* Indiana University School of Medicine, Division of Toxicology, Department of Pharmacology and Toxicology, 635 Barnhill Drive, MS 1021, Indianapolis, Indiana 46202; and
National Institute of Environmental Health Sciences, Laboratory of Experimental Pathology, 111 T. W. Alexander Drive, MD B3-06, Research Triangle Park, North Carolina 27709
Received February 26, 2001; accepted June 14, 2001
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ABSTRACT |
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Key Words: indium phosphide; oxidative stress; inflammation; lung carcinogenesis; immunohistochemistry.
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INTRODUCTION |
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Because of its recent widespread usage, IP was selected for chronic toxicity investigations by the National Toxicology Program (NTP). Three-month exposure of rats by inhalation of IP at doses of 0, 1, 3, 10, 30, or 100 mg/m3 resulted in a spectrum of inflammatory and proliferative lesions in the lungs of all exposed groups. These changes consisted of alveolar proteinosis, chronic inflammation, interstitial fibrosis, and alveolar epithelial hyperplasia (NTP, 2000).
Inhalation exposure to 0.03 mg/m3 for 2 years, or 0.1 or 0.3 mg/m3 for 21 weeks (exposure stopped at 21 weeks) was associated with increased incidences of alveolar and bronchiolar adenomas and bronchiolar carcinomas in rats of both sexes. Squamous cell carcinoma of the lung occurred in four male rats exposed to 0.3 mg/m3 (NTP, 2000). A spectrum of inflammatory and proliferative lesions of the lung was observed in all exposed groups of both sexes. This paper deals with IP-related lung pathology; additional information can be found in the NTP technical report (NTP, 2000
).
Exposure by inhalation to particles like asbestos (Muhle and Pott, 2000) or crystalline silica (Ding et al., 2000
) is known to be associated with the development of lung tumors in rats (Nehls et al., 1997
). Inflammation, in conjunction with the production of reactive oxygen species (ROS) and enhanced epithelial cell proliferation, has been proposed as the mechanism of this particle-induced tumor formation (Mossman, 2000
). Interactions of xenobiotics with several enzyme systems have resulted in the formation of free-radical intermediates, which are further able to activate molecular oxygen to superoxides (Mason, 1982
). Reactive oxygen is recognized as an important factor in gene expression by serving as a second messenger in signal transduction pathways. This function in cell signaling has been well established for H2O2 and O2-, as well as NO. At least 127 genes and signal-transducting proteins have been reported to be redox-sensitive (Allen and Tresini, 2000
; Lander, 1997
). Many of these cell-signaling pathways are known to be involved in cell proliferation, differentiation, and apoptosis. The imbalance of these biological end points is an important step in the development of cancer. The current concept of oxidative stress emphasizes the balance between oxidants and antioxidants; an imbalance of too few oxidants and too many antioxidants is considered oxidative stress. Chronic, prolonged inflammation is associated with the release of highly reactive oxygen and nitrogen species from inflammatory cells. These reactive molecules interact with DNA in proliferating epithelium to produce permanent genomic alteration (De Marzo et al., 1999
). High NO levels in inflammation are cytotoxic to viruses and bacteria, but are also able to damage cellular organelles and cause further chronic inflammation (Nathan and Xie, 1994
). Excessive amounts of NO have been found to be cytotoxic in several cell lines (Messmer et al., 1994
), but also protective against apoptosis in others (Kim et al., 1997
). Another function of NO is scavenging superoxide radicals produced by inflammation, thereby inhibiting lipid peroxidation (Rubbo et al., 2000
).
The lung is more susceptible to oxidative injury than any other organ in the body because of constant exposure to air that might contain toxic particles or oxidant gases such as nitrogen oxide or ozone (Nehls et al., 1997). Lungs have a high rate of blood perfusion, which makes them more likely to be exposed to xenobiotics (Vallyathan and Shi, 1997
). Chemical-induced carcinogenesis by inhalation was recently reviewed by Emmendoerffer et al. (2000), who examined confounding factors including the extent of the inflammatory response on lung neoplastic development.
To address our proposal that IP causes lung tumors via oxidative stress from inflammatory cells, we stained lungs of control and high-dose animals immunohistochemically to localize four different markers considered to be indicative of oxidative stress: inducible nitric oxide (i-NOS), cyclooxygenase 2 (COX-2), glutathione S-transferase Pi (GST-Pi), and 8-hydroxydeoxyguanosine (8-OHdG). Tissues used in this investigation were paraffin-embedded lungs from the 2-year NTP study (NTP, 2000).
