TWO-WEEK INHALATION TOXICITY OF POLYMERIC DIPHENYLMETHANE-4,4'-DIISOCYANATE (PMDI) IN RATS: Analysis of Biochemical and Morphological Markers of Early Pulmonary Response

The pulmonary response of Wistar rats to respirable polymeric diphenylmethane-4,4'-diisocyanate (PMDI) aerosol was examined in a 2-wk repeated nose-only inhalation exposure study. Exposure concentrations were 1.1, 3.3, and 13.7 mg PMDI/m³ (6 h/day, 15 exposures). The level of 13.7 mg/m³ was actually a combination of an initial target concentration of 10 mg/m³ in wk 1, which was raised to 16 mg/m³ in wk 2, due to a lack of signs suggestive of pulmonary irritation. An acute sensory irritation study on rats served as basis for selection of these concentrations. Shortly after the 2-wk exposure period, rats were subjected to pulmonary function and arterial blood gas measurements. Lungs were examined by light and transmission electron microscopy, and labeling indices in terminal bronchioles were measured. Bronchoalveolar lavage (BAL) was performed to assess various indicators of pulmonary inflammation, including neutrophil and macrophage numbers, protein, lactate dehydrogenase (LDH), gamma-glutamyltranspeptidase (gamma-GT), alkaline phosphatase (APh), acid phosphatase (ACPh), and beta-N-acetylglucosaminidase (beta-NAG). Phosphatidylcholine in BAL fluid and BAL cells was determined as aggregated endpoint suggestive of changes in pulmonary surfactant. Rats exposed to 3.3 and 13.7 mg/m³ experienced concentration-dependent signs of respiratory tract irritation. Determination of arterial blood gases, lung mechanics, and carbon monoxide diffusing capacity did not demonstrate specific effects. Analysis of BAL fluid and BAL cells revealed changes indicative of marked inflammatory response and/or cytotoxicity in rats exposed to 13.7 mg/m³, and the changes were characterized by statistically significantly increased activities of LDH, beta-NAG, and protein. Phospholipid concentrations were increased in rats exposed to 1.1 mg/m³ and above (elevated levels of lipid material in alveolar macrophages demonstrated by polychrome stain) and 3.3 mg/m³ and above (increased intracellular ACPh activity and intracellular phospholipids). In these groups, gamma-GT was statistically significantly increased. These findings suggest that changes in phospholipid homeostasis appear to occur at lower levels than those eliciting inflammation and cytotoxicity. Light and transmission electron microscopy suggest that exposure to 3.3 and 13.7 mg/m³ resulted in focal inflammatory lesions and an accumulation of refractile, yellowish-brownish material in alveolar macrophages with concomitant activation of type II pneumocytes. In the terminal bronchioles a concentration-dependent increase of bromodeoxyuridine (BrdU)-labeled epithelial cells was observed in all PMDI exposure groups. In summary, it appears that respirable PMDI aerosol interacts with pulmonary surfactant, which, in turn, may stimulate type II pneumocytes to increase their production of surfactant and to proliferate.     

Pulmonary fibrotic response to inhalation of ZnO nanoparticles and toluene co-exposure through directed flow nose only exposure chamber

Abstract

The increasing use of Zinc Oxide nanoparticles (ZnONPs) in paint industry is not supplemented with adequate toxicology data. This report focuses on the fibrogenic toxicity caused due to co-exposure of ZnONPs and toluene in male Wistar rats, exposed for 28 days, through directed flow nose only exposure chamber. The rats were grouped as air control, toluene control (200?ppm), zinc oxide control (10?mg/m³), low dose co-exposed (5?mg/m³ ZnO and 200?ppm of toluene) and high dose co-exposed (10?mg/m³ of ZnO and 200?ppm of toluene). Our study demonstrates that co-exposure of ZnONPs and toluene (as in paint industry), even at their respective permissible exposure level (5?mg/m³ for ZnO and 200?ppm for toluene) have the potential to produce a progressive inflammatory and fibrotic response in the alveolar tissues of the lungs. We observed a significant increase in inflammatory markers in BAL fluid and elevated malondialdehyde (MDA) levels with lower levels of intracellular reduced glutathione (GSH) in lungs of rats of co-exposed group. Significant increase in the levels of pro-inflammatory mediators (IL-6, Ikβα, Cox-II, p-NF-κB) in lung tissues also indicated pulmonary damage. To best of our knowledge this is the first study which highlights the toxicity of co-exposed ZnO NPs and toluene.     

