Effects of concurrent ozone exposure on the pathogenesis of cigarette smoke-induced emphysema in B6C3F1 mice.

Episodic elevation of air pollutants may exacerbate respiratory distress associated with chronic obstructive pulmonary disease (COPD), yet few experiments have been performed to determine how continuously polluted atmospheres may contribute to the etiology of COPD, in general and pulmonary emphysema in particular. This study describes the effects of concurrent exposure to ozone (O(3)) in the pathogenesis of cigarette smoke (CS)-induced emphysema in the mouse. Female B6C3F1 mice were whole-body exposed either to filtered air (FA) or to mainstream CS at a concentration of 250 mg total particulate material/m(3) for 6 h/day, 5 days/wk for 15 or 32 wk. Concurrently, mice were exposed either to FA or to O(3) at 0.3 ppm for 8 h/night, 5 nights/wk for the same time periods. At necropsy, mouse lungs were lavaged, and bronchoalveolar lavage fluid (BALF) was analyzed for inflammatory cell numbers, total protein, lactate dehydrogenase (LDH) and alkaline phosphatase (AP) activities, superoxide production by isolated alveolar macrophages, glutathione content, inflammatory cytokines, and proteolytic activity. Other lungs were inflated at constant pressure for 6 h with formalin for fixation, routine histopathology, and stereology. After 32 wk of exposure, CS with or without concurrent O(3) exposure produced stereologic evidence of emphysema as previously described. Concurrent O(3) exposure did not worsen any of these parameters, nor did O(3) by itself cause stereologic changes that were consistent with emphysema. The O(3) exposure caused only slight elevations of BALF macrophages, while CS exposure caused marked increases in the numbers of both BALF macrophages and neutrophils. Neutrophils in the BALF in response to CS exposure were also more numerous at 32 wk than at 15 wk. Exposure to CS caused an increase in BALF total protein, LDH, AP, and interleukin (IL)-1beta. After 32 wk, CS exposure was associated with decreased superoxide production from isolated alveolar macrophages. The CS exposure elevated BALF total glutathione primarily at 15 wk. Overall, O(3) had little effect on endpoints that were significantly affected by CS exposure. We conclude that concurrent O(3) exposure has no effect on the induction of emphysema by CS in this animal model.

Effects of subchronic inhalation exposure of rats to emissions from a diesel engine burning soybean oil-derived biodiesel fuel.

There is increasing interest in diesel fuels derived from plant oils or animal fats ("biodiesel"), but little information on the toxicity of biodiesel emissions other than bacterial mutagenicity. F344 rats were exposed by inhalation 6 h/day, 5 days/wk for 13 wk to 1 of 3 dilutions of emissions from a diesel engine burning 100% soybean oil-derived fuel, or to clean air as controls. Whole emissions were diluted to nominal NO(x) concentrations of 5, 25, or 50 ppm, corresponding to approximately 0.04, 0.2, and 0.5 mg particles/m(3), respectively. Biologically significant, exposure-related effects were limited to the lung, were greater in females than in males, and were observed primarily at the highest exposure level. There was a dose-related increase in the numbers of alveolar macrophages and the numbers of particles in the macrophages, as expected from repeated exposure, but no neutrophil response even at the highest exposure level. The macrophage response was reduced 28 days after cessation of the exposure. Among the high-level females, the group mean lung weight/body weight ratio was increased, and minimal, multifocal bronchiolar metaplasia of alveolar ducts was observed in 4 of 30 rats. Lung weights were not significantly increased, and metaplasia of the alveolar ducts was not observed in males. An increase in particle-laden macrophages was the only exposure-related finding in lungs at the intermediate and low levels, with fewer macrophages and fewer particles per macrophage at the low level. Alveolar histiocytosis was observed in a few rats in both exposed and control groups. There were statistically significant, but minor and not consistently exposure-related, differences in body weight, nonpulmonary organ weights, serum chemistry, and glial fibrillary acidic protein in the brain. There were no significant exposure-related effects on survival, clinical signs, feed consumption, ocular toxicity, hematology, neurohistology, micronuclei in bone marrow, sister chromatid exchanges in peripheral blood lymphocytes, fertility, reproductive toxicity, or teratology. This study demonstrated modest adverse effects at the highest exposure level, and none other than the expected physiological macrophage response to repeated particle exposure at the intermediate level.

