Exposure to sidestream cigarette smoke alters bronchiolar epithelial cell differentiation in the postnatal rat lung.

This study examines the effects of an ambient concentration of aged and diluted sidestream cigarette smoke (ADSS) on bronchiolar epithelial cell development and the expression of cytochrome P450 isozyme 1A1 protein in the postnatal rat lung. In control animals, the labeling indices for epithelial cells in proximal bronchi and terminal bronchioles at 7 days of age were 2.3 and 4.9%, respectively, and decreased to 0.1 and 0%, by 100 days of age. With exposure to ADSS from birth, the labeling index of epithelial cells in distal airways of rats was significantly reduced at 7 and 14 days of age, but not in epithelial cells of proximal bronchi. The expression of P450 isozyme 1A1 antigen in bronchiolar epithelial cells of control rats reached the maximal level observed at 21 days of age and subsequently decreased to low levels at 50 and 100 days of age. In contrast, exposure to ADSS significantly increased the distribution and intensity of staining for 1A1 antigen in bronchiolar epithelial cells of proximal and distal airways as early as 7 days of age and maintained elevated levels of 1A1 protein in these cells through 100 days of age. At 21 and 50 days of age, NADPH reductase protein expression was higher in the airway epithelium of rats exposed from birth to ADSS than that noted in the airways of controls. In contrast, cytochrome P450 isozyme 2B and Clara cell secretory protein expression were unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathobiology of lung tumors induced in hamsters by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and the modulating effect of hyperoxia.

Neuroendocrine lung cancer is among the most common types of lung cancers in smokers. We have recently shown that exposure of hamsters to N-nitrosodiethylamine and hyperoxia causes a high incidence of this tumor type. In this study, we show that the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone also causes neuroendocrine lung tumors in hyperoxic hamsters. Animals maintained in ambient air while being treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone developed pulmonary adenomas composed of Clara cells and alveolar type II cells. Pathogenesis experiments provide evidence for the tumors caused by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in ambient air being derived from Clara cells. In the hyperoxic hamsters, the neuroendocrine carcinogenesis appears to involve two stages: (a) transformation of focal alveolar type II cells into neuroendocrine cells and (b) development of neuroendocrine lung tumors from such foci.

A new monoclonal antibody to study mouse macrophage antigen during BHT-induced lung injury and repair.

A rat monoclonal antibody 133-13A to a mouse lung carcinoma cell line was found to react with macrophages in mouse lung [1]. This monoclonal antibody is different from previously described antibodies to macrophages. Immunogold electron-microscopy and immunoperoxidase light microscopy have been used to show that MoAb 133-13A binds specifically to macrophages in normal and in BHT treated mouse lungs. This MoAb recognizes a protein of approximately 100 kDa (P100) on cultured lung carcinoma cells and a 87 kDa protein on macrophages from lung or the peritoneal cavity which is different from other macrophage antigens. The surface glycoprotein has been purified from cultured cells using immunoaffinity chromatography. The purified protein was radioiodinated and MoAb 133-13A was used to develop a competition radioimmunoassay to quantitate P100. Spleen, intestines, lung, skin and uterus all have high levels of P100. P100 on peritoneal macrophages has been determined to be about 94,000 molecules/cell. Analyses of lung lavage and whole lung homogenates from mice treated with BHT, BHT plus 70% O2, and 70% O2 alone show that treated animals have elevated P100 content compared to corn oil treated mice.

Effects of hyperoxia on B16-F10 cells in vivo.

The in vivo effects of hyperoxia were studied in lung colonies formed by B16-F10 melanoma cells in C57BL/6 mice. Several antioxidant defenses were found to change with in vivo exposure: glutathione reductase and glucose-6-phosphate dehydrogenase activities decreased as compared with levels in the cultured cells, glutathione peroxidase activity dramatically increased, and Mn-superoxide dismutase activity and levels of total glutathione were similar in vivo and in vitro. Exposure of tumor-bearing animals to 70%, O2 for 3 weeks did not alter the antioxidant defenses measured in the tumors. One hundred percent O2 exposure did not affect either initial arrest or subsequent retention of radiolabeled B16-F10 cells in the lung. Likewise, hyperoxia did not appear to alter cell division in B16-F10 cells growing in the lung. These results are consistent with our previous studies indicating that the B16-F10 cell line is resistant to levels of O2 in vivo that adversely affect other tumor cell lines.

