Thomas C. Donaghey

Olfactory uptake of manganese requires DMT1 and is enhanced by anemia

Manganese, an essential nutrient, can also elicit toxicity in the central nervous system (CNS). The route of exposure strongly influences the potential neurotoxicity of manganese‐containing compounds. Recent studies suggest that inhaled manganese can enter the rat brain through the olfactory system, but little is known about the molecular factors involved. Divalent metal transporter‐1 (DMT1) is the major transporter responsible for intestinal iron absorption and its expression is regulated by body iron status. To examine the potential role of this transporter in uptake of inhaled manganese, we studied the Belgrade rat, since these animals display significant defects in both iron and manganese metabolism due to a glycine‐to‐arginine substitution (G185R) in their DMT1 gene product. Absorption of intranasally instilled 54Mn was significantly reduced in Belgrade rats and was enhanced in iron‐deficient rats compared to iron‐sufficient controls. Immunohistochemical experiments revealed that DMT1 was localized to both the lumen microvilli and end feet of the sustentacular cells of the olfactory epithelium. Importantly, we found that DMT1 protein levels were increased in anemic rats. The apparentfunction of DMT1 in olfactory manganese absorption suggests that the neurotoxicity of the metal can be modified by iron status due to the iron‐responsive regulation of the transporter. Thompson, K., Molina, R. M., Donaghey, T., Schwob, J. E., Brain, J. D., Wessling‐Resnick, M. Olfactory uptake of manganese requires DMT1 and is enhanced by anemia. FASEB J. 21, 223–230 (2007)

Biomechanical effects of environmental and engineered particles on human airway smooth muscle cells

The past decade has seen significant increases in combustion-generated ambient particles, which contain a nanosized fraction (less than 100 nm), and even greater increases have occurred in engineered nanoparticles (NPs) propelled by the booming nanotechnology industry. Although inhalation of these particulates has become a public health concern, human health effects and mechanisms of action for NPs are not well understood. Focusing on the human airway smooth muscle cell, here we show that the cellular mechanical function is altered by particulate exposure in a manner that is dependent upon particle material, size and dose. We used Alamar Blue assay to measure cell viability and optical magnetic twisting cytometry to measure cell stiffness and agonist-induced contractility. The eight particle species fell into four categories, based on their respective effect on cell viability and on mechanical function. Cell viability was impaired and cell contractility was decreased by (i) zinc oxide (40–100 nm and less than 44 μm) and copper(II) oxide (less than 50 nm); cell contractility was decreased by (ii) fluorescent polystyrene spheres (40 nm), increased by (iii) welding fumes and unchanged by (iv) diesel exhaust particles, titanium dioxide (25 nm) and copper(II) oxide (less than 5 μm), although in none of these cases was cell viability impaired. Treatment with hydrogen peroxide up to 500 μM did not alter viability or cell mechanics, suggesting that the particle effects are unlikely to be mediated by particle-generated reactive oxygen species. Our results highlight the susceptibility of cellular mechanical function to particulate exposures and suggest that direct exposure of the airway smooth muscle cells to particulates may initiate or aggravate respiratory diseases.

