Comparison of pulmonary inflammatory responses following intratracheal instillation and inhalation of nanoparticles

In order to examine whether intratracheal instillation studies can be useful for determining the harmful effect of nanoparticles, we performed inhalation and intratracheal instillation studies using samples of the same nanoparticles. Nickel oxide nanoparticles (NiO) and titanium dioxide nanoparticles (TiO₂) were used as chemicals with high and low toxicities, respectively. In the intratracheal instillation study, rats were exposed to 0.2 or 1?mg of NiO or TiO₂. Cell analysis and chemokines in bronchoalveolar lavage fluid (BALF) were analyzed from 3 days to 6 months following the single intratracheal instillation. In the inhalation study, rats were exposed to inhaled NiO or TiO₂ (1.65, 1.84?mg/m³, respectively) for 4 weeks. The same endpoints were examined from 3 days to 3 months after the end of exposure. Inhalation of NiO induced an increase in the number of neutrophils in BALF and concentrations of cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-2 and heme oxygenase (HO)-1. Intratracheal instillation of NiO induced persistent inflammation and upregulation of these cytokines was observed in the rats. However, inhalation of TiO₂ did not induce pulmonary inflammation, and intratracheal instillation of TiO₂ transiently induced an increase in the number of neutrophils in BALF and the concentrations of CINC-1, CINC-2 and HO-1. Taken together, a difference in pulmonary inflammation was observed between the high and low toxicity nanomaterials in the intratracheal instillation studies, as in the inhalation studies, suggesting that intratracheal instillation studies may be useful for ranking the harmful effects of nanoparticles.     

Simple in vitro models can predict pulmonary toxicity of silver nanoparticles

To study the effects of nanomaterials after inhalation, a large number of in vitro lung models have been reported in literature. Although the in vitro models contribute to the reduction of animal studies, insufficient data exists to determine the predictive value of these in vitro models for the in vivo situation. The aim of this study was to determine the correlation between in vitro and in vivo data by comparing the dose metrics of silver nanoparticles in an in vitro lung model of increasing complexity to our previously published in vivo inhalation study. In vivo, the previously published study showed that the alveolar dose expressed as particle surface area is the most suitable dose metric to describe the toxicity of silver nanoparticles after inhalation. The results of the present study show that particle surface area is a suitable dose metric to describe the effects of silver nanoparticles when using a simple monolayer of lung epithelial cells. The dose metric shifted from particle surface area to particle mass when adding an increasing number of macrophages. In addition, a co-culture of endothelial cells, epithelial cells and macrophages on a Transwell® insert correlated less well to the in vivo results compared to the epithelial monolayer. We conclude that for studying the acute pulmonary toxicity of nanoparticles simple in vitro models using an epithelial monolayer better predict the in vivo response compared to complex co-culture models.     

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.     

Comparative cardiopulmonary toxicity of exhausts from soy-based biofuels and diesel in healthy and hypertensive rats

Abstract

Increased use of renewable energy sources raise concerns about health effects of new emissions. We analyzed relative cardiopulmonary health effects of exhausts from (1) 100% soy biofuel (B100), (2) 20% soy biofuel?+?80% low sulfur petroleum diesel (B20), and (3) 100% petroleum diesel (B0) in rats. Normotensive Wistar–Kyoto (WKY) and spontaneously hypertensive rats were exposed to these three exhausts at 0, 50, 150 and 500?μg/m³, 4?h/day for 2 days or 4 weeks (5 days/week). In addition, WKY rats were exposed for 1 day and responses were analyzed 0?h, 1 day or 4 days later for time-course assessment. Hematological parameters, in vitro platelet aggregation, bronchoalveolar lavage fluid (BALF) markers of pulmonary injury and inflammation, ex vivo aortic ring constriction, heart and aorta mRNA markers of vasoconstriction, thrombosis and atherogenesis were analyzed. The presence of pigmented macrophages in the lung alveoli was clearly evident with all three exhausts without apparent pathology. Overall, exposure to all three exhausts produced only modest effects in most endpoints analyzed in both strains. BALF γ-glutamyl transferase (GGT) activity was the most consistent marker and was increased in both strains, primarily with B0 (B0?>?B100?>?B20). This increase was associated with only modest increases in BALF neutrophils. Small and very acute increases occurred in aorta mRNA markers of vasoconstriction and thrombosis with B100 but not B0 in WKY rats. Our comparative evaluations show modest cardiovascular and pulmonary effects at low concentrations of all exhausts: B0 causing more pulmonary injury and B100 more acute vascular effects. BALF GGT activity could serve as a sensitive biomarker of inhaled pollutants.     