Antibodies to mouse lung capillary endothelium.

We are interested in developing monoclonal antibodies (MoAbs) that recognize specific cell types in the lung of BALB/c mice. Normal mouse lung homogenate was used to immunize F344 rats and hybridomas were produced by fusion of rat spleen cells with mouse myeloma SP 2/0. Two hybridomas were selected which produced MoAbs active in immunohistochemistry of lung cells. MoAb 273-34A and 411-201B both show extensive peroxidase staining of capillary endothelial cells within alveolar walls of lungs at the light microscopic level. To demonstrate cell specificity, immunoelectron microscopy with gold-labeled antibody was performed. Lightly fixed lungs were frozen and thin-sectioned before staining with MoAb and 5-nm gold particles coupled to secondary antibody. Quantitative analyses of these cryosections show that both antibodies, used at optimal concentrations, are specific for binding to capillary endothelial cells. More than 95% of the gold particles are associated with capillary endothelial cells on the thin side of the alveolar wall. When capillaries adjoined thick septa containing interstitial cells, about two thirds of the gold particles were associated with endothelial cells and about one quarter with interstitial cells. These MoAbs should be useful in studying the role of endothelial cells in toxic lung injury.

An animal model for neuroendocrine lung cancer.

Neuroendocrine lung cancer is among the most common types of lung tumor in man and demonstrates a strong etiological association with cigarette smoking. However, despite numerous efforts, this cancer type has never been induced in animals. This report describes, for the first time, the reproducible induction of pulmonary neuroendocrine cancer in a readily available hamster model. The data provide evidence that deviations from pre-existing normal pulmonary oxygen levels may be an essential factor for the induction of this malignancy. This new model should greatly facilitate studies of the pathophysiology, biochemistry and therapy of neuroendocrine cancer of the lung.

Bronchoalveolar lavage in rats and mice following beryllium sulfate inhalation.

The effects of lung injury in rats and mice exposed to an aerosol of beryllium sulfate for 1 hr through nose-only inhalation were evaluated by the method of bronchoalveolar lavage. The lavage fluid of rats exposed to an aerosol of either 3.3 or 7.0 micrograms Be/liter over a 21-day period following exposure indicated lactate dehydrogenase (LDH) and alkaline phosphatase (Alk Pase) activities to be the most sensitive indicators of lung damage. LDH activity peaked at 8 days postexposure while Alk Pase activity was maximum at Day 5. Both values were 30 times greater than comparable controls at these time points. Acid phosphatase activity and albumin levels also increased over the 21-day period, but not to the same extent. The lung lavage of mice exposed to 7.2 micrograms Be/liter showed LDH activity as the most sensitive indicator of lung damage with a maximum response three times greater than that of controls at Day 5.

Skin tumorigenic potential of crude and refined coal liquids and analogous petroleum products.

The skin tumorigenic potential of seven complex hydrocarbon mixtures was determined: a coal-derived raw blend composed of light and heavy oils, a low- and high-severity hydrotreated product of that blend, and naphthas and fuel oils from the raw blend or from natural petroleum. Male and female C3H/Bdf mice were exposed three times per week to each test mixture by dermal application of 50 microliters of neat, 50, or 25% (w/v) preparations. Room, vehicle, and benzo[alpha]pyrene control groups were run concurrently. The raw blend produced an almost 100% incidence of skin tumors at all three doses while tumorigenicity was considerably decreased by hydrotreating the blend both in terms of incidence and onset. The tumorigenicities of the naphthas and fuel oils derived from the raw blend or from petroleums were low relative to that of the parent mixture. Although tumorigens in the raw blend were much reduced by hydrotreatment, tumorigenicity of the other agents did not parallel the content of polycyclic aromatic hydrocarbons known to be good tumor initiators.

