Time-course Analysis of Pulmonary Inammation Induced by Intratracheal Instillation of Fine Crystalline Silica Particles

Background: Crystalline silica is an important cause of serious pulmonary diseases. The relationship between this hazard and various physicochemical parameters of crystalline silica has been studied for more than a century, and the toxic potential of silica is known to be associated with its surface properties. However, no in vivo dataset has yet been accumulated to clarify the relevance of this, especially regarding longer term effects. To provide new insights into these longer term effects, we performed single intratracheal instillation testing of ve different crystalline silicas and assessed the time-course changes in pulmonary inammation, lung burden, and thoracic lymph node loads. The tested silicas were prepared from two commercial products [Min-U-Sil5 (MS5, US Silica Company) and SIO07PB (SPB, Kojundo Chemical)] by using three different pretreatment steps: centrifugation (C), grinding (G), and surface dissolving (D). The ve types of silica particles—MS5, MS5_C, SPB_C, SPB_G, and SPB_D—were suspended in puried water and intratracheally instilled into male F344 rats. Bronchoalveolar lavage, lung burden analysis, and histopathological examination were performed 3, 28, and 91 days after instillation. Results: MS5 and MS5_C provoked similar biphasic inammation that differed in severity: MS5 induced granuloma generation 91 days after instillation, whereas the smaller particles of MS5_C elicited only macrophage aggregation. SPB_C induced severe and persistent inammation, whereas SPB_G and SPB_D induced only slight and transient acute inammation. All of the silicas were dose-dependently retained in the lung. Silicas that induced prolonged responses were translocated to the thoracic lymph nodes as early as 28 days after instillation. Those that induced transient inammation were removed effectively from the lung with very little lymph node retention. No quantitative association was identied between lung retention and physicochemical properties, although common features of the retained materials seemed to be reduced solubility,


Zeta potential and point of zero charge
The zeta potential of the silica suspension was measured by means of laser doppler electrophoresis with the same instrument used for the particle size measurement (Zetasizer). Each 2 mg/mL suspension was prepared and the point of zero charge was measured or estimated by using HCl to titrate the pH close to 1 (see Fig. 2).
The data were summarized as mean values of duplicate measurements.

Crystallinity percentage
It is well known that crystalline silica is easily deformed to amorphous silica by mechanical damage; therefore, we needed to determine the crystallinity percentages. Conventional XRD estimation methods that use amorphous halos and crystalline re ections are poor, because the halos of nanosized particles are too broad and weak to analyze. Crystallinities were therefore estimated by using internal standard samples (39). The values indicate mass-basis crystalline component percentages.
In advance, reference XRD data (a series of integrated intensities of 18 peaks) were obtained from a 1:1 powder mixture of standard reference materials of α-quartz (SRM 1878a) and α-alumina (SRM 676a) produced by the National Institute of Standards and Technology (NIST Gaithersburg, MD, USA), with respective certi ed purities in the crystalline phase of 93.7% and 99.02%. These reference data were then used to derive the unknown degree of crystallinity of a targeted crystalline silica sample from XRD data (the same series of peaks) collected from a 1:1 mixture of the α-quartz powder and NIST's α-alumina (SRM 676a).

Solubility analysis
Time-course changes in the solubility of the tested silica in saline or ALF were assessed (see Fig. 3). ALF was prepared by using the method described in a previous study (30). Ten milliliters of suspension (2.0 mg/mL) and 30 mL of ALF were mixed and shaken at 200 rpm. At each time point, 2.5 mL of the uid was sampled and ltered at 6000g for 30 min with an ultra lter (10,000 MW; VS2032, Sartorius Stedim Biotech GmbH, Göttingen, Germany) to separate the dissolved Si ions. Ultrapure water (1 mL) was then added to the ultra lter, and the samples were centrifuged at 6000g for 15 min; these steps were performed twice. HNO 3  Animal experiment F344/DuCrlCrlj rats (Charles River Laboratories Japan, Inc., Kanagawa, Japan; male, 12 weeks old) were used.
The animals were housed in barrier-system animal rooms maintained at 21 to 25 °C and a relative humidity of 40% to 70%, with 10 to 15 air changes each hour and a photoperiod of 12 h of light each day.
One vehicle control group (puri ed water) and three dose groups were set up for each silica. The dose levels of the MS5 derivatives were set at 0.67, 2, and 6 mg/kg, whereas the SPB derivatives were dosed at 0.22, 0.67, and 2 mg/kg: 10 rats per dose were used for each material. The doses were set according to the results of preliminary studies: a single intratracheal instillation study was performed by using MS5 and SPB_C. We con rmed that evident in ammation was inducible by 3 days post-instillation, and we then set the highest doses tolerable. Details of the intratracheal instillation procedure were given in our previous study (40). Brie y, with the guidance of a laryngoscope, each dose suspension or vehicle was intratracheally instilled via the oral cavity under iso urane anesthesia (Day 0). The rats' clinical condition was then examined daily, and body weights were measured once or more weekly. On Days 3, 28, and 91, the animals were anesthetized and then humanely killed by being bled from the abdominal aorta. BALF and lung burden analysis were then performed in half the animals in each group (n = 5), and histopathological examination was performed in the other half (n = 5).