NMR-based metabolomics to determine acute inhalation effects of nano- and fine-sized ZnO particles in the rat lung

Zinc oxide (ZnO) particles induce acute occupational inhalation illness in humans and rats. However, the possible molecular mechanisms of ZnO particles on the respiratory system remain unclear. In this study, metabolic responses of the respiratory system of rats inhaled ZnO particles were investigated by a nuclear magnetic resonance (NMR)-based metabolomic approach. Male Sprague–Dawley rats were treated with a series of doses of nano-sized (35?nm) or fine-sized (250?nm) ZnO particles. The corresponding control groups inhaled filtered air. After 24?h, bronchoalveolar lavage fluid (BALF) and lung tissues were collected, extracted and prepared for ¹H and J-resolved NMR analysis, followed by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). PCA and PLSDA models from analysis of BALF and hydrophilic lung NMR spectra demonstrated that dose response trends were restricted to the 250?nm ZnO particle exposure group and were not observed in the 35?nm ZnO particle exposure group. Increased isoleucine and valine, as well as decreased acetate, trimethylamine n-oxide, taurine, glycine, formate, ascorbate and glycerophosphocholine, were recorded in the BALF of rats treated with moderate and high dose 250?nm ZnO exposures. Decreases in taurine and glucose, as well as an increase of phosphorylcholine-containing lipids and fatty acyl chains, were detected in the lung tissues from 250?nm ZnO-treated rats. These metabolic changes may be associated with cell anti-oxidation, energy metabolism, DNA damage and membrane stability. We also concluded that a metabolic approach provides more complete measurements and suggests potential molecular mechanisms of adverse effects.     

Expression of cytokine-induced neutrophil chemoattractant in rat lungs by intratracheal instillation of nickel oxide nanoparticles

Since nanoparticles easily agglomerate to form larger particles, it is important to maintain the size of their agglomerates at the nano-level to evaluate the harmful effect of the nanoparticles. We prevented agglomeration of nickel oxide nanoparticles by ultrasound diffusion and filtration, established an acute exposure model using animals, and examined inflammation and chemokine expression. The mass median diameter of nickel oxide nanoparticle agglomerates suspended in distilled water for intratracheal instillation was 26?nm (8.41?nm weighted average surface primary diameter). Male Wistar rats received intratracheal instillation of nickel oxide nanoparticles at 0.1?mg (0.33?mg/kg) or 0.2?mg (0.66?mg/kg), and were dissected 3 days, 1 week, 1 month, 3 months, and 6 months after the instillation. The control group received intratracheal instillation of distilled water. Three chemokines (cytokine-induced neutrophil chemoattractant-1 (CINC-1), CINC-2αβ, and CINC-3) in the lung tissue and bronchoalveolar lavage fluid (BALF) were determined by quantitative measurement of protein by ELISA. Both CINC-1 and CINC-2αβ concentration was elevated from day 3 to 3 months in lung tissue and from day 3 to 6 months in BALF. On the other hand, CINC-3 was elevated on day 3 in both lung tissue and BALF, and then decreased. The total cell and neutrophil counts in BALF were increased from day 3 to 3 months. In lung tissue, infiltration of mainly neutrophils and alveolar macrophages was observed from day 3 to 6 months in alveoli. These results suggest that CINC was involved in lung injury by nickel oxide nanoparticles.     

