Charles H. Hobbs

New approaches for the evaluation of pulmonary toxicity: bronchoalveolar lavage fluid analysis☆

Analysis of bronchoalveolar lavage fluid (BAL) is an effective method of detecting an inflammatory response in the lungs of animals in toxicological studies. Alterations in BAL that are the most sensitive indications of an inflammatory response are an increased content of serum proteins and an influx of neutrophils (PMNs). Elevation of the cytoplasmic enzyme lactate dehydrogenase (LDH) is a useful indicator of cytotoxicity. The pulmonary inflammatory response to particles (either mineral dusts or soot) in the lung includes greatly increased activities of such lysosomal enzymes as beta-glucuronidase and beta-N-acetylglucosaminidase in BAL. Examination of alterations in BAL in rats and mice during chronic exposure to high levels of diluted diesel exhaust revealed that steadily increasing levels of LDH, beta-glucuronidase, and hydroxyproline in BAL correlated better with the development of pulmonary fibrosis than did measures of an inflammatory response (protein, PMNs). Analysis of BAL has proven useful, both for detection of lung injury in toxicological screening tests and for determination of the mechanisms of developing chronic lung disease. Future work shows promise of developing assays for BAL analysis to identify the specific site or type of pulmonary injury present.

Comparative inhalation toxicity of nickel subsulfide to F344/N rats and B6C3F1 mice exposed for 12 days.

Groups of F344/N rats and B6C3F1 mice were exposed to aerosols of nickel subsulfide (Ni3S2) 6 hr/day for 12 days not including weekends. Actual exposure concentrations were within 3% of target (target = 10.0, 5.0, 2.5, 1.2, 0.6, and 0.0 mg Ni3S2/m3). Nickel lung burdens of exposed rats and mice increased linearly with exposure concentration. Two male rats and all mice exposed to 10.0 mg Ni3S2/m3 died before the end of the exposures. Exposure to Ni3S2 had no effect on the natural killer cell activity of mouse spleen cells. Lesions in rats and mice related to inhalation of Ni3S2 were found in the nasal epithelium, lung, and bronchial lymph nodes. The most extensive lesions were found in the lung and included necrotizing pneumonia. Emphysema developed in rats exposed to 5.0 or 10.0 mg Ni3S2/m3, while fibrosis developed in mice exposed to 5.0 mg Ni3S2/m3. Degeneration of the respiratory epithelium and atrophy of the olfactory epithelium of the nose occurred in rats exposed to as low as 0.6 mg Ni3S2/m3 and mice exposed to 1.2 mg/m3. Results indicate that inhalation exposure of rats and mice to Ni3S2 aerosol concentrations near the current threshold limit value (TLV) for nickel compounds (1 mg/m3 for Ni metal and roasting fume and dust and 0.1 mg/m3 as Ni for soluble compounds) can produce lesions in the respiratory tract. Atrophy of lymphoid tissues (spleen, thymus, and bronchial lymph nodes) was found in animals of the highest exposure concentration. Degeneration of the testicular germinal epithelium was also observed in mice and rats that survived 5.0 or 10.0 mg/m3 exposure concentrations.

Lung toxicity after 13-week inhalation exposure to nickel oxide, nickel subsulfide, or nickel sulfate hexahydrate in F344/N rats and B6C3F1 mice.

The relative toxicity of nickel oxide (NiO), nickel sulfate hexahydrate (NiSO4.6H2O), and nickel subsulfide (Ni3S2) was studied in F344/N rats and B6C3F1 mice after inhalation exposure for 6 hr/day, 5 days/week, for 13 weeks. Exposure concentrations used (as mg Ni/m3) were 0.4-7.9 for NiO, 0.02-0.4 for NiSO4.6H2O, and 0.11-1.8 for Ni3S2. No exposure-related effects on mortality and only minor effects on body weight gain were seen in rats or mice. The most sensitive parameter for nickel toxicity was histopathologic change in the lungs of exposed animals were chronic active inflammation, fibrosis, and alveolar macrophage hyperplasia were associated with nickel exposure. There was an exposure-related increase in lung weight in rats and mice. Equilibrium levels of nickel in the lung were reached by 13 weeks of nickel sulfate and nickel subsulfide exposure, whereas lung levels of nickel continued to increase throughout exposure to nickel oxide. Additional exposure-related histopathologic lesions in treated animals included atrophy of the olfactory epithelium after nickel sulfate and nickel subsulfide exposure. No nasal lesions were seen after nickel oxide exposure. Lymphoid hyperplasia of the bronchial lymph nodes developed in animals exposed to all three nickel compounds. The order of toxicity corresponded to the water solubility of the nickel compounds, with nickel sulfate being most toxic, followed by nickel subsulfide and nickel oxide.

