Multi-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.
At 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.
The 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).
Multi-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).
At 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.
The 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.
Prior studies have demonstrated a rapid and progressive acute phase response to bolus aspiration of multi-walled carbon nanotubes (MWCNTs). In this study we sought to test the hypothesis that ...inhalation exposure to MWCNT produces a fibrotic response and that the response is chronically persistent. To address the hypothesis that inhaled MWCNTs cause persistent morphologic changes, male C57BL/6 J mice were exposed in a whole-body inhalation system to a MWCNT aerosol and the fibrotic response in the alveolar region examined at up to 336 days after termination of exposure.
Inhalation exposure was to a 5 mg/m3 MWCNT aerosol for 5 hours/day for 12 days (4 times/week for 3 weeks). At the end of inhalation exposures, lungs were either lavaged for analysis of bronchoalveolar lavage (BAL) or preserved by vascular perfusion of fixative while inflated with air at 1, 14, 84, 168 and 336 days post inhalation exposure. Separate, clean-air control groups were also studied. Light microscopy, enhanced darkfield microscopy and field emission electron microscopy (FESEM) of tissue sections were used to analyze the distribution of lung burden following inhalation exposure. Morphometric measurements of Sirius Red staining for fibrillar collagen were used to assess the connective tissue response. Serial section analysis of enhanced darkfield microscope images was used to examine the redistribution of MWCNT fibers within the lungs during the post-exposure period.
At day 1 post-exposure 84 ± 3 and 16 ± 2 percent of the lung burden (Mean ± S.E., N = 5) were in the alveolar and airway regions, respectively. Initial distribution within the alveolar region was 56 ± 5, 7 ± 4 and 20 ± 3 percent of lung burden in alveolar macrophages, alveolar airspaces and alveolar tissue, respectively. Clearance reduced the alveolar macrophage burden of MWCNTs by 35 percent between 1 and 168 days post-exposure, while the content of MWCNTs in the alveolar tissue increased by 63 percent. Large MWCNT structures containing greater than 4 fibers were 53.6 percent of the initial lung burden and accounted for the majority of the decline with clearance, while lung burden of singlet MWCNT was essentially unchanged. The mean linear intercept of alveolar airspace, a measure of the expansion of the lungs, was not significantly different between groups. Pulmonary inflammation and damage, measured as the number of polymorphnuclear leukocytes (PMNs) or lactate dehydrogenase activity (LDH) and albumin in BAL, increased rapidly (1 day post) after inhalation of MWCNTs and declined slowly with time post-exposure. The fibrillar collagen in the alveolar region of MWCNT-exposed mice demonstrated a progressive increase in thickness over time (0.17 ± 0.02, 0.22 ± 0.02, 0.26 ± 0.03, 0.25 ± 0.02 and 0.29 ± 0.01 microns for 1, 14, 84, 168 and 336 days post-exposure) and was significantly different from clean-air controls (0.16 ± 0.02) at 84 and (0.15 ± 0.02) at 336 days post-exposure.
Despite the relatively low fraction of the lung burden being delivered to the alveolar tissue, the average thickness of connective tissue in the alveolar region increased by 70% in the 336 days after inhalation exposure. These results demonstrate that inhaled MWCNTs deposit and are retained within the alveolar tissue where they produce a progressive and persistent fibrotic response up to 336 days post-exposure.
Inhalation exposure studies of mice were conducted to determine if multi-walled carbon nanotubes (MWCNT) distribute to the tracheobronchial lymphatics, parietal pleura, respiratory musculature and/or ...extrapulmonary organs. Male C57BL/6 J mice were exposed in a whole-body inhalation system to a 5 mg/m3 MWCNT aerosol for 5 hours/day for 12 days (4 times/week for 3 weeks, lung burden 28.1 ug/lung). At 1 day and 336 days after the 12 day exposure period, mice were anesthetized and lungs, lymph nodes and extrapulmonary tissues were preserved by whole body vascular perfusion of paraformaldehyde while the lungs were inflated with air. Separate, clean-air control groups were studied at 1 day and 336 days post-exposure. Sirius Red stained sections from lung, tracheobronchial lymph nodes, diaphragm, chest wall, heart, brain, kidney and liver were analyzed. Enhanced darkfield microscopy and morphometric methods were used to detect and count MWCNT in tissue sections. Counts in tissue sections were expressed as number of MWCNT per g of tissue and as a percentage of total lung burden (Mean ± S.E., N = 8 mice per group). MWCNT burden in tracheobronchial lymph nodes was determined separately based on the volume density in the lymph nodes relative to the volume density in the lungs. Field emission scanning electron microscopy (FESEM) was used to examine MWCNT structure in the various tissues.
