Analysis of pulmonary surfactant in rat lungs after inhalation of nanomaterials: Fullerenes, nickel oxide and multi-walled carbon nanotubes.

http://informahealthcare.com/nan ISSN: 1743-5390 (print), 1743-5404 (electronic) Nanotoxicology, Early Online: 1–10 ! 2015 Informa UK Ltd. DOI: 10.3109/17435390.2015.1039093

ORIGINAL ARTICLE

Analysis of pulmonary surfactant in rat lungs after inhalation of nanomaterials: Fullerenes, nickel oxide and multi-walled carbon nanotubes Chikara Kadoya, Byeong-Woo Lee, Akira Ogami, Takako Oyabu, Ken-ichiro Nishi, Makoto Yamamoto, Motoi Todoroki, Yasuo Morimoto, Isamu Tanaka, and Toshihiko Myojo Nanotoxicology Downloaded from informahealthcare.com by Universitaet Zuerich on 05/07/15 For personal use only.

Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Kitakyushu, Japan

Abstract

Keywords

The health risks of inhalation exposure to engineered nanomaterials in the workplace are a major concern in recent years, and hazard assessments of these materials are being conducted. The pulmonary surfactant of lung alveoli is the first biological entity to have contact with airborne nanomaterials in inhaled air. In this study, we retrospectively evaluated the pulmonary surfactant components of rat lungs after a 4-week inhalation exposure to three different nanomaterials: fullerenes, nickel oxide (NiO) nanoparticles and multi-walled carbon nanotubes (MWCNT), with similar levels of average aerosol concentration (0.13–0.37 mg/m3). Bronchoalveolar lavage fluid (BALF) of the rat lungs stored after previous inhalation studies was analyzed, focusing on total protein and the surfactant components, such as phospholipids and surfactant-specific SP-D (surfactant protein D) and the BALF surface tension, which is affected by SP-B and SP-C. Compared with a control group, significant changes in the BALF surface tension and the concentrations of phospholipids, total protein and SP-D were observed in rats exposed to NiO nanoparticles, but not in those exposed to fullerenes. Surface tension and the levels of surfactant phospholipids and proteins were also significantly different in rats exposed to MWCNTs. The concentrations of phospholipids, total protein and SP-D and BALF surface tension were correlated significantly with the polymorphonuclear neutrophil counts in the BALF. These results suggest that pulmonary surfactant components can be used as measures of lung inflammation.

C60, inhalation, MWCNTs, pulmonary surfactant, phospholipid, SP-D

Introduction Nanomaterials, consisting of nano-objects measuring 5100 nm, have been increasingly used in many industrial sectors. Along with the development of many kinds of nanomaterials, their biological effects are also a matter of concern. Nanomaterials have a remarkably larger specific surface area than submicronsized powders of the same composition. Oberdo¨rster et al. (1992, 1994) reported that the biological effects of inhaled titanium dioxide (TiO2) particles were dependent on the surface area dose rather than the mass dose; that is, the TiO2 nanoparticles showed much higher toxicity than the same amount of submicron-sized TiO2 particles. Many in vivo experimental studies have been conducted to assess the potential effects of nanomaterials. In a Japanese New Energy and Industrial Technology Development Organization (NEDO) project, fullerenes, MWCNTs and SWCNTs were evaluated in inhalation exposure tests and/or intratracheal instillation tests. The overview of the project has been published by Nakanishi (2011) and the specific results have been published by Morimoto et al. (2010, 2011), Ogami et al. (2011), Oyabu et al. (2011) and Shinohara et al. (2010). The

Correspondence: Chikara Kadoya, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Kitakyushu, 807-8555, Japan. E-mail: [email protected]

