pulmonary toxicity of well-dispersed single-wall carbon nanotubes after inhalation
TRANSCRIPT
Nanotoxicology, November 2012; 6(7):766–775© 2012 Informa UK, Ltd.ISSN: 1743-5390 print / 1743-5404 onlineDOI: 10.3109/17435390.2011.620719
Pulmonary toxicity of well-dispersed single-wall carbon nanotubes afterinhalation
Yasuo Morimoto1, Masami Hirohashi1, Norihiro Kobayashi4, Akira Ogami1, Masanori Horie1, Takako Oyabu1,Toshihiko Myojo1, Masayoshi Hashiba1, Yohei Mizuguchi1, Tatsunori Kambara1, Byeong Woo Lee1,Etsushi Kuroda2, Manabu Shimada3, Wei-Ning Wang3, Kohei Mizuno4, Kazuhiro Yamamoto4,Katsuhide Fujita4, Junko Nakanishi4 & Isamu Tanaka1
1Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health Japan, Kitakyushu, Japan,2Department of Immunology and Parasitology, School of Medicine, University of Occupational and Environmental HealthJapan, Kitakyushu, Japan, 3Hiroshima University, Hiroshima, Japan and 4National Institute of Advanced Industrial Scienceand Technology, Tsukuba, Japan
AbstractSingle-wall carbon nanotubes (SWCNTs) were well-dispersed byultrasonication to conduct an inhalation study. SWCNTs weregenerated using a pressurised nebuliser with liquid suspensionof SWCNTs. Wistar rats were exposed to the well-dispersedSWCNT (diameter of bundle: 0.2 mm; length of bundle: 0.7 mm)for 4 weeks. The low and high mass concentrations of SWCNTswere 0.03 ± 0.003 and 0.13 ± 0.03 mg/m3, respectively. The ratswere sacrificed at 3 days, 1 month, and 3 months after the end ofexposure. There were no increases of total cell or neutrophilcounts in the bronchoalveolar lavage fluid (BALF), or theconcentration of cytokine-induced neutrophil chemoattractantin the lungs or BALF in both the high and lowconcentration-exposed groups. Pulmonary infiltration ofneutrophils was not observed in either exposed groupthroughout the observation period. Well-dispersed SWCNTdid not induce neutrophil inflammation in the lung underthe conditions in the present study.
Keywords: Nanomaterial, single-wall carbon nanotube, inhalation,dispersion
Introduction
Single-wall carbon nanotubes (SWCNTs) are a cylindricalnanostructure substance derived from wrapping a singlegraphene sheet. Demand for SWCNTs has increased as anew material for the next generation, and the effect ofSWCNT on the human body is not clear. Many in vitrostudies report that SWCNTs have cyto- and genotoxicities,and in vivo studies have shown that SWCNTs also havepulmonary toxicities (Card et al. 2008; Casey et al. 2008;Pacurari et al. 2008). Some reports also state that SWCNTs do
not have toxicities (Warheit et al. 2004). The discrepancies inthe reports about toxicity and SWCNTs are related to thephysicochemical properties of SWCNTs, including disper-sion states, dimensions, metal components, and purificationtreatment, which affect SWCNT toxicity (Card et al. 2008;Sohaebuddin et al. 2010). Before evaluating the response toSWCNTs, a major problem, nanoparticle agglomeration,must be faced. Among nanomaterials, SWCNTs are espe-cially difficult to be dispersed due to their relatively highsurface energy (Card et al. 2008; Morimoto et al. 2010a).In vitro and in vivo experiments have been performed usingSWCNTs with insufficient dispersion, including referenceswithout any documents describing their dispersion state(Card et al. 2008). In vivo and in vitro studies show thatthere are differences in the response induced by dispersedand non-dispersed nanoparticles (Liu et al. 2009; Wang et al.2010a, b; Mercer et al. 2008). In particular, agglomerationcould diminish the effective dose of carbon nanotubes in thelungs. Research shows that individually dispersed carbonnanotubes have stronger bacterial toxicity than agglomer-ated nanotubes (Liu et al. 2009). Dispersed SWCNTs stim-ulated more proliferation of bronchial epithelial cells andfibroblasts in in vitro studies and greater production ofcollagen in an in vivo study than did non-dispersed SWCNTs(Wang et al. 2010a, b). Ground SWCNTs were deposited inthe distal alveolar space and induced interstitial fibrosis inmice lungs, whereas unground SWCNTs were deposited inthe proximal alveolar space and induced granulomas(Mercer et al. 2008). Furthermore, dispersed carbon nano-tubes were also observed in the work environment (Methneret al. 2010; Han et al. 2008).
