Abstract
To study the acute and subacute lung toxicity of low dose of ultrafine colloidal silica particles (UFCSs), mice were intratracheally instilled with 0, 0.3, 3, 10, 30 or 100 μg of UFCSs. Cellular and biochemical parameters in bronchoalveolar lavage fluid (BALF), histological alteration and the body weight were determined at 3 days after instillation. Exposure to 30 or 100 μg of UFCSs produced moderate to severe pulmonary inflammation and tissue injury. To investigate the time response, mice were instilled with 30 μg of UFCSs and sacrificed at intervals from 1 to 30 days post-exposure. UFCSs induced moderate pulmonary inflammation and injury on BALF indices at acute period; however, these changes gradually regressed until recovery during the experiment. Concomitant histopathological and laminin immunohistochemical findings generally correlated to BALF data. TUNEL analyses in UFCSs-treated animals showed a significant increase of the apoptotic index in lung parenchyma at all observation times. 8-OHdG expression occurred in lung epithelial cells and activated macrophages, which correlated to lung lesions in UFCSs-treated mice. These findings suggest that instillation of a small dose of UFCSs causes transient acute moderate lung inflammation and tissue damage. Oxidative stress and apoptosis may underlie the lung tissue injury induction.
Keywords
Introduction
Colloidal silica, one of synthetically made amorphous silica that became widely used in many industries and available for various applications, is known to be far less active in producing pulmonary damage when compared to crystalline silica (Warheit et al., 1995). Subchronic inhalation toxicity studies in rats on colloidal silica reported that inhalation exposure of 50 mg/m3 (2418 μg/lung) or 150 mg/m3 (7378 μg/lung) Ludox® colloidal silica, 2.9–3.7 μm of mass median aerodynamic diameter (MMAD) ranges, produced transient pulmonary inflammatory responses. The severity and incidence of pulmonary lesions decreased progressively after a 3-month recovery period. The no-observable-effect level (NOEL) was 10 mg/m3 (489 μg/lung) (Lee and Kelly, 1992; Warheit et al., 1991).
Many toxicological studies made it clear that ultrafine particles (diameter < 100 nm) of various types can cause lung inflammatory responses, epithelial cell hyperplasia, inhibit phagocytosis, increased chemokine expression, lung fibrosis, increased oxidant-generating abilities, and lung tumors (Brown et al., 2000; Donaldson and MacNee, 2001; Warheit, 2004). Ultrafine particles have been shown to have a greater inflammatory lung responses and the development of particle-mediated lung diseases than the fine particles per given mass (Li et al., 1999; Nemmar et al., 2003). In our previous study, we have described acute pulmonary pathological effects caused by single intratracheal exposure to colloidal silica and compared the size effects in light and electron microscopy in mice. Our results showed that intratracheal instillation of high dose (3 mg/lung) of colloidal silica caused severe acute pulmonary inflammation and tissue injury; ultrafine colloidal silica particles (UFCSs) induced more severe changes than fine colloidal silica particles (FCSs). The UFCSs used in our study had a primary particle diameter of 14 nm and 10–150 nm of particle size distribution ranges in the lung. Moreover, the surface area of UFCSs was almost fifteen times greater than FCSs (Kaewamatawong et al., 2005). The increased toxicity of ultrafine particles can be related to their greater surface area per given mass, high number concentration, surface property, chemical composition and unique deposition in the lung (Brown et al., 2001; Jaques and Kim, 2000; Oberdorster, 2001; Pandurangi et al., 1990). Moreover, reactive oxygen species (ROS) also play an important role in ultrafine particle-induced pulmonary damage (Gilmour et al., 1997).
The aim of this study was to clarify biological and pathological events of intratracheally instilled low dose of UFCSs on the lungs of mice during the acute and subacute stages using bronchoalveolar lavage techniques and histopathological evaluations. In addition, factors that could be important in the induction of pulmonary toxicity of UFCSs were investigated with the use of immunohistochemistry.
Materials and Methods
Experimental Animal
Male ICR mice, weighing 35–38 g and 7–8 weeks of age, were purchased from CLEA Japan, INC. The mice were housed in an animal facility under 12/12 hr light/dark cycle, temperature of 24 ± 1°C, relative humidity of 55 ± 10% and negative atmospheric pressure. They were provided with mouse chow and filtered tap water ad libitum throughout the experiment. All animal experiments were performed according to the National Institute for Environmental Studies Guidelines for Animal Welfare.
