In vivo genotoxicity evaluation of lung cells from ...

http://informahealthcare.com/iht ISSN: 0895-8378 (print), 1091-7691 (electronic) Inhal Toxicol, 2014; 26(4): 222–234 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/08958378.2013.878006

RESEARCH ARTICLE

In vivo genotoxicity evaluation of lung cells from Fischer 344 rats following 28 days of inhalation exposure to MWCNTs, plus 28 days and 90 days post-exposure

1

Bioconvergence Department, Korea Conformity Laboratories, Incheon, Korea, 2Occupational Lung Diseases Institute, Korea Workers’ Compensation Welfare Service, Ansan, Korea, 3Institute of Nanoproduct Safety Research, Hoseo University, Asan, Korea, and 4Department of Mechanical Engineering, Hanyang University, Ansan, Korea Abstract

Keywords

Despite their useful physico-chemical properties, carbon nanotubes (CNTs) continue to cause concern over occupational and human health due to their structural similarity to asbestos. Thus, to evaluate the toxic and genotoxic effect of multi-wall carbon nanotubes (MWCNTs) on lung cells in vivo, eight-week-old rats were divided into four groups (each group ¼ 25 animals), a fresh air control (0 mg/m3), low (0.17 mg/m3), middle (0.49 mg/m3), and high (0.96 mg/m3) dose group, and exposed to MWCNTs via nose-only inhalation 6 h per day, 5 days per week for 28 days. The count median length and geometric standard deviation for the MWCNTs determined by TEM were 330.18 and 1.72 nm, respectively, and the MWCNT diameters ranged from 10 to 15 nm. Lung cells were isolated from five male and five female rats in each group on day 0, day 28 (only from males) and day 90 following the 28-day exposure. The total number of animals used was 15 male and 10 female rats for each concentration group. To determine the genotoxicity of the MWCNTs, a single cell gel electrophoresis assay (Comet assay) was conducted on the rat lung cells. As a result of the exposure, the olive tail moments were found to be significantly higher (p50.05) in the male and female rats from all the exposed groups when compared with the fresh air control. In addition, the high-dose exposed male and middle and high-dose exposed female rats retained DNA damage, even 90 days post-exposure (p50.05). To investigate the mode of genotoxicity, the intracellular reactive oxygen species (ROS) levels and inflammatory cytokine levels (TNF-a, TGF- b, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-g) were also measured. For the male rats, the H2O2 levels were significantly higher in the middle (0 days post-exposure) and high- (0 days and 28 days post-exposure) dose groups (p50.05). Conversely, the female rats showed no changes in the H2O2 levels. The inflammatory cytokine levels in the bronchoalveolar lavage (BAL) fluid did not show any statistically significant difference. Interestingly, the short-length MWCNTs deposited in the lung cells were persistent at 90 days post-exposure. Thus, exposing lung cells to MWCNTs with a short tube length may induce genotoxicity.

Multi-wall carbon nanotubes, nose-only inhalation, primary genotoxicity, single cell gel electrophoresis assay (Comet assay)

Introduction Carbon nanotubes exist as either multi-wall carbon nanotubes (MWCNTs), consisting of many hollow cylinders of carbon atoms inside one another, or single-wall carbon nanotubes (SWCNTs) with a single graphite lattice rolled into a perfect cylinder (Iijima, 1991; Vittorio et al., 2009). The special physico-chemical properties of carbon nanotubes (CNTs) make them useful for the medical, electronics, automotive and construction industries and other commercial processes, which has resulted in a dramatic increase in CNT production Address for correspondence : Il Je Yu, Institute of Nanoproduct Safety Research, Hoseo University, 165 Sechul-ri, Baebang-myun, Asan, 336-795, Korea. Tel: 82-41-540-9630. Fax: 82-41-540-9630. E-mail: [email protected]

History Received 6 May 2013 Revised 25 November 2013 Accepted 18 December 2013 Published online 25 February 2014

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Inhalation Toxicology Downloaded from informahealthcare.com by Hanyang University on 08/21/14 For personal use only.

Jin Sik Kim1, Jae Hyuck Sung1, Byung Gil Choi1, Hyeon Yeol Ryu1, Kyung Seuk Song1, Jae Hoon Shin2, Jong Seong Lee2, Joo Hwan Hwang2, Ji Hyun Lee3, Gun Ho Lee4, Kisoo Jeon4, Kang Ho Ahn4, and Il Je Yu3

and industrial applications over the last few years (Donaldson & Poland, 2012; Endo et al., 2008; Fujitani et al., 2009; Lin et al., 2004; Tenne et al., 2008). However, the growth of this global market enhances the chances of exposure to CNTs, not only during manufacture, but also with the usage, degradation and disposal of commercial products and through biomedical treatment (Donaldson et al., 2006; Gwinn & Vallyathan, 2006; Stem & McNeil, 2008). Yet, despite the increasing potential for indirect or direct exposure to CNTs, very little is still known about the effects of such exposure, thereby raising serious concerns about human and environmental health and safety issues related to CNTs (Pacurari et al., 2010; Porter et al., 2010). While exposure to CNTs can occur via inhalation, dermal contact, oral consumption or intravenous injection (for pharmacological application or

Genotoxicity of MWCNTs after sub-acute inhalation

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Table 1. Characterization data of multi-wall carbon nanotubes. Characteristics

MWCNTs (CM-95)

MWCNTs (CM-100)

Appearance Length Diameter Carbon purity Residual

Black powder 20 mm 10–15 nm 495 wt% Iron5wt 2%, Mo5wt 0.125% Al2O35wt 2.88% 201.18 Avg. IG/ID ¼ 1.12

Black powder 200 mm 10–15 nm 495 wt% Iron5wt 2%, cobalt5wt 2%, Al2O35wt 4% 224.9 Avg. IG/ID ¼ 0.92