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MATERIALS AND METHODS |
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Aerosol generation and exposure system.
The study laboratory designed stainless steel inhalation exposure chambers (Hartford Systems Division of Lab Products, Inc., Aberdeen, MD), ensuring the maintenance of uniform aerosol concentrations throughout. The total active mixing volume of each chamber was 1.7 m3. Uniformity of aerosol chamber concentration was evaluated every 2 to 4 months and found acceptable throughout the study. Additional technical details concerning IP exposure are given in the NTP report (NTP, 2000).
Animals.
Sixty male and 60 female F344/N 4-week-old rats (Taconic Farms, Germantown, NY) per exposure group were quarantined for approximately 14 days. Their health was monitored during the studies according to the protocols of the NTP Sentinel Animal Program. Animals were housed individually; feed (except during exposure) and water were available ad libitum.
The rats were exposed to aerosols of IP at levels of 0, 0.03, 0.1, or 0.3 mg/m3 for 6 h/day, 5 days/week, for 21 weeks (0.1 or 0.3 mg/m3 groups) or 105 weeks (0 or 0.03 mg/m3 groups), with interim groups (10/group/sex) evaluated at 3 months. Animals in the 0.1 and 0.3 mg/m3 groups were maintained on filtered air from exposure termination at week 21 until the end of the studies. Exposure to IP was stopped in these dosed groups because of a 1.6- to 2.1-fold increase in lung weights and the severity of the lung lesions after 3-month exposure (NTP, 2000).
Pathology.
Complete necropsies and microscopic examinations were performed on 0 and 0.3 mg/m3 interim study rats at 3 months, and all remaining rats at the end of the study. Heart, right kidney, liver, lung (left and right lobe), right testis, and thymus were examined and weighed from 0.03 and 0.1 mg/m3 interim study rats at 3 months. At necropsy, all organs and tissues were examined for grossly visible lesions, and all major tissues were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 46 µm, and stained with hematoxylin and eosin (H&E) for microscopic examination. A quantitative assessment for lung pathology was performed to evaluate the severity of the different findings (Cherniack et al., 1991).
Immunohistochemical investigation.
For the immunohistochemical evaluations, paraffin-embedded samples were taken from the lungs of female rats only, as the pulmonary damage appeared virtually the same in both sexes. To investigate the most severe lesions, only lungs of the high-dose animals were stained immunohistochemically. Samples from each of the following groups were used:
Immunohistochemistry was performed using the avidin-biotin-peroxidase method. Sections were stained with one of the following antibodies: i-NOS (Transduction Laboratories, Lexington, KY), COX-2 (Caymen Chemical, Ann Arbor, MI), GST-Pi (BioGenex, San Ramon, CA), or 8-OHdG (Japan Institute for the Control of Aging, Haruoka, Fukuroi City, Japan).
i-NOS and COX-2.
Following dehydration through a series of graded alcohols, all slides were placed in 1X Automation Buffer (AB) (Biomeda, Foster City, CA) and blocked for endogenous peroxidase activity with 3% H2O2 for 15 min. Heat-induced epitope retrieval for all slides began by incubation in 1X citrate buffer, pH 6.0 (Biocare Medical, Walnut Creek, CA). Slides stained for i-NOS were placed in a steamer (Black & Decker, Brockville, Ontario, Canada) for 30 min, cooled for 10 min, rinsed in water, and placed in buffer. Slides stained for COX-2 were heated for 5 min in a microwave oven at 50% power for a total of two cycles. Between cycles, fresh citrate buffer (50 ml) was added. After microwaving, the slides were allowed to cool for 15 min, rinsed in distilled water, and placed in 1X AB buffer. All incubations were carried out in a humidified chamber at room temperature (RT°). Slides were blocked with 5% normal goat serum for 20 min (Jackson Immunoresearch, West Grove, PA), then incubated with the primary antibody, i-NOS 1:100 or COX-2 1:300, for 1 h. Normal rabbit serum (Jackson Immunoresearch, West Grove, PA) was used as the negative control. For i-NOS staining, the positive control was LPS-treated rat liver. For COX-2 staining, the positive control was mouse vas deferens (ductus deferens). Slides were adequately washed in 1X AB buffer; incubated with the secondary antibody, biotinylated goat anti-rabbit IgG (for i-NOS 1:400, for COX-2 1:500; Vector Laboratories, Burlingame, CA) for 30 min, washed again, then labeled with an avidin-biotin complex (Vector Elite Kit) for 30 min. Slides were washed in 1X AB buffer, counterstained with Harris hematoxylin (Harelco, Gibbstown, NJ), rinsed in water, rehydrated through alcohol to xylene, and coverslipped with Permount (Surgipath, Richmond, IL).
