Robert C. Lindenschmidt

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.

Pulmonary response to inhaled silica or titanium dioxide

The pulmonary response to mineral dust inhalation was investigated by characterizing markers of lung injury and inflammation, macrophage activation, dust clearance, and histopathology. Rats were exposed (6 hr/day x 5 days) to air or 50 mg/m3 crystalline silica (SiO2) or titanium dioxide (TiO2). At 7, 14, 28, and 63 days after exposure, bronchoalveolar lavage fluid (BALF) was analyzed for lactate dehydrogenase (LDH), total protein, and N-acetylglucosaminidase, as well as cell number, type, and viability. Alveolar macrophages (AM) obtained in BALF were cultured with or without LPS and release of interleukin-1 (IL-1) and fibronectin was determined. Histopathology was conducted at 28 and 63 days. The exposure protocol resulted in 1.8-1.9 mg of mineral dust being deposited in the pulmonary region. Clearance of SiO2 was significantly less than TiO2. SiO2 increased BALF neutrophils (Days 14, 28, and 63), total protein (Days 28 and 63), and LDH and lymphocytes (Day 63). SiO2 increased AM-derived fibronectin release (Day 63) and LPS-induced IL-1 release (all time points), but not spontaneous release of IL-1. TiO2 did not change BALF biochemical or cellular parameters or AM secretory activity. Histopathology revealed minimal interstitial inflammation with SiO2 and no significant response in control or TiO2 rats. These results demonstrate the pulmonary response to inhaled SiO2 can be differentiated from the relatively innocuous TiO2 by changes in BALF markers of injury and inflammation further supporting the use of BALF analysis to make relative assessments of pulmonary toxicity. The stimulation of macrophage fibronectin release by the fibrogenic dust SiO2 and not TiO2 is consistent with a role for this glycoprotein in lung injury and repair. Last, the early and persistent effect of SiO2 on LPS-induced AM IL-1 release indicates this response may represent a sensitive early marker of dust-induced changes in the AM population.

Intratracheal versus intravenous administration of bleomycin in mice: Acute effects

We examined whether intratracheal instillation (IT) of bleomycin would produce similar or dissimilar lesions when compared to lung damage following intravenous (iv) injection of the drug. BALB/c mice were treated with either 4 U/kg IT or 100 U iv bleomycin and killed at intervals up to 21 days after treatment. Cell proliferation, histopathology, lung lavage, and hydroxyproline content were examined. There was a biphasic response in the cell proliferation in the IT-treated mice, while the iv-treated mice showed a single delayed peak in proliferation. The histopathologic features of interstitial pneumonitis, elevation of lung lavage enzyme activities, and lung hydroxyproline content were qualitatively similar between the two routes of administration, although the IT mice response was always greater in magnitude. Differences exist between the lung reaction to these two routes of administration, but these differences reflect nonspecific inflammatory response and magnitude of initial injury. We conclude that the response to bleomycin administered IT is basically similar to the changes produced by intravenous injection of the drug.

The Many Faces of an Increase in Lung Collagen

Many toxic chemicals have been shown to produce an increase in lung collagen. Biochemical measurements of total lung hydroxyproline are a convenient method to quantitate and to follow disease processes associated with such an increase. However, biochemical measurements alone do not distinguish various pathological lesions and histology is needed to provide additional important information. Cell kinetic studies enhance histopathology in showing the dynamics of cell proliferation and tissue renewal. When these methods of studying lung collagen are applied to different toxic agents, such as butylated hydroxytoluene, methylcyclopentadienyl manganese tricarbonyl, CdCl2, or anticancer drugs, different patterns of hydroxyproline accumulation and cellular kinetics are revealed. It is speculated that the development of fibrotic lung disease may be at least partially determined by the initial lesion and that quantitative analyses of cell kinetic patterns help to understand and to predict the nature and evolution of disease processes.

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.

Pulmonary toxicity of cytostatic drugs: cell kinetics.

Mice were treated with three cytostatic drugs: cyclophosphamide, busulfan, or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The alveolar labeling index was measured following drug administration with a pulse of 3H-labeled thymidine and autoradiography. In cyclophosphamide-treated animals, peak alveolar cell proliferation was seen 5 days after injection of the drug. In animals treated with busulfan or BCNU, proliferation was even more delayed (occurring 2-3 weeks after administration). In contrast, with oleic acid, the highest alveolar cell labeling was found 2 days after intravenous administration. In animals exposed to a cytostatic drug, proliferation of type II alveolar cells was never a prominent feature whereas in animals treated with oleic acid there was an initial burst of type II cell proliferation. It is concluded that the patterns of pulmonary repair vary between chemicals designed to interfere with DNA replication as compared to agents which produce acute lung damage such as oleic acid.

Attenuation of pulmonary fibrosis in mice by aminophylline

Cyclic nucleotides have been shown in vitro to regulate fibroblast proliferation and/or collagen production. We have reported previously that propranolol, which decreases the cAMP/cGMP ratio, potentiates the amount of fibrosis produced in a damaged lung. The purpose of this study was to determine if elevations in the cAMP/cGMP ratio may attenuate collagen production by fibroblasts following lung damage. Lung injury was induced in mice by either butylated hydroxytoluene (BHT) (350 or 400 mg/kg intraperitoneally) or bleomycin (4 units/kg intratracheally). The mice were treated with a phosphodiesterase inhibitor, aminophylline (20 mg/kg twice daily), prior to induction of lung injury and for the duration of the study. Cyclic nucleotide changes in the lung were also determined during lung injury, with and without aminophylline. The administration of aminophylline, which increased the cAMP/cGMP ratio, resulted in attenuation of the increase in total lung collagen normally seen after injury, while having no effect on collagen levels in the undamaged lung. The results are compatible with the hypothesis that elevation of whole lung cAMP/cGMP ratio early in the damage and repair process correlates with decreased hydroxyproline deposition.

