Alison Elder

Translocation of Inhaled Ultrafine Particles to the Brain

Ultrafine particles (UFP, particles <100 nm) are ubiquitous in ambient urban and indoor air from multiple sources and may contribute to adverse respiratory and cardiovascular effects of particulate matter (PM). Depending on their particle size, inhaled UFP are efficiently deposited in nasal, tracheobronchial, and alveolar regions due to diffusion. Our previous rat studies have shown that UFP can translocate to interstitial sites in the respiratory tract as well as to extrapulmonary organs such as liver within 4 to 24 h postexposure. There were also indications that the olfactory bulb of the brain was targeted. Our objective in this follow-up study, therefore, was to determine whether translocation of inhaled ultrafine solid particles to regions of the brain takes place, hypothesizing that UFP depositing on the olfactory mucosa of the nasal region will translocate along the olfactory nerve into the olfactory bulb. This should result in significant increases in that region on the days following the exposure as opposed to other areas of the central nervous system (CNS). We generated ultrafine elemental 13C particles (CMD = 36 nm; GSD = 1.66) from [13C] graphite rods by electric spark discharge in an argon atmosphere at a concentration of 160 μg/m3. Rats were exposed for 6 h, and lungs, cerebrum, cerebellum and olfactory bulbs were removed 1, 3, 5, and 7 days after exposure. 13C concentrations were determined by isotope ratio mass spectroscopy and compared to background 13C levels of sham-exposed controls (day 0). The background corrected pulmonary 13C added as ultrafine 13C particles on day 1 postexposure was 1.34 μg/lung. Lung 13C concentration decreased from 1.39 μg/g (day 1) to 0.59 μg/g by 7 days postexposure. There was a significant and persistent increase in added 13C in the olfactory bulb of 0.35 μg/g on day 1, which increased to 0.43 μg/g by day 7. Day 1 13C concentrations of cerebrum and cerebellum were also significantly increased but the increase was inconsistent, significant only on one additional day of the postexposure period, possibly reflecting translocation across the blood–brain barrier in certain brain regions. The increases in olfactory bulbs are consistent with earlier studies in nonhuman primates and rodents that demonstrated that intranasally instilled solid UFP translocate along axons of the olfactory nerve into the CNS. We conclude from our study that the CNS can be targeted by airborne solid ultrafine particles and that the most likely mechanism is from deposits on the olfactory mucosa of the nasopharyngeal region of the respiratory tract and subsequent translocation via the olfactory nerve. Depending on particle size, >50% of inhaled UFP can be depositing in the nasopharyngeal region during nasal breathing. Preliminary estimates from the present results show that ∼20% of the UFP deposited on the olfactory mucosa of the rat can be translocated to the olfactory bulb. Such neuronal translocation constitutes an additional not generally recognized clearance pathway for inhaled solid UFP, whose significance for humans, however, still needs to be established. It could provide a portal of entry into the CNS for solid UFP, circumventing the tight blood–brain barrier. Whether this translocation of inhaled UFP can cause CNS effects needs to be determined in future studies.

