Anatase type nano-titanium dioxide, TiO2 NP (aNTiO2) (Additional file 11: Fig. S11) was purchased from Tayca co. (primary particle size: 30 nm). TiO2 NP characteristics are summarized in Additional file 23: Table S11. A list of all primary antibodies used in these studies is shown in Additional file 23: Table S12. Other reagents used in the study were of the highest grade available commercially.
Male and female F344 rats at 4 weeks old were purchased from Charles River Laboratories Japan, Inc. (Kanagawa, Japan). The rats were housed in an air-conditioned room under a 12 h light/12 h dark (8:00–20:00, light cycle) photoperiod, and fed a general diet (CR-LPF, Oriental Yeast Co. Ltd., Tokyo, Japan) and tap water ad libitum. After a 1 week quarantine and acclimation period, they were exposed to TiO2 NP. All animal experiments were approved by the Animal Experiment Committee of the Japan Bioassay Research Center.
Generation of TiO2 NP aerosol
The generation of TiO2 NP aerosol into the inhalation chamber was performed using our established method (cyclone sieve method) [72, 73] with some modifications. Briefly, TiO2 NP was fed into a dust feeder (DF-3, Shibata Scientific Technology, Ltd., Soka, Japan) to generate TiO2 NP aerosol, and the aerosol was introduced into a particle generator (custom-made by Seishin Enterprise Co., Ltd., Saitama, Japan) to separate the aerosol and feed it into the inhalation chamber. The concentration of the TiO2 NP aerosol in the chamber was measured and monitored by an optical particle controller (OPC; OPC-AP-600, Shibata Scientific Technology), and the operation of the dust feeder was adjusted by feedback control based on upper and lower limit signals to maintain a steady state.
The mass concentration of TiO2 NP aerosol in the chamber was measured every two weeks during the exposure period. Aerosols collected on a fluoropolymer binder glass fiber filter (T60A20, φ55 mm, Tokyo Dylec, Corp., Tokyo, Japan) were weighed for each target concentration at 1, 3, and 5 h after the start of exposure. Using the mass per particle (K-value) calculated using the measured mass results (mg/m3) and the particle concentration data (particles/m3) obtained from the OPC, the particle concentration for each group during the exposure period was converted into mass concentration. The particle size distribution and morphology of the TiO2 NPs were measured at the 1st, 6th, and 13th weeks of exposure. The particle size distribution was measured using a micro-orifice uniform deposit cascade impactor (MOUDI-II, MSP Corp., Shoreview, MN). The MMAD and σg were calculated by cumulative frequency distribution graphs with logarithmic probability (Additional file 1: Fig. S1E). The TiO2 NPs in the inhalation chamber were collected on a 0.2 μm polycarbonate filter (φ47 mm, Whatman plc, Little Chalfont, UK), and observed using SEM (SU8000, Hitachi High-Tech, Tokyo, Japan) (Additional file 1: Fig. S1C).
13-week inhalation study
This experiment was conducted with reference to the OECD Guideline for Testing of Chemicals (TG 413) . Based on the results of a dose-finding study conducted previously and OECD TG 413, target concentrations for TiO2 NP aerosols were set at 6.3, 12.5, 25, and 50 mg/m3, and the exposure schedule was 6 h per day, 5 days per week, for 13 weeks (Additional file 12: Fig. S12). One hundred rats (10 males and 10 females in each group) were transferred to individual stainless steel cages and exposed to TiO2 NP for 6 h with access to food and water. Animals were autopsied on two separate days beginning the day after the final exposure date (approximately 50 animals/day). All animals were fasted from the day before the autopsy date. Rats were exsanguinated, and the following sampling was performed: BALF was collected from 5 males and 5 females from each group sacrificed on the first day and blood was collected from 5 males and 5 females from each group sacrificed the next day, as described below. For histopathological analysis, all tissues were collected from all of the rats in each group, and fixed in 10% neutral phosphate buffered formalin solution.