History Received 15 February 2015 Revised 6 April 2015 Accepted 6 April 2015 Published online 7 May 2015

observed biological effect of inhaled nanomaterials is mainly lung inflammation, but translocation of the nanomaterials in the whole body was discussed by Elder et al. (2006). The amount of exposure to nanomaterials in workplaces does not look very high compared with the doses in the laboratory animal studies, but the American Heart Association provided a statement (Brook et al., 2010) on air pollution stating that exposure to particulate matters 52.5 mm in diameter (PM2.5) over a few hours to weeks can trigger cardiovascular disease-related mortality and non-fatal events; longer-term exposure increases the risk for cardiovascular mortality to an even greater extent than exposures over a few days. Fine and ultrafine particles inhaled with air have been shown to have a higher deposition rate (20–40% for nanoparticles) in the pulmonary region than micron-size particles (ICRP, 1994). The pulmonary surfactant is a component of the lining fluid of lung alveoli and one of the first points of contact with inhaled particles in the respiratory system. The main component of surfactant secreted into the alveolar space is a lipoprotein complex produced in type II alveolar epithelial cells and Clara cells. The pulmonary surfactant plays a significant role in the stability of alveoli, such as the functions that regulate their surface tension. When this function is compromised due to lung damage, the alveoli and small airways collapse, and inflammation and fibrosis develop. The major components of pulmonary surfactant are phospholipids and about 10% of surfactant consists of protein; four surfactant proteins have been defined: SP-A, SP-B, SP-C and SP-D.

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Nanotoxicology, Early Online: 1–10

The main function of the phospholipid fraction in conjunction with the SP-B and SP-C is to decrease surface tension during the breathing cycle. SP-A and SP-D are large hydrophilic proteins and participate in immune responses (Schleh & Hohlfeld, 2009; Wright, 2005). Bronchoalveolar lavage fluid (BALF) is widely used to investigate lung damage caused by inhalation exposure to particulate matter in laboratory animals such as rats. The number of alveolar macrophages and neutrophils and the concentrations of phospholipids and total protein in BALF are useful biomarkers of the severity of inflammation in the lungs (Henderson, 2005). In our previous study of rats administered crystalline silica and nickel oxide (NiO) by intratracheal instillation, the BALF surface tension, the concentrations of phospholipids and total protein were effective biomarkers for assessing the hazardous property of these substances (Kadoya et al., 2011; Kuroda et al., 2006). In rats instilled with multi-walled carbon nanotubes (MWCNT), the changes in the BALF surface tension correlated well with SP-D levels, neutrophil counts and inflammatory scores in the lungs, as determined using the pointcounting method to analyze histopathological images (Lee et al., 2013). In intratracheal instillations, particulate suspension is not uniformly dispersed in the rat lungs, in contrast to the more uniform deposition of inhaled particles making contact with the surfactant. In this study, we try to clarify the role of the pulmonary surfactants in inflammation induced by inhalation exposure to nanomaterials based on the correlations between surfactant components and PMN counts in BALF. We analyzed the pulmonary surfactant in BALF samples obtained from rat lungs exposed to NiO nanoparticles, fullerenes or MWCNT of the past two inhalation studies by the NEDO project mentioned above. These data were compared to the previously reported neutrophil counts in BALF and pathological changes in lung tissue. In addition, to elucidate the role of BALF surface tension, we performed an in vitro study using sample solutions containing different concentrations of phospholipids, SP-D and SP-B to measure the surface tension of the solutions.

Materials and methods Sample nanoparticles Nanoparticles of NiO, fullerene and MWCNT were suspended in water for use in this study. The physicochemical profiles of this sample are shown in Table 1. The data of these samples have been published by Nakanishi (2011), and the details of the results have been reported separately (Mizuguchi et al., 2013; Oyabu et al., 2011; Shinohara et al., 2010). NiO was purchased from Nanostructured & Amorphous Materials Inc. (Houston, TX). The NiO particles were dispersed without any detergents by an ultrasonic homogenizer.