Surface area (m2/g) Raman intensity

translocation via the blood stream), the lungs are still the most likely route of exposure to CNTs, which makes inhalation studies the best method for determining the toxicity of CNTs, as inhaled CNTs that reach the distal region of the lungs are more dispersed and less agglomerated than an instilled bolus dose in an aqueous liquid (Liu et al., 2012). It is already wellknown that harmful particles and nanoparticles produce oxidative stress, inflammation and genotoxicity, which are all pathobiologically linked to cancer (Card et al., 2008; Donaldson & Poland, 2012). In addition, many studies have reported that CNTs induce reactive oxygen species (ROS) generation, inflammatory reactions and genotoxic effects. Although previous studies have been performed based on in vitro systems using micro-organisms or cell lines, the resulting data can be studied further using in vivo animal studies (Fischer & Chan, 2007; Sharma et al., 2012). Accordingly, the present study performed a sub-acute noseonly inhalation exposure study using Fischer 344 rats and evaluated the genotoxicity and mechanism of the toxic effects in the rat lung cells. The genotoxicity effects of the CNTs were analyzed using a single cell gel electrophoresis assay (Comet assay), along with the effects of the reactive oxygen species level and inflammatory cytokine level on the genotoxic outcomes. In addition, the biopersistency of the MWCNTs deposited in the lungs was evaluated using an imaging technique. Previous CNT exposure assessment studies of workers showed that exposure can involve various forms of MWCNTs, such as dispersed, single or fused fibers (Han et al., 2008) and rarely found single and fused fibrous shapes (Lee et al., 2010). Furthermore, several inhalation toxicity studies have already been conducted using tangled particle shaped MWCNTs (Ma-Hock et al., 2009; Pauluhn, 2010). Thus, taking account of the realistic doses, including the dose and shapes occurring in CNT manufacturing and handling workplaces, the present study conducted an in vivo sub-acute toxicity inhalation test combined with a genotoxicity evaluation of the lung cells.

Materials and methods Multi-wall carbon nanotube characterization The MWCNTs (product name: CM-100, diameter 10 15 nm, length 20 microns) were obtained from Hanwha Nanotech, Inc (Incheon, Korea). Also, MWCNTs have been designated as an alternative reference material in the nanomaterial safety testing program sponsored by the OECD WPMN (Working Party on Manufactured

Characterization method Scanning electron microscope Transmission electron microscope Thermogravimetric Analysis Thermogravimetric Analysis Brunauer–Emmett–Teller (BET) surface area analysis Raman spectroscopy analysis

Nanomaterials) (Kim et al., 2011). The physico-chemical properties of the MWCNTs are summarized in Table 1. Unfortunately, the MWCNTs (CM-95) used in our previous 5-day inhalation study (Kim et al., 2012b) are no longer being manufactured, and there were not enough available for a subacute study. However, the physicochemical properties of CM-95 and CM-100 were almost identical. Generation of MWCNT aerosol The MWCNT aerosol was generated using the method developed by Ahn et al. (2011). Briefly, 4 g of MWCNTs were mixed with 1 L of deionized water up to a final volume of 10 L. This mixture was then heated to 80 C to disperse the MWCNTs in the water. To further increase the dispersion, the bottle of water-dispersed MWCNTs was also submerged in an ultrasonic bath for 6 h. The well-dispersed morphology, rather than tangled shapes and occasional multiple structures, was confirmed using a transmission electron microscope (TEM) (Figure 1). After sonication, atomization was initiated, where the ultrasonic power was maintained at 160 W and the water bath temperature kept at 80 C. The generation system consisted of three parts; dilution, generation and neutralization. The dilution system included a temperature controller and ultrasonic generator to mix the MWNCTs with the deionized water at a constant temperature. To disperse the MWCNTs without the use of a surfactant, the temperature was increased, thereby increasing the vapor pressure, while lowering the water density and viscosity. Meanwhile, the generation system involved atomizing a constant concentration of MWCNTs using three atmosphere (ATM) of pressure to induce a capillary phenomenon. A tube with a diameter of less than 1 mm was used to make droplets measuring less than several microns to allow easy vaporization. To reduce the loss of diffusion of the generated MWCNTs and maintain the humidity, an 80 L/min air flow was also introduced. Plus, a high electric field was applied to the end of the capillary tube to make the MWCNTs into a single fiber. Finally, the neutralization system supplied a high electric field to eliminate static and make the MWCNTs into straight fibers. To eliminate any static or residue voltage from the generation, a soft X-ray was included to neutralize the MWCNTs before introducing them to the inhalation chambers. Thus, singlefiber-shaped MWCNTs were supplied to the inhalation chambers at low, middle and high concentrations using an orifice flow meter to ensure a constant flow rate. The aerosolized MWCNTs were collected on a transmission electron microscopy (TEM) grid using a sampler, and the


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Figure 1. Transmission electron microscope (TEM) image (A: 3000, B: 20 000, C: 100 000) and energy dispersive spectroscopy (EDS) analysis (D) of MWCNTs collected from inhalation chambers after generation. Well-dispersed MWCNTs in A, and multiple MWCNTs in B.

