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Magnéli phases were synthesized using a tube furnace (diameter = 8.9 cm) with a heating and cooling rate of 5°C min⁻¹ and an N₂ atmosphere (flow rate = 0.28 m³ min⁻¹) as previously described (1). Heating and cooling processes were isothermal at the target temperature for 2 hours (h). The process includes heating pulverized coal with TiO₂ nanoparticles. Magnéli phases were produced using commercial P25 nanoparticles, which is a mixture of the 80% anatase and 20% rutile forms of TiO₂. Magnéli phase samples were characterized using a scanning transmission electron microscope operating at 200 kV and equipped with a silicon drift detector-based Energy Dispersive X-ray Spectroscopy (EDS) system as previously described (1).

All mouse studies were approved by the respective Institutional Animal Care and Use Committees (IACUC) at Virginia Tech and East Carolina University, and were conducted in accordance with the Federal NIH Guide for the Care and Use of Laboratory Animals. All studies utilized age and gender matched wild type C57Bl/6J mice.

BMDMs were harvested from mice using standard protocols (6). Briefly, harvested bone marrow cells were cultured for 6 days in Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% fetal bovine serum (FBS), L-Glutamine, 1 × penicillin/streptomycin, and 20% L929-conditioned medium. After this 6-day incubation, BMDMs were re-plated at 250,000 cells/well and were allowed to adhere to the plate for 24 h. Cells were then treated with different concentrations of Magnéli phase particles (0, 1, 10, 100, or 1,000 ppm) overnight. Following these treatments, cell-free supernatant was collected, and cytokine levels were assessed via enzyme-linked immunosorbent assay (ELISA). Lipopolysaccharide (LPS) was used at 1 μg/mL as a positive control for cytokine release. LDH activity was measured in the supernatant to assess cytotoxicity.

Following treatment with Magnéli phases, treatment media was discarded and BMDMs (1 × 10⁶ cells/well) were fixed with 2.5% Gluteraldehyde in 0.1 M Sodium Cacodylate directly in the cell culture plate for at least 1 h. Following fixation in 2.5% glutaraldehyde in 0.1 M Na cacodylate, samples were washed two times in 0.1 M Na cacodylate for 15 min each, post-fixed in 1% OsO₄ in 0.1 M Na cacodylate for 1 h and washed two times in 0.1 M Na cacodylate for 10 min each. Fixative was removed and cells were dehydrated with increasing concentrations of ethanol for 15 min each as follows (15, 30, 50, 70, 95, and 100%). The dehydration was completed with propylene oxide for 15 min. The samples were then infiltrated with a 50:50 solution of propylene oxide:Poly/Bed 812 (Polysciences Inc., Warrington, Pennsylvania, USA) for 6–24 h followed by complete infiltration with a 100% mixture of Poly/Bed 812 for 6–12 h. The samples were embedded in fresh 100% Poly/Bed 812 using Beem embedding capsules (Ted Pella Inc., Redding, California, USA) overnight. These were then placed into a 60°C oven for at least 48 h to cure and harden. The embedded samples were trimmed and thin (60–90 nm) sections were cut and collected on copper grids (Electron Miscroscopy Sciences). These sections were stained with aqueous uranyl acetate and lead citrate and examined and photographed using a JOEL JEM 1400 transmission electron microscope.

Oxygen Consumption Rate (OCR) of BMDMs after exposure to nanoparticles was determined using a Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies). Seahorse 96-well cell culture microplates were seeded at 100,000 cells per well and subjected to a 24-h incubation with Magnéli phases. Following the incubation, cells were washed with OCR assay media (1 mM pyruvate, 2 mM glutamine, and 10 mM glucose) twice and then immersed in a total volume of 180 μL of media in each well immediately before the assay. Plates were then placed in the XF96 to establish a basal level of respiration. Afterwards, three separate injections were performed: the ATPase inhibitor oligomycin (1 μM), the mitochondrial uncoupler carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP) (3 μM) and the mitochondrial electron transport chain complex III inhibitor antimycin A (2 μM), respectively. Each condition was measured three times before moving to the next injection. Data is represented as pmol of oxygen per minute.

Cells were grown following the conditions described above and plated at 100,000 cells per well in black walled, clear, flat bottom Corning Costar 96-well microplates. Images were collected in three different channels with a 60 × objective using Hoechst 33258 (100 nM) to counterstain nuclei, dichlorofluorescein (DCF) (100 μM) to monitor reaction to H₂O₂ injury, and tetramethylrhodamine, methyl ester (TMRM) (10 nM) to assess mitochondrial membrane potential. The following excitation/emission filters were selected to collect the fluorescence signal for each channel on the instrument; DAPI (Excitation 390.0 nm/18.0 nm bandpass, Emission 435.0/48.0 nm bandpass), FITC_511 (Excitation 475.0 nm/28.0 nm bandpass, 511.0 nm/23.0 nm bandpass), Cy3 (Excitation 542.0 nm/27.0 bandpass, Emission 587.0 nm/45.0 nm bandpass), respectively. Prior to the start of the imaging protocol, wells were treated with TMRM and DCF for 30 min, protected from light, in a 37°C/5% CO₂ incubator and washed twice with phosphate buffered saline (PBS). Sequential images were taken in 10 fields of view for each channel in each well containing cells. After a basal level of fluorescence was obtained for each field, all wells were treated with 500 μM H₂O₂ to induce injury and a second round of images were obtained following the same procedure. Images were analyzed in an automated fashion using GE's InCarta software version 1.6. Multiple parameters were collected from each plate by creating custom “masks” that incorporated the fluorescent target of interest. Data are expressed as the mean pixel value under the mask minus mean pixel value for the local background or, more simply, Intensity-Background.

