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Abstract |
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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Sprague-Dawley rats were exposed to 0.8 ppm ozone (O3), to 14.4 ppm nitrogen dioxide (NO2), or to both gases simultaneously for 6 h per day for up to 90 d. The extent of histopathologic changes within the central acinus of the lungs was compared after 7 or 78 to 90 d of exposure using morphometric analysis by placement of concentric arcs radiating outward from a single reference point at the level of the bronchiole- alveolar duct junction. Lesions in the lungs of rats exposed to the mixture of gases extended approximately twice as far into the acinus as in those exposed to each individual gas. The extent of tissue involvement was the same at 78 to 90 d as noted at 7 d in all exposure groups. At the end of exposure, in situ hybridization for procollagen types I and III demonstrated high levels of messenger RNA within central acini in the lungs of animals exposed to the combination of O3 and NO2. In contrast, animals exposed to each individual gas had a similar pattern of message expression compared with that seen in control animals, although centriacinar histologic changes were still significantly different from control animals. We conclude that the progressive pulmonary fibrosis that occurs in rats exposed to the combination of O3 and NO2 is due to sustained, elevated expression of the genes for procollagen types I and III. This effect at the gene level is correlated with the more severe histologic lesions seen in animals exposed to both O3 and NO2 compared with those exposed to each individual gas. In contrast, the sustained expression of the procollagen genes is not associated with a shift in the distribution of the lesions because the area of change in each group after 7 d of exposure was the same as after 78 to 90 d of exposure.
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Introduction |
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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We recently described a new model of rapidly progressive pulmonary inflammation and fibrosis in rats that were simultaneously exposed to the mixture of 0.8 ppm ozone (O3) and to 14.4 ppm nitrogen dioxide (NO2) (1). Exposure for less than 3 mo to these oxidant gases was associated with greater than 40% mortality. Marked increases in collagen content in survivors were noted biochemically, as were dramatic parenchymal changes including epithelial injury and abnormal remodeling, and interstitial thickening with obvious increases in stainable collagen and inflammatory cell infiltrate. Inhalation of O3 or NO2 alone at these same concentrations did not result in mortality and was associated with lesser degrees of histologic change.
A 9-wk chronological study of morphologic changes in animals exposed to the same concentration and mixture of gases revealed an interesting triphasic pattern of lesion development. Lesions developed and worsened over the first 3 wk of exposure, partially resolved during the middle 3 wk, and then dramatically progressed in severity in the final 3 wk (2). For the present study, we were interested in studying the differences in outcome between the single-gas and mixed-gas exposures, including more thorough evaluations of the development of lesions, and in elucidating differences in extracellular matrix production. We report here the results of a morphometric study to determine the extent of involvement of pulmonary parenchyma with each of these three exposure scenarios at both early and late time points. To determine whether morphologic differences among the exposure groups are mirrored by changes in extracellular matrix production at the gene level, we also evaluated expression of the genes for procollagen types I and III by examining the distribution of messenger RNA (mRNA) expression using in situ hybridization.
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Materials and Methods |
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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Animals
Specific pathogen-free, 10- to 12-wk old, male Sprague- Dawley rats (Charles River, Portage, MI) were used for these experiments. Random screening of two animals for serology and cultures revealed no bacterial, mycoplasmal, or viral infections or the presence of any known pulmonary pathogens. All exposures were conducted at the California Primate Research Center Exposure Facility (Davis, CA), and all animals were given a minimum 6-d acclimatization period before exposures were begun. Rats were housed in wire-mesh cages and kept on a 12-h light (7:00 A.M. to 7:00 P.M.) and 12-h dark (7:00 P.M. to 7:00 A.M.) cycle with food (Purina Rat Chow; Ralston-Purina, St. Louis, MO) and water freely available. All exposure conditions and animal care conditions were approved by the Animal Use and Care Administration Advisory Committee of the Office of Environmental Health and Safety at the University of California, Davis, CA.
Experimental Design
The rats used for in situ hybridization and morphometric studies reported here are from the same experiment as previously reported (1). Briefly, animals inhaled O3 (0.8 ppm), or NO2 (14.4 ppm), or both gases simultaneously at the same concentrations. Control animals breathed filtered air. Some rats of all treatment groups were killed after exposure for 7 d. Additionally, rats in the combined exposure group were killed after 78 d of exposure, and those in the individual gas exposure groups after 90 d of exposure, along with age-matched control animals. Morphometric studies were performed on animals from both the early and late time points. In situ hybridization studies were performed on animals only from the late time points.