morphology of the MWCNTs photographed using TEM. The TEM analysis was carried out as follows. The filters on which the MWCNT aerosols were sampled were mounted on an electron microscope grid (200 mesh, Veco, Eerbeek, Holland) and visualized under a transmission electron microscope (TEM, Hitachi 7100, Tokyo, Japan). The length distribution of 300 randomly selected tubes was measured at a 100 000 magnification, plus the MWCNTs were analyzed using an energy-dispersive X-ray analyzer (EDX-200, Horiba, Japan) at an accelerating voltage of 75 kV. The exposure chamber environments were automatically measured using an inhalation toxicity monitoring system (NITC 30, HCT, Icheon, Korea) at the main control center. Monitoring of inhalation chambers and analysis of MWNCTs The nose-only inhalation chamber system included 30 holders, where the general flow rate of 30 L/min was equally distributed at a rate of 1 L/min air at each holder. The air samples were taken by drawing air through polyvinylidene fluoride membrane filters in sampling cassettes (37 mm diameter and 50.8 mm cowl) obtained from Pall Corp. (P/N 64678; Ann Arbor, MI). The filter samples were collected from the middle portion of a port located in the third row of holders (total six rows of holders) from the top using MSA (Escort Elf pump, MSA, Cranberry Township, PA) -operated sampling pumps at a flow rate of 1.0 L/min. The samples were taken every day during 1 h and weighed in the monitoring room (20 ± 1 C, humidity 40 ± 2%). The data

obtained during the 28 days (20 samples) were averaged. A particle sensor (PS-3034, HCT, Icheon, Korea) with two channels (0.3 mm and 1 mm) was used to monitor the control chamber that was provided with HEPA filtered air. Animals and conditions Six-week-old male and female, specific-pathogen-free (SPF) Fischer 344 rats (F344/N Slc) were purchased from Central Lab. Animal Inc. (Seoul, Korea) and acclimated for 1 week before starting the experiments. The experimental animals were trained and adapted to the nose-only inhalation chamber holders for 6 h/day for 1 week. During the acclimation and experimental periods, the rats were housed in polycarbonate cages (five rats per cage) in a room with controlled temperature (21.6 ± 1.2 C), humidity (43.7 ± 6.8%), and a 12-h light/dark cycle. The rats were fed a rodent diet (Harlan Teklab, Plaster International Co., Seoul, Korea) and filtered water ad libitum. The eight-week-old rats, weighing about 155 g (male) and 130 g (female), were then divided into four groups (each group consisted of 15 male and 10 female rats (total 60 male and 40 female rats): five male and five female rats per dose were analyzed following 28 days (6 h/day, 5 days/week, 28 days) of nose-only exposure, five male rats per dose were analyzed 28 days post-exposure, and five male and five female rats per dose were analyzed 90 days post-exposure). The dose groups consisted of a fresh-air control, low-dose group (target concentration, 0.2 mg/m3), middle-dose group (target concentration, 0.5 mg/m3), and high-dose group (target concentration, 1.0 mg/m3), where the

DOI: 10.3109/08958378.2013.878006


nose-only exposure to MWCNTs was 6 h/day, 5 days/week for 28 days. The animals were examined daily on weekdays for any evidence of exposure-related effects, including respiratory, dermal, behavioral, nasal or genitourinary changes suggestive of irritancy. The body weights were measured at the time of purchase, at the time of grouping, one day prior to exposure, once a week during the inhalation exposure and recovery, and before necropsy. Plus, the animal autopsies were conducted on day 0, day 28 and day 90 post-exposure. The experiment was approved by the Korea Conformity Laboratories (KCL) Institutional Animal Care and Use Committee and compliant with OECD test guideline 412 (OECD, 2009). Analysis of ROS level and inflammatory cytokine level in BAL fluid The rats were deeply anesthetized with an overdose of sodium pentobarbital, and then exsanguinated by severing the abdominal aorta. The lungs were lavaged four times with 3-ml aliquots of cold saline (0.9% NaCl). The samples were also centrifuged for 7 min at 500 g and the cell-free bronchoalveolar lavage (BAL) fluid used for immunological tests, such as albumin, protein, lactate dehydrogenase (LDH), H2O2 and inflammatory cytokines level analyses. The cell pellets from all the washes for each rat were then combined, washed and resuspended in 1 mL of saline and evaluated (Sung et al., 2009). Using the BAL fluid, an H&E quick stain was performed, and the total number of cells, macrophages, polymorphonuclear cells and lymphocytes was then counted using a hemocytometer at a 400 magnification. The H2O2 concentrations in the BAL fluid were measured using a flow injection analysis method (Svensson et al., 2004). Briefly, 105 mL of the prepared BAL fluid or standard fluid was mixed with 7 mL of a derivatization reagent (1.5 mmol/L p-hydroxyphenyl acetic acid 200 mL + 2.5 U/mL horseradish peroxidase 500 mL). The BAL fluid H2O2 was then analyzed using an Agilent 1200 HPLC (Agilent Technologies, Santa Clara, CA). The Glutathione (GHS) was measured using the method of Norris et al. (2001). 100 mL of ice cold 20% m-phosphoric acid was added to the BAL fluid and the mixture stored at 80 C until use. The mixture was then thawed at 37 C and centrifuged for 5 min. 5 mL of the supernatant was analyzed by LC-ESI-MS/MS (3200QTRAP, AB SCIEX, Framingham, MA). The analysis of the aldehyde levels, such as MDA (malondialdenyde), 4-HHE (4-hydroxy-2-hexenal) and n-hexanal, in the BAL fluid samples was performed using liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS), as previously described (Svensson et al., 2004). Briefly, the samples were derivatized using a 2,4dinitrophenylhydrazine solution (12 mM in acetonitrile and 2% formic acid) at room temperature for 60 min, and then 20 mL of the derivatives injected onto the LC-APCI-MS/MS system (3200QTRAP, AB SCIEX, Framingham, MA). The chromatography of the derivatives was performed on a C18 reverse-phase column (Triart-C18; 2.0 mm 100 mm, 3 mm, YMC Co. Ltd, Japan) using 20 mM aqueous acetic acid and methanol under a gradient elution condition at a flow rate of 0.2 mL/min. The gradient program was as follows: from 45%