Influence of exposure concentration or dose on the distribution of particulate material in rat and human lungs.

Differences among species in the anatomic sites of particle retention could influence responses to inhaled particles. In this study, we used morphometric techniques to examine the influence of exposure concentration on particle retention in histologic sections from rats and humans. The rats had been exposed for 24 months to diesel exhaust at 0.35, 3.5, or 7.0 mg soot/m(3). The human subjects were nonsmokers who did not work as miners, nonsmoking coal miners who worked under the current standard of 2 mg dust/m(3) for 10-20 years (mean = 14 years), and nonsmoking coal miners who worked under the former standard of < 10 mg dust/m(3) for 33-50 years (mean = 40 years). The distribution of retained particles within the lung compartments was markedly different between species. In all three groups of rats, 82-85% of the retained particulate material was located in the alveolar and alveolar duct lumens, primarily in macrophages. In humans, 57, 68, and 91% of the retained particulate material was located in the interstitium of the lung in the non-miners, coal miners under the current standard, and coal miners under the former standard, respectively. These results show that chronically inhaled diesel soot is retained predominantly in the airspaces of rats over a wide range of exposures, whereas in humans, chronically inhaled particulate material is retained primarily in the interstitium. In humans, the percentage of particles in the interstitium is increased with increased dose (exposure concentration, years of exposure, and/or lung burden). This difference in distribution may bring different lung cells into contact with the retained particles or particle-containing macrophages in rats and humans and may account for differences in species response to inhaled particles.

Animal models of pulmonary infection in the compromised host: potential usefulness for studying health effects of inhaled particles.

Pulmonary infection leading to pneumonia is a significant cause of morbidity and mortality worldwide. Airborne particles have been associated with pneumonia through epidemiological research, but the mechanisms by which particles affect the incidence of pneumonia are not well established. The purpose of this review is to examine the potential of animal models to improve our understanding of the mechanisms by which inhaled particles might affect the incidence and resolution of pulmonary infection. The pathogenesis of pneumonia in most animal models differs from that in humans because humans frequently have underlying diseases that predispose them to infection with relatively low doses of pathogens. Normal, healthy animals lack the underlying pathology often found in humans and clear bacteria and viruses rapidly from their lungs. To overcome this, animals are administered large inocula of pathogens, are treated with agents that cause mucosal lesions, or are treated with immunosuppressive drugs. Alternatively, pathogenic bacteria are protected from phagocytosis by encasing them in agar. No one animal model will replicate a human disease in its entirety, and the choice of model depends upon how well the animal infection mimics the particular human response being examined. The advantages and disadvantages of animal models in current use for bacterial and viral infections important in the etiology of human pneumonia are reviewed in detail. Considerable data indicate that prior exposure to particles compromises the ability of experimental animals to resolve a subsequent infection. In addition, information is available on the effects of particle exposure on various portions of respiratory defense including phagocytic function, ciliary movement, inflammation, and antibody response in the absence of infection. In contrast, little research to date has examined the consequences of particle exposure on the host defense mechanisms of animals already infected or on their ability to resolve their infection.

Particle clearance and histopathology in lungs of C3H/HeJ mice administered beryllium/copper alloy by intratracheal instillation.