EXTRAPULMONARY TRANSLOCATION OF ULTRAFINE CARBON PARTICLES FOLLOWING WHOLE-BODY INHALATION EXPOSURE OF RATS

Studies with intravenously injected ultrafine particles have shown that the liver is the major organ of their uptake from the blood circulation. Measuring translocation of inhaled ultrafine particles to extrapulmonary organs via the blood compartment is hampered by methodological difficulties (i.e., label may come off, partial solubilization) and analytical limitations (measurement of very small amounts). The objective of our pilot study was to determine whether ultrafine elemental carbon particles translocate to the liver and other extrapulmonary organs following inhalation as singlet particles by rats. We generated ultrafine 13 C particles as an aerosol with count median diameters (CMDs) of 20-29 nm (GSD 1.7) using electric spark discharge of 13 C graphite electrodes in argon. Nine Fischer 344 rats were exposed to these particles for 6 h. in whole-body inhalation chambers at concentrations of 180 and 80 w g/m 3 ; 3 animals each were killed at 0.5, 18, and 24 h postexposure. Six unexposed rats served as controls. Lung lobes, liver, heart, brain, olfactory bulb, and kidney were excised, homogenized, and freeze-dried for analysis of the added 13 C by isotope ratio mass spectrometry. Organic 13 C was not detected in the 13 C particles. The 13 C retained in the lung at 0.5 h postexposure was about 70% less than predicted by rat deposition models for ultrafine particles, and did not change significantly during the 24-h postexposure period. Normalized to exposure concentration, the added 13 C per gram of lung on average in the postexposure period was ~9 ng/g organ/ w g/m 3 . Significant amounts of 13 C had accumulated in the liver by 0.5 h postinhalation only at the high exposure concentration, whereas by 18 and 24 h postexposure the 13 C amount of the livers of all exposed rats was about fivefold greater than the 13 C burden retained in the lung. No significant increase in 13 C was detected in the other organs which were examined. These results demonstrate effective translocation of ultrafine elemental carbon particles to the liver by 1 d after inhalation exposure. Translocation pathways include direct input into the blood compartment from ultrafine carbon particles deposited throughout the respiratory tract. However, since predictive particle deposition models indicate that respiratory tract deposits alone may not fully account for the hepatic 13 C burden, input from ultrafine particles present in the GI tract needs to be considered as well. Such translocation to blood and extrapulmonary tissues may well be different between ultrafine carbon and other insoluble (metal) ultrafine particles.

Translocation of inhaled ultrafine manganese oxide particles to the central nervous system

Background Studies in monkeys with intranasally instilled gold ultrafine particles (UFPs; < 100 nm) and in rats with inhaled carbon UFPs suggested that solid UFPs deposited in the nose travel along the olfactory nerve to the olfactory bulb. Methods To determine if olfactory translocation occurs for other solid metal UFPs and assess potential health effects, we exposed groups of rats to manganese (Mn) oxide UFPs (30 nm; ~ 500 μg/m3) with either both nostrils patent or the right nostril occluded. We analyzed Mn in lung, liver, olfactory bulb, and other brain regions, and we performed gene and protein analyses. Results After 12 days of exposure with both nostrils patent, Mn concentrations in the olfactory bulb increased 3.5-fold, whereas lung Mn concentrations doubled; there were also increases in striatum, frontal cortex, and cerebellum. Lung lavage analysis showed no indications of lung inflammation, whereas increases in olfactory bulb tumor necrosis factor-α mRNA (~ 8-fold) and protein (~ 30-fold) were found after 11 days of exposure and, to a lesser degree, in other brain regions with increased Mn levels. Macrophage inflammatory protein-2, glial fibrillary acidic protein, and neuronal cell adhesion molecule mRNA were also increased in olfactory bulb. With the right nostril occluded for a 2-day exposure, Mn accumulated only in the left olfactory bulb. Solubilization of the Mn oxide UFPs was < 1.5% per day. Conclusions We conclude that the olfactory neuronal pathway is efficient for translocating inhaled Mn oxide as solid UFPs to the central nervous system and that this can result in inflammatory changes. We suggest that despite differences between human and rodent olfactory systems, this pathway is relevant in humans.

Does nanoparticle activity depend upon size and crystal phase

A method to investigate the dependence of the physicochemical properties of nanoparticles (e.g., size, surface area and crystal phase) on their oxidant generating capacity is proposed and demonstrated for TiO2 nanoparticles. Gas phase synthesis methods that allow for strict control of size and crystal phase were used to prepare TiO2 nanoparticles. The reactive oxygen species (ROS) generating capacity of these particles was then measured. The size dependent ROS activity was established using TiO2 nanoparticles of nine different sizes (4–195 nm) but the same crystal phase. For a fixed total surface area, an S-shaped curve for ROS generation per unit surface area was observed as a function of particle size. The highest ROS activity per unit area was observed for 30 nm particles, and observed to be constant above 30 nm. There was a decrease in activity per unit area as size decreased from 30–10 nm; and again constant for particles smaller than 10 nm. The correlation between crystal phase and oxidant capacity was established using TiO2 nanoparticles of 11 different crystal phase combinations but similar size. The ability of different crystal phases of TiO2 nanoparticles to generate ROS was highest for amorphous, followed by anatase, and then anatase/rutile mixtures, and lowest for rutile samples. Based on evaluation of the entire dataset, important dose metrics for ROS generation are established. The implications of these ROS studies on biological and toxicological studies using nanomaterials are discussed.

