David G. Frazer

Nanoparticle inhalation augments particle-dependent systemic microvascular dysfunction.

BackgroundWe have shown that pulmonary exposure to fine particulate matter (PM) impairs endothelium dependent dilation in systemic arterioles. Ultrafine PM has been suggested to be inherently more toxic by virtue of its increased surface area. The purpose of this study was to determine if ultrafine PM (or nanoparticle) inhalation produces greater microvascular dysfunction than fine PM. Rats were exposed to fine or ultrafine TiO2 aerosols (primary particle diameters of ~1 μm and ~21 nm, respectively) at concentrations which do not alter bronchoalveolar lavage markers of pulmonary inflammation or lung damage.ResultsBy histopathologic evaluation, no significant inflammatory changes were seen in the lung. However, particle-containing macrophages were frequently seen in intimate contact with the alveolar wall. The spinotrapezius muscle was prepared for in vivo microscopy 24 hours after inhalation exposures. Intraluminal infusion of the Ca2+ ionophore A23187 was used to evaluate endothelium-dependent arteriolar dilation. In control rats, A23187 infusion produced dose-dependent arteriolar dilations. In rats exposed to fine TiO2, A23187 infusion elicited vasodilations that were blunted in proportion to pulmonary particle deposition. In rats exposed to ultrafine TiO2, A23187 infusion produced arteriolar constrictions or significantly impaired vasodilator responses as compared to the responses observed in control rats or those exposed to a similar pulmonary load of fine particles.ConclusionThese observations suggest that at equivalent pulmonary loads, as compared to fine TiO2, ultrafine TiO2 inhalation produces greater remote microvascular dysfunction.

Acute pulmonary dose–responses to inhaled multi-walled carbon nanotubes

Abstract This study investigated the in vivo pulmonary toxicity of inhaled multi-walled carbon nanotubes (MWCNT). Mice-inhaled aerosolized MWCNT (10 mg/m3, 5 h/day) for 2, 4, 8 or 12 days. MWCNT lung burden was linearly related to exposure duration. MWCNT-induced pulmonary inflammation was assessed by determining whole lung lavage (WLL) polymorphonuclear leukocytes (PMN). Lung cytotoxicity was assessed by WLL fluid LDH activities. WLL fluid albumin concentrations were determined as a marker of alveolar air–blood barrier integrity. These parameters significantly increased in MWCNT-exposed mice versus controls and were dose-dependent. Histopathologic alterations identified in the lung included (1) bronciolocentric inflammation, (2) bronchiolar epithelial hyperplasia and hypertrophy, (3) fibrosis, (4) vascular changes and (5) rare pleural penetration. MWCNT translocated to the lymph node where the deep paracortex was expanded after 8 or 12 days. Acute inhalation of MWCNT induced dose-dependent pulmonary inflammation and damage with rapid development of pulmonary fibrosis, and also demonstrated that MWCNT can reach the pleura after inhalation exposure.

Pulmonary Nanoparticle Exposure Disrupts Systemic Microvascular Nitric Oxide Signaling

We have shown that pulmonary nanoparticle exposure impairs endothelium dependent dilation in systemic arterioles. However, the mechanism(s) through which this effect occurs is/are unclear. The purpose of this study was to identify alterations in the production of reactive species and endogenous nitric oxide (NO) after nanoparticle exposure, and determine the relative contribution of hemoproteins and oxidative enzymes in this process. Sprague-Dawley rats were exposed to fine TiO2 (primary particle diameter approximately 1 microm) and TiO2 nanoparticles (primary particle diameter approximately 21 nm) via aerosol inhalation at depositions of 4-90 microg per rat. As in previous intravital experiments in the spinotrapezius muscle, dose-dependent arteriolar dilations were produced by intraluminal infusions of the calcium ionophore A23187. Nanoparticle exposure robustly attenuated these endothelium-dependent responses. However, this attenuation was not due to altered microvascular smooth muscle NO sensitivity because nanoparticle exposure did not alter arteriolar dilations in response to local sodium nitroprusside iontophoresis. Nanoparticle exposure significantly increased microvascular oxidative stress by approximately 60%, and also elevated nitrosative stress fourfold. These reactive stresses coincided with a decreased NO production in a particle deposition dose-dependent manner. Radical scavenging, or inhibition of either myeloperoxidase or nicotinamide adenine dinucleotide phosphate oxidase (reduced) oxidase partially restored NO production as well as normal microvascular function. These results indicate that in conjunction with microvascular dysfunction, nanoparticle exposure also decreases NO bioavailability through at least two functionally distinct mechanisms that may mutually increase local reactive species.