ICP-MS analysis was performed on Agilent 7900 ICP-MS operating in helium collision mode to reduce/remove polyatomic interferences. Data was acquired in spectrum mode and utilized a 3-point peak pattern for quantitative analysis. Each data point acquisition had three replicates and utilized 250 sweeps per replicate. Titanium levels were monitored at both 47 and 49 m/z to additionally check for potential interferences at either value. The instrumental parameters for ICP-MS are shown in Supplemental Figure S2A.

Scandium (45 m/z) was used as an on-line addition internal standard. A calibration curve that ranged from 0.04 to 10 ppb titanium in solution was used to quantify titanium in the samples according to their response ratio with the scandium internal standard (Ti/Sc). An Anton-Parr Multiwave GO microwave system that utilized modified Teflon (PTFE-TFM) microwave vessels was used in the microwave digestions. This microwave oven provides feedback from the digestion conditions based on temperatures and whether excessive venting was detected.

Ultrapure water was purified with a Millipore MilliQ water purification system set at 18.2 MOhm. Single element stock solutions of 1,000 ppm titanium (Ti) and scandium (Sc) for ICP-MS analysis were obtained from Inorganic Ventures. Nitric acid (67–70 w/w%) and hydrofluoric acid (47–51 w/w%) were both trace metals grade and obtained from Fisher Scientific. Suprapur hydrogen peroxide (30 w/w%) was also trace metals grade and obtained from Merck. Lung tissue samples were stored in a freezer at −20°C until analysis. When ready, the samples were removed from the freezer and allowed to come to room temperature. The entirety of the provided samples was weighed directly into PTFE-TFM microwave vessels and ranged from approximately 200–300 mg.

One milliliter of ultrapure water (18.2 MΩ), 1.5 mL of 70% (w/w) nitric acid (HNO₃), 0.3 mL of 10% (w/w) hydrofluoric acid (HF), and 0.2 mL of 30% w/w hydrogen peroxide (H₂O₂) were added to each vessel. The vessels were then capped appropriately and loaded into the carousel of the microwave digestion system. The samples were digested using a temperature gradient that ramped from room temperature to 180°C over 20 min, then held at 180°C for 10 min before cooling back to 60°C over 10 min. All samples digested clear and colorless, with no visible particulates in solution.

Each of the digested samples was decanted into fresh, separate 15 mL polypropylene (PP) tubes, and their corresponding digestion vessels were rinsed with 2 mL of 2% (w/v) HNO₃ and decanted into their respective tubes for a 5 mL total volume. The concentrated samples were then vortexed and shaken to mix immediately before being diluted 1:100 in 2% (w/v) HNO₃ to ensure homogeneity of the dilute samples. The 1:100 dilution was necessary to reduce the total concentration of HF within the samples themselves so that it did not attack the quartz portions of the ICP-MS instrumentation.

Wild-type C57Bl/6 mice were treated with one dose of 100 ppm of Magnéli phases using i.t. administration, following anaesthetization with isoflurane. Mice were euthanized using CO₂ and lungs were harvested 7 days following i.t. administration. To assess chronic effects of Magnéli phases in the lung, mice were treated i.t. with 100 ppm of Magnéli phases three times per week. Lungs were harvested at 6 weeks after the initial treatment. Hematoxylin and eosin (H&E) stained lung slides were evaluated using darkfield microscopy (Cytoviva, Auburn, AL, USA) and images were collected at a magnification of 100X. Magnéli phase localization and lung histopathology were also evaluated by a board-certified veterinary pathologist using H&E stained sections prepared from formalin fixed, paraffin embedded tissues. Immunohistochemistry was conducted on the formalin fixed, paraffin embedded tissues using an APAF1 polyclonal antibody (Invitrogen; PA5-85121) and rabbit specific HRP/DAB (ABC) Detection IHC kit (Abcam).

Total RNA was harvested from whole lungs using FastRNA Pro Green Kit following manufacturer's protocols (MP Biomedicals). Total RNA was pooled from 3-8 individual mice per group for RT² Profiler PCR Array Platform (QIAgen) cDNA reaction. Gene expression was evaluated using PAMM-212Z and PAMM-052Z arrays (Qiagen) following manufacturer's protocols. The gene list for these arrays and functional categories are available through the manufacturer (Qiagen). Ingenuity Pathways Analysis (IPA) and the manufacturer's array software (Qiagen) was used to analyze gene expression data and conduct pathway analysis. IPA data were ranked and evaluated based on z-score as previously described by the research team (7–9).