Exposure Regimens
NO2 was generated by bubbling nitrogen through the liquid dimer dinitrogen tetroxide (N2O5), held at 0°C (3). NO2 in nitrogen was conveyed to the mixing inlet of the exposure chamber through stainless-steel lines. Chamber concentrations were monitored with a chemiluminescent monitor (Model 2108; Dasibi Corp., Glendale, CA). The monitor was calibrated by gas-phase titration of O3 with nitric oxide and was periodically checked against a nitric oxide span gas of known concentration.
O3 was produced from vaporized liquid medical-grade oxygen with a silent arc discharge ozonizer (Erwin Sander Corp., Giessen, Germany). Both the O3 and the oxygen were conveyed through Teflon lines to the mixing inlet of the exposure chamber. O3 concentrations in the chambers were monitored by ultraviolet (UV) photometry with calibrated Dasibi UV ozone monitors (Model 1003-AH), with data recorded every 2 min directly onto an IBM-AT computer for analysis. Calibration was checked against an absolute O3 monitor (Dasibi Model 1008-PC), a primary standard for O3 measurement. Each chamber was monitored for O3 and/or NO2 concentration for a minimum of 15 min per hour. Nominal exposure concentrations were within 5% of target values (Table 1).
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Rats were exposed for 6 h per night, 7 d per week (1:00 to 7:00 A.M.) to O3, NO2, or both gases simultaneously in 4.2 m3 glass and stainless-steel chambers with 30 changes of air per hour, as described in detail elsewhere (4). No levels of ammonia were detectable in the chambers. For rats exposed to the combination of O3 and NO2 , generator parameters were adjusted to ensure desired nominal exposure concentrations in the chambers; the concentrations were sampled through ports at cage rack level by first stabilizing the NO2 concentration, then adjusting the O3 generator as required to maintain the desired concentrations.
At the end of each exposure period, rats were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital. The trachea was cannulated and the lungs were perfused in situ for 60 min with ribonuclease (RNase)- free 4% paraformaldehyde at 30 cm of pressure. Lungs were then placed in 70% ethanol and embedded in paraffin within 24 h, with care taken to avoid exposure of lung tissue to RNases. Representative sections of the paraformaldehyde-fixed, paraffin-embedded lungs were sectioned at 6 µm. Lungs used for morphometric studies were perfused in situ for 30 min with 330 mOsm Karnovsky's solution at 30 cm of pressure. After 30 min the trachea was tied off and transected, and the heart and lungs were removed from the thorax and immersed in fixative. After a period of 24 h or more, the heart, mediastinum, and other soft tissues were dissected away from the lungs, and the fixed lung volume was measured by the displacement method of Scherle (7). Transverse sections cranial and caudal to the hilum were embedded in paraffin and sectioned.
Morphometry
The extent of lesion development in centriacinar areas was examined morphometrically using a modification of a concentric arc overlay method. This method is described in detail elsewhere (8, 9). All bronchiole-alveolar duct junctions (BADJs) were identified on a minimum of two paraffin-embedded tissue sections per animal. Each region was captured as a digital image using a 2.5 objective on the microscope interfaced to a Macintosh IIci computer (Apple Computer, Inc., Cupertino, CA) with a Dage video camera (Dage, Michigan City, IN). A pattern of concentric circles with 100-µm interval spacing was placed over each digital image, with the geometric center of the circles placed at the level of the first alveolar outpocketing. An average of 6 to 10 BADJs per animal from four to five animals per group was used for analysis.
To define oxidant-induced alterations within the centriacinus, the following criteria were used: (1) thickening of alveolar septal walls and tips along alveolar duct paths, (2) increased cellularity of the alveolar wall, (3) increased numbers of alveolar macrophages within the airspaces, (4) extension of bronchiolar epithelial cells from the airway into alveoli, and (5) increased numbers of epithelial type II cells lining alveoli. Thickening of alveolar walls was defined as a 50% or greater increase in alveolar septal tissue volume. The degree of septal wall thickening was confirmed by overlaying a series of 21 evenly spaced lines of known length (Stereology Toolbox, Davis, CA) over each captured field and counting the number of times the ends of the lines fell on alveolar tissue, as well as the number of intercepts the lines made with the air-tissue interface of the alveolar wall. From these measurements a simple determination of tissue volume normalized to surface area could be made. Increased cellularity of the septal wall was defined by the number of nuclei present in alveolar septal tips. Macrophage number was considered to be increased above control with the presence of two or more cells per alveolus.