Molecular mechanism of tumor necrosis factor-alpha production in 1-->3-beta-glucan (zymosan)-activated macrophages.

The molecular details of 1→3-β-glucans, a fungal cell wall component, induced inflammatory responses are not well understood. In the present study, we conducted a systematic analysis of the molecular events leading to tumor necrosis factor (TNF)-α production after glucan stimulation of macrophages. We demonstrated that activation of nuclear factor κB (NF-κB) is essential in zymosan A (a source of 1→3-β-glucans)-induced TNF-α production in macrophages (RAW264.7 cells). Zymosan A-induced TNF-α protein production was associated with an increase in the TNF-α gene promoter activity. Activation of the TNF-α gene promoter was dependent on activation of NF-κB. Time course studies indicated that DNA binding activity of NF-κB preceded TNF-α promoter activity. Inhibition of NF-κB activation led to a dramatic reduction in both TNF-α promoter activity and TNF-α protein production in the response to zymosan A. Mutation of a major NF-κB binding site (κ3) in the gene promoter resulted in a significant decrease in the induction of the gene promoter by zymosan A, while mutation of Egr or CRE sites failed to inhibit the response to zymosan. Together, these results strongly suggest that NF-κB is involved in signal transduction of 1→3-β-glucans-induced TNF-α expression.

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.

Carbon nanotube dosimetry: from workplace exposure assessment to inhalation toxicology

BackgroundDosimetry for toxicology studies involving carbon nanotubes (CNT) is challenging because of a lack of detailed occupational exposure assessments. Therefore, exposure assessment findings, measuring the mass concentration of elemental carbon from personal breathing zone (PBZ) samples, from 8 U.S.-based multi-walled CNT (MWCNT) manufacturers and users were extrapolated to results of an inhalation study in mice.ResultsUpon analysis, an inhalable elemental carbon mass concentration arithmetic mean of 10.6 μg/m3 (geometric mean 4.21 μg/m3) was found among workers exposed to MWCNT. The concentration equates to a deposited dose of approximately 4.07 μg/d in a human, equivalent to 2 ng/d in the mouse. For MWCNT inhalation, mice were exposed for 19 d with daily depositions of 1970 ng (equivalent to 1000 d of a human exposure; cumulative 76 yr), 197 ng (100 d; 7.6 yr), and 19.7 ng (10 d; 0.76 yr) and harvested at 0, 3, 28, and 84 d post-exposure to assess pulmonary toxicity. The high dose showed cytotoxicity and inflammation that persisted through 84 d after exposure. The middle dose had no polymorphonuclear cell influx with transient cytotoxicity. The low dose was associated with a low grade inflammatory response measured by changes in mRNA expression. Increased inflammatory proteins were present in the lavage fluid at the high and middle dose through 28 d post-exposure. Pathology, including epithelial hyperplasia and peribronchiolar inflammation, was only noted at the high dose.ConclusionThese findings showed a limited pulmonary inflammatory potential of MWCNT at levels corresponding to the average inhalable elemental carbon concentrations observed in U.S.-based CNT facilities and estimates suggest considerable years of exposure are necessary for significant pathology to occur at that level.

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.

Design, Construction, and Characterization of a Novel Robotic Welding Fume Generator and Inhalation Exposure System for Laboratory Animals