BALF collection and analysis
The left bronchus was tied with a thread, and the right lung was lavaged: 4–5 ml of saline was injected into the lung through the trachea, in and out twice, and collected as BALF. The total cell numbers in the BALF were counted using an automatic cell analyzer (ADVIA120, Siemens Healthcare Diagnostics Inc. Tarrytown, NY). Cell populations were prepared on glass slides using Cytospin 4 (Thermo Fisher Scientific, Inc., Waltham, MA). After May-Grunwald-Giemsa staining, differential white blood cell count was made by visual observation. BALF cytospin specimens were carefully examined under a microscope to classify the status of AMs phagocytosing TiO2 NPs. All AMs were divided into TiO2 NPs-laden AMs and normal AMs. The TiO2 NPs-laden AMs were then classified as Over-stuffed AMs, which had phagocytosed TiO2 NPs until the nucleus was no longer visible and Burst AMs, which were disintegrated into particles and cellular debris, and the number of each type of AM was counted.
The BALF was centrifuged at 1,960 rpm (800 × g) for 10 min at 4 °C, and the activity of LDH, ALP and γ-GTP, and the level of total protein and albumin in the supernatant was measured using an automatic analyzer (Hitachi 7080, Hitachi, High-Tech Corp., Tokyo, Japan).
Titanium burden analysis
To determine the lung burden of Ti in TiO2 NP-exposed rats, approximately 0.1 g of lung tissue was collected and weighed. The lung tissue was put into a glass vessel, treated with 3 mL of distilled water, 3 mL of sulfuric acid, and 1 mL of nitric acid at 270 °C for 1 h. Samples were then diluted to 30–50 mL with 3% sulfuric acid. The samples were further diluted 2 to 50 fold to keep the concentration within the calibration curve, and TiO2 concentration in the samples was determined by Zeeman atomic absorption spectrometry (Z-5010; Hitachi High-Tech Corporation, Tokyo, Japan) with a Hitachi High-Tech lamp for Ti (part#207–2012 Serial 0,490,158,100). Absorbance of the digested samples was detected at 364.3 nm. Quantification was performed using a seven point calibration curve prepared by diluting appropriate volumes of a 1000 mg/L stock solution (Kanto Chemical Co., Inc., Tokyo, Japan) to 0.025, 0.05, 0.1, 0.15, 0.2, 0.3, and 0.4 µg/ml. TiO2 concentrations were calculated from the corresponding molecular weight ratio of TiO2 to Ti. The values obtained were calculated as the amount of Ti per gram. The correlation between the lung burden and several toxicological markers was calculated using the Pearson correlation coefficient (Pearson’s r) using GraphPad Prism 5 (GraphPad Software, San Diego, CA).
Hematological and blood chemistry tests
For hematological examination, blood samples collected at the time of each autopsy were analyzed with an automated hematology analyzer (ADVIA120, Siemens Healthcare Diagnostics Inc. Tarrytown, NY). For biochemical tests, the blood was centrifuged at 3,000 rpm (2,110 × g) for 20 min, and the supernatant was analyzed with an automated analyzer (Hitachi 7080, Hitachi, Ltd., Tokyo, Japan).
Serial tissue sections were cut from paraffin-embedded lung specimens, and the first Sect. (2-μm thick) was stained with H&E for histological examination and the remaining sections were used for immunohistochemical analysis. The histopathological findings in this study were determined by certified pathologists from the Japanese Society of Toxicologic Pathology, based on terms adopted by International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice (INHAND). Pathological diagnosis was performed blindly by three pathologists and summarized. Each non-neoplastic lesion was evaluated for its severity and scored on a scale of “slight” to “severe” with reference to the criteria by Shackelford et al. .
Masson’s Trichrome staining
Details of this procedure have been described previously . Briefly, the slides were deparaffinized, washed with water, and then reacted with an equal volume of a mixture of 10% potassium dichromate and 10% trichloroacetic acid for 60 min at room temperature. The specimens were then washed with water and stained with Weigelt’s iron hematoxylin solution (C.I.75290, Merck-Millipore) for 10 min at room temperature. Specimens were then successively stained with 0.8% orange G solution (C.I.16230, Merck-Millipore) for 10 min at room temperature, Ponceau (C.I.14700, FUJIFILM-Wako Pure Chemical Corp., Osaka, Japan) acid fuchsin (C.I.42685, Merck-Millipore) azofloxine (C.I.18050, Chroma Germany GmbH, Augsburg, Germany) mixture for 40 min at room temperature, 2.5% phosphotungstic acid for 10 min at room temperature, and blue aniline solution (C.I.42755, Chroma Germany GmbH) under a microscope until color developed. Between each staining solution the slides were washed lightly with 1% acetic acid in water. Then, dehydration, permeabilization, and sealing were performed.