Acute respiratory failure induced by bleomycin and hyperoxia: pulmonary edema, cell kinetics, and morphology.

The acute manifestations of experimental bleomycin- and hyperoxia-induced lung damage were examined. Hamsters were treated with 5 U/kg bleomycin intratracheally followed by exposure to 80% oxygen (O2). As little as 12 hr of O2 exposure potentiated the bleomycin injury; however, the onset of mortality was 72 hr after treatment. The onset of pulmonary edema, measured by radiolabeled tracers, also occurred 72 hr after treatment. Cell kinetics studies showed that 24 hr exposure to 80% O2 did not alter early alveolar cell proliferation. Treatment with bleomycin alone did result in an early increase in alveolar macrophage and type II pneumocyte labeling. Animals treated with both bleomycin and hyperoxia had an increase in macrophage labeling, but not in type II pneumocyte labeling. We conclude that increased macrophage numbers associated with suppressed type II pneumocyte proliferation may play key roles in the potentiation and development of lung damage caused by bleomycin and hyperoxia treatment.

Protection by parenteral iron administration against the inhalation toxicity of beryllium sulfate.

Animals exposed to an aerosol of BeSO4 showed a significant reduction in mortality with iron treatment. Rats were exposed for 2 h in a nose-only inhalation chamber for 14 days to an aerosol of 2.59 micrograms/Be/l. The cumulative mortality of animals concurrently treated with iron salt was significantly reduced (P less than 0.05) compared to animals which had not received iron treatment.

Enhanced tumour development by butylated hydroxytoluene (BHT) in the liver, lung and gastro-intestinal tract.

Continuous feeding of 0.5 or 0.05% butylated hydroxytoluene (BHT) enhances the development of spontaneously occurring liver tumours in C3H mice, but not in BALB/c mice. In mouse lung, the tumour-enhancing effects of BHT vary with the carcinogen used. In the gastro-intestinal tract of mice and rats, BHT enhances the development of dimethylhydrazine-induced tumours, but is without effect on tumours produced by methylnitrosourea. Strain differences, the effect upon various carcinogens, paradoxical dose responses and mechanisms of action remain major questions in the toxicology of BHT.

Surfactant alterations following acute bleomycin and hyperoxia-induced lung damage.

Hamsters treated with 0.5 U/kg intratracheal bleomycin and exposed for 24 h to 80% O2 develop acute respiratory failure 72 h after treatment. To examine indirectly the lung epithelial type II cell changes, alterations in pulmonary surfactant was measured. Presence and amount of dipalmitoyl phosphatidyl choline (DDPC) and sphingomyelin (SM) were measured, and the DPPC/SM ratio was determined in brochoalveolar lavage samples from treated and control animals 24, 48, 72, and 96 h after treatment. Hamsters treated with bleomycin and O2 had a significantly decreased DPPC/SM ratio at 72 h, which is the time of onset of respiratory failure. The decreased DPPC/SM ratio may reflect type II cell damage and inhibition of surfactant function by the edema fluid.

Modification of lung tumor growth by hyperoxia.

The effects of hyperoxia on lung tumor development were examined in mice and rats. In mice, exposure to 70% O2 prevented the development of urethan- or 3-methylcholanthrene-induced lung tumors. Dietary antioxidants [butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA)] were unable to prevent the inhibition of tumor development by oxygen, although BHT retained its capability to enhance tumor development in mouse lung. In visible-size tumors, oxygen did not depress DNA synthesis. Oxygen also reduced the number of pulmonary metastatic nodules after i.v. injection of mammary gland-derived carcinoma cells, but failed to inhibit growth of murine lung carcinoma or murine melanoma-derived cell lines. Rats treated with one single intratracheal instillation of 3-methylcholanthrene developed multiple lung lesions; their growth could be prevented by exposure of the animals to 40 or 70% O2. It is concluded that hyperoxia prevents development of transformed cells in vivo in the lung and may affect adversely the growth of selected cell lines metastatic to the lung.

Acute pulmonary toxicity of beryllium sulfate inhalation in rats and mice: cell kinetics and histopathology.