Male C57Bl/6J mice were exposed to PBS or Magnéli phases three times/week for 6 weeks (18 total doses). Briefly, mice were anesthetized with isoflurane and received Magnéli phases (100 ppm) in 50 μl by i.t. administration as described above for the chronic exposure assessments. Pulmonary function testing was performed 24 h following the last exposure. All mice were anesthetized with tribromoethanol (TBE) (400 mg/kg), tracheostomized, and baseline pulmonary function was recorded using the FlexiVent system (SCIREQ, Montreal, QC, Canada) (10, 11).

Mice were ventilated with a tide volume of 10 ml/kg at a frequency of 150 breaths/min and a positive end expiratory pressure (PEEP) of 3 cm H₂O to prevent alveolar collapse. In addition, mice were paralyzed with pancuronium bromide (0.8 mg/kg) to prevent spontaneous breathing. Electrocardiogram (EKG) was monitored for all mice to determine anesthetic depth and potential complications that could have arisen during testing. A deep inflation perturbation was used to maximally inflate the lungs to a standard pressure of 30 cm H₂O followed by a breath hold of a few seconds to establish a consistent volume history. A snapshot perturbation maneuver consisting of a three-cycle sinusoidal wave of inspiration and expiration to measure total respiratory system resistance (R), dynamic compliance (C), and elastance (E). A Quickprime-3 perturbation, which produced a broadband frequency (0.5–19.75 Hz) over 3 s, was used to measure Newtonian resistance (Rn), which is a measure of central airway resistance. All perturbations were performed until three acceptable measurements with coefficient of determination (COD) ≥0.9 were recorded in each individual subject.

After three baseline measurements of lung function using “snapshot” and “Quickprime-3” perturbations, mice were challenged with aerosolized saline or cumulative doses of 1.5, 3, 6, 12, and 24 mg/ml methacholine (MCh) (Sigma-Aldrich, St. Louis, MO) generated by an ultrasonic nebulizer for 10 s without altering the ventilation pattern (10–12). These challenges were followed by assessments with “snapshot” and “Quickprime-3” perturbations every 30 s. Between each dose of methacholine, a deep inflation perturbation was initiated to reset lung hysteresis. For each animal, all perturbations were performed until six acceptable measurements with COD ≥0.9 were recorded.

All studies were repeated at least three independent times unless noted.

Single data point comparisons were evaluated by Student's two-tailed t-test. Multiple comparisons were evaluated for significance using Analysis of Variance (ANOVA) followed by either Tukeys or Newman–Keuls post-test, as appropriate. Graphs and statistical analyses were performed and generated using GraphPad Prism software 8 (GraphPad, San Diego, CA). All data are presented as mean ± the standard error of the mean (SEM) with p ≤ 0.05 considered significant. Data shown are representative of at least three independent studies.

In initial toxicity studies, Magnéli phases (Ti₆O₁₁) were found to be toxic to dechorionated zebrafish embryos at 100 ppm (1). Thus, we chose this formulation and dose as the focus of our subsequent studies. Utilizing previously described methods, we generated Magnéli phases that were predominately composed of Ti₆O₁₁ (1) (Figure 1A). The resultant nanoparticles were confirmed by electron microscopy analysis and X-ray diffraction patterns, as previously described (1) (Figures 1B,C). These nanoparticles have excellent light absorption in the near-infrared, UV, and visible light range (1). Likewise, the Magnéli phases also display a low amount of photocatalytic activity, as previously reported (1). The resultant nanoparticles used in our studies ranged in size from a few tens of nm to hundreds of nm (1).

Once inhaled, nanoparticles are rapidly phagocytosed by resident macrophages in the lungs, which represent the predominate cell type responsible for neutralization and clearance (13). To evaluate the effect of Magnéli phases on these cells, we generated primary bone marrow derived macrophages (BMDMs) using established protocols (13). The BMDMs were treated with varying doses of Ti₆O₁₁ Magnéli phases across a 0–1,000 ppm range and over a 24-h time course (Figures 1D–H). Transmission electron microscopy (TEM) assessments revealed Magnéli phases, concentrated in phagolysosomes within treated macrophages at all concentrations and timepoints evaluated (Figures 1D,E). Morphology assessments did not appear to show high levels of macrophage activation following phagocytosis. However, at 100 ppm, a significant number of macrophages demonstrated morphology consistent with increased cell death, specifically apoptosis (Figure 1F). Several features were noted, including cell shrinkage, loss of membrane integrity, and membrane blebbing (Figure 1F). To quantify cell death, we utilized trypan blue exclusion and manual counting using a hemacytometer. Here, we observed a dose dependent increase in cytotoxicity between 10 and 1,000 ppm, ranging from 19.75 ± 2.63% to 44.25 ± 4.787%, respectively (Figure 1G). The average cell death over the same 24-h time range in the mock treated macrophages was 16.25 ± 2.062% (Figure 1G). Macrophage cell death did not initiate immediately, requiring 24 h post-exposure to peak (Figure 1H). Using 100 ppm, cell death was assessed over a time course, with almost double the number of macrophages undergoing cell death at 24 h post-exposure (15.25 ± 0.957% mock vs. 31.25 ± 3.403% Ti₆O₁₁ Magnéli phase treatment) (Figure 1G). Together, these data show that Ti₆O₁₁ Magnéli phases, at levels observed in the environment as incidental nanoparticles generated through the industrial burning of coal, are cytotoxic to mammalian macrophages.