We examined the pulmonary toxicities of well-dispersedSWCNTs after 4-week inhalation in order to estimate theirharmful effects.
Correspondence: Yasuo Morimoto, Department of Occupational Pneumology, Institute of Industrial Ecological Sciences, University of Occupational andEnvironmental Health Japan, Yahatanishiku Iseigaoka 1-1, Kitakyushu, Fukuoka 807-8555, Japan. Tel: +81 93 691 7136. E-mail: [email protected]
(Received 13 February 2011; accepted 16 August 2011)
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Materials and methods
AnimalsMale Wistar rats (8 weeks old) were purchased from KyudoCo., Ltd. (Kumamoto, Japan). The animals were kept in theLaboratory Animal Research Center of the University ofOccupational and Environmental Health for a week withaccess to free-feeding of commercial diet and water. Allprocedures and animal handling were done according tothe guidelines described in the Japanese Guide for the Careand Use of Laboratory Animals as approved by the AnimalCare and Use Committee, University of Occupational andEnvironmental Health Japan, Kitakyushu, Japan.
Single-wall carbon nanotubeThe SWCNT material used in the present study was synthe-sised by water-assisted chemical vapour deposition deve-loped by the National Institute of Advanced IndustrialScience and Technology (AIST), Japan, using an iron catalyst.This method enabled a highly efficient synthesis of SWCNTs,where adding water vapour enhanced their catalyst activityand lifetime (Hata et al. 2004). Transmission electron micro-scope (TEM) image analysis showed that the diameters of theSWCNTswere 3± 1.1 nm.TheBrunauer, Emmett, Teller (BET)specific surface area of the SWCNT bulk material was deter-mined to be 1064 ± 37 m2/g. The total contents of the metalimpurities in the raw material were 0.05 ± 0.16 wt%, whichwas determined by thermogravimetric analysis (TGA). Ind-uctively coupledplasma-mass spectrometry (ICP-MS)analysisdetermined the individual metal impurities, which included12 ppm of Al, 145 ppm of Fe, 103 ppm of Ni, and 34 ppm ofCr. The total contents of the metal impurities determined byICP-MS analysis were 294 ppm, which was comparable withthat determined by TGA analysis. The D/G ratios of the bulkmaterial and the dispersion solution were calculated to be 0.14and 0.19, respectively, suggesting that there was only a slightdrop in SWCNT quality of the dispersion solution (Table I).
Preparation of SWCNT suspensionPrior to the inhalation study, the raw SWCNT material wascarefully dispersed into an aqueous medium (Mizuno et al.
2009). Briefly, the raw material was dispersed in 1% Tween80 solution with a bath sonicator (40 kHz, 240W) for 9 h. Thedispersed SWCNT fibres were then collected on a membranefilter and re-dispersed into distilled water in order to reducethe Tween 80 concentration in the dispersion medium. TEMobservation showed that the SWCNT fibres were suspendedin bundled form. Each bundle consisted of several to a fewtens of single SWCNT fibres, creating a rope-like shape. Thelength of the bundle was determined to be 0.2 mm in medianand ~1 mm in the maximum by atomic force microscope(AFM) image analysis. This suspension was the same as theone that was used in an intratracheal instillation study ofSWCNT (Kobayashi et al. unpublished data).