Inhaled silica-coated TiO2 nanoparticles induced airway irritation,airflow limitation and inflammation in mice

Abstract

The wide use of nanotechnology is here to stay. However, the knowledge on the health effects of different engineered nanomaterials (ENMs) is lacking. In this study, irritation and inflammation potential of commercially available silica-coated TiO₂ ENMs (10?×?40?nm, rutile) were studied. Single exposure (30?min) at mass concentrations 5, 10, 20 and 30?mg/m³, and repeated exposure (altogether 16?h, 1?h/day, 4 days/week for 4 weeks) at mass concentration of 30?mg/m³ to silica-coated TiO₂ induced first phase of pulmonary irritation (P1), which was seen as rapid, shallow breathing. During repeated exposures, P1 effect was partly evolved into more intense pulmonary irritation. Also sensory irritation was observed at the beginning of both single and repeated exposure periods, and the effect intensified during repeated exposures. Airflow limitation started to develop during repeated exposures. Repeated exposure to silica-coated TiO₂ ENMs induced also pulmonary inflammation: inflammatory cells infiltrated in peribronchial and perivascular areas of the lungs, neutrophils were found in BAL fluids, and the number of CD3 and CD4 positive T cells increased significantly. In line with these results, pulmonary mRNA expression of chemokines CXCL1, CXCL5 and CXCL9 was enhanced. Also expression of mRNA levels of proinflammatory cytokines TNF-α and IL-6 was elevated after repeated exposures. Taken together, these results indicated that silica-coated TiO₂ ENMs induce pulmonary and sensory irritation after single and repeated exposure, and airflow limitation and pulmonary inflammation after repeated exposure.     

Suppression of Pulmonary Antibacterial Defenses Mechanisms and Lung Damage in Mice Exposed to Fluoride Aerosol

In endemic fluorosis areas in China associated with coal burning, indoor airborne fluoride pollution is severe. To determine the effects of fluoride aerosols on pulmonary antibacterial defense mechanisms and lung damage, mice were exposed to various concentrations of fluoride aerosol (2, 5, or 10 mg/m³) or filtered air (control) for 14 d, 4 h/d in an inhalation chamber. Bactericidal activity against Staphylococcus aureus in the lung and the number and profile of free pulmonary cells, protein content, and lactate dehydrogenase (LDH) activity in bronchoalveolar lavage (BAL) fluid were assessed. Urinary fluoride concentration, an indicator of fluoride exposure, increased in proportion to fluoride aerosol concentration in the chamber. Wet lung weight was significantly higher on d 14 in mice exposed to 10 mg/m³ than in controls. Pulmonary bactericidal activity against S. aureus was concentration-dependently suppressed at 5 and 10 mg/m³ fluoride. The number of alveolar macrophages (AMs) in the BAL fluid of the mice not bacterially challenged decreased significantly at 10 mg/m³ fluoride. The number of polymorphonuclear leukocytes and lymphocytes increased significantly at 10 mg/m³ fluoride exposure. The concentration of total protein (TP) and albumin in BAL supernatant increased significantly at 5 and 10 mg/m³ fluoride exposure, and LDH activity rose markedly at the higher fluoride concentration. Data indicate that fluoride inhalation produces pulmonary cellular alterations that are associated with a diminished ability to cope with infectious bacteria.     

Acute Head-Only Exposure of Dogs to Phosgene. Part III. Comparison of Indicators of Lung Injury in Dogs and Rats

To better understand the relevance of phosgene-induced changes in bronchoalveolar lavage (BAL) fluid protein observed in acutely exposed rats, groups of beagle dogs were similarly exposed for 30 min to phosgene using a head-only mode of exposure. The actual exposure concentrations were 9, 16.5, and 35 mg/m³, with resultant C × t products of 270, 495, and 1050 mg/m³ × min. In rats, a C × t product of 270 mg/m³ × min caused a significant elevation of protein in the bronchoalveolar lavage (BAL) fluid, while the nonlethal threshold concentration (LCt₀₁) was estimated to be 1075 mg/m³ × min. The endpoints examined in dogs focused on changes in BAL, lung weights, arterial blood gases, and lung histopathology approximately 24 h postexposure. Mortality did not occur at any C × t product. Increased lung weights and elevations in protein, soluble collagen, and polymorphonuclear leukocyte (PMN) counts in BAL were observed at 1050 mg/m³ × min with borderline changes at 495 mg/m³ × min. Following exposure to 1050 mg/m³ × min, the analysis of arterial blood gases provided evidence of a significantly decreased arterial pO₂. Histopathology revealed a mild, although distinctive, inflammatory response at the bronchoalveolar level at 495 mg/m³ × min, whereas serofibrinous exudates and edema were observed at 1050 mg/m³ × min. The magnitude of effects correlated with the individual dogs' respiratory minute volume and breathing patterns (panting). Collectively, phosgene-induced indicators of acute lung injury appeared to be characterized best by protein in BAL fluid. With regard to both the inhaled dose and the associated increase of protein in BAL, the responses obtained in dogs appear to be more similar to humans. In contrast, elevations in BAL protein occurred in rats at three-fold lower concentrations when compared to dogs. The results of this study demonstrate that the magnitude of elevations of plasma exudate in BAL fluid following acute exposure to the pulmonary irritant phosgene is markedly more pronounced in rats when compared to the dog which is considered more human-like than rats. This is believed to be associated with the higher ventilation of small rodents and with rodent-specific sensory bronchopulmonary defense reflexes.     

