Grantee Research Project Results
2014 Progress Report: Risk Assessment for Manufactured Nanoparticles Used in Consumer Products (RAMNUC)
EPA Grant Number: R834693Title: Risk Assessment for Manufactured Nanoparticles Used in Consumer Products (RAMNUC)
Investigators: Zhang, Junfeng , Tetley, Teresa D , Chung, Kian Fan , Georgopoulos, Panos G. , Lioy, Paul J. , Schwander, Stephan K. , Ryan, Mary P. , Isukapalli, Sastry S. , Di Giulio, Richard T. , Porter, Alexandra , Garfunkel, Eric , Mainelis, Gediminas , Kipen, Howard , Lee, Ki-Bum
Institution: University of Medicine and Dentistry of New Jersey , Imperial College , Duke University
Current Institution: University of Medicine and Dentistry of New Jersey , Duke University , Imperial College
EPA Project Officer: Aja, Hayley
Project Period: April 1, 2011 through June 30, 2014 (Extended to June 30, 2016)
Project Period Covered by this Report: July 1, 2013 through June 30,2014
Project Amount: $1,999,995
RFA: Environmental Behavior, Bioavailability and Effects of Manufactured Nanomaterials - Joint US – UK Research Program (2009) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Objective:
The U.S.-U.K. consortium, Risk Assessment for Manufactured Nanoparticles Used in Consumer Products (RAMNUC), provides a systematic, multidisciplinary approach for predicting human and environmental risks associated with the use of selected consumer products that incorporate zinc oxide and silver as well as a diesel fuel additive containing cerium dioxide nanoparticles. The overall hypothesis of the RAMNUC project is that “manufactured nanoparticles (MNPs) at the point of exposure for both humans and aquatic animals will substantially differ in both physicochemical and toxicological properties from the MNPs at the source (synthesized in the laboratory or acquired commercially)”. By testing this hypothesis, the RAMNUC project will assess toxicity and bio-reactivity of exposures resulting from using consumer products that contain MNPs.
Our project includes both experimental and computational tools to analyze the selected MNPs in consumer products and a diesel fuel additive using both in vitro and in vivo experiments to assess intra- and extra-cellular bioavailability, bioreactivity and toxicity. We have been characterizing MNPs for their physical (e.g., size, shape, electric charge and state of agglomeration) and chemical properties (e.g., composition and surface functionalization). Thus, our in vitro and in vivo studies are producing mechanism-based results relating toxic effects to the physicochemical properties of the MNPs. Another aspect of our project includes an exposure simulation study that will produce realistic estimates of MNP exposures to consumers.
Data generated from these experiments are being integrated into mechanism-based computational modules of two existing source-to-exposure-to-dose-to-effects modeling systems, allowing for rational extrapolation and generalization in MNP risk assessment. Built upon an inter-institutional structure, the RAMNUC consortium serves as a model for systematically addressing complex problems associated with MNP risk assessment. The findings will contribute to the limited knowledge about health risks associated with the use of nanotechnology-based consumer products.
Progress Summary:
This center project has eight specific aims. Below we highlight major accomplishments for each aim of Project Year 3.
Aim 1 (Risk Assessment Framework):
This Aim relates to the development of a generalized conceptual risk assessment framework for risks from MNPs and adaptation of these modules using data from Aims 2 through 7. In Years 1 and 2, this framework has been developed by adapting existing frameworks and incorporating MNP life cycle analysis approaches for the following stages of the MNPs’ life cycle: (a) sources, (b) environmental fate and transport, (c) concentrations in environmental media, (d) human exposure, and (e) dose at target organs and bioavailability. We are expanding upon this framework by defining a computational structure for studying toxicodynamic responses following exposures to MNPs. This is being actively developed in collaboration with the RAMNUC team and utilizes other data and mechanistic information from ongoing related research efforts.
Computational Implementation and Application of the Risk Assessment Framework for MNPs:
- Modules for estimating realistic environmental and occupational exposures to MNPs are being developed: (a) modules for environmental levels of CeO2 nanoparticles used as a fuel additive have been developed by evaluating and applying this framework in complementary research efforts, and (b) modules for environmental levels of zinc oxide (ZnO) are being developed as part of Aim 1.
- Modules for population exposures to CeO2 nanoparticles (used as a fuel additive) have been developed incorporating exposure simulation studies (Aim 4) to support risk assessments of the MNP. These modules have been coupled with the existing human airway dosimetry model, Multiple-Path Particle Dosimetry Model (MPPD v2.11), to characterize the population-wide distributions of alveolar, tracheobronchial and pharyngeal depositions considering the particle size measurements of DEP (Aim 4) and the age/gender distribution of the CONUS (contiguous U.S.) population.
- Modules for estimating population exposures to silver nanoparticles from consumer usage of nanospray products and from ambient sources have undergone further refinement and development in Year 3 to: (a) expand the calculation of intake fractions to include exposures to silver nanoparticles in non-residential buildings; (b) incorporate county-level consumer expenditure data on personal care and cleaning products into the exposure modules of silver nanoparticles for the CONUS population; (c) benchmark the population-wide distributional estimates of intake values against individual-based estimates from regulatory European consumer exposure models; and (d) perform preliminary risk-relevant calculations by comparing estimated population-wide intake values against published human indicative no-effect levels (INEL).
