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Toxicology Letters 182 (2008) 97–101

Contents lists available at ScienceDirect

Toxicology Letters

journa l homepage: www.e lsev ier .com/ locate / tox le t

ransient pulmonary fibrogenic effect induced by intratracheal instillation ofltrafine amorphous silica in A/J mice

ina Choia, Wan-Seob Choa, Beom Seok Hana, Minjung Choa, Seung Yeul Kima, Jung-Yeon Yia,yeongwoo Ahnb, Seung Hee Kima, Jayoung Jeonga,∗

Department of Toxicological Research, Korea Food and Drug Administration, National Institute of Toxicological Research, Seoul 122-704, Republic of KoreaDepartment of Veterinary Pathology, College of Veterinary Medicine, Chungbuk National University, Chungbuk 361-763, Republic of Korea

r t i c l e i n f o

rticle history:eceived 31 March 2008eceived in revised form 16 July 2008ccepted 30 August 2008vailable online 13 September 2008

eywords:

a b s t r a c t

In order to evaluate the degree of pulmonary fibrosis and to identify the fibrogenic mechanisms inducedby ultrafine amorphous silica (UFAS), UFAS suspensions (∼50 �l) were instilled intratracheally into A/Jmice at doses of 0, 2, 10 and 50 mg/kg (n = 5 per group). Mice were sacrificed at 24 h, 1, 4 and 14 weeksafter exposure. Gomori’s trichrome staining revealed that UFAS induced severe alveolar epithelial thick-ening and pulmonary fibrosis at 1 week, though animals almost recovered at 4 and 14 weeks. The mRNAand protein levels of cytokines (IL-4, IL-10, IL-13 and IFN-�), matrix metalloproteinases (MMP-2, MMP-9

ltrafine amorphous silicaulmonary fibrosisytokinesMPs

IMP-1

and MMP-10) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in lung tissues were signif-icantly elevated at 24 h and 1 week post-treatment, though these levels decreased to near the controlrange at 4 and 14 weeks except IFN-� and MMP-2. These results demonstrate that UFAS can inducepulmonary fibrosis in the same way as crystalline silica. However, the degree of fibrosis observed wastransient. This study shows that cytokines (IL-4, IL-10, IL-13 and IFN-�), MMPs (MMP-2, MMP-9 andMMP-10) and TIMP-1 play important roles in the fibrosis induced by the intratracheal instillation of

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UFAS.

. Introduction

Individuals may be exposed to substantial quantities of syn-hetic amorphous silica in the workplace, as these materials areidely used in many industries and for various applications, e.g.,llers in the rubber industry, in tyre compounds, as free-flow andnti-caking agents in powder materials and as liquid carriers, par-icularly in the manufacture of animal feed and agrochemicals. Inddition, synthetic amorphous silicas are found in toothpaste addi-ives, paints, silicon rubber, insulation material, liquid systems inoatings, adhesives, printing inks, plastisol car undercoats and cos-etics (Merget et al., 2002). However, despite their widespread

se in industry, their toxicities and toxic mechanisms are not wellnderstood.

Animal inhalation studies have shown that ultrafine silicas mayause reversible pulmonary inflammation, emphysema and alveo-

∗ Corresponding author at: Division of Toxicologic Pathology, Department of Tox-cology Research, National Institute of Toxicological Research, Korea Food and Drugdministration, 194 Tongil-ro, Eunpyung-ku, Seoul 122-704, Republic of Korea.el.: +82 2 380 1821; fax: +82 2 388 6451.

E-mail address: jjy kfda@kfda.go.kr (J. Jeong).

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378-4274/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.toxlet.2008.08.019

© 2008 Elsevier Ireland Ltd. All rights reserved.

ar hyperinflation (Warheit et al., 1995). Moreover, amorphous silicas much more acutely toxic than the crystalline form (quartz), buthe recruitments of leukocytes and neutrophils in bronchoalveo-ar lavage (BAL) fluid seem to be somewhat lower than quartz, andheir levels may decrease faster than the case for quartz (Chen etl., 2004; Ernst et al., 2002). This can be attributed to the rapidlimination of amorphous silica from the lungs to other organsGeys et al., 2006; Nemmar et al., 2001; Oberdorster et al., 2002).urthermore, inflammation, fibrogenic effects and silicotic noduleormation are less pronounced for ultrafine silica than for quartzowders (Chen et al., 2004; Reuzel et al., 1991; Warheit et al., 1995).ata on the pulmonary effects of ultrafine amorphous silica (UFAS)

n experimental animals are limited and the mechanisms underly-ng pathological changes are not clearly understood. To date, fewathological reports have been issued on the fibrogenic effects ofFAS.