Correlating Physico-Chemical with Toxicological Properties of Nanoparticles: The Present and the Future

Nanotoxicology is still a new discipline. In this Perspective, both its origins and its future trends are discussed. In particular, we note several issues we consider important for publications in this field.

Concept of Assessing Nanoparticle Hazards Considering Nanoparticle Dosemetric and Chemical/Biological Response Metrics

Engineered nanoparticles (NP) are being developed and incorporated in a number of commercial products, raising the potential of human exposure during manufacture, use, and disposal. Although data concerning the potential toxicity of some NP have been reported, validated simple assays are lacking for predicting their in vivo toxicity. The aim of this study was to evaluate new response metrics based on chemical and biological activity of NP for screening assays that can be used to predict NP toxicity in vivo. Two cell-free and two cell-based assays were evaluated for their power in predicting in vivo toxicity of eight distinct particle types with widely differing physicochemical characteristics. The cell-free systems comprised fluorescence- and electron spin resonance-based assays of oxidant activity. The cell-based systems also used electron spin resonance (ESR) as well as luciferase reporter activity to rank the different particle types in comparison to benchmark particles of low and high activity. In vivo experiments evaluated acute pulmonary inflammatory responses in rats. Endpoints in all assays were related to oxidative stress and responses were expressed per unit NP surface area to compare the results of different assays. Results indicated that NP are capable of producing reactive species, which in biological systems lead to oxidative stress. Copper NP had the greatest activity in all assays, while TiO2 and gold NP generally were the least reactive. Differences in the ranking of NP activity among the assays were found when comparisons were based on measured responses. However, expressing the chemical (cell-free) and biological (cells; in vivo) activity per unit particle surface area showed that all in vitro assays correlated significantly with in vivo results, with the cellular assays correlating the best. Data from this study indicate that it is possible to predict acute in vivo inflammatory potential of NP with cell-free and cellular assays by using NP surface area-based dose and response metrics, but that a cellular component is required to achieve a higher degree of predictive power.

The outdoor air pollution and brain health workshop

Accumulating evidence suggests that outdoor air pollution may have a significant impact on central nervous system (CNS) health and disease. To address this issue, the National Institute of Environmental Health Sciences/National Institute of Health convened a panel of research scientists that was assigned the task of identifying research gaps and priority goals essential for advancing this growing field and addressing an emerging human health concern. Here, we review recent findings that have established the effects of inhaled air pollutants in the brain, explore the potential mechanisms driving these phenomena, and discuss the recommended research priorities/approaches that were identified by the panel.

Validation of an LDH assay for assessing nanoparticle toxicity.

Studies showed that certain cytotoxicity assays were not suitable for assessing nanoparticle (NP) toxicity. We evaluated a lactate dehydrogenase (LDH) assay for assessing copper (Cu-40, 40nm), silver (Ag-35, 35nm; Ag-40, 40nm), and titanium dioxide (TiO(2)-25, 25nm) NPs by examining their potential to inactivate LDH and interference with β-nicotinamide adenine dinucleotide (NADH), a substrate for the assay. We also performed a dissolution assay for some of the NPs. We found that the copper NPs, because of their high dissolution rate, could interfere with the LDH assay by inactivating LDH. Ag-35 could also inactivate LDH probably because of the carbon matrix used to cage the particles during synthesis. TiO(2)-25 NPs were found to adsorb LDH molecules. In conclusion, NP interference with the LDH assay depends on the type of NPs and the suitability of the assay for assessing NP toxicity should be examined case by case.

