Distribution of 2,2',5,5'-Tetrachlorobiphenyl (PCB52) Metabolites in Adolescent Rats after Acute Nose-Only Inhalation Exposure.

Inhalation of PCB-contaminated air is increasingly recognized as a route for PCB exposure. Because limited information about the disposition of PCBs following inhalation exposure is available, this study investigated the disposition of 2,2',5,5'-tetrachlorobiphenyl (PCB52) and its metabolites in rats following acute, nose-only inhalation of PCB52. Male and female Sprague-Dawley rats (50-58 days of age, 210 ± 27 g; n = 6) were exposed for 4 h by inhalation to approximately 14 or 23 µg/kg body weight of PCB52 using a nose-only exposure system. Sham animals (n = 6) were exposed to filtered lab air. Based on gas chromatography-tandem mass spectrometry (GC-MS/MS), PCB52 was present in adipose, brain, intestinal content, lung, liver, and serum. 2,2',5,5'-Tetrachlorobiphenyl-4-ol (4-OH-PCB52) and one unknown monohydroxylated metabolite were detected in these compartments except for the brain. Liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis identified several metabolites, including sulfated, methoxylated, and dechlorinated PCB52 metabolites. These metabolites were primarily found in the liver (7 metabolites), lung (9 metabolites), and serum (9 metabolites) due to the short exposure time. These results demonstrate for the first time that complex mixtures of sulfated, methoxylated, and dechlorinated PCB52 metabolites are formed in adolescent rats following PCB52 inhalation, laying the groundwork for future animal studies of the adverse effects of inhaled PCB52.

Use of a polymeric implant system to assess the neurotoxicity of subacute exposure to 2,2',5,5'-tetrachlorobiphenyl-4-ol, a human metabolite of PCB 52, in male adolescent rats.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants (POPs) that ubiquitously exist in the environment. PCB exposure has been linked to cancer and multi-system toxicity, including endocrine disruption, immune inhibition, and reproductive and neurotoxicity. 2,2',5,5'-Tetrachlorobiphenyl (PCB 52) is one of the most frequently detected congeners in the environment and human blood. The hydroxylated metabolites of PCB 52 may also be neurotoxic, especially for children whose brains are still developing. However, it is challenging to discern the contribution of these metabolites to PCB neurotoxicity because the metabolism of PCB is species-dependent. In this study, we evaluated the subacute neurotoxicity of a human-relevant metabolite, 2,2',5,5'-tetrachlorobiphenyl-4-ol (4-52), on male adolescent Sprague Dawley rats, via a novel polymeric implant drug delivery system grafted subcutaneously, at total loading concentrations ranging from 0%, 1%, 5%, and 10% of the implant (w/w) for 28 days. Y-maze, hole board test, open field test, and elevated plus maze were performed on exposure days 24-28 to assess their locomotor activity, and exploratory and anxiety-like behavior. 4-52 and other possible hydroxylated metabolites in serum and vital tissues were quantified using gas chromatography with tandem mass spectrometry (GC-MS/MS). Our results demonstrate the sustained release of 4-52 from the polymeric implants into the systemic circulation in serum and tissues. Dihydroxylated and dechlorinated metabolites were detected in serum and tissues, depending on the dose and tissue type. No statistically significant changes were observed in the neurobehavioral tasks across all exposure groups. The results demonstrate that subcutaneous polymeric implants provide a straightforward method to expose rats to phenolic PCB metabolites to study neurotoxic outcomes, e.g., in memory, anxiety, and exploratory behaviors.

Immunomodulatory Effects of Subacute Inhalation Exposure to Copper Oxide Nanoparticles in House Dust Mite-Induced Asthma.

