* Departments of Environmental Medicine and Pediatrics, The University of Rochester, Rochester, New York 14642; and
Procter and Gamble Pharmaceuticals, Health Care Research Center, Mason, Ohio 45040
Received January 18, 2000; accepted April 17, 2000
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ABSTRACT |
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Key Words: biopersistence; cytotoxicity; inhalation; mutagenicity; pulmonary inflammation; silica..
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INTRODUCTION |
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High cytotoxicity and long pulmonary retention of crystalline forms of silica are major factors responsible for its long-term effects (Langer, 1978; Quinot et al., 1979
). 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, 1992
; Reuzel et al., 1991
). 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, 1987; Kanematsu et al., 1980
). 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, 1995
). The degree of inflammation and its duration appear to be key elements for the secondary genetic response (Driscoll et al., 1995
). 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, 1994
; Nikula et al., 1995
; Nolte et al., 1994
). 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., 1996a
).
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.
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MATERIALS AND METHODS |
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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., 1992). 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., 1993a,b
). 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., 1996a
) and GAPDH (Nudel et al., 1983
) 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 2230 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., 1995). 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 1421 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.
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RESULTS |
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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 4. 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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DISCUSSION |
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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., 1994). 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., 1998; Hahon and Castranova, 1989
; Lee and Rannels, 1996
; Standiford et al., 1991
). 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
-quartz. The same authors also found that pre-treatment with an anti-MIP-2 antiserum before intratracheal instillation of
-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, 1979) 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.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Baggs, R., Ferin, J., and Oberdörster, G. (1997). Regression of pulmonary lesions produced by inhaled titanium dioxide in rats. Vet. Path. 34, 592597.[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, L979L988.
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, 149161.[ISI][Medline]
Chomczynski P, and Sacchi N. (1987). Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156159.[ISI][Medline]
Daniel, L. N., Mao, Y., and Saffiotti, U. (1993). Oxidative DNA damage by crystalline silica. Free Radic. Biol. Med. 14, 463472.[ISI][Medline]
Driscoll, K. E., Carter, J. M., Howard, B. W., Hassenbein, D. G., Pepelko, W., Baggs, R. B., and Oberdörster, G. (1996a). Pulmonary inflammatory, chemokine, and mutagenic responses in rats after subchronic inhalation of carbon black. Toxicol. Appl. Pharmacol. 136, 372380.[ISI][Medline]
Driscoll, K. E., Deyo, L. C., Carter, J. M., and Howard, B. W. (1994). Mutagenesis in rat lung epithelial cells after in vivo exposure to silica or ex vivo exposure to inflammatory cells. Resp. Crit. Care Med. 149, A553.
Driscoll, K. E., Deyo, L. C., Carter, J. M., Howard, B. W., Hassenbein, D. G., and Bertram, T. A. (1997). Effects of particle exposure and particle-elicited inflammatory cells on mutation in rat alveolar epithelial cells. Carcinogenesis 18, 423430.[Abstract]
Driscoll, K. E., Deyo, L. C., Howard, B. W., Poynter, J., and Carter, J. M. (1995). Characterizing mutagenesis in the hprt gene of rat alveolar epithelial cells. Exp. Lung Res. 21, 941956.[ISI][Medline]
Driscoll, K. E., Hassenbein, D. G., Carter, J., Poynter, J., Asquith, T. N., Grant, R. A., Whitten, J., Purdon, M. P., and Takigiku, R. (1993a). Macrophage inflammatory proteins 1 and 2: Expression by rat alveolar macrophages, fibroblasts, and epithelial cells and in rat lung after mineral-dust exposure. Am. J. Respir. Cell. Mol. Biol. 8, 311318.[ISI][Medline]
Driscoll, K. E., Howard, B. W., Carter, J. M., Asquith, T., Johnston, C., Detilleux, P., Kunkel, S. L., and Isfort, R. (1996b). Alpha-quartz-induced chemokine expression by rat lung epithelial cells: Effects of in vivo and in vitro particle exposure. Am. J. Pathol. 149, 16271637.[Abstract]
Driscoll, K. E., Simpson, L., Carter, J., Hassenbein, D., and Leikauf, G. D. (1993b). Ozone inhalation stimulates expression of a neutrophil chemotactic protein: Macrophage inflammatory protein 2. Toxicol. Appl. Pharmacol. 119, 306309.[ISI][Medline]
Dungworth, D. L., Mohr, U., Heinrich, U., Ernst, H., and Kittle, B. (1994). Pathologic effects of inhaled particles in rat lung: Associations between inflammatory and neoplastic processes. In Toxic and Carcinogenic Effects of Solid Particles in the Respiratory Tract (D. Dungworth, U. Mohr, J. Mauderly, and G. Oberdörster, Eds.), pp. 7598. ILSI Press, Washington, DC.