Aim 2 (Nanoparticle Synthesis and Characterization):
Under this Aim, our team members from material science at Imperial College London and from chemistry/biological chemistry at Rutgers University have continued to characterize physiochemical properties of materials being evaluated in the in vitro and in vivo experiments described in Aims 5-7. In addition, the following work has been performed to examine the interaction between MNPs and components of the pulmonary surfactant. (This component of the work is jointly funded by a grant from NIEHS and other funding sources.)
We have provided insights into the impact of the lung surfactant and its individual components on the stability of silver nanowires (selected for studying the effect of MNP shape) with well-controlled physicochemical properties. In this work, the influence of phospholipids and surfactant-associated proteins was investigated at different pH values, representative of environments found in the lung. Phospholipids, as well as each class of surfactant-associated proteins, the hydrophobic SP-B and SP-C and the hydrophilic SP-A and SP-D, were separately added to silver nanowires in an effort to delineate the effects of various components of the lung surfactant.
Inductively Coupled Plasma–Optical Emission Spectroscopy (ICP-OES) and a combination of analytical transmission electron microscopy (TEM) techniques were used to investigate the dissolution, colloidal stability and surface chemistry of silver nanowires. Furthermore, we examined if the interaction with phospholipids changes the dispersion of Ag nanowires and whether SP-B/C or SP-A/D induce agglomeration of Ag nanowires. TEM has enabled us to characterize the impact of the lung surfactant on the surface chemistry of the nanowire as well as the structure of the lipid corona.
The presence of phospholipids, in either DPPC or Curosurf, was found to induce a retarding effect on the release of Ag+ ions at pH 5. This was evident at the early time points of incubation but after 1 week in the case of DPPC or 2 weeks in the case of Curosurf, there was no significant difference in the total amount of Ag+ dissolved. In fact, there was slightly higher Ag+ ion release from Ag NWs incubated with DPPC after 2 weeks, in both pH 7 and pH 5, compared to Ag NWs alone. TEM imaging confirmed that Ag NWs were coated by phospholipids. Our findings confirm the ability of phospholipids to form bilayers on the surface of Ag nanomaterials and to slow down the kinetics of dissolution, at least at the early stages of incubation. DPPC also was found to improve the dispersion of Ag NWs to some degree. The slightly higher Ag+ ion release from Ag NWs incubated with DPPC at pH 5 after 2 weeks could be, therefore, explained by a lower degree of aggregation in the presence of DPPC and by the fact that no coarsening was observed. In the present work, no evidence was found for aggregation of the Ag NWs in Curosurf, which contains sp-B as well as PG. However, the improved dispersion observed for Ag NWs in DPPC was diminished when using Curosurf.
The addition of the small aggregate (SA) fraction of lung surfactant to both DPPC and Curosurf induced a further delay in the Ag+ ion release kinetics. SA contains non-specific lung proteins, such as IgG and albumin, as well as the collectins SP-A and SP-D. However, the presence of SP-A did not change the dispersion of Ag nanowires, therefore, the delay in Ag+ release is not due to aggregation. Moreover, no changes in the chemistry of the nanowire were observed and sulfidation of Ag nanowire by the sulfur containing SP-A and SP-D was ruled out. We infer from these findings that incubation of the nanowire in the presence of SA might lead to the adsorption of SP-A and/or SP-D on the surface of the Ag NWs. This could lead to passivation of the nanowire surface and consequently a slower ion release. Similar observations have been made with cysteine, whose adsorption on Ag nano-sphere surface prevented the oxidative dissolution of Ag nano-spheres and reduced their toxicity.
The most prominent observation regarding the stability of Ag nanowire was the generation of a secondary population of silver nanoparticles from the original Ag nanowires, following their incubation at pH 7 and pH 5, both in the presence and absence of different components of the lung surfactant. Formation of this secondary population was probably caused by reduction of Ag+ by PVP.
We also have provided insights into the dissolution of ZnO nanowires in various biological media. The kinetics of ZnO dissolution was found to be greatly affected by pH. ZnO nanowires were relatively stable at extracellular pH (7.4) but dissolved rapidly at lysosomal pH (5.2). The kinetics of dissolution also depended on medium components, with different profiles being observed for commonly used biological media (e.g., RPMI, DMEM and DCCM-1). This emphasizes the need for appropriate controls when performing in vitro or in vivo experiments. The impact of the lung surfactant on the stability of ZnO nanowires also is being investigated. The influence of phospholipids and surfactant-associated proteins on the dissolution kinetics, aggregation state and chemistry of ZnO nanowires is being examined. Futhermore, we have developed protocols in the confocal microscope to map the dissolution of ZnO nanowires and the release of Zn2+ ions inside live cells. We have demonstrated that the dissolution of ZnO NPs (30 nm) in human lung type-I epithelial cells precedes cell death and we currently are comparing ZnO NWs of two different lengths.