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to 98% methanol for 4 min, linear gradient, and then hold for 4.5 min. The level of inflammatory cytokines (TNF-a, TGF-b, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-g) in the BAL fluid was measured using a Bio-Plex Rat 23-Plex assay (BioRad Laboratories Inc., Hercules, CA) and Mouse/Rat/Porcine/ Canine TGF-beta 1 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Deposition of MWCNTs in lungs The MWCNTs were suspended in 0.5% bovine serum albumin (BSA, Roche Diagnostics, Germany) in neutral phosphatebuffered saline (PBS) (Noble Bio, Gyeonggi-do, Korea) and dispersed at 250 W for 3 h in an ultrasonic bath (Branson Ultrasonics 8510, Danbury, CT). Around 10 mL of the dispersed MWCNTs was placed on an ultrafine slide glass and coverslipped (Schott Nexterion, Mainz, Germany). The morphology of the MWCNTs was observed using a High Resolution Adaptor (CytoViva, Inc., Auburn, AL) and the unique spectra of the MWNCTs scanned using a Hyperspectral Imaging System (CytoViva, Inc., Auburn, AL) attached to a light microscope (Olympus, Tokyo, Japan). The lungs were weighed and fixed in a 10% formalin solution containing neutral PBS. The lungs were then embedded in paraffin, and stained with hematoxylin and eosin for a routine pathological assessment. Meanwhile, unstained and deparaffinized slides were examined for the deposition of MWCNTs in the lungs. The target regions of the lungs were observed using a high resolution illuminator and scanned at a 400 magnification (10 ocular lens and 40 objects) using a Hyperspectral Imaging System (Cytoviva, Auburn, AL). When merging the scanned images of the lungs with the MWCNT spectra using ENVI 4.4 software (Exelis Visual Information Solutions, McLean, VA), the MWCNTs in the lungs were revealed as red spots (Kim et al., 2012b). Lung cell isolation Lungs were obtained from five male and five female rats in each test group on day 0, day 28 and day 90 post-exposure, respectively. The lungs were minced and suspended in chilled PBS, and gently homogenized in ice using a tissue grinder (Kontes, Vineland, NJ). The cell suspensions were then transferred to a nylon cell strainer (BD Falcon, Franklin Lakes, NJ) in sterile tubes. The viable cell counts for the cell suspensions were determined using the trypan blue dye exclusion method. Single cell gel electrophoresis assay (Comet assay) For the first layer, 1.0% normal-melting agarose was dropped onto a frosted microscope slide. The cell resuspensions (1 105/10 mL) were then mixed with 85 mL of 0.7% lowmelting agarose and rapidly spread on the first layer. Finally, 85 mL of 0.7% low-melting agarose was used as the top layer. The prepared slides were then soaked in an alkaline lysing solution (2.5 M NaCl, 100 mM Na2-EDTA, 10 mM Tris-HCl, 1% Triton X-100, and 10% DMSO, pH 10.0) for 1 h at 4 C. Thereafter, the slides were washed in distilled water for 10 min, placed in a horizontal electrophoresis chamber, and

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Table 2. Environments of animal exposure chambers (mean ± S.E.). Group

Temperature ( C)

Humidity (%RH)

Pressure (Pascal)

Oxygen (%)

Carbon dioxide (ppm)

Flow rate (LPM)

Control Low Middle High

22.78 ± 0.74 21.80 ± 0.13 22.02 ± 0.08 22.28 ± 0.06

45.26 ± 0.39 43.76 ± 0.33 46.63 ± 0.31 55.87 ± 1.06

299.11 ± 2.50 292.99 ± 2.93 294.77 ± 2.20 296.63 ± 2.08

20.39 ± 0.10 20.59 ± 0.01 20.52 ± 0.01 20.41 ± 0.01

310.46 ± 3.14 341.50 ± 3.56 350.53 ± 4.21 344.02 ± 2.26

11.41 ± 0.78 11.45 ± 0.90 12.48 ± 1.06 11.59 ± 0.90

300

Test substance

Group

Target concentration (mg/m3)

Actual concentration (mg/m3) (n ¼ 20)

MWCNTs

Control Low Middle High

0 0.2 ± 0.06 0.5 ± 0.15 1.0 ± 0.3

0.00 ± 0.00 0.17 ± 0.00 0.49 ± 0.00 0.96 ± 0.01

electrophoresed in an alkaline buffer (1 mM Na2-EDTA, 300 mM NaOH, pH 13) for 25 min at 20 V and 275 mA. Next, the slides were gently washed in a neutralization buffer (0.4 M Tris-HCl, pH 7.5) and immersed in 100% ethanol for 1 h. The slides were then stained with 30 mL EtBr (10 mg/mL) and the Olive Tail Moment (OTM) analyzed using a fluorescent microscope (Leica, Wetzlar, Germany) and image program (Kinetic Imaging, Nottingham, UK). The OTM is a commonly used parameter and provides a good correlation with the dose of the genotoxic agent in a Comet assay (Kumaravel & Jha, 2006). Statistical analysis The statistical analyses were performed using SPSS 12.1 (Chicago, IL), and the data expressed as the mean ± standard error (S.E.). A one-way analysis of variance (ANOVA) and T-test were also applied to test all the data. A value of p50.05 indicated statistical significance.

