Pulmonary Chemokine and Mutagenic Responses in Rats after Subchronic Inhalation of Amorphous and Crystalline Silica

Carl J. Johnston*, Kevin E. Driscoll{dagger}, Jacob N. Finkelstein*, R. Baggs*, Michael A. O'Reilly*, Janet Carter{dagger}, Robert Gelein* and Günter Oberdörster*,1

* Departments of Environmental Medicine and Pediatrics, The University of Rochester, Rochester, New York 14642; and {dagger} Procter and Gamble Pharmaceuticals, Health Care Research Center, Mason, Ohio 45040

Received January 18, 2000; accepted April 17, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic inhalation of crystalline silica can produce lung tumors in rats whereas this has not been shown for amorphous silica. At present the mechanisms underlying this rat lung tumor response are unknown, although a significant role for chronic inflammation and cell proliferation has been postulated. To examine the processes that may contribute to the development of rat lung tumors after silica exposure, we characterized the effects of subchronic inhalation of amorphous and crystalline silica in rats. Rats were exposed for 6 h/day, on 5 days/week, for up to 13 weeks to 3 mg/m3 crystalline or 50 mg/m3 amorphous silica. The effects on the lung were characterized after 6.5 and 13 weeks of exposure as well as after 3 and 8 months of recovery. Exposure concentrations were selected to induce high pulmonary inflammatory-cell responses by both compounds. Endpoints characterized after silica exposure included mutation in the HPRT gene of isolated alveolar cells in an ex vivo assay, changes in bronchoalveolar lavage fluid markers of cellular and biochemical lung injury and inflammation, expression of mRNA for the chemokine MIP-2, and detection of oxidative DNA damage. Lung burdens of silica were also determined. After 13 weeks of exposure, lavage neutrophils were increased from 0.26% (controls) to 47 and 55% of total lavaged cells for crystalline and amorphous silica, with significantly greater lavage neutrophil numbers after amorphous silica (9.3 x 107 PMNs) compared to crystalline silica (6.5 x 107 PMNs). Lung burdens were 819 and 882 µg for crystalline and amorphous silica, respectively. BAL fluid levels of LDH as an indicator of cytotoxicity were twice as high for amorphous silica compared to those of crystalline silica, at the end of exposure. All parameters remained increased for crystalline silica and decreased rapidly for amorphous silica in the 8-month recovery period. Increased MIP-2 expression was observed at the end of the exposure period for both amorphous and crystalline silica. After 8 months of recovery, those markers remained elevated in crystalline silica-exposed rats, whereas amorphous silica-exposed rats were not significantly different from controls. A significant increase in HPRT mutation frequency in alveolar epithelial cells was detected immediately after 13 weeks of exposure to crystalline, but not to amorphous silica. A significant increase in TUNEL staining was detected in macrophages and terminal bronchiolar epithelial cells of amorphous silica-exposed rats at the end of the exposure period; however, crystalline silica produced far less staining. The observation that genotoxic effects in alveolar epithelial cells occurred only after crystalline but not amorphous silica exposure, despite a high degree of inflammatory-cell response after subchronic exposure to both types of silica, suggests that in addition to an inflammatory response, particle biopersistence, solubility, and direct or indirect epithelial cell cytotoxicity may be key factors for the induction of either mutagenic events or target cell death.

Key Words: biopersistence; cytotoxicity; inhalation; mutagenicity; pulmonary inflammation; silica..


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Crystalline silica particles can cause both acute and chronic pulmonary inflammatory responses (Bowden and Adamson, 1984Go; Morgan et al., 1980Go). Chronic inhalation studies with crystalline silica in rats have induced pulmonary fibrosis and cancer (IARC, 1997Go; Reiser and Last, 1979Go; Saffiotti, 1992Go). In contrast, amorphous silicas are regarded as rather innocuous materials that have been reported to cause little or no chronic adverse pulmonary effects (Groth et al., 1986Go). Amorphous silica particles (fumed silica, precipitated silica) are widely used in pharmaceutical products, paints, cosmetics, and foods, and consequently, there is a widespread occupational exposure from these substances during manufacturing processes (Groth et al., 1979Go).