Nanoparticle exposure typically drives macrophage activation and the production of inflammatory mediators (13). The cells generated using the methods detailed here are skewed toward M1 polarized, pro-inflammatory macrophages (6). Likewise, the cell death induced by the Magnéli phases were originally predicted to generate damage associated molecular patterns, such as aberrant ATP production or Calcium efflux similar to that described for A549 cells (14), that should drive innate immune system signaling in the macrophages. Here, we monitored the generation of several potent pro-inflammatory mediators, including IL-1β, IL-6, and TNF (Figures 1I–K). Consistent with electron microscopy findings indicating apoptosis as the method of cell death and the lack of morphological features associated with macrophage activation, we did not observe any statistically significant increases in either gene transcription by rtPCR or at the protein level by ELISA for any pro-inflammatory mediators evaluated (Figures 1I–K). Indeed, at the gene transcription level, we observed several mediators and transcription factors actively down-regulated (Figure 2). These data indicate that cell death and inflammation induced by Magnéli phases are not linked. Despite higher levels of cytotoxicity, cell death and Magnéli phase exposure are not sufficient to significantly activate elements of the innate immune system associated with inflammation.

Previous studies have shown that nanoparticle phagocytosis by macrophages can impair their function, resulting in greater susceptibility to a myriad of pathogens (15, 16). To evaluate macrophage functionality 24 h following either mock or Magnéli phase exposure, live cells containing nanoparticles were sorted and co-exposed to fluorescently labeled E. coli. In macrophages lacking the Magnéli phases, we observed a significant increase in mean fluorescence intensity (MFI: 10,630 ± 290) compared to those containing the nanoparticles (7,826 ± 114.2) (Figure 1L). Thus, cells containing Magnéli phases were significantly less efficient at bacterial phagocytosis, implying that nanoparticle sequestration has a deleterious impact on macrophage function.

To better define mechanisms underlying Magnéli phase cytotoxicity, we profiled gene expression and utilized Ingenuity Pathway Analysis to decipher changes in gene networks and pathways associated with a range of biological functions in macrophages. BMDMs were collected 24 h post-exposure to 100 ppm Ti₆O₁₁. Total RNA was collected and gene expression was profiled using rtPCR based gene expression arrays (Qiagen). Changes in gene expression were defined as significant if the change was ±2-fold different between the mock and treated cells. Based on the pattern of gene expression changes, apoptosis signaling was the top canonical pathway that was significantly increased following Magnéli phase exposure (Figure 2; Supplemental Figure S1A). Individually, there were no clear networks or relationships identified between the top 10 dysregulated genes (Supplemental Figure S1B). However, analysis of the congregate gene expression patterns and networks revealed a strong relationship between genes associated with apoptosis signaling with a focus on mitochondria (Figure 2A). Here, we observed significant increases in gene expression associated with both pro-apoptotic BCL-2 family members, specifically Bax (Figure 2A). Up-stream analysis revealed a significant increase in p53 signaling and down-stream analysis revealed a significant increase in Apaf1 (Figure 2A). Consistent with these findings, Caspases-3,−6, and−9 were all found up-regulated following Magnéli phase exposure, while Caspase-7 was significantly downregulated (Figure 2A). We also observed a significant up-regulation in the gene encoding ICAD, which is associated with DNA fragmentation during apoptosis (Figure 2A). In addition to apoptosis, pathway analysis further revealed that inflammation and inflammatory forms of cell death, such as necroptosis, were significantly down-regulated following Magnéli phase exposure (Figures 2A,B). While we realize the general limitations of utilizing gene expression data to define cell death pathways, the data indicates significant changes in genes associated with apoptosis pathways and is consistent with the electron microscopy and inflammation data (Figure 1). Beyond cell death, very few additional pathways were found to be significantly impacted following Magnéli phase phagocytosis at the time points and conditions evaluated here, with a notable exception being a general downregulation of genes associated with NF-κB signaling (Figure 2A). This finding is consistent with the lack of inflammation and cytokine responses observed following exposure (Figures 1I,J). To better evaluate our findings compared to those previously reported for TiO₂ exposure, we compared our findings to gene expression patterns associated for TiO₂ in the IPA database (Supplemental Figure S1C). In general, many of the same pathways are altered for both Magnéli phases and TiO₂, including the down-regulation of Tnf and the up-regulation of Ifng (Supplemental Figure S1C). However, the major difference between Magnéli phases and TiO₂ is associated with Caspase-3/-7 regulation. Caspase-7 is down regulated in both cases; but, Caspase-3 is significantly up-regulated following Magnéli phase exposure and is central in the apoptosis pathways identified (Figure 2A; Supplemental Figure S1C). This is in contrast to the down-regulation of Caspase-3 and cell death mechanisms previously described following TiO₂ exposure (17). Together, these data suggest that macrophage cell death induced by Magnéli phase exposure occurs though the mitochondrial pathway of apoptosis and is associated with the activation of pro-apoptotic BCL-2 family members, including a potential p53-BAX-APAF1 axis.