Inhalation systemThe whole-body exposure system, used to expose rats toSWCNT suspended in the air, consisted of a pressurisednebuliser and amist dryer and was connected to an exposurechamber (volume: 0.52 m3). SWCNT was suspended in theair using the pressurised nebuliser, and its aqueous com-ponent (distilled water including Tween 80) was removed bya mist dryer. The scanning electron microscope image andtransmission electron microscope image of the SWCNT inthe exposure chamber are shown in Figures 1B and 1C,respectively. The size and number concentrations ofSWCNTs in the chamber were analysed in-line using aparticle spectrometer consisting of a differential mobilityanalyser and a condensation particle counter (Model1000XP WPS, MSP Corp., Shoreview, MN, USA) throughoutthe exposure period. The average diameter and numberconcentration of the particles in the chamber weremeasuredthroughout all exposure days.
Inhalation study of SWCNTsNine-week-old male Wistar rats were divided into threegroups: low SWCNT concentration, high SWCNT concentra-tion, and Tween 80. There were 10 rats in each group. Therats inhaled the aerosol 6 h/day, 5 days/week, for 4 weeks ina whole-body exposure chamber. The SWCNTs and Tween80 aerosol in the chamber were collected with quartz fibrefilter (QAT-UP, Pall Sciences, Ann Arbor, MI, USA), theelemental carbon amounts were determined by an organiccarbon (OC)/elemental carbon (EC) aerosol analyser (SunsetLaboratory, Forest Grove, OR, USA), and the concentrationsin the chamber were calculated (Table II).
Carbon analysis of the particles showed that the massconcentration of SWCNTs in the aerosol particles deliveredto the low and high concentration rat groups was 0.03 ±0.003 and 0.13 ± 0.03 mg/m3, respectively. The particlenumber concentrations in the two groups were 5.0 ±0.7 � 104 and 6.6 ± 2.1 � 104 particles/cm3, respectively.The geometric mean value and geometric standard deviationof the particle length, the length measured along the particleshape, were 0.7 and 1.7 mm, respectively. Those for the widthof the particles, the maximum size perpendicular to thelength, were 0.2 and 1.7 mm, respectively. The Tween-exposed rats, the negative control, were designed to inhaleTween aerosol particles (average diameter: 62 ± 3 nm),prepared by using Tween 80 solution and another set of a
Table I. Characterisation of raw SWCNTs.
Characteristic SWCNT
Primary diameter 3.0 ± 1.1 nma
BET specific surface area 1064 ± 37 m2/ga
D/G ratio 0.14
Amorphous carbon content <2.3 ± 0.56%a
Total metal content 0.05 ± 0.16%a
Each metal content Fe 145 ppm
Ni 103 ppm
Cr 34 ppm
Mn 2 ppm
Al 12 ppm
Bundle diameter in suspension 12.0 ± 6.5 nma
Bundle length in suspension 0.32 mm (1.76)b
D/G ratio in suspension 0.19
pH in suspension 7.2aValues are expressed as mean ± SD.bValues are expressed as geometric mean (geometric SD).SD: standard deviation; SWCNT: single-wall carbon nanotube.
Pulmonary toxicity induced by well-dispersed SWCNT
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nebuliser and mist dryer, in a same-sized chamber at anaverage mass and particle number concentration of 0.38 ±0.06 mg/m3 and 5.6 ± 0.8 � 104 particles/cm3, respectively.After the exposure period and recovery, rats were injectedintraperitoneally with a fatal overdose of phenobarbital at3 days, 1 month, and 3 months, and dissected.
Animals after inhalation studiesEach group of 10 animals was divided into 2 subgroups of5 animals for lung tissue analysis.
The first subgroup provided bronchoalveolar lavage,which was collected using physiological saline that wasinjected through a cannula inserted into the respiratory tract,into the right lung. Three to 10 ml of physiological saline wasinfused in the right lung each time and, up to 50 ml in total oflavage fluid was collected. On the other hand, the left lungwas inflated and fixed by 4% paraformaldehyde at 25 cmH2Opressure.
