Nose-only Exposure System Research Articles

Toxicity assessment of metal oxide nanomaterials using in vitro screening and murine acute inhalation studies

Characterizations and in vitro toxicity screening were performed on metal oxide engineered nanomaterials (ENMs) independently comprising ZnO, CuO, CeO2, Fe2O3, WO3, V2O5, TiO2, Al2O3 and MgO. Nanomaterials that exhibited the highest toxicity responses in the in vitro screening assays (ZnO, CuO, and V2O5) and the lesser explored material WO3 were tested for acute pulmonary toxicity in vivo. Female and male mice (C57Bl/6J) were exposed to aerosolized metal oxide ENMs in a nose-only exposure system and toxicity outcomes (biomarkers of cytotoxicity, immunotoxicity, inflammation, and lung histopathology) at 4 and 24 h after the start of exposure were assessed. The studies were performed as part of the NIEHS Nanomaterials Health Implications Research consortium with the purpose of investigating the effects of ENMs on various biological systems. ENMs were supplied by the Engineered Nanomaterials Resource and Coordination Core. Among the ENMs studied, the highest toxicity was observed for CuO and ZnO NPs in both in vitro and in vivo acute models. Compared to sham-exposed controls, there was a significant increase in bronchoalveolar lavage neutrophils and proinflammatory cytokines and a loss of macrophage viability at both 4 h and 24 h for ZnO and CuO but not seen for V2O5 or WO3. These effects were observed in both female and male mice. The cell viability performed after in vitro exposure to ENMs and assessment of lung inflammation after acute inhalation exposure in vivo were shown to be sensitive endpoints to predict ENM acute toxicity.

Development, Characterization, and Standardization of a Nose-Only Inhalation Exposure System for Exposure of Rabbits to Small-Particle Aerosols Containing Francisella tularensis.

Inhalation of Francisella tularensis causes pneumonic tularemia in humans, a severe disease with a 30 to 60% mortality rate. The reproducible delivery of aerosolized virulent bacteria in relevant animal models is essential for evaluating medical countermeasures. Here we developed optimized protocols for infecting New Zealand White (NZW) rabbits with aerosols containing F. tularensis We evaluated the relative humidity, aerosol exposure technique, and bacterial culture conditions to optimize the spray factor (SF), a central metric of aerosolization. This optimization reduced both inter- and intraday variability and was applicable to multiple isolates of F. tularensis Further improvements in the accuracy and precision of the inhaled pathogen dose were achieved through enhanced correlation of the bacterial culture optical density and the number of CFU. Plethysmograph data collected during exposures found that respiratory function varied considerably between rabbits, was not a function of weight, and did not improve with acclimation to the system. Live vaccine strain (LVS)-vaccinated rabbits were challenged via aerosol with human-virulent F. tularensis SCHU S4 that had been cultivated in either Mueller-Hinton broth (MHB) or brain heart infusion (BHI) broth. LVS-vaccinated animals challenged with SCHU S4 that had been cultivated in MHB experienced short febrile periods (median, 3.2 days), limited weight loss (<5%), and longer median survival times (∼18 days) that were significantly different from those for unvaccinated controls. In contrast, LVS-vaccinated rabbits challenged with SCHU S4 that had been cultivated in BHI experienced longer febrile periods (median, 5.5 days) and greater weight loss (>10%) than the unvaccinated controls and median survival times that were not significantly different from those for the unvaccinated controls. These studies highlight the importance of careful characterization and optimization of protocols for aerosol challenge with pathogenic agents.

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.

Mass spectrometry-based analysis of murine bronchoalveolar lavage fluid following respiratory exposure to 4,4'-methylene diphenyl diisocyanate aerosol

