Janet M. Benson

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.

Recreational exposure to aerosolized brevetoxins during Florida red tide events

Abstract During two separate Karenia brevis red tide events, we measured the levels of brevetoxins in air and water samples, conducted personal interviews, and performed pulmonary function tests on people before and after they visited one of two Florida beaches. One hundred and twenty-nine people participated in the study, which we conducted during red tide events in Sarasota and Jacksonville, FL, USA. Exposure was categorized into three levels: low/no exposure, moderate exposure, and high exposure. Lower respiratory symptoms (e.g. wheezing) were reported by 8% of unexposed people, 11% of the moderately exposed people, and 28% of the highly exposed people. We performed nasal–pharyngeal swabs on people who experienced moderate or high exposure, and we found an inflammatory response in over 33% of these participants. We did not find any clinically significant changes in pulmonary function test results; however, the study population was small. In future epidemiologic studies, we plan to further investigate the human health impact of inhaled brevetoxins.

Response of Rodents to Inhaled Diluted Diesel Exhaust: Biochemical and Cytological Changes in Bronchoalveolar Lavage Fluid and in Lung Tissue

The effect of long-term (24 months) inhalation of diesel exhaust on the bronchoalveolar region of the respiratory tract of rodents was assessed by serial (every 6 months) analysis of bronchoalveolar lavage fluid (BALF) and of lung tissue from F344/Crl rats and CD-1 mice (both sexes) exposed to diesel exhaust diluted to contain 0, 0.35, 3.5, or 7.0 mg soot/m3. The purpose of the study was twofold. One was to assess the potential health effects of inhaling diluted exhaust from light-duty diesel engines. The second was to determine the usefulness of BALF analysis in detecting the early stages in the development of nononcogenic lung disease and differentiating them from the normal repair processes. No biochemical or cytological changes in BALF or in lung tissue were noted in either species exposed to the lowest, and most environmentally relevant, concentration of diesel exhaust. In the two higher levels of exposure, a chronic inflammatory response was measured in both species by dose-dependent increases in inflammatory cells, cytoplasmic and lysosomal enzymes, and protein in BALF. Histologically, after 1 year of exposure, the rats had developed focal areas of fibrosis associated with the deposits of soot, while the mice, despite a higher lung burden of soot than the rats, had only a fine fibrillar thickening of an occasional alveolar septa in the high-level exposure group. Higher increases in BALF beta-glucuronidase activity and in hydroxyproline content accompanied the greater degree of fibrosis in the rat. BALF levels of glutathione (GSH) and glutathione reductase activity increased in a dose-dependent fashion and were higher in mice than in rats. Lung tissue GSH was depleted in a dose-dependent fashion in rats but was slightly increased in mice. This depletion may have played a role in the greater fibrogenic response observed in rats. Other tissue changes in enzymatic activity were small compared to changes observed in BALF. The exposure did not increase the cytochrome P-450 content of the lung in either species. The results suggest that, for the noncarcinogenic health effects reported in this paper, there is a threshold of exposure below which adverse effects were not observed. This threshold was well above environmentally relevant levels of diesel exhaust but may be in the range of some occupational exposures. The analysis of BALF proved a useful adjunct to the chronic toxicity study to quantitate the inflammatory changes accompanying the development of pulmonary disease.

Cytochrome P450 2E1 is the primary enzyme responsible for low-dose carbon tetrachloride metabolism in human liver microsomes

We examined which human CYP450 forms contribute to carbon tetrachloride (CCl(4)) bioactivation using hepatic microsomes, heterologously expressed enzymes, inhibitory antibodies and selective chemical inhibitors. CCl(4) metabolism was determined by measuring chloroform formation under anaerobic conditions. Pooled human microsomes metabolized CCl(4) with a K(m) of 57 microM and a V(max) of 2.3 nmol CHCl(3)/min/mg protein. Expressed CYP2E1 metabolized CCl(4) with a K(m) of 1.9 microM and a V(max) of 8.9 nmol CHCl(3)/min/nmol CYP2E1. At 17 microM CCl(4), a monoclonal CYP2E1 antibody inhibited 64, 74 and 83% of the total CCl(4) metabolism in three separate human microsomal samples, indicating that at low CCl(4) concentrations, CYP2E1 was the primary enzyme responsible for CCl(4) metabolism. At 530 microM CCl(4), anti-CYP2E1 inhibited 36, 51 and 75% of the total CCl(4) metabolism, suggesting that other CYP450s may have a significant role in CCl(4) metabolism at this concentration. Tests with expressed CYP2B6 and inhibitory CYP2B6 antibodies suggested that this form did not contribute significantly to CCl(4) metabolism. Effects of the CYP450 inhibitors alpha-naphthoflavone (CYP1A), sulfaphenazole (CYP2C9) and clotrimazole (CYP3A) were examined in the liver microsome sample that was inhibited only 36% by anti-CYP2E1 at 530 microM CCl(4). Clotrimazole inhibited CCl(4) metabolism by 23% but the other chemical inhibitors were without significant effect. Overall, these data suggest that CYP2E1 is the major human enzyme responsible for CCl(4) bioactivation at lower, environmentally relevant levels. At higher CCl(4) levels, CYP3A and possibly other CYP450 forms may contribute to CCl(4) metabolism.