8-OHdG and GST-Pi.
Following dehydration through a series of graded alcohols, the slides for 8-OHdG determination were placed in Bouin's fixative for 3 h at RT°, washed in 80% ethanol four times, then left therein overnight. Tissues were blocked for endogenous peroxidase activity with 0.3% H2O2 in methanol for 30 min and rinsed in PBS buffer. For denaturation of DNA, the slides were placed in 0.05 N NaOH in 40% ethanol for 12 min, rinsed in PBS, and incubated with 250 µg/ml RNase for 1 h. The RNase was rinsed off with PBS, and an incubation in 5% skim milk in PBS for 90 min followed. An avidin/biotin block (Vector Laboratories, Burlingame, CA) was applied for 20 min. The primary 8-OHdG antibody (IgG1, 20 µg/ml, 1:50) was then applied to the slides overnight at 4°C.
The GST-Pi slides were placed in PBS after dehydration; endogenous peroxidase was blocked by 3% H2O2 for 15 min, and slides were incubated with 5% skim milk for 90 min at RT°. The primary polyclonal rabbit anti-GST-Pi antibody (BioGenex, San Ramon, CA) was applied at 1:200 for 3 h at 37°C. All slides (GST-Pi and 8-OHdG) were then washed adequately in PBS and incubated with BioGenex biotin-labeled antimouse antibody followed by BioGenex streptavidin-conjugated peroxidase, each at 1:20 for 20 min at RT°. The peroxidase binding was demonstrated by the reaction with 3-amino-9-ethylcarbazole. Rinsing in deionized water and counterstaining with Meyer's hematoxylin followed. Tissue was covered with BioGenex SuperMount overnight and coverslipped with Eukitt® (Calibrated Instruments, Hawthorne, NY).
As a control for the specifity of the reaction for 8-OHdG, some slides were incubated with formamidopyrimidine DNA glycosylase (Fpg) protein, a trifunctional DNA base excision repair enzyme that removes a wide range of oxidatively damaged bases via N-glycosylase activity and cleaves both the 3`- and 5`-phosphodiester bonds of the resulting apurinic/apyrimidinic site. By application of the enzyme for 1 h prior to that of the primary antibody, a large amount of 8-OHdG in the tissue should be repaired and so not react with the primary antibody.
As a positive control for GST-Pi staining, livers were used from rats that were treated with diethylnitrosamine (DEN) over a period of 2 weeks, kept without treatment for 7 months for the development of liver foci, then treated with lipopolysaccharide for 1 week.
Grading of labeling.
The immunopositivity of the sections was graded by two pathologists using a scale ranging from 0 () to 4 (++++). Only a strong specific immunohistochemical reaction was considered and graded as follows, with approximate percentages indicating those numbers of relevant cells showing a strong positive reaction: (0) = No specific immunohistologic reaction visible; (1) = 1 to 10% of relevant cells showing a strong positive reaction; (2) = Up to approximately 25%; (3) = Up to approximately 50%; (4) = More than approximately 50%.
In case of disagreement of single grades, an average was taken. The immunohistochemical reactivity of each histological category (normal tissue, atypical hyperplasia, chronic active inflammation, alveolar epithelium hyperplasia, squamous cyst, alveolar/bronchiolar carcinoma, alveolar/bronchiolar adenoma) was assessed separately.
Statistical analysis.
For evaluation of the statistical significance of the immunopositivity, we used a new test procedure recently introduced by Peddada et al. (manuscript submitted) to detect a broad range of trends. This procedure is based upon a general methodology developed by Hwang and Peddada (1994). We first converted the data in terms of ranks, then estimated the mean ranks under the hypothesized trend. Using these trend estimates we obtained our test statistic, which is the difference between the largest mean rank and the smallest mean rank. The null distribution of this statistic was obtained by the bootstrap trend test, conservative for correlated data (Peddada et al., manuscript submitted). For COX-2, 8-OHdG, and GST-Pi labelings, the hypothesis was that the immunopositivity increases from hyperplasia to adenoma to carcinoma to atypical hyperplasia to squamous cyst. For the i-NOS stain, the hypothesis asserted that the distribution of immunopositivity increases from adenoma to carcinoma to atypical hyperplasia. For i-NOS evaluation, we did not include hyperplasia and squamous cyst due to sparsity of data.