1. Diisocyanates are highly reactive electrophiles utilized in the manufacture of a wide range of polyurethane products and have been identified as causative agents of occupational allergic respiratory disease. However, in spite of the significant occupational health burden associated with diisocyanate-induced asthma, the mechanism of disease pathogenesis remains largely unknown.2. To better understand the fate of inhaled diisocyanates, a nose-only aerosol exposure system was constructed and utilized to expose a BALB/c mouse model to an aerosol generated from 4,4'-methylene diphenyl diisocyanate (MDI). Tissue and bronchoalveolar lavage samples were evaluated 4 and 24 h post-exposure for evidence of diisocyanate-protein haptenation, and a label-free quantitative proteomics strategy was employed to evaluate relative changes to the protein content of the cellular fraction of the lavage fluid.3. Following MDI aerosol exposure, expression of the number of proteins with immunological or xenobiotic metabolism relevance is increased, including endoplasmin, cytochrome P450 and argininosuccinate synthase. Western blot analysis indicated MDI-conjugated protein in the lavage fluid, which was identified as serum albumin.4. Tandem mass spectrometry analysis of MDI-albumin revealed MDI conjugation occurs at a dilysine motif at Lys525, as well as at a glutamine-lysine motif at Lys414, in good agreement with previously published in vitro data on diisocyanate-conjugated serum albumin.

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System.

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and bronchodilator drugs. Despite the availability of multiple treatments, both diseases are growing public health concerns. The majority of asthma patients are well controlled on current inhaled therapies but a substantial number of patients with severe asthma are not. Asthma affects an estimated 300 million people worldwide and approximately 20 percent have a severe form of the disease. In contrast to asthma, there are few effective therapies for COPD. An estimated 10% of the population has COPD and the trend in death rates is increasing for COPD while decreasing for other major diseases. Although developing drugs for inhaled delivery is challenging, the nose-only inhalation unit enables direct delivery of novel drugs to the lung of rodents for pre-clinical efficacy and safety/toxicology studies. Inhaled drug delivery has multiple advantages for respiratory diseases, where high concentration in the lung improves efficacy and minimizes systemic side effects. Inhaled corticosteroids and bronchodilators benefit from these advantages and inhaled delivery may also hold potential for future biologic therapies. The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs of rodents, key data required before initiating clinical development.

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and bronchodilator drugs. Despite the availability of multiple treatments, both diseases are growing public health concerns. The majority of asthma patients are well controlled on current inhaled therapies but a substantial number of patients with severe asthma are not. Asthma affects an estimated 300 million people worldwide and approximately 20 percent have a severe form of the disease. In contrast to asthma, there are few effective therapies for COPD. An estimated 10% of the population has COPD and the trend in death rates is increasing for COPD while decreasing for other major diseases. Although developing drugs for inhaled delivery is challenging, the nose-only inhalation unit enables direct delivery of novel drugs to the lung of rodents for pre-clinical efficacy and safety/toxicology studies. Inhaled drug delivery has multiple advantages for respiratory diseases, where high concentration in the lung improves efficacy and minimizes systemic side effects. Inhaled corticosteroids and bronchodilators benefit from these advantages and inhaled delivery may also hold potential for future biologic therapies. The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs of rodents, key data required before initiating clinical development.

Design and Characterization of an Inhalation System to Expose Rodents to Nanoaerosols

ABSTRACTThe number of workers potentially exposed to nanoparticles (NPs) in industrial processes is constantly increasing, even though the toxicological effects of these compounds have not yet been fully characterized. The hazards associated with this exposure can be assessed most relevantly by toxicology studies involving inhalation of nanoaerosols by animals. In this paper, we describe and characterize an aerosol generated in a nose-only exposure system used to study the respiratory effects of NPs in rat; this system was designed to meet the most stringent requirements for animal testing in terms of protection of operators against risks associated with NPs and biohazards and to comply with the OECD guidelines for chemical testing. The inhalation facility was fully validated by exposing Fisher 344 rats to TiO2 P25 aerosols at 10 mg m–3. Aerosol monitoring and in-depth characterization were ensured by real-time devices (condensation particle counter, optical particle sizer, scanning mobility particle sizer, aerodynamic particle sizer and electrical low pressure impactor) and samples taken for off-line analyses (gravimetric analysis, mass size distribution from cascade impactor, TEM observations). The test atmosphere was stable in terms of concentrations and distributions (mass or number) between different inhalation towers on a given day and between days (intra-experiment), as well as between inhalation campaigns (between experiments). In terms of the respiratory deposition profile, preliminary results after exposure for one month indicate that this system is relevant, and should therefore be appropriate for in vivo inhalation toxicity studies.