Biokinetics and effects of barium sulfate nanoparticles

BackgroundNanoparticulate barium sulfate has potential novel applications and wide use in the polymer and paint industries. A short-term inhalation study on barium sulfate nanoparticles (BaSO4 NPs) was previously published [Part Fibre Toxicol 11:16, 2014]. We performed comprehensive biokinetic studies of 131BaSO4 NPs administered via different routes and of acute and subchronic pulmonary responses to instilled or inhaled BaSO4 in rats.MethodsWe compared the tissue distribution of 131Ba over 28 days after intratracheal (IT) instillation, and over 7 days after gavage and intravenous (IV) injection of 131BaSO4. Rats were exposed to 50 mg/m3 BaSO4 aerosol for 4 or 13 weeks (6 h/day, 5 consecutive days/week), and then gross and histopathologic, blood and bronchoalveolar lavage (BAL) fluid analyses were performed. BAL fluid from instilled rats was also analyzed.ResultsInhaled BaSO4 NPs showed no toxicity after 4-week exposure, but a slight neutrophil increase in BAL after 13-week exposure was observed. Lung burden of inhaled BaSO4 NPs after 4-week exposure (0.84 ± 0.18 mg/lung) decreased by 95% over 34 days. Instilled BaSO4 NPs caused dose-dependent inflammatory responses in the lungs. Instilled BaSO4 NPs (0.28 mg/lung) was cleared with a half-life of ≈ 9.6 days. Translocated 131Ba from the lungs was predominantly found in the bone (29%). Only 0.15% of gavaged dose was detected in all organs at 7 days. IV-injected 131BaSO4 NPs were predominantly localized in the liver, spleen, lungs and bone at 2 hours, but redistributed from the liver to bone over time. Fecal excretion was the dominant elimination pathway for all three routes of exposure.ConclusionsPulmonary exposure to instilled BaSO4 NPs caused dose-dependent lung injury and inflammation. Four-week and 13-week inhalation exposures to a high concentration (50 mg/m3) of BaSO4 NPs elicited minimal pulmonary response and no systemic effects. Instilled and inhaled BaSO4 NPs were cleared quickly yet resulted in higher tissue retention than when ingested. Particle dissolution is a likely mechanism. Injected BaSO4 NPs localized in the reticuloendothelial organs and redistributed to the bone over time. BaSO4 NP exhibited lower toxicity and biopersistence in the lungs compared to other poorly soluble NPs such as CeO2 and TiO2.

Biologic responses to nanomaterials depend on exposure, clearance, and material characteristics

Nanomaterials create challenges for the toxicology and risk assessment communities. While some nanomaterials are not new, such as colloids and the nano fractions of polydisperse aerosols such as welding fume, others are novel and unexplored. In addition to elucidating biologic responses, we also need to characterize human exposure, and to better understand the pharmacokinetics and translocation potential of nanomaterials. For example, do the smallest nanoparticles cross the air-blood barrier more readily, and can they enter cells via non-endocytic mechanisms? To what extent do nanofibers share the remarkable properties of asbestos fibers? Can carbon nanotubes find their way to the pleural space or not? More generally, we need to craft the laws of nanotoxicology. How do the size, shape, and surface chemistry of nanoparticles influence their anatomic fate and biologic consequences? Parallel to the rapid growth of nanotechnology must be a thoughtful development of the field of nanotoxicology.

Influence of DMT1 and iron status on inflammatory responses in the lung

Divalent metal transporter 1 (DMT1) is the major iron transporter responsible for duodenal dietary iron absorption and is required for erythropoiesis. Recent studies suggest that loss of DMT1 activity could be involved in metal-related lung injury, but little is known about the effects of iron status and DMT1 function on pulmonary inflammation. To better define the role of DMT1 and iron status in pulmonary inflammatory responses, we performed bronchoalveolar lavage (BAL) following intratracheal instillation of lipopolysaccharide (LPS) to the Belgrade rat, an animal model deficient in DMT1 function. In the basal state, the BAL fluid of Belgrade rats had more macrophages and higher lactate dehydrogenase, myeloperoxidase, albumin, and hemoglobin levels compared with heterozygote control rats. Following LPS instillation, the macrophage fraction relative to total BAL cell content and levels of albumin and IgM were increased in Belgrade rats compared with controls. In contrast, heterozygote Belgrade rats made anemic by diet-induced iron deficiency exhibited attenuated inflammatory responses to LPS. These combined results show that pulmonary inflammation can be modified by both DMT1 and iron status. Loss of DMT1 alters pulmonary responses necessary for lung homeostasis in the basal state and enhances LPS-induced inflammation and therefore would contribute to progression of lung injury.

