William T. Goldsmith

An in vivo and in vitro toxicological characterisation of realistic nanoscale CeO2 inhalation exposures

Abstract Nanoscale CeO2 is increasingly used for industrial and commercial applications, including catalysis, UV-shielding and as an additive in various nanocomposites. Because of its increasing potential for consumer and occupational exposures, a comprehensive toxicological characterisation of this nanomaterial is needed. Preliminary results from intratracheal instillation studies in rats point to cytotoxicity and inflammation, though these studies may not accurately use realistic nanoscale exposure profiles. By contrast, published in vitro cellular studies have reported limited toxicological outcomes for the case of nano-ceria. Here, the authors present an integrative study evaluating the toxicity of nanoscale CeO2 both in vitro, using the A549 lung epithelial cell line, and in vivo using an intact rat model. Realistic nano-ceria exposure atmospheres were generated using the Harvard Versatile Engineered Nanomaterial Generation System (VENGES), and rats were exposed via inhalation. Finally, the use of a nanothin amorphous SiO2 encapsulation coating as a means of mitigating CeO2 toxicity was assessed. Results from the inhalation experiments show lung injury and inflammation with increased PMN and LDH levels in the bronchoalveolar lavage fluid of the CeO2-exposed rats. Moreover, exposure to SiO2-coated CeO2 did not induce any pulmonary toxicity to the animals, representing clear evidence for the safe by design SiO2-encapsualtion concept.

Respiratory toxicologic pathology of inhaled diacetyl in sprague-dawley rats.

Inhalation of butter flavoring vapors by food manufacturing workers causes an emerging lung disease clinically resembling bronchiolitis obliterans. Diacetyl, an α-diketone, is a major component of these vapors. In rats, we investigated the toxicity of inhaled diacetyl at concentrations of up to 365 ppm (time weighted average), either as six-hour continuous exposures or as four brief, intense exposures over six hours. A separate group inhaled a single pulse of ~1800 ppm diacetyl (92.9 ppm six-hour average). Rats were necropsied 18 to 20 hours after exposure. Diacetyl inhalation caused epithelial necrosis and suppurative to fibrinosuppurative inflammation in the nose, larynx, trachea, and bronchi. Bronchi were affected at diacetyl concentrations of 294.6 ppm or greater; the trachea and larynx were affected at diacetyl concentrations of 224 ppm or greater. Both pulsed and continuous exposure patterns caused epithelial injury. The nose had the greatest sensitivity to diacetyl. Ultrastructural changes in the tracheal epithelium included whorling and dilation of the rough endoplasmic reticulum, chromatin clumping beneath the nuclear membrane, vacuolation, increased inter-cellular space and foci of denuded basement membrane. Edema and hemorrhage extended into the lamina propria. These findings are consistent with the conclusion that inhaled diacetyl is a respiratory hazard.

Viable Influenza A Virus in Airborne Particles from Human Coughs

Patients with influenza release aerosol particles containing the virus into their environment. However, the importance of airborne transmission in the spread of influenza is unclear, in part because of a lack of information about the infectivity of the airborne virus. The purpose of this study was to determine the amount of viable influenza A virus that was expelled by patients in aerosol particles while coughing. Sixty-four symptomatic adult volunteer outpatients were asked to cough 6 times into a cough aerosol collection system. Seventeen of these participants tested positive for influenza A virus by viral plaque assay (VPA) with confirmation by viral replication assay (VRA). Viable influenza A virus was detected in the cough aerosol particles from 7 of these 17 test subjects (41%). Viable influenza A virus was found in the smallest particle size fraction (0.3 μm to 8 μm), with a mean of 142 plaque-forming units (SD 215) expelled during the 6 coughs in particles of this size. These results suggest that a significant proportion of patients with influenza A release small airborne particles containing viable virus into the environment. Although the amounts of influenza A detected in cough aerosol particles during our experiments were relatively low, larger quantities could be expelled by influenza patients during a pandemic when illnesses would be more severe. Our findings support the idea that airborne infectious particles could play an important role in the spread of influenza.