High cytotoxicity and long pulmonary retention of crystalline forms of silica are major factors responsible for its long-term effects (Langer, 1978Go; Quinot et al., 1979Go). Amorphous silica, in the form of fumed silica, consists of ultra-fine primary particles with a very large surface area, which are less persistent in the lung (Lee and Kelly, 1992Go; Reuzel et al., 1991Go). However, high doses of amorphous silica may also result in acute pulmonary inflammatory responses, which could conceivably trigger long-term effects, despite a low biopersistence of the particles.

Many poorly soluble particulate materials producing lung tumors in rats are not primary genotoxic agents (IARC, 1987Go; Kanematsu et al., 1980Go). The lack of inherent genotoxic activity of poorly soluble particles, and their association with the development of rat lung tumors after chronic inhalation exposure, implies a secondary genotoxic mechanism for their response. With respect to rat lung tumors induced by chronic high inhalation exposure of poorly soluble, low-toxicity particles, lung particle overload-related persistent inflammation, and increased epithelial cell proliferation in the pulmonary region of the rat lung represent generic mechanisms to induce secondary genotoxicity via oxidants released from inflammatory cells (Oberdörster, 1995Go). The degree of inflammation and its duration appear to be key elements for the secondary genetic response (Driscoll et al., 1995Go). Driscoll et al. (1995, 1997) determined HPRT mutations in rat alveolar epithelial cells after in vivo exposure to inflammatory doses of poorly soluble particles. This is the cell type from which the rat lung tumors most likely derive after exposure to these types of particles (Dungworth et al, 1994Go; Nikula et al., 1995Go; Nolte et al., 1994Go). Our earlier study has confirmed the utility of this assay for inhalation studies showing increased HPRT mutation frequencies in isolated type II epithelial cells after subchronic inhalation of high concentrations of carbon black particles (Driscoll et al., 1996aGo).

In the present study, we compared the pulmonary responses of amorphous and crystalline silica inhaled by rats for 3 months at concentrations that resulted in a high inflammatory response by both compounds at the end of exposure. We also measured pulmonary silica retention and the mutagenic effects on rat alveolar epithelial cells by using an ex vivo assay to test the hypothesis that persistent alveolar inflammation is associated with a significant increase in alveolar epithelial-cell-mutation frequency independent of the biopersistence of particles. Our results demonstrate that after 3 months of exposure, only crystalline silica, and not amorphous silica exposure resulted in an increased epithelial cell mutation frequency. Since exposures to both particles induced an active and persistent inflammatory response, the results suggest that particle-induced inflammation is not solely responsible for mutagenic effects.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Experimental design.
Three groups of male Fischer-344 rats, weighing between 200 and 250 g, were exposed 6 h/day, 5 days/week for 13 weeks to filtered air or aerosols of silica (crystalline or amorphous) in compartmentalized 300-liter horizontal laminar flow, whole-body chambers. Cristobalite (C and E Minerals, King of Prussia, PA; gift from David R. Hemingway, University of Vermont) was used as crystalline silica and precipitated silica (Aerosil 200 Degussa) as amorphous silica. Silica aerosols were generated using a screw-feed mechanism (ACCURate, Whitewater, WI) in combination with a Venturi-type dust feeder. The aerosol was brought to Boltzman equilibrium by passing the airborne particles across a 20-mCi 85K source. The mean (± SD) aerosol concentrations for the amorphous and cristobalite groups were 50.4 ± 19.0 (amorphous silica) and 3.0 ± 1.0 (crystalline silica) mg/m3. The mass median aerodynamic diameter (geometric standard deviation) was 0.81 and 1.3 µm, respectively, for the amorphous and crystalline silica groups. After 6.5 and 13 weeks of exposure and 3 and 8 months of recovery, groups of rats were euthanized by intraperitoneal (ip) injection of Na-pentobarbital (50 mg/kg) followed by exsanguination via the abdominal aorta, for analysis of lung silica burdens, cellular and biochemical bronchoalveolar lavage fluid markers of lung injury and inflammation, histopathology, inflammatory cytokine gene expression, and mutagenesis in alveolar epithelial cells.