Impact:
By synthesizing and characterizing Ag and ZnO nanowires with well controlled physicochemical properties, we are able to assess how these properties change over time in different biological environments, such as cell culture media or the lung surfactant. This allows making more accurate predictions about the reactivity of these nanomaterials at the point of exposure. The effects of Ag or ZnO NWs on human lung cells in vitro will help us elucidate the mechanisms of toxicity for these materials and determine which physicochemical properties affect their bioreactivity.
Aim 3 (Exposure Simulation Studies):
The main goal of this Aim is to investigate exposure to nanoparticles from commercially available consumer sprays that incorporate ZnO and Ag MNPs. We identified a set of 19 nanotechnology products for evaluation, of which 13 contained silver and 6 contained zinc oxide. Because inhalation exposures depend on the aerosolization method of a particular product, we used three different ways to aerosolize each product and then determined the size distribution and concentration of the released particles. The tested aerosolization methods included C-Flow® and Collison® commercial nebulizers as well as sprayers included with each product. Scanning Mobility Particle Sizer (SMPS) and Aerodynamic Particle Sizer (APS) were used to analyze particle size distribution and concentration of the aerosols generated from the products. To analyze the agglomeration status and fraction of nanoparticles in the produced aerosol, the researchers constructed an electrostatics-based collector to capture aerosolized particles on TEM grids. The device allows capturing wide particle size distribution as well as particles of only a certain size when used in conjunction with SMPS. Because the main focus of the project is the consumer exposures to silver and zinc compounds, all spray products (in liquid form) were analyzed using Inductively Coupled Plasma–Mass Spectrometry (ICP-MS) technique to determine mass concentrations of metal.
Nano-sized and micron-sized particles were produced by all three aerosolization methods, but their concentrations and size distributions showed dependence on the aerosolization method. The concentrations in the nano-sized region (1-100 nm) ranged from 102 to 106 particles/cm3 for Ag and 102 to 105 particles/cm3 for Zn products. Use of sprayers supplied with the products tended to produce higher concentration of particles above 1 μm and, equally, higher mass concentration of particles compared to the other two methods. These larger particles are likely nanoparticle agglomerates or aggregates with particles from product matrix.
Analysis of the airborne particles that were captured by an electrostatic collector and analyzed by TEM (1 silver and 1 zinc product) showed the presence of individual nano-sized particles and micron-sized agglomerates. These data confirm the measurements of airborne particles indicating that consumers using sprays with nanoparticles would be exposed to individual nanoparticles and their agglomerates.
The ICP-MS analysis of the products showed the presence of silver (Ag 107) in all sprays labeled as containing silver nanoparticles. The concentrations ranged from ~1 ppm to ~16 ppm. Presence of other metals also was detected. All five zinc products contained Zn 66 in concentrations ranging from 13 ppm to 97,000 ppm. Interestingly enough, all zinc products also contained Ag 107 at approximately 1 ppm concentration. These data were used to estimate mass concentration of airborne silver and zinc to which a consumer using the products would be exposed via inhalation.
The data described above serve as an input for Aim 8 of the project to model population-based exposure to silver and zinc MNPs due to consumer products. In addition, the data obtained in Aim 3 formed a basis for selecting silver and zinc products for in vitro and in vivo experiments and for interpreting results from in vitro and in vivo studies.
Aim 4 (Diesel Exhaust Characterization and Particle Collection):
The purpose of this aim was to collect and characterize diesel exhaust from an internal combustion engine combusting diesel fuels with varying amount of Envirox added to an ultralow sulfur diesel purchased in New Jersey. Envirox was added at 0, 0.1, 1, and 10 times the manufacturer recommended concentration, corresponding to 0, 0.9, 9.0, and 90 CeO2 μg/mL, respectively, in the fuel. A comprehensive characterization of gaseous and particulate emissions has been completed, resulting in a publication in Environmental Science & Technology. Under this aim, diesel exhaust particles (DEPs) also have been collected from burning the diesel fuel with various amount of Envirox added. In Year 3, DEP collection has been completed; and physicochemical properties of the DEP samples have been characterized. These DEP samples have been provided for in vitro and in vivo experiments.
Aim 5 (In Vitro Studies):
The in vitro studies have been conducted in parallel in two laboratories (the Tetley Lab in London and the Schwander Lab in New Jersey).
Tetley Lab
In vitro studies of raw ZnO Nanoparticles: effect on human alveolar cells. In these studies of raw ZnO nanoparticles (ZnO NP), we have exposed our human alveolar epithelial type-1-like cells (TT1) and primary human lung fibroblasts (HLF) to increasing concentrations (0.1-50 μg/mL) of 15 and 250 nm ZnO for up to 24 hours; ionic Zn (ZnCl2) was used as control. We studied viability using the MTT assay, mediator release using ELISA, superoxide generation (DFE), activation of MAPK signalling pathways (phosphorylation of P38, JnK and ErK MAP kinases), DNA damage and the effect of Curosurf, a pig surfactant, on cell death.
