2.1 Zinc oxide nanoparticles. ZnO-NPs (MKN-ZnO-020) with primary diameter of 20 nm were purchased from mkNano (Mississauga, ONT, Canada). The surface area was measured by Brunauer-Emmett-Teller (BET) (Macsorb HM model-1201, MOUNTECH, Tokyo, Japan). Endotoxin analysis was conducted using Pierce LAL Chromogenic Endotoxin Quantification Kit (Thermo Scientific, Waltham, MA). A biocompatible dispersion medium (DM) was used to disperse the nanoparticles, which was Ca2+- and Mg2+-free phosphate buffered saline (PBS, pH 7.4), supplemented with 5.5 mM D-glucose, 0.6 mg/ml mouse serum albumin, and 0.01 mg/ml 1, 2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) [19, 20]. The nanoparticles were dispersed in the DM to prepare a suspension with a concentration of 0.25 µg NP/ml DM for the low-dose group (10 µg/mouse), another with a concentration of 0.75 NP µg/ml DM for the high-dose group (30 µg/mouse), and a suspension with only DM, without nanoparticles, for the control group. The particles were dispersed using a cup-type sonicator (Branson Sonifier, cup horn), at 100 W, 80% pulse mode for 10 minutes. The hydrodynamic size was determined using dynamic light scattering (DLS) (Zetasizer Nano-S; Malvern Instruments, Worcestershire, UK).
2.2 Animals.Nrf2−/− female mice were generated as described by Itoh et al. [21] and backcrossed six times at the Central Institute for Experimental Animals (Kanagawa, Japan) and then further backcrossed seven times at the Division of Experimental Animals, Nagoya University Graduate School of Medicine (Nagoya, Japan). The genotypes of mice were confirmed by PCR amplification of genomic DNA isolated from the tail. PCR amplification was carried out using three different primers, 5#-TGGACGGGACTATTGAAGGCTG-3# (Nrf2-sense for both genotypes), 3#-GCCGCCTTTTCAGTAGATGGAGG-5# (Nrf2-antisense for wild-type), and 5#-GCGGATTGACCGTAATGGGATAGG-3# (Nrf2-antisense for LacZ). Another 24 pathogen-free age-matched C57BL/6JJcl female mice (Nrf2+/+) weighing 22-27 g were purchased from CLEA Japan Inc. (Tokyo). All mice were housed and acclimatized in a clean environment for 1 week before the start of exposure experiments. Food and water were provided ad libitum. The animal room was light- and temperature-controlled with a 12-h light-dark cycle (lights on at 9 am and off at 9 pm), room temperature of 23-25°C and relative humidity at 57-60%. One day before the start of the experiment, mice of the two genotype groups were weighed and divided at random into three exposure groups (n=8 each); the control (0 µg ZnO-NPs), low-dose (10 µg ZnO-NPs) and high-dose (30 µg ZnO-NPs) groups. The latter two selected exposure doses are equivalent to 0.5 or 1.5 mg/kg body weight. The lower concentration of 0.5 mg/kg is comparable to deposition of 0.48 mg/kg in adult human lung from inhalation to ZnO for one week at the threshold limit value of 2 mg/m3 (time-weighted average), as proposed by the American Conference of Governmental Industrial Hygienists (ACGIH), based on the values of 500 mL air/breath, 12 breath/min, 40 h/week [22].
The guide of the Japan Government Laws concerning the protection and control of animals, and the guide of animal experimentation of Nagoya University School of Medicine were followed throughout the experiments. The experiment protocol was approved by Nagoya University Animal Experiment Committee.
2.3 Pharyngeal aspiration of ZnO-NPs. Pharyngeal or oropharyngeal aspiration is proved to be an effective convenient alternative to inhalation exposure for the hazard assessment of nanomaterials [23]. For this purpose, the mouse was first anesthetized by intraperitoneal injection of pentobarbital, then suspended with a rubber band anchored around the upper incisors and placed on its back on an inclined board. ZnO-NP suspensions were vortexed for 10 seconds first, then the tongue was gently extended outside the oral cavity using blunt forceps, and 40 µl aliquot of the selected concentration was pipetted into the back of the tongue, which was pulled for 1 minute after pipetting then released. With the tongue protruded, the mouse was unable to swallow, and the liquid trickled down slowly into the lungs. Following release of the tongue, the mouse was gently lifted off the board, placed on its left side, and monitored for recovery from anesthesia.