The acute response in the lung of mice and rats exposed to an aerosol of BeSO4 was characterized by cell kinetics and histopathology. Animals were exposed for 1 hour in a nose-only chamber to a 13 microgram/liter chamber concentration of BeSO4 or to H2SO4 as control aerosol, and killed thereafter over a period of 21 days. Lung cell kinetics were evaluated as the labeling index (LI), defined as the percentage of cells labeled with tritiated thymidine. The labeled cells were also differentiated as to type. In rats, the LI showed a peak response on Day 8 following exposure, while the LI of mice showed a maximum response at Day 5. In rats, the proliferative response involved type II alveolar epithelial cells, interstitial and capillary endothelial cells. Histopathology showed type II alveolar cell hyperplasia and vacuolization of their cytoplasm, a thickened interstitium with infiltrates of interstitial macrophages and segmental leukocytes; a prominent feature was alveolar macrophages with ragged membranes. Three weeks after exposure, the interstitial response was largely resolved. In mice, the proliferative response was mainly found in the alveolar macrophage population and the interstitial and endothelial cells. Histopathological changes were similar to those found in rats, although less severe.

Separation of early diffuse alveolar cell proliferation from enhanced tumor development in mouse lung.

A/J mice given urethan (1000 mg/kg) followed by four injections of butylated hydroxytoluene (BHT) (400 mg/kg) developed within 4 mo approximately 40% more lung tumors than mice treated with urethan and given four injections of corn oil. Administration of the metabolic inhibitor piperonyl butoxide prior to BHT injection abolished overall alveolar cell proliferation although it did not completely suppress type II alveolar cell proliferation. Tumor multiplicity in those animals remained significantly higher (29%) than in corresponding controls. In mice treated with 3-methylcholanthrene repeated injections of BHT (300 mg/kg) increased tumor multiplicity by a much larger factor (500-800). Pretreatment of mice with BHT reduced the number of tumors produced by methylcholanthrene and at the same time blocked all cell proliferation following additional injections of BHT; nevertheless, BHT treatment given after methylcholanthrene again increased tumor multiplicity by the same factor (500-800%) seen in animals not made tolerant to BHT. It is concluded that the enhancing effect of BHT on lung tumor development is not due to the production of diffuse alveolar cell hyperplasia.

Strain A mouse pulmonary tumor test results for chemicals previously tested in the National Cancer Institute carcinogenicity tests.

Sixty-five chemicals were coded and examined for their ability to induce lung tumors in strain A/St (laboratory A) or strain A/J (laboratory B) mice. Thirty-five chemicals were tested in laboratory A only, 6 in laboratory B only, and 24 in both laboratories. Two-year carcinogenicity test results as well as genotoxicity test data are available for most of these chemicals. There was poor interlaboratory agreement in strain A test results for the 24 chemicals tested in both laboratories. In addition, there was poor agreement between strain A test results from either laboratory and 2-year carcinogenicity test results or genotoxicity results. Possible explanations for these findings include selection of a large number of aromatic amines in the group of chemicals submitted for strain A testing, differences in strain A testing protocols and in statistical analysis of results from the two laboratories, low sensitivity of the strain A/St mice used in this particular study, and general problems inherent in comparing any relatively short-term animal tumor model with 2-year carcinogenicity tests. Since there is no absolute reference for carcinogenicity, no one test system is better than another. Carcinogenicity test data are relevant only to the test model employed.

Inhibition of mouse lung tumor development by hyperoxia.