Size-dependent deposition of inhaled nanoparticles in the rat respiratory tract using a new nose-only exposure system

ABSTRACTConcerns about the potential health effects of exposure to nanomaterials have led to a growing number of in vivo inhalation toxicity studies using nanoparticle aerosols. Estimates of aerosol deposition within the respiratory tract are important for these studies to enable: (a) the interpretation of the results, in particular, the evaluation of dose–response relationships; (b) comparison with the results of other related studies; and (c) the extrapolation of results from animal models to human. Unfortunately, only a limited number of studies have been undertaken to investigate respiratory tract deposition efficiencies for nano-sized aerosol particles. This is of particular importance as deposition efficiencies are predicted to vary significantly over the nano-size range for some elements of the respiratory tract. In this study, female Wistar-Kyoto rats were exposed in a new design nose-only inhalation exposure system to spark generated radioactive iridium-192 nanoparticle aerosols of four particle ...

Nose-only cigarette smoke exposure plus airway lipopolysaccharide inhalation induced chronic obstructive pulmonary disease and associated pulmonary hypertension in mice

To establish a mouse model of chronic obstructive pulmonary disease (COPD) and associated pulmonary hypertension (COPD-PH) induced by nose-only cigarette smoking exposure plus airway lipopolysaccharide (LPS) inhalation. There were 24 male C57B6 mice divided into a control group and a model group at random. The model group was given LPS by intranasal inhalation on day 1 and day 14 and exposed to the cigarette smoke in a nose-only exposure system, while the control group was given physiological saline and exposed to normal air. The model establishment was evaluated according the following parameters: the lung function and the right heart pressure, the total and differential cell numbers in bronchial alveolar lavage fluid (BALF), and the pathological changes of lung tissues. The functional residual capacity data of the model group and the control group were (0.402 ± 0.057) and (0.243 ± 0.064) ml respectively (P<0.05). The inspiratory resistance data of the model group and the control group were (1.056 ± 0.121) and (0.789 ± 0.063) cmH(2)O · ml(-1) · s(-1) (1 cmH(2)O=0.098 kPa) respectively (P<0.05). The static lung compliance data of the model group and the control group were (0.084 ± 0 .007) and (0.056 ± 0.004) cmH(2)O/ml respectively (P<0.05). The right ventricular mean pressure of the model group and the control group were (11.3 ± 1.3) and (7.9 ± 1.1) mmHg (1 mmHg=0.133 kPa) respectively (P<0.05), while the right ventricular hypertrophy index of the model group and the control group were (0.267 ± 0.019) and (0.195 ± 0.023) respectively (P<0.05). Moreover, the histological staining showed that goblet cell hyperplasia, lung inflammation and thickening of smooth muscle layers of bronchial and pulmonary small vessels occurred in the model group, which indicated ongoing airway and blood vessel remodeling. A COPD-PH mouse model was established by nose-only cigarette smoking exposure plus airway LPS inhalation in a short period of time, and this method was more similar to the smoking behavior of human.

Polyhexamethylene guanidine phosphate aerosol particles induce pulmonary inflammatory and fibrotic responses.

