Two types of CNT were used in this study. DWCNT (brand name Tocana; Toray Industries ,Inc., Tokyo, Japan) with an iron content below detectable limits and water content equal to 0.025% by weight (information provided by the company) and MWCNT-7 (Mitsui Chemicals Inc., Tokyo, Japan) with an iron content of 0.3% by weight . Table 6 shows the water and iron content of the fibers.
Nine-week old male F344 rats were purchased from Charles River Japan Inc. (Yukohama, Japan). The animals were housed in the Center for Experimental Animal Science of Nagoya City University Medical School, maintained on a 12 h light–dark cycle, and received Oriental MF basal diet (Oriental Yeast Co., Tokyo, Japan) and tap water ad libitum. The experimental protocol was approved by the Animal Care and Use Committee of Nagoya City University Medical School, and the research was conducted according to the Guidelines for the Care and Use of Laboratory Animals of Nagoya City University. The experiment was started after a 2-week acclimation and quarantine period.
A total of 180 rats 11 weeks old were divided into six groups of 30 animals each: Group 1, no treatment; Group 2, vehicle (saline with 0.5 % Pluronic F-68: Sigma-Aldrich Merck); Group 3, DWCNT (0.125 mg/rat); Group 4, DWCNT (0.25 mg/rat); Group 5, DWCNT (0.5 mg/rat); and Group 6, MWCNT-7 (0.5 mg/rat): MWCNT-7 was used as the reference material. Rats were administered the test solutions by TIPS as previously described . Briefly, rats were anesthetized with 3% isoflurane and administered 0.5 ml vehicle or test material suspensions (15.6 ug , 31.25, or 62.5 μg of test material in 0.5 ml vehicle) using a micro sprayer (series IA–1B Intratracheal Aerosolizer Penn-century, Philadelphia, PA). Rats were administered one dose every other day over a 15-day period (8 administrations for total doses of 0.125, 0.25, or 0.5 mg DWCNT per rat and 0.5 mg MWCNT per rat). The amount of DWCNT administered to the rats was approximately equivalent to or less than the doses used in the studies by Crouzier et al., Tian et al., Sager et al., and El-Gazzar et al [24, 25, 26, 28]. The dose of MWCNT-7 was one third the amount of MWCNT-7 that caused mesothelioma in rats . At week 52 an interim sacrifice was performed on 5 rats from the Untreated and Vehicle groups, 5 rats from the DWCNT 0.125mg/rat group, 7 rats from the DWCNT 0.25mg/rat group, 6 rats from the DWCNT-0.5 mg/rat group, and 5 rats from the MWCNT-7 group. After 52 weeks, animals that were found moribund were sacrificed, and the final sacrifice was at week 104.
Preparation of the test materials
Test materials were weighed and dispersed in tert-butyl alcohol by sonication for 10 min and stored frozen at -20˚C. Shortly before administration, the T-butyl alcohol was removed using an Eyela Freeze Drying machine (FDU-2110; Tokyo Rikakikai Co., Ltd., Tokyo, Japan), and the DWCNT and MWCNT-7 were suspended in saline containing 0.5% Pluronic F-68 (PF68, Sigma-Aldrich Merck) at 31.25, 62.5, and 125 μg/ml. After suspension in Saline + PF68, test materials were sonicated for 2 min four times at 3000 rpm using a polytron PT 1600E bench top homogenizer (Kinematika AG, Lattau, Switzerland). Immediately prior to administration, the suspensions were sonicated for 30 min using a Tomy Ultrasonic disruptor, UD-211, equipped with a TP-040 micro tip (Tomy Seiko Co., Ltd., Tokyo, Japan) at a power setting of 4.
Characterization of the test materials before and after TIPS administration
After sonication and before administration to the rats' lungs, 20 μl of each test material suspension was placed on a micro grid membrane pasting copper mesh (EMS 200-Cu, Nisshin EM Co., Ltd., Tokyo, Japan) for measurement of DWCNT and MWCNT-7 prior to TIPS administration. For measurement of DWCNT and MWCNT-7 in the lungs of rats at 52 and 104 weeks after instillation about 1 gm of paraformaldehyde fixed lung tissue was digested as previously described . Briefly, tissues were incubated in Clean 99-K200® (Clean chemical Co., Ltd., Ibraki, Japan) overnight or until complete dissolution of lung tissues. The digested solution was then centrifuged at 12,000 rpm for 30 min and the supernatant was discarded. The pellet was resuspended in distilled water and lightly sonicated. The pellet was collected by centrifugation and washed two more times. After a final centrifugation, the pellet was resuspended in 200 μl of distilled water and the specimens were collected on EMD MilliporeTM Polycarbonate Membrane Filters (Millipore, Tokyo, Japan). Fibers were viewed by SEM (Field Emission Scanning Electronic Microscope; Hitachi High Technologies, Tokyo, Japan) at 5–10 kV and TEM (Transmission electron microscope; EDAX, Tokyo, Japan) at 15K-50K. Photos were analyzed by NIH image analyzer software (NIH, Bethesda, Maryland, USA). At least 200-300 fibers of each type of CNT were measured.