Previous studies have revealed that nanoparticles can significantly impair mitochondria function and the convergence of signaling pathways on the mitochondria identified in the gene expression profiling and pathway analysis together warrant higher resolution functional assessments. We first utilized an Agilent Seahorse XF cell mitochondrial stress test to evaluate the oxygen consumption rate (OCR) in BMDMs treated for 24 h with either mock, 100 ppm, or 1,000 ppm Magnéli phases (Figure 3A). At both doses, the basal respiration and the maximal respiratory capacity (MRC) were significantly increased in BMDMs treated with Magnéli phases compared to mock (Figure 3A). Cells treated with 1,000 ppm of Magnéli phases did not show the typical decline in OCR after the addition of oligomycin (Oligo), a known ATP synthase inhibitor, indicating an increase in proton leak in the mitochondria (Figure 3A). Proton leak is typically associated with mitochondrial damage and stress (18, 19). Likewise, these cells only showed a minimal change in MRC following the addition of the proton ionophore FCCP (Figure 3A), which likely reflects the significant increase in cells undergoing cell death. The BMDMs treated with 100 ppm Magnéli phases showed the most dramatic shifts, with the highest level of basal respiration and the largest MRC increase, following FCCP treatment (Figure 3A). The impressive increase in MRC suggests that Magnéli phase exposure resulted in a substantially higher reserve capacity in BMDMs, which is also likely associated with the increased energy demands of the cells following increased damage, stress, and cell death. Complementing the OCR data, we also evaluated the extracellular acidification rate (ECAR), which is an assessment of glycolysis. While we did not observe significant differences in ECAR following exposure to Magnéli phases at 1,000 ppm, we did observe a significant increase following exposure to 100 ppm (Figure 3B). These data indicate a shift in the substrate utilization, likely due to increased cellular metabolism in cells treated with 100 ppm Magnéli phases. Together, these data indicate that Magnéli phase exposure results in altered mitochondrial bioenergetics that can significantly contribute to damage associated processes, such as reactive oxygen species (ROS) generation and the activation of cell death cascades.

Because proton leak is also a sign of mitochondrial damage and stress, we next investigated mitochondrial membrane potential (MMP), as decreases in MMP are associated with mitochondrial membrane damage and changes in mitochondrial function. The cell-permeant dye, tetramethylrhodamine (TMRM) accumulates in active mitochondria with healthy MMPs. A decrease in TMRM fluorescence indicates a loss of mitochondrial membrane potential. BMDMs treated with 100 ppm Magnéli phases had significantly lower TMRM fluorescence (86.26 ± 13.06 AU) compared to mock (613.60 ± 51.89 AU) (Figure 3C), indicating treatment significantly altered mitochondrial membrane potential. The subsequent addition of H₂O₂ to each sample was used as a positive control and resulted in significant decreased TMRM fluorescence compared to basal fluorescence in the mock treated cells, with a much smaller decrease in the cells treated with Magnéli phases (Figure 3C). Together, these data suggest that Magnéli phase exposure results in a significant decrease in mitochondrial membrane potential, and confirms severe mitochondrial membrane damage.

The production and modulation of ROS is a common biological response in macrophages to nanoparticle exposure, is associated with cytotoxicity, and is commonly involved in mitochondrial stress (20–22). Increased ROS production is a significant driver of cell death and stress following TiO₂ exposure (20). A previous study assessed ROS levels in A549 alveolar epithelial cell lines following TiO₂ and undefined Magnéli phase exposure (14). Under the conditions evaluated, the TiO_x nanoparticles increased intracellular Ca²⁺ that was associated with low levels of cytotoxicity, but no significant changes on ROS production were observed (14). Due to the inherent differences in nanoparticle responses between different cell types and the potent cytotoxic effects of dysregulated ROS production, we next evaluated this response following exposure to Ti₆O₁₁ nanoparticles in macrophages. For this we utilized another cell-permeant dye, 2′,7′-dichlorofluorescin diacetate (DCFDA), which is oxidized by ROS into the fluorescent DCF. BMDMs challenged with 100 ppm Magnéli phases for 24 h had a significantly higher level of ROS production (1,105 ± 94.17) compared to mock exposed cells (76.39 ± 7.22) (Figure 3D). Taken together, these data suggest that macrophages treated with Magnéli phases show increased ROS production and loss of mitochondrial membrane potential after 24 h. All of which are indications that these particles induce mitochondrial stress and are consistent with the induction of mitochondria-mediated apoptosis following Magnéli phase exposure.