In a previous study on the pulmonary effects of intratrachealxposure to UFAS, we found that UFAS induced severe pulmonary

nflammation, and pulmonary neutrophils infiltration at 24 h andweek and chronic granulomatous inflammation at 4 and 14 weeks

Cho et al., 2007). The purpose of this study is to describe pul-onary fibrogenic effects caused by intratracheal exposure to UFAS.

n addition, we studied the expression of several fibrogenic medi-

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tors in lungs after exposure in mice by intratracheal instilla-ion.

. Materials and methods

.1. Animals

Male A/J mice, 5 weeks of age, were purchased from Charles River Laborato-ies (Japan). Five mice were housed in polycarbonate cages and acclimatized for4 days. An AIN76A diet (Dyets, Bethlehem, PA) and water were provided ad libitum.nvironmental conditions (temperature, 23 ± 1 ◦C; relative humidity, 55 ± 5%; 12 hight/dark cycle) were monitored at 4-h intervals 24-h/day throughout the experi-

ent. Body weight changes were recorded weekly to assess general health status.ice were housed in a Korea FDA accredited animal facility in accordance with

AALAC International Animal Care Policies (accredited unit: Korea Food and Drugdministration; unit number: 000996).

.2. Rationale for the selection of doses

In a previous study, instillation of UFAS at doses up to 50 mg/kg showed noignificant decreases in body weights and clinical signs (Cho et al., 2007). In addition,aewamatawong et al. performed intratracheal instillation of UFAS at dose about5 mg/kg and Rao et al. instilled silica at dose of 20 mg/kg. Therefore, we instilledhe UFAS at doses of 0, 2, 10 and 50 mg/kg.

.3. Intratracheal UFAS exposure

Ultrafine silica (particle size 0.014 �m and surface area 200 ± 25 m2/g) was pur-hased from Sigma and suspended in PBS for intratracheal instillation. Suspensionsere shaken vigorously and sonicated prior to intratracheal instillation. Possible

ndotoxin contaminations of suspensions and BAL fluid were checked using Limu-us Amebocyte Lysate Assays (Cambrex, Walkersville, MD). UFAS suspensions (50 �l)repared in PBS at doses of 0, 2, 10 and 50 mg/kg (n = 5 per group), and deliveredsing a 24-gauge blunted needle by intratracheal instillation under light isofluranenesthesia (Cho et al., 2007; Kaewamatawong et al., 2005; Pryhuber et al., 2003; Raot al., 2004). At 24 h and 1, 4 and 14 weeks after instillation, mice were sacrificed andungs were removed for further examination.

.4. Gomori’s trichrome staining

Lung sections were deparaffinized, hydrated with a graded alcohol series andistilled water, placed in preheated Bouin’s solution at 56 ◦C for 15 min. The slidesere then cooled in water and washed in running water to remove the yellow dis-

oloration. Sections were stained in working Weigert’s iron hematoxylin solutionor 5 min, washed under running water for 5 min, stained with Gomori’s trichrome

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ig. 1. Gomori’s trichrome staining and quantitative morphometric analysis of mouse luere taken at 24 h (A), 1 week (B), 4 weeks (C) and 14 weeks (D) after administering 50 mguantitative analysis of Gomori’s trichrome positive signal areas versus total lesion areas

ers 182 (2008) 97–101

olution (Sigma–Aldrich, St. Louis, MO) for 5 min, placed in 0.5% acetic acid for 1 min,nd then mounted for light microscopy.

.5. Quantitative morphometric analysis

Quantitative morphometric analysis was carried out using a computer imagenalysis system comprised an Olympus DP71 digital camera and the VideoTestaster 4.0 program. Image analysis was performed to determine average areas ofomori’s trichrome positive connective tissues in alveolar walls and areas of posi-

ive signals in connective tissues and cells in foci of granulomatous inflammation. Sixisual fields were analyzed per section and three sections were examined per ani-al at a magnification of 400×. Areas containing airways or blood vessels >25 mm

n diameter were excluded from the analysis.