It has been shown that inhalation exposure to copper oxide nanoparticles (CuO NPs) results in pulmonary inflammation. However, immunomodulatory consequences after CuO NP inhalation exposure have been less explored. We tested the effect of CuO NP aerosols on immune responses in healthy, house dust mite (HDM) asthmatic, or allergen immunotherapy (AIT)-treated asthmatic mice (BALB/c, females). The AIT consisted of a vaccine comprising HDM allergens and CpG-loaded nanoparticles (CpG NPs). AIT treatment involved mice being immunized (via subcutaneous (sc) injection; 2 doses) while concomitantly being exposed to CuO NP aerosols (over a 2 week period), starting on the day of the first vaccination. Mice were then sensitized twice by sc injection and subsequently challenged with HDM extract 10 times by intranasal instillation. The asthmatic model followed the same timeline except that no immunizations were administered. All mice were necropsied 24 h after the end of the HDM challenge. CuO NP-exposed healthy mice showed a significant decrease in TH1 and TH2 cells, and an elevation in T-bet+ Treg cells, even 40 days after the last exposure to CuO NPs. Similarly, the CuO NP-exposed HDM asthma model demonstrated decreased TH2 responses and increased T-bet+ Treg cells. Conversely, CuO NP inhalation exposure to AIT-treated asthmatic mice resulted in an increase in TH2 cells. In conclusion, immunomodulatory effects of inhalation exposure to CuO NPs are dependent on immune conditions prior to exposure.

Cerium Oxide Enhances the Toxicity of Zinc Oxide Nanoparticles in Human Lung Epithelial Cell Cultures.

Recently, many approaches have been developed to improve the performance of nanomaterials. Combining more than one nanomaterial is one such approach that achieves superior results. However, during the fabrication of nanomaterials or formulation of end products, materials can be released into the ambient air and be inhaled by workers. The adverse health outcomes of inhaling such compounds are unknown. In this study, we examined such effects in combining two of the most utilized nanomaterials in several industrial sectors: zinc oxide (ZnO) and cerium oxide (CeO2). These materials can be found together in sunscreens, polyvinyl alcohol (PVA) films, and construction products. The aim of this study was to assess the adverse biological outcomes of CeO2-ZnO nano-mixtures in human lung epithelial cells. A549 human lung epithelial cells were treated with increasing concentrations of ZnO or CeO2 NPs alone, or as a mixture of both, under submerged conditions for 24 h. After treatment, cell viability, reactive oxygen species (ROS) formation, cell membrane integrity, and cytokine production were examined. ZnO NPs showed a dose-dependent trend for all endpoints. CeO2 NPs did not exhibit any toxic effect in any individual concentrations. When higher doses of ZnO were combined with increasing doses of CeO2, loss of cell viability and an elevation in cell membrane leakage were observed. Interleukin 8 (IL-8) and ROS generation were higher when ZnO NPs were combined with CeO2 NPs, compared to cells that were treated with ZnO alone. The release of monocyte chemoattractant protein-1 (MCP-1) was reduced in the cells that were treated with higher doses of ZnO and CeO2. Thus, the presence of CeO2 enhanced the toxicity of ZnO in A549 cells at non-toxic levels of CeO2. This suggests an additive toxicity of these two nanomaterials.

Time course of pulmonary inflammation and trace element biodistribution during and after sub-acute inhalation exposure to copper oxide nanoparticles in a murine model.

BACKGROUND: It has been shown that copper oxide nanoparticles (CuO NPs) induce pulmonary toxicity after acute or sub-acute inhalation exposures. However, little is known about the biodistribution and elimination kinetics of inhaled CuO NPs from the respiratory tract. The purposes of this study were to observe the kinetics of pulmonary inflammation during and after CuO NP sub-acute inhalation exposure and to investigate copper (Cu) biodistribution and clearance rate from the exposure site and homeostasis of selected trace elements in secondary organs of BALB/c mice. RESULTS: Sub-acute inhalation exposure to CuO NPs led to pulmonary inflammation represented by increases in lactate dehydrogenase, total cell counts, neutrophils, macrophages, inflammatory cytokines, iron levels in bronchoalveolar lavage (BAL) fluid, and lung weight changes. Dosimetry analysis in lung tissues and BAL fluid showed Cu concentration increased steadily during exposure and gradually declined after exposure. Cu elimination from the lung showed first-order kinetics with a half-life of 6.5 days. Total Cu levels were significantly increased in whole blood and heart indicating that inhaled Cu could be translocated into the bloodstream and heart tissue, and potentially have adverse effects on the kidneys and spleen as there were significant changes in the weights of these organs; increase in the kidneys and decrease in the spleen. Furthermore, concentrations of selenium in kidneys and iron in spleen were decreased, pointing to disruption of trace element homeostasis. CONCLUSIONS: Sub-acute inhalation exposure of CuO NPs induced pulmonary inflammation, which was correlated to Cu concentrations in the lungs and started to resolve once exposure ended. Dosimetry analysis showed that Cu in the lungs was translocated into the bloodstream and heart tissue. Secondary organs affected by CuO NPs exposure were kidneys and spleen as they showed the disruption of trace element homeostasis and organ weight changes.