Elastica Van Gieson staining
Briefly, the slides were deparaffinized, washed with water, reacted with Maeda Modified Resorcinol-Fuchsin Staining Solution (Mutoh Chemical, Part No. 40321, Japan) for 30 min at room temperature, and rinsed with 100% ethanol to remove excess stain. The slides were then washed with running water, stained with Weigelt's iron hematoxylin solution (C.I.75290, Merck-Millipore, US) for 10 min at room temperature, and washed with running water for 10 min. The slides were then reacted with 1% Sirius red solution (Mutoh Chemical, Part No. 33061, Japan) for 3–5 min at room temperature, washed with water, dehydrated with 90%-100% ethanol, permeabilized, and sealed.
Immunohistological multiple staining analyses
Details of the multiple staining method have been described previously . Briefly, lung tissue sections were deparaffinized with xylene, hydrated through a graded ethanol series, and incubated with 0.3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. Slides were then incubated with 10% normal serum at room temperature (RT) for 10 min to block background staining, and then incubated for 2 h at RT with the first primary antibody. After washing with PBS, the slides were incubated with histofine simple stain rat MAX-PO (MULTI) (414,191, Nichirei, Tokyo, Japan) for 30 min at RT. After washing with PBS, slides were incubated with DAB EqV Peroxidase Substrate Kit, ImmPACT (SK-4103, Vector laboratories) for 2–5 min at RT. Importantly, after washing with dH2O after color detection, the sections were treated with citrate buffer at 98 °C for 30 min before incubation with the next primary antibody to denature the antibodies already bound to the section. This procedure was repeated for the second and then the third primary antibody. HighDef red IHC chromogen (ADI-950–142, Enzo Life Sciences, Inc., Farmingdale, NY) was used for the second coloration and Histogreen chromogen (AYS-E109, Cosmo Bio, Tokyo, Japan) for the third coloration. Coloration was followed by hematoxylin staining for 30–45 s. The slides were then processed for light microscopy. The sections were observed under an optical microscope ECLIPSE Ni (Nikon Corp., Tokyo, Japan) or BZ-X810 (Keyence, Osaka, Japan).
To perform various morphometric measurements on PDFs, only the 50 mg/m3 group of both sexes, which could ensure a sufficient number of PDF occurrences to be analyzed, were used in this study.
For measurement of Ki67 and γ-H2AX positive indices, the male and female 0 mg/m3 groups (n = 5) and the 50 mg/m3 groups (n = 5) were used for analysis. For the 50 mg/m3 exposure groups, positive indexes were counted separately for pulmonary dust foci (PDF) and tissue surrounding a lesion (SUR). In all animals, at least ten fields of view were measured using a 40 × objective lens. More than 500 LPCAT1-positive AEC2 per individual were measured for Ki67 and 1000 LPCAT1-positive AEC2 per individual were measured for γ-H2AX, and the mean value per individual was used for statistical analysis.
For the Tm4sf1 positive index in PDF and Agglomeration lesions, 50 PDF and 50 Agglomeration lesions were randomly selected from each 50 mg/m3 exposure group of each sex, and the percentage of TTF1/Tm4sf1 double positive AEP and TTF1-single positive AEC2 were measured.
Except for the incidence and integrity of histopathological lesions, the data comparisons among multiple groups were performed as follows: when homogeneous variance and normal distribution were observed in samples without sex differences, a one-way ANOVA was used to compare the exposure and control groups. When the one-way ANOVA was significant, Dunnett’s multiple comparisons test was used to compare the control and exposure groups. If variances were significantly different, the control and exposure groups were evaluated using Kruskal–Wallis non-parametric analysis of variance. If the Kruskal–Wallis analysis was significant, the control and exposure groups were compared using Dunn’s test. The samples with sex differences were analyzed by two-way ANOVA with Tukey’s multiple comparison test. All statistical analyses were using performed GraphPad Prism 5 (GraphPad Software). The incidences and integrity of lesions were analyzed by the chi-square test using GraphPad Prism 5 (GraphPad Software). All statistical significance was set at p < 0.05.