BALF examination
Detailed procedures of the BALF examination were also given in our previous study (40). After euthanasia of each rat, the whole lung was lavaged twice with 7 mL of saline (n = 5). Note that, because of a technical error, the BALF could not be obtained from one rat 28 days after it had received MS5_C at 2 mg/kg group (n = 4).
Total cell counts (ADVIA 120 hematology analyzer, Siemens Healthcare Diagnostics, Tarrytown, NY, USA) were determined. Then smear slides were prepared and Giemsa stained, and a differential cell count of 200 cells was performed. Additionally, total protein content and lactate dehydrogenase activity were measured (Clinical Analyzer 7170, Hitachi Science Systems, Ibaraki, Japan).

Lung burden and lymph node load analyses
After the lavage, the SiO 2 contents of the lungs and thoracic lymph nodes (parathymic lymph node and bilateral posterior mediastinal lymph nodes) were analyzed. The lungs and lymph nodes were homogenized in 2 mL of ultrapure water (Milli-Q Advantage A10 Ultrapure Water Puri cation System, Merck Millipore, Billerica, MA).

Pathological examination
The lungs, trachea, parathymic lymph nodes, posterior mediastinal lymph nodes, liver, kidneys, spleen, and brain were removed. The lungs, liver, kidneys, spleen, and brain were weighed, and the relative weights were calculated on the basis of body weight. All organs or tissues were xed in 10% (v/v) neutral phosphate-buffered formalin solution. Para n-embedded specimens were prepared, sectioned, stained with hematoxylin and eosin, and examined histopathologically. The histological changes and terminology of the lung and lymph nodes underwent peer-review externally by two expert pathologists.

Statistical analysis
For body weights, organ weights, the results of the BALF examinations, lung burden and lymph node loads,

Scanning electron micrographs
We prepared eld-emission scanning electron microscopy (FE-SEM) images of the tested silicas ( Fig. 1). FE-SEM revealed that MS5, MS5_C, SPB_C, and SPB_G had clumping particulates, whereas the SPB_D particles fused with each other to such an extent that we could no longer identify representative particulate shapes or particle sizes (see following section.) Characterization of the tested silica particles Particle size The primary particle sizes of the tested silicas were calculated from the SEM images (MS5, MS5_C, SPB_C, and SPB_G) or from the speci c surface area (all the silicas) ( Table 1). Secondary particle sizes in the dose suspensions, as measured by dynamic light scattering, ranged from 58.8 to 481 nm in the following order: SPB_D << MS5_C < SPB_C < SPB_G << MS5. As intended, these sizes were considered to be in the respirable range (15).

Speci c surface area
The speci c surface area values, as measured by means of N 2 gas absorption, are shown in Table 1. The speci c surface areas of the tested silicas ranged from 6.1 m 2 /g to greater than 25 times more at 153.6 m 2 /g, in the following order: MS5 < SPB_C < MS5_C < SPB_G < SPB_D.
[Please place Table 1 here.] Zeta potential and point of zero charge We plotted a zeta potential titration curve, which re ects the distribution of surface acidic moieties (Fig. 2). MS5 and MS5_C, as well as SPB_C and SPB_G, had similar curves, implying that they had similar surface properties.
The titration curve of SPB_D differed slightly from those of the others but was fairly close in pattern to those of SPB_C and SPB_G. The pH points at which the zeta potential was zero (i.e. the point of zero charge) were calculated as 1.31 for SPB_G and 1.23 for SPB_D (Table 1). Those for MS5, MS5_C, or SPB_C could not be determined (<1), although they were estimated as the following order: MS5 ≈ MS5_C < SPB_C.

