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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Exposure to quartz and high concentrations of other poorly soluble particles can lead to the development of lung tumors in the rat. The mechanisms involved in particle-induced carcinogenesis seem to include inflammation-associated production of reactive oxygen species (ROS) and DNA damage. ROS induce 8-oxoguanine (8-oxoGua) and a panel of other oxidation products in DNA. In proliferating cells such DNA lesions can lead to various types of mutations, which might be critical for cancer-related genes with respect to tumor formation. Quartz is known to mediate the induction of 8-oxoGua in the nuclear DNA of lung cells when applied to the lung of rats. We have investigated the time- and dose-dependent biologic effects of quartz and, as a control, corundum, on cell proliferation and various pulmonary inflammation and toxicity markers in rat bronchoalveolar lavage fluid (BALF); on the induction of 8-oxoGua in the DNA of rat lung cells; and on the cellular levels of p53 wild-type and p53 mutant (mut) protein. Rats were exposed by intratracheal instillation to various amounts of quartz (0.3, 1.5, or 7.5 mg/rat) or corundum (0.3, 1.5, or 7.5 mg/rat) and measured at Days 7, 21, and 90 after exposure. Corundum had no adverse effects except a slight elevation of 8-oxoGua at a dose of 7.5 mg/rat. However, significant changes in the BALF were detected at all quartz doses. 8-oxoGua was significantly increased only at 1.5 and 7.5 mg quartz/rat. The amount of cells with detectable p53 wild-type protein levels was increased at 1.5 and 7.5 mg quartz/rat at 7 and 21 d. Elevated amounts of cells with enhanced p53 mut protein levels were measured at all time points after instillation of 7.5 mg quartz/rat.
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Introduction |
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Materials and Methods
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Tumorigenesis induced by chemical or physical carcinogens is a multistep process involving defined mutations in cancer-related genes of target cells at all stages of development (1). Genetic alterations can be triggered by a variety of DNA reactive agents, such as chemical carcinogens, ultraviolet light, ionizing radiation, and reactive oxygen species (ROS).
Recent in vitro and in vivo studies have demonstrated that the mutagenic and tumorigenic effects of particles in the lung are closely linked to the formation of ROS (2, 3). ROS generate different types of DNA alterations including single-strand breaks; 8-oxoguanine (8-oxoGua), the most frequently occurring mutagenic base modification; and various other DNA oxidation products (4). ROS derive from various exogenous sources but are also involved in physiologic reactions in the cell. To counteract this continuous burden most cells possess potent defense mechanisms that protect DNA from oxidative damage. These mechanisms comprise antioxidant systems for the deactivation of ROS molecules and efficient repair proteins for the elimination of oxidative DNA damage (5). Normally, formation of ROS and the potency of the cellular defense mechanisms lead to a basic steady-state level of 8-oxoGua and other oxidation products in DNA. Formation of large amounts of ROS can overload the cellular defense mechanisms and lead to elevated DNA alterations, increasing the mutation rates of proliferation competent cells (e.g., pneumocytes II, Clara cells, and bronchial epithelial cells) in the lung. Critical mutations for the genome of these cell types are, for example, mutations in proliferation control genes as the p53 tumor suppressor gene. Mutations in this gene frequently promote uncontrolled growth and lead to neoplastic transformation (8). Wild-type p53 accumulates in cells exposed to DNA-damaging agents. The most important functions of p53 are the mediation of growth arrest, to enable the cellular repair machine to eliminate mutagenic DNA modifications before replication and subsequent manifestation of mutations, or, alternatively, the mediation of apoptosis. p53 mutations are commonly missense mutations that exchange amino acids. These changes can alter the protein conformation, leading to increased stability of p53 protein or alteration of sequence-specific DNA binding and transcription factor activity of the protein (9). Both may lead to inactivation of the p53 control mechanisms. These mechanisms of particle-induced tumor formation in the rat, particularly the existence of scavenging and DNA repair, implicate nonlinear dose response and thresholds in critical DNA damage even for highly toxic particles such as quartz.
However, little is known about the amount and quality of particles that is necessary to overcome the cellular defense.