Comparative toxicity of nickel oxide, nickel sulfate hexahydrate, and nickel subsulfide after 12 days of inhalation exposure to F344/N rats and B6C3F1 mice

The relative toxicity of nickel oxide (NiO), nickel sulfate hexahydrate. (NiSO4.6H2O), and nickel subsulfide (Ni3S2) was studied in F344/N rats and B6C3F1 mice after inhalation exposure for 6 h/day, 5 days/week for 12 exposure days. Exposure concentrations used (as mg Ni/m3) were 0.9-23.6 for NiO; 0.8-13.3 for NiSO4.6H2O, and 0.4-7.3 for Ni3S2. For each compound there were 5 exposure groups plus a control group. NiSO4.6H2O was the most toxic compound with exposure related mortality seen at exposure concentrations of 13.3 mg/m3 in rats and 1.6 mg/m3 and above in mice. For Ni3S2, mortality was seen in mice (but not in rats) at the highest exposure concentration (7.3 mg/m3). No mortality was seen after NiO exposure. Lesions of the lung and nasal cavity were seen in both rats and mice after exposure to NiSO4.6H2O and Ni3S2 at the 4 highest exposure concentrations. Lesions of the lung were seen primarily at the highest exposure concentrations after NiO exposure. The amount of nickel in the lungs at the end of exposure varied in relation to the water solubility of the compounds. Based on these 2-week studies, the toxicity ranking was NiSO4.6H2O greater than Ni3S2 much greater than NiO. Additional studies are in progress to assess the relative toxicities of these three nickel compounds after 90-day exposures.

Biochemical responses of rat and mouse lung to inhaled nickel compounds

Nickel subsulfide (Ni3S2), nickel sulfate (NiSO4), and nickel oxide (NiO) are encountered occupationally in the nickel refining and electroplating industries, with inhalation being a common route of exposure. The purposes of this study were to evaluate the biochemical responses of lungs of rats and mice exposed for 13 weeks to occupationally relevant aerosol concentrations of Ni3S2, NiSO4, and NiO, to correlate biochemical responses with histopathologic changes, and to rank the compounds by toxicity. Biochemical responses were measured in bronchoalveolar lavage fluid (BALF) recovered from lungs of exposed animals. Parameters evaluated in BALF were lactate dehydrogenase (LDH), beta-glucuronidase (BG), and total protein (TP). Total and differential cell counts were performed on cells recovered in BALF. All compounds produced an increase in LDH, BG, TP, and total nucleated cells, and an influx of neutrophils, indicating the presence of a cytotoxic and inflammatory response in the lungs of exposed rats and mice. Increases in BG were greater than increases in LDH and TP for both rats and mice. Chronic active inflammation, macrophage hyperplasia, and interstitial phagocytic cell infiltrates were observed histologically in rats and mice exposed to all compounds. Statistically significant increases in BG, TP, neutrophils, and macrophages correlated well with the degree of chronic active inflammation. Results indicated a toxicity ranking of NiSO4 greater than Ni3S2 greater than NiO, based on toxicities of the compounds at equivalent mg Ni/m3 exposure concentrations.

Comparative inhalation toxicity of nickel sulfate to F344/N rats and B6C3F1 mice exposed for twelve days.

Groups of F344/N rats and B6C3F1 mice were exposed to aerosols of nickel sulfate hexahydrate (NiSO4.6H2O) 6 hr/day for 12 days to determine the short-term inhalation toxicity of this compound. Target exposure concentrations were 60, 30, 15, 7, 3.5, and 0 mg NiSO4.6H2O/m3. Endpoints evaluated included clinical signs, mortality, quantities of Ni in selected tissues, effect on mouse resistance to tumor cells, and pathological changes in tissues of both rats and mice. All mice exposed to 7 mg NiSO4.6H2O/m3 or greater and 10 rats exposed to 15 mg NiSO4.6H2O/m3 or greater died before the termination of exposures. Quantities of Ni remaining in lungs of rats at the end of the exposure were independent of exposure concentration. Lung burdens of Ni in mice were approximately one-half that in lungs of rats. Exposure of female mice to 3.5 mg NiSO4.6H2O/m3 had no effect on resistance to tumor cells as determined by spleen natural killer cell activity. Histopathological changes were seen in tissues of rats and mice exposed to as low as 3.5 mg NiSO4.6H2O/m3. Lesions related to NiSO4.6H2O exposure occurred in lung, nose, and bronchial and mediastinal lymph nodes. Results indicated that exposure of rats and mice to amounts of NiSO4.6H2O aerosols resulting in Ni exposure concentrations only eight times greater than the current threshold limit value for soluble Ni (0.1 mg/m3) for as little as 12 days can cause significant lesions of the respiratory tract.