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RESULTS |
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Survival and Body Weights
The survival rates of chamber controls and treated animals were approximately equal (NTP, 2000). The average body weights of exposed rats compared to chamber controls were similar (NTP, 2000
).
Pulmonary Pathology
The lung weights of all groups of exposed female rats were significantly (1.2- to 2.2-fold) greater than those of the chamber controls after 3 months (NTP, 2000). Most exposed animals showed dose-dependent increased incidences of chronic active inflammation (Table 1
), which is characterized by numerous histiocytes and mononuclear inflammatory cells and fewer pleomorphonuclear (PMNL) inflammatory cells within the alveoli and interstitia (Figs. 1A
D). Other changes included the presence of foreign bodies (indium phosphide particles) visible as black dots within the alveoli or macrophages (not shown), alveolar epithelial hyperplasia, and alveolar proteinosis (Table 1
).
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Chronic inflammation (Figs. 1EF) consisted of accumulations of alveolar macrophages with foamy cytoplasm, occasional multinucleated giant cells and cholesterol clefts, cell debris, and some neutrophils. A thickening of the alveolar interstitium and the overlying pleura was observed frequently in connective tissues. Alveolar proteinosis, characterized by diffuse homogeneous to granular deposition of eosinophilic material, was noted within the alveolar lumen, mostly around the areas of chronic inflammation. Scattered black dots (IP particles) less than 1 µm in diameter were found within alveoli and phagocytic cells.
Atypical proliferation of alveolar/bronchiolar epithelium with fibrous components (Figs. 1I and 1J and 2AD) was also found in these animals. The lesions, whose size ranged from a few hundred micrometers to more than 1 cm in diameter, were round with a central fibrous core containing dispersed alveolar structures lined with uniformly cuboidal epithelial cells. Aggregates of chiefly necrotic inflammatory cells were present in adjacent alveoli and often within the glandular structures. At the periphery, the proliferative lesions contained one to many layers of epithelium, which sometimes extended into neighboring alveoli. These cells showed pleomorphism and sometimes contained mitotic figures. Small lesions were seen close to areas of chronic inflammation. Small lesions with some peripheral epithelial proliferation were diagnosed as "atypical hyperplasia," whereas larger lesions with more epithelial proliferation and cellular polymorphism and/or local invasion were diagnosed as alveolar/bronchiolar adenoma or carcinoma.
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Alveolar/bronchiolar adenomas consisted of clearly outlined masses compressing and surrounding normal lung tissue and exhibited acinar, papillary, and/or compact structure. These epithelial cells within adenomas were mostly of one form and resembled hyperplastic epithelial cells.
Alveolar/bronchiolar carcinomas (Figs. 2I and 2J) displayed a more heterogeneous pattern, cellular pleomorphism, and atypia with local invasion or metastasis.
Immunohistological Evaluation
The immunohistochemical markers revealed distinct patterns of localization (Tables 3 and 4). Staining for i-NOS resulted in a cytoplasmic expression, mainly in macrophages, not in epithelial cells (Figs. 1A, 1B, 1E, 1F, 1I, and 1J
). COX-2 immunolabeling demonstrated a cytoplasmic concentration of chromogen in macrophages and epithelial cells included within lesions (Figs. 1C and 1D and Figs. 2D, 2G, 2H, and 2J
). GST-Pi was expressed mainly in the cytoplasm and partially in the nuclei of epithelial cells (Figs. 1G and 2A, 2B, 2E, and 2I
); few macrophages expressed GST-Pi. 8-OHdG was localized within nuclei of epithelial cells (Figs. 1H and 2C and 2F
); few macrophages expressed 8-OHdG.
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After 3 months, treated animals (Table 3) of the highest-dose group (0.3 mg/m3) manifested relatively extensive i-NOS (average grade 4.0; Figs. 1A and 1B
) and COX-2 staining (average grade 2.8; Figs. 1C and 1D
) within infiltrating macrophages in sites of chronic active inflammation, the major lesion in these animals. Although portions of some lesions showed high grades of staining of GST-Pi, its overall expression was only present in a small percentage of cells in chronic active inflammation and epithelial hyperplasia. 8-OHdG, expressed only in some animals, appeared in a small percentage of cells within chronic active inflammation. Alveolar proteinosis, diagnosed in all treated animals of this group, showed a relatively high percentage of COX-2positive area within the proteinaceous material (average grade 4.0).