The lungs of the second subgroup (five rats) were homo-genised to extract protein. The lung tissue was homogenisedwith a T-PER tissue protein extraction reagent, and thencentrifuged (1500� g for 10 min). The protein concentrationof the supernatant was measured by a BCA Protein AssayKit (PIERCE, Rockford , IL USA) using bovine serum albuminas a standard. The total protein concentration was adjustedto a final concentration of 500 mg/ml for cytokine-inducedneutrophil chemoattractant-1 (CINC-1) and CINC-2, 4000mg/ml for CINC-3, and 2000 mg/ml for heme oxygenase-1 (HO-1). Chemokine and HO-1 concentration in the lungtissue was determined by Quantikine Rat CINC-1, CINC-2,CINC-3 (R&D Systems, Minneapolis, MN, USA) (Cat.
#RCN100, #RCN200, and #RCN300) and an HO-1 ELISAKit (Stressgen Bioreagents, Ann Arbor, MI, USA). Thesechemokines and the HO-1 concentration in the bronchoal-veolar lavage fluid (BALF) supernatant were also measured.Alkaline phosphatase (ALP) released in the BALF superna-tant was determined by a LabAssayTM ALP (Wako PureChemical Industries, Ltd., Japan).
Tissue preparation for H&E stainThe lungs (total left lobe), which were inflated and fixed by4% paraformaldehyde, and the trachea were resected fromthe surrounding tissue. The lung tissue was embedded inparaffin and 5-mm thick sections were cut from the lobe. Thesamples were then sectioned and stained with H&E.
Processing of lung tissue for TEMThe lung tissues were fixed using a glutaraldehyde andosmium tetroxide solution, dehydrated in ethanol, andembedded in epoxy resin. The specimens were stainedwith a 2% uranyl acetate solution and 0.5% lead citratesolution at room temperature. Conventional TEM observa-tion was performed within an H-7000 (Hitachi, Japan) at anacceleration voltage of 80 kV. Energy-filtering TEM obser-vation was performed by an EM922 (Carl Zeiss SMT, GmbH,Oberkochen, Germany), which was equipped with anOMEGA energy filter. Zero-loss filtering, which can increasethe scattering and phase contrast of the TEM image, wasimplemented.
Statistical analysisStatistical analysis was carried out using the Mann–Whitney U test, with differences of p < 0.05 considered tobe statistically significant.
Results
Characterisation of SWCNTsAlthough thicker than the fibres in the suspension inFigure 1A, these aerosol SWCNTs mainly exhibited arope-like shape similar to the SWCNTs in the suspension.Electron micrograph images of the SWCNTs sampled from
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Figure 1. Electron micrograph image of SWCNT. (A) Transmission electron microscope image of SWCNT in suspension before aerosol generation.(B) Scanning electron microscope image of SWCNT in exposure chamber; arrow: SWCNT with rope-like shape; arrowhead: SWCNT with tangledappearance. Approximately 90% of SWCNTs were dispersed as rope-like shape. (C) Transmission electronmicroscope image of SWCNT in exposurechamber; arrowhead: individual SWCNT. Many SWCNTs with a rope-like shape were observed.
Table II. Characterisation of SWCNT in exposure chamber.
Characteristic SWCNT
Mass concentration (mg/m3) Low: 0.03 ± 0.003High: 0.13 ± 0.03
Number concentration (particles/cm3) Low: 5.0 ± 0.7 � 104
High: 6.6 ± 2.1 � 104
Geometric mean length (geometric SD) 0.7 mm (1.7)
Geometric mean width (geometric SD) 0.2 mm (1.7)
SD: standard deviation; SWCNT: single-wall carbon nanotube.
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the rat chamber are shown in Figures 1B and 1C. Bundles ofSWCNTs were observed in the rope-like shape (Figure 1C).There were coarser SWCNTs with a tangled appearance inthe aerosol particles, but the fraction of such SWCNTs wasonly about 10%. The high-resolution TEM image indicatedthat the SWCNTs with the rope-like shape consisted ofbundles of several to tens of SWCNTs, indicating that theSWCNT structure in the suspension was preserved in thosein the aerosol. Considering the gas-phase growth process ofthe SWCNTs in this study and the magnitude of the adhesionforce between adjacent SWCNT fibres, SWCNTs are thoughtto form a substantially similar shape if they are dispersed inthe air by some sort of dry-base handling for manufacturing,storage, and use (Tables I and II).