The suspension was centrifuged to separate coarse particles, and then the recovered supernatant was filtered through a membrane filter with 1 mm diameter pores. The concentration of the aqueous suspension of NiO was adjusted to 0.5 mg/ml for the inhalation study. Bulk C60 fullerenes were purchased from Frontier Carbon Corporation (Japan). The fullerene was dispersed in 0.1 mg/ml Tween-80 (Wako Pure Chemical Industries, Ltd., Japan) in an agate mortar under a nitrogen atmosphere to avoid oxidization, then the fullerene suspension was dispersed with zirconium particles (50 mm) using a high-performance dispersion machine (UAM-15, Kotobuki Industries Co., LTD., Japan). After centrifugation of the suspension, the recovered supernatant was used for the inhalation tests. The concentration of the aqueous suspension of C60 fullerenes was adjusted to 0.17 mg/ml of fullerene with 0.5 mg/ml Tween-80 (Morimoto et al., 2011; Shinohara et al., 2010). MWCNTs were provided by Nikkiso Co., Ltd (Tokyo, Japan). The MWCNTs were kneaded with fructose, and the solidified body was ground using a planetary ball mill. After soaking the ground product in hot water, the MWCNTs were separated from the fructose by filtration and rinsing. The recovered MWCNTs were dispersed in a dispersing medium (an aqueous suspension of 0.5 mg/ml Triton X-100, Wako Pure Chemical Industries, Ltd., Osaka) by a homogenizer. The suspension of MWCNTs was classified by centrifugation for use in the inhalation tests. The concentration of the aqueous suspension of MWCNTs was adjusted to 0.25 mg/ml of MWCNTs with 0.125 mg/ml Triton X-100 (Morimoto et al., 2011). Inhalation exposure tests Male Wistar rats (9 weeks old) were purchased from Kyudo Co., Ltd. (Kumamoto, Japan) and divided into three groups of 30 rats each. Two aerosol inhalation exposure studies were conducted for 6 h/day, 5 days/week for 4 weeks. In the first study, aerosols of NiO and fullerene, and clean air (control 1) were supplied to the rats. In the second study, aerosols of MWCNT and Triton X-100, and clean air (control 2) were supplied to the rats. The results of the aerosols of Triton X-100 will not be discussed in this report because they were negative the same as clean air (Morimoto et al., 2011). The sample suspensions of NiO, fullerene and MWCNT were spray-dried by a pressurized atomization apparatus to prepare each aerosol. The aerosol generator (Nanomaster, JSR Corp., Tokyo, Japan) dispersed the suspension and dried the mist particles (Shimada et al., 2009). The aerosol particles were then introduced into inhalation exposure chambers. The size and average concentration of each kind of aerosol particle are shown in Table 1. Scanning electron microscope (SEM) photos of the generated aerosol particles are shown in Figure 1. Each group of rats was whole-body exposed to one of the test aerosols in the

Table 1. Sample characterization of 4-week inhalation exposure.

Sample NiO C60 MWCNT a

Average concentration (mg/m3)

Deposited amount in lung (mg)a

0.20 0.13 0.37

0.017 0.010 0.076

DLSb 50% diameter (nm) 26 33 –

SMPSc 50% diameter (nm) 54 92 d

Density (g/cm3)

Specific surface area (m2/g)

Reference

6.8 1.72 1.72

172.0 105.7 69

Mizuguchi et al. (2013) Shinohara et al. (2010) Oyabu et al. (2011)

Measured amount of deposited particles in lung 3 days after termination of inhalation. Suspension size distribution measured by dynamic light scattering method. c Aerosol size distribution measured by a differential mobility analyzing system (Shimada et al., 2009). d Average length (geometric standard deviation) of MWCNT fiber is 1.1 mm (2.7) and average diameter is 63 nm (1.5) measured by scanning electron microscopy (SEM) observation. b


DOI: 10.3109/17435390.2015.1039093

exposure system. Another two groups of 15 rats each were kept in a filtrated clean air chamber as negative controls. Ten rats for each group exposed to the aerosols and five rats for each group of clean air were dissected at 3 days, 1 month and 3 months after termination of inhalation. The rats were sacrificed by exsanguination from the heart under deep anesthesia by intraperitoneal injection of pentobarbital. BALF collection and histopathological analysis were conducted using the removed lungs of five rats from each group. The deposited amounts of inhaled particles in the lungs of the other five rats exposed to the aerosols were analyzed using digested lung tissues by an inductively coupled plasma-atomic emission spectrometer for the NiO (Mizuguchi et al., 2013), an HPLC system equipped with a UV detector for the C60 (Shinohara et al., 2010) and X-ray diffraction method for the MWCNTs (Oyabu et al., 2011). All procedures and animal handling were performed according to the guidelines described in the Japanese Guide for the Care and Use of Laboratory Animals as approved by the Animal Care and Use Committee, University of Occupational and Environmental Health, Japan. Bronchoalveolar lavage fluid Bronchoalveolar lavage fluid (BALF) was collected by inserting a cannula into the right lung via the respiratory tract, with the left main bronchus clamped, and pouring in a physiological saline (15 ml for each rat). After centrifuging the BALF (1500 rpm 10 min), the supernatant was frozen and kept at 30 C before measurements. Recovered cells in the BALF, such as alveolar macrophages and polimorphonuclear neutrophils (PMN), were also analyzed with an automatic blood cell counter to determine cell numbers. Smears from the cell samples were also prepared on glass slides by the CytoSpin method and stained. These slides were observed by optical microscopy to determine the number of PMN in 200 cells per slide.