Particles
Ultrafine colloidal silica (Grade PL-1) was obtained as a gift from Fuso Chemical Co., Ltd., Japan (Lot No. R2Z007). The silica particles had a spherical configuration with a relatively uniform size distribution averaging approximately 14 nm. The surface area, which measured by Brunauer, Emmett and Teller (BET) method, was 194 m2/g. The UFCSs had very low levels of metal compositions (potassium, aluminium, magnesium < 0.01 ppm; iron < 0.005 ppm; sodium = 0.05 ppm; calcium < 0.01 ppm) (Kaewamatawong et al., 2005). The silica particles were suspended in water at a concentration of 120 mg/ml and then sterilized at 0.5 kgf/cm2 and 120°C for 20 min.
Experimental Design
Dose response
Sixty mice were divided randomly into 6 groups of 10 animals each. The mice were anesthetized by intraperitoneal (IP) injection of 3 mg/kg xylazine (2% solution, Celactal™; Bayer, Tokyo, Japan) and 75 mg/kg ketamin (Ketalar® 50, Sankyo, Tokyo, Japan). 50 μl aqueous suspensions of 0.3, 3, 10, 30 or 100 μg of UFCSs suspended in 0.01 M phosphate-buffered saline (PBS) were instilled intratracheally via a small canule, followed by 0.1 ml of air. After intratracheal instillation, the mice were kept in an upright position for 15 min to allow the fluid to spread throughout the lung. The control groups of mice were instilled with 50 μl of 0.01 M PBS. At 3 days after instillation, the body weights were collected and the animals in each group were sacrificed for pulmonary tissue or BALF sample collections. The body weights of mice after instillation with UFCSs 0.3 and 10 μg significantly decreased on day 1, and then increased from 2 days after instillation. In mice exposed to 30 and 100 μg UFCSs, body weight was significantly decreased compared to controls throughout 3 days post-exposure.
Time effect
Eighty mice were divided randomly into 10 groups of 7–9 animals each. The mice were intratracheally instilled with 50 μl of 30 μg of UFCSs suspended in 0.01 M PBS as described above. The control groups of mice were instilled with 50 μl of 0.01 M PBS. Animals in each group were killed at 1, 3, 7, 15 and 30 days after instillation, respectively. Body weights were collected daily from 1 day after exposure through the end of the post-exposure period. A significant decrease in body weight was noted in the exposure groups compared to the control groups from 1 day after instillation, and then recovery occurred over the next 5 days post-exposure.
Bronchoalveolar Lavage
The trachea was exposed with a midline incision and cannulated with a modified 21 gauge needle. Bronchoalveolar lavage (BAL) was accomplished by 3 × 1 ml sterile 0.01 M PBS. The recovery of BALF for each mouse was measured, and the recovery rate was calculated. The average fluid recovery was greater than 90%. The BALF was centrifuged at 8,000 rpm for 8 min at 4°C and the supernatants were stored at −80°C until analysis.
Biochemical and Cytological Evaluation of BALF
The first tubes of BAL supernatant were used to measure total concentration of protein using the micro-BCA method (Pierce Chemical Co. Rockford, IL). BALF cells were quantified by hemocytometric counting, and cell viability was determined by exclusion of tryphan blue dye. Cell differentiation was evaluated for BALF pooled from each mouse by Diff-Quik stain (Sysmex, Kobe, Japan).
Histopathology
After gross examination of respiratory organs such as lungs and hilar lymph nodes, the lungs and trachea were removed en bloc and instilled with 10% buffered neutral formalin. Whole lungs and hilar lymph nodes were processed according to routine histological techniques. After paraffin embedding, 3 μm sections were cut and stained with hematoxylin and eosin (H&E) and Azan stain.