Results Monitored results for inhalation chambers The conditions inside the animal exposure chambers, including the temperature, humidity, pressure, oxygen, carbon dioxide and flow rate are all presented in Table 2. The particle concentrations in the control chamber measured using the particle sensor were 0.10 ± 0.01 particles/cm3 for the 0.3 mm particles and 0.004 ± 0.001 particles/cm3 for the 1.0 mm particles, respectively. While the target concentrations for the low, middle and high concentration chambers were 0.2 ± 0.06, 0.5 ± 0.15 and 1.0 ± 0.3 mg/m3, respectively, the actual concentrations were 0.17 ± 0.00, 0.49 ± 0.00 and 0.96 ± 0.01 mg/m3, respectively (Table 3). The count median length distribution of the MWNCTs was measured based on 300 MWCNTs using TEM photographs. The length distribution was 68–1517 nm, and the count median length and geometric standard deviation were 330.18 and 1.72 nm, respectively (Figure 2). When comparing the average MWCNT length of 2.57 mm for CM-95 (Kim et al., 2012b) with 330 nm for CM-100, it is surprising that CM-100 with nearly the same physicochemical properties as CM-95 showed a totally different aerosol generation pattern. This

250 Cumulative frequency (#)


Table 3. Chamber concentrations of MWCNTs.

200

CML

150

100

50

0 100

150

200

300

500

1000

1500

Length (nm)

Figure 2. Length distribution of MWCNTs. CML: cumulative mean length.

shortened tube length phenomenon could have been caused by the sonication process in the MWCNT aerosol generation. Sonication has already been shown to shorten the MWCNT tube length by approximately 1/3 of the original length (Huang & Terentjev, 2008). Thus, CM-100 may have been more susceptible to sonication than CM-95, which was previously used for generation. Measurement of DNA damage It is already well-known that single cell gel electrophoresis (Comet assay) is a very useful DNA damage detection technique for eukaryotic cells (Singh et al., 1988; Zegura & Filipic, 2004). Thus, a Comet assay was conducted to evaluate the in vivo genotoxicity effect of the MWCNTs in the Fischer 344 rat lung cells. The animals were nose-only exposed to various concentrations of MWCNTs for 28 days and the recovery periods were 0 days and 90 days. During the experiment, no mortality, clinical abnormalities or significant body weight changes were observed before the autopsy (data not shown). For the male rats, the OTMs 0 days post-exposure were 13.62 ± 1.44, 28.43 ± 1.44 (p50.05), 34.17 ± 1.67 (p50.05) and 44.22 ± 2.24 (p50.05) for the fresh air control, low, middle and high-dose groups, respectively (Figure 3A), while 90 days post-exposure, the OTMs were 14.89 ± 0.82, 17.52 ± 0.65, 17.16 ± 0.69 and 18.36 ± 0.74 (p50.05) for the fresh air control, low, middle and high-dose groups, respectively (Figure 3B). For the female rats, the OTMs 0 days post-exposure were 19.32 ± 1.10, 27.93 ± 1.42 (p50.05), 26.13 ± 1.09 (p50.05) and 33.72 ± 1.76 (p50.05) for the fresh air control, low, middle and high-dose groups, respectively (Figure 3C), while 90 days post-exposure, the OTMs were 14.07 ± 0.60,


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Figure 3. Quantitative assessment of DNA damage in male and female rat lung cells using single cell gel electrophoresis (Comet assay). The slides were stained with EtBr (10 mg/ml) and analyzed using a fluorescent microscope and image program. (A) Male rats 0 days post-exposure, (B) male rats 90 days post-exposure, (C) female rats 0 days post-exposure and (D) female rats 90 days post-exposure. Values are expressed as mean ± S.E. (*p50.05, when compared with negative control group).

16.54 ± 0.64, 20.57 ± 0.76 (p50.05) and 21.44 ± 0.80 (p50.05) for the fresh air control, low, middle and highdose groups, respectively (Figure 3D). Measurement of ROS levels in BAL fluid To detect the reactive oxygen species production, the H2O2 level was measured in the BAL fluid. Samples were collected from the male and female F-344 rats on day 0, day 28 (only from males) and day 90 post-exposure to the MWCNTs. For the male rats, the middle (on day 0 post-exposure) and highdose groups (on day 0 and day 28 post-exposure) exhibited a significantly increased level of H2O2 (p50.05) when compared with the fresh air control group (Figure 4). However, for the female rats, there was no significant change in the H2O2 level with any of the MWCNT concentrations when compared with the fresh air control group at all the time points (data not shown). The levels of MDA, 4-HHE, hexanal and GSH did not show any significant differences between the control and exposed groups. Measurement of inflammatory cytokine level in BAL fluid To investigate the inflammatory response stimulation, the total number of cells, macrophages, polymorphonuclear cells (PMNs), and lymphocytes was first measured in the BAL fluid following the exposure to MWCNTs. Next, the concentrations of natural immunity mediator cytokines (TNF-a,

IL-1, IL-10, IL-12 and IFN-g) and adaptive immunity mediator cytokines (IL-2, IL-4, IL-5, IL-10, TGF-b and IFN-g) were measured in the BAL fluid (data not shown). As a result, no significant increase in the total number of cells, macrophages, PMNs and lymphocytes was observed on day 0 and day 28 post-exposure following 28 days of MWCNT exposure (Tables 4 and 5). Furthermore, the TNF-a levels were found to be below the detection limit, while the other levels showed no statistically significant difference in the BAL fluid for all the MWCNT concentrations at the different sampling points (data not shown). Potential lung deposition of MWCNTs The MWCNT-exposed lungs showed MWCNTs deposited in the pleural after 28 days of inhalation exposure (red spots) (Figure 5). The MWCNT deposition also persisted, even at 28 days post-exposure (only in males) (Figure 6), giving more clear images, and 90 days post-exposure (Figure 7). The distinct images at 28 days post-exposure may indicate aggregation/agglomeration of the MWCNTs inside the cells (Figure 6). However, the distinct images were resolved by 90 days post-exposure (Figure 7).

Discussion Carbon nanotubes are fiber-shaped and analogous to asbestos, which is known to cause pulmonary fibrosis, malignant


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Figure 4. Concentrations of hydrogen peroxide (H2O2) in male rat bronchoalveolar lavage (BAL) fluid. (A) 0 days post-exposure, (B) 28 days post-exposure and (C) 90 days post-exposure. Values are expressed as mean ± S.E. (*p50.05, when compared with negative control group).