Lung burden analysis.
A modification of the method of Hemenway et al. (1990) was used to determine lung-silica burdens. The lavaged lung was cut into small pieces and placed in the bottom of a platinum crucible. The lung was dried overnight in a 68°C oven and then ashed for 24 h using a plasmod asher. Gold Label sodium carbonate (0.5 g) was added to the ashed lung, and the lung and NaCO3 fused for 30 min. Crucibles were then cooled to room temperature. Ten (10) ml of double distilled water was added to the sample, which was placed in a 90°C water bath for 15 min and sonicated for 20 min. Samples were analyzed for Si, using emission spectroscopy at a wavelength of 251.612.

Bronchoalveolar lavage fluid analysis.
Bronchoalveolar lavage (BAL) was performed as described in detail previously (Ferin et al., 1992Go). Briefly, the lungs and heart were excised, the trachea cannulated, and the lungs lavaged, 10 times with 5 ml of sterile saline each time, at room temperature. The BAL fluid was centrifuged (10 min. at 400 x g) and the acellular supernatant was analyzed for total protein, lactate dehydrogenase (LDH), and glucuronidase levels or activities. Total protein was determined using the micro-BCA method (Pierce Chemical Co., Rockford, IL). LDH was assayed using a Sigma diagnostic kit (DG 1340-UU, Sigma, St. Louis, MO). The enzyme glucuronidase was measured by the release of p-nitrophenol from the substrate 4-nitrophenylglucuronide, determined at 420 nm on a Cary 219 spectrophotometer (Varian Instruments, Sunnydale, CA). BAL fluid cells were quantified by hemocytometric counting, and cell viability was determined by exclusion of trypan blue dye. Cell differentials were performed on cytocentrifuge preparations that were fixed in methanol and stained with Diff-Quik (Sigma).

Histopathology.
After lavage, a lobe of lung was inflated, with 10% neutral buffered formalin (NBF), to the original size. The bronchus was ligated, and the entire lobe immersed in 10% NBF. After a minimum 24-h fixation, the lung was grossly slabbed in a radial pattern, with the bronchus as the central point. The lung slab was processed through paraffin, sectioned at 5 microns, and stained with Gormor's trichrome. All sections were coded by accession number, which "blinded" the observer from the treatment. The sections were examined for alveolitis: the number of neutrophils and macrophages and the amounts of proteinaceous fluid and alveolar type-II epithelial cell proliferation. In addition, the severity of inflammation in bronchioles and bronchi was noted, as was the extent of fibrosis and the relative amount of lung parenchyma affected (diffuseness). All were ranked on a severity score of form 0.0 (no significant lesions) to 4.0 (very severe process) and then summed to yield a summary toxicity score.

RNA isolation/polymerase chain reaction (PCR).
Expression of MIP-2 mRNA in lungs was assessed, as described in detail elsewhere (Driscoll et al., 1993aGo,bGo). Briefly, the left lung lobes from 2 animals/exposure group/time were quick-frozen in liquid nitrogen for later isolation of RNA. RNA was extracted as described by Chomczynski and Sacchi (1987), and mRNA transcript levels were assessed by PCR amplification of the MIP-2 cDNA. GAPDH mRNA was evaluated concurrently with MIP-2 mRNA as a control. PCR primers were designed from the published sequences for MIP-2 (Driscoll et al., 1996aGo) and GAPDH (Nudel et al., 1983Go) and were as follows:

MIP-2: 5'-GGCACATCAGGTACGATCCAG-3'

5'-ACCCTGCCAAGGGTTGACTTC-3'

GAPDH: 5'-CAGGATGCATTGCTGACAATC-3'

5'-GGTCGGTGTGAACGGATTTG-3'

PCR reactions were overlaid with mineral oil and amplification was carried out through 22–30 cycles of denaturation at 94°C for 1 min, oligo-annealing at 55°C for 1 min, and extension at 72°C for 2 min. Reactions were electrophoresed in 1.5% agarose gels containing ethidium bromide in Tris-acetate/EDTA buffer to visualize the MIP-2 and GAPDH PCR products. We confirmed that the PCR products obtained with the primer sequences were MIP-2 or GAPDH by Southern analysis and using oligonucleotide probes complementary to mRNA sequences internal to the PCR primer sequences used (data not shown).