Beryllium/copper (BeCu) alloys are commonly used in the electronics, automotive, consumer, defense, and aerospace industries. Some individuals exposed occupationally to BeCu alloys have developed chronic beryllium disease. However, little is known of the toxicity and fate of BeCu alloys in the respiratory tract. To begin to address this question, we investigated the pulmonary toxicity and clearance of BeCu alloy (2% Be; 98% Cu) in mice. Groups of 40 female C3H/HeJ mice were administered 12.5, 25, and 100 microg BeCu alloy or 2 and 8 microg Be metal by intratracheal instillation. Mice were sacrificed at 1 h and 1, 7, 14, and 28 days postinstillation. Left lungs were evaluated for histopathological change. Right lungs were analyzed for Be and Cu content. Twenty-five percent of the high-dose BeCu mice and 7.5% of the mid-dose BeCu mice died within 24 h of dosing. Acute pulmonary lesions included acute alveolitis and interstitial inflammation. Type II epithelial cell hyperplasia and centriacinar fibrosis were present by 7 days after dosing. Lesions persisted through 28 days after instillation. No lesions attributable to alloy exposure were present in liver or kidney. Be metal instillation caused no deaths and minimal pulmonary changes over the time studied, indicating that the pulmonary lesions were due to Cu rather than Be. Cu cleared the lung with a half-time of 0. 5-2 days. Be cleared with a half-time of several weeks or longer. Results of this study suggest that exposure to BeCu alloy is more acutely toxic to lung than Be metal. The results of tissue analyses also indicate that, while the Cu component of the alloy clears the lung rapidly, Be is retained and may accumulate upon repeated exposure.

Rat lung tumors induced by exposure to selected poorly soluble nonfibrous particles.

Rodent bioassays have been used to assess the carcinogenicity of several inhaled, poorly soluble, nonfibrous particles that vary in toxicity and carcinogenic potency. There is substantial published information from chronic inhalation bioassays of diesel exhaust, carbon black, titanium dioxide, talc, and coal dust. This review summarizes data from studies with exposures for 2 yr or more using these 5 materials. The review has four objectives: (1) to summarize the current information available from these bioassays concerning exposure-dose-carcinogenic response in rats, (2) to summarize the pathologic and phenotypic features of the neoplastic response in rats, (3) to examine possible strain- and gender-related differences, and (4) to compare the neoplastic responses of rat to those of other species exposed to these materials.

Animal models of chronic bronchitis and their relevance to studies of particle-induced disease.

Chronic bronchitis is a significant cause of morbidity and mortality. Chronic irritation of the conducting airways by inhaled substances, most importantly cigarette smoke, air pollution, and occupational exposures, is thought to be a key factor in the pathogenesis of chronic bronchitis. Microbial infections have been implicated in acute exacerbations of bronchitis and in its progression. Several animal models of chronic bronchitis have been developed. This review examines similarities and dissimilarities among commonly used animal models of bronchitis and the human disease. The most commonly used animal models of chronic bronchitis are those employing SO2, tobacco smoke, lipopolysaccharide (endotoxin), proteases, and secretagogues. Bronchiolitis induced by nickel and nitric acid have also been reported. Rats, hamsters, and dogs are the species most frequently used; sheep and monkeys have been used less frequently. These models vary in the extent or location of mucous-cell hyperplasia and metaplasia, airway inflammation, chronicity, ease of induction, and reproducibility. Frequently, the deficiencies in these models are attributable to anatomic differences between human and animal airways, differences in the severity or chronicity of inflammation or fibrosis, or lack of complete characterization of the responses and their time course in the animal model. These animal models may be useful for investigating how, and under what exposure conditions, ambient pollutants might exacerbate airway inflammation, mucus hypersecretion, and airflow limitation.

Animal models of emphysema and their relevance to studies of particle-induced disease.

Emphysema is a pulmonary disease that may be exacerbated by inhaled particles. Over the years, many animal models of emphysema have been developed that may be useful in studying the effects of inhaled particles on humans with emphysema. Models have been described in many species, and many approaches have been described for inducing emphysema. Emphysema in humans is a parenchymal component of chronic obstructive pulmonary disease and frequently coexists in a complex with disease of the airways such as bronchitis. Animal models of emphysema usually recapitulate only one or a few aspects of this complex disease. Thus, the emphysema model must be selected carefully in order to answer specific questions about the interactive effects of particles and emphysema.

Multiple modes of responses to air pollution particulate materials in A549 alveolar type II cells.