Respiratory effects observed in welders have included lung function changes, metal fume fever, bronchitis, and a possible increase in the incidence of lung cancer. Many questions remain unanswered regarding the causality and possible underlying mechanisms associated with the potential toxic effects of welding fume inhalation. The objective of the present study was to construct a completely automated, computer-controlled welding fume generation and inhalation exposure system to simulate real workplace exposures. The system comprised a programmable six-axis robotic welding arm, a water-cooled arc welding torch, and a wire feeder that supplied the wire to the torch at a programmed rate. For the initial studies, gas metal arc welding was performed using a stainless steel electrode. A flexible trunk was attached to the robotic arm of the welder and was used to collect and transport fume from the vicinity of the arc to the animal exposure chamber. Undiluted fume concentrations consistently ranged from 90–150 mg/m3 in the animal chamber during welding. Temperature and humidity remained constant in the chamber during the welding operation. The welding particles were composed of (from highest to lowest concentration) iron, chromium, manganese, and nickel as measured by inductively coupled plasma atomic emission spectroscopy. Size distribution analysis indicated the mass median aerodynamic diameter of the generated particles to be approximately 0.24 μm with a geometric standard deviation (σ g ) of 1.39. As determined by transmission and scanning electron microscopy, the generated aerosols were mostly arranged as chain-like agglomerates of primary particles. Characterization of the laboratory-generated welding aerosol has indicated that particle morphology, size, and chemical composition are comparable to stainless steel welding fume generated in other studies. With the development of this novel system, it will be possible to establish an animal model using controlled welding exposures from automated gas metal arc and flux-cored arc welding processes to investigate how welding fumes affect health.

Impairment of Coronary Arteriolar Endothelium-Dependent Dilation after Multi-Walled Carbon Nanotube Inhalation: A Time-Course Study

Engineered nanomaterials have been developed for widespread applications due to many highly unique and desirable characteristics. The purpose of this study was to assess pulmonary inflammation and subepicardial arteriolar reactivity in response to multi-walled carbon nanotube (MWCNT) inhalation and evaluate the time course of vascular alterations. Rats were exposed to MWCNT aerosols producing pulmonary deposition. Pulmonary inflammation via bronchoalveolar lavage and MWCNT translocation from the lungs to systemic organs was evident 24 h post-inhalation. Coronary arterioles were evaluated 24–168 h post-exposure to determine microvascular response to changes in transmural pressure, endothelium-dependent and -independent reactivity. Myogenic responsiveness, vascular smooth muscle reactivity to nitric oxide, and α-adrenergic responses all remained intact. However, a severe impact on endothelium-dependent dilation was observed within 24 h after MWCNT inhalation, a condition which improved, but did not fully return to control after 168 h. In conclusion, results indicate that MWCNT inhalation not only leads to pulmonary inflammation and cytotoxicity at low lung burdens, but also a low level of particle translocation to systemic organs. MWCNT inhalation also leads to impairments of endothelium-dependent dilation in the coronary microcirculation within 24 h, a condition which does not fully dissipate within 168 h. The innovations within the field of nanotechnology, while exciting and novel, can only reach their full potential if toxicity is first properly assessed.

Comparison of stainless and mild steel welding fumes in generation of reactive oxygen species

BackgroundWelding fumes consist of a wide range of complex metal oxide particles which can be deposited in all regions of the respiratory tract. The welding aerosol is not homogeneous and is generated mostly from the electrode/wire. Over 390,000 welders were reported in the U.S. in 2008 while over 1 million full-time welders were working worldwide. Many health effects are presently under investigation from exposure to welding fumes. Welding fume pulmonary effects have been associated with bronchitis, metal fume fever, cancer and functional changes in the lung. Our investigation focused on the generation of free radicals and reactive oxygen species from stainless and mild steel welding fumes generated by a gas metal arc robotic welder. An inhalation exposure chamber located at NIOSH was used to collect the welding fume particles.ResultsOur results show that hydroxyl radicals (.OH) were generated from reactions with H2O2 and after exposure to cells. Catalase reduced the generation of .OH from exposed cells indicating the involvement of H2O2. The welding fume suspension also showed the ability to cause lipid peroxidation, effect O2 consumption, induce H2O2 generation in cells, and cause DNA damage.ConclusionIncrease in oxidative damage observed in the cellular exposures correlated well with .OH generation in size and type of welding fumes, indicating the influence of metal type and transition state on radical production as well as associated damage. Our results demonstrate that both types of welding fumes are able to generate ROS and ROS-related damage over a range of particle sizes; however, the stainless steel fumes consistently showed a significantly higher reactivity and radical generation capacity. The chemical composition of the steel had a significant impact on the ROS generation capacity with the stainless steel containing Cr and Ni causing more damage than the mild steel. Our results suggest that welding fumes may cause acute lung injury. Since type of fume generated, particle size, and elapsed time after generation of the welding exposure are significant factors in radical generation and particle deposition these factors should be considered when developing protective strategies.