Measurement of the extent of changes in the lung parenchyma radiating from the BADJ was based on the confirmation of changes described previously to the furthest distance from the terminal bronchiole, estimated to the nearest 10 µm. These measurements were facilitated by using the concentric-circle grid as a uniform method for measuring the greatest linear distance of change from the BADJ. In this manner, all centriacinar profiles were analyzed in an identical unbiased manner.
This method of morphometric analysis was performed on tissues from rats in the filtered air and in all three exposure groups after 7 and 78 d (mixture of O3 and NO2) or 90 d (O3 or NO2 alone) of exposure.
Probes
The complementary DNA (cDNA) probes for procollagen types I and III used in this study were synthesized
and characterized by Armstrong and associates as previously reported; characterization included sequence analysis and Northern blotting (10). These identical probes were
used in this study. A 1,074-base pair (bp) 
1 (I) procollagen cDNA probe for procollagen type I and a 449-bp 1
(III) procollagen cDNA probe for procollagen type III
were used to prepare sense and antisense riboprobes for in
situ hybridization.
In Situ Hybridization
In situ hybridization to detect mRNA for procollagen types I and III was performed on lung tissue from rats of all three exposure groups on the last day of the exposure period (78 d for the mixed-exposure group, and 90 d for animals exposed to the individual gases). To synthesize 35S-labeled RNA, the plasmid was linearized downstream from the cDNA sequence to produce transcripts from the insert region only. The template DNA was linearized with the appropriate restriction endonuclease, then purified by phenol/chloroform extraction and ethanol precipitation.
The 35S-labeled RNA was synthesized using the Riboprobe Gemini II System (Promega Corp., Madison, WI).
Using the standard transcription protocol, the reagents
were added to a 20-µl final volume in the following order:
300 µCi of 35S- uridine triphosphate (UTP) (specific activity 1,000 to 1,500 Ci/mMol) dried down in a microfuge
tube; 4 µl 5× transcription buffer containing spermidine;
2 µl 100 mM dithiothreitol (DTT); 20 units RNasin RNase
inhibitor; 4 µl each of 2.5 mM adenosine triphosphate, cytidine triphosphate, and guanosine triphosphate; 2.4 µl 100 mM UTP; 1 µg linearized template DNA; and RNase-free water to make up final reaction volume. Finally, 1 µl of the
appropriate RNA polymerase enzyme (to produce sense
and antisense RNA) was added to the reaction mixture,
and the mixture was incubated for 1 h at 37°C. To digest
plasmid DNA, RNase-free deoxyribonuclease was added
to a final concentration of 1 U/mg DNA and incubated at
37°C for 15 min. The riboprobe was then purified by phenol/chloroform extraction and ethanol precipitation.
For complementary RNA probe hybridization, lung tissue sections on slides were deparaffinized, rehydrated, and sequentially pretreated as follows: 4% paraformaldehyde fixation for 10 min; two washes in phosphate-buffered saline (PBS) for 3 min each; treated with 0.3% Triton-X-100 for 15 min at 37°C; washed twice in PBS for 3 min each, proteinase K (1 µg/ml) for 30 min at 37°C, PBS-glycine for 30 s, and 4% paraformaldehyde for 5 min; two washes with PBS for 3 min each; acetylated with 0.25% acetic anhydride/0.1 M triethanolamine for 10 min, and 50% deionized formamide/2× saline sodium citrate (SSC) for 10 min at 37°C. The slides were then placed in RNase-free humidified chambers, 1.5 × 106 cpm/ml 35S-labeled RNA was applied to each tissue section, and the slides were coverslipped and incubated overnight at 50°C.
After overnight incubation, the coverslips were removed and the slides washed in the following solutions: 4× SSC containing 10 mM DTT for 15 min at room temperature; 30 s in increasing ethanol concentrations (70, 95, and 100%, each containing 0.3 M ammonium acetate); hybridization buffer without probe for 20 min at 65°C (this temperature is 5°C below the in situ melt temperature values and significantly reduces background without loss of signal); 2× SSC for 15 min; 40 µg/ml RNase A in RNase buffer (0.3 M NaCl, 10 mM Tris-HCl [pH 7.5], 5 mM ethylenediamenetetraacetic acid) for 30 min at 37°C; RNase buffer for 30 min at 37°C; 2× SSC for 30 min at 37°C; and two washes in 0.1× SSC for 15 min each, followed by dehydration in a graded ethanol series containing 0.3 M ammonium acetate. All slides were air-dried for at least 30 min before proceeding to the next step of autoradiography.