Crystallinity percentage
The mass-basis rates of the crystalline silica contents were calculated by using the X-ray diffraction (XRD) method (Table 1). Crystallinity percentages ranged from 59.1% to 89.0% in the following order: SPB_G < SPB_D < MS5_C < SPB_C < MS5.

Solubility
We determined the solubilities of the silica materials in arti cial lysosomal uid (ALF) and physiological saline (Fig. 3). MS5 was slightly soluble in either solution, whereas MS5_C was almost insoluble. This indicated that the insoluble compartment of MS5 was collected as MS5_C during the centrifugation pretreatment. Among the SPB derivatives, SPB_G was the most soluble in ALF and in saline; the solubilities of SPB_C and SPB_D to ALF were similar to each other, and that of SPB_C to saline was higher than that of SPB_D after long time mixing.

Animal experiment
Actual dose and initial lung burden The actual doses of the silica suspensions are shown in

Clinical signs and body weights
No mortalities or abnormal clinical signs were observed in either control animals or treated animals after instillation of any of the silica materials. No marked treatment-related body weight changes (Additional le 1) were noted in any of the control or treated animals throughout the observation periods.

Bronchoalveolar lavage uid (BALF) examinations
We examined the BALF cytological parameters (Fig. 4 BALF chemistry parameters, including total protein content and lactate dehydrogenase activity, showed responses similar to those of the cytological parameters (Additional les 2 and 3). No signi cant changes compared with in the controls were noted in any dose group at any time point in the SPB_G treatments.

Lung burden and lymph node load
We examined time-course changes in lung burden and thoracic lymph node loads ( Thus the silica particles that induced only transient in ammation-namely, SPB_G and SPB_D (see Fig. 4)were removed from the lung effectively via pathways other than lymph node drainage, whereas those that induced prolonged in ammation-namely, MS5, MS5_C, and SPB_C-tended to remain in the lung for at least 91 days after instillation, during which period they were slowly translocated to the thoracic lymph nodes.

Pathological examination
Organ weight We determined absolute and relative (g/100 g body weight) lung weights (Figs. 6 and 7). With administration of the MS5 derivatives, absolute and relative lung weights were signi cantly increased in the 6 mg/kg groups on Day 3 compared with in the controls, and the absolute weight of animals receiving 2 mg/kg of MS5_C was also

Histopathological examination
Histopathological ndings in the lung are summarized in Table 3, and representative changes are shown in Figures 8 and 9 and Additional le 4. On Day 3, in ammatory cell in ltration was observed in the high-dose MS5 and MS5_C groups (Fig. 8A, 8B). By Day 28, the in ammatory responses had tended to recover: slight alveolar macrophage aggregation only was seen (Fig. 9A, 9C); this was also the case on Day 91 for MS5_C (Fig.   9D). However, in the MS5-treated group, nodular granulomas had been generated by Day 91 (Fig. 9B). Among the groups that received SPB derivatives, moderate in ammatory cell in ltration and degeneration and necrosis of alveolar macrophages were observed in the high-dose SPB_C group on Day 3 (Fig. 8C). The in ammation induced in the SPB_C group remained active on Days 28 and 91 and was accompanied by macrophage degeneration (Fig. 9E, 9F, arrowheads) and type II cell hyperplasia. Only slight to mild in ammatory cell in ltration was observed in the high-dose SPB_D group on Day 3 (Fig. 8E), and no marked changes were noted in the SPB_G groups (Fig. 8D). No neoplastic lesions were noted in any silica group at any of the doses at any time point.
[Please place Table 3 here.] Histological ndings in the posterior mediastinal lymph nodes are summarized in Table 4, and representative changes are shown in Figure 10 and Additional le 5. Granuloma formation was seen in the MS5, MS5_C, and SPB_C groups on Day 28 and thereafter (Fig. 10). Severity and incidence tended to increase over time (Table 4).
No abnormal changes were noted in the SPB_G or SPB_D groups.
[Please place Table 4 here.] No toxicologically relevant lesions were noted in the other organs or tissues examined at any time point (Additional les 6 8).