Pulmonary Effects after Acute Inhalation of Oil Dispersant (COREXIT EC9500A) in Rats

COREXIT EC9500A (COREXIT) was used to disperse crude oil during the 2010 Deepwater Horizon oil spill. While the environmental impact of COREXIT has been examined, the pulmonary effects are unknown. Investigations were undertaken to determine whether inhaled COREXIT elicits airway inflammation, alters pulmonary function or airway reactivity, or exerts pharmacological effects. Male rats were exposed to COREXIT (mean 27 mg/m3, 5 h). Bronchoalveolar lavage was performed on d 1 and 7 postexposure. Lactate dehydrogenase (LDH) and albumin were measured as indices of lung injury; macrophages, neutrophils, lymphocytes, and eosinophils were quantified to evaluate inflammation; and oxidant production by macrophages and neutrophils was measured. There were no significant effects of COREXIT on LDH, albumin, inflammatory cell levels or oxidant production at either time point. In conscious animals, neither breathing frequency nor specific airway resistance were altered at 1 hr, 1 d and 7 d postexposure. Airway resistance responses to methacholine (MCh) aerosol in anesthetized animals were unaffected at 1 and 7 d postexposure, while dynamic compliance responses were decreased after 1 d but not 7 d. In tracheal strips, in the presence or absence of MCh, low concentrations of COREXIT (0.001% v/v) elicited relaxation; contraction occurred at 0.003–0.1% v/v. In isolated, perfused trachea, intraluminally applied COREXIT produced similar effects but at higher concentrations. COREXIT inhibited neurogenic contractile responses of strips to electrical field stimulation. Our findings suggest that COREXIT inhalation did not initiate lung inflammation, but may transiently increase the difficulty of breathing.

Acute effects of COREXIT EC9500A on cardiovascular functions in rats.

These studies characterized cardiovascular responses after an acute inhalation exposure to COREXIT EC9500A, the oil dispersant used in the Deepwater Horizon oil spill. Male Sprague-Dawley rats underwent a single 5-h inhalation exposure to COREXIT EC9500A (average exposure level 27.12 mg/m3) or air. On d 1 and 7 following the exposure, rats were implanted with indwelling catheters and changes in heart rate and blood pressure were assessed in response to increasing levels of adrenoreceptor agonists. A separate group of rats was euthanized at the same time points, ventral tail arteries were dissected, and vascular tone along with dose-dependent responses to vasoconstricting and dilating factors were assessed in vitro. Agonist-induced dose-dependent increases in heart rate and blood pressure were greater in COREXIT EC9500A-exposed than in air-exposed rats at 1 d but not 7 d after the exposure. COREXIT EC9500A exposure also induced a rise in basal tone and reduced responsiveness of tail arteries to acetylcholine-induced vasodilation at 1 d but not 7 d following the exposure. These findings demonstrate that an acute exposure to COREXIT EC9500A exerts transient effects on cardiovascular and peripheral vascular functions.

Neurotoxicity Following Acute Inhalation Exposure to the Oil Dispersant COREXIT EC9500A

Consequent to the 2010 Deepwater Horizon oil spill in the Gulf of Mexico, there is an emergent concern about the short- and long-term adverse health effects of exposure to crude oil, weathered-oil products, and oil dispersants among the workforce employed to contain and clean up the spill. Oil dispersants typically comprise of a mixture of solvents and surfactants that break down floating oil to micrometer-sized droplets within the water column, thus preventing it from reaching the shorelines. As dispersants are generally sprayed from the air, workers are at risk for exposure primarily via inhalation. Such inhaled fractions might potentially permeate or translocate to the brain via olfactory or systemic circulation, producing central nervous system (CNS) abnormalities. To determine whether oil dispersants pose a neurological risk, male Sprague-Dawley rats were exposed by whole-body inhalation exposure to a model oil dispersant, COREXIT EC9500A (CE; approximately 27 mg/m3 × 5 h/d × 1 d), and various molecular indices of neural dysfunction were evaluated in discrete brain areas, at 1 or 7 d postexposure. Exposure to CE produced partial loss of olfactory marker protein in the olfactory bulb. CE also reduced tyrosine hydroxylase protein content in the striatum. Further, CE altered the levels of various synaptic and neuronal intermediate filament proteins in specific brain areas. Reactive astrogliosis, as evidenced by increased expression of glial fibrillary acidic protein, was observed in the hippocampus and frontal cortex following exposure to CE. Collectively, these findings are suggestive of disruptions in olfactory signal transduction, axonal function, and synaptic vesicle fusion, events that potentially result in an imbalance in neurotransmitter signaling. Whether such acute molecular aberrations might persist and produce chronic neurological deficits remains to be ascertained.