In the control group after 2 years (Table 4), i-NOS was expressed in macrophages of two animals with chronic inflammation (grade 2.8) and also in a single carcinoma (grade 3.0). COX-2 was localized in two animals in epithelium of alveolar epithelial hyperplasia (grade 2.5), in macrophages of one animal showing chronic inflammation (grade 1.0), and in epithelium of one carcinoma (grade 3.0). GST-Pi was localized in the epithelium of alveolar hyperplasia (grade 1.5) in two animals, chronic inflammation (grade 0.5) in four animals, and carcinoma in one animal (grade 4.0) in which the macrophages expressed GST-Pi (grade 2.5). 8-OHdG was localized in epithelium of alveolar hyperplasia (grade 0.5) in two animals and chronic inflammation (grade 0.6) in four animals.
After 2-year exposure (Table 4), i-NOS was expressed mostly in lesions containing macrophages, including a few adenomas, most carcinomas, atypical hyperplasias (Figs. 1I and 1J
), and, most prominently, chronic inflammatory lesions (Figs. 1E and 1F
). COX-2 was expressed in most adenomas in epithelium and macrophages, all carcinomas in epithelium and macrophages (Fig. 2J
), squamous cysts in epithelium (Figs. 2G and 2H
), atypical hyperplasias in epithelium and macrophages (Fig. 2D
), chronic active inflammation in epithelium and macrophages, alveolar epithelial hyperplasias in epithelium, and alveolar proteinosis. GST-Pi was observed chiefly in epithelial cells but seldom in macrophages. It was localized in the epithelium of all adenomas, squamous cysts (Fig. 2E
), atypical hyperplasias (Figs. 2A and 2B
), and chronic active inflammations (Fig. 1G
), all but one carcinoma (Fig. 2I
), and half of the alveolar epithelial hyperplasias. 8-OHdG, rarely expressed in macrophages, was seen mainly in the epithelium of a few carcinomas, most adenomas, atypical hyperplasias (Fig. 2C
) and alveolar epithelial hyperplasias, all chronic active inflammations (Fig. 1H
), and all squamous cysts but one (Fig. 2F
).
Staining patterns were noted for each type of lesion. Adenomas expressed all four markers in average grades of about 2, whereas carcinomas expressed all except 8-OHdG to a slightly higher degree, indicating that the relative percentage of stained area in carcinomas was higher. Atypical hyperplasias (Figs. 1I and 1J and 2AD) expressed i-NOS, COX-2, and GST-Pi with grades of about 3, and 8-OHdG staining was graded about 2. Squamous cysts (Figs. 2EH
) exhibited, respectively, grades 4, 3, 4, and 2 for i-NOS, COX-2, GST-Pi, and 8-OHdG. Chronic active inflammations (Figs. 1EH
) expressed i-NOS and COX-2 with an average grade of 3, and GST-Pi and 8-OHdG with an average grade of 2. Alveolar epithelial hyperplasias expressed COX-2 at grade 3, and GST-Pi and 8-OHdG at grade 1. Alveolar proteinosis demonstrated COX-2 expression, on average, at grade 4, but no labeling for the other markers.
Statistical Evaluation
The staining data for the 3-month interim study and the control group of the 2-year study did not indicate the necessity of any statistical testing because of the sparsity of data in these groups. Results of the analyses performed on the other groups are summarized in Figure 3AD). The trends were highly significant for 8-OHdG (Fig. 3D
) and GST-Pi (Fig. 3C
), with p values less than 0.05 in both cases, revealing an increase in immunopositivity from hyperplasia to adenoma to carcinoma to atypical hyperplasia to squamous cyst. We did not find a statistically significant trend for the i-NOS or COX-2 immunopositivity. Due to the paucity of data, we were unable to test for any correlation between the distribution of immunopositivity of GST-Pi and i-NOS. We did test for the equality of the two distributions in adenoma, carcinoma, and atypical hyperplasia using standard nonparametric rank tests for paired data. We concluded that there was no statistically significant difference among immunopositivity distributions of i-NOS and GST-Pi in adenoma (n = 5, p = 0.75), carcinoma (n = 7, p = 1.00), and atypical hyperplasia (n = 15, p = 0.56).
