyxiation in a large percentage of exposed animals. They concluded that the high doses and large agglomerates resulted in an artifactual response that was not relevant to likely human exposure. In a subsequent pharyngeal aspiration study, Shvedova et al. (19) demonstrated that purified SWCNT (low-iron contamination) produce a significant granulomatous response associated with deposition sites of agglomerates. This study also demonstrated a progressive fibrotic response in the alveolar parenchyma that did not appear to rely on inflammatory cell mechanisms in the later stages of development and that was not associated with the sites exhibiting granulomatous response. These results suggest that a dispersed or submicrometer form of SWCNT, which was not detectable by light microscopic means, could be responsible for the alveolar wall connective tissue response. More recently, Mangum et al. (13) conducted similar experiments that also demonstrated the fibrotic response and found that SWCNT were able to form focal adhesions or bridges between alveolar macrophages, which may serve as a biomarker for nanotube exposure.
As evident from the studies described above, the pulmonary response to SWCNT is subject to debate, varying from a non-dose-dependent reaction to a dose-dependent progressive interstitial fibrotic response. The hypothesis to be tested in the present study is whether the deposition pattern and pulmonary reaction to SWCNT structure depend on the degree of dispersion in the exposure suspension.
In the present study, we report results using a combination of a number of different approaches to determine whether a population of dispersed SWCNT is present in suspensions used for pharyngeal aspiration in the study by Shvedova et al. (19) study and to test the possible health effects of a more dispersed suspension of purified SWCNT. To provide a direct test of the effect of improved SWCNT dispersion on toxicity, dispersed SWCNT (DSWCNT) were prepared and used for comparisons to the responses obtained with the less dispersed SWCNT preparation. Two different labeling processes were used to identify the distribution of both the less dispersed SWCNT and the more dispersed SWCNT preparations: labeling with 10-nm colloidal gold and labeling with fluorescent quantum dots. With the use of these labels, it was possible to identify the distribution of the more dispersed SWCNT form in the lungs, to determine whether the less dispersed SWCNT preparation also contained finer structures, and to determine where those finer structures were distributed.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Carbon nanotube source and dose. SWCNT purified from a high-pressure carbon monoxide production process (HiPCO) with <2 wt% of contaminants (CNI, Houston, TX) were used for preparation of DSWCNT. A dose of 10 µg/mouse was used for both SWCNT and DSWCNT exposures. This dose is the lowest of those used in our prior studies of responses to SWCNT (19).
Experimental groups and time points. Animals were studied at 1 h after pharyngeal aspiration to determine the initial deposition pattern and at 1 day, 7 day, and 1 mo postexposure to determine the acute inflammatory response, chronic fibrotic response, and fate of the aspirated carbon nanotube (CNT). A PBS-treated group was used as the control. Six mice, given DSWCNT or SWCNT, were studied at each time point for measurement of connective tissue and alveolar responses by morphometry. Bronchoalveolar lavage (BAL) analysis was done at each time point with six animals per group given DSWCNT. Additional groups were given gold-labeled DSWCNT (n = 4) or quantum dot-labeled DSWCNT (n = 4). These groups were studied at 1 h and at 1, 3, and 7 days after aspiration. To control for the possibility of unbound label, additional groups of mice were given aspirations of 0.1 µg of colloidal gold or 0.25 µg of quantum dots. The doses were chosen based on the amount of bound gold or quantum dots in the 10 µg/mouse gold-labeled DSWCNT and 10 µg/mouse quantum dot-labeled DSWCNT studies, respectively. These animals were studied 1 day after aspiration. A morphometric analysis of the fate of these unbound labels was conducted to identify how the lung processed unbound label.
Pharyngeal aspiration. This alternative to inhalation has been described in detail elsewhere (17). Briefly, after anesthetization, the animal was placed on a board in a near vertical position and the animal's tongue was extended with padded forceps. A suspension of the material to be aspirated (50 µl/mouse) at the desired concentration or PBS vehicle was placed on the back of the tongue. A slight pull of the tongue results in a reflex gasp and aspiration of the droplet. The tongue was held, and the animal was monitored for a few breaths after aspiration. To eliminate the possibility of incidental food aspiration, food was removed 4 h before the procedure; water was removed 1 h before the procedure.
