Preparation of MWCNTs, FL and FLW

MWCNT-N and MWCNT-7 were supplied by NIKKISO (Tokyo, Japan), and Mitsui Chemicals Inc. (Tokyo, Japan), respectively. C60 FL and FLW, composed of C60 FL were synthesized at room temperature by the liquid–liquid interfacial precipitation method (LLIP method) at National Institute for Materials Science. Briefly, FNWs were precipitated and grown by forming a liquid–liquid interface between a C60-saturated toluene solution and isopropyl alcohol (IPA) (Miyazawa et al. 2014). All of the particles (FL, FLW, MWCNT-7, MWCNT-N) were dispersed and diluted into 5% poloxamer 188 solution (Sigma-Aldrich, St. Louis, Mo, USA) at concentrations of 62.5 and 125 μg/mL. The suspensions were homogenized at 20 kHz, 3000 × g for 20 min using an ultrasonic homogenizer (UD-211, TOMY, Tokyo, Japan) before treatment to minimize aggregation of the administered materials.

Animals

Ten-week-old male F344 rats were purchased from Charles River Laboratories Japan, Inc., Yokohama, Japan. They were housed in the Laboratory Animal Facility of Nagoya City University Medical School, maintained on a 12:12 h light: dark cycle, and received Oriental MF Basal diet (Oriental Yeast, Tokyo, Japan) and water ad libitum. The research protocol was recommended by the Animal Care and Use Committee of Nagoya City University Medical School, and the animals were taken care of according to the Guidelines for the Care and Use of Laboratory Animals of Nagoya City University (no. H30M-008).

Experimental design

We used a total of 224 male F344 rats for the experiment. The animals were quarantined and acclimated for 2 weeks before the start of the experiment. The animals were divided by stratified randomization into 10 body weight-matched groups. Group 1 (25 rats) No treatment; Group 2 (22 rats) Vehicle, saline with 0.5% poloxamer-188 solution; Group 3a (20 rats) FL 0.25 mg/rat; Group 3b (24 rats) FL 0.5 mg/rat; Group 4a (20 rats) FLW 0.25 mg/rat; Group 4b (23 rats) FLW 0.5 mg/rat; Group 5a (20 rats) MWCNT-7 0.25 mg/rat; Group 5b (25 rats) MWCNT-7 0.5 mg/rat; Group 6a (20 rats) MWCNT-N 0.25 mg/rat; Group 6b (25 rats) MWCNT-N 0.5 mg/rat. Rats were administered the test solutions by Intra-tracheal intra-pulmonary spraying (TIPS) as previously described (El-Gazzar et al. 2019; Sato et al. 2023). Briefly, rats were anesthetized with 4% isoflurane and given 0.5 ml of vehicle or test substance suspension (62.5 or 125 μg/ml) using a DIMS-type microsprayer aerosolizer (for rats) that was connected to a 1-mL disposable syringe (OSAKA CHEMICAL Co., Ltd., Osaka, Japan). Rats were administered test solutions once every other day for 15 days for a total of 8 doses. The body weight was measured on the weekly basis. Animals were sacrificed by exsanguination from the abdominal aorta under deep isoflurane anesthesia 1 week and 104 weeks after the final TIPS administration. Five rats from the no treatment, vehicle, and high dose groups were sacrificed at 1 week, and the remaining rats were sacrificed after 104 weeks. Rats began to die after 52 weeks, mostly from causes of death unrelated to the procedure. Treatment-related cause of death was mesothelioma, seen only in the MWCNT-7 0.5 mg/rat group, with the earliest death occurring at 80 weeks post-treatment. Animals that died before the final sacrifice were immediately necropsied for quantitative analysis of neoplastic lesions in the lung and pleura. At 104 weeks, 3 animals from each test substance exposure group were used to determine the amount of residual nanomaterial (NM) remaining in the whole lung. Animals that survived for more than 52 weeks and the animals sacrificed at 104 weeks that were not used for determination of residual NM remaining in the lung were used to evaluate carcinogenic lesions in the lungs and pleura.

Collection of tissue samples and pleural lavage

Blood samples collected from the abdominal aorta were centrifuged at 3000×g, 4 °C for 10 min and then the serum was collected. 10 ml of RPMI1640 (Thermo Fisher Scientific, Rockford, IL) was injected into the pleural cavity through the left side of the diaphragm, the entire chest cavity was shaken from side to side, and the lavage was collected. The pleural lavage was centrifuged at 1000 × g, 4 °C for 10 min and the supernatant were used for biochemical analysis. Precipitates after centrifugation were fixed with 4% paraformaldehyde solution and paraffin-embedded for histological analysis. Myeloperoxidase (MPO) levels were measured using an Amplite Fluorimetric Myeloperoxidase Assay Kit (AAT Bioquest, Inc., Sunnyvale, CA) and ROS levels were measured using the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay (Thermo Fisher Scientific) as previously described (Naiki et al. 2012). The left lobe of the lung was cut into small pieces and immediately frozen at -80⁰C for RNA extraction or ROS detection, and the 4 right lobes were fixed with 4% paraformaldehyde solution and paraffin-embedded for light microscopic examination, scanning electron microscopy (SEM), and immunohistochemical analysis. SEM was performed as previously described (Saleh et al. 2020). The heart, trachea, paratracheal lymph node, liver, kidney, spleen, and chest wall were removed and fixed with 10% formalin, paraffin-embedded and used for histological evaluation.