Table 4. Cell count in bronchoalveolar lavage (BAL) fluid from male rats at day 0, day 28 and day 90 post-exposure following 28 days of MWCNT exposure. Exposure 28 days GROUP: (mean ± S.E.) a

Total cell # Macrophagesb PMNsc Lymphocytesd TPe ALBf LDHg Recovery 28 days Total cell #a Macrophageb PMNsc Lymphocytesd TPe ALBf LDHg Recovery 90 days Total cell #a Macrophagesb PMNsc Lymphocytesd TPe ALBf LDHg

Control

Low

Medium

High

0.42 ± 0.03 0.41 ± 0.03 0.01 ± 0.00 0.01 ± 0.00 3.64 ± 0.36 7.54 ± 0.54 19.40 ± 3.14

(5) (5) (5) (5) (5) (5) (5)

0.43 ± 0.09 0.39 ± 0.08 0.02 ± 0.01 0.01 ± 0.01 4.02 ± 0.29 9.42 ± 1.09 24.20 ± 4.39

(5) (5) (5) (5) (5) (5) (5)

0.49 ± 0.11 0.44 ± 0.09 0.03 ± 0.02 0.01 ± 0.00 4.66 ± 0.43 9.46 ± 0.94 25.60 ± 3.89

0.48 ± 0.09 0.46 ± 0.09 0.01 ± 0.00 0.01 ± 0.00 4.52 ± 0.75 8.18 ± 0.37 17.20 ± 0.37

(5) (5) (5) (5) (5) (5) (5)

0.53 ± 0.05 0.49 ± 0.05 0.02 ± 0.00 0.01 ± 0.00 4.50 ± 0.60 8.34 ± 0.36 18.40 ± 1.08

(5) (5) (5) (5) (5) (5) (5)

0.39 ± 0.03 (5) 0.36 ± 0.04 (5) 0.02 ± 0.01 (5) 0.01 ± 0.00 (5) 6.30 ± 1.08 (5) 10.26 ± 1.50 (5) 27.40 ± 10.19 (5)

0.47 ± 0.07 (5) 0.44 ± 0.06 (5) 0.01 ± 0.00(5) 0.01 ± 0.00 (5) 3.86 ± 0.71 (5) 8.84 ± 0.50 (5) 18.80 ± 1.39 (5)

0.80 ± 0.06 0.74 ± 0.06 0.04 ± 0.01 0.01 ± 0.00 4.66 ± 0.43 9.46 ± 0.94 25.60 ± 3.89

0.74 ± 0.04 0.71 ± 0.04 0.02 ± 0.00 0.01 ± 0.00 4.10 ± 0.15 9.34 ± 0.94 26.20 ± 4.92

0.76 ± 0.06(5) 0.73 ± 0.06 (5) 0.02 ± 0.00 (5) 0.01 ± 0.00 (5) 3.64 ± 0.36 (5) 7.54 ± 0.54 (5) 19.40 ± 3.14 (5)

0.74 ± 0.06 (5) 0.72 ± 0.05 (5) 0.02 ± 0.00 (5) 0.01 ± 0.00 (5) 4.02 ± 0.29 (5) 9.42 ± 11.09 (5) 24.20 ± 4.39 (5)

(5) (5) (5) (5) (5) (5) (5)

(5) (5) (5) (5) (5) (5) (5)

0.39 ± 0.08 0.34 ± 0.07 0.03 ± 0.01 0.02 ± 0.00 4.10 ± 0.15 9.34 ± 0.94 26.20 ± 4.92

(5) (5) (5) (5) (5) (5) (5)

(5) (5) (5) (5) (5) (5) (5)

( ): animal number. Total cell count (103/ml)ml; bMacrophages (103/ml); cPolymorphonuclear cells (PMNs) (103/ml); dLymphocytes (103/ml); eTotal protein (TP) (mg/dl); fAlbumin (ALB) (mg/dl); gLactate Dehydrogenase (IU/l).

a


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Table 5. Cell count in BAL fluid from female rats on day 0 and day 90 post-exposure following 28 days of MWCNT exposure. Exposure 28 days GROUP: (mean ± S.E.) a


Total cell # Macrophagesb PMNsc Lymphocytesd TPe ALBf LDHg Recovery 90 days Total cell #a Macrophagesb PMNsc Lymphocytesd TPe ALBf LDHg

Control

Low

Medium

High

0.24 ± 0.02 0.23 ± 0.02 0.01 ± 0.00 0.00 ± 0.00 9.18 ± 2.07 4.22 ± 0.93 16.00 ± 1.30

(5) (5) (5) (5) (5) (5) (5)

0.24 ± 0.03 0.23 ± 0.03 0.01 ± 0.00 0.01 ± 0.00 5.38 ± 0.55 4.82 ± 0.73 17.00 ± 0.32

(5) (5) (5) (5) (5) (5) (5)

0.34 ± 0.03 0.33 ± 0.03 0.01 ± 0.00 0.01 ± 0.00 6.50 ± 1.37 4.74 ± 0.79 17.80 ± 1.20

(5) (5) (5) (5) (5) (5) (5)

0.26 ± 0.03 0.24 ± 0.03 0.01 ± 0.00 0.01 ± 0.00 4.72 ± 0.42 4.62 ± 0.81 19.20 ± 3.01

(5) (5) (5) (5) (5) (5) (5)

0.56 ± 0.03 0.53 ± 0.03 0.02 ± 0.00 0.00 ± 0.00 9.18 ± 2.07 4.22 ± 0.93 16.00 ± 1.30