Short-term inhalation study of graphene oxide nanoplates

Graphene oxides possess unique physicochemical properties with important potential applications in electronics, pharmaceuticals, and medicine. However, the toxicity following inhalation exposure to graphene oxide has not yet been clarified. Therefore, this study conducted a short-term graphene oxide inhalation toxicity analysis using a nose-only inhalation exposure system and male Sprague–Dawley rats. A total of four groups (15 rats per group) were exposed: (1) control (fresh air), (2) low concentration (0.76?±?0.16?mg/m³), (3) moderate concentration (2.60?±?0.19?mg/m³), and (4) high concentration (9.78?±?0.29?mg/m³). The rats were exposed to graphene oxide for 6?h/day for 5 days, followed by recovery for 1, 3, and 21 days. No significant body or organ weight changes were noted after the short-term exposure or during the recovery period. Similarly, no significant systemic effects of toxicological importance were noted in the hematological assays, bronchoalveolar lavage fluid (BAL) inflammatory markers, BAL fluid cytokines, or blood biochemical assays following the graphene oxide exposure or during the post-exposure observation period. Moreover, no significant differences were observed in the BAL cell differentials, such as lymphocytes, macrophages, or polymorphonuclear cells. Graphene oxide-ingested alveolar macrophages as a spontaneous clearance reaction were observed in the lungs of all the concentration groups from post 1?day to post 21 days. Histopathological examination of the liver and kidneys did not reveal any significant test-article-relevant histopathological lesions. Importantly, similar to previously reported graphene inhalation data, this short-term nose-only inhalation study found only minimal or unnoticeable graphene oxide toxicity in the lungs and other organs.     

Pulmonary and systemic effects of short-term inhalation exposure to ultrafine carbon black particles

While environmental particles are associated with mortality and morbidity related to pulmonary and cardiovascular (CV) disease, the mechanisms involved in CV health effects are not known. Changes in systemic clotting factors have been associated with pulmonary inflammation. We hypothesized that inhaled ultrafine particles result in an inflammatory response which may stimulate systemic clotting factor release. Adult male Wistar rats were exposed to either fine or ultrafine carbon black (CB) for 7 h. The attained total suspended particle concentrations were 1.66 mg/m³ for ultrafine CB and 1.40 mg/m³ for fine CB. Particle concentration of ultrafine particles was more than 10 times greater than that of fine particles and the count median aerodynamic diameter averaged 114 nm for the ultrafine and 268 nm for the fine carbon particles. Data were collected immediately, 16 and 48 h following exposure. Only ultrafine CB caused an increase in total bronchoalveolar lavage (BAL) leukocytes, whereas both fine (2-fold) and ultrafine (4-fold) carbon particles caused an increase in BAL neutrophils at 16 h postexposure. Exposure to the ultrafine, but not fine, carbon was also associated with significant increases in the total numbers of blood leukocytes. Plasma fibrinogen, factor VII and von Willebrand factor (vWF) were unaffected by particle treatments as was plasma Trolox equivalent antioxidant status (TEAC). Macrophage inflammatory protein-2 mRNA was significantly increased in BAL cells 48 h following exposure to ultrafine CB. The data show that there is a small but consistent significant proinflammatory effect of this exposure to ultrafine particles that is greater than the effect of the same exposure to fine CB.     

Pulmonary Toxicity Assessments Of Inhaled Ethylene Oxide/Propylene Oxide Copolymer Lubricants In Rats

Abstract

The pulmonary toxicities of 5 different ethylene oxide/propylene oxide (EO/PO) copolymer commercial lubricant candidates were assessed by exposing groups of rats for 3 consecutive days (6 hlday) to aerosols of the different EO/PO test materials and evaluating pulmonary parameters at selected postexposure time periods. Because all 5 compounds could not be evaluated simultaneously, these studies were conducted over a period of 2 wk. During wk 1 of the study, rats were exposed either to 22 mg/m³ (mean value for the 3 days) of UCON 50-HB-5100 (50-HB-5100), to 110 mg/m³ of Pluronic L31 (131), or to 99.4 mg/m³ of Pluronic L64 (L64). The mass median aerodynamic diameters (MMADs) for all 3 compounds were < 2.6 μm. In the second group of studies, rats were exposed to 42 mg/m³ of UCON 50-HB-2000 (50-HB-2000), or to 111 mg/m³ of UCON 75-H-1400 (75-H-1400), with MMADs < 1.8 μm. Sham controls were exposed to room air. One rat in the UCON 50-HB-5W0 group died within 7 days postexposure. Similarly, 1 rat in the UCON 50-HB-2000 group died within 8 days postexposure. Within 48 h after exposure, the lungs of rats exposed to UCON 50-HB-5W0 and 50-HB-2000 were edematous. The lungs of rats were lavaged at 0 h (i.e., immediately after), 2 days, 1 wk, 1 and 3 mo postexposure. Cellular and biochemical data on samples recovered from bronchoalveolar lavage (BAD demonstrated a substantial pulmonary inflammatory response concomitant with increases in BAL fluid levels of lactate dehydrogenase (LDH), protein, alkaline phosphatase, and N-acetylglucosaminidase in the lungs of rats exposed to UCON 50-HB-5100. Similarly, the BAL biochemical and pulmonary cell differential data for 50-HB-2000-exposed rats were similar but less severe to that previously measured in 50-HB-5100-exposed rats. In contrast, the lungs of rats exposed to Pluronic 131 and L64 and UCON 75 H-1400 demonstrated only slight and reversible pulmonary inflammatory effects. The results from this study validate this inhalation bioassay technique for predicting the pulmonary toxicity of inhaled aerosols by confirming the effects measured in a previous 2-wk inhalation toxicity study with these same compounds. In the earlier study, UCON 50-HB-5W0 and UCON 50-HB-2000 produced severe pulmonary toxicity in rats. The cellular and biochemical results presented here confirm the earlier findings of significant pulmonary toxicity produced by inhalation of the UCON 50-HB-5W0 and UCON 50-HB-2000 compounds. In contrast, the three other compounds (Pluronic L31, Pluronic L64, UCON 75-H-1400) produced only weak pulmonary inflammatory effects following 3-day exposures at high aerosol concentrations.     