Polyhexamethylene guanidine (PHMG) phosphate was used as a disinfectant for the prevention of microorganism growth in humidifiers, without recognizing that a change of exposure route might cause significant health effects. Epidemiological studies reported that the use of humidifier disinfectant containing PHMG-phosphate can provoke pulmonary fibrosis. However, the pulmonary toxicity of PHMG-phosphate aerosol particles is unknown yet. This study aimed to elucidate the toxicological relationship between PHMG-phosphate aerosol particles and pulmonary fibrosis. An in vivo nose-only exposure system and an in vitro air-liquid interface (ALI) co-culture model were applied to confirm whether PHMG-phosphate induces inflammatory and fibrotic responses in the respiratory tract. Seven-week-old male Sprague-Dawley rats were exposed to PHMG-phosphate aerosol particles for 3 weeks and recovered for 3 weeks in a nose-only exposure chamber. In addition, three human lung cells (Calu-3, differentiated THP-1 and HMC-1 cells) were cultured at ALI condition for 12 days and were treated with PHMG-phosphate at set concentrations and times. The reactive oxygen species (ROS) generation, airway barrier injuries and inflammatory and fibrotic responses were evaluated in vivo and in vitro. The rats exposed to PHMG-phosphate aerosol particles in nanometer size showed pulmonary inflammation and fibrosis including inflammatory cytokines and fibronectin mRNA increase, as well as histopathological changes. In addition, PHMG-phosphate triggered the ROS generation, airway barrier injuries and inflammatory responses in a bronchial ALI co-culture model. Those results demonstrated that PHMG-phosphate aerosol particles cause pulmonary inflammatory and fibrotic responses. All features of fibrogenesis by PHMG-phosphate aerosol particles closely resembled the pathology of fibrosis that was reported in epidemiological studies. Finally, we expected that PHMG-phosphate infiltrated into the lungs in the form of aerosol particles would induce an airway barrier injury via ROS, release fibrotic inflammatory cytokines, and trigger a wound-healing response, leading to pulmonary fibrosis. A simultaneous state of tissue destruction and inflammation caused by PHMG-phosphate had whipped up a "perfect storm" in the respiratory tract.

A dynamic smoke generation and nose-only inhalation exposure system for rats: preliminary results from studies of selected transportation materials

Context: Smoke inhalation injury is the main cause of fatalities for fire victims. Understanding in the pathophysiology of the injury has not been fully explored in recent years. To further explore the pathophysiological mechanism, a dynamic and controllable animal model is necessary.Objective: To develop a rat model of smoke inhalation injury to simulate human victims in air-restricted vehicle cabin fires.Materials and methods: Smoke concentration, including CO, O2, VOCs and smoke temperature under different combustion conditions, were detected. Levels of COHb, respiratory function, lung wet-to-dry weight ratio and protein concentration in BALF and blood were measured. Pathological evaluations of lung in tissues were conducted at 1, 6, 24 and 48 h post-exposure.Results: Smoke concentration rose with the increase of combustion temperature and decrease of oxygen flow. Further, 215 kinds of VOCs in the smoke were detected, and the concentrations of benzene, methylbenzene, ethylbenzene, dimethylbenzene, phenylethylene and trimethylbenzene was 32.93, 402.06, 764.03, 113.73, 1006.61 and 89.28 mg/m3, respectively. Significant hypoxemia and CO poisoning occurred in rats. The FCOHb after exposure for 14 min immediately rose to (44.2 ± 12.3) % and then gradually decrease to a normal level at 300 min post-exposure. At 24 h post-exposure, Penh increased significantly (p < 0.05), and high pulmonary vascular permeability and significant lung edema (p < 0.05) were observed in the smoke inhalation group.Discussion and conclusion: In summary, the novel rat model of smoke inhalation injury system used in the study is dynamic and controllable, and appropriate for use in smoke inhalation injury studies of air-restricted cabins in vehicles.

Antenatal exposure of maternal secondhand smoke (SHS) increases fetal lung expression of RAGE and induces RAGE-mediated pulmonary inflammation.

BackgroundReceptors for advanced glycation end-products (RAGE) are immunoglobulin-like pattern recognition receptors abundantly localized to lung epithelium. Our research demonstrated that primary tobacco smoke exposure increases RAGE expression and that RAGE partly mediates pro-inflammatory signaling during exposure. However, the degree to which RAGE influences developing lungs when gestating mice are exposed to secondhand smoke (SHS) has not been determined to date.MethodsTimed pregnant RAGE null and wild type control mice were exposed to 4 consecutive days of SHS from embryonic day (E) 14.5 through E18.5 using a state of the art nose-only smoke exposure system (Scireq, Montreal, Canada). RAGE expression was assessed using immunofluorescence, immunoblotting, and quantitative RT-PCR. TUNEL immunostaining and blotting for caspase-3 were performed to evaluate effects on cell turnover. Matrix abnormalities were discerned by quantifying collagen IV and MMP-9, a matrix metalloprotease capable of degrading basement membranes. Lastly, TNF-α and IL-1β levels were assessed in order to determine inflammatory status in the developing lung.ResultsPulmonary RAGE expression was elevated in both dams exposed to SHS and in fetuses gestating within mothers exposed to SHS. Fetal weight, a measure of organismal health, was decreased in SHS-exposed pups, but unchanged in SHS-exposed RAGE null mice. TUNEL assessments suggested a shift toward pulmonary cell apoptosis and matrix in SHS-exposed pups was diminished as revealed by decreased collagen IV and increased MMP-9 expression. Furthermore, SHS-exposed RAGE null mice expressed less TNF-α and IL-1β when compared to SHS-exposed controls.ConclusionsRAGE augmentation in developing pups exposed to maternal SHS weakens matrix deposition and influences lung inflammation.

