Preparation of the test material
The MWCNT, MWNT-7 (also called as Mitsui-7, XRNI-7, or MWCNT-7; lot, 060 125-01 k), was obtained from Mitsui Chemicals (Tokyo, Japan) and was fully characterized in our previous reports [24, 34]. Since intratracheal instillation bypasses the upper respiratory tract, it may deliver fiber aggregates, which are presumed to be entrapped in the nasal cavity in inhalation tests, to the lung parenchyma. Thus, the bulk materials were pretreated with a filtration method, the Taquann method, which removes the agglomerates and aggregates of fibers without changing the size distribution of the fibers by employing a fine filtration (a 53-µm mesh) and a critical point drying technique [24] (Fig. 1 and Additional File. 2: Fig. S3). Thereafter, the Taquann treated-MWCNT was baked at 200 °C for 2 h in a dry heat sterilizer for the elimination of endotoxin. The MWCNT was suspended in saline containing 0.1% Tween 80 at a concentration of 0.125 (low-dose group) and 0.5 mg/mL (high-dose group) and then sonicated for 30 min using an ultrasonic bath (100 W; US-702, SND, Nagano, Japan). To minimize aggregation, the suspension was again sonicated in the animal room immediately before the administration.
MWCNT characterization
The suspension used for administration was diluted at a concentration of 0.01 mg/mL with pure water containing 0.1% Triton X-100, and 1 µL of the diluted suspension was placed on an inorganic aluminum oxide membrane filter (Whatman® Anodisc, Cytiva, Marlborough, MA, USA). The filter was coated with gold and viewed by SEM (Quanta™ FEG250; Thermo Fisher Scientific, Waltham, MA, USA) at 10–20 kV. A total of 1000 fibers were randomly photographed at magnifications of 5,000 and 10,000 for length measurements and 60,000 for width measurements. The morphological classification and length and width measurements of the MWCNT fibers were performed by ImageJ software (NIH, Bethesda, MD, USA).
The secondary diameter in the suspension containing MWCNT at 0.01 mg/mL was analyzed by DLS (Zetasizer Nano, Malvern, Worcestershire, UK).
Animals and treatment
A total of 150 of 5-week-old male specific pathogen-free Fischer 344 (F344/DuCrlCrlj) rats were purchased from Jackson Laboratories Japan (Kanagawa, Japan). Male rats were chosen because of their susceptivity to the development of lung tumors [11] and for comparisons with TIPS studies [20,21,22]. The rats were housed in a polycarbonate cage (3 rats per cage) in a room maintained at a temperature of 23 ± 0.1 °C and 53.1 ± 7.9% relative humidity on a 12 h light–dark photophase cycle, and given standard CE-2 basal diet (CLEA Japan, Tokyo, Japan) and drinking water via a bacterial filter ad libitum. After a 2-week quarantine and acclimation period, the animals were randomly divided into 3 groups: vehicle control group, low-dose group, and high-dose group, which consisted of 40, 55, and 55 animals, respectively (Additional File 1 Table S1).
Intratracheal administration of MWCNT to rats was performed 26 times at intervals of 4 weeks from 9 weeks of age, according to previous studies [18, 59]. Briefly, the rats were deeply anesthetized by inhalation of 3% isoflurane (Pfizer, New York, NY, USA), held on a holder inclined at 45°, and then the vehicle or the MWCNT suspension was instilled at 1 mL/kg body weight through the larynx into the lung using a feeding cannula (20-gage; Natsume Seisakusho, Tokyo, Japan) connected to a 1.0-mL syringe (Terumo, Tokyo, Japan). The animal was kept in the holder until its usual respiratory rhythm was recovered and then placed back in the housing cage. The MWCNT dosage, i.e., 0 (control), 0.125 (low dose) or 0.5 (high dose) mg/kg body weight, was decided based on a pilot study to examine the time course of the lung burden.
General conditions were observed twice daily, and body weight was measured every week.