Toxicity Assessment of 91-Day Repeated Inhalation Exposure to an Indoor School Air Mixture of PCBs.

School indoor air contaminated with polychlorinated biphenyls (PCBs) released from older building materials and paint pigments may pose health risks to children, as well as teachers and staff, by inhalation of PCBs. The health effects of long-term inhalation exposure to PCBs are poorly understood. We conducted a comprehensive toxicity assessment of 91-day repeated inhalation exposure to a lab-generated mixture of PCBs designed to emulate indoor school air, combining transcriptomics, metabolomics, and neurobehavioral outcomes. Female Sprague-Dawley rats were exposed to school air mixture (SAM+) at a concentration of 45.5 ± 5.9 µg/m3 ∑209PCB or filtered air 4 h/day, 6 days/week for 13 weeks using nose-only exposure systems. The congener-specific PCB body burden was quantified in major tissues using GC-MS/MS. The generated SAM+ vapor recapitulated the target school air profile with a similarity coefficient, cos θ of 0.91. PCB inhalation yielded 875-9930 ng/g ∑209PCBlipid weight levels in tissues in the following ascending order: brain < liver < lung < serum < adipose tissue. We observed that PCB exposure impaired memory, induced anxiety-like behavior, significantly reduced white blood cell counts, mildly disrupted metabolomics in plasma, and influenced transcription processes in the brain with 274 upregulated and 58 downregulated genes. With relatively high exposure and tissue loading, evidence of toxicity from half the end points tested was seen in the rats.

Redox sensitive miR-27a/b/Nrf2 signaling in Cr(VI)-induced carcinogenesis.

Hexavalent chromium [Cr(VI)] is a well-known carcinogen that can cause several types of cancer including lung cancer. NF-E2-related factor 2 (Nrf2), the redox sensitive transcription factor, can protect normal cells from a variety of toxicants and carcinogens by inducing the expression of cellular protective genes and maintaining redox balance. However, Nrf2 also protects cancer cells from radio- and chemo-therapies and facilitates cancer progression. Although Cr(VI) treatment has been demonstrated to upregulate Nrf2 expression, the mechanisms for Nrf2 regulation upon chronic Cr(VI) exposure remain to be elucidated. We found that Nrf2 was upregulated in BEAS-2B cells exposed to Cr(VI) from 1 to 5 months, and also in Cr(VI)-induced transformed (Cr-T) cells with Cr(VI) treatment for 6 months. We showed that KEAP1, the classic negative regulator of Nrf2, was downregulated after Cr(VI) exposure for 4 months, suggesting that Nrf2 induction by Cr(VI) treatment is through KEAP1 decrease at late stage. To further decipher the mechanisms of Nrf2 upregulation at early stage of Cr(VI) exposure, we demonstrated that miR-27a and miR-27b were redox sensitive miRNAs, since reactive oxygen species (ROS) scavengers induced miR-27a/b expression. After Cr(VI) exposure for 1 month, the expression levels of miR-27a/b was dramatically decreased. The changes of miR-27a/b and their target Nrf2 were confirmed in vivo by mouse model intranasally exposed to Cr(VI) for 12 weeks. Nrf2 was a direct target of miR-27a/b, which acted as tumor suppressors in vitro and in vivo to inhibit tumorigenesis and cancer development of Cr-T cells. The results suggested that the inhibition of miR-27a/b was responsible for Nrf2 upregulation at both early stage and late stage of Cr(VI) exposure. This novel regulation of Nrf2 upon chronic Cr(VI) exposure through redox-regulated miR-27a/b will provide potential targets for preventing and treating Cr(VI)-induced carcinogenesis in the future.

Validation of blood arsenic and manganese assessment from archived clotted erythrocyte fraction in an urban cohort of mother-child dyads.