Tracheobronchial lymph nodes were found to contain 1.08 and 7.34 percent of the lung burden at 1 day and 336 days post-exposure, respectively. Although agglomerates account for approximately 54% of lung burden, only singlet MWCNT were observed in the diaphragm, chest wall, liver, kidney, heart and brain. At one day post exposure, the average length of singlet MWCNT in liver and kidney, was comparable to that of singlet MWCNT in the lungs 8.2 ± 0.3 versus 7.5 ± 0.4 um, respectively. On average, there were 15,371 and 109,885 fibers per gram in liver, kidney, heart and brain at 1 day and 336 days post-exposure, respectively. The burden of singlet MWCNT in the lymph nodes, diaphragm, chest wall and extrapulmonary organs at 336 days post-exposure was significantly higher than at 1 day post-exposure.
Inhaled MWCNT, which deposit in the lungs, are transported to the parietal pleura, the respiratory musculature, liver, kidney, heart and brain in a singlet form and accumulate with time following exposure. The tracheobronchial lymph nodes contain high levels of MWCNT following exposure and further accumulate over nearly a year to levels that are a significant fraction of the lung burden 1 day post-exposure.
Engineered carbon nanotubes are currently used in many consumer and industrial products such as paints, sunscreens, cosmetics, toiletries, electronic processes and industrial lubricants. Carbon ...nanotubes are among the more widely used nanoparticles and come in two major commercial forms, single-walled carbon nanotubes (SWCNT) and the more rigid, multi-walled carbon nanotubes (MWCNT). The low density and small size of these particles makes respiratory exposures likely. Many of the potential health hazards have not been investigated, including their potential for carcinogenicity. We, therefore, utilized a two stage initiation/promotion protocol to determine whether inhaled MWCNT act as a complete carcinogen and/or promote the growth of cells with existing DNA damage. Six week old, male, B6C3F1 mice received a single intraperitoneal (ip) injection of either the initiator methylcholanthrene(MCA, 10 μg/g BW, i.p.), or vehicle (corn oil). One week after i.p. injections, mice were exposed by inhalation to MWCNT (5 mg/m³, 5 hours/day, 5 days/week) or filtered air (controls) for a total of 15 days. At 17 months post-exposure, mice were euthanized and examined for lung tumor formation.
Twenty-three percent of the filtered air controls, 26.5% of the MWCNT-exposed, and 51.9% of the MCA-exposed mice, had lung bronchiolo-alveolar adenomas and lung adenocarcinomas. The average number of tumors per mouse was 0.25, 0.81 and 0.38 respectively. By contrast, 90.5% of the mice which received MCA followed by MWCNT had bronchiolo-alveolar adenomas and adenocarcinomas with an average of 2.9 tumors per mouse 17 months after exposure. Indeed, 62% of the mice exposed to MCA followed by MWCNT had bronchiolo-alveolar adenocarcinomas compared to 13% of the mice that received filtered air, 22% of the MCA-exposed, or 14% of the MWCNT-exposed. Mice with early morbidity resulting in euthanasia had the highest rate of metastatic disease. Three mice exposed to both MCA and MWCNT that were euthanized early had lung adenocarcinoma with evidence of metastasis (5.5%). Five mice (9%) exposed to MCA and MWCNT and 1 (1.6%) exposed to MCA developed serosal tumors morphologically consistent with sarcomatous mesotheliomas, whereas mice administered MWCNT or air alone did not develop similar neoplasms.
These data demonstrate that some MWCNT exposures promote the growth and neoplastic progression of initiated lung cells in B6C3F1 mice. In this study, the mouse MWCNT lung burden of 31.2 μg/mouse approximates feasible human occupational exposures. Therefore, the results of this study indicate that caution should be used to limit human exposures to MWCNT.