Immunohistochemistry.
It is conceivable that cytotoxic events can increase cell apoptosis and/or cell necrosis. This in turn may result in a decrease in mutation frequencies when affected epithelial cells have died. In order to identify the extent of potential cell apoptosis/necrosis as a consequence of DNA damage, we performed terminal transferase dUTP nick-end-labeling (TUNEL)-staining on lung sections. Sections were deparaffinized and hydrated before blocking of endogenous hydrogen peroxide with hydrogen peroxide-methanol. TUNEL staining was performed using an ApopTag kit obtained from Oncor (Gaithersburg, MD), according to the manufacturer's recommendations. Stained sections were photographed using color-slide film. All images were digitally color balanced using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA).

Type-II cell isolation and HPRT assay.
The rat alveolar type-II cell isolation and the HPRT clonal selection assay were performed as described in detail previously, with the exception that alveolar epithelial cells were harvested from the right lung only in the present study (Driscoll et al., 1995Go). Briefly, animals were injected with sodium heparin (40 U, ip) before euthanization by ip injection with sodium pentobarbital. The lungs and trachea were removed and the lungs were perfused, via the pulmonary artery, with a buffered salt solution (BSS: 125 mM NaCl, 5 mM KCl, 2.5 mM NaHPO4, 17 mM HEPES, 0.1% glucose, 1.0% nystatin, and 1.0% antibiotic-antimycotic, pH 7.4) at 9 ml/min, using a Harvard infusion pump. The right lung lobe was lavaged 5 times with 5 ml sterile BSS and twice with 5 ml sterile BSS containing 2.5 mM CaCl2 and 1.2 mM MgSO4 (Ca-Mg BSS). Five ml of a pronase (protease E; Sigma) solution (0.25% in Ca-Mg BSS) was instilled into the right lung every 5 min for a total of 3 times. The lung tissue was placed in beakers containing 1 mg DNase, 4 ml saline, and 5 ml fetal bovine serum (FBS; Gibco, Gaithersburg, MD) and minced into ~1- to 4-mm pieces. Lung tissue was filtered (120-µm sterile nylon filters) and the resultant cell suspensions were centrifuged (500 x g). Cell pellets were resuspended in Ham's F12 medium (Gibco) containing 2% FBS, layered over a Nycodenz gradient (Accurate Chemical, Westbury, NY), and centrifuged for 20 min at 1500 x g. The cell layer just beneath the interface was removed, washed twice with saline, and resuspended in RluE medium (Biological Research Facility and Faculty, Ijamsville, MD). Staining for alkaline phosphatase activity routinely identified the epithelial cells. Cell counting was performed using a hemocytometer and trypan blue dye exclusion was used to determine cell viability.

Freshly isolated alveolar type-II cells were seeded at 2 x 105 epithelial cells/flask into 6 T25 flasks, the cells were allowed to attach overnight (37°C, 5% CO2), and then the culture dishes were washed with RluE cell culture medium to remove non-adherent cells. The cell cultures were fed with medium containing 6TG (40 µM; Sigma) to select for mutation in the HPRT gene; cultures were re-fed every other day with 6TG-containing medium. After 14–21 days in culture the cells were fixed and immunostained with an antibody to cytokeratins 8, 18 and 19 (Biogenex, San Ramon, CA) and 6TG-resistant cytokeratin staining colonies of greater than 50 cells counted. Mutation frequencies were calculated as (number of colonies/treatment)/(plating efficiency)/(106 cells) = mutants/106 cells.

Statistical analysis.
Results were evaluated for statistical significance by analysis of variance. Differences from the air control group were determined using Dunnett's test. Statistical significance was considered at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lung Silica Burdens
Table 1Go summarizes the silica lung burden determined after 6.5 and 13 weeks of exposure and 3 and 8 months of recovery in clean air. At the end of exposure, the group-mean lung burdens were 819 and 882.7 µg/lung for the crystalline and amorphous silica, respectively. By 3 months after exposure, amorphous silica burdens were significantly decreased relative to the end of exposure and decreased further by 8 months of exposure. The crystalline silica burdens post-exposure were relatively unchanged from the end of exposure.