BAL cell counting.
The degree of inflammatory response induced by the pharyngeal-aspirated particulates was estimated by the total cells, macrophages, neutrophils, and lymphocytes recruited into the mouse lungs and recovered by BAL. BAL of mice was conducted as previously described by our laboratory (28). Mice were weighed and killed with an intraperitoneal injection of pentobarbital sodium (>100 mg/kg). The trachea was cannulated with a blunted 22-gauge needle, and BAL was performed using cold sterile Ca2+- and Mg2+-free PBS at a volume of 1 ml/lavage for six lavages with a total of 
5 ml of BAL fluid per mouse collected and pooled. Centrifuge slide preparations were then prepared from the lavage fluid. Cell counts were performed to identify alveolar macrophages and polymorphonucleocytes (PMN) based on their characteristic cell shape in centrifuge slide preparations stained with the Hema-3 kit (Fisher Scientific, Pittsburgh, PA), and differential counts of BAL cells were conducted. Three hundred cells per slide were counted.
Vascular perfusion fixation of the lungs. Preservation of the lungs was done by vascular perfusion of fixative. This method of fixation was chosen to eliminate the possible disturbance of deposited materials or cells in the air spaces. Methods for vascular perfusion fixation were as previously reported (16) with the basic goal being to preserve the lungs at a state comparable to that present at end expiratory volume. For this procedure, the animal was anesthetized (75 mg/kg Nembutal) by intraperitoneal injection. Once a deep level of anesthesia was obtained, a tracheal cannula was inserted and a laparotomy was performed. The animal was then euthanized by exsanguination. The pulmonary artery was cannulated via the ventricle, and an outflow cannula was inserted into the left atrium. In quick succession, the tracheal cannula was connected to a 5 cmH2O pressure source to inflate the lungs with air, and clearing solution was perfused via the pulmonary artery. After blood was cleared from the lungs, the perfusate was switched to fixative consisting of 2% glutaraldehyde, 1% formaldehyde, and 1% tannic acid with sucrose as an osmotic agent. Tannic acid was included to produce an electron-dense staining of connective tissue fibers and to assist in preservation of lipid structures in the lungs. Clearing solution consisted of a physiological salt solution containing heparin (100 U/ml) and adjusted to 350 mosM with sucrose.
Fixed lung volume was measured by water displacement. Longitudinal sections were cut from the lungs. The lungs were embedded in paraffin and sectioned at a thickness of 5 µm for light microscopy. For electron microscopy, tissue blocks were en bloc stained with uranyl acetate and embedded in epoxy resin. Ultra-thin sections (60 nm) were cut, stained with uranyl acetate and lead citrate, and examined by a transmission electron microscope (JEOL JEM-1220).
Cryo fixation. Animals were anesthetized with pentobarbital sodium (75 mg/kg) by intraperitoneal injection and euthanized by exsanguination. The trachea was cannulated. An incision was made just below the sternum, and both hemidiaphragms were punctured to deflate the lungs. The lungs were then slowly inflated with 0.8 ml of a cryomix over a 1-min period. After inflation, the lungs were excised from the chest, placed in a cryomold, and covered with chilled OCT (Tissue-Tek; Sakura Finetek, Torrance, CA). The lungs-cryomold were then frozen in dry ice-cooled isopentane (–40°C to –50°C) and stored at –80°C until sectioned. A cryomicrotome was used to cut sections 4 µm thick. The lung tissue sections were mounted on slides and vapor fixed with vapors from a heated 2% paraformaldehyde solution. The cryomix consisted of 70% by volume OCT and 30% by volume of a 30% by weight sucrose-PBS solution.
Sirius red staining for collagen detection. Collagen in the lungs was detected with Sirius red staining (8), which has been demonstrated to be a quantitative morphometric method for collagen determination in the lungs (1, 12). Paraffin sections were deparaffinized and rehydrated with xylene-alcohol series to distilled water. The slides were then stained with 0.1% Picrosirius solution (100 mg of Sirius red F3BA in 100 ml of saturated aqueous picric acid, pH 2) for 1–2 h, washed for 1 min in 0.01 N HCl, counterstained with Mayer's hematoxylin for 2 min, dehydrated, and mounted with a coverslip.