Satellite animals were sacrificed at week 26 and week 52 after the first administration to monitor histological features and lung burden levels. At each time point, 5, 10, and 10 animals were sacrificed for the control, low, and high-dose groups, respectively. For each group, 30 animals were included for carcinogenic assessment (Additional File. 1: Table S1).
Autopsy and sample collection
At the interim sacrifices (weeks 26 and 52) and termination of the study (week 104), animals were killed by exsanguination through the abdominal aorta under 3% isoflurane anesthesia, and macroscopically examined. Animals that died or were humanely euthanized before the end of the experiments were similarly treated.
The number of animals used in each analysis is summarized in Additional File. 1: Table S1. BALFs were collected from 5 animals from each group in the interim sacrifices and 10 animals from each group at the terminal necropsy. To lavage only the right lung, the left bronchus was tied with a thread, and the right lung was lavaged 2 times with 4 mL of phosphate-buffered saline at a water pressure of 30 cm. The washout was centrifuged at 402 g at 4 °C for 10 min. The supernatant was collected for biochemical analyses, and the pellet was gently resuspended for cytological analysis. For measurement of the lung burden, a whole lung was obtained from 4 or 5 animals from both the MWCNT-treated groups at the interim and terminal necropsies. PLFs were collected from the animals used for measuring the lung burden at the interim and from 15 animals of the terminal necropsies. The thorax of the rat was lavaged once with 8 mL of saline using a syringe with a needle, and the washout was collected for SEM analyses.
At the terminal necropsy, major organs, trachea, lungs, parietal pleura (diaphragm and chest wall), heart, spleen, bone marrow, mediastinal lymph nodes, thymus, tongue, salivary glands, esophagus, stomach, small intestine, large intestine, liver, pancreas, kidneys, urinary bladder, adrenal glands, pituitary gland, thyroid glands with parathyroids, testes, epididymis, seminal vesicles, prostate, mammary glands, muscle, bone, brain, spinal cord, eyes, Harderian glands, Zymbal’s glands, and skin, were collected from all animals for histopathological examination. The brain, heart, lungs, liver, spleen, kidneys, adrenal glands, and testes from 10 animals from each group were weighed.
Hematological analysis
The blood samples obtained from 10 animals in each group at the terminal necropsy were analyzed with an automatic blood cell analyzer (KX-21NV, Sysmex, Hyogo, Japan). Differential counts of leukocytes were made by a light microscopic observation of smeared specimens stained following a routine May–Grunwald–Giemsa protocol.
Histopathology
All dissected organs and tumor masses were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned (4 μm thickness), and stained with hematoxylin and eosin. All 5 separate lung lobes were evaluated. The entire mediastinum including the pericardium, heart, mediastinal pleura, and RPF were embedded in agarose, and then trimmed, following the routine procedure for paraffin sections. Among a series of samples along the cranial–caudal axis, 3 levels or more were histologically examined (Additional File. 2: Figs. S 1D and S1E).
The severity of non-neoplastic lesions was graded on a 5-point scale of no/minimal (0), slight (1), mild (2), moderate (3), or marked (4). Proliferative lesions of the lung and pleura were blindly examined by more than 4 pathologists, and the final diagnosis was made by Dai Nakae, one of the authors, who is a board-certified pathologist of the Japan Society of Toxicologic Pathology (Diplomate of JSTP) and the Japan Society of Pathology. Pleural mesotheliomas were classified into 3 types, epithelioid, sarcomatoid, and biphasic. Biphasic mesothelioma was diagnosed when both epithelioid and sarcomatoid components were > 10% of the tumor masses collected from the thoracic cavity.
For immunostaining, antigen retrieval was performed in Tris–EDTA buffer (pH 9.0) using an autoclave for 60 min or 10 mM citrate buffer (pH 6.0) using a microwave for 15 min, followed by the inactivation of endogenous peroxidase by immersion in H2O2. After blocking with Protein Block (X0909; Agilent technologies, Santa Clara, CA, USA) for 20 min at room temperature, the sections were treated with primary antibodies: thyroid transcription factor-1 (TTF-1, ab72876, abcam, Cambridge, UK), or mesothelin/c-ERC (28001, Immuno Biological Laboratories, Gunma, Japan) for 1 h at room temperature. Diaminobenzidine signals were detected with a horseradish peroxidase-secondary antibody conjugate (K4061, Agilent Technologies) according to the manufacturer’s instructions.