Characterization and pharmacokinetic analysis of crystalline versus amorphous rapamycin dry powder via pulmonary administration in rats

The pharmacokinetics of inhaled rapamycin (RAPA) is compared for amorphous versus crystalline dry powder formulations. The amorphous formulation of RAPA and lactose (RapaLac) was prepared by thin film freezing (TFF) using lactose as the stabilizing agent in the weight ratio 1:1. The crystalline formulation was prepared by wet ball milling RAPA and lactose and posteriorly blending the mixture with coarse lactose (micronized RAPA/micronized lactose/coarse lactose=0.5:0.5:19). While both powders presented good aerosolization performance for lung delivery, TFF formulation exhibited better in vitro aerodynamic properties than the crystalline physical mixture. Single-dose 24h pharmacokinetic studies were conducted in Sprague–Dawley rats following inhalation of the aerosol mist in a nose-only inhalation exposure system. Lung deposition was higher for the crystalline group than for the TFF group. Despite higher pulmonary levels of drug that were found for the crystalline group, the systemic circulation (AUC0–24) was higher for the amorphous group (8.6ngh/mL) than for crystalline group (2.4ngh/mL) based on a five-compartmental analysis. Lung level profiles suggest that TTF powder stays in the lung for the same period of time as the crystalline powder but it presented higher in vivo systemic bioavailability due to its enhanced solubility, faster dissolution rate and increased FPF at a more distal part of the lungs.

Ventilatory and metabolic changes in a mouse model of chronic lung disease (713.9)

We measured changes in ventilatory and metabolic responses to acute hypoxia and hypercapnia in a mouse model of chronic lung disease. Baseline measurements were made before any treatment. Then we deleted vascular endothelial growth factor (VEGF) in the lungs using the Cre‐loxP strategy. Lungs of VEGF‐lung null mice were treated with adeno‐associated virus (AAV) expressing Cre‐recombinase; control mice were treated with AAV‐LacZ. Measurements were made 1 month later. Then we exposed mice to cigarette smoke (CS, 10 cigarettes/day, 5 days/wk via a nose‐only exposure system) and repeated measurements after 2 and 4 months. VEGF‐lung null and control mice gained weight for 1 month but both groups lost weight during the first 2 months of CS and gained weight by 4 months of CS. Ventilation decreased in normoxia or hypercapnia similarly in VEGF‐lung null and control mice after 4 months CS mainly because of decreased tidal volume. Ventilation normalized for CO2 production shows the same trend so arterial PCO2 should increase. More ventilatory and arterial blood gas measurements are planned after 6 months of CS to quantify the effects of this treatment on pulmonary gas exchange.Grant Funding Source: Supported by NIH RO1 HL‐081823, P01 HL‐091830

Generation and delivery of nanoaerosols from biological and biologically active substances

A device for the generation of nanoaerosols from biological substances was designed and the resulting nanoaerosols were characterized. The function of the generator is based on the electrospray-neutralization process in which nanoclusters, obtained by electrohydrodynamic atomization, are neutralized by counter-ions generated by the electrospraying of ethanol at the opposite potential. The device is capable of generating nanoaerosol from suspensions of nanoparticles and solutions of non-volatile organic substances (e.g., antibiotics), proteins, and other biomolecules. This device produces nanoaerosols (10–300nm in size) with a concentration up to 2×107cm−3. A quartz crystal microbalance based device was designed to monitor the performance of the generator. Using magnetic Fe3O4 nanoparticles as reporters, it has been demonstrated that approximately 0.4µg of nanoaerosol was deposited into a mouse lung in 1h in a nose-only exposure system. Thus, we present an approach to both generate nanoaerosol from biological and therapeutic substances and to estimate the doses delivered to mice exposed to these nanoaersolized substances.