Morphometric analysis. Quantitative morphometric methods were used to measure the average thickness of Sirius red-positive connective tissues in the alveolar regions. Volume and surface densities were measured by standard morphometric analyses (15, 25). This consisted of basic point and intercept counting. Volume density was determined from counting the number of points over the Sirius red-positive connective tissues in the alveolar regions and points over Sirius red-positive connective tissues and cells in the granulomatous regions. Surface density of the alveolar wall and granulomatous regions were determined from intercepts between a line overlay and the alveolar wall. These point and intercept counts were made with a 121-point, 11-line overlay graticule (12.5 mm2 with 100 divisions) at x100 magnification taken at six locations equally spaced across each section (one section per animal). This process was repeated twice for each animal. To limit the measurements to alveolar parenchyma, areas containing airways or blood vessels >25 mm in diameter were excluded from the analysis. Average thickness of the Sirius red-positive connective tissues of the alveolar wall was computed from two times the ratio of volume density of point to the surface density of the alveolar wall.
Preparation of dispersed SWCNT. Two hundred milligrams of purified SWCNT (low iron) were initially suspended in 60 ml of distilled water, passed through a 200-mesh stainless steel screen to grossly separate the material, and sonicated for 10 min. The solution was then expanded to 200 ml with acetone and placed in an ultrasonic bath maintained at room temperature for 24 h. When dispersed in acetone, there was an approximately 150-fold increase in the volume of the SWCNT sample compared with that in distilled water. This was due to the dissociation of nanotubes as the nonionic solvent, acetone, reduced the van der Waals forces binding the CNTs (27). The dispersed SWCNT were then filtered from the solution using two stages of a 20-µm nylon mesh screen, which had a 4-µm hole diameter (CMN-0029-C, Small Parts, Miami Lakes, FL). The acetone solution containing the DSWCNT was then passed through a 0.2-µm polytetrafluoroethylene (PTFE) filter, which had been weighed before use. After collection on the filter, the DSWCNT were washed several times with distilled water to remove residual acetone. The filter was dried overnight in vacuum and weighed to determine the quantity of DSWCNT (typically 5–10 mg). Before use, the filter containing the DSWCNT was placed in PBS and DSWCNT were released into solution by brief sonication. Acetone and PBS were prefiltered with a 0.2-µm PTFE filter before use. To assess the size distribution of the DSWCNT and SWCNT samples, a sample of each was taken, diluted to decrease loading density, and filtered onto a 25-mm diameter, 0.1-µm pore polycarbonate filter (VCTP02500 isopore membrane; Millipore, Billerica, MA). The polycarbonate filter was then washed with distilled water and dried. Micrographs of the CNT particles on the filter were then taken with a scanning electron microscope. The area equivalent diameter of the DSWCNT and SWCNT was then determined. The distribution of average area equivalent diameter of the DSWCNT was approximately normally distributed with a mean of 0.69 µm and a variance of 0.49 with a maximum area equivalent diameter of 3.7 µm. The area equivalent diameter of the SWCNT was approximately normally distributed with a mean of 15.2 µm and a variance of 3.2 µm. The improvement in dispersion was demonstrated by microscopic evaluation of the suspensions before aspiration, as shown in Fig. 1.
|
7.0. The CNT-PTFE filter was incubated in a 10-nm colloidal gold solution, pH 9.0, (polygold GC; Polysciences, Warrington, PA), which was monodispersed before use by sonication and filtration on a submicrometer filter. After 15 min, filtered BSA was added to a final concentration of 5% BSA to stabilize the gold particles and to assist in removal of unbound gold particles. Tris·HCl-saline with 1% BSA at pH 8.2 was then passed through the CNT-PTFE filter five or six times to remove any unbound colloidal gold. The filter was rinsed with Tris·HCl-saline and then rinsed with distilled water and stored dried on the filter until it was used. Before aspiration, the filter was rinsed with PBS and CNT were released from the PTFE filter by brief sonication. Silver enhancement of gold-labeled CNT in sections. For light microscope visualization of the gold-labeled CNT, paraffin sections were deparaffinized and dehydrated. The sections were then washed with multiple changes of deionized water. Sections were developed with a silver enhancement kit designed for gold labels (BB International, Cardiff, UK). The reaction was terminated after 15 min of development by washing in deionized water, and then the sections were lightly stained with Sirius red to improve detection of the silver deposits. Sections from PBS-treated controls and lungs given unlabeled SWCNT were used to control for background silver development in sections.