Pulmonary toxicity of printer toner following inhalation and intratracheal instillation

Abstract

The pulmonary effects of a finished toner were evaluated in intratracheal instillation and inhalation studies, using toners with external additives (titanium dioxide nanoparticles and amorphous silica nanoparticles). Rats received an intratracheal dose of 1?mg or 2?mg of toner and were sacrificed at 3 days, 1 week, 1 month, 3 months and 6 months. The toner induced pulmonary inflammation, as evidenced by a transient neutrophil response in the low-dose groups and persistent neutrophil infiltration in the high-dose groups. There were increased concentrations of heme oxygenase-1 (HO-1) as a marker of oxidative stress in the bronchoalveolar lavage fluid (BALF) and the lung. In a 90-day inhalation study, rats were exposed to well-dispersed toner (mean of MMAD: 3.76?µm). The three mass concentrations of toner were 1, 4 and 16?mg/m³ for 13 weeks, and the rats were sacrificed at 6 days and 91 days after the end of the exposure period. The low and medium concentrations did not induce neutrophil infiltration in the lung of statistical significance, but the high concentration did, and, in addition, upon histopathological examination not only showed findings of inflammation but also of fibrosis in the lung. Taken together, the results of our studies suggest that toners with external additives lead to pulmonary inflammation and fibrosis at lung burdens suggest beyond the overload. The changes observed in the pulmonary responses in this inhalation study indicate that the high concentration (16?mg/m³) is an LOAEL and that the medium concentration (4?mg/m³) is an NOAEL.     

Toxicological investigations on the respirable fraction of silicon carbide grain products by the in vitro vector model

Abstract

Increased lung cancer incidence with workers at the production site of crude silicon carbide (SiC) using the Acheson process has been reported. Several agents derived from the process were discussed as causative factors. Recently concern had been expressed about the presence of cleavage fragments (CFs) in commercial products fulfilling the WHO criteria for fibers. This study has focused on the toxicological significance of such CFs. The test samples were respirable fractions of five different commercial samples of SiC grains. The CF content (scanning electron microscopy) was in the range 17–493?fibers/µg. Crystalline silica and whiskers could not be detected. Quartz DQ12, cristobalite, SiC whisker, UICC crocidolite and electrocorundum were used as control reference samples. Biological activity was assessed with the in vitro vector model (VM) on ex vivo rat and guinea pig alveolar macrophages (AMs). The dose range of the VM is derived from calculated AM loads from intratracheal instillation experiments and confirmed by measured AM loads from inhalation studies on alumina monohydrate particles with low biological activity: ≤120?pg/AM. The response of the references was clearly different from that of the SiC grains which yielded low toxicity overall. However, the parameter reactive oxygen species secreted by AMs was elevated at the higher SiC doses, but not related to the CF content of these samples. Our data showed that CFs seem to have no biological relevance. This is in agreement with results from recent studies in which no carcinogenic activity had been demonstrated for CFs.     

Toxicologic evaluation of tungsten: 28-day inhalation study of tungsten blue oxide in rats

The toxicity and toxicokinetics of tungsten blue oxide (TBO) were examined. TBO is an intermediate in the production of tungsten powder, and has shown the potential to cause cellular damage in in vitro studies. However, in vivo evidence seems to indicate a lack of adverse effects. The present study was undertaken to address the dearth of longer-term inhalation toxicity studies of tungsten oxides by investigating the biological responses induced by TBO when administered via nose-only inhalation to rats at levels of 0.08, 0.325, and 0.65?mg TBO/L of air for 6?h/day for 28 consecutive days, followed by a 14-day recovery period. Inhaled TBO was absorbed systemically and blood levels of tungsten increased as inhaled concentration increased. Among the tissues analyzed for tungsten levels, lung, femur and kidney showed increased levels, with lung at least an order of magnitude greater than kidney or femur. By exposure day 14, tungsten concentration in tissues had reached steady-state. Increased lung weight was noted for both terminal and recovery animals and was attributed to deposition of TBO in the lungs, inducing a macrophage influx. Microscopic evaluation of tissues revealed a dose-related increase in alveolar pigmented macrophages, alveolar foreign material and individual alveolar foamy macrophages in lung. After a recovery period there was a slight reduction in the incidence and severity of histopathological findings. Based on the absence of other adverse effects, the increased lung weights and the microscopic findings were interpreted as nonadverse response to exposure and were not considered a specific reaction to TBO.     