In the only in vivo toxicity study conducted to date, Magnéli phases were shown to be bioactive and toxic to zebrafish at 100 ppm (1). These studies provided the first indication that these incidental nanoparticles could be biologically active and potentially toxic to a variety of animal species, including humans. However, the impact of Magnéli phases in mammals and the mechanism of toxicity were not explored (1). Thus, we next sought to extend these findings using mice and a respiratory exposure model. Mice were exposed to 100 ppm Ti₆O₁₁ Magnéli phases in 1 × PBS via intra-tracheal administration using methods previously described to evaluate nanoparticle toxicity and trafficking in the respiratory tract (13, 23). To quantify the concentration of Magnéli phases in the lungs over time, we utilized inductively coupled plasma mass spectrometry analysis to detect titanium levels (Figure 4A; Supplemental Figure S2A). Immediately following i.t. administration, tissues including the lungs, liver, spleen, and kidneys were removed, digested, and processed for ICP-MS. These data represent the maximum deposition control (Figure 4). As expected, no titanium was detected in tissues outside of the lungs or in any tissues that were not exposed to the Magnéli phases. However, in exposed lungs, we observed 14.46 ± 4.32 μg/g of lung tissue immediately after deposition (Figure 4A). Within 24 h post-exposure, titanium levels detected in the lungs dropped to 3.40 ± 1.19 μg/g of lung tissue (Figure 4A). However, surprisingly, after this single exposure to the Ti₆O₁₁ Magnéli phases, the levels of titanium in the lungs were maintained over the course of a week with levels holding steady at 5.30 ± 1.23 μg/g (Day 3) and 5.91 ± 1.30 μg/g (Day 7) (Figure 4A). Titanium levels were below the level of detection in all of the other tissues evaluated at the time points assessed (Supplemental Figure S2B). Together, these data emphasize the potential consequences of even a single exposure to Magnéli phases, which are retained in the lungs at significant levels for an extended duration following inhalation.

We next sought to identify where in the lungs the Magnéli phases were being sequestered. We initially utilized histopathology assessments of H&E stained lung sections to evaluate both nanoparticle deposition and pathology. Lungs were fixed by gravity inflation with formalin and paraffin embedded for staining as previously described (13, 23). Lung pathology was evaluated by a board-certified veterinary pathologist (S.C.O.). Control mice received either vehicle or were challenged with a 1 mg/ml dose of LPS (Figures 4B,C). As anticipated, lung histopathology was normal in the vehicle treated animals (Figure 4B). Conversely, we observed significant airway inflammation in the LPS treated animals, characterized by predominately macrophage and neutrophil infiltration concentrated around the airways and vasculature (Figure 4C). In the experimental animal groups, we did not observe significant evidence of inflammation in the mice exposed to Magnéli phases at 1, 3, or 7 days post-exposure (Figure 4D). However, Magnéli phases were readily observed as punctate foci within cells in the lungs under light microscopy conditions (Figure 4D). Further assessments at higher magnification revealed that the vast majority of Magnéli phases were sequestered in macrophages, with no nanoparticles observed under light microscopy conditions associated with or within any other cell type in the lung or observed in the vehicle control specimens (Figures 4B,D). To confirm the association of Magnéli phases with the macrophages in the lungs, we conducted enhanced darkfield microscopy (Cytoviva) (Figures 4E–G). A reference image of Magnéli phases was generated and used to identify the nanoparticles in vivo (Figure 4E). As anticipated, we did not detect any Magnéli phases in the vehicle control specimens (PBS) (Figure 4F). However, consistent with our pathology assessments, Magnéli phases were confirmed in macrophages throughout the lungs and were readily identified by their distinctive punctate staining (Figure 4G; green arrow). Together, these data show that Magnéli phases are effectively phagocytosed by lung-associated macrophages; however, the nanoparticles are not effectively cleared from the tissue, remaining at steady levels over the course of at least 7 days following a single exposure.