ZnO NP induced 50% cytoxicity in TT1 cells at 10 µg/mL and above, which was lethal at 24 hours. Pre-incubation with the antioxidants, 0.5 mM and 5 mM glutathione (GSH) and n-acetylcysteine (NAC), had a protective effect on ZnO NP toxicity; 5 mM GSH was most effective – completely restoring viability at 4 hours at ZnO NP concentrations above 10 µg/mL and partially restoring viability (~40-60%) at these doses at 24 hours. ZnO NPs displayed similar reaction kinetics to ZnCl2 (equimolar for Zn ion concentrations) implying that Zn ion dissolution is the main mechanism of toxicity. ZnO NPs induced super oxide formation in a concentration dependant manner at 2 and 4 hours in TT-1 cells and induced double stranded DNA damage. Pre-incubation of NPs with Curosurf had a mild protective effect on ZnO NP toxicity against TT-1 cells at 4 hours, although there was no significant protective effect of Curosurf at 24 hours.
Sub-lethal doses of ZnO NP and ZnCl2 (below 10 µg/mL) stimulated release of IL-6 and IL-8 from TT-1 cells in a dose-dependent manner at both 4 and 24 hours. When TT-1 cells were pre-incubated with the antioxidants GSH or NAC, the reduced release of IL-6 and IL-8 (due to cell death) at ZnO NP exposure concentrations above 10 µg/mL returned to levels of stimulation previously seen with sub-lethal (less than 10 µg/mL) doses of ZnO NPs. 10 µg/mL ZnO NP and equimolar ZnCl2 activate P38, JnK and ErK MAP kinases, important signalling molecules in production of cytokine and chemokine mediators, in TT-1 cells, suggesting activation of MAPK as one mechanism of enhanced mediator production. However, inhibition of P38, JnK or ErK MAP kinases had no significant effect on ZnO NP TT1 cell cytotoxicity.
HLF cells were slightly more resistant to ZnO NP toxicity, thus at the same threshold dose of 10 µg/mL and above, the most marked reduction in viability was ~50% at 24 hours following exposure. ZnO NP stimulated a dose-dependent release of IL-8 and IL-6 by HLF cells at 4 and 24 hours. This work suggests that dissolution of ZnO NP could contribute to its toxicity. Enhanced TT1 cell mediator release was accompanied by enhanced MAPK phosphorylation/activity.
Interestingly, while antioxidant treatment could prevent cytotoxicity and prevented increased mediator production, specific inhibition of MAPK had no effect, suggesting different mechanistic processes involved in ZnO NP-stimulated inflammatory mediator release. HLF were more resistant to the effects of ZnO NP.
Effects of diesel exhaust particles (DEP) generated in the absence and presence of Envirox on human alveolar cells in vitro. DEP were generated using the same engine and fuel containing no Envirox and Envirox at concentrations 0.1x, 1x and 10x that used in the UK. TT1 cells and HLF were exposed to increasing concentrations of each sample (10-100 μg/mL) for up to 72 hours. We measured cell viability (MTT), superoxide formation (DFE), inflammatory mediator release (ELISA), MAPK activation. There was no significant change in TT1 cell viability with either increasing DEP concentration (up to 100 μg/mL) or increasing Envirox concentration at 24 and 72 hours, and there was no DNA damage at 2 hours. Nevertheless, there was an increase in IL-6 and IL-8 release, particularly with 0x and 0.1x Envirox, which fell as the concentration of Envirox increased, but not to the level of untreated control cells. 100 μg/mL of DEP with low levels of Envirox (0x and 0.1x) activated P38, ErK and JnK MAPK at 2 hours, probably triggering the observed inflammatory mediator release, and thus MAPK activation also fell with increasing concentrations of Envirox.
DEP generated with 0x and 0.1x Envirox induced a dose-dependent increase in superoxide formation in TT-1 cells at 4 hours. This effect was significantly supressed with increasing concentrations of Envirox, so that there was little or no superoxide production detected at a high dose (100 μg/mL) of DEP in the presence of 1x and 10x Envirox.
Using HLF, similarly, there was no loss of viability in DEP/Envirox exposed HLF cells at 24 and 72 hours. Indeed, increased levels of mitochondrial activity (MTT viability assay) at high concentrations of Envirox were detected at 72 hours, which was shown to be due to increased levels of fibroblast proliferation. 100 μg/mL DEP inhibited collagen production by HLF cells at 72 hours, which was not related to the concentration of Envirox.
Effect of nanoparticles in consumer products on human alveolar cells in vitro. A number of products were originally chosen for characterization. Subsequently, four products, two silver containing and two zinc containing were chosen for in vitro studies of TT1 cells. Each product was studied as the whole product, the particle-containing fraction and non-particle containing fraction (following the establishment of a suitable protocol to do this; these fractions currently are being further characterized at Rutgers University). We have performed some preliminary cytotoxicity studies using a range of dilutions from the original product. The actual concentrations of nanoparticles and other reagents will be determined following full characterization.