Exposure to components of air pollution may cause adverse effects on lung cellular and organ functions through several mechanisms. Cell death, altered gene expression including production of cytokines, and modifications of normal cellular processes are possible outcomes that may be independent or coupled. To assess the effects of materials representative of a variety of particulate components of air pollution on lung epithelium, a human cell line of type II origin (A549 cells) was exposed to these materials in vitro. Materials tested included carbon black (CB), diesel soot from two sources (DS), residual oil fly ash (ROFA), Ottowa Ambient Air particulate (OAA), silicon dioxide (SiO2), and nickel subsulfide (Ni3S2). Endpoints included loss of adherence measured by crystal violet staining (CV), lactate dehydrogenase release (LDH), release of interleukin-8 (IL-8) measured by ELISA, and alkaline phosphatase activity in the cells (APc) and released into the supernatant (APS). Nuclear morphology was also examined. SiO2 and Ni3S2 both caused dose-dependent acute toxicity as assessed by LDH and CV, and caused alterations in nuclear morphology consistent with apoptosis. However, much more IL-8 was released into the tissue culture supernatant by SiO2 at the same levels of cytotoxicity than by Ni3S2. Neither of these acutely toxic materials increased APc or APS, but the less cytotoxic materials caused very significant release of AP in the order OAA > DS > ROFA >> SiO2 = Ni3S2. OAA and, to a lesser extent, DS caused increases in mitotic fraction and increased CV staining, consistent with stimulation of proliferation. These results suggest multiple modes of responses to toxic materials and imply that a toxicological screening process should address these and possibly other endpoints.

Enhanced pulmonary epithelial replication and axial airway mucosubstance changes in F344 rats exposed short-term to mainstream cigarette smoke.

Cigarette smoking is associated with respiratory diseases that may be caused by injury to specific pulmonary cells. The injury may manifest itself as site-specific enhanced cellular replication. In this study, rats were exposed either to mainstream cigarette smoke (CS; 250 mg total particulate matter/m(3)) or to filtered air (FA) for 6 h/day, 5 days/week, for 2 weeks. In one group, cells in S-phase were labeled over 7 days by bromodeoxyuridine (BrdU) released from implanted osmotic pumps (pump labeled), while another group received BrdU by injection 2 h prior to necropsy (pulse labeled). Morphometry showed that the type II epithelial BrdU labeling index (LI) was significantly elevated in the CS-exposed animals of both labeling groups. The axial airway and terminal bronchiolar LIs were enhanced by CS only in the pump-labeled group. In a third group (pulse labeled), 2 weeks of recovery following exposure to CS allowed a normalization in the type II LI. In the pump-labeled rats, the CS-induced elevation of the type II LI was greater than the LI elevation in conducting airways, suggesting that the parenchyma may have been injured more than the conducting airways. The terminal bronchiolar LI in the pump-labeled group, regardless of exposure, was significantly greater than the axial airway LI. Pump labeling, in contrast to pulse labeling, could therefore discern differences among replication rates of conducting airway epithelium in different regions of the lung. Mucosubstance (MS) within the axial airway epithelium was quantified by morphometry. The CS exposure did not increase the total number of MS-containing cells or the total number of axial airway epithelial cells, but there was a phenotype change in the MS cells. Neutral MS cells (periodic acid-Schiff-positive) were significantly decreased, while acid MS cells (alcian blue-positive) were slightly increased by CS exposure. Either cell replication and differentiation or differentiation alone may have changed the phenotype in the MS cell population.

Cigarette smoke exposure produces more evidence of emphysema in B6C3F1 mice than in F344 rats.

