Ramon M. Molina

Gadolinium induces macrophage apoptosis

Gadolinium (Gd) suppresses reticuloendothelial functions in vivo by unknown mechanisms. In vitro exposure of rat alveolar macrophages to GdCl3·6H2O caused cell death, as measured by trypan blue permeability, in both dose‐ and time‐dependent fashions. Even a 10‐min exposure to Gd caused significant cell death by 24 h. The morphology of Gd‐treated cells, pyknosis and karyorrhexis prior to loss of membrane integrity, suggested apoptosis. Upon flow cytometric examination, Gd‐treated propidium iodide‐excluding cells demonstrated light scatter changes characteristic of apoptotic cells (decreased forward and increased right angle scatter). Gel electrophoresis of DNA from Gd‐treated macrophages clearly showed the ladder pattern unique to apoptotic cells. Electron‐dense structures containing Gd were observed via electron spectroscopic imaging within phagosomes and also within nuclei (associated with condensed chromatin). Gadolinium, endocytosed by macrophages and distributed to nuclei, causes apoptosis of macrophages in vitro.

Pulmonary intravascular macrophages: their contribution to the mononuclear phagocyte system in 13 species

The organ uptake of intravenously injected particles was examined in 13 species. All animals were injected intravenously with 198Au colloid and magnetic iron oxide particles. Vascular clearance kinetics of 198Au colloid was similar in all species. Pulmonary uptake of 198Au colloid ranged from 17 to 60% in sheep, calves, pigs, and cats but was <1.1% in monkeys, hyraxes, rabbits, guinea pigs, rats, mice, and chickens. For iron oxide particles, pulmonary uptake ranged from 80 to 99% in sheep, calves, pigs, goats, and cats and 15 to 18% in hamsters, hyraxes, and monkeys and was <10% in rabbits, chicken, mice, rats, and guinea pigs. In all species, the bulk of the remainder of particle uptake was in the liver. Pulmonary intravascular macrophages are the cellular site of lung uptake in calves, cats, pigs, goats, and sheep, whereas monocytes and neutrophils predominate in other species. Kupffer cells were the site of uptake in the liver. Our data show marked species differences in the fate of circulating particles; ruminants, pigs, and cats have extensive pulmonary localization due to phagocytosis by pulmonary intravascular macrophages.The organ uptake of intravenously injected particles was examined in 13 species. All animals were injected intravenously with 198Au colloid and magnetic iron oxide particles. Vascular clearance kinetics of198Au colloid was similar in all species. Pulmonary uptake of 198Au colloid ranged from 17 to 60% in sheep, calves, pigs, and cats but was <1.1% in monkeys, hyraxes, rabbits, guinea pigs, rats, mice, and chickens. For iron oxide particles, pulmonary uptake ranged from 80 to 99% in sheep, calves, pigs, goats, and cats and 15 to 18% in hamsters, hyraxes, and monkeys and was <10% in rabbits, chicken, mice, rats, and guinea pigs. In all species, the bulk of the remainder of particle uptake was in the liver. Pulmonary intravascular macrophages are the cellular site of lung uptake in calves, cats, pigs, goats, and sheep, whereas monocytes and neutrophils predominate in other species. Kupffer cells were the site of uptake in the liver. Our data show marked species differences in the fate of circulating particles; ruminants, pigs, and cats have extensive pulmonary localization due to phagocytosis by pulmonary intravascular macrophages.

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)

Noninfectious entry of HIV-1 into peripheral and brain macrophages mediated by the mannose receptor

Although protein receptors on the plasma membrane involved in the initial steps of productive HIV-1 infection have been well characterized, little is known about interactions between cellular carbohydrate receptors and HIV-1. Here, we report the involvement of a carbohydrate receptor, the macrophage mannose receptor (MR), and its role in supporting HIV-1 binding and entry. HIV-1 can enter the cytoplasm of human macrophages and microglia as well as murine macrophages by MR, although no subsequent viral replication was observed. Correspondingly, HIV-1 entry into Cos-7 cells after induction of expression of MR by transfection with MR-cDNA did not demonstrate viral replication. Our studies suggest that whereas MR may serve as a binding and an entry site, the MR-mediated pathway does not lead to productive HIV-1 infection. In addition, we report that recombinant HIV-1 gp120 blocks MR-mediated phagocytosis in human and murine alveolar macrophages and microglial cells. Therefore, characterization of the HIV-1 noninfectious MR-mediated phagocytic pathway may foster advances in HIV-1 vaccine design and an improved understanding of HIV-1/AIDS pathogenesis and host defenses.

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.

Development and characterization of a Versatile Engineered Nanomaterial Generation System (VENGES) suitable for toxicological studies

A novel system for generation of engineered nanomaterials (ENMs) suitable for in situ toxicological characterization within biological matrices was developed. This Versatile Engineered Nanomaterial Generation System (VENGES) is based on industry-relevant, flame spray pyrolysis aerosol reactors that can scaleably produce ENMs with controlled primary and aggregate particle size, crystallinity, and morphology. ENMs are produced continuously in the gas phase, allowing their continuous transfer to inhalation chambers, without altering their state of agglomeration. Freshly generated ENMs are also collected on Teflon filters for subsequent physicochemical and morphological characterization and for in vitro toxicological studies. The ability of the VENGES system to generate families of ENMs of pure and selected mixtures of iron oxide, silica, and nanosilver with controlled physicochemical properties was demonstrated using a range of state-of-the-art-techniques. Specific surface area was measured by nitrogen adsorption using the Brunauer–Emmett–Teller method, and crystallinity was characterized by X-ray diffraction. Particle morphology and size were evaluated by scanning and transmission electron microscopy. The suitability of the VENGES system for toxicological studies was also shown in both in vivo and in vitro studies involving Sprague–Dawley rats and human alveolar-like monocyte derived macrophages, respectively. We demonstrated linkage between physicochemical ENM properties and potential toxicity.