Immunohistochemistry
Laminin
Tissue sections from lung were immunostained by using avidin-biotin complex (ABC) method, in which labeled Streptavidin biotin (LSAB) kit (DAKO, Glostrup, Denmark) was included. After deparaffinization of the sections, the sections were treated with proteinase K for 30 min at 39°C. The sections were incubated with 3% H2O2 to quench endogenous peroxidase for 15 min at room temperature and then with 10% normal goat serum for 5 min in microwave oven 250 w to inhibit nonspecific reactions. Thereafter the sections were reacted over night at 4°C with Rabbit anti-laminin monoclonal antibody diluted 1:200 (DAKO, Glostrup, Denmark). The peroxidase conjugated goat anti-rabbit IgG diluted 1:400 (DAKO) was reacted to sections as a secondary antibody in microwave oven 200 w for 7 min. The positive reactions resulted in brown staining with the substrate 3,3′-diaminobenzidine tetrahydrochloride (DAB), and the sections were counterstained with hematoxylin for 30 sec.
8-hydroxy-2-deoxyguanosine (8-OHdG)
After deparaffinization and rehydration, sections were pretreated with 3% hydrogen peroxide in absolute methanol for 10 min and then washed with PBS. Non-specific background was eliminated by incubating the sections with Reagent 1A (ZYMED® LAB-SA SYSTEM HISTOMOUSE™–PLUS KITS) for 30 min and rinsed well with distilled water (DW). The section were incubated with Reagent 1B (ZYMED® LAB-SA SYSTEM HISTOMOUSE™–PLUS KITS) for 10 min and rinsed well with DW and PBS. 8-OHdG monoclonal antibody (Japan Institute for the Control of Aging, Shizuoka, Japan) (1:200 in PBS) was incubated for 1 hr on the tissue. The section were washed with PBS and incubated with biotinylated secondary antibody, Reagent 1C (ZYMED® LAB-SA SYSTEM HISTOMOUSE™–PLUS KITS) for 30 min and then washed with PBS. Streptavidin-peroxidase, Reagent 2 (ZYMED® LAB-SA SYSTEM HISTOMOUSE™–PLUS KITS) was then added to the section for 20 min. The sections were washed with PBS. The peroxidase activity was visualized by addition of substrate-chromogen solution, Reagent 3 (ZYMED® LAB-SA SYSTEM HISTOMOUSE™–PLUS KITS). Peroxidase will catalyze the substrate (hydrogen peroxidase) and convert the chromogen (AEC) to a red deposit, which demonstrates the location of the antigen. The section were rinsed in DW and counterstained with hematoxylin, Reagent 4 (ZYMED® LAB-SA SYSTEM HISTOMOUSE™–PLUS KITS) for 30 sec.
Detection of Apoptosis
The lung and hilar lymph node tissues fixed in 10% neutral buffered formalin and embedded in paraffin were processed for a Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) assay. Apop Tag® Peroxidase In situ Apoptosis Detection Kit (Chemicon International, Temecula, CA) was used following the manufacturer’s instruction. Briefly, the slides were pretreated with proteinase K, applied to H2O2 and incubated with the reaction mixture containing Terminal Deoxynucleotidyl Transferase (TdT) and digoxigenin-conjugated dUTP for 1 hr at 37°C. Labeled DNA was visualized with peroxidase-conjugated anti-digoxigenin antibody with DAB as the chromogen and then counterstained the slides with hematoxylin for 30 sec. Apoptotic cells were identified by TUNEL in conjunction with characteristic morphological changes, such as cell shrinkage, membrane blebbing, and chromatin condensation, to distinguish apoptotic cells and apoptotic bodies from necrotic cells. For each sample, 1000 cells/organ were examined and scored at a magnification of ×400. The results represented apoptotic index indicating percentages of cells affected by apoptosis.
Statistical Analysis
Data were expressed as means ± standard error (SE). Statistical analysis was evaluated using Student’s t-test. Differences between means were regarded as significant at p value of less than 0.05.
Results
Dose Response
Bronchoalveolar lavage fluid analysis
Table 1 presents the results for cellular and biochemical constituents in BALF after instillation of various doses of UFCSs. The total cell numbers in BALF were significantly increased after 3 days post-exposure for 10, 30 and 100 μg UFCSs-exposed groups. Cell differential analyses of BALF of mice exposed to 30 and 100 μg UFCSs demonstrated significant increases in the numbers of neutrophils and lymphocytes at 3 days after instillation. Total protein value in lung lavage fluid is considered to be a sensitive marker of alterations in the permeability of alveolar-capillary barrier. All exposure groups showed significant increase in total protein values in BALF above controls following a 3-day post-exposure.