Cigarette smoke (CS) causes pulmonary emphysema in humans, but results of previous studies on CS-exposed laboratory animals have been equivocal and have not clearly demonstrated progression of the disease. In this study, morphometry and histopathology were used to assess emphysema in the lungs of B6C3F1 mice and Fischer-344 rats. The animals were exposed, whole-body, to CS at a concentration of 250 mg total particulate matter/m3 for 6 h/day, 5 days/week, for either 7 or 13 months. Morphometry included measurements of parenchymal air space enlargement (alveolar septa mean linear intercept [Lm], volume density of alveolar air space [VVair]), and tissue loss (volume density of alveolar septa [VVspt]). In addition, centriacinar intra-alveolar inflammatory cells were counted to assess species differences in the type of inflammatory response associated with CS exposure. In mice, many of the morphometric parameters indicating emphysema differed significantly between CS-exposed and control animals. In CS-exposed rats, only some of the parameters differed significantly from control values. The Lm in both CS-exposed mice and rats was increased at 7 and 13 months, indicating an enlargement of parenchymal air spaces, but the VVair was increased significantly only in CS-exposed mice. The VVspt was decreased at both time points in mice, but not in rats, indicating damage to the structural integrity of parenchyma. Morphologic evidence of tissue destruction in the mice included alveoli that were irregular in size and shape and alveoli with multiple foci of septal discontinuities and isolated septal fragments. Morphometric differences in the mice at 13 months were greater than at 7 months, suggesting a progression of the disease. Inflammatory lesions within the lungs of mice contained significantly more neutrophils than those lesions in rats. These results suggest that B6C3F1 mice are more susceptible than F344-rats to the induction of emphysema by this CS exposure regimen and that in mice the emphysema may be progressive. Furthermore, the type of inflammatory response may be a determining factor for species differences in susceptibility to emphysema induction by CS exposure.

CT imaging of intrathoracic lymph nodes in dogs with bronchoscopically administered iodinated nanoparticles.

RATIONALE AND OBJECTIVES: The purpose of the study was to determine if airway instillation of iodinated nanoparticles results in contrast material enhancement of tracheobronchial lymph nodes in dogs. MATERIALS AND METHODS: Eight dogs underwent intrabronchial instillation of iodinated nanoparticles; six dogs received 900 mg each, and two dogs received 450 mg each. Spiral computed tomography (CT) was then performed 2-34 days later. RESULTS: CT scans obtained 2 days after instillation showed the presence of contrast material within the lung parenchyma but no nodal enhancement. Scans obtained 6-34 days after instillation showed enhancement of the right, left, and middle tracheobronchial lymph nodes (analogous to the mediastinal nodes in humans). Mean nodal attenuation on CT images was 117 HU +/- 43, and the mean nodal volume was 129 mm3 +/- 113. Histologic specimens of the nodes showed macrophage hyperplasia. CONCLUSION: Iodinated nanoparticles instilled into small airways are transported to the tracheobronchial lymph nodes, where they result in contrast enhancement.

Experimental hypersensitivity pneumonitis: influence of Th2 bias.

Cultured murine CD4+ cells from Saccharopolyspora rectivirgula sensitized C3H/HeJ (Th1 bias) donors can adoptively transfer murine experimental hypersensitivity pneumonitis (EHP). We sensitized BALB/c mice (Th2 bias) with S. rectivirgula, obtained spleen and lung associated lymph node (LALN) cells, cultured the cells with specific antigen, and attempted adoptive transfer of EHP. We also treated both C3H/HeJ and BALB/c donor mice with IL4 and anti-IFNgamma before exposure to S. rectivirgula and then cultured cells from both spleen and LALN before attempted transfer of EHP. We found that cultured spleen and lung associated lymph node cells can adoptively transfer EHP in both C3H/HeJ and BALB/c mice as demonstrated by infiltration of the recipient lungs with CD4+ lymphocytes. Treatment of both mouse strains with IL4 and anti-IFNgamma did not change the ability of cultured cells to adoptively transfer EHP. We conclude that EHP induced by S. rectivirgula can occur in animals with either a Th1 or a Th2 bias and is not altered by treatment with IL4 and anti-IFNgamma. This suggests that attributes of the antigen and not genetic background or cytokine environment at the site of initial sensitization determines the results of exposure to S. rectivirgula.

Chronic cigarette smoke exposure increases the pulmonary retention and radiation dose of 239Pu inhaled as 239PuO2 by F344 rats.