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TABLE 1 Lung Burdens after Subchronic Exposure of Rats to Crystalline and Amorphous Silicas (µg SiO2/lung)
 
Bronchoalveolar Lavage Fluid Analysis
Table 2Go presents the results of the BAL fluid analysis. Exposure to 3-mg/m3 crystalline silica produced significant changes in all BAL parameters examined. These changes in BAL endpoints persisted throughout the post-exposure period. Exposure to 50-mg/m3 amorphous silica also produced significant changes in all BAL parameters examined through the end of exposure. Total cell numbers, PMN, and protein and glucuronidase levels in BAL at the end of exposure were greater than for crystalline silica. The changes in BAL endpoints in the amorphous silica group returned to near sham-exposed levels by 3 months post-exposure, and values were not significantly different from controls by 8 months post-exposure.


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TABLE 2 Changes in Bronchoalveolar Lavage Fluid Contents in Rats after 6.5 and 13 Weeks of Inhalation Exposure to SiO2 Particles and 12 or 32 Weeks of Recovery ( ± SD; n = 4)
 
Histopathology
The extent of the inflammatory process in the various compartments of the lung (alveolus, bronchiole, bronchus) was determined by the microscopic examination of appropriately stained sections. Identity as to treatment and time was made only after completion of the examination. The control animals had virtually no excessive number of neutrophils or macrophages in the alveoli, and the extent of type-II cell proliferation remained essentially normal (Fig. 1AGo). By comparison, animals exposed to crystalline silica had increased numbers of neutrophils and macrophages demonstrable at 90 days of treatment, with persistence of elevated levels through the 8-month post-exposure period (Fig. 1BGo). The amorphous silica-exposed animals likewise had elevated numbers of neutrophils and macrophages, but the elevation was first detectable at the first observation point (45 days of treatment), and tended to decrease in the post-exposure period. These observations are consistent with the BAL data. The crystalline silica-exposed animals had a very mild proliferative response at 90 days of exposure, but demonstrated continued elevation from 3 to 8 months post-exposure. By comparison, the amorphous silica proliferative response was evident at the earliest time point (45 days of exposure), and decreased precipitously at the 8-month post- exposure point (Fig. 1CGo). Fibrosis, as detected by Gormor's trichrome staining, was present in the alveolar septa of lungs from silica-treated animals, with a greater persistence in the crystalline-silica group. This is consistent with previous published observations on the persistence of fibrosis with crystalline silica (Hemenway et al., 1986Go) and other poorly soluble particles (Baggs et al. 1997Go).



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FIG. 1. Histopathology of the lungs from adult rats exposed to room air (A), crystalline silica (B), or amorphous silica (C) for 3 months and examined 8 months after completion of exposure. Note the greatly thickened alveolar septa in B (open arrow) and the normal or slightly thickened septa in C (solid arrow). In both crystalline- and amorphous-exposed animals, there was a persistence of increased numbers of hypertrophic alveolar macrophages (M). All illustrations are of the same magnification; bar = 50 µm.

 
MIP-2 Gene Expression
Figure 2Go presents results of the RT-PCR analysis of MIP-2 mRNA expression in lung. Minimal or no detectable MIP-2 mRNA was found in rat lungs from the air control. MIP-2 was clearly present at the end of the 13-week exposure in amorphous and crystalline silica-exposed rats. At the end of 8 months of recovery, MIP-2 mRNA was detected in rat lungs exposed to crystalline silica. However, minimal MIP-2 mRNA was detected in rat lungs exposed to amorphous silica.



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FIG. 2. Effect of subchronic inhalation of SiO2 particles on expression of mRNA for MIP-2 and GAPDH in rat lung. Shown are negatives of ethidium bromide-stained gel with the Rt-PCR products for MIP-2 and GAPDH amplified for rat lung RNA. Rats were exposed to air or amorphous (Ae) or crystalline (Cr) silica at concentrations of 50 mg/m3 and 3 mg/m3, respectively, for 13 weeks, followed by an 8-month recovery period.