After drying, the slides were dipped in Kodak NTB-2 emulsion diluted 1:1 with H2O warmed to 40°C, then transferred to autoradiography slide boxes containing fresh desiccant. After exposure for 4 to 10 d at 4°C, the slides were developed in D19 Developer for 4 min, rinsed in H2O, and fixed in sodium thiosulfate. The slides were stained with Lee's methylene blue/basic fuchsin, air-dried, and coverslipped. The distribution of message in sections hybridized with each riboprobe was examined using conventional brightfield microscopy and confocal laser scanning microscopy operated in the reflectance mode. A Bio-Rad MRC6000 laser scanning confocal system (Bio-Rad, Watford, UK) mounted on an Olympus BH-2 microscope permitted the same microscope to be used for both imaging methods.
Statistics
Data from the morphometric analysis were analyzed using the Dunnet's two-sided test to compare the control group with each treatment group at each time point. If significant differences were noted, Duncan's multiple range test was then used to compare differences between treatment groups at each time point (11).
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Results |
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Abstract
Introduction
Materials and methods
Results
Discussion
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Histopathology
Histopathologic changes in the lungs of the animals chronically exposed to the combination of gases have been described elsewhere (1). In the animals exposed to the mixture of gases for 7 d, there was marked histiocytic and neutrophilic inflammation of the centriacinar regions, with inflammatory cells in airspaces and in the interstitium. The interstitium was thickened with inflammatory cells and edema. Epithelial cells in affected areas were hypertrophied, and there was patchy epithelial necrosis. The changes in animals exposed to O3 or NO2 alone were similar in quality to those of the mixed-exposure groups but were much less severe, particularly following exposure to NO2.
After 78 d of exposure, the lesions in the mixed-exposure animals were much more severe than at 7 d, with marked architectural remodeling and distortion of the centriacinar regions. There were inappropriate cell types lining adjacent alveoli, including mucus goblet cells and tall columnar ciliated cells. The interstitium in affected areas was markedly thickened, with increased numbers of haphazardly arranged collagen bundles. In addition to the inflammatory cell types as previously described, there were increased numbers of mast cells in the interstitium. The lesions in the animals exposed to either O3 or NO2 alone for 90 d were much less severe than those of the mixed-exposure group, and they were also milder at 90 d than at 7 d. The residual lesions in the animals exposed to O3 were slightly worse than in the animals exposed to NO2, and included some areas of architectural remodeling, including bronchiolarization of alveolar ducts and mild interstitial fibrosis. End-point changes in NO2-exposed animals included mild epithelial hypertrophy and very slight interstitial thickening.
Morphometry
Figure 1 shows the placement of the concentric arcs over the BADJ. Results of morphometric analysis of the extent of lesions caused by each type of oxidant gas exposure are presented in Table 2. Lesions caused by inhalation of the mixture of O3 and NO2 (Figure 1d) extended approximately twice as far (an average of 550 µm) into the alveolar ducts as those caused by exposure to either individual gas (an average of 250 µm; Figures 1b and 1c). There was no significant difference in the extent of involvement when O3 alone was compared with NO2 alone. When the extent of lesions at 7 d was compared with the extent at 78 or 90 d, there was no significant difference in any of the treatment groups. In other words, the extent of involvement at 7 d was predictive of the extent of involvement at termination of exposure for each exposure regimen.
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In Situ Hybridization
A consistent finding in the control animals as well as in all treatment groups was the expression of mRNA for procollagen types I and III by fibroblasts in the adventitia surrounding larger bronchioles, bronchi, and large blood vessels (Figure 2). The number of positive cells and the intensity of grains appeared to be directly proportional to the size of the airways and vessels. This expression is indicative of a low level of collagen production even in normal lungs, and is further evidence of the specificity of these probes. Expression in the parenchyma of control animals, including in BADJ regions, was uniformly negative (Figure 3a).
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In animals exposed to the mixture of gases, expression of mRNA for procollagen type I was markedly increased in the remodeled centriacinar areas, with large numbers of heavily labeled fibroblasts within the interstitium (Figures 3b and 4a). This was spatially consistent with areas we had previously determined to have increased stainable collagen (2). Expression was less intense around large airways and blood vessels than in remodeled areas (expression was similar to controls), and was uniformly negative in unaffected areas of parenchyma. Expression for procollagen type III was seen in the same areas as for type I, but labeling appeared less intense and was seen in fewer cells (Figure 4b). This apparent difference in expression of the two procollagen types was consistently seen. However, since our aim was to identify the spatial expression of the two procollagen genes in the different treatment groups, quantification was not done.