Histopathology
Histopathological changes (Data not shown) induced by intratracheal instillation of 30 or 100μg at 3 days post-exposure showed moderate to severe focal alveolitis especially at the terminal bronchiolar and alveolar duct regions. In the foci, infiltration and accumulation of numerous particle-laden alveolar macrophages (AMs), neutrophils and fewer lymphocytes were observed. Increasing numbers of active AMs and neutrophils in alveolar spaces were also observed together with swelling and regenerative hyperplasia of type II epithelial cell. The other lower doses (0.3, 3 and 10μg) of UFCSs induced similar histopathological patterns. However, the lesions were milder and occupied a small area of the lung specimens.
Time Effect
Bronchoalveolar lavage fluid analysis
Following instillation of 30 μg UFCSs, the numbers of total cells, macrophages, neutrophils and lymphocytes in BALF at 1, 3, 7, 15 and 30 days post-exposure showed a transient increase pattern and then returned toward control levels (Table 2). Total numbers of lung cells were significantly increased and persisted up to 15 days before returning to control levels by 30 days post-exposure. Significantly elevated numbers of AMs were evident at 1 day after instillation and remained significant increased for over 7 days. The numbers of neutrophils increased markedly 1 day after instillation, followed by a further increase over 3 days. Lymphocytes showed a changing pattern different from the other inflammatory cells. Significant differences over corresponding controls of lymphocytes were gradual increase over the 7 days of the experiment and then returned to control levels by 30 days post-exposure (Table 2).
The concentrations of total protein in BALF in UFCSs-treated mice were greater than those of control animals at 1 day after exposure and gradually returned to control levels at 15 days post-exposure (Table 2).
Histopathology
In the controls, no significant lesions were observed in all time points (Figure 1A). By contrast, at 1 day after instillation, moderate increases of neutrophils sharply demarcated from the remaining normal alveoli were noted in UFCSs treated groups. A number of nodular aggregates of neutrophils and particle-laden AMs were observed in some alveolar regions adjacent to the bronchioles. The nodular lesions consisted of neutrophils, active AMs, particle-laden AMs and some cell debris (Figure 1B). Thickened alveolar wall with increased number of inflammatory cells was also seen. By 3 days after instillation, moderate focal alveolitis characterized by accumulation of numerous particle-laden AMs, neutrophils and fewer lymphocytes was observed at the terminal bronchiolar and alveolar duct regions (Figure 1C). Alveolar septal walls surrounding the foci were thickened with occasional type II epithelial cell regenerative hyperplasia. Particle-laden AMs and neutrophils infiltration into bronchial associated lymphoid tissue (BALT) was also observed. Changes in the lungs of mice killed at 7 days after UFCSs instillation were restricted to the appearance of the aggregated foci consisting of particle-laden AMs, lymphocytes and fibroblasts with occasional deposition of minimal volume of collagen fibers (Figure 1D). The lesions were located around blood vessels adjacent to terminal bronchioles and alveolar ducts. Peribronchiolar and perivascular lymphoid proliferation was present. The hilar lymph nodes were slightly enlarged associated with accumulation of particle-laden macrophages and hyperplastic histiocytes in subcapsular and medullary sinus.
At 15 days post-exposure, the inflammatory foci in lung parenchyma were markedly reduced in number and more focally concentrated, which were characterized by loose accumulation of particle-laden AMs, lymphocytes and fibroblasts with increased collagen fiber deposition (Figure 1E). Some alveolar walls enclosing to the foci were thickened due to hyperplasia of type II epithelial cells and interstitial accumulation of macrophages. Peribronchiolar and perivascular lymphoid tissues which were activated at the acute stage showed inactive structure. In 30 days after instillation, inflammatory lesions of lung and lymph node were almost recovered except for slight thickening of alveolar septal walls with some areas of interstitial fibrosis (Figure 1F).
Immunohistological Evaluation
Laminin
Thin string-like lines of intense brown positive stains were observed in bronchial basement membrane, blood vessel basement membrane, around bronchial gland and along alveolar septa of mice from control groups (Figure 2A). Lung tissues from UFCSs-treated mice at 1, 3 and 7 days after instillation showed extensive patchy areas of non-stain or weak positive reaction; the basement membranes of the alveoli in site of the inflammatory foci showed interruptions with a patchy distribution of the immunoreactivity (Figure 2B). Lung tissues from UFCSs-treated mice at 15 and 30 days after instillation showed mostly similar positive pattern as in the controls. However, brown faint discontinuous lines were observed in the areas of alveolar wall thickening (Figure 2C).