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Measurement of pulmonary surfactant in BALF We measured the concentrations of phospholipids, total protein, SP-D and BALF surface tension in order to evaluate any change in pulmonary surfactant in the rats exposed to the nanoparticles. The measurement methods for the surfactant analysis were basically the same as the method described in previous papers (Lee et al., 2013). Phospholipid concentration Phosphatidylcholines and phosphatidylglycerols are major species of surfactant phospholipids. We used the enzymatic method to measure the total phospholipid concentration in the BALF. A standard curve was made using a standard sample of NESCAUTO PL Kit-K (Alfresa Pharma Corporation, Tokyo, Japan), and the phospholipid concentration was determined by measuring BALF absorption (at 595 nm) with a spectrophotometer (Spectramax Plus 384; Molecular Devices, Sunnyvale, CA). Total protein concentration The method for measuring the total protein concentration was based on the same principle as the Bradford protein assay. A standard curve was made using the Bovine Serum Albumin (BSA) standards (Thermo Scientific, Waltham, MA) as the standard sample, and the protein concentration was determined by measuring BALF absorption (at 595 nm) with the a spectrophotometer, as above. SP-D concentration SP-D measured in this study is a largest molecule (43 kDa) among the four surfactant proteins, and modulates immune and inflammatory responses in the lung (Wright, 2005). The SP-D concentration in the BALF was measured with an enzyme-linked immunosorbent assay (ELISA) kit for rat and mouse (Yamasa Co.,

Figure 1. SEM photographs (30 000) of aerosol particles in inhalation chamber (A) C60, (B) NiO, and (C) MWCNT.

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Chiba, Japan). The BALF samples were diluted 300 times with a dilution buffer supplied by the manufacturer. The absorbance of the samples was measured at 450 nm using the spectrophotometer. All assays were performed in strict accordance with the manual procedures of the kit.


BALF surface tension BALF surface tension directly indicates the functioning level of the pulmonary surfactant. We measured the surface tension with a du Nouy surfactometer (Taihei Rikakogyo, Tokyo, Japan) using the du Nouy ring method after diluting a 1.8-ml of BALF sample with 10 parts physiological saline. The platinum ring was 13 mm in ring diameter and 0.2 mm in wire diameter. The BALF sample was poured into disposable petri dish (constant surface area; 58 cm2). The correction factor was obtained from the water temperature and surface tension (72 mN/m at 20 C) of the distilled water on the day of the experiment. The actual surface tension was calculated by the correction factor to the value obtained for each sample. Surface tension of artificial BALF 1,2-Dipalmitoyl-sn-glycero-3-phosphocholines (hereafter DPPC, Wako Pure Chemical Industries, Ltd., Osaka) as a typical component of surfactant phospholipids and SP-B and SP-D (both standard solutions enclosed in the ELISA kit, EIAab Science Co., Ltd, Wuhan, China) in saline were mixed to make artificial BALF solutions. The surface tension of the solutions was measured with the du Nouy surfactometer following the same procedure as the BALF analysis. Histopathology of rat lungs The left lung of each rat, that is, the clamped side at BALF collection, was fixed with 10% buffered formalin at 25 cmH2O overhead pressure. Paraffin sections of the left lung (3 mm thickness) were stained with hematoxylin–eosin (HE) by Hist Science Laboratory Co., Ltd. (Tokyo, Japan). Statistical analysis Results are presented as mean ± standard deviation. The results were compared by the Mann–Whitney U-test, with differences of p ¼ 0.05 or less considered to be statistically significant. Construct validity between phospholipid concentration, total protein concentration, SP-D concentration, surface tension and PMN counts was measured by Spearman’s rank correlation coefficients. Statistical analysis was performed using IBM SPSS Statistics 19 (Chicago, IL).