Pathological features of rat lung following inhalation and intratracheal instillation of C60 fullerene

We evaluated the pulmonary pathological features of rats that received a single intratracheal instillation and a 4-week inhalation of a fullerene. We used fullerene C₆₀ (nanom purple; Frontier Carbon Co. Ltd, Japan) in this study. Male Wistar rats received intratracheal dose of 0.1, 0.2, or 1?mg of C₆₀, and were sacrificed at 3 days, 1 week, 1 month, 3 months, 6 months, and 12 months. In the inhalation study, Wistar rats received C₆₀ or nickel oxide by whole-body inhalation for 6?h/day, 5 days/week, 4 weeks, and were sacrificed at 3 days, 1 month, and 3 months after the end of exposure. During the observation period, no tumors or granulomas were observed in either study. Histopathological evaluation by the point counting method (PCM) showed that a high dose of C₆₀ (1?mg) instillation led to a significant increase of areas of inflammation in the early phase (until 1 week). In the inhalation study of the C₆₀-exposed group, PCM evaluation showed significant changes in the C₆₀-exposed group only at 3 days after exposure; after 1 month, no significant changes were observed. The present study demonstrated that the pulmonary inflammation pattern after exposure to well-characterized C₆₀ via both intratracheal and inhalation instillation was slight and transient. These results support our previous studies that showed C₆₀ has no significant adverse effects in intratracheal and inhalation instillation studies.     

Comparative short-term inhalation toxicity of five organic diketopyrrolopyrrole pigments and two inorganic iron-oxide-based pigments

Diketopyrrolopyrroles (DPP) are a relatively new class of organic high-performance pigments. The present inhalation and particle characterization studies were performed to compare the effects of five DPP-based pigments (coarse and fine Pigment Red 254, coarse and fine meta-chloro DPP isomer and one form of mixed chlorinated DPP isomers) and compare it to coarse and fine inorganic Pigment Red 101. Wistar rats were exposed head-nose to atmospheres of the respective materials for 6?h/day on 5 consecutive days. Target concentrations were 30?mg/m³ as high dose for all compounds and selected based occupational exposure limits for respirable nuisance dust. Toxicity was determined after end of exposure and after 3-week recovery using broncho-alveolar lavage fluid (BALF) and microscopic examinations of the entire respiratory tract. Mixed chlorinated DPP isomers and coarse meta-chloro DPP isomer caused marginal changes in BALF, consisting of slight increases of polymorphonuclear neutrophils, and in case of coarse meta-chloro DPP increased MCP-1 and osteopontin levels. Mixed chlorinated DPP isomers, Pigment Red 254, and meta-chloro DPP caused pigment deposits and phagocytosis by alveolar macrophages, slight hypertrophy/hyperplasia of the bronchioles and alveolar ducts, but without evidence of inflammation. In contrast, only pigment deposition and pigment phagocytosis were observed after exposure to Pigment Red 101. All pigments were tolerated well and caused only marginal effects in BALF or no effects at all. Only minor effects were seen on the lung by microscopic examination. There was no evidence of systemic inflammation based on acute-phase protein levels in blood.     

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.     

Biopersistence of potassium hexatitanate in inhalation and intratracheal instillation studies

An inhalation study and an intratracheal instillation study were conducted to evaluate the biological effects of the new chemical, potassium hexatitanate (PH). For the inhalation study, Wistar male rats were exposed to PH for 6?h a day, 5 days a week for a period of 3 months. The mass median aerodynamic diameter of PH in the exposure chamber was 4.9?µm (1.8) and the mean concentration during the exposure was 2.3?±?0.1?mg/m³. After the 3-month inhalation period, rats were dissected at 3 days, 1 month, 3 months, 6 months, and 12 months. The initial PH burden was 0.17?±?0.03?mg/lung, and this decreased exponentially up to 6 months after inhalation. After 6 months, the rate at which the burden decreased slowed. The biological halftime up to 6 months after exposure was 2.3 months. No difference was found in the dimension of PH fibers in the lung during the observation period and the histopathological examination found no remarkable inflammation or fibrosis. For the intratracheal instillation study, the rats were given a single 2-mg dose of PH suspended in a 0.4?ml saline solution. The geometric mean diameter was 4.3?µm (2.3). After instillation, the rats were dissected at 3 days to 12 months. The PH burden in the lungs decreased exponentially and the biological halftime was 3.1 months. The results of the dimension of PH and histopathological findings were the same as those for the inhalation study. These data suggest that the toxicity of PH in the lung is low in these doses.     