Cell count and ALP release in BALFFigure 2 shows the number of total cells, neutrophils, andALP released in BALF. The total cell and neutrophil counts inthe high and low concentrations of SWCNTs did not increasesignificantly more than in the Tween-exposed groups (neg-ative control) during the observation period. No significantrelease of ALP was observed in the SWCNT-exposed groups.Figure 3 shows alveolar macrophages in the lung. SWCNTswere dose dependently found as black spots in the alveolarmacrophage in both the low (Figure 3B) and high (Figure 3C)concentration-exposed groups at 3 days. Phagocytosis ofthe SWCNTs in alveolar macrophages (Figures 3E and 3F)
was also observed at 3 months, and decreased according toobservation times.
Concentration of CINCs in lungThere was no difference in the CINC-1 concentration in thelung tissue between the SWCNT-exposed groups and theTween-exposed group during the observation period.
As with the CINC-1 concentration, the CINC-2 con-centration in the lung tissue was not significantly increasedin the SWCNT-exposed groups. No significant differencein CINC-3 concentration in lung tissue was observedamong the three groups throughout the observation period(Figures 4A–C).
HO-1 concentration in lung and BALFRegarding the concentration of HO-1 in lung tissue, therewere no significant increases in HO-1 level in either con-centration of SWCNT during the observation period.
Although transient decreases in the concentration ofHO-1 in BALF in the SWCNT-exposed groups were observedin the time course, no consistent changes in the concentra-tion of HO-1 were found during the observation period(Figure 5).
Histopathological changes in lungsFigures 6A–F show pathological features in the lung at 3 daysand 1 month. No neutrophil infiltrations into the alveolar
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Figure 2. Analysis of BALF after inhalation of SWCNT. (A) Total cell count; (B) neutrophil cell count; (C) release of ALP. Each column and barrepresents the mean ± standard deviation of five rats. An asterisk indicates a statistically significant difference of p < 0.05 compared with eachunexposed group. Inhalation of SWCNT did not induce inflammatory response in the rat lungs.BALF: bronchoalveolar lavage fluid; SWCNT: single-wall carbon nanotube.
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space, granulomatous lesion, interstitial collagen depositionor emphysematous changes were found during the obser-vation period in either of the SWCNT-exposed groups.Alveolar macrophages that ingested SWCNTs were seen toa slight extent. There were no pathological changes in theTween-exposed group throughout the period.
Morphological feature of alveolar macrophage by TEMFigure 7 shows alveolar macrophages in the SWCNT-exposed lungs using TEM at 3 days after inhalation. SWCNTswere found in a plurality of phagolysozomes in an alveolarmacrophage. Individual and agglomerated SWCNTs were
also identified in the alveolar macrophages. No SWCNTswere observed in the nuclei or organelles.
Discussion
The characteristics of the present inhalation study of SWCNTare that: 1) SWCNTs with small amounts of metal contam-ination without acid treatment for purification were usedand 2) aerosol from SWCNTs was generated using the wet-based method. For metal contamination, the concentrationof metal in the SWCNTs was approximately 300 ppm asmeasured by TGA and ICP-MS, and was a very low
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Figure 3. Phagocytosis of SWCNT in alveolar macrophages. (A) Alveolar macrophages of negative control (Tween-exposed) at 3 days; (B) alveolarmacrophages exposed to low concentration of SWCNT at 3 days; (C) alveolar macrophages exposed to high concentration of SWCNT at 3 days; (D)alveolar macrophages of negative control (Tween-exposed) at 3 months; (E) alveolar macrophages exposed to low concentration of SWCNT at3 months; (F) alveolar macrophages exposed to high concentration of SWCNT at 3 months. The black spots are assumed to be SWCNTs. SWCNTswere found in the alveolar macrophage in both low and high concentration-exposed groups at not only 3 days but also at 3 months. Thephagocytosis of SWCNTs in alveolar macrophages decreased according to observation times.