In the present study, proliferation, inflammation, and toxicity markers in bronchoalveolar lavage (BAL) fluids (BALFs) have been investigated after the exposure of the animals to increasing amounts of quartz (10) or to the low-nuisance dust corundum (11) and physiologic saline as a control. In addition, an immunocytologic assay (ICA) has been used for the quantification of 8-oxoGua in the DNA of individual cells in rat lungs. Finally, p53 wild-type and p53 mutant (mut) protein levels were determined as indicators for a general disturbance of the genome (p53) and as a hint for early-occurring mutations in cancer-related genes.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Exposure
Female Wistar rats (180 to 220 g body weight) were exposed by intratracheal instillation to quartz (DQ12; 0.3, 1.5, or 7.5 mg/rat) or corundum (0.3, 1.5, or 7.5 mg/rat) suspended in physiologic saline (0.5 ml) under slight ether anesthesia; control animals were exposed to physiologic saline or left untreated. Animals were killed at Day 7, 21, or 90 after treatment (10 animals/exposure and time point). Frozen lung-tissue sections were used for the immunohistochemical assays.
Inflammation and Toxicity Markers
BALs were performed at Day 7, 21, or 90 after exposure. Cells
contained in BALF were sedimented (400 × g, 10 min, 4°C), resuspended in phosphate-buffered saline (PBS), and counted. Differential cell counts were made from cytocentrifuge smears
stained with Pappenheim. Protein concentrations of the BALF
supernatants were measured by an assay based on the method of
Lowry and colleagues (12). The activity of tumor necrosis factor
(TNF)-
in BALF was determined by a cell lytic assay according
to Aggarwal and associates (13).
ICA
The ICA was performed essentially as described for the quantification of DNA alkylation products (14). In brief, frozen lung sections were fixed for 15 min in methanol and rehydrated in 2× SSC (300 mm sodium chloride and 30 mm sodium citrate, pH 7.2). After treatment with RNAse A (200 µg/ml 2× SSC) and RNAse T1 (50 units/ml 2× SSC) for 1 h at 37°C, sections were washed in 0.14 M NaCl. Cellular DNA was denatured by incubation in a solution containing 70 mM NaOH, 40% EtOH, and 0.14 M NaCl for 5 min at 4°C, followed by incubation with proteinase K (2 µg/ml) for 10 min at 37°C. To avoid nonspecific antibody binding, sections were incubated first with PBS containing 20% bovine serum albumin (BSA) for 20 min at room temperature (RT), and then with a rabbit anti-8-oxoGua antibody (0.5 µg antibody/ ml PBS containing 1% BSA; Squarix Biotechnology, Marl, Germany) for 16 h at 4°C. Unbound antibodies were removed by extensive washing with PBS containing 0.1% BSA. Goat antirabbit-immunoglobulin (Ig) G F(ab)2 fragments conjugated to rhodamine isothiocyanate (2 µg/ml PBS containing 1% BSA; Dianova, Hamburg, Germany) were added for 45 min at 37°C. After washing with PBS, nuclear DNA was stained with 4,6-diamidino-2-phenylindole (DAPI) (0.3 µM in PBS). Samples were then mounted in an antifading medium (PBS, pH 8.2, containing 20% glycerol, 10% polyvinylalcohol, and 0.03 M dithiothreitol).
Image Analysis
Fluorescence images were recorded and quantitated by a Hammamatsu cooled CCD video camera and a multiparameter image analysis program (Ahrens ACAS cytometry analysis system; Ahrens, Bargteheide, Germany) as described (14). Each value represents the average fluorescence intensity of about 100 individual cell nuclei.
p53-Protein Staining Procedure
Frozen tissue sections were fixated with EtOH (30 min), rehydrated with PBS, and treated for two 5-min periods in a 650-W microwave (in citrate buffer, pH 6.0). The sections were cooled to RT and then washed three times with destilled water (A. dest.) and three times with PBS. After preincubation with PBS/1% BSA (30 min, 37°C) anti-p53-protein antibodies were applied for 1 h at 37°C. After washing, the secondary antibody was added for 45 min, at 37°C, followed by DAPI counterstaining and mounting in antifading medium. To determine the amount of positive cells, at least 300 cells per section in four to six microscopic visual fields were examined.
Anti-p53-protein antibodies. (1) Pan (wild-type and mutant-specific): Sheep anti-p53 antiserum (Roche, Mannheim, Germany). Dilution: 1:40 in PBS containing 1% BSA. Secondary antibody: Rabbit antisheep-IgG antibody conjugated with rhodamine isothiocyanate (Dianova), diluted to 2 µg/ml in PBS/1% BSA.