In vitro biodegradation
DWCNT and MWCNT-7 fibers were added to cultures of RAW cells, a mouse macrophage cell line, and to plates without cells (control). Cultures were incubated overnight with the CNTs at 37˚C in a humidified incubator. The cells were washed twice with PBS to remove materials not associated with the cells. Control plates were not handled. The cultures were then incubated for an addition 4 days. After 5 days incubation with CNTs, culture media was removed from the RAW cell cultures, and the cells were washed twice with PBS to remove materials not associated with the cells. The cells were then harvested and the CNTs recovered from the cells. For the control plates, the CNTs were collected from the cell-free culture media. The collected CNTs were measured using far-infrared absorption to estimate the effective CNT length .
Electron microscopic viewing of fibers in lung tissues
For high magnification viewing, H&E stained slides were immersed in xylene to remove the cover glass, dried, and processed for SEM (Model S Field Emission SEM; Hitachi High Technologies, Tokyo, Japan). For ultrafine viewing, a small piece of paraformaldehyde fixed lung tissue was imbedded in epoxy resin and processed for TEM (EDAX, Tokyo, Japan).
Measurement of DWCNT and MWCNTs in the lung
Measurement of the amount of CNT fibers in the lung tissue was performed as described previously [19, 65].
Tissue sample collection and histopathological examination
At necropsy, blood samples were collected via the abdominal aorta under deep isoflurane anesthesia and serum samples were stored at -80 °C. Organs, including lung, liver, kidney, spleen, brain, heart, and testes were examined for any macroscopic lesions. The trachea, esophagus, lymph nodes (including mediastinal lymph nodes), diaphragm including the diaphragmatic region of the parietal pleura were examined macroscopically and then processed and examined histopathologically.
The 4 right lobes of the lung of each rat were excised at necropsy, frozen in liquid nitrogen, and sored at – 80 degrees for further biochemical analysis. The remaining left lung was inflated and fixed with 4% paraformaldehyde solution in phosphate-buffered saline (PBS) adjusted to pH 7.3 and processed for light microscopic examination. H&E stained tissue sections were evaluated by two board-certified Pathologists of the Japanese society of Toxicologic pathology, Drs. Hiroyuki Tsuda and Satoru Takahashi, and diagnosis of hyperplasia, adenoma, adenocarcinoma, and mesothelioma was done according to the INHAND criteria .
PCNA staining of deparaffinized slides processed for antigen retrieval and blockade of endogenous peroxidase activity was performed as previously described . For each lung specimen more than 1000 pulmonary epithelial cells and more than 500 visceral pleural and parietal pleural mesothelial cells were counted blindly in random fields. All nuclei showing brown staining of more than half of the nucleus were considered to be positive.
To determine the degree of inflammation the number of macrophages per cm2 of lung tissue was determined: Deparaffinized slides processed for antigen retrieval and blockade of endogenous peroxidase activity were incubated with PBS containing 5% BSA and 5% goat serum for 1 h, then incubated with the macrophage marker anti-CD68 (BMA Biomedicals, August, Switzerland) diluted 1:2000 in PBS containing 1% BSA and 1% goat serum over- night at 4 °C. After overnight incubation, the slides were incubated with secondary antibody (Nichirei Biosciences, Tokyo, Japan) for 1 h, visualized with DAB (Nichirei Bio- sciences, Tokyo, Japan), and counterstained with hematoxylin. Light microscopic images representing at least one cm2 from each lung were used to determine the density of the macrophages in the lungs.