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DISCUSSION |
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IP inhalation resulted in severe pulmonary inflammation, consisting of abundant, varied inflammatory cell infiltrations associated with free radicals (Emmendoerffer et al., 2000), which, if not scavenged, can affect cellular organelles and cause the formation of ROS. This oxidative stress, an important factor in gene expression, serves as a second messenger in signal transduction pathways (Allen and Tresini, 2000
; Lander, 1997
) and can induce cellular proliferation (Klaunig et al., 1998
) that may lead to the induction of alveolar and bronchioalveolar tumors (Emmendoerffer et al., 2000
; Mossman, 2000
).
After 3 months of inhalation exposure, IP particles induced chronic inflammation, alveolar epithelial hyperplasia, and alveolar proteinosis. The severity of the lesions correlated with increasing concentrations of IP. Immunohistochemical staining of lungs exposed to 0.3 mg/m3 showed i-NOS expression in inflammatory cells associated with minimal to mild alveolar hyperplasia. Accompanying inflammation, severe alveolar proteinosis developed, which showed a relatively large area of COX-2 positivity. In lung tumorigenesis, COX-2 expression has been found in mouse, rat, and humans (Bauer et al., 2000).
Pulmonary lesions changed in character with prolonged, 2-year exposure. Inflammatory lesions became extensive, severe, and associated with alveolar squamous metaplasia and development of atypical hyperplasias, squamous cysts, and alveolar and bronchiolar tumors. A similar development followed 2-year inhalation of cobalt sulfate (Bucher et al., 1999). The atypical fibroproliferative lesions consisted of a central fibrous core containing dispersed alveolar structures filled with inflammatory cells and exhibiting peripheral papillary epithelial proliferation. Typically, the squamous cysts were located at the periphery of atypical hyperplastic foci. Extensive chronic inflammation was either an integral part of these foci or occurred within a short distance. Atypical hyperplastic lesions seemed to progress to carcinomas. Depending on size, intrusion into neighboring alveoli, and presence of mitosis, these lesions were diagnosed as atypical hyperplasia or carcinoma. Rats administered IP intratracheally showed severe pulmonary inflammation and epithelial cell damage (Oda, 1997
); similar effects occurred with intratracheal instillation of IP or indium arsenide to Syrian hamsters (Tanaka et al., 1996
). Intratracheal administration of indium chloride to rats resulted in severe lung damage and fibrosis (Blazka et al., 1994
). We postulate an inflammation-carcinoma sequence in which chronic, prolonged inflammation is associated with the release of highly reactive oxygen and nitrogen species from inflammatory cells (Fig. 4
).
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Cyclooxygenase catalyzes arachidonate metabolism and exists in two isoforms, COX-1 and COX-2, expressed in normal lung tissue of humans and experimental animals (Ermert et al., 1998). Prostanoids produced from arachidonic acid function in maintenance of airway smooth muscle tone, surfactant production, pulmonary hypertension, and vascular leakage. COX-1 is the "housekeeping" gene and COX-2 the inducible gene involved in inflammation and cellular proliferation (Herschman, 1994
). COX-2 overexpression occurs in colorectal cancer, gastric cancer, breast cancer, and some lung cancers (Ochiai et al., 1999
). Cyclooxygenase inhibitors, e.g., nonsteroidal antiinflammatory drugs (NSAIDS), reduce the size and number of colon adenomas (Reddy et al., 2000
), and other COX-2-specific inhibitors prevent lung carcinogenesis (Rioux and Castonguay, 1998
). In our investigation, the 3-month-exposure animals demonstrated a high percentage of COX-2positive area within alveolar proteinosis (average grade 3.9). After 2-year exposure, COX-2 immunopositivity remained high (average grade 4.0) and showed elevation (approximately 3.0) in all lesions in epithelial cells and macrophages. These findings may confirm an association between COX-2 expression and lung carcinogenesis.
Two explanations could account for the failure of the trend test to show statistical significance for immunopositivity increase from hyperplasia to adenoma to carcinoma to atypical hyperplasia to squamous cyst in i-NOS and COX-2 localizations: bootstrap methodology, as implemented, may be conservative for correlated data, and small sample size can contribute to lower testing resolution.