Based on our ex vivo findings that Magnéli phases are cytotoxic, we next evaluated the in vivo impact of nanoparticle sequestration on macrophages and lung tissue. Bronchoalveolar lavage fluid (BALF) was collected following euthanasia and cellularity was evaluated following differential staining. In both the PBS and Magnéli phase treated specimens, macrophages were the dominate population of cells recovered (Figure 5A). This is in contrast to the highly inflammatory conditions observed following LPS treatment (data not shown) or following exposure to nanoparticles with immunostimulatory properties, where neutrophils are the dominate cell type observed (24–27). Magnéli phases were readily identified in macrophages collected from the lungs of exposed mice at all time points evaluated, with 71 ± 10.16% of the macrophages containing at least 1 readily observable nanoparticle under 40 × light microscopy (Figure 5A). Consistent with our in vitro cytotoxicity data, many of the macrophages recovered from the airways demonstrated morphological features consistent with cellular stress and death (Figure 5A). The cell populations recovered in the BALF were further quantified using a hemocytometer (Figure 5B). Macrophages, neutrophils, and lymphocytes were recovered in the BALF from mice exposed to either PBS or Magnéli phases (Figure 5B). The quantity and populations of cells recovered in BALF are routinely utilized as surrogates to characterize lung inflammation (28). Compared to findings commonly reported following exposure to immunostimulatory agents, such as LPS, and consistent with the pathology evaluations shown in Figures 4B–D, the number and populations of cells recovered following exposure to Magnéli phases indicate that these nanoparticles do not induce significant lung inflammation (Figure 5B). Indeed, assessments of inflammatory mediator levels, such as IL-6, in the BALF following either Magnéli phase exposure or LPS exposure, further confirm these findings (Supplemental Figure S3A). Consistent with the increased cytotoxicity, we observed a significant decrease in the number of macrophages recovered in the BALF from mice exposed to Magnéli phases (Figure 5B). No other cell populations appeared to be impacted (Figure 5B). Together, these data indicate that airway exposure to a single dose of Magnéli phases at a range previously found in environmental samples following incidental nanoparticle release and cytotoxic to zebrafish embryos indeed facilitates macrophage cell death in the mammalian lung. Consistent with the apoptosis findings (shown in Figures 1, 2), neither in vivo macrophage cell death nor exposure to the Magnéli phases result in significant inflammation.

To better define the mechanisms underlying the in vivo response to Magnéli phase exposure, lung tissue was collected 7 days post-exposure. Total RNA was isolated and processed for gene expression profiling using the SuperArray platform and IPA, as previously described (7–9). The gene expression profiling analysis revealed significant changes in several pathways associated with cell death and lung function. The three top pathways predicted by IPA analysis to be significantly activated 7 days post-exposure to the Magnéli phases were (1) apoptosis—specifically apoptosis of monocytes; (2) formation of ROS; and (3) fibrosis. In all three cases the combination of genes up-regulated and down-regulated were predicted to have an activating effect on the pathways identified (Figure 5C). Also, of note, no pathways were predicted to be significantly inhibited based on the gene expression patterns evaluated and no pathways associated with inflammation were significantly modulated following Magnéli phase exposure. The predicted activation of apoptosis of monocytes and formation of ROS in vivo are highly consistent with the findings from our in vitro studies and further emphasize the detrimental impact that the Magnéli phases are having on the macrophage populations in the lungs following exposure. The fibrosis gene expression signature is intriguing. The 7-day timeframe of the exposure model is far too soon for lung fibrosis to present in the mice and no pathologic features associated with fibrosis were detected in our specimens by histopathology, collagen, or fibronectin assessments (Figure 4). It is possible that we are identifying gene signatures associated with fibrosis at a very early time in the lungs, prior to pathological disease onset. However, this gene expression pattern would also be consistent with increased regeneration and repair processes in the lungs, perhaps associated with the increased cell death.

In most cases of incidental nanoparticle release into the environment, biological exposure to Magnéli phases are expected to occur repeatedly and for an extended duration. Thus, we next sought to determine the impact of chronic airway exposure to the Ti₆O₁₁ nanoparticles over a 30-day time period. Under these conditions, we observed a significant increase in titanium concentrations in the lungs, which accumulated to levels that were ~6-fold higher (35.82 μg/g of lung) than levels observed after a single exposure (5.92 μg/g of lung) (Figure 6A). Similar to the findings from the single exposures, pathology assessments of the lungs again revealed that the majority of the Magnéli phases detectable under bright-field microscopy were sequestered in lung-associated macrophages (Figure 6B). Despite the increased concentrations of nanoparticles in the lungs, we did not observe any pathologic features associated with increased inflammation or differences in pro-inflammatory mediators, such as IL-6, IL-1β, or TNF levels, in the lungs (Figure 6B; Supplemental Figures S3A–F). Pathology assessments revealed no evidence of interactions with other cell types beyond the macrophages under bright-field microscopy. To better define nanoparticle-cell interactions, we evaluated specimens using enhanced dark field microscopy (Cytoviva) (Figure 6C). Consistent with our bright field data and similar to what we observed following the single exposure, the Magnéli phases are overwhelmingly concentrated in macrophages in the lung (Figure 6C). It should be noted that we did identify punctate staining associated with alveolar epithelial cells, indicating that under chronic exposure conditions, there are some interactions between other cell types in the lungs and the Ti₆O₁₁ nanoparticles (Figure 6C). However, compared to the macrophages, Magnéli phase interactions with these cells were more sporadic and involved far fewer nanoparticles per cell (Figure 6C). Using cells recovered following BALF and evaluated following differential staining assessments, the number of macrophages containing Magnéli phases per 100 cells was determined and used to generate a phagocytic index (29). Following the single exposure to Magnéli phases, we calculated the phagocytic index to be 71.14 ± 3.84 on day 7, with the majority of macrophages recovered containing >1 nanoparticle per cell (Figure 6D). However, following the multiple exposures, the phagocytic index significantly increased to 91.00 ± 2.17 on day 30, again with almost all macrophages recovered containing >1 nanoparticle per cell (Figure 6D). Together, these data are consistent with the Magnéli phases being sequestered in the macrophages. Following cell death, the nanoparticles likely continue to concentrate in the lungs as cells undergoing apoptosis and associated debris (including the Magnéli phases) are further phagocytosed by additional macrophages, creating a positive feedback loop.