Discussion
Crystalline silica is a cause of the most prevalent occupational disease in mining industry workers, and numerous toxicological studies have been conducted over more than a century (6,8). The toxic mechanism has not yet been concluded although, some predictors has been gured out until now. The hazard potential is known to associated with the surface hemolytic activity (9,10), and the hemolytic activity is associated with the surface silanol distribution (16). Turci et al. (12) estimated that there is heterogeneity of the surface silanol acidic sites, and that this is re ected in the behavior of the zeta potential titration curve. Generally, in water suspensions, silica particles are negatively charged. As the pH value decreases, the zeta potential of homogeneous silanol silica sharply approaches zero (12). In contrast, that of heterogeneous quartz silanol gradually moves toward zero, and these heterogeneous silanol surfaces are a more potent cause of cellular stress in macrophages (12). Accordingly, here, we examined the zeta potential behavior of ve silica suspensions. Although we were unable clearly to distinguish their silanol disorder statuses, we surmised from the zeta potential patterns that the surface properties of the tested silicas differed between the MS5 derivatives and the SPB derivatives.
MS5-one of the silica products most frequently tested in toxicological research (6,8)-is well known to cause biphasic in ammation, consisting of an early-stage response and later granuloma formation, in rats (17).
These granulomas, which are similar to those seen in silicosis, can be induced in rats by inhalation of, or intratracheal exposure to, MS5 (18)(19)(20), as occurred in our study. Interestingly, our results revealed that granuloma formation in the lung was suppressed by centrifugation pretreatment: MS5_C did not cause granuloma formation in this organ. The zeta potential analysis revealed that the surface properties of MS5 and MS5_C were similar to each other, so the contributions of their surface silanols are presumably comparable. In the lymph nodes however, granulomatous changes were detected in both the MS5-and MS5_C-treated groups.
This is because the lymphatic system is a major route of excretion of exogenous materials, as determined by toxicity studies (21). Especially in the case of crystalline silica, Friedetzky et al. (22) have reported that lymph node silicosis, which is characterized by disorganization within the lymph nodes, occurs before the pulmonary lesions. Correspondingly, here, the amount of translocated MS5_C was much less than that of MS5 (see Fig. 5), and this was con rmed by histological grading (Table 4 and Fig. 10). This implies that pulmonary granulomas are generated when a silica accumulates beyond the capacity or threshold of the draining lymph nodes.
Because the initial lung burdens of MS5 and MS5_C were comparable to each other, less MS5_C than MS5 was translocated to the lymph nodes. One possible cause of the difference is the occurrence of another clearance pathway from the lung, namely the mucociliary escalator (23). Mucociliary transport occurs predominantly for undissolved and relatively small particles (24). It is conceivable that the less soluble and smaller MS5_C was excreted to a much greater extent than MS5 via the mucociliary system. Therefore, our results suggest that e cient mucociliary excretion leads to suppression of the pulmonary granuloma formation induced by MS5 derivatives; moreover, a key nding in MS5 derivative toxicity is lymph node accumulation.
Among the SPB derivatives, SPB_G and SPB_D were excreted rapidly from the lung with only minor translocation to the lymph nodes, but SPB_C was not rapidly excreted. As mentioned above, e cient mucociliary transport-a main excretion pathway other than the lymphatic system-may be involved in excretion, predominantly for undissolved and relatively small particles. Accordingly, the rapid excretion of SPB_D-the smallest and less soluble particles-seemed reasonable, although those of SPB_C and SPB_G were less straightforward. These two silicas were of comparable particle sizes, but SPB_C was less soluble than SPB_G (see Fig. 3). Therefore, SPB_C could be expected to be excreted effectively via the mucociliary escalator, but in fact it was predominantly excreted to the lymph nodes (see Fig. 5). One possible mechanism of the reduced mucociliary clearance is the serious macrophage toxicity that was speci cally detected in SPB_Ctreated animals. Mucociliary excretion is triggered by macrophage engulfment of foreign bodies (24,25), but in SPB_C-treated animals the macrophages showed moderate to severe degeneration or necrosis, or both, with decreasing numbers (see Fig. 4). In this situation, macrophage function cannot be maintained. The mechanism by which silica is toxic to alveolar macrophages is triggered by lysosomal membrane destabilization and rupture, which eventually causes cell death and necrosis (26)(27)(28). Pavan and Fubini (29) have reported that this membrane destabilization is caused by surface silanol heterogeneity. In light of this hypothesis, macrophage toxicity should also occur in SPB_G-treated animals, because SPB_C and SPB_G have similar surface properties (see Fig. 2). However, SPB_G induced barely detectable neutrophil in ltration in the BALF examination. Another marked difference between the properties of SPB_C and SPB_G was their solubility in ALF: SPB_G was much more soluble (see Fig. 3). This may have contributed to the detoxi cation of SPB_G. Previously we reported in an intratracheal instillation study that soluble NiO particles were rapidly excreted from the lung via the urine and digestive tract (30). NiO instillation was accompanied by severe pulmonary in ammation, possibly because of the toxic potential of Ni + per se, but in the case of silica instillation the dissolved ion form is regarded as non-toxic (31,32). Thus, SPB_G may be disorganized in the lysosomes and then excreted without translocation to the lymph nodes, possibly via the digestive tract or in the urine. In contrast, SPB_C was aggressively translocated to the lymph nodes, despite the severe macrophage toxicity. It is uncertain whether the lymph node accumulation was the cause or the result of macrophage toxicity, but notably SPB_C-a silica that induced prolonged toxicity-was also accumulated in the lymph nodes.