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In animals exposed to NO2 alone, expression of mRNA for both types of procollagen was similar to that seen in controls, with the exception of infrequent labeled cells in centriacinar regions (Figure 3d). The same was generally true for animals exposed to O3 alone. However, in more markedly affected and remodeled areas in these animals, there was some increased labeling for type I procollagen (Figure 3c) and less for type III (not shown). Still, the intensity of label was less and the number of cells labeled was fewer in these animals than in animals exposed to both O3 and NO2.
In all cases, hybridization with a labeled sense riboprobe gave negative results, with only diffusely scattered background grains (Figures 4c and 4d).
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Discussion |
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Materials and methods
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Morphometric analysis showed that the extent of parenchymal involvement is the same after long-term exposure as it is after short-term exposure for the three gas-inhalation scenarios studied. O3 or NO2 alone had a similar extent of effect at 7 or 90 d of exposure, with tissue and cellular changes noted 250 µm into the acinus. Simultaneous exposure to both O3 and NO2 resulted in changes that penetrated far deeper into the acinus, to an average of 550 µm. Investigators have subjectively judged that the lesions of chronic O3 exposure shift distally over time, but morphometric studies such as ours were not performed (12). We originally made the same subjective assessment of increasing extent of involvement with time in our mixed-exposure animals, but it did not hold up with morphometric evaluation.
The reason for the much greater extent of the lesions in the mixed-exposure animals as compared with those exposed to single gases is not known. We have argued previously that the greater toxicity caused by the mixture of gases is due to the reaction of O3 and NO2 to form a more toxic species, perhaps N2O5. This is based on our findings with acute exposures, which showed a significant synergistic, not additive, effect when the two gases are combined (4, 13). The combination of O3 and NO2 may lead to a less efficient depletion of these gases through fluid and/or tissue uptake as they pass down the airways, resulting in delivery of a higher concentration of the gases deeper into each of the ventilatory units of the lungs.
As we reported previously, the severity of lesions in the animals exposed to each individual gas lessened over time, whereas the lesions became progressively more severe in animals exposed to the mixture of gases (1, 2). The reason for the discrepancy between changes in severity and lack of change in extent is unclear. One may speculate that cells along the ventilatory pathway develop increased ability to absorb and/or detoxify the oxidant gases over time, which prevents gases from reaching greater depths. We have found by immunohistochemistry that the antioxidant enzyme copper zinc superoxide dismutase is present in abundance in the affected areas of lung in the animals exposed to the mixture of gases (unpublished data). This enzyme and others may somehow prevent increasing extent of damage over time, but the amounts of enzyme present simply may not be enough to prevent increasing severity of injury from the overwhelming amounts of oxidant gas present with mixed exposure. Alternatively, adaptations in breathing patterns (for example: rapid, shallow breathing) may prevent extension of lesions further into the acinus over time. If such adaptations have occurred within the first 7 d of exposure in all treatment groups, that may explain why there is no change in the extent of lesions when comparing animals at 7 d with those who have had chronic exposure. Such studies of breathing patterns are planned and should help answer these questions.
In situ hybridization revealed that in rats chronically exposed to a mixture of O3 and NO2 there is sustained, increased expression of mRNA for procollagen types I and III, and this increased expression is seen in injured and remodeled centriacinar areas. These results indicate that exposure to this mixture of gases leads to transcriptionally regulated changes in the production of extracellular matrix components, and that such changes are localized to sites of injury. In contrast, in animals chronically exposed to either O3 or NO2 alone, expression of both procollagen types appears to be the same as in control animals, despite continual exposure to these gases and despite the fact that there is some mild residual remodeling in the lungs of these animals. The only exception to this observation was in O3-exposed rats, in which there was some labeling for both procollagen types in scattered areas that were more severely affected and remodeled. On the basis of these findings, we theorize that the severity of injury may be at least partially responsible for the increased expression of the two procollagen genes examined in this study.