(2) Mutant-specific (epitope aa 212-217): Ab-1 mouse monoclonal antibody (Lab Vision Corp., Fremont, CA). Dilution: 1:250 in PBS containing 1% BSA. Secondary antibody: Goat antimouse-IgG F(ab)2 fragments conjugated with fluorescein isothiocyanate (Dianova), diluted to 2 µg/ml in PBS/1% BSA.
Proliferation Marker Ki-67
For the detection of Ki-67-positive cells, frozen tissue sections were fixated with EtOH (30 min), rehydrated with PBS, and treated twice for 5 min each time in a 650-W microwave (in citrate buffer, pH 6.0). The sections were cooled to RT and then washed three times with A. dest. and three times with PBS. After preincubation with PBS/1% BSA (30 min, 37°C) Ki-67 mouse monoclonal antibodies (Dianova), diluted to 20 µg/ml in PBS/1% BSA, were applied for 2 h at RT. After washing, the secondary antibody (rabbit antimouse-tetramethylrhodamine isothiocyanate; Dianova) was added for 45 min at 37°C, followed by DAPI counterstaining and mounting in antifading medium. To determine the amount of positive cells, at least 300 cells per section in four to six microscopic visual fields were examined.
Statistics
Results are given as means ± standard deviation (SD). Significant differences were calculated using a Mann-Whitney U test on data. A value of P < 0.05 was considered significant.
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Analysis of BALFs
Exposure of Wistar rats to quartz resulted in significant, concentration-dependent changes in BALF parameters; whereas after exposure to corundum, only in a few cases were statistically significant changes in the corresponding BALF parameters detected.
Total cell numbers. After 1.5 mg of quartz/rat the total cell numbers were elevated 3- to 4-fold at Days 3 and 21, and 5-fold at Day 90 in comparison with untreated or NaCl-treated animals. At 7.5 mg, cell numbers were increased to 4-fold at Day 3, 6-fold at Day 21, and more than 30-fold at Day 90. Instillation of 0.3 mg quartz/rat or corundum did not lead to statistically significant elevation of cell numbers (Figure 1).
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Absolute amount of neutrophils. After instillation of 0.3 mg quartz/rat, the absolute amount of neutrophils in BALF was elevated 4-fold at Day 21 and 38-fold at Day 90. After 1.5 mg, the amount of neutrophils was increased 23-fold at Day 3 to 153-fold at Day 90. At 7.5 mg, the values increased from 57-fold at Day 3 to more than 500-fold at Day 90. Instillation of corundum significantly increased the absolute amount of neutrophils (4- to 10-fold at 7.5 mg only at Day 3) (Figure 2).
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Percentage of neutrophils. After instillation of 0.3 mg quartz/rat, the percentage of neutrophils of all cells in the BALF was about 3% at Day 3 and increased to 35% at Day 90. After 1.5 mg, the percentage increased from 15% at Day 3 to 46% at Day 90. At 7.5 mg, they increased from 33% at Day 3 to 53% at Day 90. Instillation of corundum resulted in a slightly increased percentage of neutrophils (4 to 8%) only at 7.5 mg (Figure 3).
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Protein concentration in BALFs. The protein concentration after 0.3 mg of quartz was significantly increased (1.5 to 2 times controls) only after 90 d. At 1.5 mg, the protein concentration increased at all time points (2 to 3 times), whereas after 7.5 mg the protein concentration steadily increased from Day 7 (3 times control) to Day 90 (6 times control). After corundum, no statistically significant increase of protein content was measured (Figure 4).
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Activity of TNF-. Significant elevated activity of TNF-
was observed in BALFs at Days 3 and 90 after instillation
of 7.5 mg of quartz (Figure 5), with a characteristic reduction at Day 21. After instillation of corundum, a significant
elevation after 3 d, and a clear reduction (compared with
controls) after 90 d were measured.
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Quantification of 8-oxoGua in Single Cells of Lung Tissue Sections
For the ICA, an immunoaffinity-selected rabbit anti-8-oxoGua antibody was used. As shown in Figure 6B (1.5 mg DQ12/rat 90 d after instillation), binding of the antibody molecules (Figure 6B iii, red fluorescence) was restricted to the nuclear DNA of the alveolar cells (Figure 6B ii, blue fluorescence) and exhibited a clearly differing fluorescence intensity compared with untreated control tissue (Figure 6A iii).