Taken together, our results showed that a key nding in prolonged silica toxicity was translocation to the thoracic lymph nodes. Unfortunately, some dose groups did not show time-dependent translocation, so unlike in our previous work (30) we were unable to determine a translocation constant. This may have been because of the low clearance rates of MS5 and SPB_C. Comparison of the properties of the effectively excreted silicas (SPB_G and SPB_D) and the poorly cleared ones (MS5, MS5_C, and SPB_C) revealed a distinct difference in two parameters, namely the point of zero charge and the crystallinity (see Table 1). The points of zero charge of the safer two silicas were able to be determined (SPB_G, 1.31; SPB_D, 1.23), but they were incalculable for the others, in the following order: MS5 ≈ MS5_C < SPB_C < 1. This may re ect differences in the surface properties under acidic conditions, such as intralysosomal after engulfment by macrophages, and it may thus be one possible predictor of toxicity. However, considering the severity and speci city of the macrophage toxicity in the SPB_C treatments, the contribution of properties associated with the point of zero charge is likely to be limited.
The toxicity of amorphous silica has not yet been clari ed, although mechanical damage such as grinding can cause partial conversion of crystalline to amorphous silica in the surface layer of the particles, thus weakening toxicity (29). Here, conversion seemed to have occurred to some extent in SPB_G, as suggested by the low crystallinity percentage. However, the amorphous component was not concentrated in the surface layer of SPB_G, because that of the alkaline-treated SPB_D was not dramatically increased but remained comparable.
Moreover, the intensity of the in ammatory response did not correspond to the crystallinity order. Therefore, crystallinity was unlikely to have contributed to the toxic potential.
Additionally, we analyzed the contribution of other quantitative parameters-initial lung burden, lung burden on the basis of secondary particle size, and lung burden on the basis of speci c surface area-to BALF neutrophil numbers on Day 3 (Fig. 11). Neutrophil numbers showed signi cant increases in the case of all the tested silicas (see Fig. 4): the BALF and initial lung burden data were obtained from identical animals so as to enable us to precisely analyze the dose responses. Seemingly, neutrophil in ltration was dependent on mass-basis (mg/kg) dose levels (Fig. 11A) rather than on a secondary particle basis (mg/kg/nm) (Fig. 11B) or speci c surface area basis (m 2 /kg) (Fig. 11C) in the MS5, MS5_C, SPB_G, and SPB_D groups. Notably, SPB_C induced excessive neutrophil in ltration, and the neutrophils may have been recruited by factors different from those for the others silicas, such as cytokines leaked from degenerated or ruptured alveolar macrophages (33).
Both absolute and relative weights increased dose-dependently in the cases of MS5, MS5_C, SPB_C, and SPB_D treated animals on Day 3. Notably, in our study, no treatment-related body weight losses occurred (Additional le 1), so the absolute and relative weight values tended to be aligned with each other. Porter (19) also reported that lung weight increased with inhalation of MS5, re ecting the pulmonary in ammatory status. Wahlström et al. (34) reported the usefulness of lung weight measurement in inhalation toxicity studies, and they showed some correlation between the weight increases (fold change) and histological in ammatory changes, including perivascular in ammation, macrophage in ltration, alveolitis, pneumonitis, and pneumonia. Our results also indicated that lung weight responded closely to acute pulmonary in ammation (Day 3) as detected in the BALF examination and histopathology. Additionally, signi cant increases of the lung weights remained in the SPB_Ctreated groups on Days 28 and 91-with persist in ammation. Notably, on Day 91, MS5-treated animals showed neutrophil in ltration and even protein leakage into the alveoli (Fig. 4 and Additional le 2), indicating the presence of pulmonary edema with granuloma formation, but the lung weight change was not signi cant.
Nevertheless, on a fold-change basis, the absolute lung weights of the low-, medium-, and high-dose MS5treated animals were 1.02, 1.04, and 1.10 times those of the corresponding controls. Therefore, lung weight may be useful as a nonspeci c indicator of the in ammation induced by intratracheal instillation.
In terms of dosing method, care needs to be taken in conducting instillation studies and in interpreting their results. Instillation bypasses the upper respiratory tract, so coarse particles that would normally be ltered out in the nasal cavity, can reach the alveoli. This therefore leads to excessive deposition compared with that in real-life inhalation exposure (13). Also, the dose rates we used here were very much higher than the slow buildup seen in occupational settings (35). Therefore, instillation data cannot be used directly in human risk assessment, and the method must be regarded as a hazard identi cation method (5). Although real-life dose rates cannot be replicated in intratracheal instillation studies, our intention was to apply nely dispersed silica suspensions, all of which were within a respirable size (i.e. secondary particle size < 0.5 μm) (15). Notably, silica does not induce toxic lesions in the nasal cavity, so our nasal bypass delivery method does not raise concern about underestimation of the effects on the upper respiratory tract. Regarding the dose levels, the silica concentration of lung tissue para n blocks from silicosis patients has been reported at 2.24 mg/mg lung (36).
The highest dose applied here was 6 mg/kg, in the high-dose MS5 and MS5_C groups; this was converted to about 1.25 mg/lung (calculated from a provisional body weight of 250 g at dosing, and an absolute lung weight of 1 g)-much less than in the silicosis patients. Therefore, the dose levels that we used here were not excessive. An additional limitation of instillation studies has recently been raised. Holzwarth et al. (37) reported that some instillation studies are not able to achieve nominal dose amounts because of the unexpected retention of dosing syringes; the authors pointed out that numerous misleading results have been published.
We agree with this perception, and we therefore believe that it is very important to analyze the lung burden at all time points, as we did here. We believe that our data, taken together, are su ciently reliable and will help to further understanding of the toxicity of crystalline silica.
Our reliable intratracheal instillation study revealed variable hazard pro les of crystalline silica. Additionally, we revealed that translocation to the lymph node is important for predicting silica toxicity by using a commonly tested MS5 derivative. Furthermore, to the best of our knowledge, this is the rst report to show variable SPBderivative toxicity. Although the main drivers of the translocation from lung to lymph nodes have not yet been clari ed, our data suggested that silica solubility, surface properties, and particle size may be contributors. To gain a comprehensive understanding of silica toxicity we need further, well-planned investigations.