The hypothesis was tested that continuous hyperoxia would enhance the development of lung tumors in mice. In strain A/J mice treated with a single dose of urethan (1000 mg/kg) and exposed to 70% O2 for 16 wk, an average of 5 tumors per lung developed, whereas in animals kept in air, an average of 20 tumors per lung was found. When the animals were returned to air after oxygen exposure, it was found that a difference of 15 tumors per lung between the two groups persisted up to 1 yr later, indicating that O2 was tumoricidal. The shortest duration of O2 exposure to be effective was 4 wk, and delay of O2 exposure up to 12 wk after urethan still was effective in reducing the number of developing tumors. Histopathology showed that continued exposure to 70% O2 produced some hyperplasia of the bronchiolar epithelium and only very discrete changes in the pulmonary parenchyma. Analysis of cell proliferation patterns with a continuous [3H]thymidine labeling technique showed a persistent high cell labeling in the bronchiolar epithelium and a temporary increase in alveolar wall cell labeling. Chronic hyperoxia failed to alter the activities of pulmonary superoxide dismutase or glucose-6-phosphate dehydrogenase. Ornithine decarboxylase, on the other hand, was increased as long as the animals remained exposed to oxygen. It was concluded that hyperoxia kills developing tumor cells in mouse lung.

Ferritin and in vivo beryllium toxicity.

Beryllium (Be+2), a divalent metal ion, is toxic to both man and animal. Although the molecular basis for its toxicity is unknown, it is well established that micromolar concentrations of beryllium specifically inhibit certain enzymes. Previous in vitro studies have shown that the presence of ferritin, an iron-storage protein, reactivated these enzymes by sequestering beryllium (Price and Joshi, 1984). In the present study we demonstrate in vivo that beryllium and zinc are bound by ferritin in greater amounts than Pb+2, Cu+2, and Cd+2. Beryllium did not induce the synthesis of metallothionein. In animals pretreated with an iron salt (ferric ammonium citrate, 40 mg/kg body wt), liver ferritin was elevated approximately five times and the toxicity of intravenously injected beryllium was significantly attenuated. Excretion and deposition studies suggested that iron salt treatment resulted in a reduction of liver beryllium. Thus the protection against beryllium toxicity by ferric ammonium citrate may be due to increased production of ferritin which binds beryllium and its subsequent elimination in the feces.

The effects of dietary butylated hydroxytoluene on liver and colon tumor development in mice.

Male and female C3H mice were fed a diet containing 0.5% or 0.05% of the antioxidant butylated hydroxytoluene (BHT). After 10 months, male but not female animals had a significantly increased incidence of liver tumors compared to animals kept on a BHT-free control diet. In a second experiment, male BALB/c mice were treated subcutaneously with the carcinogens dimethylhydrazine (DMH) or intrarectally with methylnitrosourea (MNU). A diet containing 0.5% BHT significantly increased the incidence of colon tumors in DMH treated animals but had no effect in mice given MNU. It is concluded that the effect of BHT on tumor development depends on strain and target organ examined and possibly also on the chemical carcinogen used.

Cadmium-induced lung injury: cell kinetics and long-term effects.

The effects of exposure to CdCl2 aerosols followed by hyperoxia were studied in mouse lung. Special emphasis was placed on analysis of cell proliferation following injury. Male Balb/c mice were exposed to aerosols of 4.9 micrograms Cd/liter for 1 hr while controls were exposed to water aerosols. Immediately after, half of each group was placed in 80% O2 for 6 days, while the rest were left in room air. Three endpoints were used to assess lung injury; measurement of hydroxyproline, [14C]thymidine incorporation into DNA, and histopathology. Parenchymal and bronchiolar labeling indices were determined following autoradiography. A 1-hr exposure to CdCl2 aerosols caused marked cell proliferation in the lung with the peak of cell labeling occurring at Day 5. In animals exposed to both CdCl2 + 80% O2, the cell labeling peak was delayed until Day 9. Cell differentiation studies showed a delay in the peak of type II epithelial cell and endothelial cell division when CdCl2 exposure was followed by the 80% O2 treatment. On Day 15 most of the labeled cells were identified as interstitial cells in both treated groups. Bronchiolar cell labeling was suppressed at the early time period in the Cd + O2 group. With time, the histologically visible lung lesions tended to resolve in animals exposed to CdCl2 or CdCl2 and 80% O2, whereas total pulmonary hydroxyproline remained at all times (3, 6, and 12 months) significantly higher in Cd-treated animals when compared to controls. It was concluded that acute lung injury by a toxic inhalant can be amplified if there is an initial delay in pulmonary cell proliferation following an acute insult.