Ann F. Hubbs

Single-walled carbon nanotube-induced mitotic disruption

Carbon nanotubes were among the earliest products of nanotechnology and have many potential applications in medicine, electronics, and manufacturing. The low density, small size, and biological persistence of carbon nanotubes create challenges for exposure control and monitoring and make respiratory exposures to workers likely. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to 24, 48 and 96 μg/cm(2) single-walled carbon nanotubes (SWCNT). To investigate mitotic spindle aberrations at concentrations anticipated in exposed workers, primary and immortalized human airway epithelial cells were exposed to SWCNT for 24-72 h at doses equivalent to 20 weeks of exposure at the Permissible Exposure Limit for particulates not otherwise regulated. We have now demonstrated fragmented centrosomes, disrupted mitotic spindles and aneuploid chromosome number at those doses. The data further demonstrated multipolar mitotic spindles comprised 95% of the disrupted mitoses. The increased multipolar mitotic spindles were associated with an increased number of cells in the G2 phase of mitosis, indicating a mitotic checkpoint response. Nanotubes were observed in association with mitotic spindle microtubules, the centrosomes and condensed chromatin in cells exposed to 0.024, 0.24, 2.4 and 24 μg/cm(2) SWCNT. Three-dimensional reconstructions showed carbon nanotubes within the centrosome structure. The lower doses did not cause cytotoxicity or reduction in colony formation after 24h; however, after three days, significant cytotoxicity was observed in the SWCNT-exposed cells. Colony formation assays showed an increased proliferation seven days after exposure. Our results show significant disruption of the mitotic spindle by SWCNT at occupationally relevant doses. The increased proliferation that was observed in carbon nanotube-exposed cells indicates a greater potential to pass the genetic damage to daughter cells. Disruption of the centrosome is common in many solid tumors including lung cancer. The resulting aneuploidy is an early event in the progression of many cancers, suggesting that it may play a role in both tumorigenesis and tumor progression. These results suggest caution should be used in the handling and processing of carbon nanotubes.

Mouse pulmonary dose- and time course-responses induced by exposure to multi-walled carbon nanotubes

Carbon nanotubes (CNT) come in a variety of types, but one of the most common forms is multi-walled carbon nanotubes (MWCNT). MWCNT have potential applications in many diverse commercial processes, and thus human exposures are considered to be likely. In order to investigate the pulmonary toxicity of MWCNT, we conducted an in vivo dose-response and time course study of MWCNT in mice in order to assess their ability to induce pulmonary inflammation, damage, and fibrosis using doses that approximate estimated human occupational exposures. MWCNT were dispersed in dispersion medium (DM) and male C57BL/6J mice (7 weeks old) received either DM (vehicle control), 10, 20, 40 or 80mug MWCNT by aspiration exposure. At 1, 7, 28 and 56 days post-exposure, MWCNT-induced pulmonary toxicity was investigated. Bronchoalveolar lavage (BAL) studies determined pulmonary inflammation and damage was dose-dependent and peaked at 7 days post-exposure. By 56 days post-exposure, pulmonary inflammation and damage markers were returning to control levels, except for the 40mug MWCNT dose, which was still significantly higher than vehicle control. Histopathological studies determined that MWCNT exposure caused rapid development of pulmonary fibrosis by 7 days post-exposure, that granulomatous inflammation persisted throughout the 56-day post-exposure period, and also demonstrated that MWCNT can reach the pleura after pulmonary exposure. In summary, the data reported here indicate that MWCNT exposure rapidly produces significant adverse health outcomes in the lung. Furthermore, the observation that MWCNT reach the pleura after aspiration exposure indicates that more extensive investigations are needed to fully assess if pleural penetration results in any adverse health outcomes.

Systemic Microvascular Dysfunction and Inflammation after Pulmonary Particulate Matter Exposure

