Fullerenes
Bulk high-purity (>99.5%) C60 fullerenes were purchased from Frontier Carbon Corporation (Japan). The manufacturer's specifications indicated a specific surface area of 0.92 m2/g. Bulk fullerene material roughly dispersed in 0.1 mg/ml polyoxyethylen sorbitan monooleate (Tween-80, Wako Pure Chemical Industries, Ltd., Japan) was milled in an agate mortar for 30 minutes under a nitrogen atmosphere. The milled fullerene material was suspended with zirconium particles (50 μm) using a high-performance dispersion machine (UAM-15, Kotobuki Industries Co., LTD., Japan) and centrifuged at 8000 × g for 60 min. The concentration was determined by an HPLC system (#1100, Agilent Technologies, Santa Clara, CA). The fullerenes were individually dispersed in this suspension [28]. The mean diameter based on volume and mass by the dynamic light scattering technique (UPA, NIKKISO Co., LTD., Japan) was 33 nm (Figure. 1). This fullerene suspension was used as the directly injected solution in the intratracheal instillation study and for the generation of fullerene nanoparticles in the inhalation study.
Nickel Oxide
Nickel oxide (20 nm nominal primary diameter, 99.8% purity) was used as a reference material for nanoparticles [31], and was purchased from Nanostructured & Amorphous Materials Inc. The BET specific surface area of the measured sample was 104.6 m2/g and the weighted average surface primary diameter (Sauter diameter) was 8.41 nm. The nickel oxide particles were dispersed without any detergents by ultrasonication for 90 minutes using an ultrasonic homogenizer (Model 450, Branson Ultrasonics Corporation, Danbury, USA). The particles were suspendedby centrifugation at 10,000G for 20 min and the recovered supernatant was filtered through a membrane filter with 1 μm diameter pores. The particle size distribution was determined by the dynamic light scattering technique, and the mean diameter based on volume and mass was 26 nm (Figure 1).
Intratracheal instillation of fullerenes
In the instillation liquid (distilled water containing 0.1% Tween 80), the diameter of fullerene nanoparticles was confirmed as 33 nm (Figure 1). 0.1 mg (0.33 mg/kg) or 0.2 mg (0.66 mg/kg) or 1 mg (3.3 mg/kg) of fullerenes was suspended in 0.4 ml of distilled water including 0.1% Tween 80. 0.2 mg (0.66 mg/kg) and nickel oxide nanoparticles (average agglomerate diameter in the suspension: 26 nm) were suspended in 0.4 ml distilled water. Each material suspension was intratracheally instilled once in Wistar male rats (9 weeks old). The negative control group was exposed to distilled water including 0.1% Tween 80. The animals were dissected at 3 days, 1 week, 1 month, 3 months, and 6 months after instillation.
Inhalation study of fullerenes
The whole-body exposure system, used to expose rats to fullerenes or nickel oxide nanoparticles, consisted of a pressurized nebulizer and a mist dryer, connected to an exposure chamber (volume: 0.52 m3). Same fullerenes and nickel oxide suspensions in the intratracheal instillation study were used for the inhalation study. They were put into the nebulizer and used for the generation of the aerosols [32]. The size and number concentrations of aerosol particles at the exit of the nebulizer and inside the exposure chamber were analyzed in-line using a particle spectrometer consisting of a differential mobility analyzer (DMA) and a condensation particle counter (CPC) (Model 1000XP WPS, MSP Corp., Shoreview, MN) throughout the exposure period. Rats were exposed to the aerosol continuously for 4 weeks (6 hours/days, 5 days/week), and the size distribution and number concentration in the chamber were measured throughout the exposure period. The fullerene aerosol in the chamber had an average mass concentration of 0.12 × 0.03 mg/m3 (0.5 × 0.1 mg/m3: including Tween 80) maintained by the isokinetic suction of air through a glass fiber filter, a particle concentration of 4.1 × 104 particles/cm3, and an average geometric diameter of 96 ± 5 nm by DMA and CPC. The nickel oxide nanoparticle aerosol in the chamber had an average mass concentration of 0.2 ± 0.1 mg/m3, a particle concentration of 9.2 × 104 particles/cm3, and an average geometric diameter of 59 ± 3 nm. We found through high-resolution transmission electron microscopy that the structure of the fullerene crystals in the aerosol particles remained identical with that in the fullerene suspension. We also confirmed that particle size of fullerenes used in intratracheal instillation and inhalation studies was less than 100 nm by TEM [5, 32].
Nine-week-old male Wistar rats were divided into 3 groups: fullerene, nickel oxide nanoparticle, and control groups. The rats inhaled the aerosol for 6 hours a day, 5 days a week, for 4 weeks in a whole-body exposure chamber. The control rats were exposed to only clean air in a same-sized chamber located in the same air-conditioned room. After an exposure period of 4 weeks, the rats were dissected at 3 days, 1 month, and 3 months of recovery.