Acute Nose-Only Exposure of Rats to Phosgene. Part I: Concentration × Time Dependence of LC50s,Nonlethal-Threshold Concentrations,and Analysis of Breathing Patterns

Groups of young adult Wistar rats were acutely exposed to phosgene gas using a directed-flow nose-only mode of exposure. The exposure durations used were 10, 30, 60, and 240 min and the corresponding C × t products bracketed a range from 1538 to 2854 mg/m³× min. The postexposure period was 2 wk. Subgroups of rats were subjected to respiratory function measurements. With few exceptions, mortality occurred within 24 h after exposure. The median lethal concentration (LC₅₀) and the estimated nonlethal threshold concentrations (LC₀₁) for 10, 30, 60, and 240 min were 253.3 (105.3), 54.5 (29.2), 31.3 (21.1), and 8.6 (5.3) mg/m³, respectively. With regard to the fixed outcome Cⁿ× t product, the exponent n was found to be ～0.9 for both the LC₅₀ and the LC₀₁. Due to an apparent rodent-specific transient depression in ventilation, results from 10-min exposures were excluded for the calculation of average C × t products. The average LCt₅₀ (and confidence interval 95%) and LCt₀₁ were 1741 (1547–1929) mg/m³× min and 1075 mg/m³× min, respectively, with a LCt₅₀/LCt₀₁ ratio of 1.6. Respiratory function measurements revealed an increased apnea time (AT), which is typical for lower respiratory tract irritants. This response was associated with transiently decreased respiratory minute volumes. Borderline, although distinct, changes in AT occurred at 1.2 × 30 mg/m³ × min and above, which did not show evidence of recovery during a 30-min postexposure period at 47.6 × 30 mg/m³× min and above. In an ancillary study, one group of rats was exposed to 1008 mg/m³× min (at 4.2 mg/m³ for 240 min; postexposure period 4 wk). Emphasis was on the time course of nonlethal endpoints (bronchoalveolar lavage, BAL) and histopathology of the lungs of rats sacrificed at the end of the 4-wk postexposure period. The climax of BAL protein was on the first postexposure day and exceeded approximately 70 times the control without causing mortality. The changes in BAL protein resolved within 2 wk. Histopathology did not show evidence of lung remodeling or progressive, potentially irreversible changes 4 wk postexposure. In summary, the analysis of the C × t dependent mortality revealed a steep C × t mortality relationship. The C × t product in the range of the nonlethal threshold concentration (1008 mg/m³ × min) caused pulmonary injury as indicated by markedly increased protein in BAL. Changes resolved almost entirely within the 4-wk postexposure period.     

Siderite (FeCO3) and magnetite (Fe3O4) overload-dependent pulmonary toxicity is determined by the poorly soluble particle not the iron content