Conclusions
Here, we evaluated the comparative toxicities of ve different silica particles, including a classical MS5 derivative and novel SPB derivatives. Variable toxicity pro les were noted, and the key nding of translocation to the lymph nodes, which involved prolonged pulmonary toxicity, was observed. Although further knowledge needs to be accumulated for a comprehensive understanding of these complex silica toxicities, we believe that our results give valuable insights, not only into the mechanisms of silica toxicity but also into the design of future tests by particle toxicologists.

Declarations
Ethics approval and consent to participate All the in vivo experiments were preapproved by the Institutional Animal Care and Use Committee of our laboratory. All procedures were conducted in compliance with the guidelines for the animal experiment at the test facility.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.      Figure 1 Scanning electron micrographs of tested silica particles.

Figure 2
Page 24/32 Zeta potential titration curve of silica particles. Zeta potential was measured in duplicate and mean values were plotted.    Absolute lung weights (g) on Days 3, 28, and 91 after instillation. *: P < 0.05 (Dunnett's test); **: P < 0.01 (Dunnett's test) compared with vehicle control. Representative histopathological changes in the lung 3 days after intratracheal instillation of silicas.
Arrowheads: degenerated or necrotic alveolar macrophages. Hematoxylin and eosin stain.   Dose-response analysis of BALF in ltrating neutrophil numbers on Day 3. Neutrophil numbers were plotted to compare the initial lung burden; lung burden on a secondary particle size basis-horizontal line of B shows (lung burden [mg/kg] / secondary particle size [nm]); and lung burden on a speci c surface area basishorizontal line of C shows (lung burden [mg/kg] × speci c surface area [m2/mg]).