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The results obtained by quantitative evaluation of the fluorescence signals with an image analysis system are summarized in Figure 7. Exposure to quartz induced significantly increased 8-oxoGua levels in lung cells after 21 and 90 d at concentrations higher than 0.3 mg, whereas exposure to 0.3 mg resulted in 8-oxoGua levels that were not significantly higher than those determined in control animals. At 90 d after instillation of 7.5 mg of quartz, the 8-oxoGua content was significantly increased compared with 21 d; whereas at 1.5 mg exposure, 8-oxoGua did not increase after 21 d. Exposure to corundum at 0.3 and 1.5 mg induced 8-oxoGua levels undistinguishable from untreated controls and physiologic saline. Instillation of 7.5 mg generated elevated levels of 8-oxoGua at all time points, but the elevation was not statistically significant. Cell type and region-specific analysis of samples from animals exposed to quartz revealed that no cells were detected which significantly differed from the average 8-oxoGua levels calculated for alveolar cells.
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p53 Immunostaining
Unexposed animals and animals treated with 0.3 and 1.5 mg of corundum were negative for p53 immunostaining throughout the lung. Exposure to 7.5 mg corundum led to a positive staining in less than 0.1% of the lung cells during the whole experiment. At 0.3 mg quartz, positive-stained cells could be detected only at 21 d after instillation. Treatment with 1.5 and 7.5 mg of quartz induced clearly elevated amounts of p53-positive cells, with the maximum (2% positive cells) after 3 d. After 1.5 mg of quartz, positive cells decreased to control amounts at 90 d; whereas after 7.5 mg of quartz at 90 d, about 0.6% of the lung cells were still p53-positive (Figure 8). Mut p53 immunostaining was positive only for animals treated with 1.5 and 7.5 mg quartz. At 7.5 mg of quartz, mut p53 remained constant during the whole experiment (Figure 9); whereas at 1.5 mg of quartz, only after 3 d was a significant amount of positive cells detected.
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Cell Proliferation
Only in quartz-treated animals could a significant increase of Ki-67-positive cells be found, indicating enhanced cell proliferation activity. At 3 d after exposure the Ki-67 staining had a maximum of about 20% positive cells (Figure 10). Nontreated and corundum-treated animals exhibited a basal level of 5 to 10% positive cells, consistent with our previous findings using bromodeoxyuridine (BrdU) labeling (3).
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Exposure of rats to quartz can induce lung tumors (15, 16). In addition, high concentrations of other poorly soluble or insoluble particles, including titanium dioxide, carbon black, and diesel exhaust, also result in an enhanced frequency of lung tumors (17). The detailed route by which these materials can exert their tumorigenic potential still remains to be clarified. It is, however, evident that tumor development is preceded by an inflammatory response in the lung caused by the deposition of such particles. Though inflammation does not necessarily lead to enhanced rates of tumors as does, for example, neurodegenerative or cardiovascular disease (18), persistent pulmonary inflammation seems to be the major risk factor for carcinogenesis. Pulmonary inflammation in the rat is associated with a series of processes including recruitment of macrophages and neutrophils, the release of large amounts of genotoxic ROS (hydrogen peroxide, hydroxy radicals, nitric oxide, and other oxidants) from these cell types, cytotoxic effects leading to cell leakage and apoptotic cell death, and enhanced synthesis of surfactant phospholipids in pneumocytes II (19).
In particular, formation of ROS and cell proliferation may play a key role in the formation of particle-induced lung tumors: ROS can produce 8-oxoGua and various other mutagenic lesions in genomic DNA which, if not repaired before replication, can activate proto-oncogenes or inactivate tumor-suppressor genes (19, 23, 24).