The two poorly soluble iron containing solid aerosols of siderite (FeCO₃) and magnetite (Fe₃O₄) were compared in a 4-week inhalation study on rats at similar particle mass concentrations of approximately 30 or 100?mg/m³. The particle size distributions were essentially identical (MMAD ≈1.4 μm). The iron-based concentrations were 12 or 38 and 22 or 66?mg Fe/m³ for FeCO₃ and Fe₃O₄, respectively. Modeled and empirically determined iron lung burdens were compared with endpoints suggestive of pulmonary inflammation by determinations in bronchoalveolar lavage (BAL) and oxidative stress in lung tissue during a postexposure period of 3 months. The objective of study was to identify the most germane exposure metrics, that are the concentration of elemental iron (mg Fe/m³), total particle mass (mg PM/m³) or particle volume (μl PM/m³) and their associations with the effects observed. From this analysis it was apparent that the intensity of pulmonary inflammation was clearly dependent on the concentration of particle-mass or -volume and not of iron. Despite its lower iron content, the exposure to FeCO₃ caused a more pronounced and sustained inflammation as compared to Fe₃O₄. Similarly, borderline evidence of increased oxidative stress and inflammation occurred especially following exposure to FeCO₃ at moderate lung overload levels. The in situ analysis of 8-oxoguanine in epithelial cells of alveolar and bronchiolar regions supports the conclusion that both FeCO₃ and Fe₃O₄ particles are effectively endocytosed by macrophages as opposed to epithelial cells. Evidence of intracellular or nuclear sources of redox-active iron did not exist. In summary, this mechanistic study supports previous conclusions, namely that the repeated inhalation exposure of rats to highly respirable pigment-type iron oxides cause nonspecific pulmonary inflammation which shows a clear dependence on the particle volume-dependent lung overload rather than any increased dissolution and/or bioavailability of redox-active iron.     

Pulmonary hypofunction due to calcium carbonate nanomaterial exposure in occupational workers: a cross-sectional study

Calcium carbonate nanomaterials (nano-CaCO₃) are widely used in both manufacturing and consumer products, but their potential health hazards remain unclear. The objective of this study was to survey workplace exposure levels and health effects of workers exposed to nano-CaCO₃. Personal and area sampling, as well as real-time and dust monitoring, were performed to characterize mass exposure, particle size distribution, and particle number exposure. A total of 56 workers (28 exposed workers and 28 unexposed controls) were studied in a cross-sectional study. They completed physical examinations, spirometry, and digital radiography. The results showed that the gravimetric nano-CaCO₃ concentration was 5.264?±?6.987?mg/m³ (0.037–22.192?mg/m³) at the workplace, and 3.577?±?2.065?mg/m³ (2.042–8.161?mg/m³) in the breathing zone of the exposed workers. The particle number concentrations ranged from 8193 to 39?621 particles/cm³ with a size range of 30–150?nm. The process of packing had the highest gravimetric and particle number concentrations. The particle number concentration positively correlated with gravimetric concentrations of nano-CaCO₃. The levels of hemoglobin, creatine phosphokinase (CK), lactate dehydrogenase, and high-density lipoprotein cholesterol (HDL-C) in the nano-CaCO₃ exposure group increased significantly, but the white blood cell count (WBC), Complement 3 (C3), total protein (TP), uric acid, and creatinine (CREA) all decreased significantly. The prevalence rate of pulmonary hypofunction was significantly higher (p?=?0.037), and the levels of vital capacity (VC), forced vital capacity (FVC), forced expiratory volume in one second (FEV1), FEV1/FVC, peak expiratory flow and forced expiratory flow 25% (FEF 25%), FEF 25–75% were negatively correlated with gravimetric concentrations of nano-CaCO₃ (p?3 exposure level was associated with pulmonary hypofunction (p?=?0.005). Meanwhile, a dose-effect relationship was found between the accumulated gravimetric concentrations of nano-CaCO₃ and the prevalence rate of pulmonary hypofunction (p?=?0.048). In conclusion, long-term and high-level nano-CaCO₃ exposure can induce pulmonary hypofunction in workers. Thus, lung function examination is suggested for occupational populations with nano-CaCO₃ exposure. Furthermore, future health protection efforts should focus on senior workers with accumulation effects of nano-CaCO₃ exposure.     

Hexamethylene Diisocyanate (HDI)-Induced Lung Impairment: Studies in Isolated Perfused and Ventilated Guinea Pig Lungs

Abstract: Isolated, perfused and ventilated guinea pig lungs were exposed to hexamethylene diisocyanate via the air passages. Two air concentrations of hexamethylene diisocyanate were studied (3.5 and 11 mg/m³). There was a statistically significant (P<0.05 ? 0.001) dose-related reduction in both conductance and compliance but no effects were noted on the pulmonary circulation. With 3.5 mg/m³ hexamethylene diisocyanate the conductance capacity was reduced with 38% and compliance with 30% after 60 min. exposure. Eleven mg/m³ hexamethylene diisocyanate reduced the conductance and compliance capacity with 86 and 69 %, respectively, on an average. The reduction in lung function (with 11 mg/m³) was abolished when 100 μM diclofenac, a cyclooxygenase inhibitor, was added to the perfusate (P<0.01). The thromboxane A₂ antagonist L-670,596 (20 μM) exerted a partial protective effect. The capacity of conductance and compliance decreased with 46 and 32% respectively, on an average, after preperfusion with L-670,596 and a following exposure of 11 mg/m³ hexamethylene diisocyanate for 60 min. Statistically significant protection (P<0.05) was obtained on compliance and the P-value was<0.1 for conductance. Thus, these data indicate that hexamethylene diisocyanate-induced bronchoconstriction is mediated via arachidonic acid release and thromboxane formation, in isolated, perfused and ventilated guinea pig lungs.     

