We used nickel oxide (NiO), cerium dioxide (CeO2), titanium dioxide (TiO2), and zinc oxide (ZnO) as industrial nanomaterials in the present study. Commercially available NiO (US3355, US Research Nanomaterials, Houston, TX), CeO2 (Wako Chemical, Ltd. Japan), TiO2 (Rutile) (MT-150AW, Teyca Co. Ltd., Osaka, Japan), and ZnO (Sigma-Aldrich Co. LLC., Tokyo, Japan) were dispersed in 0.4 ml distilled water. The physicochemical profiles of these samples are shown in Table 4 [21, 39, 42-44]. We defined the toxicity of the chemicals as follows: the chemicals which induced either sustained inflammation, fibrosis or tumors were set as having high pulmonary toxicity, and the chemicals that did not induce any of those pathological lesions were set as having low pulmonary toxicity. Accordingly, NiO and CeO2 were classified as nanomaterials with high pulmonary toxicity [10, 21, 42], and TiO2 and ZnO were classified as nanomaterials with low pulmonary toxicity [21, 39, 44, 45].
Male Fischer 344 rats (9–11 weeks old) used for exposure to nanomaterials were purchased from Charles River Laboratories International, Inc., Kanagawa, Japan. The animals were kept in the Laboratory Animal Research Center of the University of Occupational and Environmental Health for 2 weeks with free access to a commercial diet and water. All procedures and animal handling were done according to the guidelines described in the Japanese Guide for the Care and Use of Laboratory Animals as approved by the Animal Care and Use Committee, University of Occupational and Environmental Health, Japan.
The NiO, TiO2, CeO2, and ZnO nanomaterials were suspended in 0.4 ml distilled water. Doses of 0.2 mg (equivalent to 0.8 mg/kg BW) or 1 mg (equivalent to 4 mg/kg BW) were administered to rats (12 weeks old) in a single intratracheal instillation. Each of the negative control groups received distilled water.
Animals following intratracheal instillation
In the exposure to the 4 different nanomaterials and the control, there were 5 rats in each group at each time point. Animals were dissected at 3 days, 1 week, 1 month, 3 months and 6 months after intratracheal instillation and the lung was divided into right and left lungs. Analysis of cDNA microarray and qRT-PCR was performed with the homogenized third lobe of the right lung, and histopathological evaluation was performed with the left lung inflated and fixed by 4% paraformaldehyde or 10% formaldehyde.
Total RNA extraction
The third lobes of the right lungs (n=5 per group per time point) were homogenized using a QIAzol lysis reagent with a TissueRupotor (Qiagen, CA, USA). Total RNA from the homogenates was extracted using a miRNeasy Mini Kit (Qiagen, CA, USA) following the manufacturer’s instructions. RNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., MA, USA), and the quality of the samples was analyzed by a Bioanalyzer 2100 (Agilent Technologies, CA, USA).
We used a 3D-Gene Rat Oligo Chips 20K (version 1.1) (Toray Industries, Tokyo, Japan), which could mount 20,174 genes, for the DNA microarray analysis. Total RNA extracted from the lungs of the 5 rats in the NiO-high dose group was mixed in equal amounts to make one sample, and that was amplified by the use of an Amino Allyl MessageAmp II aRNA Ampliﬁcation Kit (Ambion, Inc., CA, USA). The control group was treated in the same manner. The antisense RNA (aRNA) were labeled with Cy5, using Amersham Cy5 Mono-Reactive Dye (GE Healthcare, Buckinghamshire, UK), and the labeled aRNA were hybridized at 37℃ for 16 hours. The hybridization was performed according to the supplier's protocols . The chips were washed and dried, and then scanned in an ozone-free environment using a 3D-Gene Scanner 3000 (Toray Industries, Tokyo, Japan) and analyzed by use of 3D-Gene Extraction Software (Toray Industries, Tokyo, Japan). The digitalized ﬂuorescent signals provided by the above-described software were regarded as the raw data. All of the normalized data were globally normalized per microarray, such that the median of the signal intensity was adjusted to 25. The function of the enhanced expression genes was analysed by Database for Annotation Visualization and Integrated Discovery 6.8 .
Validation of gene expression data using quantitative real-time polymerase chain reaction
Total RNA extracted from the lungs at each observation point in each group were transcribed into cDNA (High-Capacity cDNATM Kit, Life Technologies, Tokyo, Japan). qRT-PCR assays were performed using TaqMan (TaqMan Gene Expression Assays, Thermo Fisher Scientific Inc., MA, USA) according to the manufacturer’s protocol. The Assays-on-Demand TaqMan probes and primer pairs were CXCL5 (Assay ID Rn00573587_g1), CCL2 (Assay ID Rn00580555_m1), CCL7 (Assay ID Rn01467286_m1), CXCL10 (Assay ID Rn00594648_m1), and CXCL11 (Assay ID Rn00788261_g1). All experiments were performed in a StepOnePlusTM Real-Time PCR Systems (Life Technologies, Tokyo, Japan). All expression data were normalized to endogenous control β-actin expression (Assay ID Rn00667869_m1).
Histopathology and immunohistochemistry
The obtained lung tissue, which was inflated and fixed with 4% paraformaldehyde or 10% formaldehyde under a pressure of 25 cm water, was embedded in paraffin, sectioned at a thickness of 4μm, and then stained with hematoxylin and eosin (H&E). The slides were assessed for histological changes (H&E stain) by a board-certified pathologist (New Histo. Science Laboratory Co., Ltd., Tokyo, Japan). The severity of the histological changes in the lung in the control and nanoparticle-exposed rats was scored as none (0), minimal (0.5), mild (1), moderate (2) or severe (3).
Upregulation of CXCL5, CCL2 and CCL7 was evaluated by immunostaining with rabbit anti-mouse CXCL5 polyclonal antibody (1:200 dilution, bs-2549R; Bioss Inc., Woburn, USA), goat anti-rat CCL2 polyclonal antibody (1:200 dilution, sc-1785; Santa Cruz Biotechnologies, Inc., CA, USA), and goat anti-mouse CCL7 polyclonal antibody (1:50 dilution, sc-21202; Santa Cruz Biotechnologies, Inc., CA, USA), respectively, using the lung tissue samples from the NiO-high dose group of 1 month after intratracheal instillation.
Statistical analysis was carried out using JMPR Pro software (JMP Version 14.2.0, SAS Institute Inc., NC, USA). P values <0.05 were considered to be significant. Dunnett’s tests were used appropriately to detect individual differences in the gene expression levels of each of the 5 chemokines between those exposed to the 4 nanomaterial samples and the controls. We assigned the toxicity of the exposure nanomaterials as being high or low according to the gene expression levels of each of the 5 chemokines of each sample (20 samples for both high and low toxicity at each time point), and analyzed the sensitivity and specificity for high toxicity at each time point to create the ROC curves and AUCs. Youden’s Index was used to determine the cut-off value. Youden’s Index was defined as follows: Youden’s Index = sensitivity + specificity-1, where the definitions of sensitivity and specificity are shown in Additional file 2 (Table S2) together with specific examples using a confusion matrix. In the evaluation using the combination of chemokine genes, the cases where the expression of at least one gene was equal to or higher than the cut-off value were defined as positive. Spearman's rank correlation coefficient was used to estimate the correlation between gene expression levels of CXCL5, CCL2, CCL7, CXCL10 or CXCL11 and the score of inflammatory cell infiltration of lung tissue.