Cardiovascular effects of inhaled diesel exhaust in spontaneously hypertensive rats.

Particulate matter air pollution is associated with increased cardiovascular mortality. The present study examined the cardiac effects of diesel exhaust exposure in spontaneously hypertensive rats. These rats (4 mo old, n=6 males and 4–6 females/concentration) were exposed to one of five diesel exhaust levels (0, 30, 100, 300, and 1000 μg particles/m3) for 6 h per day for 7 d. Electrocardiographic measurements were obtained by radiotelemetry beginning 3 d prior to exposure and ending 4d after exposure cessation. Control rats displayed a reduced daytime heart rate from the beginning of the protocol, whereas exposed rats maintained a significantly elevated heart rate throughout the exposure. Daytime heart rate values for male control rats averaged 265±5 beats/min (mean±standard error [SE]), whereas values for exposed rats averaged 290±7 beats/min. This difference persisted during the evenings of the exposure period but was not observed at any time during the preex-posure or postexposure periods. The PQ interval, an index of atrioventricular node sensitivity, was significantly prolonged among exposed animals in a concentration-dependent manner. Increased heart rate with prolongation of the PQ interval may represent a substrate for ventricular arrhythmias. These results concur with previous reports suggesting that realistic exposure concentrations of air pollution affect the pacemaking system of rats.

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.

Particle clearance and histopathology in lungs of F344/N rats and B6C3F1 mice inhaling nickel oxide or nickel sulfate

The goals of this study were to (1) determine the effects of repeated inhalation of relatively insoluble nickel oxide (NiO) and highly soluble nickel sulfate hexahydrate (NiSO4.6H2O) on lung particle clearance, (2) investigate the effects of repeated inhalation of NiO or NiSO4 on the pulmonary clearance of subsequently inhaled 85Sr-labeled microspheres, (3) correlate the observed effects on clearance with accumulated Ni lung burden and associated pathological changes in the lung, and (4) compare responses in F344 rats and B6C3F1 mice. Male F344/N rats and B6C3F1 mice were exposed whole-body to either NiO or NiSO4.6H2O 6 hr/day, 5 days/week for up to 6 months. NiO exposure concentrations were 0, 0.62, and 2.5 mg NiO/m3 for rats and 0, 1.25, and 5.0 mg NiO/m3 for mice. NiSO4.6H2O exposure concentrations were 0, 0.12, and 0.5 mg NiSO4.6H2O/m3 for rats and 0, 0.25, and 1.0 mg NiSO4.6H2O/m3 for mice. After 2 and 6 months of whole-body exposure, groups of rats and mice were acutely exposed nose-only to 63NiO (NiO-exposed animals only), 63NiSO4.6H2O (NiSO4.6H2O-exposed animals only), or to 85Sr-labeled polystyrene latex (PSL) microspheres (both NiO- and NiSO4.6H2O-exposed animals) to evaluate lung clearance. In addition, groups of rats and mice were euthanized after 2 and 6 months of exposure and at 2 and 4 months after the whole-body exposures were completed to evaluate histopathological changes in the left lung and to quantitate Ni in the right lung. Repeated inhalation of NiO results in accumulation of Ni in lungs of both rats and mice, but to a greater extent in lungs of rats. During the 4 months after the end of the whole-body exposures, some clearance of the accumulated Ni burden occurred from the lungs of rats and mice exposed to the lower, but not the higher NiO exposure concentrations. Clearance of acutely inhaled 63NiO was also impaired in both rats and mice, with the extent of impairment related to both exposure concentration and duration. However, the clearance of acutely inhaled 85Sr PSL microspheres was not impaired. The repeated inhalation of NiO resulted in alveolar macrophage (AM) hyperplasia with accumulation of NiO particles in both rats and mice, chronic alveolitis in rats, and interstitial pneumonia in mice. These lesions persisted throughout the 4-month recovery period after the NiO whole-body exposures were terminated. In contrast, repeated inhalation of NiSO4.6H2O did not result in accumulation of Ni in lungs of either rats or mice and did not affect the clearance of 63NiSO4.6H2O inhaled after either 2 or 6 months of NiSO4.6H2O exposure. Clearance of the 85Sr-labeled microspheres was significantly impaired only in rats exposed to the microspheres after 2 months of exposure to NiSO4.6H2O. Histopathological changes in rats were qualitatively similar to those seen in NiO-exposed rats. Only minimal histopathological changes were observed in NiSO4.6H2O-exposed mice. These results suggest that repeated inhalation of NiO at levels resulting in AM hyperplasia and alveolitis may impair clearance of subsequently inhaled NiO. The potential effects of repeated inhalation of soluble NiSO4.6H2O on the clearance of subsequently inhaled poorly soluble particles are less clear.