Subchronic inhalation toxicity of iron oxide (magnetite, Fe(3) O(4) ) in rats: pulmonary toxicity is determined by the particle kinetics typical of poorly soluble particles

Wistar rats were nose‐only exposed to pigment‐sized iron oxide dust (Fe₃O₄, magnetite) in a subchronic 13‐week inhalation study according to the OECD testing guidelines TG#413 and GD#39. A 4 week pilot study with a 6 month post exposure period served as basis for validating the kinetic modeling approaches utilized to design the subchronic study. Kinetic analyses made during this post exposure period demonstrated that a diminution in particle clearance and lung inflammation occurred at cumulative exposure levels exceeding the lung overload threshold. Animals were exposed 6 h per day, five days per week for 13 consecutive weeks at actual concentrations of 0, 4.7, 16.6 and 52.1 mg m^?3 (mass median aerodynamic diameter ≈1.3 μm, geometric standard deviation = 2). The exposure to iron oxide dust was tolerated without mortality, consistent changes in body weights, food and water consumption or systemic toxicity. While general clinical pathology and urinalysis were unobtrusive, hematology revealed changes of unclear toxicological significance (minimally increased differential neutrophil counts in peripheral blood). Elevations of neutrophils in bronchoalveolar lavage (BAL) appeared to be the most sensitive endpoint of study. Histopathology demonstrated responses to particle deposition in the upper respiratory tract (goblet cell hyper‐ and/or metaplasia, intraepithelial eosinophilic globules in the nasal passages) and the lower respiratory tract (inflammatory changes in the bronchiolo‐alveolar region). Consistent changes suggestive of pulmonary inflammation were evidenced by BAL, histopathology, increased lung and lung‐associated‐lymph node (LALN) weights at 16.6 and 52.1 mg m^?3. Increased septal collagenous fibers were observed at 52.1 mg m^?3. Particle translocation into LALN occurred at exposure levels causing pulmonary inflammation. In summary, the retention kinetics iron oxide reflected that of poorly soluble particles. The empirical no‐observed‐adverse‐effect level (NOAEL) and the lower bound 95% confidence limit on the benchmark concentration (BMCL) obtained by benchmark analysis was 4.7 and 4.4 mg m^?3, respectively, and supports an OEL (time‐adjusted chronic occupational exposure level) of 2 mg m^?3 (alveolar fraction). Copyright © 2011 John Wiley & Sons, Ltd.     

Subacute inhalation toxicity study of synthetic amorphous silica nanoparticles in Sprague-Dawley rats

Synthetic amorphous silica nanoparticles (SiNPs) are one of the most applied nanomaterials and are widely used in a broad variety of industrial and biomedical fields. However, no recent long-term inhalation studies evaluating the toxicity of SiNPs are available and results of acute studies are limited. Thus, we conducted a subacute inhalation toxicity study of SiNPs in Sprague-Dawley rats using a nose-only inhalation system. Rats were separated into four groups and target concentrations selected in this study were as follows: control (fresh air), low- (0.407?±?0.066?mg/m³), middle- (1.439?±?0.177?mg/m³) and high-concentration group (5.386?±?0.729?mg/m³), respectively. The rats were exposed to SiNPs for four consecutive weeks (6 hr/day, 5 days/week) except for control group of rats which received filtered fresh air. After 28-days of inhalation exposure to SiNPs, rats were sacrificed after recovery periods of one, seven and 28 days. Although there were minimal toxic changes such as temporary decrease of body weight after exposure, increased levels of red blood cells (RBCs) and hemoglobin (Hb) concentration, the lung histopathological findings and inflammatory markers in bronchoalveolar lavage (BAL) fluid including polymorphonuclear (PMN) leukocyte, lactate dehydrogenase (LDH), albumin and protein did not show significant changes at any recovery period. The results of this study suggest that the subacute inhalation of SiNPs had no toxic effects on the lung of rats at the concentrations and selected time points used in this study.     

Altered lung function in rats after subacute exposure to n-butyl isocyanate

The objectives of this study were to use pulmonary function tests, blood gas measurements and bronchoalveolar lung lavage (BAL) to characterize lesions in the respiratory tract of young adult male Wistar rats as a result of a 5-day exposure (6 h/day) to 0, 1.1, 6.2, 15 or 26 mg n-butyl isocyanate (n-BIC)/m³ air. Further objectives were to probe the diagnostic sensitivities of these procedures in comparison with more traditional evaluations (clinical observation, lung weight, histopathology). Measurements were performed during post-exposure weeks 2 and 5. Most rats exposed to 26 mg/m³ died or were sacrificed in a moribund state during post-exposure week 2. All other rats survived the exposure regimen. In rats exposed to 15 and 26 mg/m³ a significant decrease in body weight, laboured breathing, hypoactivity, nasal discharge, cyanosis, and hypothermia were observed. Pulmonary function measurements revealed increased total lung capacity (TLC) and residual volume (RV), decreased forced expiratory flow rates and quasistatic compliance in rats exposed to 26 mg/m³. At the end of the observation period rats exposed to 6.2 and 15 mg/m³ air were hyperresponsive to an acetylcholine bronchoprovocation aerosol. Arterial blood gas measurements revealed an arterial hypoxia and an increase in venous admixture, suggesting a severe mismatch of the ventilation-perfusion relationship, Biochemical and cellular components in BAL fluid (BALF) indicated a concentration dependent and protracted increase of polymorphonuclear leucocytes and further inflammatory parameters. In the 1.1 mg/m³ group BALF parameters were not significantly elevated. The major histopathological lesions of the lung were thickening of septa, emphysema, and intra-alveolar oedema in rats exposed to 26 mg/m³. Collectively, these results demonstrate obstructive and progressive lung disease with associated gas trapping and severe disturbance of the ventilation perfusion relationship which is considered to be the cause of delayed mortality. In terms of variability and sensitivity the increase in BALF parameters was most sensitive in indicating the diseased state of the lung.     