Our gene expression profiling data indicated that this sequestration resulted in apoptosis. To further evaluate this, we conducted immunohistochemistry targeting APAF1, which is an important mediator in mitochondria mediated apoptosis and was significantly up-regulated at the gene expression level (Figure 2A). Immunohistochemistry revealed that Ti₆O₁₁ treatment resulted in significant increases in APAF1⁺ cells in the lungs. Consistent with previous data, endothelial cells have high levels of APAF1⁺ staining, which was observed in the Saline treated animals (Figure 6E). However, we did not observe any significant staining in other cell types, including macrophages (Figure 6E). In LPS treated lungs, we observed robust staining in multiple cell types in the lungs, including endothelial cells, epithelial cells, macrophages, and neutrophils (Figure 6G). However, in the Magnéli phase treated animals, we did not observe the same level of widespread staining (Figure 6F). Rather, the APAF⁺ cells were predominately limited to the endothelial cells and macrophages containing Magnéli phases (Figure 6F). Also, of note, only the macrophages containing Magnélli phases were APAF⁺. Together, these data are consistent with our other findings localizing the in vivo effects of the Magnélli phases to the macrophage compartment in the lungs.

Based on the data above, we predicted that exposure to Magnéli phases would negatively impact mammalian lung function. To directly test this hypothesis, we determined the impact of multiple exposures of Ti₆O₁₁ nanoparticles over a 30 day period of time on basal airflow and airway mechanics utilizing a computer-controlled small-animal ventilator with highly sensitive pressure transducers (Flexivent) to quantify airway opening pressures, volume, and airflow (10, 11). In addition to basal measurements, we also determined whether exposure to these Magnéli phases modulates lung pathophysiology and airway mechanics following exposure to the bronchoconstricting agent methacholine. To evaluate airway mechanics, a snapshot perturbation maneuver that consisted of a three-cycle sinusoidal wave of inspiration and expiration was used to measure total respiratory system resistance (R), dynamic compliance (C), and elastance (E). A Quickprime-3 perturbation, which produced a broadband frequency (0.5–19.75 Hz) over 3 s, was used to measure Newtonian resistance (Rn), which is a measure of central airway resistance. For the methacholine studies, after three baseline measurements of lung function, mice were challenged with aerosolized saline or increasing doses of 1.5, 3, 6, 12, and 24 mg/ml of methacholine, generated by an ultrasonic nebulizer for 10 s without altering the ventilation pattern, followed by assessments every 30 s (30, 31). Between each dose of methacholine, a deep inflation perturbation was initiated to reset lung hysteresis.

Consistent with our hypothesis, exposure to Magnéli phases resulted in significant increases in basal resistance and Newtonian resistance (Figures 7A,B). These data indicate that the accumulation of Ti₆O₁₁ nanoparticles significantly attenuates baseline lung function. The increased basal resistance and Newtonian resistance suggests that the airway diameter and lung volumes are reduced under baseline conditions. We also observed an increase in basal elastance and the converse decrease in compliance (Figures 7C,D). These findings reflect defects in alveolar and elastic tissue in the lungs and indicate that exposure to Magnéli phases results in increased lung stiffness and reduced lung expansion abilities. Beyond baseline assessments, we also sought to evaluate lung function following methacholine challenge, which is commonly used to induce smooth muscle constriction in the lungs. Despite the increased baseline resistance and Newtonian resistance, the lungs of mice exposed to Magnéli phases showed minimal increases in airway constriction following methacholine challenge (Figures 7E,F). This is in contrast to the unexposed animals, which demonstrated the anticipated dose dependent increases (Figures 7E,F). These data indicate that the lungs of these mice lack the capacity to properly function and are likely at maximum for resistance and Newtonian resistance. Methacholine challenge also results in increased elastance and decreased compliance in untreated mice (Figures 7G,H). However, similar to the resistance measures, Magnéli phase exposure resulted in minimal changes in elastance and dynamic compliance, indicating a stiffening of the lungs (Figures 7G,H). Together, these data are consistent with Magnéli phase exposure resulting in significant detrimental changes in lung mechanics and function.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.