Interlaboratory evaluation of in vitro cytotoxicity and inflammatory responses to engineered nanomaterials: the NIEHS Nano GO Consortium.

Background: Differences in interlaboratory research protocols contribute to the conflicting data in the literature regarding engineered nanomaterial (ENM) bioactivity. Objectives: Grantees of a National Institute of Health Sciences (NIEHS)-funded consortium program performed two phases of in vitro testing with selected ENMs in an effort to identify and minimize sources of variability. Methods: Consortium program participants (CPPs) conducted ENM bioactivity evaluations on zinc oxide (ZnO), three forms of titanium dioxide (TiO2), and three forms of multiwalled carbon nanotubes (MWCNTs). In addition, CPPs performed bioassays using three mammalian cell lines (BEAS-2B, RLE-6TN, and THP-1) selected in order to cover two different species (rat and human), two different lung epithelial cells (alveolar type II and bronchial epithelial cells), and two different cell types (epithelial cells and macrophages). CPPs also measured cytotoxicity in all cell types while measuring inflammasome activation [interleukin-1β (IL-1β) release] using only THP-1 cells. Results: The overall in vitro toxicity profiles of ENM were as follows: ZnO was cytotoxic to all cell types at ≥ 50 μg/mL, but did not induce IL-1β. TiO2 was not cytotoxic except for the nanobelt form, which was cytotoxic and induced significant IL-1β production in THP-1 cells. MWCNTs did not produce cytotoxicity, but stimulated lower levels of IL-1β production in THP-1 cells, with the original MWCNT producing the most IL-1β. Conclusions: The results provide justification for the inclusion of mechanism-linked bioactivity assays along with traditional cytotoxicity assays for in vitro screening. In addition, the results suggest that conducting studies with multiple relevant cell types to avoid false-negative outcomes is critical for accurate evaluation of ENM bioactivity.

Interlaboratory Evaluation of Rodent Pulmonary Responses to Engineered Nanomaterials: The NIEHS Nano GO Consortium

Background: Engineered nanomaterials (ENMs) have potential benefits, but they also present safety concerns for human health. Interlaboratory studies in rodents using standardized protocols are needed to assess ENM toxicity. Methods: Four laboratories evaluated lung responses in C57BL/6 mice to ENMs delivered by oropharyngeal aspiration (OPA), and three labs evaluated Sprague-Dawley (SD) or Fisher 344 (F344) rats following intratracheal instillation (IT). ENMs tested included three forms of titanium dioxide (TiO2) [anatase/rutile spheres (TiO2-P25), anatase spheres (TiO2-A), and anatase nanobelts (TiO2-NBs)] and three forms of multiwalled carbon nanotubes (MWCNTs) [original (O), purified (P), and carboxylic acid “functionalized” (F)]. One day after treatment, bronchoalveolar lavage fluid was collected to determine differential cell counts, lactate dehydrogenase (LDH), and protein. Lungs were fixed for histopathology. Responses were also examined at 7 days (TiO2 forms) and 21 days (MWCNTs) after treatment. Results: TiO2-A, TiO2-P25, and TiO2-NB caused significant neutrophilia in mice at 1 day in three of four labs. TiO2-NB caused neutrophilia in rats at 1 day in two of three labs, and TiO2-P25 and TiO2-A had no significant effect in any of the labs. Inflammation induced by TiO2 in mice and rats resolved by day 7. All MWCNT types caused neutrophilia at 1 day in three of four mouse labs and in all rat labs. Three of four labs observed similar histopathology to O-MWCNTs and TiO2-NBs in mice. Conclusions: ENMs produced similar patterns of neutrophilia and pathology in rats and mice. Although interlaboratory variability was found in the degree of neutrophilia caused by the three types of TiO2 nanoparticles, similar findings of relative potency for the three types of MWCNTs were found across all laboratories, thus providing greater confidence in these interlaboratory comparisons.