A Comparison of the Inflammatory Response of the Lung to Inhaled versus Instilled Particles in F344 Rats

The potential pulmonary toxicity of poorly soluble airbornedusts generated in industrial and environmental settings isoften evaluated by inhalation studies in rodents. Studies usingintra tracheal instillation of particles have been suggestedas a less expensive alternative. We conducted a study to comparethe inflammatory response of the lung to instilled versus inhaledparticles. In one study, female F344/N rats, 11/13 weeks ofage, were exposed for 6 hr/day, 5 days/week for 4 weeks by inhalationto O, 0.1, 1.0, or 10 mg/m³ of either ß-quartz (toxicparticle) or TiO² (relatively low toxicity particle) and thelung burdens were determined at 1 week after the end of theexposure. The lungs were evaluated by analysis of bronchoalveolarlavage fluid (BALF) at 1, 8, and 24 weeks after the end of theexposure and by histopathology at 24 weeks. In a second study,rats were exposed by instillation to the lung burdens presentin the preceding study at 1 week after the inhalation exposure,and the rats were evaluated in the same manner as in the inhalationstudy. In general, the degree of alveolitis, as evaluated byhistopathology and BALF analysis, was similar by the two methodsof exposure. With lung burdens up to 750 µ/g, lung, theTi0² elicited no changes in BALF parameters at any time by eithermethod of exposure, nor was any histopathology observed. TheBALF changes elicited by -quartz were of approximately the samemagnitude and followed the same time course by either exposuremethod with the lowest dose delivered to the lung by eithermethod being a "no-effect" dose. At the highest dose, microgranulomaswere observed in bronchial-associated lymphoid tissue (BALT)in both sets of rats. However, the highest inhalation exposureinduced pleural granulomatous lesions that were not observedin the animals instilled with -quartz. The results indicatethat the relative potentials of the two materials to producebronchoalveolitis and granulomatous lesions in BALT could beappropriately evaluated using either intratracheal or inhalationexposures.     

Thirteen-week study of toxicity of fiber-like multi-walled carbon nanotubes with whole-body inhalation exposure in rats

Abstract

Cancer development due to fiber-like straight type of multi-walled carbon nanotubes (MWCNTs) has raised concerns for human safety because of its shape similar to asbestos. To set concentrations of MWCNT for a rat carcinogenicity study, we conducted a 13-week whole body inhalation study. F344 male and female rats, 6-week-old at the commencement of the study, were exposed by whole-body inhalation to MWCNT at concentrations of 0, 0.2, 1 and 5?mg/m³ with a generation and exposure system utilizing the cyclone sieve method. Measured concentrations in the exposure chambers were 0.20?±?0.02, 1.01?±?0.11 and 5.02?±?0.25?mg/m³ for 13 weeks. The MMAD (GSD) of MWCNT were 1.4–1.6?μm (2.3–3.0), and mean width and length were 94.1–98.0?nm and 5.53–6.19?μm, respectively, for each target concentration. Lung weights were increased 1.2-fold with 1?mg/m³ and 1.3-fold with 5?mg/m³ in both sexes compared to the controls. In the bronchoalveolar lavage fluid (BALF) analyses, inflammatory parameters were increased concentration-dependently in both sexes from 0.2?mg/m³. Granulomatous changes in the lung were induced at 1 and 5?mg/m³ in females and even at 0.2?mg/m³ in males. Focal fibrosis of the alveolar wall was observed in both sexes at 1?mg/m³ or higher. Inflammatory infiltration in the visceral pleural and subpleural areas was induced only at 5?mg/m³. In conclusion, we determined 0.2?mg/m³ as the low-observed-adverse-effect level (LOAEL) for respiratory tract toxicity in the present inhalation exposure study of rats.     