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concentration. Usually, an amount of metal components thatcannot be ignored is included in commercially available as-produced SWCNTs, such as <35% in HiPco SWCNTs(Lam et al. 2006). As metal contamination is an important
factor affecting pulmonary response, the contamination isnormally removed by purification processes such as thermaloxidation and/or acid treatment. These purification pro-cesses can also affect the physicochemical properties of
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Figure 4. Concentration of CINC in rat lungs after inhalation of SWCNT. (A) CINC-1; (B) CINC-2; (C) CINC-3. Each column and bar represents themean ± standard deviation of five rats. Inhalation of SWCNT did not induce concentration of CINC-1, CINC-2, or CINC-3.CINC: cytokine-induced neutrophil chemoattractant; SWCNT: single-wall carbon nanotube.
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Figure 5. Expression of HO-1 in rat lungs after inhalation of SWCNT. (A) HO-1 concentration in lung tissue; (B) HO-1 concentration in BALF. Eachcolumn and bar represents the mean ± standard deviation of five rats. An asterisk indicates a statistically significant difference of p < 0.05 comparedwith each unexposed group. Inhalation of SWCNT did not induce concentration of HO-1 in the lung or BALF.BALF: bronchoalveolar lavage fluid; HO: heme oxygenase; SWCNT: single-wall carbon nanotube.
Pulmonary toxicity induced by well-dispersed SWCNT
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SWCNTs such as crystallinity, surface chemistry, dimension,and agglomeration state (Kang et al. 2008; Coccini et al.2010). Commonly, the purification processes oxidise chem-ically active SWCNT sites, resulting in open-end caps andintroducing defect sites at the side walls, where hydroxyl,carbonyl, and carboxyl functionalities are formed (Mann &Hase 2001; Vaisman et al. 2006; Jung et al. 2006). In existingstudies in which carbon nanotubes were dispersed for inha-lation experiments, all or most of aerosol particles generatedby using a dry-based dispersion method consisted ofentangled agglomerates of several to tens of micrometresin size (Mitchell et al. 2007; Baron et al. 2008; Ma-Hock et al.2009; Pauluhn 2010). The present inhalation experimentaimed at supplying aerosol particles having a rope-likeshape that reflects well the morphology of bundles ofSWCNTs. The usefulness of wet-based dispersing methodshas been confirmed for nanosized materials. Grassian et al.(2007) and Oyabu et al. (2007) investigated methods for
aerosol generation by spray-drying of aqueous particle sus-pension using a collision nebuliser and an ultrasonic nebu-liser, respectively, and obtained titanium dioxide and nickeloxide aerosol particles of 130–140 nm in geometric meandiameter. The method in which a pressurised nebuliser anda mist dryer were used (Shimada et al. 2009; Yokoyama et al.2009) was found to be capable of generating further smallerparticles. Nickel oxide particles of about 60 nm in geometricmean diameter and fullerene particles of 90–100 nm weresuccessfully generated and supplied for inhalation experi-ments. This method was also applied to aerosolisation ofmulti-walled carbon nanotubes. In contrast to dry-basedmethods, about 70% of the generated aerosol particles con-sisted of individual nanotubes (Morimoto et al. 2011). There-fore, a generation system based on this method wasemployed for the present SWCNT aerosol generation. Inthis study, 90% of the fibres were well-dispersed, and therope-like shapes consisted of bundles of several to tens of
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Figure 6. H&E staining of lung sections exposed to SWCNT after inhalation. Magnification 100�. (A) Lung of negative control (Tween-exposed) at3 days; (B) lung exposed to low concentration of SWCNT at 3 days; (C) lung exposed to high concentration of SWCNT at 3 days. No inflammatoryresponse by neutrophils or granulomatous lesion was observed in SWCNT-exposed lungs. (D) Lung of negative control (Tween-exposed) at3 months; (E) lung exposed to low concentration of SWCNT at 3 months; (F) lung exposed to high concentration of SWCNT at 3 months. Nosignificant inflammatory or fibrotic responses were observed in SWCNT-exposed lungs.