Ingestion of Mn and Pb by rats during and after pregnancy alters iron metabolism and behavior in offspring

Manganese (Mn) and lead (Pb) exposures during developmental period can impair development by direct neurotoxicity or through interaction with iron metabolism. Therefore, we examined the effects of maternal ingestion of Mn or Pb in drinking water during gestation and lactation on iron metabolism as well as behavior in their offspring. Pregnant dams were given distilled water, 4.79mg/ml Mn, or 2.84mg/ml Pb in drinking water during gestation and lactation. Pups were studied at time of weaning for (59)Fe absorption from the gut, duodenal divalent metal transporter 1 (DMT1) expression, hematological parameters, and anxiety-related behavior using an Elevated Plus Maze (EPM) test. Metal-exposed pups had lower body weights and elevated blood and brain concentrations of the respective metal. Pb-exposed pups had lower hematocrits and higher blood Zn protoporphyrin levels. In contrast, Mn exposed pups had normal hematological parameters but significantly reduced Zn protoporphyrin. Pharmacokinetic studies using (59)Fe showed that intestinal absorption in metal-exposed pups was not different from controls, nor was it correlated with duodenal DMT1 expression. However, intravenously injected (59)Fe was cleared more slowly in Pb-exposed pups resulting in higher plasma levels. The overall tissue uptake of (59)Fe was lower in Mn-exposed and lower in the brain in Pb-exposed pups. The EPM test demonstrated that Mn-exposed, but not Pb-exposed, pups had lower anxiety-related behavior compared to controls. We conclude that gestational and lactational exposures to Mn or Pb differentially alter Fe metabolism and anxiety-related behavior. The data suggest that perturbation in Fe metabolism may contribute to the pathophysiologic consequences of Mn and Pb exposure during early development.

Effects of copy center particles on the lungs: a toxicological characterization using a Balb/c mouse model

Abstract Context: Printers and photocopiers release respirable particles into the air. Engineered nanomaterials (ENMs) have been recently incorporated into toner formulations but their potential toxicological effects have not been well studied. Objective: To evaluate the biological responses to copier-emitted particles in the lungs using a mouse model. Methods: Particulate matter (PM) from a university copy center was sampled and fractionated into three distinct sizes, two of which (PM0.1 and PM0.1–2.5) were evaluated in this study. The particles were extracted and dispersed in deionized water and RPMI/10% FBS. Hydrodynamic diameter and zeta potential were evaluated by dynamic light scattering. The toxicological potential of these particles was studied using 8-week-old male Balb/c mice. Mice were intratracheally instilled with 0.2, 0.6, 2.0 mg/kg bw of either the PM0.1 and PM0.1–2.5 size fractions. Fe2O3 and welding fumes were used as comparative materials, while RPMI/10% FBS was used as the vehicle control. Bronchoalveolar lavage (BAL) was performed 24 hours post-instillation. The BAL fluid was analyzed for total and differential cell counts, and biochemical markers of injury and inflammation. Results: Particle size- and dose-dependent pulmonary effects were found. Specifically, mice instilled with PM0.1 (2.0 mg/kg bw) had significant increases in neutrophil number, lactate dehydrogenase and albumin compared to vehicle control. Likewise, pro-inflammatory cytokines were elevated in mice exposed to PM0.1 (2.0 mg/kg bw) compared to other groups. Conclusion: Our results indicate that exposure to copier-emitted nanoparticles may induce lung injury and inflammation. Further exposure assessment and toxicological investigations are necessary to address this emerging environmental health pollutant.

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.

Bioavailability, distribution and clearance of tracheally instilled, gavaged or injected cerium dioxide nanoparticles and ionic cerium

Cerium oxide nanoparticles (NPs) have wide commercial applications. Understanding their fate in the body is fundamental to toxicological evaluations. We compared bioavailability, tissue distribution, clearance and excretion of radioactive 141Ce after intratracheal instillation (IT), gavage, or intravenous (IV) injection of neutron-activated 141CeO2 NPs and 141CeCl3 (ionic 141Ce) in Wistar Han rats. First, we evaluated pulmonary responses to IT-instilled CeO2 NPs and CeCl3 and observed dose-dependent inflammatory effects. Then, groups of rats were IT-instilled with 1 mg kg−1 of 141CeO2 NPs or 0.1 mg kg−1 141CeCl3. Sequential analyses of lungs over 28 days showed slow lung clearance of 141CeO2 NPs (half-life = ~140 days) and of ionic 141Ce (half-life = ~55 days). However, less than 1% and 6% of instilled 141Ce was measured in selected extrapulmonary organs in 141CeO2 NPs and ionic 141Ce groups, respectively. Following gavage (5 mg kg−1), nearly 100% of both test materials was excreted in the feces. Since detected 141Ce activity in tissues could be in nanoparticulate or dissolved form, we also compared the 141Ce tissue distribution post-IV injection with the IT and gavage data. Both IV-injected ionic 141CeCl3 and 141CeO2 NPs were predominantly retained in the liver, bone and spleen, all organs that typically remove circulating particles. We conclude that nanoceria is slowly cleared from the lungs but has minimal extrapulmonary accumulation. Potential risks from prolonged pulmonary retention need further investigation. Risk from ingested nanoceria is likely far lower due to very low absorption and rapid elimination of ceria not absorbed from the gastrointestinal tract.