Pulmonary responses to Stachybotrys chartarum and its toxins: mouse strain affects clearance and macrophage cytotoxicity.

We investigated differences in the pulmonary and systemic clearance of Stachybotrys chartarum spores in two strains of mice, BALB/c and C57BL/6J. To evaluate clearance, mice were intratracheally instilled with a suspension of radiolabeled S. chartarum spores or with unlabeled spores. The lungs of C57BL/6J mice showed more rapid spore clearance than the lungs of BALB/c mice, which correlated with increased levels of spore-associated radioactivity in the GI tracts of C57BL/6J as compared with BALB/c mice. To identify mechanisms responsible for mouse strain differences in spore clearance and previously described lung inflammatory responses, we exposed alveolar macrophages (AMs) lavaged from BALB/c and C57BL/6J mice to S. chartarum spores, S. chartarum spore toxin (SST), and satratoxin G (SG) in vitro. The S. chartarum spores were found to be highly toxic with most cells from either mouse strain being killed within 24 h when exposed to a spore:cell ratio of 1:75. The spores were more lethal to AMs from C57BL/6J than those from BALB/c mice. In mice, the SST elicited many of the same inflammatory responses as the spores in vivo, including AM recruitment, pulmonary hemorrhage, and cytokine production. Our data suggest that differences in pulmonary spore clearance may contribute to the differences in pulmonary responses to S. chartarum between BALB/c and C57BL/6J mice. Enhanced AM survival and subsequent macrophage-mediated inflammation may also contribute to the higher susceptibility of BALB/c mice to S. chartarum pulmonary effects. Analogous genetic differences among humans may contribute to reported variable sensitivity to S. chartarum.

Environmental Mold and Mycotoxin Exposures Elicit Specific Cytokine and Chemokine Responses

Background Molds can cause respiratory symptoms and asthma. We sought to use isolated peripheral blood mononuclear cells (PBMCs) to understand changes in cytokine and chemokine levels in response to mold and mycotoxin exposures and to link these levels with respiratory symptoms in humans. We did this by utilizing an ex vivo assay approach to differentiate mold-exposed patients and unexposed controls. While circulating plasma chemokine and cytokine levels from these two groups might be similar, we hypothesized that by challenging their isolated white blood cells with mold or mold extracts, we would see a differential chemokine and cytokine release. Methods and Findings Peripheral blood mononuclear cells (PBMCs) were isolated from blood from 33 patients with a history of mold exposures and from 17 controls. Cultured PBMCs were incubated with the most prominent Stachybotrys chartarum mycotoxin, satratoxin G, or with aqueous mold extract, ionomycin, or media, each with or without PMA. Additional PBMCs were exposed to spores of Aspergillus niger, Cladosporium herbarum and Penicillium chrysogenum. After 18 hours, cytokines and chemokines released into the culture medium were measured by multiplex assay. Clinical histories, physical examinations and pulmonary function tests were also conducted. After ex vivo PBMC exposures to molds or mycotoxins, the chemokine and cytokine profiles from patients with a history of mold exposure were significantly different from those of unexposed controls. In contrast, biomarker profiles from cells exposed to media alone showed no difference between the patients and controls. Conclusions These findings demonstrate that chronic mold exposures induced changes in inflammatory and immune system responses to specific mold and mycotoxin challenges. These responses can differentiate mold-exposed patients from unexposed controls. This strategy may be a powerful approach to document immune system responsiveness to molds and other inflammation-inducing environmental agents.