The findings based on in situ hybridization of differences in procollagen gene expression are consistent with our previous observations of marked differences in pulmonary lesions and biochemical measurements between the three groups. The lack of procollagen gene expression in animals exposed to the individual gases corresponded to a lack of change in whole-lung hydroxyproline levels, whereas in the mixed-exposure animals there was markedly increased gene expression combined with increased hydroxyproline content (1). Despite the presence of increased stainable collagen in animals of all three groups, procollagen gene expression was seen only in the mixed-exposure group, corresponding to areas of active synthesis where the collagen was being deposited. This result emphasizes that excess collagen may be present in focal areas of the lung, without concurrent measurable increases in gene expression.
In situ hybridization studies of animals exposed to combinations of O3 and NO2 have not previously been reported. Parks and colleagues evaluated the expression of mRNA for procollagen types I and III in animals chronically exposed to O3 at various concentrations (14). They found that even at the highest concentration tested (1.0 ppm for 20 mo), there was no increase in mRNA expression for either collagen type at the end point of exposure (14). Another study of a matched group of animals, as were examined in the study of Parks and coworkers, also showed that there was no increase in mRNA expression for procollagen type I at the end of the exposure, even though there was evidence of increased stainable collagen (15). These results are all consistent with our findings with respect to chronic exposure to O3 alone, and further supports the suggestion that there is a toxicologic interaction upon exposure to the mixture of gases that leads to sustained expression of procollagen genes at sites of injury. If, as discussed previously, a new species such as N2O5 is formed, it may directly or indirectly (via increased severity of tissue damage) lead to increased procollagen gene expression. Animals exposed to the mixture of gases exhibit persistent inflammation associated with continued epithelial injury and continued cell proliferation (2). Any of these changes could conceivably lead to increased expression of procollagen genes.
Many investigators have measured relative changes in amounts of isolated mRNA for procollagen types I and III or measured the amounts of the two collagen types using biochemical methods (10, 16). Such studies have given quite variable results with respect to the relative amounts and temporal changes of the two collagen types. It is therefore impossible to predict the ratio and amounts of these two procollagen types in different disease processes of the lung. Most reviews of extracellular matrix regulation in the lung agree that type I is the most abundant form in chronic pulmonary fibrosis (26, 27). However, based on the results of the above studies, it appears that much remains to be determined about these two collagen types in pulmonary injury. In addition, the results suggest that there is not one defined pathway leading to pulmonary fibrosis. Although we made the subjective observation that the expression of mRNA for procollagen type I was more intense than that for procollagen type III, our aim in this study was to use the method of in situ hybridization as a qualitative estimate of procollagen gene expression in specific anatomic sites that have been damaged by oxidant gases.
In summary, we have determined by morphometric analysis that the lesions in animals exposed to the mixture of gases extend approximately twice as far into the acinus as those in rats exposed to each individual gas, but that there is no change in extent of lesions with time in any of the groups. We have also shown that there is a difference in the response at the transcriptional level of procollagen gene expression when animals exposed to O3 or NO2 individually are compared with those exposed to a mixture of the two gases. Rats chronically exposed to the individual gases do not show increased gene expression, whereas those exposed to the mixture of gases do. This is the first study to report in situ changes in procollagen gene expression in the lungs of animals exposed to the combination of O3 and NO2, and our findings of sustained expression of these genes in these animals is in marked contrast to previous studies showing a lack of such gene expression after chronic exposure to other oxidant gases.
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Footnotes |
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Address correspondence to: Kent E. Pinkerton, Institute of Toxicology and Environmental Health, University of California, Davis, CA 95616. E-mail: kepinkerton{at}ucdavis.edu
(Received in original form December 12, 1997 and in revised form April 28, 1998).
Abbreviations: bronchiole-alveolar duct junction, BADJ; complementary DNA, cDNA; messenger RNA, mRNA; nitrogen dioxide, NO2; ozone, O3; phosphate-buffered saline, PBS; ribonuclease, RNase; saline sodium citrate, SSC.Acknowledgments: The authors thank Brian Tarkington and Tim Duvall for the operation of the exposure facilities used in these studies, Janice Peake for technical assistance in the photography of in situ hybridization results, Adam Eliot and Dan Hung for preparation of images relating to the morphometry study, and Purnima Naidu for her work in the performance of the morphometry study. This work was supported by National Institutes of Health Grants ES-00628, ES-05707, and RR-00169.
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References |
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Materials and methods
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1. Last, J. A., T. R. Gelzleichter, K. E. Pinkerton, R. M. Walker, and H. Witschi. 1993. A new model of progressive pulmonary fibrosis in rats. Am. Rev. Respir. Dis. 148: 487-494 [Medline].
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