Results Table 1 shows the average concentrations of the nanomaterials in the 4-week inhalation exposure, the average deposited amount of intrapulmonary particles 3 days after the termination of the inhalation exposure, particle sizes and other properties. Although the average concentrations of each of the nanomaterials in the chambers were within three times of each other, the amount of MWCNTs deposited in the lungs was quite high compared to the NiO and C60 fullerenes. SEM photos of the particles taken at the same magnification in the inhalation exposure chamber revealed that the morphology of the NiO and C60 particles, as dry particles of suspension mists, was almost circular (Figure 1). On the other hand, the MWCNTs existed as individual MWCNT fibers or bundles of several fibers, suggesting that the morphological


difference between the compact particles and fibrous materials contributed to the difference in the deposited amounts. Figure 2 shows the measured concentrations of total protein and surfactant components, surface tension and PMN counts in the BALF at 3 days, 1 month and 3 months after the termination of the inhalation exposure. Figure 2(A) shows the phospholipid concentrations in the BALF at each time point. Two control groups paired with the NiO and C60 fullerene-exposed groups and the MWCNT-exposed group were both exposed to clean air at the same time period as the inhalation exposure. No significant difference was observed between the fullerene-exposed groups and the control groups throughout the observation period, but significant increases were observed at 3 days and at 1 month in the NiO-exposed group and at 3 days in the MWCNT-exposed group. Figure 2(B) shows the total protein concentrations in the BALF samples at each time point. As with the phospholipid concentrations, no significant difference in total protein concentrations was observed between the fullerene-exposed group and the control group throughout the study period, but the protein concentration after the inhalation of the test aerosols showed significant increases at 3 days and at 1 month in the NiO-exposed group and at 3 days in the MWCNT-exposed group compared to each control group. Figure 2(C) shows the concentrations of SP-D in the BALF samples at each time point. In comparison with the control group, significant increases were observed at 3 days and at 1 month in the NiO-exposed group. The responses in the fullerene-exposed group and MWCNT-exposed group were similar to each control group. As the values of SP-D concentrations in the BALF were fairly higher than our previous results (Lee et al., 2013), detail will be discussed later. Figure 2(D) shows the BALF surface tension at each time point. The BALF surface tension in the fullerene-exposed group did not differ significantly in comparison with the control group throughout the study period. Although the BALF surface tension tended to decrease in the NiO group, these changes were not significant. The MWCNT and the control groups showed a significant difference only at day 3 after termination of the inhalation exposure. Figure 2(E) shows a summary of the PMN counts in BALF reported in our previous papers (Morimoto et al., 2010, 2011). Similarly to the pulmonary surfactant results, no significant difference was observed between the fullerene and the control groups. On the other hand, there was a significant increase in the PMN counts in the NiO-exposed group compared with the control group at 3 days and at 1 month after the termination of inhalation. An increase in the PMN counts in the MWCNT-exposed group compared with the control group was observed at 3 days, but it was not statistically significant. Figure 3 shows histopathological images (200 magnifications) of rat lungs obtained at 3 days and at 1 month after the termination of inhalation exposure to clean air, NiO nanoparticles, fullerenes or MWCNTs. At 3 days post-exposure, many macrophages infiltrated into the lung alveolar space and interstitium in the NiO group, but hardly any did so in the fullerene group. With regard to the MWCNTs, in addition to the infiltration into the lung alveolar space and interstitium, some macrophages in the interstitium appear to have engulfed the MWCNTs. At 1 month, the findings in the NiO group were similar to those observed at 3 days, with the additional infiltration of inflammatory cells in the lung interstitium. Hardly any changes were observed in the fullerene group. The macrophage-engulfed MWCNTs were accumulated in the lungs of the MWCNT group (see inset of Figure 3). These findings decreased 3 months after inhalation.


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Figure 2. Phospholipid (A), total protein (B) and SP-D (C) concentrations in BALF, BALF surface tension (D) and neutrophil (PMN) cell counts (E) after inhalation of three nanomaterials, fullerene (C60), nickel oxide (NiO) and multi-walled carbon nanotube (MWCNT). Each column and bar represents the mean ± standard deviation of five rats. An asterisk (*) indicates a statistically significant difference of p50.05 compared to each negative control group; double asterisk (**), of p50.01 compared to each negative control group. The data of (E) have been published in our previous papers (Morimoto et al., 2010, 2011).