Inhalation Toxicity of Brevetoxin 3 in Rats Exposed for Twenty-Two Days

Brevetoxins are potent neurotoxins produced by the marine dinoflagellate Karenia brevis. Exposure to brevetoxins may occur during a K. brevis red tide when the compounds become aerosolized by wind and surf. This study assessed possible adverse health effects associated with inhalation exposure to brevetoxin 3, one of the major brevetoxins produced by K. brevis and present in aerosols collected along beaches affected by red tide. Male F344 rats were exposed to brevetoxin 3 at 0, 37, and 237 μg/m3 by nose-only inhalation 2 hr/day, 5 days/week for up to 22 exposure days. Estimated deposited brevetoxin 3 doses were 0.9 and 5.8 μg/kg/day for the low-and high-dose groups, respectively. Body weights of the high-dose group were significantly below control values. There were no clinical signs of toxicity. Terminal body weights of both low- and high-dose-group rats were significantly below control values. Minimal alveolar macrophage hyperplasia was observed in three of six and six of six of the low- and high-dose groups, respectively. No histopathologic lesions were observed in the nose, brain, liver, or bone marrow of any group. Reticulocyte numbers in whole blood were significantly increased in the high-dose group, and mean corpuscular volume showed a significant decreasing trend with increasing exposure concentration. Humoral-mediated immunity was suppressed in brevetoxin-exposed rats as indicated by significant reduction in splenic plaque-forming cells in both low- and high-dose-group rats compared with controls. Results indicate that the immune system is the primary target for toxicity in rats after repeated inhalation exposure to relatively high concentrations of brevetoxins.

Uptake, Tissue Distribution, and Excretion of Brevetoxin-3 Administered to Mice by Intratracheal Instillation

Brevetoxins are a family of potent lipid-soluble neurotoxins produced by the dinoflagellate Karenia brevis, the organism responsible for Florida red tide. Brevetoxins aerosolized by surf and wind produce irritation of the eyes, nose, and throat in people on or near red tide-affected beaches. The effects of chronic exposures to brevetoxins on healthy and health-compromised individuals are not known. The purpose of this study was to investigate the pulmonary uptake, tissue distribution, and excretion of polyether brevetoxin-3 in mice, a rodent model for investigating the potential systemic adverse health effects associated with repeated brevetoxin inhalation. Male CBA/CaJ mice were administered [3H]brevetoxin-3 by intratracheal instillation. Groups of 3 mice were sacrificed immediately after instillation and at 0.5, 3, 6, 12, 24, 48, and 96 h postinstillation. Four additional mice were placed into metabolism cages for excreta collection up to 168 h postinstillation. Brevetoxin-3 distributed rapidly to all tissues, with the highest initial doses in the liver and gastrointestinal tract. Elimination half-times ranged from approximately 28 h for fat, heart, intestines, kidneys, liver, and muscle to approximately 90 h for brain and testes. The total dose to tissue ranged from 39 ng brevetoxin equivalents-h/g for testes to 406 ng brevetoxin equivalents-h/g for liver. Approximately 90% of excretion had occurred within 96 h, with 11 and 64% of the initial brevetoxin dose excreted in urine and feces, respectively. These results are consistent with earlier reports of rapid absorption and widespread tissue distribution of brevetoxins in rats. This research was funded under National Institutes of Health contract NIH PO1-ES10594. LRRI animal facilities are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. The authors are grateful for the assistance provided by D. Esparza, A. Dison, and D. Santistevan.