Glutathione S-transferases (GSTs) contain multiple genes involved in detoxification and chemical activation (Eaton and Bammler, 1999). GSTs decrease the reactivity of electrophilic compounds with cellular macromolecules by catalyzing the nucleophilic attack of glutathione (Armstrong, 1997
). GSTs detoxify endogenous products of lipid peroxidation and protect against oxidative stress in humans. Oxidative stress increases induced higher levels of GST-Pi whose overexpression can signify premalignant changes, like those found in rat preneoplastic hepatocytes forming GST-Pi-expressing foci. We observed that GST-Pi expression, mainly in epithelial cells, rarely macrophages, increased dramatically from 3 months to 2 years. After 3 months, the main pathological reaction was lung inflammation. Hyperplastic changes were minimal without epithelial atypia. After 2 years, GST-Pi expression occurred in all proliferative and neoplastic lesions, with average grades trending from hyperplasia to adenoma to carcinoma to atypical hyperplasia to squamous cyst. This correlation and the i-NOS and 8-OHdG staining indicated that the last three lesions are affected by oxidative stress. GST-Pi immunopositivity also indicated oxidative stress associated with premalignant or malignant characteristics of epithelial cells within lesions. GST-Pi expression in neoplasms has occurred in human lung carcinomas (Eimoto et al., 1988
).
8-hydroxydeoxyguanosine (8-OHdG), a major indicator of DNA damage (Kasai and Nishimura, 1984) and oxidation, causes G-C
T-A transversions in bacteria and mammalian cells. Treatment of mice with diesel exhaust particles resulted in a dose-dependent increase in 8-OHdG in lung DNA (Ichinose et al., 1997
). Immunohistochemical detection of 8-OHdG was first reported in paraffin-embedded rat livers (Takahashi et al., 1998
). This immunohistochemical technique evaluating 8-OHdG does not discriminate between nuclear and mitochondrial DNA damage. A positive expression of 8-OHdG could represent mitochondrial DNA changes that do not lead to somatic mutations (Hensley et al., 2000
). The progression in immunopositivity of 8-OHdG was comparable to that of GST-Pi. After 3 months, no expression of this marker occurred in epithelial cells of alveolar hyperplasia. After 2-year exposure, 8-OHdG expression trended increasingly from hyperplasia to adenoma to carcinoma to atypical hyperplasia to squamous cystsas we observed for GST-Pi staining, which showed, however, a slightly higher grading. The similar levels of expression of GST-Pi and 8-OHdG in proliferative and neoplastic lesions revealed that these indicators could be associated with oxidative stress. 8-OHdG, recently established as an immunohistochemical label in paraffin-embedded tissue (Takahashi et al., 1998
), and used first in lung tissue in our laboratory, has become a useful marker.
Statistical analysis of the immunohistochemical grading indicated a trend of significantly higher relative frequency distribution of immunopositivity of GST-Pi and 8-OHdG in the atypical hyperplastic and squamous cystic lesions. This tendency suggests a significant oxidative stressinduced response in the epithelial cell due to abundant inflammatory cell infiltration and an ensuing overwhelming release of ROS. The lower grade of GST-Pi and 8-OHdG expression in carcinoma cells suggests that they are less protected from oxidative or electrophilic DNA damage and may be targets for genetic alterations and, hence, precursors for clonal expansion.
Our data support strongly our hypothesis that IP inhalation, effecting uptake of foreign particles into the lung, causes oxidative stress progressing to induction of pulmonary cancer (Fig. 4). Severe alveolar inflammation, marked by massive infiltration of macrophages and severe alveolar proteinosis, occurs subsequently. The inflammatory cells prompt an induction of i-NOS and COX-2, which secondarily produce NO and prostanoids. NO has proinflammatory qualities; COX-2 increases inflammation and cellular proliferation and inhibits apoptosis.
When exposure was terminated at 21 weeks and the results were examined after 2 years, female rats, while still showing chronic active inflammation and the presence of foreign bodies, had also developed proliferative and neoplastic pulmonary lesions. At this time point, the expression of all four biomarkers in different cell types indicated that an inflammatory response (i-NOS, COX-2), as well as oxidative stress (GST-Pi, 8-OHdG), still existed.
All of these observations lend strong credence to the supposition that oxidative stress plays a major role in the development of lung cancer from indium phosphide inhalation. Additional investigations using fresh lung tissue and measuring 8-OHdG by liquid chromatography, for example, might provide further confirmation of our findings.
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ACKNOWLEDGMENTS |
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NOTES |
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