2.4 Bronchoalveolar lavage (BAL), total and differential cell counts. Fourteen days after exposure, the mice were euthanized by intraperitoneal injection of a lethal dose of pentobarbital. The trachea and lungs of each mouse were exposed and bronchoalveolar lavage was conducted. For this purpose, an 18-gauge needle was inserted into the trachea and both lungs were lavaged by 1 ml of 10% PBS (gentle instillation and aspiration). The instillation and aspiration of PBS was repeated 5 times, making a total volume of 5 ml. The amount of recovered bronchoalveolar lavage fluid (BALF) was measured and recorded. The average volume of the retrieved fluid was >90% of the instilled; the amounts and recovery rates were not different among the three exposure groups. The collected BALF was kept on ice until centrifuged at 1500 rpm for 5 minutes, and the supernatant was aliquoted into three tubes and kept at –80°C until further analysis. The cell pellets were re-suspended in 1 ml of ACK lysis buffer (for red blood cells lysis) and left for 5 minutes at room temperature. Then 10 ml of 10% PBS were added and the whole volume was re-centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and the cell pellet was re-suspended in 1 ml 10% PBS and kept on ice for use to determine the total and differential cell counts. Total cell count was determined using a ChemoMetec Nucleocounter (Allerød, Denmark), while differential cell count was performed under optical microscope on slides prepared by cytospin and stained with May-Grunwald-Giemsa (Merck, Darmstadt, Germany). The BALF cell types included macrophages, neutrophils, lymphocytes and eosinophils. The relative differential counts were presented as percentages of total cells counted in 10 fields of each cytospin smear. The absolute differential count was calculated as the product of the number of the total cell count and the proportion of the relative differential count.
2.5 Measurement of total protein in BALF. Total protein in BALF was measured using a Bio-Rad protein assay kit according to the instructions provided by the manufacturer (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin (BSA) as a standard.
2.6 Histopathological examination of the lung. After completion of BAL, the lungs were removed, washed in saline and the right lung was immediately frozen for further analysis. The left lung was fixed in 4% formalin, dehydrated with graded alcohol concentrations, embedded in paraffin, cut into 3 µm-thick sections, placed on slides, stained with hematoxylin and eosin (H&E) and examined under optical microscope by a pathologist blinded to the exposure. These lung sections were used to determine the degree of lung inflammation. The degree of peribronchial and perivascular inflammation was evaluated on a subjective scale of 0-3, as described previously [24–27]. A score of 0 represented no detectable inflammation, while score of 1 represented occasional cuffing with inflammatory cells. For score 2, most bronchi or vessels were surrounded by a thin layer (1-5 cells thick) of inflammatory cells. For score 3, most bronchi or vessels were surrounded by a thick layer (>5 cells thick) of inflammatory cells. Total lung inflammation was defined as the average of the peribronchial and perivascular inflammation scores. Four lung sections per mouse were scored and the inflammation score was expressed as the average value. Tissue slides were examined under an optical microscope (model DM750, Leica Microsystem, Wetzlar, Germany) and images were captured with Leica Application Suite V3 software.
2.7 Quantification of total glutathione and oxidized glutathione. The frozen lung tissue samples were homogenized with 5 volumes (w/v) of cold 50mM MES buffer (pH 6.01) containing 1mM EDTA. The protein in each sample was denatured with equal volume of 0.1% metaphosphoric acid (Sigma-Aldrich) and mixed on a vortex mixer. The mixture was allowed to stand at room temperature for 5 min and then centrifuged at 2000 x g for 3 min. The supernatant (95 µl) was kept at -20ºC until used for determination of total glutathione and oxidized glutathione (GSSG). First, 90 µl of supernatant was treated with 4.5 µl of 4M triethanolamine (Sigma-Aldrich) solution and vortexed well before assay. For the analysis of total reduced form of glutathione (GSH), 30 µl TEAM-treated sample was diluted 20-fold with MES buffer (pH 6.0) containing 2mM EDTA. An aliquot (50 µl) of the diluted solution was treated with 150 µl freshly prepared assay cocktail and assayed at 405 nm with a microplate reader (Gen5™ & Gen5 Secure, BioTek® Instruments, Inc.). For GSSG determination, 30 µl of TEAM-treated sample was diluted 10 times with MES buffer before derivatization with 2-vinylpyridine., Two µl of 1M 2-vinylpyridine was added to 200 µl of diluted solution of every sample or GSSG standard in tube, and then the tubes were mixed on a vortex mixer and incubated for 1 h at room temperature. Total GSH and GSSG concentrations were calculated from a standard curve using GSSG (Cayman; 703014) prepared according to the GSH assay kit (Cayman Chemical Company, Ann Arbor, MI; #703002), and normalized versus protein concentration. Total GSH and GSSG were expressed in micromoles of GSH (or GSSG) per milligram of protein.