Fate of Inhaled Nickel Oxide and Nickel Subsulfide in F344/N Rats

AbstractThe fates of inhaled nickel oxide (NiO, green oxide calcined at 1200°C) and nickel subsulfide (Ni3S2), two occupationally relevant nickel compounds, have been studied in male F344/N rats. Groups of rats underwent pernasal exposure to 9.9 mg NiO/m3 or to 5.7 mg Ni3S2/m3 for 70 and 120 min, respectively. The activity median aerodynamic diameters (geometric standard deviation) of the NiO and Ni3S2 aerosols were 1.3 μm (2.0) and 1.3 μm (1.5), respectively. End points evaluated included total and regional respiratory tract deposition of the aerosols, lung clearance of deposited material, distribution of solubilized material to extrarespiratory tract tissue, and pathways of Ni excretion from the body. The fractions of the inhaled NiO and Ni3S2 aerosols that deposited in the respiratory tract were 0.11 and 0.13, respectively. The fractions of the inhaled aerosol that deposited in the lungs were 0.05 for both aerosols. Inhaled NiO cleared slowly from the lungs, with a half-life of approximately 120 days. ...

The Immunotoxicity of Three Nickel Compounds following 13-Week Inhalation Exposure in the Mouse

Groups of B6C3F1 mice were exposed to aerosols of nickel subsulfide (Ni3S2), nickel oxide (NiO), or nickel sulfate hexahydrate (NiSO4.6H2O) 6 hr/day, 5 days per week for 65 days to determine the immunotoxicity of these compounds. Exposure concentrations were 0.11, 0.45, and 1.8 mg Ni/m3 for Ni3S2, 0.47, 2.0, and 7.9 mg Ni/m3 for NiO; and 0.027, 0.11, and 0.45 mg Ni/m3 for NiSO4. Thymic weights were decreased only in mice exposed to 1.8 mg Ni/m3 Ni3S2. Increased numbers of lung-associated lymph nodes (LALN), but not spleen nucleated cells, were seen with all compounds. Nucleated cells in lavage samples were increased in mice exposed to the highest concentrations of NiSO4 and NiO and to 0.45 and 1.8 mg Ni/m3 Ni3S2. Increased antibody-forming cells (AFC) were seen in LALN of mice exposed to 2.0 and 7.9 mg Ni/m3 NiO and 1.8 mg Ni/m3 Ni3S2. Decreased AFC/10(6) spleen cells were observed in mice exposed to NiO, and decreased AFC/spleen were seen for mice exposed to 1.8 mg Ni/m3 Ni3S2. Only mice exposed to 1.8 mg Ni/m3 Ni3S2 had a decrease in mixed lymphocyte response. All concentrations of NiO resulted in decreases in alveolar macrophage phagocytic activity, as did 0.45 and 1.8 mg Ni/m3 Ni3S2. None of the nickel compounds affected the phagocytic activity of peritoneal macrophages. Only 1.8 mg Ni/m3 Ni3S2 caused a decrease in spleen natural killer cell activity. Results indicate that inhalation exposure of mice to nickel can result in varying effects on the immune system, depending on dose and physicochemical form of the nickel compound. These nickel-induced changes may contribute to significant immunodysfunction.

Surface Area, Adsorption, and Desorption Studies on Indoor Dust Samples

Adsorption by dust samples from homes was studied to evaluate the extent to which household dust particles could act as carriers of vapors into the lower respiratory tract. The dust samples were examined for volatilizable and combustible materials via thermogravimetric analysis as well as for specific surface area and surface characteristics by nitrogen and formaldehyde adsorption and desorption isotherms. Particle morphology and size were examined by scanning electron microscopy (SEM) and elemental composition by energy dispersive x-ray analysis (EDXA). Thermogravimetric analysis (TGA) demonstrated marked differences between samples from different homes. The volatilizable material removed by heating dust samples in argon to 500°C ranged from 32% to 69%. The residue not combustible in oxygen at 700°C, presumably minerals, ranged from 11% to 58%. In most cases, differences between samples from the same borne were small. SEM of the samples demonstrated the presence of many fibers and of irregularly shaped p...

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 μg). Additional wild-type p53 +/- mice were exposed by inhalation to either 500 Bq of 239PuO2 or 60 μg 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 53 +/- 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.