None of the Mesosilver fractions had an effect on TT1 cell viability at 24 hours, at original product dilution ranges of 1/100 to 1/10. Neither were there any changes in pro-inflammatory cytokine release using these Mesosilver fractions. In contrast, Nanofix was extremely cytotoxic to TT-1 cells at dilutions from 1/100 to 1/10. This effect was partly due to the nanoparticles and partly due to other components in the suspension medium of the product. There were significantly increased levels of IL-6 and IL-8 production at all dose ranges observed in the nanoparticle-containing and particle-free fractions of Nanofix.
TheraZinc and Dermazinc displayed severe toxicity to TT-1 cells at all dilution ranges. This effect appears to be mainly due to the particle-free fraction and only partly due to the nanoparticle-containing fraction of the product at low dilutions (1/20 and 1/10; i.e., higher concentrations of product).
Chung Lab
Summary of toxicity of silver nanoparticles, wires and silver salts to primary human airway smooth muscle cells (ASM). Primary airway smooth muscle (ASM) samples were obtained from five individual patients and grown as a monolayer in either 6 or 96 well plates in complete DMEM. The media contained essential amino acids, glutamine and FBS. The cells were serum starved for 24 hours prior to exposure to the vehicle control (particle free), a panel of silver nanoparticle suspensions (AgNP) or a silver nitrate solution for 4, 24 and 72 hours in serum free medium. The panel of silver nanoparticles consisted of suspensions of 20 nm AgNP sphere (citrate coated), 50 nm AgNP sphere (citrate coated), short nanowires (S-NW, PVP coated, 1.3 μm long) and long nanowires (L-NW, PVP coated, 14 μm long). Cells were exposed to 5 or 25 μg/mL of these suspensions (denoted low and high dose, respectively). Cells also were exposed to two dilutions of a silver nitrate solution, either 25 μg/mL in SF media, or 1% of a 25 μg/mL AgNO3 solution, to represent the typical rate of dilution of the nanowires in the SF DMEM over 72 hours (as characterized previously at Imperial College using ICP-MS).
Results show that ASM viability (MTS assay) did not change following exposure to any of the nanoparticle treatments relative to the particle-free control at any time point, while high dose AgNO3, caused a 30-40% loss in viability at 24 and 72 hours. Effect on cellular proliferation also was assessed with the (BrdU) assay, which measures the rate of incorporation of 5-bromo-2’-deoxyuridine into cellular DNA during cell proliferation. There was a small drop in the level of BrdU incorporation at 72 hours for 20 and 50 nm citrate spheres and also nearly 100% drop for high dose AgNO3 solution. We performed a Western blot to quantify the presence of phosphorylated H2AX (indicating DNA damage) and cleavage of PARP (apoptosis marker). There was no change in PARP cleavage for any of the treatments at any of the time points. High dose Ag+ was not tested in this assay as it kills most of the cells. There also was no increase in H2AX phosphorylation or PARP cleavage at 4 and 24 hours for the particles. However, at 72 hours, there was a trend towards an increase in H2AX phosphorylation for 20 and 50 nm Ag Spheres only, although this was not statistically significant with several of the patients not responding. There also was an increase in mitochondrial ROS (JC-1) at 72 hours for the 20 and 50 nm Ag Spheres, however more n numbers need to be obtained for this marker as only n=3 so far. We also quantified the level s of the inflammatory cytokines IL-6 and Il-8 in the supernatant and only saw an increase in the levels following treatment with AgNO3. We also have taken TEM images of the interaction of the ASM cells with the AgNP and Ag+ ions and showed uptake and penetration of the wires through the cellular membranes as well as sulfidation of the nanoparticles in the cells. We also are investigating if H2S producing enzymes (CBS/CSE and MST) play a role in the sulfidation of the internalized AgNP. In addition, we are in the process of measuring lipid peroxidation (MDA) and the DNA damage marker, 8-OHdG in the cell supernatant.
Schwander Lab
In Year 3, studies focused on bioreactivity assessments of (1) DEPs from a normal diesel fuel (DEP) and the impact of the diesel additive Envirox (CeO2) on the bioreactivity in our cell model systems and (2) consumer product-derived Ag and Zn nanoparticles.
Cytotoxicity and bioreactivity of DEP in peripheral blood mononuclear cells (PBMC). Metabolic activity of cells (proportional to the number of viable cells) was evaluated by MTS assay (Figure 1). PBMC from healthy donors exposed to no particles served as control. No significant effect on the viability of PBMC was observed at any of the dosages used (0, 1, 10 μg/mL) by MTS assay.
To assess bioreactivity of DEPs in PBMC, frequencies of cytokine (IL-1ß and TNF-α [IFN-γ and IL-6 not shown])-producing cells as well as IL1b, TNFa, IL6 and IL8 mRNA expression were assessed in PBMC by ELISPOT assays and qRT-PCR, respectively (Figure 2). We found that PBMC in vitro exposure to all the DEPs (resulting from diesel with varying amount of Envirox added) resulted in very limited bioreactivity with the exception of that induced by the DEP emitted from diesel containing 0.1x Envirox. We are investigating the reasons why DEP from 0.1x Envirox fuel showed significantly higher bioreactivity compared to the DEP from diesel containing other amount of Envirox.