Correlation between pulmonary surfactant components and surface tension In this study, we used the data of phospholipids, total protein and SP-D measured as pulmonary surfactant at 3 days, 1 month and 3 months after termination of inhalation (n ¼ 60 for control, C60, NiO, MWCNT) to calculate the Spearman rank correlation coefficient to determine which substance would be influential on the surface tension. The Spearman rank correlation coefficient of the phospholipid concentration against surface tension was 0.120 (p ¼ 0.360), indicating the absence of a correlation between the two (Figure 4A). Phospholipids are well known as important components in regulating BALF surface tension, but in the present study the concentration of phospholipids had less effect on the surface tension than the other components did. A correlation was noted between the total protein concentration and surface tension, with a correlation coefficient of 0.419 (p ¼ 0.001) (Figure 4B). The surface tension decreased in

proportion to the increase in SP-D concentration, with a correlation coefficient of 0.221 (p ¼ 0.090) (Figure 4C). Surface tension of artificial BALF The surface tension of sample solutions containing DPPC, mixtures of DPPC and SP-D, and mixtures of DPPC and SP-B was measured as shown in Figure 5. The range of concentration of the DPPC was set at 0–300 mg/ml, based on the findings in Figure 2(A). The range of concentration of SP-D and SP-B was set at 1–10 ng/ml due to the limited amount of ELISA standards available to us. Throughout the range of concentration of the DPPC, the surface tension was similar to that of the physiological saline. SP-D reduced surface tension at 1 ng/ml, with no further significant reduction at 5 or 10 ng/ml (Figure 5A). On the other hand, SP-B reduced the surface tension gradually as its concentration increased from 1 to 10 ng/ml. In particular, the changes in surface tension were influenced by the amount of DPPC in the

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Figure 3. Hematoxylin and eosin staining of lung sections 3 days (A) and 1 month (B) after termination of inhalation of nanomaterials. Magnification 200 (scale bar is 50 mm). Images of NiO nanoparticles showed macrophage (white arrows) accumulation in the alveoli with infiltration of inflammatory cells. Images of MWCNT showed alveolar macrophages (black arrows) with black granules, which look like MWCNTs (see the inset). These findings decreased 3 months after inhalation.

mixture. As shown in Figure 5(B), the mixture containing 10 ng/ml SP-B and 200 mg/ml DPPC reduced the surface tension to as low as 48 mN/m.

Discussion In this study, we used NiO, C60-fullerene and MWCNT to investigate the biological effect of inhalation exposure to these nano-sized materials by measuring surface tension and the concentrations of phospholipids, total protein and SP-D in BALF. The results of the pulmonary surfactants shown in Figure 2(A–D) are similar to those in our previous studies of C60-fullerene administered to rats by intratracheal instillation or inhalation exposure, not only the PMN counts as shown in Figure 2(E) but also the related chemokines (CINC-1, -2ab and -3) (Morimoto et al., 2010) and pathological changes as determined

by the point-counting method (Ogami et al., 2011). In a study of inhalation exposure to nano- and micro-sized fullerenes in rats, Baker et al. (2008) observed the infiltration of macrophages in the lung alveoli but no signs of lung inflammation, and thereby concluded that the biological effect of inhalation exposure to fullerenes was not severe. In contrast, Sayes et al. (2007) observed inflammation in the lungs a day after the intratracheal instillation of nano-sized and soluble fullerenes (C60 (OH)24). Park et al. (2010) reported pathological changes accompanied by macrophage infiltration and granulation in the area surrounding bronchitis after the intratracheal instillation of C60 in mice. We suppose that the discrepancy in these results may be due to the differences in doses between the studies or due to responses to overdose of the samples administered by intratracheal instillation. Nickel compounds, of which NiO has extremely low solubility and is highly persistent in the lung, are classified as carcinogenic


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Figure 4. Relationship between phospholipid concentration (A), protein concentration (B), SP-D concentration (C), versus surface tension of BALF after inhalation of nanomaterials and control. Values of are Spearman’s rank correlation coefficient for all the data. The concentration of phospholipids had less effect on the surface tension than the other components did.

Figure 5. Relationship between phospholipid (DPPC) concentration and concentration of SP-D (A) or SP-B (B), versus surface tension of the solutions. SP-B reduced surface tension as its concentration increased from 1 to 10 ng/ml with the amount of DPPC in the mixture.