Inhalation toxicity study of disk-shaped potassium octatitanate particles (Terracess TF) in rats following 90 days of aerosol exposure

Since fibrous particles such as asbestos and some man-made fibers (MMF) have been known to produce carcinogenic or fibrogenic effects, disk-shaped potassium octatitanate (POT) particles (trade name: Terracess TF) were manufactured as nonfibrous particles. A 90-day inhalation toxicity study of Terracess TF was performed to evaluate comparative inhalation toxicity of the disk shape with a fibrous shape that was previously evaluated. Four groups of 20 male and 15 female rats each were exposed to Terracess TF aerosols at concentrations of 0, 2, 10, or 50?mg/m³ for 90 days. Ten male and 10 female rats per group were sacrificed at 90 days of exposure. After 90 days of exposure, 5 male rats per group were sacrificed at 3 wk of recovery period and 4–5 male rats per group or 5 female rats per group were sacrificed at 15?wk of recovery for lung clearance and histopathology. The mass median aerodynamic equivalent diameter (MMAED) of the aerosols of test materials ranged from 2.5 to 2.9?μm. There were no test-substance-related adverse effects on clinical observations. At the end of the 90-day exposure, a slight increase in lung-to-body weight ratios was observed at 50?mg/m³ in male but not in female rats. However, lung weights were within normal limits after 3- or 15-wk recovery periods. Microscopically, inhaled Terracess TF particles were mostly phagocytized by free alveolar macrophages (AMs) in the alveolar airspaces and alveolar walls maintained normal structure at 2 and 10?mg/m³. At 50?mg/m³, some alveoli were distended and filled with aggregates of particle-laden AMs. The alveolar walls showed slight type II pneumocyte hyperplasia, but neither proliferative inflammation nor alveolar fibrosis was present at 50?mg/m³. The clearance half-times for Terracess TF were estimated to be in the order of 6 to 9?mo for the 50-mg/m³ group and 2 to 3?mo for the 10- and 2-mg/m³ groups. The lung responses and lung clearance rate were comparable to those of “nuisance” type dusts at these concentrations. Based on interpretation that aggregated particle-laden AMs in alveoli was considered to be an early histopathological sign of lung overloading, an effect level was considered to be 50?mg/m³ and no-observed-adverse-effect level (NOAEL) was 10?mg/m³. This experiment clearly demonstrated that particle morphology was considered to be an important factor to determine inhaled particle toxicity.     

In vitro and ex vivo toxicological testing of sildenafil-loaded solid lipid nanoparticles

Abstract

The aim of this study was to investigate the potential cytotoxicity of solid lipid nanoparticles (SLN) loaded with sildenafil. The SLNs were tested as a new drug delivery system (DDS) for the inhalable treatment of pulmonary hypertension in human lungs. Solubility of sildenafil in SLN lipid matrix (30:70 phospholipid:triglyceride) was determined to 1% sildenafil base and 0.1% sildenafil citrate, respectively. Sildenafil-loaded SLN with particle size of approximately 180?nm and monomodal particle size distribution were successfully manufactured using a novel microchannel homogenization method and were stable up to three months. Sildenafil-loaded SLN were then used in in vitro and ex vivo models representing lung and heart tissue. For in vitro models, human alveolar epithelial cell line (A459) and mouse heart endothelium cell line (MHEC5-T) were used. For ex vivo models, rat precision cut lung slices (PCLS) and rat heart slices (PCHS) were used. All the models were treated with plain SLN and sildenafil-loaded SLN in a concentration range of 0–5000?µg/ml of lipid matrix. The toxicity was evaluated in vitro and ex vivo by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Median lethal dose 50% (LD₅₀) values for A549 cells and PCLS were found to be in the range of 1200–1900?µg/ml while for MHEC5-T cells and precision cut heart slices values were found between 1500 and 2800?µg/ml. PCHS showed slightly higher LD₅₀ values in comparison to PCLS. Considering the toxicological aspects, sildenafil-loaded SLN could have potential in the treatment of pulmonary hypertension via inhalation route.     