Exposure to arsenic (As) and manganese (Mn) from contaminated food, drinking water and dust are linked to a host of adverse health effects. The recent discovery of unmonitored community exposures to hazardous levels of metals, as seen in the Flint Water Crisis and East Chicago, have demonstrated a need for novel biomonitoring methods utilizing samples other than whole blood. Here, we present a method utilizing clotted erythrocyte fraction samples, a blood component commonly archived in biorepositories, to predict whole blood levels of As and Mn. This method would allow for innovative retrospective assessments of environmental exposures in previously unused samples. Whole blood and clotted erythrocyte fraction samples were simultaneously collected from 84 participants in the Airborne Exposure to Semivolatile Organic Pollutants (AESOP) cohort study of mother-child dyads in East Chicago. Clotted erythrocyte fraction samples were prepared by alkaline dilution and subsequently analyzed using inductively coupled plasma-mass spectrometry. A strong linear relationship was observed between whole blood and clotted erythrocyte fraction with Pearson correlation coefficients (r, p < 0.001) of 0.74, and 0.82 for As and Mn, respectively. Modeled whole blood Mn levels predicted from clotted erythrocyte fractions evaluated at a test threshold representing the NHANES median of 9.7 µg/L, were found to have diagnostic sensitivity of 88% and specificity of 71%. Clotted erythrocyte partitioning of As was tested on a wide range of oral gavage doses using a rat model. Results from this investigation demonstrate clotted erythrocyte fraction samples are a viable alternative biological sample for retrospective public health surveillance of environmental exposure to As and Mn.

Encapsulating Polyethyleneimine-DNA Nanoplexes into PEGylated Biodegradable Microparticles Increases Transgene Expression In Vitro and Reduces Inflammatory Responses In Vivo.

Encapsulating genetic material into biocompatible polymeric microparticles is a means to improving gene transfection while simultaneously decreasing the tendency for inflammatory responses; and can be advantageous in terms of delivering material directly to the lungs via aerosolization for applications such as vaccinations. In this study, we investigated the advantages of using polymeric microparticles carrying the luciferase reporter gene in increasing transfection efficiency in the readily transfectable HEK293 cell line and the difficult to transfect RAW264.7 cell line. The results indicated that there was a limit to the ratio of nitrogen in polyethylenimine (PEI) to phosphate in DNA (N/P ratio) beyond which further increases in transgene expression no longer, or only marginally, occurred. Microparticles encapsulating PEI:DNA nanoplexes induced cellular toxicity in a dose-dependent manner. PEGylation increased transgene expression, likely related to enhanced degradation of particles. Furthermore, intra-tracheal instillation in rats allowed us to investigate the inflammatory response in the lung as a function of PEGylation, porosity, and size. Porosity did not influence cell counts in bronchoalveolar lavage fluid in the absence of PEG, but in particles containing PEG, non-porous particles recruited fewer inflammatory cells than their porous counterparts. Finally, both 1 µm and 10 µm porous PLA-PEG particles recruited more neutrophils than 4 µm particles. Thus, we have shown that PEGylation and lack of porosity are advantageous for faster release of genetic cargo from microparticles and a reduced inflammatory response, respectively.

Comprehensive Subchronic Inhalation Toxicity Assessment of an Indoor School Air Mixture of PCBs.

Few in vivo inhalation studies have explored the toxicity of environmentally relevant mixtures of polychlorinated biphenyls (PCBs). The manufacture of industrial PCBs was banned in 1978, but PCBs continue to be formed in industrial and consumer products. Schools represent a significant source of airborne exposures to legacy and nonlegacy PCBs, placing children at risk. To evaluate the impact of these exposures, we generated an airborne mixture of PCBs, called the School Air Mixture (SAM), to match the profile of an older school from our adolescent cohort study. Female Sprague-Dawley rats were exposed either to SAM or filtered air in nose-only exposure systems, 4 h/day for 4 weeks. Congener-specific air and tissue PCB profiles were assessed using gas chromatography with tandem mass spectrometry (GC-MS/MS). PCB exposures recapitulated the target school air profile with a similarity coefficient, cos θ of 0.83. PCB inhalation yielded µg/g ∑209 PCB levels in tissues. Neurobehavioral testing demonstrated a modest effect on spatial learning and memory in SAM-exposed rats. PCB exposure induced oxidative stress in the liver and lungs, affected the maturational stages of hematopoietic stem cells, reduced telomerase activity in bone marrow cells, and altered the gut microbiota. This is the first study to emulate PCB exposures in a school and comprehensively evaluate toxicity.