(Group 1) in the International Agency for Research on Cancer (IARC) categories. The threshold limited value (TLV) of insoluble inorganic compounds of nickel recommended by ACGIH is 0.2 mg/m3 (as Ni). The occupational exposure level (OEL) of nickel compounds recommended by the Japan Society for Occupational Health is 0.1 mg/m3 (as Ni for insoluble compounds) (The Japan Society for Occupational Health, 2013). The average concentration of NiO aerosol particles in the present study was 0.16 mg/m3 as Ni; that is, 0.20 mg/m3 as NiO (see Table 1). The reason for the observed inflammation after a 4-week

exposure to NiO particles near the OEL is presumable, because the aerosol particles in this study were nano-sized. In our previous report, surfactant analysis showed that submicron-sized NiO particles instilled in rat lungs have stronger biological effects than micron-sized NiO particles (Kadoya et al., 2011). Many studies have reported the biological effects caused by MWCNT administration (DeLorme et al., 2012; Kasai et al., 2014; Ma-Hock et al., 2009). Depending not only on the dose, but also on the diameter and length of the MWCNT particles, significant differences compared with control groups have been


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Figure 6. Relationship between neutrophil in BALF versus phospholipid concentration (A), protein concentration (B), SP-D concentration (C), surface tension of BALF (D) after inhalation of nanomaterials and control. Values of are Spearman’s rank correlation coefficient for all the data.

reported in the number of PMN in BALF, the concentrations of LDH and total protein (Murphy et al., 2013), and the concentrations of surfactant-specific SP-D (Han et al., 2010). We previously analyzed pulmonary surfactant components, such as BALF surface tension and the concentrations of phospholipids, total protein, and SP-D, after the intratracheal instillation of short and long MWCNTs. The results of the analyses, including pathological findings, revealed that the long MWCNTs caused more severe inflammation than the short MWCNTs did (Lee et al., 2013). The sample of MWCNTs tested in the present study was the same as the sample of the short MWCNTs mentioned above. Even though the deposited amount in the lung 3 days after the termination of inhalation exposure, as shown in Table 1, was less than half that from the low dose (0.2 mg/rat) in the intratracheal instillation study, significant changes were observed in the surfactant components. The deposited amount of fullerenes 3 days after the termination of inhalation was slightly lower than that of NiO, and the fullerenes caused almost no inflammatory response in the lung. The inflammatory response in the lungs was less severe with MWCNT than with NiO, even though the pulmonary deposition of MWCNT was 4.5 times more. At 3 days after the termination of inhalation, the hazard ranking of the inhaled nanoparticles was consistently in the order of control ¼ C605MWCNT55NiO (Figure 2). None of the nanoparticles had a significant effect on the pulmonary surfactants 3 months after the termination of exposure. The results of the pulmonary surfactant analysis of the three different nanoparticles used in this study indicate that the BALF surface tension and the concentrations of total protein and surfactant components showed changes that were consistent with

the PMN and pathological findings, indicating that they should be the effective biomarkers of acute lung inflammation caused by nanoparticle inhalation. The concentrations of SP-D in the BALF shown in Figure 2(C) were a few hundred times higher than the values in our previous report (Lee et al., 2013). In our previous study, we used an ELISA kit for rat and mouse manufactured by EIAab Science Co., Ltd. Wuhan, China. The maximum concentrations of standard solution of SP-D of the Wuhan ELISA kit were 10 ng/ml, and those of the Yamasa ELISA kit were 30 ng/ml, almost similar level. The major difference between both ELISA kits was that the manual of the Yamasa ELISA kit recommended dilution of the samples before analysis, such as 100 to 400 times for BALF (Murata et al., 2010), and we chose 300 times dilution. Measured concentrations of SPD in the BALF in this study were one digit lower than those of the phospholipid concentrations in the BALF (Figure 2A) and reasonable for the portion of the phospholipid (about 90%) and surfactant proteins (about 10%) (Schleh & Hohlfeld, 2009; Wright, 2005). However, further validation study will be needed for the kits of SP-D. The correlation between the BALF surface tension and the concentrations of phospholipids, total protein or SP-D is shown in Figure 4. The correlation with the total protein concentrations was statistically significant and was the highest among the components, while the surface tension showed a weak correlation (not statistically significant) with SP-D and no correlation with phospholipids. Despite the common notion that phospholipids affect BALF surface tension, no correlation with phospholipids was observed in this study, similar to our previous results (Lee et al., 2013). This result suggests that BALF surface tension