To count the MWCNT fibers penetrating the visceral pleura, a new region of the knife blade was used to cut each section block, with a unidirectional cut from outside of the tissue toward the center of the parenchyma, to minimize the possibility that the fibers were artificially positioned on the visceral pleura. To enhance the contrast between the tissue and MWCNT fibers, the sections were stained with Kernechtrot solution.
Cytological analysis in the BALF
Cells recovered after centrifugation of the BALF were stained with Turk’s solution, and the number of leukocytes was determined using a counting chamber slide. For differential leukocyte counts, cytoslides were prepared (CF-120, Sakura Finetek Japan, Tokyo, Japan), stained with May–Grunwald–Giemsa, and microscopically examined.
Biochemical analyses in the BALF
LDH activity in the BALF was determined by an assay kit (Takara Bio, Shiga, Japan) and an LDH standard (Roche Diagnostics, Mannheim, Germany). Concentrations of total protein in the supernatant of the BALF were measured by a kit (Cytiva) following the manufacturer’s instructions. Levels of CINC-1 ( also known as KC, CXCL-1, and GRO-α) and CCL-2 in the BALF supernatant were individually analyzed using ELISA kits: CINC-1, RCN100 (R&D systems, Minneapolis, MN, USA); CCL-2, ELR-MCP-1 (RayBiotech Life, Peachtree Corners, GA, USA).
MWCNT measurement in the lung and PLF
MWCNT quantification in the lung was performed as previously described [60, 61]. Briefly, fixed whole lung samples were digested by a strong alkali solution (Clean 99, K200; Clean Chemical, Osaka, Japan), and after washing and removing organic debris with sulfuric acid, benzo[ghi]perylene (B(ghi)P) (CAS: 191–24-2, Sigma-Aldrich®, B9009, Merck, Kenilworth, NJ, USA) was added to the sample solution containing MWCNTs. The sample solution was immediately and thoroughly mixed by an ultrasonic homogenizer (UH-50, SMT, Tokyo, Japan) so that B(ghi)P was adsorbed to the MWCNT. The solution was then passed through a membrane filter (Whatman® Nuclepore, 111,109, Cytiva). The B(ghi)P that interacted with the MWCNT on the filter was desorbed into acetonitrile. Finally, the B(ghi)P was analyzed by an UHPLC system (Nexera X2, Shimadzu, Kyoto, Japan) with a reversed phase column (ACQUITY UPLC BEH C18, Waters, Milford, MA, USA).
Counting and morphological analyses of MWCNT fibers in the PLF were performed by SEM, as described above. The PLF was centrifuged at 20,000 g for 1 h, and the pellet was digested with Clean 99 for 2 h. After washing once with pure water containing 0.1% of Triton X-100, the precipitate was resuspended at a volume of 50 µL. Four or 5 animals were used to count the numbers of fibers at each time point. A total of 1,000 fibers randomly selected from these samples were further examined for length measurement and a morphological classification.
Similarly, a total of 1,000 fibers in the digested lung tissue, which was used in the lung burden measurement, were analyzed for the length distribution and morphology.
Statistical analysis
Body weight, organ weight, hematology, cell counts in the BALF, and biochemical parameters in the BALF were analyzed by Dunnett’s multiple comparison test. Survival curves were plotted according to the Kaplan–Meier method, and the log-rank test was used to detect significant differences in survival rates between the treated groups and the vehicle control group. Histological scoring of non-neoplastic lesions was analyzed by Steel’s multiple comparison test. The difference in the fiber length between the lung and PLF was analyzed by Student’s t-test. Differences in the incidence of neoplastic or non-neoplastic lesions from the control group were analyzed by Fisher’s exact test. Two-tailed tests were used for all statistical analyses. Differences in values were deemed significant when p-values were less than 0.05. Statistical analyses were performed using StatLight software (Yukms, Kanagawa, Japan).