Acute in vivo pulmonary toxicity assessment of occupationally relevant particulate matter from a cellulose nanofiber board.

Cellulose nanofibers (CNFs) are an emerging engineered nanomaterial that are utilized in a variety of applications, including as a replacement for urea-formaldehyde, and other adhesives, as the binding agent in manufactured fiber and particle boards. To ensure the health and well-being of those producing, installing, or otherwise using cellulose nanofiber boards (CNFBs) it is imperative that the particulate matter (PM) produced during CNFB manipulation be evaluated for toxicity. We developed and internally verified a generation system to examine the PM produced by sanding CNFB using aluminum oxide sandpaper. With 80-grit sandpaper our system produced a low dispersity aerosol, as determined by a scanning mobility particle sizer and an optical particle counter, with a geometric mean of 28 nm (GSD = 1.60). ICP-MS evaluation showed little difference in metal concentrations between CNFB PM and nonsanded CNFB stock. We then used the system to simultaneously generate and expose both male and female C57BL/6J mice acutely for 4 hours at a concentration of 7.9 mg/m3. Sham-exposed controls were treated similarly but without sanding the CNFB. Analysis of bronchoalveolar lavage (BAL) fluid biomarkers showed no signs of inflammatory response at either 4- or 24-hours post exposure. Further, BAL cell viability, number of total cells, and pulmonary cellular recruitment were not significantly changed between the sham-exposed controls and CNFB-exposed mice. Histology further confirmed no pulmonary toxicity as a result of CNFB PM inhalation. We conclude that inhalation of a high concentration of the PM from manipulation of a CNFB did not produce acute toxic responses within 24 hours of exposure.

Toxicity assessment of metal oxide nanomaterials using in vitro screening and murine acute inhalation studies.

Characterizations and in vitro toxicity screening were performed on metal oxide engineered nanomaterials (ENMs) independently comprising ZnO, CuO, CeO2, Fe2O3, WO3, V2O5, TiO2, Al2O3 and MgO. Nanomaterials that exhibited the highest toxicity responses in the in vitro screening assays (ZnO, CuO, and V2O5) and the lesser explored material WO3 were tested for acute pulmonary toxicity in vivo. Female and male mice (C57Bl/6J) were exposed to aerosolized metal oxide ENMs in a nose-only exposure system and toxicity outcomes (biomarkers of cytotoxicity, immunotoxicity, inflammation, and lung histopathology) at 4 and 24 h after the start of exposure were assessed. The studies were performed as part of the NIEHS Nanomaterials Health Implications Research consortium with the purpose of investigating the effects of ENMs on various biological systems. ENMs were supplied by the Engineered Nanomaterials Resource and Coordination Core. Among the ENMs studied, the highest toxicity was observed for CuO and ZnO NPs in both in vitro and in vivo acute models. Compared to sham-exposed controls, there was a significant increase in bronchoalveolar lavage neutrophils and proinflammatory cytokines and a loss of macrophage viability at both 4 h and 24 h for ZnO and CuO but not seen for V2O5 or WO3. These effects were observed in both female and male mice. The cell viability performed after in vitro exposure to ENMs and assessment of lung inflammation after acute inhalation exposure in vivo were shown to be sensitive endpoints to predict ENM acute toxicity.

Comparison of in vitro toxicity of aerosolized engineered nanomaterials using air-liquid interface mono-culture and co-culture models.

Airborne engineered nanomaterials (ENMs) can readily enter the human body through inhalation potentially leading to adverse health effects such as cardiovascular and pulmonary diseases. Our group has previously utilized and validated an integrated low flow system capable of generating and depositing airborne ENMs directly onto cells at an air-liquid interface (ALI). To further improve this ALI method for an even closer representation of the in vivo system, a co-culture model containing epithelial, endothelial and macrophage cell lines (A549, EA.hy 926, and THP-1 differentiated macrophages) was established and validated for testing ENMs toxicity. In the co-culture model, cells were exposed to citrate-capped gold (Au), 15% silver on silica (Ag-SiO2) and copper oxide (CuO) ENMs under the same protocol (4 h ALI exposure with a target concentration of 3.5 mg/m3) and compared to responses with A549 cells only or THP-1 differentiated cells only. The toxicological profile was assessed by measuring cell viability, reactive oxygen species (ROS) production, lactate dehydrogenase (LDH) release, and interleukin (IL)-8 concentration. Results showed that 15% Ag-SiO2 induced more oxidative stress-related toxicity in the co-culture than in A549 cells alone. Both 15% Ag-SiO2 and CuO exposure produced significantly higher levels of IL-8 in the co-culture compared with A549 cells alone. Citrate-capped Au was largely inert. Further exposures of CuO on macrophages alone provided evidence of cell-cell interaction in the co-culture model. In addition, the co-culture model exhibited a similar response to primary human bronchial epithelial cells in terms of ROS and IL-8 responses after CuO exposure, suggesting a more advanced refinement of the conventional model for in vitro inhalation study.