LUNG RESPONSES TO HYPOTHYROIDISM, HYPERTHYROIDISM, AND LIPOPOLYSACCHARIDE CHALLENGE IN RATS

The objectives of this investigation were to study the effects of hypo- and hyperthyroidism on some factors involved in lung injury under basal conditions (air exposure) and during an inflammatory response induced by inhalation exposure to lipopolysaccharide (LPS; 100 µg/ml; 3 h) in adult rats. Thyroid status was altered by thyroidectomy or thyroxine injections for 15 d. Hyperthyroidism alone caused a greater degree of lung cell damage, an increase in the permeability of the alveolar-capillary barrier, a rise in the total number of phagocytic cells obtained by bronchoalveolar lavage (BAL), and enhanced nitric oxide (NO) release by phagocytic cells relative to that in euthyroid control animals. Hypothyroidism alone was associated with opposite effects. Exposure of animals to LPS produced inflammatory responses, which included significant increases in lung cell damage, permeability of the alveolar-capillary barrier, number of phagocytic cells obtained by BAL, and NO production by the phagocytic cells. In general, hyperthyroidism enhanced the effects of LPS, while hypothyroidism reduced LPS-induced responses. These results suggest that thyroid status alone can affect some of the factors involved in lung injury and also modulate some of the inflammatory effects of LPS. Hyperthyroidism tends to enhance lung injury, while hypothyroidism seems to reduce lung injury.

A Computer-Controlled Whole-Body Inhalation Exposure System for the Oil Dispersant COREXIT EC9500A

An automated whole-body inhalation exposure system capable of exposing 12 individually housed rats was designed to examine the potential adverse health effects of the oil dispersant COREXIT EC9500A, used extensively during the Deepwater Horizon oil spill. A computer-controlled syringe pump injected the COREXIT EC9500A into an atomizer where droplets and vapor were formed and mixed with diluent air. The aerosolized COREXIT EC9500A was passed into a customized exposure chamber where a calibrated light-scattering instrument estimated the real-time particle mass concentration of the aerosol in the chamber. Software feedback loops controlled the chamber aerosol concentration and pressure throughout each exposure. The particle size distribution of the dispersant aerosol was measured and shown to have a count median aerodynamic diameter of 285 nm with a geometric standard deviation of 1.7. The total chamber concentration (particulate + vapor) was determined using a modification of the acidified methylene blue spectrophotometric assay for anionic surfactants. Tests were conducted to show the effectiveness of closed loop control of chamber concentration and to verify chamber concentration homogeneity. Five automated 5-h animal exposures were performed that produced controlled and consistent COREXIT EC9500A concentrations (27.1 ± 2.9 mg/m3, mean ± SD).

Model Predictions of the Recruitment of Lung Units and the Lung Surface Area–Volume Relationship During Inflation

Experimental evidence suggests that the lung behaves as if it is composed of a large population of units which are recruited and derecruited during lung expansion and contraction. This study combines two previous models in order to estimate the probability distribution function describing lung unit opening pressures and the resulting alveolar surface area–volume relationship of the excised rat lung during inflation. Results indicate that the opening pressures of lung units during inflation can be described by a normal distribution. The end-expiratory pressure (EEP) has a large effect on the number of lung units that open during inflation and the properties of the area–volume relationship of the lung, but the distribution of opening pressures of individual lung units is fairly consistent regardless of EEP. This study also presents evidence that when the normalized lung area–volume relationship is represented by the equation [AL]N = [φ VL]Nn during inflation from the closed state, the expansion coefficient n is between 0.86 and 1. This result supports the theory that, for inflation from EEPs below 4 cmH2O, lung expansion occurs in part by the recruitment of lung units and not solely by the expansion of open units.

Viable influenza A virus in airborne particles expelled during coughs versus exhalations

To prepare for a possible influenza pandemic, a better understanding of the potential for the airborne transmission of influenza from person to person is needed.