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SWCNTs. Unlike previous dry powder inhalation studies(Pauluhn 2010; Baron et al. 2008), few intertwined fibresand agglomerates consisting of rope-like-shaped particleswere observed in this experiment.
Only one previous inhalation study of SWCNT was anacute inhalation study (4 days) using the dry-based method(Shvedova et al. 2008), but it did not mention the SWCNTdispersion in the chamber. Furthermore, unpurified SWCNTswere used; the Fe component was 17.7%. Our inhalationsystem was appropriate because of the well-dispersed andpurified SWCNTs and the selection of fibrous materials.
We did not observe the pathological features of infil-tration of the inflammatory cells or fibrosis in the lungsthat inhaled SWCNTs. However, previous inhalation andintratracheal instillation studies of SWCNTs showed pul-monary inflammation and/or fibrosis, unlike our study. Ina previous study, acute inhalation of unpurified SWCNTs
showed granulomatous inflammation and the progressionof collagen deposition (Shvedova et al. 2008). In intratrachealinstillation studies, Lam et al. (2004) injected 0.1 and 0.5 mgof three kinds of SWCNTs into mice. Two kinds of lowdoses and all kinds of high doses induced granuloma inthe lung. Mangum et al. (2006) also reported that thepharyngeal aspiration of 2 mg/kg of SWCNTs inducedlocal interstitial fibrosis in mice. It has also been reportedthat in a pharyngeal aspiration study of SWCNTs, granulo-mas were not found in the lungs but interstitial fibrosis wascaused over a wide area. Warheit et al. (2004) reported thatintratracheal instillation of 1 and/or 5 mg/kg of SWCNTsinduced only transient multifocal granuloma. The dose inprevious in vivo studies was higher than that in ourexperiment. The pulmonary deposition of SWCNTs inour experiment can be calculated as follows: assumingthat the respiratory minute volume (RMV) of a rat with
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Figure 7. Alveolar macrophages in SWCNT-exposed lungs using TEM at 3 days after inhalation of SWCNT. (A) Alveolar macrophage of negativecontrol (Tween-exposed). (B) Alveolar macrophage exposed to low concentration of SWCNT. (C) Alveolar macrophage exposed to highconcentration of SWCNT. (D) Magnified image of alveolar macrophage exposed to high concentration of SWCNT; arrow: agglomerated SWCNTs.(E) Magnified image of alveolar macrophage exposed to high concentration of SWCNT; arrow: individual SWCNT. Individual and agglomeratedSWCNTs were also identified in the alveolar macrophages.
Pulmonary toxicity induced by well-dispersed SWCNT
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approximately 0.3 kg body weight (BW) is 0.19 l/min basedon the equation RMV = 0.499 � BW0.809, obtainedby Bide et al. (2000), and the deposition fraction of inhaledSWCNTs into the lungs of rats is 0.05 (5%), based on themultiple-path particle dosimetry (MPPD) model version2.1 (ARA 2009) calculation, then the pulmonary depositionof SWCNTs is calculated to be 1.9 and 8.8 mg in the low andhigh concentration group, respectively. We have intratrache-ally instilled the same SWCNTs in rats with 0.04, 0.2, 1, and2 mg/kg BW, and evaluated them until 3 or 6 months afterinstillation (Kobayashi et al. unpublished data). As a result,0.04 mg/kg (approximately 12 mg/rat) of SWCNT exposuredid not induce pulmonary inflammation at any time point,and 0.2 mg/kg (approximately 60 mg/rat) induced transientpulmonary inflammation that was recovered 1 month afterinstillation. By contrast, a dose more than 1 mg/kg (approx-imately 300 mg/rat) induced persistent pulmonary inflam-mation for at least 6 months after instillation. These resultsindicate that pulmonary inflammation was induced withincreased pulmonary deposition of SWCNTs; however,the response was slight and reversible in the group withSWCNT deposition approximately three times greaterthan that in the high concentration exposure group inthe present study. Therefore, the above-mentioned con-centration (0.13 mg/m3) is considered to be applicable toinhalation exposure for up to 3 months.