Fluorescent quantum dot labeling of CNT. SWCNT or DSWCNT were labeled with fluorescent quantum dots (30 nm in diameter). For this purpose, CNT were conjugated with an amine functionalized Fort orange fluorochrome, EviDots binding kit (T2-MP; Evident Technologies, Troy, NY). Two hundred micrograms of acid-treated CNT (SWCNT or DSWCNT) were filtered onto a 0.2-µm PTFE filter. The material was rinsed with distilled water to pH 7 and then labeled with quantum dots according to the EviTag conjugation protocol, which is a modification of a procedure first described by Grabarek and Gergely (5). Labeled CNT were then released into PBS solution by brief sonication.
Laser scanning confocal microscopy. For study of quantum dot-labeled CNT, lungs from animals treated by pharyngeal aspiration of Fort orange-labeled quantum dots were cyropreserved and sectioned. Images from the sections were recorded with a Zeiss LSM 510 confocal microscope using a x60 objective with a pixel size of 290 nm. The tissue was imaged by differential interference contrast, and the Fort orange-labeled quantum dots were imaged with a 543-nm HeNe laser for excitation, with a 560-nm long-pass filter to capture the red-orange emission of the quantum dots.
To study the collagen distribution by confocal imaging, 5-µm-thick paraffin sections were deparaffinized and transferred to ethanol. The sections were then fluorescently labeled by incubation for 24 h with a 0.1 mg/ml concentration of Lucifer yellow-CH according to manufacturer's protocol (Molecular Probes, Eugene, OR). Sections were then rinsed in ethanol for 5 min and coverslipped. Images were recorded with a x60 objective with excitation of 488 nm and emission spectra >510 nm used to image the fluorescently labeled collagen.
Statistical analyses.
Data were analyzed by ANOVA (STATGRAF). Bartlett's test was used to test for homogeneity of variances between groups. Statistical differences were determined by one-way ANOVA, with significance set at P 
0.05. When significant F-values were obtained, individual means were compared with control using two-sided Dunnett's test (3), and P < 0.05 was considered to be significant. Data are given as means ± SE.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
60% over the PBS controls occurs in response to DSWCNT at 1–7 days postexposure and returns to control at 30 days. This magnitude of alveolar macrophage response was similar to that previously observed for less dispersed SWCNT (19). Overall, the inflammatory sequence as indicated by BAL analysis and examination of lung sections was consistent with a mild, transient parenchymal inflammation that developed and was resolved within the first week of exposure.
|
|
20% of the total of less dispersed SWCNT material in the lungs based on the morphometric point counting of these sections. Silver-enhanced sections from lungs of mice given the gold-labeled DSWCNT showed a much finer and diffuse pattern of deposition, as illustrated in Fig. 5C. DSWCNT were identified throughout the interstitial spaces and in the vicinity of the pleura. Occasional deposits were also identified within alveolar macrophages, but these were <5% of the total by point counting of silver-enhanced sections. Deposition of DSWCNT in conducting airways was minimal. Figure 6 demonstrates the typical nanorope fragment interactions with lung cells, which were observed within alveolar macrophages and the alveolar interstitium within 1 day of aspiration of gold-labeled DSWCNT. Figure 6A shows two inclusions within an alveolar macrophage containing gold-labeled nanoropes (arrows indicate the gold label). Such engulfment of DSWCNT by alveolar macrophages was relatively rare. Figure 6B shows the much more typical finding of gold-labeled DSWCNT in the alveolar interstitial space. Background silver deposits per unit area of tissue section were determined in sections from PBS controls and estimated to account for <2% of the silver deposits in treated sections. In practice, it was possible to distinguish between background silver deposits and those developed from the presence of a gold label because the gold-labeled CNT generated a grapelike cluster of rounded silver deposits due to the multiple gold nucleation sites for silver binding, whereas background silver deposits were single and irregular to sharp-edged silver deposits.