 
TUNEL Staining
TUNEL staining was used to determine if exposure to amorphous or crystalline induced DNA damage would be indicative of apoptosis or necrosis. This assay uses the enzyme terminal-transferase to add digoxigenin-conjugated nucleotides to free 3'-hydroxyl groups on DNA, which can be visualized as a brown stain. At the end of exposure, lungs exposed to room air had some lightly stained TUNEL-positive nuclei among numerous TUNEL-negative nuclei (Fig. 3AGo). This low level of staining was observed throughout the parenchyma and in bronchiolar epithelial cells. These faint TUNEL-positive cells did not appear apoptotic and have been observed in adult mouse lungs exposed to room air (O'Reilly et al., 1998Go). In contrast, intensely stained TUNEL-positive cells were detected throughout the terminal bronchiolar epithelium and throughout the parenchyma of rats exposed to amorphous silica (Fig. 3BGo). In contrast, lungs exposed to crystalline silica had little TUNEL staining, similar to the room air-exposed lungs (Fig. 3CGo). Although TUNEL staining has often been used to identify apoptotic cells, it labels any DNA with a free 3'-hydroxyl group. Previous studies have shown that even necrotic cells can be TUNEL-positive (Ansari et al., 1993Go; Grasl-Kraupp et al., 1995Go). Based upon our findings and these other studies, it is likely that TUNEL staining found to a high degree after amorphous but not after crystalline silica exposure represents fragmented DNA that will be either repaired or results in cell death through necrosis or apoptosis.



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FIG. 3. Identification of DNA fragmentation in lungs of adult rats at 3 months of exposure to room air (A), amorphous (B) or crystalline (C) silica; or at 8 months post-exposure from room air (D), amorphous (E), or crystalline (F) silica. Sections were stained for the presence of free 3'-hydroxyl groups, which was detected as a brown stain. Sections were counterstained with methyl green. Bar in (A) = 100 µm. Filled arrows identify intensely TUNEL-positive cells in the parenchyma and open arrows indicate intensely TUNEL-positive cells in airway.

 
TUNEL staining was performed on sections obtained from rats recovered in room air for 8 months. Unexposed lungs had faint TUNEL-positive cells occasionally observed in the bronchioles and parenchyma (Fig. 3DGo). Similar findings were observed in lungs exposed to amorphous silica and recovered in room air (Fig. 3EGo). Some faint TUNEL-positive cells were observed in the bronchioles, endothelium of small vessels, and parenchyma. In general, TUNEL staining was indistinguishable between the amorphous-treated and control groups at this time. In contrast, lungs exposed to crystalline silica and recovered in room air had intense TUNEL staining localized to cell debris in hypertrophic areas of the parenchyma (Fig. 3FGo). The parenchyma of these lungs had large emphysematous regions that contained macrophages, cellular debris, and hypertrophic cells. Intense staining was observed in both macrophages and debris while the alveolar cells had minimal staining. The bronchioles of these lungs also had minimal TUNEL staining.

Isolation of alveolar epithelial cells and HPRT mutation frequencies.
No differences were detected between treatment groups in the yield of alveolar type-II cells or the viability of the lung cell isolates. The group-mean proportion of type-II cells in the cell isolates ranged from 70 to 79%. Cells not staining for alkaline phosphatase appeared to be macrophages and lymphocytes based on morphology. The group number of alveolar type-II cells obtained ranged from 1.5 to 3.4 x 106/right lung lobe. Cell viability was always greater than 90%.

The HPRT mutation frequencies for rat alveolar epithelial cells are summarized in Figure 4Go. Mutation frequencies in the air control group were 7.6 (± 3.4) mutants/106 epithelial cells. Exposure to 3 mg/m3 crystalline silica resulted in HPRT mutation frequencies which were 4.3-fold greater than the air control group, immediately after exposures. No significant changes in the HPRT mutation frequency compared to controls were observed for alveolar epithelial cells from rats exposed to 50-mg/m3 amorphous silica at this time point.