Fibrogenic carbon nanotubes (CNTs) induce the polarization of M1 and M2 macrophages in mouse lungs. Polarization of the macrophages regulates the production of proinflammatory and pro-resolving lipid ...mediators (LMs) to mediate acute inflammation and its resolution in a time-dependent manner. Here we examined the molecular mechanism by which multi-walled CNTs (MWCNTs, Mitsui-7) induce M1 polarization in vitro. Treatment of murine macrophages (J774A.1) with Mitsui-7 MWCNTs increased the expression of Alox5 mRNA and protein in a concentration- and time-dependent manner. The MWCNTs induced the expression of CD68 and that induction persisted for up to 3 days post-exposure. The expression and activity of inducible nitric oxide synthase, an intracellular marker of M1, were increased by MWCNTs. Consistent with M1 polarization, the MWCNTs induced the production and secretion of proinflammatory cytokines tumor necrosis factor-α and interleukin-1β, and proinflammatory LMs leukotriene B4 (LTB4) and prostaglandin E2 (PGE2). The cell-free media from MWCNT-polarized macrophages induced the migration of neutrophilic cells (differentiated from HL-60), which was blocked by Acebilustat, a specific leukotriene A4 hydrolase inhibitor, or LY239111, an LTB4 receptor antagonist, but not NS-398, a cyclooxygenase 2 inhibitor, revealing LTB4 as a major mediator of neutrophil chemotaxis from MWCNT-polarized macrophages. Knockdown of Alox5 using specific small hairpin-RNA suppressed MWCNT-induced M1 polarization, LTB4 secretion, and migration of neutrophils. Taken together, these findings demonstrate the polarization of M1 macrophages by Mitsui-7 MWCNTs in vitro and that induction of Alox5 is an important mechanism by which the MWCNTs promote proinflammatory responses by boosting M1 polarization and production of proinflammatory LMs.
Multi-walled carbon nanotubes (MWCNT) are new materials with a wide range of industrial and commercial applications. However, their nano-scaled size and fiber-like shape render them respirable and ...potentially fibrogenic if inhaled into the lungs. To understand MWCNT fibrogenesis, we analyzed the pathologic and molecular aspects of the early phase response to MWCNT in mouse lungs. MWCNT induced rapid and pronounced lesions in the lungs characterized by increased cellularity and formation of fibrotic foci, most notably near where MWCNT deposited, within 14 days post-exposure. Deposition of collagen fibers was markedly increased in the alveolar septa and fibrotic foci, accompanied by elevated expression of fibrotic genes
Col1a1
,
Col1a2
, and
Fn1
at both mRNA and protein levels. Fibrosis was induced rapidly at 40 μg, wherein fibrotic changes were detected on day 1 and reached a maximal intensity on day 7 through day 14. Induction of fibrosis was dose-dependent at the dose range of 5–40 μg, 7 days post-exposure. MWCNT elicited rapid and prominent infiltrations of neutrophils and macrophages alongside fibrosis implicating acute inflammation in the fibrotic response. At the molecular level, MWCNT induced elevated expression of proinflammatory cytokines TNFα, IL1α, IL1β, IL6, and CCL2 in lung tissues as well as the bronchoalveolar lavage fluid, in a dose- and time-dependent manner. MWCNT also increased the expression of fibrogenic growth factors TGF-β1 and PDGF-A in the lungs significantly. These findings underscore the interplay between acute inflammation and the early fibrotic response in the initiation and propagation of pulmonary fibrosis induced by MWCNT.
We 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 mum and ~21 nm, respectively) at concentrations which do not alter bronchoalveolar lavage markers of pulmonary inflammation or lung damage.
By 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.
These observations suggest that at equivalent pulmonary loads, as compared to fine TiO2, ultrafine TiO2 inhalation produces greater remote microvascular dysfunction.
Abstract 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 80 μg 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 40 μg 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.
Carbon nanotubes are commercially-important products of nanotechnology; however, their low density and small size makes carbon nanotube respiratory exposures likely during their production or ...processing. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to single-walled carbon nanotubes (SWCNT). In this study, we examined whether multi-walled carbon nanotubes (MWCNT) cause mitotic spindle damage in cultured cells at doses equivalent to 34 years of exposure at the NIOSH Recommended Exposure Limit (REL). MWCNT induced a dose responsive increase in disrupted centrosomes, abnormal mitotic spindles and aneuploid chromosome number 24 hours after exposure to 0.024, 0.24, 2.4 and 24 μg/cm² MWCNT. Monopolar mitotic spindles comprised 95% of disrupted mitoses. Three-dimensional reconstructions of 0.1 μm optical sections showed carbon nanotubes integrated with microtubules, DNA and within the centrosome structure. Cell cycle analysis demonstrated a greater number of cells in S-phase and fewer cells in the G2 phase in MWCNT-treated compared to diluent control, indicating a G1/S block in the cell cycle. The monopolar phenotype of the disrupted mitotic spindles and the G1/S block in the cell cycle is in sharp contrast to the multi-polar spindle and G2 block in the cell cycle previously observed following exposure to SWCNT. One month following exposure to MWCNT there was a dramatic increase in both size and number of colonies compared to diluent control cultures, indicating a potential to pass the genetic damage to daughter cells. Our results demonstrate significant disruption of the mitotic spindle by MWCNT at occupationally relevant exposure levels.
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