TUNEL assay
Table 3 presents the percentage of apoptotic positive cells in bronchiolar epithelium and lung parenchyma of five animals belonging to each experiment groups. No significant differences in the apoptotic index in bronchial epithelial cells between control and UFCSs-exposed groups except for transient increase at 3 days after instillation. Whereas the average numbers of apoptotic cells in lung parenchyma of exposure animals were significantly elevated above the controls at all time points examined.
8-OHdG
In control lungs, the immunohistochemical staining of 8-OHdG was barely detectable in both airway epithelium and lung parenchyma (Figure 3A). By contrast, in the UFCSs-treated mice at 1 day post exposure, positive staining for 8-OHdG appeared in a large number of cells associated with pulmonary inflammation. 8-OHdG was expressed mainly in the cytoplasm and partially in the nuclei of bronchiolar epithelial cells, alveolar epithelial cells and activated AMs (Figure 3B). After 3 and 7 days, 8-OHdG was observed chiefly in macrophages located around the sites of focal alveolitis but minimally in bronchiolar and alveolar epithelial cells (Figure 3C). At 15 and 30 days post-exposure, a cytoplasmic expression of 8-OHdG was only present in a small number of alveolar epithelial cells (Figure 3D).
Discussion
The purpose of this study was to determine the biological and pathological effects of intratracheally instilled low dose of UFCSs on the lungs of mice in terms of dose and time response during the acute and subacute stages. To study dose response, we exposed mice to different doses of UFCSs and investigated the changes in body weight, inflammatory cellular and biochemical parameters in BALF, and histopathology at 3 days after instillation. The results showed that instillation of 30 or 100 μg UFCSs produced moderate to severe pulmonary effects consistent with the development of lung injury, as evidenced by increased total cells, together with increases in leukocyte counts in BALF. This was accompanied by change in alveolar permeability, as measured by total protein in lavage fluid. To investigate time effect, bronchoalveolar lavage fluid analysis was carried out from 1 day through 30 days after a single instillation of 30 μg UFCSs. The effects of UFCSs on bronchoalveolar lavage indices suggested that UFCSs induced moderate pulmonary inflammation and injury at an early stage, but this effect gradually decreased during the experiment. Concomitant histopathological findings generally correlated to BALF data, showing moderate pulmonary inflammation and injury characterized by infiltration of neutrophils and active AMs, focal alveolitis, particle-laden AMs accumulation, and thickened alveolar wall with occasional regenerative hyperplasia of type II epithelial cells at day 1, 3 and 7 after instillation. However, the lung lesions were milder at 15 days and almost recovered at the final time point.
Subchronic inhalation toxicity studies in rats demonstrated that exposure to colloidal silica at the concentration of 50 mg/m3 (2418 μg/lung) or 150 mg/m3 (7378 μg/lung) induced transient pulmonary inflammation but changes may regress during the recovery period compared to persistent pulmonary inflammation by crystalline silica, while no toxic pulmonary effects were measured in animals exposed to 10 mg/m3 (489 μg/lung). The range of airborne particle sizes, in the form of MMAD was 2.9–3.7 μm (Lee and Kelly, 1992; Warheit et al., 1991). In our study, we exposed mice with a single intratracheal instillation of various low doses of UFCSs. Our data showed that even a small dose (30 μg/lung) of UFCSs can induce acute moderate pulmonary inflammation and injury characterized by increased cellular and biochemical indices in BALF with concomitant progressive histopathological lesions. However, these inflammatory responses receded and recovered during the experiment. From the results of dose responses, pulmonary lesions and significant differences from the controls for BALF total protein were still observed in mice exposed to 0.3, 3 or 10 μg UFCSs. Thus, the concentration that can induce no toxic pulmonary effects in our study might be less than 0.3 μg/lung.