In the present study we have analyzed dose-dependent
effects of quartz (DQ12) and corundum on the alveolar
cells in the lung of rats. Only the exposure to quartz at doses
of 1.5 and 7.5 mg/animal resulted in significant changes in
all of the five BALF parameters examined, indicating a
relevant inflammation in the lung. Thus, the concentrations of protein, as well as the number of neutrophils in
BALFs, have already been elevated 3 d after exposure and
were increased after 21 d and 3 mo. Particularly the numbers of neutrophils were markedly increased for all quartz
concentrations at the end of the study. TNF- activity reflects the release of cytokines from macrophages and neutrophils, as well as the cytokine activity of the alveolar
structure cells. Quartz application led initially to clearly elevated TNF- activities in the BALF but were reduced at
the low quartz dose to control levels after 90 d. At 1.5 and 7.5 mg of quartz, TNF- activity underwent a U-shaped
activity profile during the 90 d of observation. The reduction of the TNF- activity after the initial excessive activity response to quartz may be an attempt on the part of the
host to protect the organ against the development of uncontrolled proliferative and fibrotic processes. Recent
publications describe a downregulation of TNF- during
the course of pulmonary inflammation and fibrosis induced by inorganic particles that is associated with and at
least partly caused by an overproduction of soluble TNF-
receptor and interleukin-10 (25). Permanent excessive inflammation as observed in the rat may override these protection mechanisms, leading to high activities of TNF-. It
could be speculated that the induction of anti-inflammatoric regulatory mechanisms is dose-dependent: Low levels of inflammation do not lead to a response.
The formation of 8-oxoGua in the DNA of the lung cells was significantly increased only after 1.5 and 7.5 mg of quartz, whereas at 0.3 mg no significant increase of 8-oxoGua could be detected. After exposure to corundum, no differences were found between control animals and exposed animals in the majority of the BAL parameters. Only exposure to corundum at 7.5 mg/animal resulted, in the early stage, in slightly elevated numbers of neutrophils, and also in moderately increased levels of 8-oxoGua. The percentage of p53 protein- and p53 mut protein-positive cells was enhanced only after treatment with quartz. The number of p53-positive cells returned to normal at 90 d after exposure, except in animals treated with 7.5 mg quartz. This effect was also described by Mishra and coworkers (26) after asbestos treatment of rats. At first sight this observation seems astonishing, because DNA lesions such as 8-oxoGua are permanently formed in the cellular DNA of lung cells also after 1.5 mg quartz treatment and should induce p53 expression. The observed reduction can be explained in part by an increased elimination of the positive cell fractions via p53-controlled apoptosis. Further, it can be speculated that there is an adaptation of lung cells to the particle-induced radical reactions. After 90 d the amount of the p53 protein-positive cells detected by the pan-antibody was the same as the amount of p53 mut protein-positive cells and may represent only the p53 mut fraction detected by the pan-antibody. The p53-mut results demonstrate that mutations in a cancer-related gene occur after instillation of quartz, but seem to persist even for 3 mo only after 7.5 mg treatment.
To exclude a crossreactivity of the mut antibody with wild-type p53 protein and to analyze the species-specificity of the antibody (all p53 mut antibodies are developed against human p53 mut protein, but are described to crossreact with rat), sequence analyses for the antibody-specific mutation loci in positively stained cells are underway.
Ki-67 staining was used as a proliferation marker. Proliferation is an important prerequisite for manifestation of the genomic mutations outlined earlier. Quartz-treated animals exhibited a significant elevated proliferation 3 d after treatment which decreased during the 90 d of experiment. These findings are in some contrast to proliferation data measured after incubation with BrdU in our earlier experiments (3). In these experiments (3), alveolar cell proliferation after treatment with 2.5 mg of quartz was enhanced significantly during the whole period of 90 d after exposure. It is known that determination of DNA replication by BrdU or other proliferation markers is very often not completely consistent (27). Further experiments may have to use different methods for proliferation activity.
The present study indicates that there may exist a threshold for the formation of mutagenic DNA lesions, as represented by the major adduct 8-oxoGua, in the rat lung during the observed period. The formation of ROS induced by 0.3 mg of quartz seems to be tolerated by the lung defense mechanisms.