The epidemiologic association between pulmonary exposure to ambient particulate matter (PM) and cardiovascular dysfunction is well known, but the systemic mechanisms that drive this effect remain unclear. We have previously shown that acute pulmonary exposure to PM impairs or abolishes endothelium-dependent arteriolar dilation in the rat spinotrapezius muscle. The purpose of this study was to further characterize the effect of pulmonary PM exposure on systemic microvascular function and to identify local inflammatory events that may contribute to these effects. Rats were intratracheally instilled with residual oil fly ash (ROFA) or titanium dioxide at 0.1 or 0.25 mg/rat 24 hr before measurement of pulmonary and systemic microvascular responses. In vivo microscopy of the spinotrapezius muscle was used to study systemic arteriolar responses to intraluminal infusion of the Ca2+ ionophore A23187 or iontophoretic abluminal application of the adrenergic agonist phenylephrine (PHE). Leukocyte rolling and adhesion were quantified in venules paired with the studied arterioles. Histologic techniques were used to assess pulmonary inflammation, characterize the adherence of leukocytes to systemic venules, verify the presence of myeloperoxidase (MPO) in the systemic microvascular wall, and quantify systemic microvascular oxidative stress. In the lungs of rats exposed to ROFA or TiO2, changes in some bronchoalveolar lavage markers of inflammation were noted, but an indication of cellular damage was not found. In rats exposed to 0.1 mg ROFA, focal alveolitis was evident, particularly at sites of particle deposition. Exposure to either ROFA or TiO2 caused a dose-dependent impairment of endothelium-dependent arteriolar dilation. However, exposure to these particles did not affect microvascular constriction in response to PHE. ROFA and TiO2 exposure significantly increased leukocyte rolling and adhesion in paired venules, and these cells were positively identified as polymorphonuclear leukocytes (PMNLs). In ROFA- and TiO2-exposed rats, MPO was found in PMNLs adhering to the systemic microvascular wall. Evidence suggests that some of this MPO had been deposited in the microvascular wall. There was also evidence for oxidative stress in the microvascular wall. These results indicate that after PM exposure, the impairment of endothelium-dependent dilation in the systemic microcirculation coincides with PMNL adhesion, MPO deposition, and local oxidative stress. Collectively, these microvascular observations are consistent with events that contribute to the disruption of the control of peripheral resistance and/or cardiac dysfunction associated with PM exposure.

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.

Nanoparticle inhalation augments particle-dependent systemic microvascular dysfunction.

BackgroundWe have shown that pulmonary exposure to fine particulate matter (PM) impairs endothelium dependent dilation in systemic arterioles. Ultrafine PM has been suggested to be inherently more toxic by virtue of its increased surface area. The purpose of this study was to determine if ultrafine PM (or nanoparticle) inhalation produces greater microvascular dysfunction than fine PM. Rats were exposed to fine or ultrafine TiO2 aerosols (primary particle diameters of ~1 μm and ~21 nm, respectively) at concentrations which do not alter bronchoalveolar lavage markers of pulmonary inflammation or lung damage.ResultsBy histopathologic evaluation, no significant inflammatory changes were seen in the lung. However, particle-containing macrophages were frequently seen in intimate contact with the alveolar wall. The spinotrapezius muscle was prepared for in vivo microscopy 24 hours after inhalation exposures. Intraluminal infusion of the Ca2+ ionophore A23187 was used to evaluate endothelium-dependent arteriolar dilation. In control rats, A23187 infusion produced dose-dependent arteriolar dilations. In rats exposed to fine TiO2, A23187 infusion elicited vasodilations that were blunted in proportion to pulmonary particle deposition. In rats exposed to ultrafine TiO2, A23187 infusion produced arteriolar constrictions or significantly impaired vasodilator responses as compared to the responses observed in control rats or those exposed to a similar pulmonary load of fine particles.ConclusionThese observations suggest that at equivalent pulmonary loads, as compared to fine TiO2, ultrafine TiO2 inhalation produces greater remote microvascular dysfunction.

Efficacy of a Technique for Exposing the Mouse Lung to Particles Aspirated from the Pharynx

Recent studies have demonstrated that the mouse lung can be exposed to soluble antigens by aspiration of these antigens from the pharynx. This simple technique avoids the trauma associated with intratracheal instillation. In this study, the pharyngeal aspiration technique was validated for exposing the mouse lung to respirable particles. Using respirable fluorescent amine-modified polystyrene latex beads and beryllium oxide particles, we investigated the localization of aspirated particles within the lung and the relationship between the amount of material placed in the pharynx and the amount deposited in the lung. For exposure, mice were anesthetized with isoflurane in a bell jar, placed on a slant board, and the tongue was gently held in full extension while a 50-µl suspension of particles was pipetted onto the base of the tongue. Tongue restraint was maintained until at least two breaths were completed. Less than a minute after exposure, all mice awoke from anesthesia without visible sequela. There were no significant differences in particle distribution between the left and right side of the lung (p = .16). Particles were widely disseminated in a peribronchiolar pattern within the alveolar region. There was a linear and significant correlation (r 2 = .99) between the amount administered and the amount deposited in the lung. In beryllium-exposed mice, measurable lung beryllium was 77.5 to 88.2% of the administered beryllium. These findings demonstrate that following aspiration of pharyngeal deposited particles, exposures to the deep lung are repeatable, technically simple, and highly correlated to the administered dose.