DOI: 10.3109/17435390.2015.1039093

indicates the change of SP-B and SP-C, rather than phospholipids. The surface tension of surfactant in lung was510 mN/m (Schurch et al., 1976) and lower than that of the present results. The difference of surface tension may be caused by dilution of BALF samples recovered using saline. In this study, we prepared mixtures of DPPC and SP-B or SP-D and measured the surface tension of the mixtures, as shown in Figure 5. Although SP-D reduced the surface tension at 1 ng/ml, no further reduction was observed at 5–10 ng/ml. The mixture of DPPC and SP-B reduced the surface tension in a dose-dependent manner. The ratio of SP-B to total phospholipid in tracheal aspirates of healthy infants was ranging from 0.2 to 0.4% (Cogo et al., 2013), that is, 0.1–1 mg/ml in this study. SP-B level in artificial BALF in this study would be one order lower than the values but the change of surface tension in SP-B mixture was detected. Schleh & Hohlfeld (2009) reported that SP-B had a greater influence on the surface tension than SP-D did. The phospholipids in the surfactant are lined with hydrophobic tails at the air–liquid interface of the alveoli. Particles deposited on the surfactant are surrounded by the hydrophobic tails of phospholipids and form vesicles. SP-B and SP-C are hydrophobic proteins and interact at the surface of the surfactant, forming stable monolayers and bilayers (Whitsett & Weaver, 2002). The present in vitro data suggest that BALF surface tension was regulated by SP-B and phospholipids, such as DPPC used here (Figure 5B). The surface tension of BALF in vivo may serve as an indicator of the combination of SP-B and phospholipids in pulmonary surfactants in vitro. The measurement of the concentration of SP-B or SP-C in BALF in vivo will be made clear the relation in future. To assess the suitability of phospholipids, total protein, SP-D and BALF surface tension as biomarkers for inflammation, the Spearman rank correlation coefficients were calculated with the data of PMN counts in BALF, as shown in Figure 6. The sample number was 60, with 15 rats in each of the four groups. The correlation with the PMN counts was 0.578 (p ¼ 0.000) for phospholipid concentration, 0.645 (p ¼ 0.000) for total protein concentration, 0.443 (p ¼ 0.000) for SP-D concentration and 0.438 (p ¼ 0.000) for surface tension. All the four parameters were correlated significantly with the PMN counts in BALF, which is gold standard of lung inflammation. In our previous studies (Lee et al., 2013), we examined pulmonary surfactant after an intratracheal instillation of MWCNT in rats and reported that acute changes in BALF surface tension correlated well with the levels of neutrophil counts, and inflammatory scores of the lungs by a point-counting analysis of the histopathological images, similar to the present findings. Surfactant proteins, in particular SP-D, are recently studied for pulmonary diseases. The levels of SP-A and SP-D are known to increase in the serum of patients with interstitial pneumonia or pulmonary alveolar proteinosis (Kuroki et al., 1993; Takahashi et al., 2000). It is expected that the SP-D in the serum will increase, followed by an increase in SP-D in the BALF, as shown in this study; however, further studies are needed to elucidate the changes of SP-D in BALF and serum caused by inhaled nanoparticles.

Conclusion Three days after inhalation of NiO nanoparticles, C60 and MWCNTs at similar level of aerosol concentrations, the inflammatory responses, such as PMN counts, were in the order of of control ¼ C605MWCNT55NiO. The concentrations phospholipids, total protein and SP-D and BALF surface tension were significantly correlated with the PMN counts in the BALF in this study. Such tendencies were reported in our previous

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instillation study of MWCNTs (Lee et al., 2013) and were confirmed by this inhalation study. The pulmonary surfactant components can be used as measures of inflammatory response of lung to inhaled particles.

Declaration of interest This research was funded by a grant from the New Energy and Industrial Technology Development Organization of Japan (NEDO) titled: ‘‘Evaluating risks associated with manufactured nanomaterial: Inhalation exposure methods for developing toxicity evaluations (P06041)’’. The authors have no conflicts of interest.

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