Biopersistence of potassium hexatitanate in inhalation and intratracheal instillation studies

An inhalation study and an intratracheal instillation study were conducted to evaluate the biological effects of the new chemical, potassium hexatitanate (PH). For the inhalation study, Wistar male rats were exposed to PH for 6?h a day, 5 days a week for a period of 3 months. The mass median aerodynamic diameter of PH in the exposure chamber was 4.9?μm (1.8) and the mean concentration during the exposure was 2.3?±?0.1?mg/m(3). After the 3-month inhalation period, rats were dissected at 3 days, 1 month, 3 months, 6 months, and 12 months. The initial PH burden was 0.17?±?0.03?mg/lung, and this decreased exponentially up to 6 months after inhalation. After 6 months, the rate at which the burden decreased slowed. The biological halftime up to 6 months after exposure was 2.3 months. No difference was found in the dimension of PH fibers in the lung during the observation period and the histopathological examination found no remarkable inflammation or fibrosis. For the intratracheal instillation study, the rats were given a single 2-mg dose of PH suspended in a 0.4?ml saline solution. The geometric mean diameter was 4.3?μm (2.3). After instillation, the rats were dissected at 3 days to 12 months. The PH burden in the lungs decreased exponentially and the biological halftime was 3.1 months. The results of the dimension of PH and histopathological findings were the same as those for the inhalation study. These data suggest that the toxicity of PH in the lung is low in these doses.     

Toxicological assessment of Ricinus communis Linn root extracts

Ricinus communis Linn (Euphorbiaceae) plant parts are claimed to be used as carminative, asthma, bronchitis, leprosy, anti-inflammatory, cathartic, and aphrodisiac. The toxicological study was carried out in the root part of the plant. The collected root was extracted with methanol and water. The extracts were vacuum-dried to yield the respective aqueous (AE) and methanol (ME) extracts. Toxicological assessment sought to determine the safety of Ricinus communis root extracts. The extracts were evaluated in the acute toxicity study (OECD-423 guidelines) and 90 days repeated dose toxicological assessment in Wistar albino rats. The acute oral toxicity of the aqueous (AE) and methanol (ME) extracts did not produce any toxic symptoms or mortality at the dose level of 2000?mg/kg in rats. In the 90 days (sub-chronic toxicity) repeated dose toxicity study the extracts (AE and ME) were administered 1000?mg/kg daily through oral route. The sub-chronic toxicity study demonstrated no significant changes in body weight, food, and water intake. Hematology parameters RBC, WBC, DLC, Hb, blood clotting time, and the biochemical parameters glucose, blood urea nitrogen, creatinine, total cholesterol, total protein, total bilirubin AST, ALT, and ALP were estimated. Histopathology observation of the major vital organs (liver, kidney, heart, spleen, lungs, ovary, testis, and brain) were tested. The hematology, biochemical and histopathology evaluations did not show any adverse effects in any of the organs tested. These results demonstrate the non-toxic nature of the root extracts AE and ME can be used for long-term usage in clinical practice.     

Assessment of inhaled acute ammonia-induced lung injury in rats

This study examined acute toxicity and lung injury following inhalation exposure to ammonia. Male Sprague-Dawley rats (300–350?g) were exposed to 9000, 20?000, 23?000, 26?000, 30?000 or 35?000?ppm of ammonia for 20?min in a custom head-out exposure system. The exposure atmosphere, which attained steady state within 3?min for all ammonia concentrations, was monitored and verified using a Fourier transform infrared spectroscopy (FTIR) gas analyzer. Animals exposed to ammonia resulted in dose-dependent increases in observed signs of intoxication, including increased chewing and licking, ocular irritation, salivation, lacrimation, oronasal secretion and labored breathing. The LCt₅₀ of ammonia within this head-out inhalation exposure model was determined by probit analysis to be 23?672?ppm (16?489?mg/m³) for the 20?min exposure in male rats. Exposure to 20?000 or 23?000?ppm of ammonia resulted in significant body weight loss 24-h post-exposure. Lung edema increased in all ammonia-exposed animal groups and was significant following exposure to 9000?ppm. Bronchoalveolar fluid (BALF) protein concentrations significantly increased following exposure to 20?000 or 23?000?ppm of ammonia in comparison to controls. BAL cell (BALC) death and total cell counts increased in animals exposed to 20?000 or 23?000?ppm of ammonia in comparison to controls. Differential cell counts of white blood cells, neutrophils and platelets from blood and BALF were significantly increased following exposure to 23?000?ppm of ammonia. The following studies describe the validation of a head-out inhalation exposure model for the determination of acute ammonia-induced toxicity; this model will be used for the development and evaluation of potential therapies that provide protection against respiratory and systemic toxicological effects.