|
|
|
|
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A more difficult issue to address is the possibility that the label alters the response of SWCNT in lungs. As demonstrated in the results, both gold and quantum dots are rapidly phagocytized by macrophages. We had concerns that attachment of the label to the CNT would induce avid phagocytosis by macrophages. For this reason, we intentionally conducted the labeling reactions under conditions that favored the lowest labeling density, which would still provide the capacity for detection. In comparing the lungs given the labeled CNT with unlabeled CNT, we were unable to identify any differences in response. Gold labeling of less dispersed SWCNT produced granulomas similar to those shown for unlabeled less dispersed SWCNT. Gold-labeled DSWCNT were rapidly incorporated within the alveolar interstitium and did not produce granulomatous lesions, as was the case for unlabeled DSWCNT. Similar results were found for quantum dot labeling of both less dispersed SWCNT and DSWCNT. Thus there was no evidence for differences in how the lungs processed or responded to labeled or unlabeled CNT for either of the two distinctly different labels.
In earlier studies, Lam et al. (10) noted that areas not directly associated with the deposition of SWCNT agglomerates appear "thicker" than normal but did not identify the cause. We demonstrated that DSWCNT produce significant increases in interstitial collagen and further showed that a significant fraction of the administered less dispersed SWCNT is deposited in the lungs as smaller nanorope structures. Based on our labeling studies, we also demonstrated that the small nanorope structures penetrate into the alveolar interstitium. For the less dispersed SWCNT preparation, 80% of the material is in the large agglomerate form, which, at least initially, remains in the air spaces and ultimately develops into granulomas; 20% of the SWCNT material is small enough to enter the alveolar walls and stimulate interstitial collagen accumulation.
Typically, SWCNT have been administered in PBS and are prone to rapid agglomeration. In PBS, SWCNT agglomerate within 1 min and can even be observed to agglomerate while being vortexed. Ultrasonication immediately before use can significantly reduce this problem and can result in deposits within the lungs with a diameter of 15 µm (9, 19). Once deposited in the lungs, the action of surfactant and protein may reduce van der Waals interactions between nanotubes and disperse these agglomerates. The dispersive action of alveolar-lining fluid on nanoparticles has been demonstrated in vitro (18). Evidence exists that improved dispersion of ultrafine carbon black in alveolar-lining fluid increases the inflammatory potential of the dust in vivo (20). In the present study, improved dispersion of CNT altered the distribution from agglomerates in the proximal alveoli to dispersed structures in the alveolar walls and pulmonary responses from granulomatous to an interstitial fibrotic reaction. Our study using both gold and quantum dot labeling of less dispersed SWCNT demonstrates that a significant fraction of the aspirated SWCNT material may exist as a submicrometer fraction that can rapidly migrate into alveolar interstitial spaces. The morphological evaluation of the distribution of labeled CNT indicates that relatively little of this material is a target for macrophage engulfment and phagocytosis. Results from in vitro studies by Kagan et al. (9) found little evidence for macrophage phagocytosis, whereas the study of Cherukuri et al. (2) using cells suspended in chemical surfactant demonstrated active ingestment. These differences may reflect differences in the state of the SWCNT samples used. For instance, purified SWCNT is low in iron, whereas raw SWCNT is rich in iron particles that may stimulate phagocytosis. Alternatively, the use of chemical surfactants in the incubation may enhance membrane permeabilities.
Studies to date have been limited to the responses during the first 30–60 days of exposure to SWCNT. Freshly produced CNT have a low chemical functionalization (21). Our observed low-labeling densities obtained by both gold and quantum dot labels and the low propensity for phagocytosis (9) are consistent with the idea that freshly manufactured SWCNT are not highly functionalized. This suggests that lung responses might begin to diminish at some time after the initial deposition and dispersion. However, the lungs are known to have a high oxidant-generating potential compared with other organs and also continually produce and reprocess large quantities of surfactants that may act as dispersive agents on the SWCNT. Over longer time frames, SWCNT encased in granulomas may be altered sufficiently to be released in a form that is either more readily recognized by macrophages or incorporated into the alveolar interstitial spaces. A delayed release of dispersed and more highly functionalized SWCNT would likely have adverse consequences.