Lung cell exposure to secondary photochemical aerosols generated from OH oxidation of cyclic siloxanes.

To study the fate of cyclic volatile methyl siloxanes (cVMS) undergoing photooxidation in the environment and to assess the acute toxicity of inhaled secondary aerosols from cVMS, we used an oxidative flow reactor (OFR) to produce aerosols from oxidation of decamethylcyclopentasiloxane (D5). The aerosols produced from this process were characterized for size, shape, and chemical composition. We found that the OFR produced aerosols composed of silicon and oxygen, arranged in chain agglomerates, with primary particles of approximately 31 nm in diameter. Lung cells were exposed to the secondary organosilicon aerosols at estimated doses of 54-116 ng/cm2 using a Vitrocell air-liquid interface system, and organic gases and ozone exposure was minimized through a series of denuders. Siloxane aerosols were not found to be highly toxic.

Submucosal Gland Myoepithelial Cells Are Reserve Stem Cells That Can Regenerate Mouse Tracheal Epithelium.

The mouse trachea is thought to contain two distinct stem cell compartments that contribute to airway repair-basal cells in the surface airway epithelium (SAE) and an unknown submucosal gland (SMG) cell type. Whether a lineage relationship exists between these two stem cell compartments remains unclear. Using lineage tracing of glandular myoepithelial cells (MECs), we demonstrate that MECs can give rise to seven cell types of the SAE and SMGs following severe airway injury. MECs progressively adopted a basal cell phenotype on the SAE and established lasting progenitors capable of further regeneration following reinjury. MECs activate Wnt-regulated transcription factors (Lef-1/TCF7) following injury and Lef-1 induction in cultured MECs promoted transition to a basal cell phenotype. Surprisingly, dose-dependent MEC conditional activation of Lef-1 in vivo promoted self-limited airway regeneration in the absence of injury. Thus, modulating the Lef-1 transcriptional program in MEC-derived progenitors may have regenerative medicine applications for lung diseases.

Multifunctional nanoparticles for real-time evaluation of toxicity during fetal development.

Increasing production of nanomaterials in industrial quantities has led to public health concerns regarding exposure, particularly among pregnant women and developing fetuses. Information regarding the barrier capacity of the placenta for various nanomaterials is limited due to challenges working with ex vivo human placentas or in vivo animal models. To facilitate real-time in vivo imaging of placental transport, we have developed a novel, multifunctional nanoparticle, based on a core of mesoporous silica nanoparticles (MSN), and functionalized for magnetic resonance imaging (MRI), ultrasound, and fluorescent microscopy. Our MSN particles were tested as a tracking method for harmful and toxic nanomaterials. In gravid mice, intravenous injections of MSN were administered in the maternal circulation in early gestation (day 9) and late gestation (day 14). MRI and ultrasound were used to track the MSN following the injections. Changes in contrast relative to control mice indicated that MSN were observed in the embryos of mice following early gestation injections, while MSN were excluded from the embryo by the placenta following late gestation injections. The timing of transplacental barrier porosity is consistent with the notion that in mice there is a progressive increasing segregation by the placenta in later gestation. In addition, built-in physico-chemical properties of our MSN may present options for the therapeutic treatment of embryonic exposure. For example, if preventive measures such as detoxification of harmful compounds are implemented, the particle size and exposure timing can be tailored to selectively distribute to the maternal side of the trophoblast or delivered to the fetus.

Biocompatibility of Multi-Imaging Engineered Mesoporous Silica Nanoparticles: In Vitro and Adult and Fetal In Vivo Studies.