Collagen deposition in lung tissues and visceral and partial pleura was quantified in light microscopic images of lung tissues and pleural sections stained with Masson’s Trichrome (Abcam, Tokyo, Japan) using NIH image analyzer software (NIH, Besthesda, Meryland, USA): ten individual images were captured from 2 lung and 3 diaphragm sections from 3 rats per study group. Thresholding using pre-defined RGB criteria for collagen deposition was performed. This allowed collagen to be differentiated from alveolar tissue or pleural tissue. The surface area of fibrosed tissue was measured. Total lung alveolar and pleural tissue surface areas were measured individually by thresholding using predefined RGB criteria for lung alveolar and pleural tissues.The results are expressed as the percentage area of pulmonary collagen deposition per tissue surface area.
At the final necropsy pleural lavage fluid collection was done for all rats as previously described . High mobility group box protein 1 (HMGB1) was measured using a rat HMGB1 ELISA kit (Arigo Biolaboratories; ARG81310) according to the manufacturer's instructions.
CCL2 and CCL3 ELISA
Frozen right lung tissue samples (approximately 100 mg) were thawed and rinsed 3 times with ice-cold PBS and homogenized in 1 mL tissue protein extraction reagent (Thermo Scientific, Rockford, IL, USA) containing 1% (v/v) protease inhibitor cocktail (Sigma-Aldrich Merck). The homogenates were centrifuged at12000 g for 5 minutes at 4°C. Protein content of the supernatant was measured using the BCA Protein Assay Kit (Pierce Biotech). The levels of CCL2 and CCL3 in the supernatant were measured using a Rat MCP-1/CCL2 ELISA Kit (Sigma-Aldrich Merck; RAB0058) and a CCL3 ELISA Kit (LSBio; LS-F5526) according to the manufacturers' instructions.
Detection of DNA adductomes in the lung tissue
Rat lung DNA was extracted by Gentra® Puregene cell and tissue kit (Qiagen). The DNA (50 ug) was digested by incubation at 37°C for 12 h in 300 ul of 5 mM Tris-HCl (pH7.4) containing 50 units of DNaseⅠ, 1 unit of Nuclease P1, 2 units of alkaline phosphatase, and 0.225 units of phosphodiesterase. After digestion, internal standards (100 pmol of 2’,3’-dideoxyinosine and 2’,3’-dideoxyadenosine) were added to the DNA hydrolysates. The hydrolysates were filtered through Amicon Ultra 3kDa centrifugal filters (Sigma-Aldrich Merck; Z677094), and 500 ul of methanol was added to the purified samples. After centrifugation and removal of the methanol, residual methanol was removed by evaporated in vacuo. DNA residues were dissolved in 100 ul of 50% methanol, and 10 ul of sample was subjected to LC/MS.
UHPLC-TOF-MS analyses was performed with a Shimadzu UHPLC Nexera X2 system (Shimadzu) using a Synergi Hydro-RP column (2.5 μm, 100 mm × 2 mm, Phenomenex) and Triple TOF 5600+ (SCIEX) with an electrospray ionization device running in the positive ion mode. The detector conditions were as follows: ion spray voltage at 5500 V, source temperature of 350°C, ion source gas 1, 60 psi, ion source gas 2 60 psi, declustering potential 80 V, collision energies of 45 V, and collision energy spread 15 V. Nitrogen was used as the collision gas. DNA adducts were detected using the MRMHR mode. This strategy was designed to detect the neutral loss of 2’-deoxyribose from positively ionized 2’-deoxynucleoside adducts by monitoring the samples with [M+H]+→[M+H-116]+ transitions . In the mobile phases used for LC-TOF-MS analyses, solvent A consisted of a 0.1% (v/v) solution of formic acid in water and solvent B consisted of a 0.1% (v/v) solution of formic acid in acetonitrile. The DNA adducts were eluted from the column using a linear gradient, which started at 95% solvent A and 5% solvent B, and progressed to 100% solvent B over a period of 10 min. The system was then eluted with 100% solvent B for 10 min before being returned to the initial conditions over a period of 10 min to allow for the equilibration of the column. The system was operated at a constant flow rate of 0.2 ml/min for all of the analyses.
Survival rates were analyzed using the Kaplan-Meier method. The incidences of bronchioloalveolar hyperplasia, bronchioloalveolar adenoma, bronchioloalveolar carcinoma, and total lung tumor incidences were analyzed for difference from vehicle controls using GraphPad's Fisher’s extract test, and continuous data was analyzed using GraphPad's QuickCals t Test Calculator. All data are expressed as mean + standard deviation. p-values < 0.05 were considered to be significant.