Ferin, J., Oberdörster, G., and Penney, D. P. (1992). Pulmonary retention of ultrafine and fine particles in rats. Am. J. Respir. Cell Mol. Biol. 6, 535542.[ISI][Medline]
Grasl-Kraupp, B., Ruttkay-Nedecky, B., Koudelka, H., Bukowska, K., Bursch, W., and Schulte-Hermann, R. (1995). In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: A cautionary note. Hepatology 21, 14651468[ISI][Medline]
Groth, D. H., Moorman, W. J., Lynch, D. W., Stettler, L. E., Wagner, W. D., and Horning, R. W. (1979). Chronic effects of inhaled amorphous silicas in animals. In Proceedings Symposium on Health Effects of Synthetic Silica Particulates, pp. 118143. Marbella, Spain.
Groth, D. H., Stettler, L. E., Platek, S. F., Lal, J. B., and Burg, J. R. (1986). Lung tumors in rats treated with quartz by intratracheal instillation. Cancer Research Monographs 2, 243253.
Hahon, N., and Castranova, V. (1989). Interferon production in rat type-II pneumocytes and alveolar macrophages. Exp. Lung Res. 15, 429445.[ISI][Medline]
Hemenway, D. R., Absher, M. P., Landesman, M., Trombley, L., and Emerson, R. J. (1986). Differential lung response following silicon dioxide polymorph aerosol exposure. In Silica, Silicosis and Cancer (D. F. Goldsmith, D. M. Winn, and C. Shy, Eds.), pp. 105116. Praeger, New York.
Hemenway, D. R., Absher, M. P., Trombley, L., and Vacek, P. M. (1990). Comparative clearance of quartz and cristobalite from the lung. Am. Ind. Hyg. Assoc. J. 51, 363369.[ISI][Medline]
IARC (International Agency for Research on Cancer) (1987). Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Vol. 42, IARC, Lyon, France.
IARC (International Agency for Research on Cancer) (1997). IARC Working Group on the evaluation of carcinogenic risks to humans: Silica, some silicates, coal dust and para-aramid fibrils. Monographs on the Evaluation of the Carcinogenic Risks to Humans. Vol. 68, IARC, Lyon, France.
Iler, R. K. (1979). The surface chemistry of silica. In The Chemistry of Silica: Solubility, Polymerization, Colloid and Surface Properties and Biochemistry (R. K. Iler, Ed.), pp. 622714. Wiley-Interscience, New York.
Kanematsu, N., Hara, M., and Kada, T. (1980). REC assay and mutagenicity studies on metal compounds. Mutat. Res. 77, 109116.[ISI][Medline]
Langer, A. M. (1978). Crystal faces and cleavage planes in quartz as templates in biological processes. Q. Rev. Biophys. 11, 543575.[ISI][Medline]
Lee, K. P., and Kelly, D. P. (1992). The pulmonary response and clearance of ludox colloidal silica after a 4-week inhalation exposure in rats. Fundam. Appl. Toxicol. 19, 399410.[ISI][Medline]
Lee, Y-V., and Rannels, D. E. (1996). Alveolar macrophages modulate the epithelial cell response to coal dust in vitro. Am. J. Physiol. 270, L123L132.