2.8 Malondialdehyde assay. The malondialdehyde (MDA) assay (Life Science Specialties, LLC; NWK-MDA01) was performed according to the protocol supplied by the manufacturer. A 10% wt/vol homogenate was prepared from lung tissue in cold Assay Buffer (Phosphate buffer, pH 7.0 with EDTA). Absorbance was read at 532 nm using a PowerScan4 microplate reader (DS Pharma Medical Co., Japan) after reaction of the sample with thiobarbituric acid (TBA). Samples were analyzed in duplicate, and MDA level was expressed in micromoles of MDA per milligram of protein.
2.9 RNA isolation and real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The mRNA expression level of Nrf2-dependent genes; superoxide dismutase 1 (SOD1), catalase (CAT), glutamate-cysteine ligase catalytic subunit (GcLc), glutamate-cysteine ligase modifier subunit (GcLm), NAD(P)H quinone oxidoreductase (NQO1), heme-oxygenase 1 (HO-1) and glutathione reductase (GR), and metal-binding protein genes; metallothioneins (MT-1 and MT-2), which protect against oxidative stress, and proinflammatory cytokines; tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), transforming growth factor beta (TGF-β), interleukin-6 (IL-6), interleukin-1beta (IL-1β), monocyte chemotactic protein-1 (MCP-1), chemokine (C-X-C motif) ligand 1 (CXCL1, KC) and chemokine (C-X-C motif) ligand 2 (CXCL2, MIP-2), fibrosis-related gene: matrix metalloproteinase 2 (MMP2) were measured in lung tissues. About 15 mg of frozen lung tissue from each animal were homogenized and total RNA was extracted using ReliaPrepTM RNA Tissue Miniprep System, treated with DNase (Promega, WI) and kept at -80ºC until used. The RNA concentration was determined with a Nanodrop-1000 3.5.1 (Thermo Fisher Scientific, Waltham, MA). The quality of isolated RNA was assessed by calculating the A260/A280 ratio to ensure values between 1.7 and 2.0. For complementary DNA (cDNA) synthesis, SuperScript III Reverse transcriptase kit (Life Technologies, Carlsbad, CA) was used. The collected cDNA was kept at -30ºC until quantified by quantitative real-time PCR using AriaMx Real-Time PCR System (Agilent Technologies, Inc., Santa Clara, CA) and THUNDERBIRD SYBR qPCR Mix (Toyobo Co., Osaka, Japan) for KC, MIP-2, IL-6, IL-1β and MCP-1 or using Mx3005P QRCP System (Agilent Technologies, Waldbronn, Germany) and Universal ProbeLibrary System Assay (Roche Diagnostics) for the other genes. The mRNA expression levels were normalized to β-actin for each gene. The sequences of the primers used in this study are shown in Supplementary Material (Table S1).
2.10 Statistical analysis. Data were expressed as mean ± standard deviation. Differences between the control and exposure groups were examined using Dunnett`s multiple comparison method following one-way ANOVA or Steel multiple comparison method following Kruskal Wallis nonparametric test in each genotype. To test a trend with level of exposure to ZnO-NPs, simple regression analysis or simple ordinal logistic regression analysis on the exposure level of ZnO nanoparticles was applied in each genotype separately. Multiple regression analysis or multiple ordinal logistic regression analysis using dummy variables for genotype in full model was applied to examine effect of interaction between genotype and exposure level. When the interaction between genotype and exposure level was not significant, multiple regression analysis on exposure level and genotype in a non-interaction model was applied to test the effects of exposure level and genotype.
Statistical analysis was performed using the JMP software version 16 (SAS Institute, Cary, NC) and probability (p) value <0.05 was considered statistically significant.