Pathological examination and Immunohistochemistry

Three µm-thick sections were prepared from the fixed, paraffin-embedded specimens and stained with Hematoxylin and Eosin (H&E). Underlying sections were immunohistochemically stained with antibodies against CD68 (Bio-Rad Cat# MCA341R, RRID:AB_2291300, Cambridge, UK), Ki67 (Abcam Cat# ab16667, RRID:AB_302459, Hercules, CA), and 8-hydroxydeoxy guanosine (8-OHdG) (Japan Institute for Control of Aging Cat# MOG-020P, RRID:AB_3096485, Shizuoka, Japan). H&E slides were evaluated by two experts in pathology (A.N-I. and H.T.) and diagnosis of hyperplasia, adenoma, adenocarcinoma in lung, and mesothelioma was done according to the INHAND criteria.

Detection of ROS production

Six µm thick slices were cut from the frozen lung tissues of rats sacrificed at 1 week after TIPS administration and incubated in 5 μM dihydroethidium (Thermo Fisher Scientific) for 15 min in the dark. The slides were washed with phosphate buffered saline and ROS was detected at 518/605 nm using an image analyzer (BZ 9000 Fluorescence Microscope, RRID:SCR_015486, Keyence, Osaka, Japan). Five images per rat were randomly taken with the same exposure time at 400 × magnification, and the average fluorescence intensity in the alveolar epithelial nuclei was quantified using BZ-analysis application software (Keyence).

RNA extraction and quantitative reverse transcription–PCR

Total RNA was isolated from frozen lung tissues by phenol–chloroform extraction (Isogen; Nippon Gene Co. Ltd., Tokyo, Japan). One microgram of RNA was converted to cDNA using PrimeScript™ RT Master Mix (Takara). The cDNA was subjected to quantitative real-time PCR (qRT-PCR) using TB Green™ Premix Ex Taq™ II (Takara) and detected by the AriaMx Real-Time PCR System (g8830a, RRID:SCR_019469, Agilent Technologies). The primers used are listed in Table S1. GAPDH mRNA levels were used as internal controls.

Total protein and albumin in the pleural lavage

The levels of total protein and albumin in the pleural lavage at week 1 were measured using standard procedures on an autoanalyzer by a commercial laboratory (DIMS Institute of Medical Science, Aichi, Japan).

Measurement of FLW and MWCNTs in the lung

MWCNT concentration in the lung was measured as previously described (Ohnishi et al. 2013, 2016). Briefly, the formalin-fixed lungs (n = 3 per group) containing MWCNT were digested by a strong alkali solution (Clean 99 K200; Clean Chemical, Osaka, Japan), and after removing organic matter with sulfuric acid, benzo[ghi]perylene (B(ghi)P) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was added to the sample solution. The sample solution was immediately sonicated by an ultrasonic homogenizer (VP-30S, Taitec, Saitama, Japan) to allow the MWCNT to adsorb the B(ghi)P. The solution was filtrated through a membrane filter (Whatman® Nuclepore, 111,109, Cytiva). The B(ghi)P on the filter was desorbed from the MWCNT into acetonitrile. Finally, the B(ghi)P was analyzed by an UPLC system (Waters, Milford, MA) coupled to a fluorescence detector with a reversed phase column (ACQUITY UPLC BEH C18, Waters).

FL and FLW in the formalin-fixed lungs (n = 3 per group) was measured as below with reference to JIS Z 8981:2010 (Quantitative analysis methods for [60]fullerene and [70]fullerene by high performance liquid chromatography). The digested lung samples by a strong alkali solution passed thorough the liquid–liquid extraction column (EXtrelut NT, Merck, Darmstadt, Germany). The adsorbed FLWs in the column were eluted using toluene. The elution was concentrated by nitrogen and filled to 1 mL by toluene: methanol = 1:1. The FLW concentrations were analyzed by an HPLC system (LC-10; Shimadzu Corporation, Kyoto, Japan). Absorbance was detected at 325 nm. The flow rate of the mobile phases (toluene: methanol = 1:1) was 0.8 mL/min. A reversed phase column (Inertsil ODS-2, GL Sciences Inc., Tokyo, Japan) was used at 35 °C.

Statistical analysis

Differences in quantitative data, expressed as mean ± standard deviation (SD), between groups were compared by one-way ANOVA and the Turkey’s multiple comparison test using the software package GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA). P < 0.05 was considered significant.