Morgan, A., Moores, S. R., Holmes, A., Evans, J. C., Evans, N. H., and Black, A. (1980). The effect of quartz administered by intratracheal instillation on the rat lung: I. The cellular response. Environ. Res. 22, 112.[ISI][Medline]
Nikula, K. J., Snipes, M. B., Barr, E. B., Griffith, W. C., Henderson, R. F., and Mauderly, J. L. (1995). Comparative pulmonary toxicities and carcinogenicities of chronically inhaled diesel exhaust and carbon black in F344 rats. Fundam. Appl. Toxicol. 25, 8094.[ISI][Medline]
Nolte, T., Thiedemann, K-U., Dungworth, D. L., Ernst, H., Paulini, I., Heinrich, U., Dasenbrock, C., Peters, L., Ueberschar, S., and Mohr, U. (1994). Histological and ultrastructural alterations of the bronchioloalveolar region in the rat lung after chronic exposure to a pyrolized pitch condensate or carbon black, alone or in combination. Inhal. Toxicol. 6, 459483.[ISI]
Nudel, U., Zakut, R., Neuman, S., Levy, Z., and Yaffe, D. (1983). The nucleotide sequence of the rat cytoplasmic beta-actin gene. Nucleic Acids Res. 11, 17591771.[Abstract]
Oberdörster, G. (1994). Extrapolation of results from animal inhalation studies with particles to humans. In Toxic and Carcinogenic Effects of Solid Particles in the Respiratory Tract (D. Dungworth, U. Mohr, J. Mauderly, and G. Oberdörster, Eds.), pp. 5773. Washington, DC: ILSI Press.
Oberdörster, G. (1995). Lung particle overload: Implications for occupational exposures to particles. Regul. Toxicol. Pharmacol. 21, 123135.[ISI][Medline]
O'Reilly, M. A., Staversky, R. J., Stripp, B. R., and Finkelstein, J. N. (1998). Exposure to hyperoxia induces p53 expression in mouse lung epithelium. Am. J. Respir. Cell Mol. Biol. 18, 4350.
Quinot, E., Cavelier, C., and Merceron, M. O. (1979). Surface chemistry and cytotoxic properties of silica. Biomedicine 30, 155160.[ISI][Medline]
Reiser, K. M., and Last, J. A. (1979). Silicosis and fibrogenesis: Fact and artifact. Toxicology 13, 5172.[ISI][Medline]
Reuzel, P. G., Bruijntjes, J. P., Feron, V. J., and Woutersen, R. A. (1991). Subchronic inhalation toxicity of amorphous silicas and quartz dust in rats. Food Chem. Toxic. 29, 341354.[ISI][Medline]
Saffiotti, U. (1992). Lung cancer induction by crystalline silica. Prog. Clin. Biol. Res. 374, 5169.[Medline]
Standiford, T. J., Kunkel, S. L., Phan, S. H., Rollins, B. J., and Strieter, R. M. (1991). Alveolar macrophage-derived cytokines induce monocyte chemoattractant protein-1 expression from human pulmonary type II-like epithelial cells. J. Biol. Chem. 266, 99129918.
Warheit, D. B., McHugh, T. A., and Hartsky, M. A. (1995). Differential pulmonary responses in rats inhaling crystalline colloidal or amorphous silica dusts. Scand. J. Work Environ. Health 21(Suppl. 2), 1921.
Yuen, I. S., Hartsky, M. A., Snajdr, S. I., and Warheit, D. B. (1996). Time course of chemotactic factor generation and neutrophil recruitment in the lungs of dust-exposed rats. Am. J. Respir. Cell Mol. Biol. 15, 268274.[Abstract]