As a portion of a study to examine how chronic cigarette smoke exposure might alter the risk of lung tumors from inhaled 239puO2 in rats, the effects of smoke exposure on alpha-particle lung dosimetry over the life-span of exposed rats were determined. Male and female rats were exposed to inhaled 239PuO2 alone or in combination with cigarette smoke. Animals exposed to filtered air alone served as controls for the smoke exposure. Whole-body exposure to mainstream smoke diluted to concentrations of either 100 or 250 mg total particulate matter m(-3)(LCS or HCS, respectively) began at 6 wk of age and continued for 6 h d(-1), 5d wk(-1), for 30 mo. A single, pernasal, acute exposure to 239PuO2 was given to all rats (control, LCS and HCS) at 12 wk of age. Exposure to cigarette smoke caused decreased body weight gains in a concentration dependent manner. Lung-to-body weight ratios were increased in smoke-exposed rats. Rats exposed to cigarette smoke before the 239PuO2 exposure deposited less 239Pu in the lung than did controls. Except for male rats exposed to LCS, exposure to smoke retarded the clearance of 239Pu from the lung compared to control rats through study termination at 870 d after 239PuO2 exposure. Radiation doses to lungs were calculated by sex and by exposure group for rats on study for at least 360 d using modeled body weight changes, lung-to-body weight ratios, and standard dosimetric calculations. For both sexes, estimated lifetime radiation doses from the time of 239PuO2 exposure to death were 3.8 Gy, 4.4 Gy, or 6.7 Gy for the control, LCS, or HCS exposure groups, respectively. Assuming an approximately linear dose-response relationship between radiation dose and lung neoplasm incidence, approximate increases of 20% or 80% in tumor incidence over controls would be expected in rats exposed to 239PuO2 and LCS or 239PuO2 and HCS, respectively.

Pulmonary delivery of nanoparticles of insoluble, iodinated CT X-ray contrast agents to lung draining lymph nodes in dogs.

Lung cancer continues to be a leading cause of death around the world. Staging of this disease is critically dependent upon the involvement or noninvolvement of the lymph nodes which drain the region of lung containing the lesion/tumor. Palpation, unenhanced CT, and lymph node excision (i.e., mediastinectomy) are currently used to ascertain the status of these regional draining lymph nodes. The work reported herein details the first efforts toward the pulmonary instillation of iodinated nanoparticles for contrast-enhanced CT of lung draining lymph nodes. The data reflect the impact of dose, time post instillation, and formulation (surfactant) upon the observed CT enhancement of the tracheobronchial lymph nodes of beagle dogs. In addition, initial safety is discussed with both macroscopic and microscopic observations. The results indicate that pulmonary instillation of small volumes of iodinated nanoparticles could be successfully used to aid staging of lung cancer by CT imaging.

Aberrant methylation of p16(INK4a) is an early event in lung cancer and a potential biomarker for early diagnosis.

The p16(INK4a) (p16) tumor suppressor gene can be inactivated by promoter region hypermethylation in many tumor types including lung cancer, the leading cause of cancer-related deaths in the U.S. We have determined the timing of this event in an animal model of lung carcinogenesis and in human squamous cell carcinomas (SCCs). In the rat, 94% of adenocarcinomas induced by the tobacco specific carcinogen 4-methylnitrosamino-1-(3-pyridyl)-1-butanone were hypermethylated at the p16 gene promoter; most important, this methylation change was frequently detected in precursor lesions to the tumors: adenomas, and hyperplastic lesions. The timing for p16 methylation was recapitulated in human SCCs where the p16 gene was coordinately methylated in 75% of carcinoma in situ lesions adjacent to SCCs harboring this change. Moreover, the frequency of this event increased during disease progression from basal cell hyperplasia (17%) to squamous metaplasia (24%) to carcinoma in situ (50%) lesions. Methylation of p16 was associated with loss of expression in both tumors and precursor lesions indicating that both alleles were functionally inactivated. The potential of using assays for aberrant p16 methylation to identify disease and/or risk was validated by detection of this change in sputum from three of seven patients with cancer and 5 of 26 cancer-free individuals at high risk. These studies show for the first time that an epigenetic alteration, aberrant methylation of the p16 gene, can be an early event in lung cancer and may constitute a new biomarker for early detection and monitoring of prevention trials.