Despite potentially serious adverse effects of engineered nanoparticles on maternal health and fetal development, little is known about their transport across the placenta. Human and animal studies are primarily limited to ex vivo approaches; the lack of a real-time, minimally invasive tool to study transplacental transport is clear. We have developed functionalized mesoporous silica nanoparticles (MSN) for use in magnetic resonance, ultrasound, and fluorescent imaging. This material is designed as a model for, or a carrier of, environmental toxicants, allowing for in vivo evaluation. To establish a baseline of biocompatibility, we present data describing MSN tolerance using in vitro and in vivo models. In cultured cells, MSN were tolerated to a dose of 125 µg/mL with minimal effect on viability and doubling time. For the 42 day duration of the study, none of the mice exhibited behaviors usually indicative of distress (lethargy, anemia, loss of appetite, etc.). In gravid mice, the body and organ weights of MSN-exposed dams were equivalent to those of control dams. Embryos exposed to MSN during early gestation were underweight by a small degree, while embryos exposed during late gestation were of a slightly larger weight. The rate of spontaneous fetal resorptions were equivalent in exposed and control mice. Maternal livers and sera were screened for a complement of cytokines/chemokines and reactive oxygen/nitrogen species (ROS/RNS). Only granulocyte-colony stimulating factor was elevated in mice exposed to MSN during late gestation, while ROS/RNS levels were elevated in mice exposed during early/mid gestation. These findings may usher future experiments investigating environmental toxicants using real-time assessment of transport across the placenta.

Amine modification of nonporous silica nanoparticles reduces inflammatory response following intratracheal instillation in murine lungs.

Amorphous silica nanoparticles (NPs) possess unique material properties that make them ideal for many different applications. However, the impact of these materials on human and environmental health needs to be established. We investigated nonporous silica NPs both bare and modified with amine functional groups (3-aminopropyltriethoxysilane (APTES)) in order to evaluate the effect of surface chemistry on biocompatibility. In vitro data showed there to be little to no cytotoxicity in a human lung cancer epithelial cell line (A549) for bare silica NPs and amine-functionalized NPs using doses based on both mass concentration (below 200µg/mL) and exposed total surface area (below 14m(2)/L). To assess lung inflammation, C57BL/6 mice were administered bare or amine-functionalized silica NPs via intra-tracheal instillation. Two doses (0.1 and 0.5mg NPs/mouse) were tested using the in vivo model. At the higher dose used, bare silica NPs elicited a significantly higher inflammatory response, as evidence by increased neutrophils and total protein in bronchoalveolar lavage (BAL) fluid compared to amine-functionalized NPs. From this study, we conclude that functionalization of nonporous silica NPs with APTES molecules reduces murine lung inflammation and improves the overall biocompatibility of the nanomaterial.

PAMAM dendrimers as nano carriers to investigate inflammatory responses induced by pulmonary exposure of PCB metabolites in Sprague-Dawley rats.

Polychlorinated biphenyls (PCBs) persist and accumulate in the ecosystem depending upon the degree of chlorination of the biphenyl rings. Airborne PCBs are especially susceptible to oxidative metabolism, yielding mono- and di-hydroxy metabolites. We have previously demonstrated that 4-chlorobiphenyl hydroquinones (4-CB-HQs) acted as cosubstrates for arachidonic acid metabolism by prostaglandin H synthase (PGHS) and resulted in an increase of prostaglandin production in vitro. In the present study, we tested the capability of 4-CB-HQ to act as a co-substrate for PGHS catalysis in vivo. BQ and 4-CB-2',5'-HQ were administered intratracheally to male Sprague-Dawley rats (2.5 µmol/kg body weight) using nanosized polyamidoamine (PAMAM) dendrimers as carriers. We found that 24 h post application, PGE2 metabolites in kidney of rats treated with 4-CB-2',5'-HQ were significantly increased compared to the controls. The increase of PGE2 metabolites was correlated with increased alveolar macrophages in lung lavage fluid. The elevation of PGE2 synthesis is of great interest since it plays a crucial role in balancing homeostasis and inflammation where a chronic disturbance may increase risk of cancer. PAMAM dentrimers proved to be an effective transport medium and did not stimulate an inflammatory response themselves.