Effects of consumer product (Mesosilver, Nanofix, Derma Zinc, Thera Zinc) -derived Ag and Zn nanoparticles and suspension fractions thereof (whole product, NP-free and Concentrated NP fractions) on toxicity and IL-1β and TNF-α expression (ELISPOT assays) in human PBMC. In toxicity studies, PBMC were exposed to different fractions of Meso Silver (upper panel) and Nanofix Silver (lower panel) at 1:10, 1:20, 1:40, 1:80 dilution for 24 hours (at 37ºC in a humidified 5% CO2 environment). PBMC cultured in complete culture media without NP exposure (none) were used as unexposed controls. Cell viability was measured by MTS assay. The results are summarized in Table 1. Because of the extreme toxic nature of some of the products, we currently are testing their bioreactivity with further dilutions (50-400 fold).
Table 1: Toxicity and Bioreactivity of Consumer Product-Derived NPs in Human PBMC
Spark generated silver inhalation. We have completed our study of the inhalation of spark-generated silver nanospheres. Sprague-Dawley and Brown-Norway rats (n = 6-12 per group) were exposed by nose only inhalation to air only or spark generated silver (1.1 mg/m3) for either 3 hours only or for 3 hours on 4 consecutive days. Deposited silver in the lungs of the rats was estimated, by MMAD model, to be 0, 10 or 30 µg, respectively. Rats were returned to their home cages for 1 or 7 days following which lung function testing by the forced oscillation technique was conducted. All Ag NP exposed rats had a dose-dependent increase in total BAL cells, neutrophils and macrophages at 1 day following exposure. BN rats also had an increase in BAL eosinophils. BN rats also had altered lung mechanics at the highest dose with increased resistance in the large airway (Rn) and peripheral lung tissues (tissue elastance, H). Additionally, total protein and MDA in the BAL increased for both rat strains. Responses were transient and for the majority of parameters, had returned to baseline levels by 7 days post inhalation. Further analysis such as multiplex analysis BAL chemokines and cytokines, histology and surfactant activity of BAL fluid are being completed. Gene arrays have been performed on the lungs and results will be analyzed.
Diesel exhaust particles (DEP)/DEP+Envirox instillation.We also have completed our study of the DEP particles collected from a diesel motor engine using diesel fuel containing the recommended amounts of cerium oxide (Envirox at 1X), and at other concentrations of 0.1X and 10X recommended amount). These DEPs are denoted as 0X, 0.1X, 1X and 10X. The DEP on the filters were extracted into endotoxin free water, using sonication in an ice water bath for 16 hours, to give final concentrations ranging between 1,450 and 1,780 µg/mL. All suspensions were diluted to a final concentration of 1450 µg/mL prior to exposure in mice. C57BL/6 mice n = 6-9 per group, were anaesthetized by isofluorane inhalation and immediately exposed to 50 µL of DEP in suspensions by intra-tracheal instillation, which is equivalent to 73 µg of DEP per mouse. Anaesthetized mice were hung by their incisors on a wire attached to a board at an angle of 45°. Non-surgical instillations of 50 µL of the 0X, 0.1X, 1X or 10X DEP suspensions in water, were made directly into the trachea, via a 22-gauge gavage ball tipped needle bent at an angle, followed by 100 µL of air to aid distribution of the liquid in the lung. Similarly, instillations of 50 µL vehicle only (water) also were performed in parallel. Mice were returned to their home cages for either 1 or 7 days after the instillation, following which lung function testing by the forced oscillation technique was conducted. All mice receiving 0X, 0.1X, 1X or 10X DEP, had significant increases in total BAL cells, neutrophils and macrophages compared with the vehicle only mice. However, as the concentration of Envirox in the fuel increased, the number of total cells decreased, mainly due to a reduction in the number of macrophages. Mice instilled with 10X DEP also showed an improvement in lung mechanics (Rn, H and AHR) compared with the mice instilled with 0X. Total cells were much lower at 7 days compared with 1 day for all DEP exposed mice, although numbers were still significantly higher than the vehicle only group. There was a still small but significant reduction in cellular infiltrate for the mice receiving the DEP made with higher concentrations of Envirox (10X). However, lung mechanics had returned to baseline levels by 7 days post exposure for all mice.