Mineralogy affects geoavailability, bioaccessibility and bioavailability of zinc

We correlated mineralogical and particle characteristics of Zn-containing particles with Zn geoavailability, bioaccessibility, and bioavailability following gavage and intranasal (IN) administration in rats. We compared samples of Zn/Pb mine waste and five pulverized pure-phase Zn minerals (<38 μm). Particles were neutron-activated to produce radioactive (65)Zn. We assessed geoavailability using sequential extractions and bioaccessibility using in vitro extraction tests simulating various pH and biological conditions. Zn in vivo bioavailability and in vitro bioaccessibility decreased as follows: mine waste > hydrozincite > hemimorphite > zincite ≈ smithsonite >> sphalerite. We found significant correlations among geoavailability, bioaccessibility and bioavailability. In particular, Zn bioavailability post-gavage and post-IN was significantly correlated with bioaccessibility in simulated phagolysosomal fluid and gastric fluid. These data indicate that solid phase speciation influences biological uptake of Zn and that in vitro tests can be used to predict Zn bioavailability in exposure assessment and effective remediation design.

Surface modification of zinc oxide nanoparticles with amorphous silica alters their fate in the circulation

Abstract Nanoparticle (NP) pharmacokinetics and biological effects are influenced by many factors, especially surface physicochemical properties. We assessed the effects of an amorphous silica coating on the fate of zinc after intravenous (IV) injection of neutron activated uncoated 65ZnO or silica-coated 65ZnO NPs in male Wistar Han rats. Groups of IV-injected rats were sequentially euthanized, and 18 tissues were collected and analyzed for 65Zn radioactivity. The protein coronas on each ZnO NP after incubation in rat plasma were analyzed by SDS-PAGE gel electrophoresis and mass spectrometry of selected gel bands. Plasma clearance for both NPs was biphasic with rapid initial and slower terminal clearance rates. Half-lives of plasma clearance of silica-coated 65ZnO were shorter (initial – <1 min; terminal – 2.5 min) than uncoated 65ZnO (initial – 1.9 min; terminal – 38 min). Interestingly, the silica-coated 65ZnO group had higher 65Zn associated with red blood cells and higher initial uptake in the liver. The 65Zn concentrations in all the other tissues were significantly lower in the silica-coated than uncoated groups. We also found that the protein corona formed on silica-coated ZnO NPs had higher amounts of plasma proteins, particularly albumin, transferrin, A1 inhibitor 3, α-2-hs-glycoprotein, apoprotein E and α-1 antitrypsin. Surface modification with amorphous silica alters the protein corona, agglomerate size, and zeta potential of ZnO NPs, which in turn influences ZnO biokinetic behavior in the circulation. This emphasizes the critical role of the protein corona in the biokinetics, toxicology and nanomedical applications of NPs.

Manganese Uptake and Distribution in the Brain after Methyl Bromide-Induced Lesions in the Olfactory Epithelia

Manganese (Mn) is an essential nutrient with potential neurotoxic effects. Mn deposited in the nose is apparently transported to the brain through anterograde axonal transport, bypassing the blood-brain barrier. However, the role of the olfactory epithelial cells in Mn transport from the nasal cavity to the blood and brain is not well understood. We utilized the methyl bromide (MeBr) lesion model wherein the olfactory epithelium fully regenerates in a time-dependent and cell type-specific manner over the course of 6-8 weeks postinjury. We instilled (54)MnCl(2) intranasally at different recovery periods to study the role of specific olfactory epithelial cell types in Mn transport. (54)MnCl(2) was instilled at 2, 4, 7, 21, and 56 days post-MeBr treatment. (54)Mn concentrations in the blood were measured over the first 4-h period and in the brain and other tissues at 7 days postinstillation. Age-matched control rats were similarly studied at 2 and 56 days. Blood and tissue (54)Mn levels were reduced initially but returned to control values by day 7 post-MeBr exposure, coinciding with the reestablishment of sustentacular cells. Brain (54)Mn levels also decreased but returned to control levels only by 21 days, the period near the completion of neuronal regeneration/bulbar reinnervation. Our data show that Mn transport to the blood and brain temporally correlated with olfactory epithelial regeneration post-MeBr injury. We conclude that (1) sustentacular cells are necessary for Mn transport to the blood and (2) intact axonal projections are required for Mn transport from the nasal cavity to the olfactory bulb and brain.