(5) (5) (5) (5) (5) (5) (5)

0.53 ± 0.03 0.51 ± 0.03 0.01 ± 0.00 0.01 ± 0.00 5.38 ± 0.55 4.82 ± 0.73 17.00 ± 0.32

(5) (5) (5) (5) (5) (5) (5)

0.56 ± 0.03 0.54 ± 0.03 0.01 ± 0.00 0.01 ± 0.00 6.50 ± 1.37 4.74 ± 0.79 17.80 ± 1.20

(5) (5) (5) (5) (5) (5) (5)

0.64 ± 0.10 0.61 ± 0.10 0.02 ± 0.00 0.01 ± 0.00 4.72 ± 0.42 4.62 ± 0.81 19.20 ± 3.01

(5) (5) (5) (5) (5) (5) (5)

( ): animal number. Total cell count (103/ml); bMacrophages (103/ml); cPolymorphonuclear cells (PMNs) (103/ml); dLymphocytes (103/ml); eTotal protein (TP) (mg/dl); fAlbumin (ALB) (mg/dl); gLactate Dehydrogenase (IU/l).

a

mesothelioma, pleural plaque and bronchogenic lung cancer (Manning et al., 2002; Mossman et al., 1996; Vallyathan et al., 1992). Asbestos generates ROS production, which results in the release of pro-inflammatory cytokines and chemokines, DNA damage, the phosphorylation of MAPK, activation of transcription factors and induction of early response genes. These cascades then lead to cell proliferation and culminate in fibrinogenesis and carcinogenesis (Pacurari et al., 2010). While the toxicity and genotoxic effects of CNTs, such as ROS generation, inflammatory responses and DNA damage, have already been extensively investigated in in vitro systems (Uno, 2006; Vallyathan et al., 1992), in vitro studies cannot reflect the cell–cell and cell–matrix interaction and hormonal effects (Sharma et al., 2012) occurring in in vivo systems. Thus, the importance of in vivo studies has already been highlighted in nanotoxicology fields (Fischer & Chan, 2007). Plus, when taking account of the real exposure situation for MWCNTs, an in vivo inhalation exposure study can provide more human-exposure-relevant information. The MWCNTs detected in workplaces can have a diverse range of sizes and shapes, including highly-agglomerated structures or particle shapes (Ma-Hock et al., 2009; Pauluhn, 2010), well-dispersed MWCNTs (Han et al., 2008), and rarely found single or fused fibrous shapes (Lee et al., 2010). The present MWCNT sub-acute inhalation study attempted to simulate the dispersed MWNCT exposure experienced in the workplace, as previously described by Han et al., where the maximum exposure level of well-dispersed MWCNTs was 0.43 mg/m3 (Han et al., 2008). Comparing dispersed MWCNT exposure with the particle shape or agglomerated MWCNTs can also provide a clearer understanding of the toxicity outcomes in the case of a high aspect ratio nanoparticle exposure situation. For the genotoxicity evaluation of MWCNTs, the OTMs were measured on day 0 and day 90 post-exposure after 28 days of nose-only inhalation exposure. The high-dose exposed male and middle and highdose exposed female rats showed persistent DNA damage even 90 days post-exposure, corresponding to an incomplete clearance of MWNCTs from the pleural. It has been

previously reported that genotoxicity effects can result from the direct interaction of nanoparticles with genetic material or secondary damage resulting from particle-induced reactive oxygen species production (Kisin et al., 2011). According to the recent in vitro studies, SWCNTs and MWCNTs induce ROS production in mammalian cells (Sharma et al., 2007; Shvedova et al., 2003; Ye et al., 2009, 2011). In a previous in vivo 5-day inhalation study by the current authors, a 2.57 mm average tube length induced an increased concentration of intracellular H2O2 with a similar trend of DNA damage (Kim et al., 2012b). In this study, the intracellular ROS levels were also measured to determine the cause of genotoxic effects following MWCNT exposure. The H2O2 levels were significantly higher in the middle (0 days postexposure) and high (0 days and 28 days post-exposure) -dose male rats. However, the female rats did not show a statistically significant difference at any of the concentrations and time points (data not shown). The oxidative stress caused by MWCNT exposure is also known to mediate the release of pro-inflammatory cytokines and cause the translocation of transcription factors, such as nuclear factor (NF)-kB, to the nucleus, which regulates the pro-inflammatory genes, such as tumor necrosis factor (TNF)-a and interleukins (ILs) (Ye et al., 2009). Murphy et al. (2012) reported that CNTs stimulate the release of acute cytokines, such as TNF-a, IL-1b, IL-6 and IL-8, in Met5a mesothelial cells and THP-1 macrophages (Murphy et al., 2012). In a previous in vitro toxicogenomic study, the current authors observed that TNF-a, IL-6 and IL-8 were up-regulated by MWCNTs and asbestos in normal human bronchial epithelia cells, although asbestos had a stronger effect than MWCNTs (Kim et al., 2012a). However, in the present study, there was no induction of natural immunity mediators (TNF-a, IL-1, IL-10, IL-12 and IFN-g) or adaptive immunity mediator cytokines (IL-2, IL-4, IL-5, IL-10, TGF-b and IFN-g) by the sub-acute MWCNT inhalation exposure. Fiber length is generally an important factor in pathogenicity and associated with a slower clearance from respiratory airways (Davis, 1994; Riganti et al., 2003). The biopersistence

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Figure 5. Enhanced dark field images of MWCNT-exposed rat lungs. MWCNTs scatter light with a high efficiency, producing bright-red structures in enhanced dark field images for 0 days post-exposure following 28 days of MWCNT exposure. Red arrows direct the MWCNT (red spots) in the circle.