.6. RNA extraction, cDNA construction and quantitative real-time PCR

Total RNA was extracted from lung tissues using a single-step techniquesing TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA), according to theanufacturer’s instructions. RNA quantities in samples were determined by elec-

rophoresis in 2% agarose gels stained with ethidium bromide. 18S and 28S bandsere visualized under UV light. Total RNA was then treated with DNAse I (Invitrogen

ife Technologies, Carlsbad, CA) before further processing.First-strand cDNA synthesis of total RNA was performed using a High-capacity

DNA Archive kit (Applied Biosystems, Foster City, CA), according to the manufac-urer’s instructions. Quantitative real-time PCR was performed by adding UniversalCR Buffer and Taqman primer/probe assay reagent specific for IL-4, IL-10, IL-13,FN-�, MMP-2, MMP-7, MMP-9, MMP-10 and TIMP-1, according to the manufac-urer’s instructions. GAPDH primer/probes were used for control purposes. Allrimer/probes were purchased from Applied Biosystems as Assay-on-DemandTM

ene Expression Products (Taqman MGB probes, FAM dye-labeled). The PCR condi-ions used were as follows: 50 ◦C for 2 min, 95 ◦C for 10 min, and 40 cycles of 95 ◦Cor 15 s and 60 ◦C for 1 min on an ABI Prism 7500 unit (Applied Biosystems, Fosterity, CA). CT (threshold cycle) values obtained for genes of interest were normalizedersus GAPDH (house-keeping gene) and fold increases were compared with vehicleontrols.

.7. Immunohistochemistry

Paraffin-embedded lung sections were used for the immunohistochemicaletection of IL-4, IL-10, IL-13, MMP-9 and TIMP-1. For immunohistochemistry,ections were mounted on silanized slides (Dako, Glostrup, Denmark) and deparaf-

nized and hydrated with a xylene–alcohol series to distilled water. Endogenouseroxidase activity was quenched with 3% hydrogen peroxide–methanol solutiont room temperature for 15 min. For antigen retrieval, slides were then placedor 20 min in proteinase K. The slides were then blocked with 10% normal don-ey (IL-4, IL-10, IL-13 and TIMP-1) or goat (MMP-9) serum, and then placed inrimary antibody or an equivalent amount of normal donkey or goat IgG as a

ngs following the intratracheal instillation of UFAS. Representative lung sections/kg of UFAS intratracheally (collagen expression is shown by bright green staining).showed that fibrosis peaked at 1 week post-treatment (E).

M. Choi et al. / Toxicology Letters 182 (2008) 97–101 99

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ig. 2. Time courses of the gene expression of cytokines (IL-4, IL-10, IL-13 and IFN-�ith UFAS: (A) IL-4, (B) IL-10, (C) IL-13, (D) IFN-�, (E) MMP-2, (F) MMP-9, (G) MMP-

egative control for 1 h at room temperature and incubated at 4 ◦C overnight.mmunocomplexes were detected using the ImmunoCruz Staining system (Santaruz Biotechnology, Santa Cruz, CA) and visualized with 3,3′-diaminobenzidineetrahydrochloride (Zymed Laboratories Inc., San Francisco, CA) as chromogen.ections were then counterstained with Mayer’s hematoxylin and examinednder a light microscope. IL-4, IL-10, IL-13 and TIMP-1 primary antibodies werebtained from R&D Systems (Minneapolis, MN) and MMP-9 was obtained fromHEMICON (Temecula, CA).

.8. Statistical analysis

Experimental results are expressed as means ± standard deviations. Data werenalyzed using the JMP software package (version 4.0; SAS Institute, Cary, NC).ll results were compared using Dunnett’s t-test after ANOVA analysis. For allomparisons, P values of less than 5% (P < 0.05) were considered statisticallyignificant.

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ig. 3. Time courses of the protein expressional patterns of cytokines (IL-4, IL-10 and IL-week (B), 4 weeks (C) and 14 weeks (D) after treatment with 50 mg/kg UFAS. IL-10 proteith 50 mg/kg of UFAS. IL-13 protein expression at 24 h (I), 1 week (J), 4 weeks (K) and 14 w

rown color (arrow). (For interpretation of the references to color in this figure legend, th

MMPs (MMP-2, MMP-9 and MMP-10) and TIMP-1 in the lungs of A/J mice treatedd (H) TIMP-1 (*P < 0.05 versus control, n = 3 mice each).