Impact of emerging pollutants on pulmonary inflammation in asthmatic rats: ethanol vapors and agglomerated TiO2 nanoparticles

Context: Titanium dioxide nanoparticles (nano-TiO₂) and ethanol vapors are air contaminants with increasing importance. The presence of a pathological pulmonary condition, such as asthma, may increase lung susceptibility to such contaminants.

Objective: This study aimed to investigate if exposure to inhaled ethanol vapors or nano-TiO₂ can modulate the rat pulmonary inflammatory response resulting from an allergic asthmatic reaction.

Materials and methods: Brown Norway rats were sensitized (sc) and challenged (15?min inhalation, 14 days later) with chicken egg ovalbumin (OVA). Leukocytes were counted in bronchoalveolar lavages (BAL) performed at 6, 24, 36, 48 and 72?h following the challenge and either after ethanol exposures (3000 ppm, 6?h/day, daily) or at 48?h (peak inflammation) for nano-TiO₂ exposures (9.35?mg/m³ aerosol for 6 and 42?h after the OVA challenge). For the nano-TiO₂ exposures, plasma and BAL cytokines were measured and lung histological analyzes were performed.

Results: Exposure to ethanol did not significantly affect BAL leukocytes after OVA challenge. Exposure to nano-TiO₂ significantly decreased BAL leukocytes compared to OVA-challenged controls. Plasma and BAL IL-4, IL-6, and INF-γ levels were also decreased in the nano-TiO₂ group.

Discussion: While ethanol vapors do not modify the pulmonary inflammation in rats during an asthmatic response, a surprising protective effect for agglomerated nano-TiO₂ was observed. A putative mechanistic basis involving a decrease in the Th2 response caused by OVA is proposed.

Conclusion: Allergic pulmonary inflammation is not up-regulated by inhalation of the pollutants ethanol and nano-TiO₂. On the contrary, nano-TiO₂ decreases lung inflammation in asthmatic rats.     

Host and environmental factors affect pulmonary responses measured in bronchoalveolar lavage

Abstract

Context: Bronchoscopy with bronchoalveolar lavage (BAL) is used to measure pulmonary effects in inhalational exposure studies.

Objectives: To determine how host and background environmental factors may affect pulmonary responses in BAL.

Materials and methods: We retrospectively analyzed 77 healthy non-smoking volunteers (38 males and 39 females, age 18–35) who participated in a bronchoscopy study to donate cells for in vitro studies. BAL was performed by lavaging one subsegment of both the lingular segment of the left upper lobe and the right middle lobe with 250?ml of sterile normal saline each. We obtained temperature, relative humidity, ambient O₃, PM_2.5 and PM₁₀ levels from monitor stations in Durham area in North Carolina. We correlated concentrations of leptin, adiponectin, monocyte chemotactic protein-1 (MCP-1), interleukin (IL)-8, ferritin and total lavaged cells in BAL samples with body mass index (BMI), age, ambient O₃, PM_2.5, PM₁₀, temperature and relative humidity.

Results: Increased BMI was associated with higher lavage leptin. Males had higher MCP-1 and total lavaged cells than females. Average PM_2.5, PM₁₀ and O₃ concentrations before bronchoscopy were 13.7?µg/m³, 21.2?µg/m³ and 0.029?ppm, respectively. Using stepwise multiple linear regression, we found positive associations of MCP-1 with BMI, and of total lavaged cells with humidity and O₃. There were inverse associations of IL-8 and total lavaged cells with temperature.

Discussion and conclusions: Background environmental and host factors may affect some pulmonary responses to ambient pollutants. Interpretation of pulmonary effects in inhalational exposure studies may need to consider the effects of some host and environmental factors.     