Progressive pulmonary fibrosis in rats: a biochemical, cell kinetic, and morphologic analysis☆

The concomitant treatment of rats with bleomycin and hyperoxia results in synergistic development of pulmonary injury. We exposed rats to 70% oxygen for 72 hr following an intratracheal instillation of bleomycin (0.2 U/kg body wt). Animals were killed 15, 30, 60 and 90 days after treatment for hydroxyproline, cell kinetics, and histopathologic analysis. A 16% increase in hydroxyproline over controls was seen 15 days after treatment which was manifested by the proliferation phase of diffuse alveolar damage and an increase in cell labeling by tritiated thymidine. Thirty days after treatment the hydroxyproline remained elevated while lung injury appeared to be healing with a residual focal interstitial pneumonitis and a drop in cell labeling. Between 60 and 90 days, there was an additional significant increase in hydroxyproline to 44% over controls. Diffuse interstitial pneumonitis with fibrosis was observed. Cell labeling remained constant between 60 and 90 days. We conclude that the treatment of rats with bleomycin and hyperoxia results in slowly progressive pulmonary fibrosis. The increase in hydroxyproline in the chronic phase was not accompanied by an increase in cell proliferation, and therefore may have resulted from an increase in cellular production of hydroxyproline rather than increased number of cells producing collagen.

Modification of gastrointestinal tumor development in rats by dietary butylated hydroxytoluene

Male Fischer 344 rats were given two or four injections of 1,2-dimethylhydrazine (DMH), 40 mg/kg sc, and then fed a diet containing 0.5% butylated hydroxytoluene (BHT). Five months later, the animals treated with two doses of DMH had a significantly higher incidence of colon tumors than the animals fed a BHT-free control diet. In animals treated with four injections of DMH, the increase in colon tumor incidence was statistically not significant, but BHT appeared to produce a shift in tumor distribution. In a second experiment, Fischer 344 rats were treated with 2 X 40 mg/kg of DMH and fed a diet of 0.5 or 0.1% BHT for 6 months; these animals had a significantly increased incidence of small intestinal tumors (duodenum, jejunum, and ileum) compared with animals fed the control diet. In rats treated with DMH and given a diet of 0.5% butylated hydroxyanisole (BHA), overall incidence of gastrointestinal tract tumors was higher than in control animals, although the difference was statistically not significant. Administration of N-nitroso-N-methylurea (NMU; 90 mg/kg given orally) produced stomach and colon tumors; 0.5% BHT in the diet did not modulate tumor incidence. It is concluded that dietary BHT may enhance development of gastrointestinal tumors produced by DMH, but not by NMU, provided exposure to BHT occurs after exposure to the carcinogen.

O,S,S,-Trimethyl phosphorodithioate-induced lung damage in rats and mice

O,S,S,-Trimethyl phosphorodithioate (OSS) is a contaminant of various organophosphorus insecticides which induces delayed damage to rat lung bronchiolar and alveolar epithelial cells. Whether lung damage occurs in mice has not been tested. Changes in DNA synthesis, an index of cell division after the induction of damage, were monitored by measuring thymidine incorporation into pulmonary DNA. Mice, treated with 45 mg/kg OSS, exhibited a significant increase in pulmonary thymidine incorporation on Day 5. Maximal increases occurred on Days 7-10 and were followed by a gradual decline to control levels by Day 15. The labeling index of mouse lung cells, determined following autoradiography, exhibited a similar time course. Differential cell counts indicated that maximal division of type II cells occurred before that of interstitial cells, although interstitial cells were the predominant type labeled at all times. Pulmonary DNA synthesis was significantly increased in rats 2 days after treatment with 90 mg/kg OSS. Maximal thymidine incorporation was measured on Day 3, followed by a decline to control levels on Day 5. Thymidine incorporation into total lung DNA was dose related in both species. Maximal increases appeared after 45 and 90 mg/kg OSS in mice and rats, respectively. The histopathological changes in mouse lung tissue were similar, but somewhat less severe than those seen in rats. Rats exhibited a severe interstitial pneumonitis with type I alveolar cell destruction followed by type II cell proliferation. Mice exhibited a mild to moderate alveolitis with only slight damage to type I cells. Necrosis of bronchiolar Clara cells was evident in both species but was more extensive in rats. SKF 525a and piperonyl butoxide prevented OSS-induced increases in pulmonary DNA synthesis in rat lung suggesting that metabolic activation was necessary to elicit damage. Piperonyl butoxide treatments had no effect, however, on thymidine incorporation after OSS in mouse lung tissue, and the highest dose of SKF 525a had only a moderate inhibitory effect on this parameter while increasing animal mortality. These data indicate that systemic treatment with OSS results in damage to mouse, as well as rat, lung tissue at both the alveolar and bronchiolar levels.