. Results

.1. Gomori’s trichrome staining and image analysis

At 24 h after particle instillation, alveolar wall thickening andeutrophils influx were clearly evident (Fig. 1A). Positive signals forbrosis were most severe at 1 week after 50 mg/kg UFAS instillation,

.e., alveolar epithelial thickness and the amount of septal fibrosisere greater in this group than in the other groups (Fig. 1B). How-

ver, the amount of septal fibrosis was markedly reduced at 4 weeksnd had returned to the steady state at 14 weeks post-treatmentFig. 1C and D). Morphometric analysis findings were closely corre-ated with representative Gomori’s staining figures (Fig. 1E). Mean

13) in the lungs of A/J mice treated with UFAS. IL-4 protein expression at 24 h (A),in expression at 24 h (E), 1 week (F), 4 weeks (G) and 14 weeks (H) after treatmenteeks (L) after treatment with 50 mg/kg of UFAS. The positive labeling was stained

e reader is referred to the web version of the article.)

100 M. Choi et al. / Toxicology Letters 182 (2008) 97–101

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ig. 4. Time courses of the protein expression patterns of MMP-9 and TIMP-1 in the lC) and 14 weeks (D) after treatment with 50 mg/kg of UFAS. TIMP-1 protein expresf UFAS. The positive labeling was stained brown color (arrow). (For interpretation ohe article.)

ollagen areas peaked at 1 week and had decreased to control levelst 14 weeks post-treatment.

.2. mRNA expressions of fibrogenesis mediators induced by UFAS

IL-4, IL-10 and IL-13 mRNA levels peaked at 1 week after0 mg/kg UFAS instillation at 22-, 8- and 21-times baseline, respec-ively (Fig. 2A–C). IL-4, IL-10 and IL-13 mRNAs were significantlyncreased at 24 h and 1 week post-treatment and returned to nearontrol levels at 14 weeks. Moreover, the expression of IL-10 wasecreased at 14 weeks in the 50 mg/kg UFAS group and IL-13 mRNAas decreased at 4 and 14 weeks in all treatment groups comparedith vehicle controls. IFN-� mRNA expression peaked at 24 h post-

reatment and the expression was persisted until 14 weeks afterdministering 10 mg/kg of UFAS (Fig. 2D). MMP-2 mRNA expres-ion levels decreased at 24 h post-treatment and then increased atand 14 weeks (Fig. 2E). MMP-9 mRNA expression was elevated at4 h and 1 week post-treatment and was significantly lower thanhese levels at 4 and 14 weeks (Fig. 2F). MMP-10 mRNA levels werelevated from 24 h to 4 weeks post-treatment (Fig. 2G). Interest-ngly, the instillation of a 2 mg/kg of UFAS induced the expressionsf MMP-9 and MMP-10 mRNA later than 10 and 50 mg/kg of UFAS.IMP-1 mRNA expression was elevated at 24 h and 1 week post-reatment in a dose-dependent manner (Fig. 2H). However, MMP-7

RNA was not expressed in any treatment group.

.3. Inductions of fibrogenesis mediators by UFAS

In accordance with their mRNA expression profiles, IL-4, IL-10nd IL-13 protein levels were elevated at 1 week post-treatmentFig. 3). In addition, the time courses of MMP-9 and TIMP-1 mRNAxpressions were in-line with their immunohistochemical expres-ions (Fig. 4). Strong positive signals for MMP-9 and TIMP-1 proteinere observed in alveolar macrophages and lower signal levelsere observed in alveolar walls in the 50 mg/kg dose group. The

xpression of fibrogenic mediators increased in a dose-dependentanner. The overall protein expression pattern of these fibrogenicediators was well correlated with their mRNA expression pattern.

. Discussion

The present study described the induction of pulmonary fibro-enesis by intratracheal instillation of UFAS, and the time courses

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f A/J mice treated with UFASs. MMP-9 protein levels at 24 h (A), 1 week (B), 4 weekst 24 h (E), 1 week (F), 4 weeks (G) and 14 weeks (H) after treatment with 50 mg/kgreferences to color in this figure legend, the reader is referred to the web version of

ene and protein expression patterns of fibrogenic mediators in A/Jice. The intratracheal instillation of UFAS in mice has been previ-

usly demonstrated to cause severe alveolar epithelial thickeningnd pulmonary septal fibrosis at 1 week post-treatment and almostecovered at 14 weeks post-treatment (Cho et al., 2007). Accordingo a previous study, the effect of fibrogenesis induced by nanosizedilica might be milder than that induced by microsized silica inats, perhaps because nanoparticles tend to be easily diffused andranslocated (Chen et al., 2004). Furthermore, several studies havehown that nanoparticles might translocate from deposition sitesn the lungs to extrapulmonary organs through the systemic circu-ation, which would reduce the amounts stored in alveoli (Kreylingt al., 2002; Nemmar et al., 2001; Oberdorster et al., 2002; Semmlert al., 2004).