Effect of nanotechnology-enabled consumer silver sprays inhaled in rats. Mesosilver is a commercially available cleaning product, which contains ‘silver nanoparticles suspended in water’. Brown Norway rats (n= 6 per group) were exposed by nose only inhalation to air only or to the nebulized Mesosilver product for 3 hours on 4 consecutive days. Two nebulizers were used and concentrations in the air were around (0.3 mg/m3). Deposited silver in the lungs of the rats was estimated, by the MMAD model, to be approximately 10-20 µg. Final estimate calculations currently are being completed. Rats were returned to their home cages for 1 or 7 days following which lung function testing by the forced oscillation technique was conducted. There were significant differences in the means of the number of total BAL cells for the four groups tested and neutrophilla for some groups. The study is blinded and codes are not broken at this time. However, it can be said the Mesosilver is less inflammatory than similar doses of the spark generated silver or the instilled silver from our previous studies. Analysis of BAL parameters including differential cells, total protein, MDA, multiplex analysis for chemokines and cytokines, surfactant proteins and histology as well as analysis of lung function data currently are being completed for this study. The inhalation toxicity of the Mesosilver product will be compared with a commercial product containing zinc nanoparticles.
Aim 7 (Ecotoxicity Studies):
This is the first year of work for Aim 7 under the direction of Dr. Di Giulio. This aim was designed to assess via fish embryo and larval assays the toxicity and bioavailability of consumer nanoparticles selected earlier by other working groups. Embryos and larvae of the freshwater zebrafish (Danio rerio) were used as initial screening subjects with follow up testing of significantly toxic products to be done in embryos and larvae of the Atlantic killifish (Fundulus heteroclitus). Initial experiments focused entirely on diesel emission particles (DEPs, batch 6) produced from diesel fuel spiked with various concentrations (0X, 0.1X, 1X, and 10X the manufacturer’s recommended concentration) of Envirox and performed both developmental toxicity assays and gene expression assays. All exposures were performed in 30% Danieau zebrafish medium, which has elevated ionic strength over pure water. Consequently, almost all of the DEPs fell out of suspension within 1 hour of dosing, thus potentially elevating the local concentration of DEPs at the bottom of the well where the embryos and hatchlings were located.
Developmental toxicity assays were performed with zebrafish embryos that were dosed intact at 4-5 hours post fertilization (hpf), dechorionated and dosed at 24 hpf, or allowed to hatch and dosed at 5 dpf. Acute mortality, hatching, swim bladder inflation, cardiac and yolk edema, and skeletal deformities were monitored every 24 hr for up to 144 hours. 0X DEP was significantly more acutely lethal to dechorionated embryos than 10X DEP; however, mortality barely reached 40% even at up to 125 μg/mL and no difference in the rate of deformity was seen among any of the treatments. No significant differences were seen in any recorded endpoint with intact embryos or hatchlings, suggesting that the zebrafish embryonic chorion is an effective barrier to DEP toxicity and hatchlings are more resistant than embryos. Cytochrome P450 activity (via in ovo EROD assay) in intact embryos showed no significant differences among DEP treatments both with and without exposure to UV light. This may be due to CYP inhibitor PAHs such as fluoranthene present in all of the DEP suspensions.
Due to the presence of several PAHs as well as CeO2 in the DEP preparation, the expression of genes involved with PAH toxicity and the AHR pathway (CYP1a), oxidative stress and the NRF2 pathway (NRF2, MnSOD, CuZnSOD, HMOX1, NQO1, GSTp2, GCLC, GCLM) and metal toxicity (zMT) were measured with QPCR visualized with SYBR green. Intact or dechorionated embryos were exposed to 50 μg/mL DEP at 4-5 hpf and dechorionated (if needed) and flash frozen at 12 or 24 hr. The NRF2 pathway genes MnSOD, CuZnSOD, and GPx1 were generally upregulated with 10X DEP the highest and 0X the second highest. However, with the remaining antioxidant genes tested (NRF2, GCLC, NQO1, HMOX1, and zMT), 1X DEP showed the highest expression with often significantly downregulated expression with 0x and 0.1X DEP exposure. The AHR pathway gene CYP1a were downregulated upon exposure to 0X, 0.1X, and 10X, but not 1X DEP.
No significant differences in gene expression were seen between exposed intact or dechorionated embryo or between 12 and 24 hr exposures, though variability within treatments was high. Taken together, DEP appears to influence the NRF2 antioxidant and AHR pathways with mixed effects of added Envirox concentration.
Aim 8 (Refinement of Computational Modules):
This aim was initiated in Year 2 and involves the refinement of the risk assessment framework by parameterization of computational modules developed as part of Aim 1 with data collected from Aims 2–7. We currently are modifying the already developed modules addressing the specific experimental scenarios from Aims 2 through 6. We:
- Incorporated additional measurements of particle size distributions of consumer nanospray products (exposure simulation studies of Aim 3) into the population exposure modules of nAg.
- Parameterized particle size distributions of DEP for the exposure modules of CeO2 according to data from Aim 4.
Future Activities:
Aim 1 Plans for Year 4:
This information is incorporated in the plans for Aim 8.
Aim 2 Plans for Year 4:
The major tasks for the final year of Aim 2 include (i) to test whether there is a threshold length for uptake and toxicity of ZnO nanowires by type 1 lung epithelial cells (ii) to continue providing sample characterizations when needed by other aims, and (iii) to complete data analysis and prepare manuscripts for publications in the peer-reviewed literature.