The exposure level of DSWCNT was 10 µg/mouse lung in the present investigation. Maynard et al. (14) monitored respirable dust levels during laboratory handling of SWCNT and reported peaks of 53 µg/m3. With an assumption of a minute ventilation of 20,000 ml/min during light work (4) and 21% alveolar deposition of 0.69 µm of DSWCNT (7), 106.8 µg of DSWCNT would be predicted to deposit on the alveolar epithelial surface of a worker per day. Normalizing to the equivalent alveolar epithelial surface area in the human (102 m2/lung) and mouse (0.05 m2/lung) from published morphometric analysis (23), one can calculate that workers would receive a unit alveolar lung burden equivalent to pharyngeal aspiration of 10 µg of DSWCNT in the mouse in 200 workdays. Therefore, one can conclude that the exposure level of DSWCNT used in the present study is relevant to potential occupational exposures.
In conclusion, there are at least three principal pathways for dissemination of SWCNT in the lungs. In general, 80% of the less dispersed material deposits in the lungs as large agglomerates that result in rapidly forming granulomatous lesions in the alveolar region immediately proximal to the small airways. The remaining portion of less dispersed nanotubes appears to be aspirated as smaller structures and/or is dispersed by the lung milieu into a finer form, which is widely distributed throughout the air spaces of the lung parenchyma and is rapidly incorporated into the alveolar interstitial wall, inducing increased collagen production that progresses in the absence of persistent inflammation. A relatively small fraction of the purified SWCNT was found to be engulfed or otherwise processed by alveolar macrophages. Improved dispersal of SWCNT results in aspiration of smaller structures that are more prone to enter the alveolar walls. The pulmonary response to DSWCNT is a more potent interstitial fibrotic reaction in the absence of granuloma formation.
|
ACKNOWLEDGMENTS |
|---|
The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health.
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  J. Muller, M. Delos, N. Panin, V. Rabolli, F. Huaux, and D. Lison Absence of Carcinogenic Response to Multiwall Carbon Nanotubes in a 2-Year Bioassay in the Peritoneal Cavity of the Rat Toxicol. Sci., August 1, 2009; 110(2): 442 - 448. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Legramante, F. Valentini, A. Magrini, G. Palleschi, S. Sacco, I. Iavicoli, M. Pallante, D. Moscone, A. Galante, E. Bergamaschi, et al. Cardiac autonomic regulation after lung exposure to carbon nanotubes Human and Experimental Toxicology, June 1, 2009; 28(6-7): 369 - 375. [Abstract] [PDF]
|
|
|
|
|
|
U. C. Nygaard, J. S. Hansen, M. Samuelsen, T. Alberg, C. D. Marioara, and M. Lovik Single-Walled and Multi-Walled Carbon Nanotubes Promote Allergic Immune Responses in Mice Toxicol. Sci., May 1, 2009; 109(1): 113 - 123. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. P. Ryman-Rasmussen, E. W. Tewksbury, O. R. Moss, M. F. Cesta, B. A. Wong, and J. C. Bonner Inhaled Multiwalled Carbon Nanotubes Potentiate Airway Fibrosis in Murine Allergic Asthma Am. J. Respir. Cell Mol. Biol., March 1, 2009; 40(3): 349 - 358. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Shvedova, E. Kisin, A. R. Murray, V. J. Johnson, O. Gorelik, S. Arepalli, A. F. Hubbs, R. R. Mercer, P. Keohavong, N. Sussman, et al. Inhalation vs. aspiration of single-walled carbon nanotubes in C57BL/6 mice: inflammation, fibrosis, oxidative stress, and mutagenesis Am J Physiol Lung Cell Mol Physiol, October 1, 2008; 295(4): L552 - L565. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. W. Card, D. C. Zeldin, J. C. Bonner, and E. R. Nestmann Pulmonary applications and toxicity of engineered nanoparticles Am J Physiol Lung Cell Mol Physiol, September 1, 2008; 295(3): L400 - L411. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