In this study, the expressions of cytokines (IL-4, IL-10, IL-13 andFN-�), matrix metalloproteinases (MMP-2, MMP-9 and MMP-10)nd TIMP-1 were found to be involved in the regulation of the pul-onary fibrogenic process at both the mRNA and protein levels.ur results indicate that the induction of fibrogenesis in response toFAS is closely associated with the expressions of these cytokines,MPs and TIMP-1 genes. T helper (Th) 2 cytokines, including IL-

, IL-10 and IL-13, are known to promote fibroblast proliferation,ollagen gene expression and collagen synthesis (Ingram et al.,003; Sime and O’Reilly, 2001). A previous study showed that IL-0 overexpression significantly contributes to silica-induced lungbrosis by exacerbating Th2 response and by increasing the pro-uctions of the profibrotic cytokines IL-4 and IL-13 (Barbarin etl., 2005). However, Th1 cytokines, including IFN-�, have beenound to the opposite effect, as they ameliorate the fibrotic processSempowski et al., 1996). For example, IFN-� was found to haven antifibrotic effect in a bleomycin mouse model of lung fibrosisecause it down-regulated TGF-�1 and collagen gene expressionsGurujeyalakshmi and Giri, 1995). INF-� has also been shown tonhibit the growth of collagen synthesis by fibroblasts derived fromormal and fibrotic human lungs (Narayanan et al., 1992). Further-ore, our results show that the instillation of UFAS significantly and

ose-dependently increased the expressions of IL-4, IL-10 and IL-3 at the mRNA and protein levels, and that these peaked at 1 week

ost-treatment.

MMP family enzymes are responsible for the degradation ofxtracellular matrix components. MMP-2 (gelatinase A) and MMP-(gelatinase B) were found to be produced by alveolar macrophages

n response to silica exposure (Perez-Ramos et al., 1999), and to

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M. Choi et al. / Toxicolog

ave substrate affinities for basement membrane type IV colla-en, denatured collagens (gelatin) and elastin. Moreover, MMP-10stromelysin-2) is known to digest proteoglycans and some gly-oproteins, and to be expressed in fibroblasts, keratinocytes, Tells, menstrual endometrium and in some tumor cells (Ishiharat al., 2003). In addition, TIMP-1 stimulates fibroblast prolifera-ion and inhibits MMPs (Perez-Ramos et al., 1999), is produced by

acrophages and epithelial cells, and is strongly associated withulmonary fibrosis (Manoury et al., 2006). In the present study, thexpressions of MMP-2, MMP-9 and MMP-10 at the mRNA and pro-ein levels were found to be increased by UFAS, and these genes arenown to play important roles in the fibrosis induced by UFAS. Inddition, TIMP-1 mRNA and protein peaked at 24 h post-treatmentnd then gradually decreased. Furthermore, these gene and pro-ein expressional patterns were found to be closely correlated withomori’s trichrome staining results.

In summary, IL-4, IL-10 and IL-13, known as fibrogenic media-ors in lung, were increased from 24 h to 1 week by treatment ofFAS, and the collagen synthesis was inhibited by INF-�. In addi-

ion, MMP-2, MMP-9 and MMP-10, known as degradators in lungbrosis, increased at 24 h and 1 week. TIMP-1, known as inhibitorf MMPs, expression was up-regulated, so that MMP-9 was dimin-shed after 4 weeks. Overexpression of MMP-2 was involved in theecovery of lung fibrosis at the later stage (14 weeks) (Yang et al.,007). Furthermore, these expressional changes and the fibrogeniculmonary lesions observed were transient and recovered to nearontrol levels at 4 and 14 weeks post-treatment. Further research isequired to identify the other mediators involved in the lung injurynduced by UFAS.

onflict of interest

None declared.

cknowledgements

This work was supported by grants from the Korea Food andrug Administration, the Nanotoxicology Program (07141KFDA761nd 08161KFDA542).

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