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of fibers is limited by two key mechanisms (Davis, 1994). The first is removal by macrophages after phagocytosis. The macrophage clearance efficiency is greatest with short fibers (55 mm long) and reduced with longer fibers. Fibers longer than 50 mm cannot be cleared by macrophages and remain in the lungs permanently. The second mechanism is removal by actual dissolution. Long fibers can fracture into shorter fibers with the aid of chemical dissolution, making them more susceptible to macrophage clearance. Consequently, fiber dimensions are a critical measure of carcinogenicity, the most potent fibers being 48 mm in length and 50.25 mm in diameter (Pott & Friedrichs, 1972; Pott, 1978; Stanton & Wrench, 1972; Stanton et al., 1977). It has already been reported that long CNTs and long asbestos cause instillationinduced acute inflammation and progressive fibrosis of the parietal pleura, yet not short fibers due to clearance (Murphy et al., 2011). Interestingly, the present study found a persistent presence in the pleura and lung parencheyma at 90 days following sub-acute (28 days) inhalation exposure to shortlength MWCNTs (330.18 ± 1.72). Although the fiber length is critical to the lung pathology development, leading to inflammation, fibrosis and potentially mesothelioma (Donaldson et al., 2006; Murphy et al., 2011), the biodurability of MWCNTs in the lungs could be different. Several recent findings suggested that lung fibers or tangled forms of MWCNTs were engulfed by macrophages, modified and processed into shorter MWCNTS (Elgrabli et al., 2008) or the smaller MWCNT structures were rapidly incorporated into the alveolar interstitium where clearance is low (Mercer et al., 2013). TEM observation of lung macrophages after 15 days of MWCNT instillation revealed a significant diminution of the MWCNT length (Elgrabli et al., 2008). In addition, when evaluating inhalation exposure for 12 weeks at 5 mg/m3 MWCNT, 65% of the MWCNTs remained in the lungs even at 336th day post-exposure (Mercer et al., 2013). These MWCNTS were either smaller MWCNT structures that were rapidly incorporated into the alveolar interstitium, where clearance is lower, or larger MWCNT structures associated with macrophages and a smaller yet significant component due to MWCNTs in the airways at 336 days post-exposure following inhalation. Plus, the clearance of singlet fibers was not changed, even at 168 days postexposure (Mercer et al., 2013). Therefore, the persistence of the short fibers in the present study was consistent with the findings of other studies that short fibers are not easily cleared and retained in the alveolar interstitium and macrophages. The short-length MWCNTs also induced DNA damage in the exposed lung cells that persisted at 90 days post-exposure. Furthermore, the lung cells from the high-dose male rats maintained ROS generation for 28 days post-exposure, although a significant change of cytokines was not observed. Two principle modes of genotoxic action have been reported for particles, known as primary and secondary genotoxicity (Schins & Knaapen, 2007). Primary genotoxicity is defined as genetic damage elicited by particles in the absence of pulmonary inflammation, while secondary genotoxicity implies a pathway of genetic damage resulting from an oxidative DNA attack by reactive oxygen/nitrogen species (ROS/RNS), generated during particle-elicited inflammation.

Inhal Toxicol, 2014; 26(4): 222–234

In the present study, the MWCNTs-induced ROS generation and a persistent deposit of short-length MWCNTs was found in the rat lung cells at 90 days post-exposure. The narrow diameter and longer length of SWCNTs suggest a potential to interact with critical biological structures. For these reasons, the sub-acute inhalation exposure of the Fischer 344 rats to MWCNTs seemed to induce both primary and secondary genotoxic effects. These results also suggest the need for subchronical inhalation toxicity data for further assessment. Dose-dependent increases in the frequency of DNA damage in a Comet assay have been seen after exposing lung fibroblasts to CNF, asbestos or SWCNTs (V79) (Kisin et al., 2011). In this case, two possible mechanisms were suggested: the production of ROS that react with the DNA or physical interference with the DNA/chromosomes and/or mitotic apparatus. SWCNTs have been found to interact with the mitotic spindle apparatus, including mitotic tubulin and chromatin (Sargent et al., 2009, 2010). The similarity of CNTs and microtubules may facilitate interaction with the centrosome and mitotic spindle, instead of the physical interference with the spindle that occurs with asbestos (Cortez & Machado-Santelli, 2008; Sargent et al., 2009). The induction of DNA damage by the short-fiber-length MWCNTs in the present study would also seem to suggest some kind of interaction with the cell mitotic apparatus. Genotoxicity observed in female rats without induction of inflammation and ROS generation may also indicates that male rats were susceptible than the female rats. Lung function changes after 28 days of MWCNT exposure in this study also showed the male rats were more susceptible than female rats (data not shown). Some animal study data suggest that female sex hormones may influence the lung function, airway responsiveness and inflammation (Degano et al., 2001; Kline et al., 1999; Ligeiro de Oliveira et al., 2004; Shirai et al., 1995). The male sex hormone testosterone has also been suggested to play a key role in LPS-induced airway hyper-responsiveness, as the airway response is decreased in castrated male rats and increased in female rats administered exogenous testosterone (Card et al., 2006). Finally, changes in the aerosol generation behavior of MWCNTs according to the manufacturing method should also be considered when designing MWCNT inhalation toxicity experiments. While the generator used in the present study was previously effective in generating CM-95 MWCNTs with the desired length, it was unable to generate CM-100 MWCNTs with a similar tube length to CM-95 MWCNTs. Also, since many manufacturing methods are confidential business information, it is really hard to identify the cause of different aerosol generation behavior. In addition, a further challenge is to generate MWCNT aerosols using new MWCNT manufacturing methods.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This research was supported by the Nano R&D program through the National Research Foundation of Korea funded by the Korean Ministry of Education, Science and Technology (2011-0019171).

DOI: 10.3109/08958378.2013.878006


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