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).

Acute pulmonary dose–responses to inhaled multi-walled carbon nanotubes

Abstract This study investigated the in vivo pulmonary toxicity of inhaled multi-walled carbon nanotubes (MWCNT). Mice-inhaled aerosolized MWCNT (10 mg/m3, 5 h/day) for 2, 4, 8 or 12 days. MWCNT lung burden was linearly related to exposure duration. MWCNT-induced pulmonary inflammation was assessed by determining whole lung lavage (WLL) polymorphonuclear leukocytes (PMN). Lung cytotoxicity was assessed by WLL fluid LDH activities. WLL fluid albumin concentrations were determined as a marker of alveolar air–blood barrier integrity. These parameters significantly increased in MWCNT-exposed mice versus controls and were dose-dependent. Histopathologic alterations identified in the lung included (1) bronciolocentric inflammation, (2) bronchiolar epithelial hyperplasia and hypertrophy, (3) fibrosis, (4) vascular changes and (5) rare pleural penetration. MWCNT translocated to the lymph node where the deep paracortex was expanded after 8 or 12 days. Acute inhalation of MWCNT induced dose-dependent pulmonary inflammation and damage with rapid development of pulmonary fibrosis, and also demonstrated that MWCNT can reach the pleura after inhalation exposure.

Induction of aneuploidy by single-walled carbon nanotubes

Engineered carbon nanotubes are newly emerging manufactured particles with potential applications in electronics, computers, aerospace, and medicine. The low density and small size of these biologically persistent particles makes respiratory exposures to workers likely during the production or use of commercial products. The narrow diameter and great length of single‐walled carbon nanotubes (SWCNT) suggest the potential to interact with critical biological structures. To examine the potential of nanotubes to induce genetic damage in normal lung cells, cultured primary and immortalized human airway epithelial cells were exposed to SWCNT or a positive control, vanadium pentoxide. After 24 hr of exposure to either SWCNT or vanadium pentoxide, fragmented centrosomes, multiple mitotic spindle poles, anaphase bridges, and aneuploid chromosome number were observed. Confocal microscopy demonstrated nanotubes within the nucleus that were in association with cellular and mitotic tubulin as well as the chromatin. Our results are the first to report disruption of the mitotic spindle by SWCNT. The nanotube bundles are similar to the size of microtubules that form the mitotic spindle and may be incorporated into the mitotic spindle apparatus. Environ. Mol. Mutagen., 2009. Published 2009 Wiley‐Liss, Inc.

Pulmonary Nanoparticle Exposure Disrupts Systemic Microvascular Nitric Oxide Signaling

We have shown that pulmonary nanoparticle exposure impairs endothelium dependent dilation in systemic arterioles. However, the mechanism(s) through which this effect occurs is/are unclear. The purpose of this study was to identify alterations in the production of reactive species and endogenous nitric oxide (NO) after nanoparticle exposure, and determine the relative contribution of hemoproteins and oxidative enzymes in this process. Sprague-Dawley rats were exposed to fine TiO2 (primary particle diameter approximately 1 microm) and TiO2 nanoparticles (primary particle diameter approximately 21 nm) via aerosol inhalation at depositions of 4-90 microg per rat. As in previous intravital experiments in the spinotrapezius muscle, dose-dependent arteriolar dilations were produced by intraluminal infusions of the calcium ionophore A23187. Nanoparticle exposure robustly attenuated these endothelium-dependent responses. However, this attenuation was not due to altered microvascular smooth muscle NO sensitivity because nanoparticle exposure did not alter arteriolar dilations in response to local sodium nitroprusside iontophoresis. Nanoparticle exposure significantly increased microvascular oxidative stress by approximately 60%, and also elevated nitrosative stress fourfold. These reactive stresses coincided with a decreased NO production in a particle deposition dose-dependent manner. Radical scavenging, or inhibition of either myeloperoxidase or nicotinamide adenine dinucleotide phosphate oxidase (reduced) oxidase partially restored NO production as well as normal microvascular function. These results indicate that in conjunction with microvascular dysfunction, nanoparticle exposure also decreases NO bioavailability through at least two functionally distinct mechanisms that may mutually increase local reactive species.