Driscoll and colleagues (28) suggested that there is a strict correlation between the amount of neutrophils, ROS release, and oxidative damage. Our experiments revealed, at least for quartz exposure, a correlation between 8-oxoGua burden and the number of neutrophils. Further, our results indicate an adaptive response resulting in a (limited) induction of defense mechanisms. Application of 7.5 mg of quartz after 3 d or 1.5 mg after 21 d resulted in increased amounts of neutrophils (35% of the cellular fraction in the BALF) and markedly elevated contents of 8-oxoGua in the DNA of alveolar cells, whereas 0.3 mg of quartz resulted in comparable amounts of neutrophils at Day 90 but no significant increase of 8-oxoGua in the DNA. At Day 90 after instillation of 1.5 mg, enhancement of neutrophils to 43% of the cellular fraction did not correlate with a further increase of 8-oxoGua, indicating a new steady-state situation between production of ROS and cellular defense. After 7.5 mg of quartz the enhancement of neutrophils to 50 to 60% in the cellular fraction seems to exceed the defense, resulting in a continuous increase of 8-oxoGua. A higher level of antioxidant defenses after oxidative stress can be observed in both prokaryotes and eukaryotes in rodents and humans and has been documented by different authors (e.g., 29). Antioxidant defense can occur via two general mechanisms: the scavenging of ROS and the repair of DNA damage that derives from the interaction of these radicals with the cellular DNA. Recent studies in different species demonstrated the induction of scavenging molecules after exposure to reactive oxygen or other mutagenic agents that are thought to act via O-radical reactions: In the lung tissue of rats exposed to increased amounts of oxygen (30) an increase in the content of glutathione and an upregulation of manganese-, copper-, and zinc-superoxide dismutase gene expression after exposure to asbestos fibers and quartz (31) were shown. In an interspecies study between the reaction of rat and hamster to quartz and carbon black particle exposure, induction of the antioxidant proteins catalase and metallothionine was measured (32). In human lungs, a reduced persistence of oxidative DNA lesions was observed after adaptive hyperbaric oxygen therapy (33) that may depend on enhancement of either scavenging or repair mechanisms. Until now it was not known whether chronic application of DNA-reactive ROS leads to induction of repair enzymes in eukaryotic cells. Increased levels of a DNA repair enzyme have been demonstrated in prokaryotic cells (34); and recently it was shown that the DNA glycosylase OGG1 (7), which is involved in 8-oxoGua repair, was induced by the treatment of rats with diesel exhaust (35). In contrast, instillation of high amounts of corundum did not induce markedly elevated numbers of neutrophils and other inflammation-associated markers, but slightly elevated the contents of 8-oxoGua in the DNA of alveolar cells. These findings indicate that after the application of high amounts of particles there may also be other, neutrophil-independent, mechanisms contributing to a higher production of 8-oxoGua and other mutagenic DNA lesions in the genome of target cells. After application of 1.5 and 7.5 mg of quartz, as well as 7.5 mg of corundum, the mutagenic lesion 8-oxoGua persists at higher levels in the genome of lung cells at least for 3 mo. Driscoll and associates (28) demonstrated that significantly elevated mutation frequencies in the hprt gene occured 15 mo after the instillation of at least 2 mg of quartz/animal. At this time the percentage of neutrophils in BALFs was still higher than 40%, indicating a permanent inflammation during the whole period. The elevated mutation rates only after 2 mg of quartz/animal are consistent with our data for the occurrence of lung cells positive for the mut p53 protein at a concentration higher than 1.5 mg/rat during the 90 d of the experiment.
At present, it is not yet clear for how long and to what extent 8-oxoGua and other ROS-induced DNA lesions have to be elevated in target cells to induce sufficient mutations in cancer-related genes to initiate the multistep process of carcinogenesis; however, it seems evident that quartz-induced tumor formation is triggered only by a permanent and significant inflammatory response in the lung. Our results indicate that low doses of quartz do not lead to such inflammation and consequently may not increase the cancerogenic risk in rats.
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Address correspondence to: Dr. Joachim Bruch, Institute of Hygiene and Occupational Medicine, University of Essen Medical School, Hufelandstrasse 55, 45 122 Essen, Germany.
(Received in original form March 21, 2000 and in revised form November 30, 2000).
Abbreviations: bronchoalveolar lavage, BAL; BAL fluid, BALF; bromodeoxyuridine, BrdU; bovine serum albumin, BSA; 4,6-diamidino-2-phenylindole, DAPI; immunocytologic assay, ICA; mutant, mut; 8-oxoguanine, 8-oxoGua; phosphate-buffered saline, PBS; reactive oxygen species, ROS; room temperature, RT; standard deviation, SD; tumor necrosis factor, TNF.Acknowledgments: This work was supported by the Minister für Wirtschaft, Mittelstand und Technologie des Landes Nordrhein-Westfalen, Arbeitsgemeinschaft Staub- und Silikosebekämpfung, Deutsche Forschungsgemeinschaft (DFG) Project No. BR875/3-1, and Stockhausen GmbH and Co.KG, Krefeld, Germany.
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