Carcinogenic responses of transgenic heterozygous p53 knockout mice to inhaled 239PuO2 or metallic beryllium.

The transgenic heterozygous p53+/- knockout mouse has been a model for assessing the tumorigenicity of selected carcinogens administered by noninhalation routes of exposure. The sensitivity of the model for predicting cancer by inhaled chemicals has not been examined. This study addresses this issue by acutely exposing p53+/- mice of both sexes by nose-only inhalation to either air (controls), or to 1 of 2 levels of 239PuO2 (500 or 100 Bq 239Pu) or beryllium (Be) metal (60 or 15 micrograms). Additional wild-type p53+/+ mice were exposed by inhalation to either 500 Bq of 239PuO2 or 60 micrograms of Be metal. These carcinogens were selected because they operate by differing mechanisms and because of their use in other pulmonary carcinogenesis studies in our laboratory. Four or 5 of the 15 mice per sex from each group were sacrificed 6 mo after exposure, and only 2 pulmonary neoplasms were observed. The remainder of the mice were held for life-span observation and euthanasia as they became moribund. Survival of the p53+/- knockout mice was reduced compared to the p53+/+ wild-type mice. No lung neoplasms were observed in p53+/- mice exposed to air alone. Eleven of the p53+/- mice inhaling 239PuO2 developed pulmonary neoplasms. Seven p53+/+ mice exposed to 239PuO2 also developed pulmonary neoplasms, but the latency period for pulmonary neoplasia was significantly shorter in the p53+/ mice. Four pulmonary neoplasms were observed in p53+/- mice exposed to the higher dose of Be, whereas none were observed in the wild-type mice or in the heterozygous mice exposed to the lower dose of Be. Thus, both p53+/- and p53+/+ mice were susceptible to 239Pu-induced carcinogenesis, whereas the p53+/- but not the p53+/+ mice were susceptible to Be-induced carcinogenesis. However, only 2 pulmonary neoplasms (1 in each of the 239PuO2 exposure groups) were observed in the 59 p53+/ mice that were sacrificed or euthanatized within 9 mo after exposure, indicating that the p53+/- knockout mouse might not be appropriate for a 6-mo model of carcinogenesis for these inhaled carcinogens.

Effect of cigarette smoke on CYP1A1, CYP1A2 and CYP2B1/2 of nasal mucosae in F344 rats.

Enzymes of the nasal tissue, one of the first tissues to contact inhaled toxicants, are relatively resistant to induction by traditional inducers. Because tobacco smoke has been shown to induce cytochrome P450 1A1 (CYP1A1) in rat and human lung tissue, we hypothesized that it would also alter levels of xenobiotic-metabolizing enzymes in nasal mucosae. In the present study, the effect of mainstream cigarette smoke (MCS) on nasal CYP1A1, CYP1A2 and CYP2B1/2 was explored. Four groups of 30 F344 rats were exposed to MCS (100 mg total particulate matter/m3) or filtered air for 2 or 8 weeks. Western analysis of microsomes from nasal tissue of MCS-exposed rats showed an induction of CYP1A1 in respiratory and olfactory mucosae, as well as liver, kidney and lung. Relative to controls, CYP1A2 levels increased slightly in the liver and olfactory mucosa. CYP2B1/2, which increased in the liver, appeared to decrease in upper and lower respiratory tissues. Little to no immunoreactivity with CYP1A1 antibody was observed in fixed nasal sections of control rats, yet intense immunoreactivity was seen in epithelia throughout the nasal cavity of MCS-exposed rats. Ethoxyresorufin O-deethylase activity (associated with CYP1A1/2) decreased approximately 2-fold in olfactory mucosa, but increased in non-nasal tissues of rats exposed to MCS. Methoxy- and pentoxyresorufin O-dealkylase activities (associated with CYP1A2 and CYP2B1/2, respectively) decreased in olfactory and respiratory mucosae, as well as lung (CYP2B1/2), yet increased in liver. These data suggest that xenobiotic-metabolizing enzymines of the nasal mucosae may be regulated differently than other tissues.