Impact of Non-constant Concentration Exposure on Lethality of Inhaled Hydrogen Cyanide

The ten Berge model, also known as the toxic load model, is an empirical approach in hazard assessment modeling for estimating the relationship between the inhalation toxicity of a chemical and the exposure duration. The toxic load (TL) is normally expressed as a function of vapor concentration (C) and duration (t), with TL equaling C(n) × t being a typical form. Hypothetically, any combination of concentration and time that yields the same "toxic load" will give a constant biological response. These formulas have been developed and tested using controlled, constant concentration animal studies, but the validity of applying these assumptions to time-varying concentration profiles has not been tested. Experiments were designed to test the validity of the model under conditions of non-constant acute exposure. Male Sprague-Dawley rats inhaled constant or pulsed concentrations of hydrogen cyanide (HCN) generated in a nose-only exposure system for 5, 15, or 30 min. The observed lethality of HCN for the 11 different C versus t profiles was used to evaluate the ability of the model to adequately describe the lethality of HCN under the conditions of non-constant inhalation exposure. The model was found to be applicable under the tested conditions, with the exception of the median lethality of very brief, high concentration, discontinuous exposures.

Abstract 683: Toxicity of aerosolized azacytidine (Vidaza) in mice.

Abstract Background: Azacytidine, a prototypical demethylation agent, has been approved to treat myelodysplastic syndrome. Commercial azacytidine (Vidaza) is delivered to patients by subcutaneous or intravenous administration. In a previous study, we found that aerosolized azacytidine can 1) inhibit human orthotopic lung cancers in a mouse xenograft model, 2) demethylate several promoters of tumor suppressor genes reported to be hypermethylated in human lung cancer, and 3) be better tolerated and more effective than systemic administration. These studies specifically addressed toxicity associated with aerosolized delivery of Vidaza to the lungs; specifically hematologic and pulmonary toxicity. Methods: Vidaza was dissolved into sterile water for injection and aerosolized in a clinical standard aerosol system. Swiss mice male and female 6/group were restricted into a nose-only exposure system for receiving the aerosol. We treated the mice at 2 different doses, 7.5 mg/m2 daily x 7 days and 2.5 mg/m2 daily x 7 days. Blood, sternum (for bone marrow), and lung were collected at 3 and 6 weeks after the final aerosol treatment. The bone and lung were fixed in 10% neutral buffered formalin and routinely processed to paraffin blocks, sectioned, stained, and evaluated histologically. In a preliminary pharmacokinetic study, azacytidine concentrations in the blood and lung samples from mice receiving 2.5 mg/m2 of aerosolized azacytidine were determined by a MS-HPLC method previously described by others. Results: Neither dose of aerosolized Vidaza resulted in bone marrow changes. A mild pneumonitis was present in lungs from mice treated with 7.5 mg/m2 at 3 weeks but was not evident at six weeks (reversible). No pulmonary toxicity was observed at the 2.5 mg/m2 dose. The preliminary pharmacokinetic study data indicate very low drug concentrations in the blood and much higher concentrations in the lungs. Conclusion: Our study demonstrates that low dose aerosolized azacytidine can be safely administered to mice. Reversible pulmonary toxicity (pneumonitis) was observed at 7.5 mg/m2 at 3 weeks post treatment. The recommended starting dose of aerosolized azacytidine in human clinical phase I trial should be 2.5 mg/m2. Supported by NIH grant 5R01CA154755 to Roman Perez-Soler and Yiyu Zou. Citation Format: Xuan Qiu, Rani Sellers, Yuanxin Liang, Bilal Piperdi, Roman Perez-Soler, Yiyu Zou. Toxicity of aerosolized azacytidine (Vidaza) in mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 683. doi:10.1158/1538-7445.AM2013-683

Inhalation of uranium nanoparticles: Respiratory tract deposition and translocation to secondary target organs in rats