Laminin, a noncollagenous glycoprotein with an approximate molecular weight of 900000, is an intrinsic component of all basement membranes. Laminin plays a central role in the formation, the architecture, and the stability of basement membranes as well as control of cellular interactions. Because it is present in all pulmonary basement membranes, it can be used as a marker for these structures (Aumailley and Smythe, 1998; Gil and Hernandez, 1984). In mice exposed to UFCSs, weak and discontinuous positive stainings of laminins were observed along basement membranes in both airway epithelium and lung parenchyma especially in the site of the inflammatory foci. The distribution of the basement membrane lesions associated with pulmonary inflammation and damage in the acute phase and gradually declined until recovery at the end time point. The severity of the lesions correlated with the increasing concentration of total protein in BALF. These results suggest that UFCSs exposure induced pulmonary basement membrane destruction, leading to alterations in the permeability of the alveolar-capillary barrier resulted in the leakage of the transudation of serum proteins from the vasculature into alveolar lumens.
Johnston et al. (2000) reported that inhalation exposure to high dose of amorphous silica induced fragmented DNA damage of bronchiolar epithelial cells and alveolar macrophages that will be either repaired or results in cell death through apoptosis or necrosis. In the present study, many TUNEL positive cells were observed in lungs of UFCSs-treated mice compared to those of the control groups. Apoptotic cells were determined with careful observation of TUNEL-stained sections and serial H&E-stained sections because some necrotic cells could be also TUNEL-positive. If TUNEL-positive cells did represent histological features of necrosis in H&E-stained sections, they were not considered to be apoptotic cells. The TUNEL-positive stains were found in bronchiolar epithelium, lung parenchymal cells and active AMs. There was a significant and considerable increase of the apoptotic index, as assessed by TUNEL assay, in the lung parenchyma at all observation time points but transient increase in bronchial epithelial cells after instillation with 30 μg UFCSs. These results suggest that even a small dose (30 μg) of UFCSs can cause apoptosis of lung parenchymal cells.
Ultrafine particles have been reported to cause oxidative stress as a result of generation of reactive oxygen species in a number of in vivo and in vitro studies (Brown et al., 2001; Dick et al., 2003; Donaldson and Stone, 2003). 8-hydroxydeoxyguanosine (8-OHdG) is one of the most specific and representative of base modification among oxidative DNA damage products (Kasai, 1997). Hydroxyl radical, singlet oxygen, and peroxynitrite are proposed to produce 8-OHdG (Warita et al., 2001). There is evidence to suggest that 8-OHdG is a major mutagenic lesion, producing predominately G→ T transversion mutations (Kuchino et al., 1987). Immunohistochemical detection of 8-OHdG was recently established as a useful marker indicative of oxidative stress in various paraffin-embedded tissues (Gottschling et al., 2001; Takahashi et al., 1998). In this study, positive stains for 8-OHdG antibody were observed mainly in the cytoplasm and partially in the nuclei of bronchiolar epithelial cells, alveolar epithelial cells and activated AMs. The progression of immunopositivity of 8-OHdG was correlated to pulmonary lesions induced by exposure to UFCSs. These results suggest that oxidative damage may play an important role in the development of pulmonary inflammation and injury after instillation of UFCSs. The mechanism of the generation of the oxidative stress is not understood, but appears to be related to the large surface area of particles. Silicon functionalities as well as traces of iron impurities on the silica surface are implicated in free radical release at the surface and in subsurface layers of particles (Vallyathan et al., 1988; Fubini et al., 1990). Furthermore, a specific binding of silanol groups (SiOH) of silica to the phosphate groups of DNA was reported (Mao et al., 1994). This close proximity between DNA and the active sites of the silica surface would enable the short-lived radicals to induce DNA damage (Saffiotti et al., 1994). Free radicals from the particle surface can cause transcripts of pro-inflammatory gene products via oxidative stress responsive transcript factors (Donaldson et al., 1996). This could lead to the inflammatory response. Moreover, reactive oxygen species may be generated during phagocytosis of particles, leading to enhancement of oxidative stress.
In summary, this study demonstrated the pulmonary biological and pathological responses after intratracheal instillation of low dose of UFCSs in mice during the acute and subacute stages. Low dose of UFCSs produced moderate inflammation and tissue damage on the lungs of mice during the acute period, but these responses were not sustained through a 30-day period after instillation and almost recovery at the subacute stage. Furthermore, our current study found that UFCSs can induce oxidative damage and apoptosis, which may be underlying causes of the lung tissue injury. The data from the dose and time responses in this study may be useful in predicting the acute and subacute effects of UFCSs on lungs.