Effects of inhaled aerosolized carfentanil on real-time physiological responses in mice: a preliminary evaluation of naloxone

This study examined the real-time exposure–response effects of aerosolized carfentanil (CRF) on opioid-induced toxicity, respiratory dynamics and cardiac function in mice. Unrestrained, conscious male CD-1 mice (25–30?g) were exposed to 0.4 or 4.0?mg/m³ of aerosolized CRF for 15?min (Ct?=?6 or 60?mg?min/m³) in a whole-body plethysmograph chamber. Minute volume (MV), core body temperature (T_c), mean arterial blood pressure (MAP) and heart rate (HR) were evaluated in animals exposed to CRF or sterile H₂O. Loss of consciousness and Straub tail were observed in before 1?min following initiation of exposure to 6 or 60?mg?min/m³ of CRF. Clinical signs of opioid-induced toxicity were observed in a dose-dependent manner. Exposure to 6 or 60?mg?min/m³ of CRF resulted in significant decrease in MV as compared to the controls. MAP, HR and T_c decreased 24?h in animals exposed to either 6 or 60?mg?min/m³ of CRF as compared to the controls. Post-exposure administration of naloxone (NX, 0.05?mg/kg, i.m.) did not increase the MV of animals exposed to CRF to control levels within 24?h, but decreased clinical signs of opioid-induced toxicity and the duration of respiratory depression. This is the first study to evaluate real-time respiratory dynamics and cardiac function during exposure and up to 24?h post-exposure to CRF. The evaluation of toxicological signs and respiratory dynamics following exposure to CRF will be useful in the development of therapeutic strategies to counteract the ongoing threat of abuse and overuse of opioids and their synthetic variants.     

In vitro and in vivo genotoxic effects of straight versus tangled multi-walled carbon nanotubes

Some multi-walled carbon nanotubes (MWCNTs) induce mesothelioma in rodents, straight MWCNTs showing a more pronounced effect than tangled MWCNTs. As primary and secondary genotoxicity may play a role in MWCNT carcinogenesis, we used a battery of assays for DNA damage and micronuclei to compare the genotoxicity of straight (MWCNT-S) and tangled MWCNTs (MWCNT-T) in vitro (primary genotoxicity) and in vivo (primary or secondary genotoxicity). C57Bl/6 mice showed a dose-dependent increase in DNA strand breaks, as measured by the comet assay, in lung cells 24?h after a single pharyngeal aspiration of MWCNT-S (1–200?μg/mouse). An increase was also observed for DNA strand breaks in lung and bronchoalveolar lavage (BAL) cells and for micronucleated alveolar type II cells in mice exposed to aerosolized MWCNT-S (8.2–10.8?mg/m³) for 4 d, 4?h/d. No systemic genotoxic effects, assessed by the γ-H2AX assay in blood mononuclear leukocytes or by micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow or blood, were observed for MWCNT-S by either exposure technique. MWCNT-T showed a dose-related decrease in DNA damage in BAL and lung cells of mice after a single pharyngeal aspiration (1–200?μg/mouse) and in MNPCEs after inhalation exposure (17.5?mg/m³). In vitro in human bronchial epithelial BEAS-2B cells, MWCNT-S induced DNA strand breaks at low doses (5 and 10?μg/cm²), while MWCNT-T increased strand breakage only at 200?μg/cm². Neither of the MWCNTs was able to induce micronuclei in vitro. Our findings suggest that both primary and secondary mechanisms may be involved in the genotoxicity of straight MWCNTs.     

Cardiopulmonary responses in spontaneously hypertensive and Wistar-Kyoto rats exposed to concentrated ambient particles from Detroit,Michigan

Toxicological effects have been observed in rats exposed to concentrated ambient particles (CAPs) from different regions of the United States. The objective of this study was to evaluate the cardiopulmonary and systemic effects of CAPs in Detroit. The authors stationed a mobile concentrator at a location near major traffic and industrial sources. Spontaneously hypertensive (SH) and Wistar-Kyoto (WKY) rats were exposed to fine CAPs (diameter <?0.1–2.5?μm) 8?h/day for 13 consecutive days. Animals were implanted with telemeters, and electrocardiogram data were recorded continuously. Bronchoalveolar lavage (BAL) fluid and plasma were analyzed. Comprehensive exposure monitoring was conducted, including CAPs components. CAPs exposure concentrations were 103–918?μg/m³ (mean?=?502?μg/m³). The authors found no statistically significant differences in heart rate or SDNN (standard deviation of the normal-to-normal intervals), a measure of heart rate variability, between CAPs-exposed and control rats. The authors found significantly higher levels of C-reactive protein in the serum of CAPs-exposed SH rats compared with air-exposed animals. Protein in BAL fluid was elevated in WKY rats exposed to CAPs. Measurement of trace metals in lung tissue showed elevated concentrations of V, Sb, La, and Ce in CAPs-exposed SH animals versus controls. These elements are generally associated with oil combustion, oil refining, waste incineration, and traffic. Examination of wind rose data from the exposure period confirmed that the predominant wind direction was SSW, the direction of many of the aforementioned sources. These results indicate that ambient particles in Detroit can cause mild pulmonary and systemic changes in rats, and suggest the importance of local PM_2.5 sources in these effects.