The concentrations of CINC1–3, representative of ratchemokines, did not significantly increase in the lungsthat inhaled SWCNT. The CINC-1 and CINC-2 expressionwas increased in a lung injury model using nickel oxidenanoparticles, and the CINC-1 expression was increased in apulmonary inflammation model using hazardous particlessuch as diesel particles (Nishi et al. 2009; Yokota et al. 2005).The results of an intratracheal instillation study of titaniumdioxide (micron-size), which is less hazardous to the lung,revealed a mild and transient increase in CINC-1 andCINC-2 expression only at an acute phase (Nishi et al.2009). We had previously analysed comprehensive geneexpression by microarrays in a 4-week inhalation study ofnickel oxide nanoparticles and fullerene (Morimoto et al.2010), finding that a highly increased expression ofCINC-1 and CINC-3 was observed from the exposure tonickel oxide nanoparticles, while a low increased expressionwas observed from fullerene exposure. In the previousinhalation study, neutrophil inflammations were related toCINC expression, and those data were also consistent withthe data in this inhalation study.
As free radical generation can induce inflammation, wealso examined HO-1 expression as an oxidative stressmarker. Inhaled SWCNTs did not induce a concentrationof HO-1 in the lung or BALF. We previously reported thatintratracheal instillation of crystalline silica and crocidolite(Nagatomo et al. 2007, 2006), materials with high toxicity,induced persistent increased expression of HO-1 in rat lung,and that micron-size titanium dioxide, a material with lowtoxicity, did not (Nagatomo et al. 2007). An inhalation studyof diesel engine exhaust showed that HO-1 mRNA levelswere elevated in mice lungs (Risom et al. 2003). In lungdisorders in humans, the levels of HO-1 in serum were
elevated in patients with silicosis (Sato et al. 2006). Thatreport states that an increase in HO-1 and the formation of8-hydroxydeoxyguanosine was observed and that those pos-itive cells were also found in the centre and peripheral areasof the silicotic nodule in patients with silicosis (Sato et al.2006). Therefore, a weak or no free radical generation ininhaled SWCNTs might be partially related to not inducinginflammation in the lung.
In order to examine whether or not SWCNTs contribute tolung injuries, we examined their capability to release ALPfrom alveolar epithelial cells. No released ALP in BALF wasfound during the observation time. We also found thatpulmonary inflammation and the ALP release level increasedin rats exposed to nickel oxide nanoparticles (Nishi et al.2009; Morimoto et al. 2010b, c). Fullerene, which did notshow inflammation or fibrosis, caused no release of ALP(Morimoto et al. 2010b). Sayes et al. (2007) also found asignificant amount of ALP in BALF in an intratrachealinstillation study with crystalline silica, but did not findthe same results in a study with fullerene. Consideringour and previous data, the inhalation of SWCNT also maynot induce alveolar damage at this exposure level.
In conclusion, we examined a 4-week inhalation study ofwell-dispersed purified SWCNTs. Neither low nor high con-centrations of SWCNTs induced inflammation of mainlyneutrophils or the concentration of CINCs or HO-1 in thelung. Well-dispersed SWCNTs did not induce neutrophilinflammation in the lung under the conditions in thepresent study.
Acknowledgements
This research was funded by a grant from the New Energyand Industrial (NEDO) titled: “Evaluating risks associatedwith manufactured nanomaterials; Developing toxicityevaluating methods by inhalation exposure (P06041)”.
Declaration of interest
The authors report no conflicts of interest. The authors aloneare responsible for the content and writing of the paper.Technology Development Organization of Japan.
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Pulmonary toxicity induced by well-dispersed SWCNT
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