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FIG. 4. HPRT mutation frequencies observed for rat alveolar epithelial cells isolated from the lungs of rats exposed subchronically to air or to silica particles. Statistically significant increases in mutation frequencies were detected from epithelial cells obtained immediately after 13 weeks of exposure to 3-mg/m3 crystalline silica (p < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic inhalation of crystalline silica dust has been shown to induce pulmonary inflammation, development of fibrosis, and tumors in experimental animals and occupationally exposed humans (Bowden and Adamson, 1984Go; IARC, 1987Go; Morgan et al., 1980Go). It has been suggested that rat lung tumors occurring after exposure to poorly soluble particulate materials develop secondary to genotoxic events due to persistent inflammation and increased epithelial cell proliferation (Driscoll, 1995; Oberdörster, 1994Go), and that this inflammatory mechanism of pulmonary carcinogenesis is also responsible for crystalline silica-induced lung tumors (Driscoll et al., 1996bGo). To better understand mutagenic events in the lung in response to increased and persistent inflammation, we exposed rats to fibrogenic and tumor-inducing crystalline silica and non-fibrogenic amorphous silica particles and characterized pulmonary inflammation, cytotoxicity, biopersistence,and mutagenic effects of subchronic silica inhalation on rat lung.

Our approach was to use doses of amorphous and crystalline silica, which generated a similar neutrophil influx into the alveolar space, over 13 weeks to determine whether there is a relationship between prolonged inflammatory cell recruitment and epithelial cell mutations. Our results demonstrate that after 3 months of increased inflammatory cell levels, only crystalline-silica exposure resulted in a significant increase in HPRT mutation frequency in rat alveolar epithelial cells, despite significantly greater levels of lavage neutrophils in the amorphous silica groups. LDH levels in lung lavage, as indicators of cytotoxicity, were twice as high after amorphous exposure compared to crystalline silica. Since lavaged-cell viability was not significantly different between the two types of silica, it is likely that the source of the increase in LDH is epithelial cells. The increased TUNEL staining in epithelial cells of the amorphous silica-exposed rats supports this suggestion. The increased numbers of cells showing positive TUNEL staining after 90 days of amorphous-silica inhalation suggest significant increase of intracellular damage, which may lead to cell death. Cells that were predominantly affected were macrophages and epithelial cells lining the terminal bronchioles. In contrast, only a small population of cells stained TUNEL-positive after exposure to crystalline silica. Pulmonary retention of the two particulate silica compounds was also significantly different, showing a rapid post-exposure decrease for amorphous silica and prolonged retention for crystalline silica. Increased particle biopersistence may, therefore, be an additional factor in vivo for crystalline silica-induced mutagenicity, whereas increased cytotoxicity due to the very high administered dose of amorphous silica may have caused necrosis or apoptosis of mutated epithelial cells. The increased cytotoxicity could be a consequence of the very high numbers of alveolar neutrophils and their activation in the amorphous silica group.

It is conceivable that a direct genotoxic effect of persistent particles such as crystalline silica is involved in causing the observed mutations in epithelial target cells. For example, Daniel et al. (1993) found that incubation of DNA with very high doses of quartz resulted in DNA strand breaks. However, this is unlikely to occur in vivo since Driscoll et al. could not demonstrate such direct effect when dosing rat lung epithelial cells with crystalline silica in vitro (Driscoll et al., 1994Go). In contrast, they did observe increased mutation frequencies in the same assay after exposure of the rat lung epithelial cells to crystalline silica-elicited inflammatory cells.

A proposed mechanism in the generation of lung tumors in rats, after chronic particle exposure, is that of chronic inflammation-induced secondary genotoxic response. At the end of exposure, MIP-2 mRNA was increased to similar levels and coincided with lung neutrophils. During recovery, the persistence of MIP-2 expression and increased neutrophils was associated with crystalline silica exposure only. These results are consistent with those of Warheit et al. (1995) who demonstrated that short-term exposure to crystalline silica (50 and 150 mg/m3) produced persistent pulmonary inflammatory responses characterized by neutrophil recruitment and persistence for over one month post-exposure, whereas, amorphous silica (100 mg/m3) produced a transient pulmonary inflammatory response and most biochemical markers quickly returned to control levels once exposure had stopped.