Animals after inhalation and intratracheal instillation studies
Each group of 10 animals was divided into 2 subgroups of 5 animals for lung tissue analysis. The first subgroup (5 rats) provided bronchoalveolar lavage, which was collected using physiological saline that was poured through a cannula inserted in the respiratory tract into the right lung, while the left lung was clamped. Three - 10 ml of physiological saline was infused per time and lavage fluid was collected up to 50 ml in total. The left lung was inflated and fixed by intratracheal instillation of 4% paraformaldehyde at 25 cm H2O pressure.
The lungs of the second subgroup (5 rats) were homogenized to extract protein and mRNA.
All procedures and animal handling were done 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.
Chemokine measurement of lung tissue and BALF
Lung tissue was homogenized with a T-PER tissue protein extraction reagent, and then centrifuged (1500 × g for 10 min). The protein concentration of the supernatant was measured by the BCA Protein Assay Kit (PIERCE) using Bovine serum albumin. Total protein concentration was adjusted with a final concentration of 500 μg/ml for CINC-1 and CINC-2αβ and 4000 μg/ml for CINC-3. Chemokine concentration was determined by Quantikine Rat CINC-1, CINC-2αβ, and CINC-3 (R&D Systems) (Cat. #RCN100, #RCN200, and #RCN300, respectively) and absorbance at 450 nm was measured by a microplate reader. CINC-1, CINC-2αβ, and CINC-3 in the lung tissue were determined. Alkaline phosphatase (ALP) released in the BALF supernatant was determined by LabAssayTM ALP (Wako Pure Chemical Industries, Ltd. Japan).
Semiquantitative real-time PCR
RNA was extracted from the lung using RNeasy(R) Mini Kit (50) (QIAGEN Hilden, Germany). Single-strand cDNA was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems. CA.).
Real-time PCR and subsequent calculations were performed with the 7500 Real-Time PCR System (Applied Biosystems. Forster. CA. USA), which detects the signals emitted from fluorogenic probes during PCR. Primers and probes were designed according to guidelines from Applied Biosystems with the Primer Express 3.0 software (Applied Biosystems. Forster. CA.). The primer sets were as follows: rat CINC-1; Assay ID Rn00578225_m1, rat CINC-2αβ; Assay ID Rn00593435_m1, rat CINC-3 (MIP-2); Assay ID Rn00586403_m1, and for rat β-actin (RatACTB) Accession number: NM_031144.
Real-time PCR was performed with TaqMan Universal PCR Master Mix reagents. The PCR mixture contained 25 μl of TaqMan Universal PCR Master Mix (2×), 2.5 μl of TaqMan Gene Expression Assays and 17.5 μl of d-water in a total volume of 45 μl. PCR was performed using 5 μl of the first strand cDNA mix. After 2 min at 50°C, to permit UNG cleavage, AmpliTaq Gold was activated by a 10 min incubation at 95°C. Each of the 60 PCR cycles consisted of a 15 s denaturation step at 95°C and a hybridization step, with probes and primers and for DNA synthesis, for 1 min at 60°C.
The average cycle threshold (CT) was determined for each group of animals at each time point. Relative gene expression was calculated using the comparative CT (ΔΔCT: the difference of the average ΔCT value of the exposed group and the average ΔCT of the control group) method which assesses the difference in gene expression (ΔCT: difference between the threshold cycle) between CINC-1, CINC-2αβ and CINC-3 as the target gene and β-actin as the internal standard gene for each sample to generate the ΔΔCT. Relative gene expression in the Y axis was then determined by the formula 2-ΔΔCT. The relative expression of the average in each group was calculated with respect to the control group in each time point, and the relative expression of the control group was set at 1. Statistical analyses involved comparison of the cycle difference of CINC and β-actin between the exposed and the control groups, and were performed at the ΔCT stage.
Tissue preparation for HE stain
The lungs, which were inflated and fixed by 4% paraformaldehyde, and trachea were resected from the surrounding tissue. The lung tissue was embedded in paraffin, and 5 μm-thick sections were cut from the lobe. The samples were then sectioned and stained with hematoxylin and eosin.
Processing of lung tissue for transmission electronmicroscope (TEM)
The lung tissues were fixed using glutaraldehyde and osmium tetroxide solution, and then dehydrated in ethanol, and embedded in epoxy resin. The specimens were stained with a 2% uranyl acetate solution and 0.5% lead citrate solution at room temperature. Conventional TEM observation was performed within an H-7000 (Hitachi, Japan) at the acceleration voltage of 80 kV. Energy-filtering TEM observation was performed with an EM922 (Carl Zeiss SMT, Germany), which was equipped with an OMEGA energy filter. Zero-loss filtering, which can increase the scattering and phase contrast of the TEM image was carried out.
Statistical analysis
Statistical analysis was carried out using the Mann-Whitney test with differences of p < 0.05 considered to be statistically significant.