Uranium nanoparticles (<100nm) can be released into the atmosphere during industrial stages of the nuclear fuel cycle and during remediation and decommissioning of nuclear facilities. Explosions and fires in nuclear reactors and the use of ammunition containing depleted uranium can also produce such aerosols. The risk of accidental inhalation of uranium nanoparticles by nuclear workers, military personnel or civilian populations must therefore be taken into account. In order to address this issue, the absorption rate of inhaled uranium nanoparticles needs to be characterised experimentally. For this purpose, rats were exposed to an aerosol containing 107 particles of uranium per cm3 (CMD=38nm) for 1h in a nose-only inhalation exposure system. Uranium concentrations deposited in the respiratory tract, blood, brain, skeleton and kidneys were determined by ICP-MS. Twenty-seven percent of the inhaled mass of uranium nanoparticles was deposited in the respiratory tract. One-fifth of UO2 nanoparticles were rapidly cleared from lung (T1/2=2.4h) and translocated to extrathoracic organs. However, the majority of the particles were cleared slowly (T1/2=141.5d). Future long-term experimental studies concerning uranium nanoparticles should focus on the potential lung toxicity of the large fraction of particles cleared slowly from the respiratory tract after inhalation exposure.

Short-Term Systemic Effects of Nose-Only Cigarette Smoke Exposure in Mice: Role of Oxidative Stress

Background/Aims: Long–term cigarette smoking (CS) is a major risk factor for respiratory and cardiovascular diseases, and is also known to adversely affect other organs. However, data on the systemic effects of short-term CS exposure (STCSE) are scarce. Presently, using a nose-only exposure system, we evaluated the systemic effects of STCSE in mice. Methods: We assessed the effects of CS generated by 9 consecutive cigarettes per day for 4 days in a nose-only exposure system on cardiovascular, hepatic and renal endpoints evaluated on day 5 in mice. Control mice were exposed to air only. Results: CS significantly increased systolic blood pressure and decreased total nitric oxide plasma concentration. Circulating platelets and erythrocyte numbers were also increased. However, STCSE did not significantly increase thrombosis in pial arterioles and venules. STCSE significantly raised plasma alanine aminotransferase and gamma glutamyl transpeptidase activities, but did not affect urea or creatinine concentrations. Interestingly, while STCSE enhanced the production of reactive oxygen species in heart and kidney and lipid peroxidation in heart, liver and kidneys, it also enhanced the antioxidant activity of superoxide dismutase, probably indicating that STCSE causes adaptive reactions to counterbalance the potentially damaging action of oxygen radicals induced by STCSE. Conclusion: These results suggest that STCSE causes blood pressure increase, hepatotoxicity and oxidative stress in the heart, liver and the kidneys. These data provide information on the initial steps leading to the systemic effects of STCSE, a stage at which the diseases may likely be reversed.

Evaluation of the pulmonary effects of short-term nose-only cigarette smoke exposure in mice

Much is known about the chronic effects of cigarette smoke (CS) on lung function and inflammation and development of chronic obstructive pulmonary disease. However, the underlying pathophysiological mechanisms related to the short-term exposure to CS are not fully understood. Here, we assessed the effect of CS generated by nine consecutive cigarettes per day for four days in a nose-only exposure system on airway resistance measured using forced oscillation technique, lung inflammation and oxidative stress in BALB/c mice. Control mice were exposed to air. Mice exposed to CS showed a significant increase of neutrophils and lymphocytes numbers in bronchoalveolar lavage (BAL). The total protein and endothelin levels in BAL fluid were significantly augmented suggesting an increase of alveolar-capillary barrier permeability. Similarly, airway resistance was significantly increased in the CS group compared with controls. Furthermore, reactive oxygen species and lipid peroxidation levels in lung tissue were significantly increased. The antioxidant activities of reduced glutathione, glutathione S transferase and superoxide dismutase were all significantly increased following CS exposure, indicating that CS could trigger adaptive responses that counterbalance the potentially damaging activity of oxygen radicals induced by CS exposure. In conclusion, our data indicate that short-term nose-only exposure to CS causes lung inflammation and increase of airway resistance mediated at least partly through the oxidative stress.