Yuen et al. (1996) had also shown that crystalline and amorphous silica elicited increased pulmonary inflammation after intratracheal instillation; however, the response was transient for amorphous silica but was sustained post-crystalline-silica exposure. MIP-2 mRNA was increased to a similar magnitude and time course for both forms of silica. This suggests that cell types other than neutrophils are involved in mediating the MIP-2 response.

Increasing evidence implicates the type-II cell as a potential mediator of pulmonary recruitment and activation of inflammatory cells through the release of a variety of chemokines (Barrett et al., 1998Go; Hahon and Castranova, 1989Go; Lee and Rannels, 1996Go; Standiford et al., 1991Go). Recently, Driscoll and co-workers (1996b) reported that rat type-II cells express mRNA for the chemokines MIP-2 and CINC in response to direct interaction with {alpha}-quartz. The same authors also found that pre-treatment with an anti-MIP-2 antiserum before intratracheal instillation of {alpha}-quartz in rats reduced by 60% the accumulation of neutrophils in bronchoalveolar lavage fluid. Barrett et al. (1998) extended these findings, demonstrating increased mRNA levels for the chemokines MCP-1, MIP-2, and RANTES in a time- and dose-dependent fashion after cristobalite exposure of mouse lung epithelial cells (MLE-15) in vitro. Concurrence between increased MIP-2 expression and increased neutrophils further support a role for this chemokine in the inflammatory response to particle exposure.

Biopersistence of inhaled particles is a crucial endpoint to determining long-term lung damage in exposed animals. The clearance rate of amorphous silica from rat lungs at 50 mg/m3 appeared to be relatively rapid, considering the lung burdens of 756 and 883 µg/lung after 6.5 and 13 weeks of exposure, respectively. The rapid clearance may be explained by the fact that amorphous silica does demonstrate slight solubility in water (Iler, 1979Go) and is soluble in the lung. The degree of solubility of solid particles in general depends on the particle size and surface area. The amorphous silica used in this study has a small primary particle size (12 nm) and a large (200 ± 25 m2/g) surface area. In contrast, the clearance rate from rat lung of crystalline silica at 3 mg/m3, which demonstrated a comparable lung burden to the amorphous silica at the end of exposure, was relatively low, with lung burdens remaining at 657 and 743 µg/lung after 12 and 32 weeks of recovery, respectively. The biopersistence of SiO2 appears to be most important for long-term effects.

In summary, our results suggest that inflammation alone is not responsible for the development of mutagenic effects in rat lungs after high doses of particle exposures. The observation of mutagenic effects in our study only after crystalline silica exposures suggests additional factors, including biopersistence of particles and direct or indirect cytotoxicity to target cells, to be important determinants of secondary genotoxic events.


    ACKNOWLEDGMENTS
 
The authors thank Brian Howard, Nancy Corson, Pamela Mercer, Kiem Nguyen, and Rhonda Staversky for their excellent technical assistance. Also, we thank Judy Havalack for outstanding clerical assistance. These studies were partially supported by NIEHS grants ESO1247 and ESO4872.


    NOTES
 
1 To whom correspondence should be addressed at the University of Rochester Medical Center, Department of Environmental Medicine, 575 Elmwood Avenue, Box EHSC, Rochester, NY 14642. Fax: (716) 256-2631. E-mail: Gunter_Oberdorster{at}urmc.rochester.edu. Back


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ansari, B., Coates, P. J., Greenstein, D. B., and Hall, P. A. (1993). In situ end-labelling detects DNA strand breaks in apoptosis and other physiological and pathological states. J. Pathol. 170, 1–8.[ISI][Medline]

Baggs, R., Ferin, J., and Oberdörster, G. (1997). Regression of pulmonary lesions produced by inhaled titanium dioxide in rats. Vet. Path. 34, 592–597.[Abstract]

Barrett, E. G., Johnston, C., Oberdörster, G., and Finkelstein, J. N. (1998). Silica-induced chemokine expression in alveolar type-II cells is mediated by TNF-alpha-induced oxidant stress. Am. J. Physiol. 276, L979–L988.

Bowden, D. H., and Adamson, I. Y. (1984). The role of cell injury and the continuing inflammatory response in the generation of silicotic pulmonary fibrosis. J. Pathol. 144, 149–161.[ISI][Medline]

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