Dose-response relationships between inhaled beryllium metal and lung toxicity in C3H mice.

Inhaled beryllium (Be) can induce a range of adverse pulmonary responses in animals and humans including acute pneumonitis, chronic granulomatous lung disease, and cancer. To facilitate comparisons with our previous data describing Be toxicity in rats, we evaluated the toxic effects of inhaled Be metal in mice. Groups of 34 strain C3H/HeJ mice were acutely exposed by the nose-only route to aerosolized Be metal to achieve measured initial lung burdens of 0, 1.7, 2.6, 12, or 34 microg. All mice received aerosolized 85 Sr-labeled fused aluminosilicate particles (85 Sr-FAPs) immediately before their Be exposure so that the influence of Be on lung retention of these poorly soluble tracer particles could be externally quantitated. Groups of mice were euthanized at 8, 15, 40, 90, 210, and 350 days after exposure for evaluation of histopathological changes and for cytologic and biochemical indicators of lung damage measured in bronchoalveolar lavage fluid. Clearance of 85 Sr-FAP tracer particles through 196 days after exposure was delayed in mice receiving the 12 and 34 microg Be lung burdens, but not the 1.7 or 2.6 microg lung burdens. Increased total cell numbers, increased percentage of neutrophils, and elevated levels of total protein and the activities of beta-glucuronidase and lactate dehydrogenase in bronchoalveolar lavage fluid were observed in the two highest Be lung burden groups compared with controls. Lung lesions included particle-containing macrophages, granulomatous pneumonia, lymphocytic interstitial aggregates, and mononuclear interstitial infiltrates. These lesions were occasionally seen in mice receiving the 2.6 microg lung burden, were present in most of the mice receiving 12 or 34 microg lung burdens, and were generally increased in severity with time and lung burden. Thus, we have demonstrated that a single, acute inhalation exposure to Be metal can chronically retard particle clearance and induce lung damage in mice. The initial lung burdens used caused responses ranging from no apparent effects to significant Be-induced responses. A comparison of these data with our previous data from rats indicates that the mass of Be metal required to induce lung damage in mice is similar to that needed for rats. When expressed on a lung weight-normalized basis, mice appeared to be more resistant to the toxic effects of inhaled Be than rats.

Apoptosis is a pathway responsible for the resolution of endotoxin-induced alveolar type II cell hyperplasia in the rat.

Previous studies showed that intratracheal instillation of endotoxin induces transient type II cell hyperplasia in the rat lung and described some of the mechanisms involved in the proliferative response of type II cells. The purpose of the present study was to investigate how long the type II cell hyperplasia persists and how it is resolved. The portion of epithelial cells in hyperplastic lesions of the rat lung expressing cyclin D1, an indicator for cells in the G1 phase of the cell cycle, was greatest at 3 d post instillation and decreased after 4 and 6 d. The fate of the proliferating epithelial cells was traced by injecting the rats with 5-bromo-2' deoxy uridine (BrdU) 2 d post instillation, the peak time point for maximum incorporation of BrdU. Exfoliated BrdU-positive epithelial cells were detected in the alveolar spaces in tissue sections from rats 4, 5, and 6 d post instillation. BrdU-positive epithelial cells showed flattened nuclei at 6 and 10 d post instillation. Expression of the 116 kD poly(ADP-ribose) polymerase (PARP) was low in type II cells from control rats, and was increased at 3, 4, and 6 d post instillation. In cells obtained by lavage, only a 35 kD cleavage product of PARP was detected, which is an indicator of necrotic cell death. In isolated type II cells from rats 3, 4, and 6 d post endotoxin instillation, progressive cleavage of the PARP to its 89 kD residual fragment was detected, which is a direct evidence for the activation of caspases. Furthermore, apoptotic epithelial cells with condensed nuclei were identified by electron microscopy in rats 4 d post instillation. These results indicate that apoptosis is an additional mechanism for the resolution of endotoxin-induced lung epithelial hyperplasias.