Aim 3 Plans for Year 4:
The major task for the final year of Aim 3 is to complete data analysis and prepare manuscripts for publications in the peer-reviewed literature. Complete more detailed studies using TEM, SEM, Zeta sizer and other characterizations will be conducted to correlate the physical chemical properties of MNPs in cloths with their biological and environmental effects.
Aim 4 Plans for Year 4:
We plan to complete a manuscript reporting physicochemical properties of DEPs emitted from burning diesel with and without CeO2 nanoparticles (Envirox) added.
Aim 5 Plans for Year 4:
DEP in vitro experiments have been completed so we plan to complete manuscripts reporting findings from these experiments. We will complete in vitro studies with the consumer products in the first half of the final project year, complete data analysis, and then prepare manuscripts. The Tetley lab will finalize studies of effects of zinc oxide in TT1 cells and HLF studies on DEP/Envirox. The Schwander lab will continue assessing the effects of consumer product-derived nanoparticles on cellular toxicity and bioreactivity in primary human immune cells (blood and lung cells). The Chung lab will complete the toxicity and TEM studies of silver nanoparticles in primary airway smooth muscle cells and initiate mechanistic studies on the role of sulfidation of internalized AgNPs.
Aim 6 Plans for Year 4:
We are in the process of completing the DEP instillation study and will complete a manuscript on this study in the final year. We plan to complete the AgNP consumer spray in vivo exposure experiments and will compare the results from the effects of AgNP in consumer products and the effects of AgNP generated via the spark generation method. We plan to initiate studies of Zn consumer products.
Aim 7 Plans for Year 4:
We are wrapping up the DEP experiments and are ready to start consumer product materials experiments. In the final project year, we plan to complete a manuscript on DEP experiments results and prepare a manuscript on the consumer product Ag and/or ZnO results.
Aim 8 Plans for Year 4:
The major task of this Aim in the final year is to facilitate the integration of all project results by using the modeling system and its modules that have been optimized in the previous years. We plan to submit several manuscripts led by the modeling team.
Journal Articles on this Report : 12 Displayed | Download in RIS Format
| Other project views: | All 47 publications | 28 publications in selected types | All 28 journal articles |
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Chen S, Theodorou IG, Goode AE, Gow A, Schwander S, Zhang JJ, Chung KF, Tetley TD, Shaffer MS, Ryan MP, Porter AE. High-resolution analytical electron microscopy reveals cell culture media-induced changes to the chemistry of silver nanowires. Environmental Science & Technology 2013;47(23):13813-13821. |
R834693 (2014) R834693 (2015) R834693 (Final) |
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Chen S, Goode AE, Sweeney S, Theodorou IG, Thorley AJ, Ruenraroengsak P, Chang Y, Gow A, Schwander S, Skepper J, Zhang JJ, Shaffer MS, Chung KF, Tetley TD, Ryan MP, Porter AE. Sulfidation of silver nanowires inside human alveolar epithelial cells: a potential detoxification mechanism. Nanoscale 2013;5(20):9839-9847. |
R834693 (2014) R834693 (2015) R834693 (Final) |
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Leo BF, Chen S, Kyo Y, Herpoldt KL, Terrill NJ, Dunlop IE, McPhail DS, Shaffer MS, Schwander S, Gow A, Zhang J, Chung KF, Tetley TD, Porter AE, Ryan MP. The stability of silver nanoparticles in a model of pulmonary surfactant. Environmental Science & Technology 2013;47(19):11232-11240. |
R834693 (2014) R834693 (2015) R834693 (Final) |
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Nazarenko Y, Zhen H, Han T, Lioy PJ, Mainelis G. Potential for inhalation exposure to engineered nanoparticles from nanotechnology-based cosmetic powders. Environmental Health Perspectives 2012;120(6):885-892. |
R834693 (2011) R834693 (2012) R834693 (2013) R834693 (2014) R834693 (2015) R834693 (Final) |
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Nazarenko Y, Zhen H, Han T, Lioy PJ, Mainelis G. Nanomaterial inhalation exposure from nanotechnology-based cosmetic powders: a quantitative assessment. Journal of Nanoparticle Research 2012;14(11):1229 (14 pp.). |
R834693 (2014) R834693 (2015) R834693 (Final) |
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Nazarenko Y, Lioy PJ, Mainelis G. Quantitative assessment of inhalation exposure and deposited dose of aerosol from nanotechnology-based consumer sprays. Environmental Science: Nano 2014;1(2):161-171. |
R834693 (2013) R834693 (2014) R834693 (2015) R834693 (Final) |
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Royce SG, Mukherjee D, Cai T, Xu SS, Alexander JA, Mi Z, Calderon L, Mainelis G, Lee K, Lioy PJ, Tetley TD, Chung KF, Zhang J, Georgopoulos PG. Modeling population exposures to silver nanoparticles present in consumer products. Journal of Nanoparticle Research 2014;16(11):2724. |
R834693 (2013) R834693 (2014) R834693 (2015) R834693 (Final) |
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Sarkar S, Zhang L, Subramaniam P, Lee KB, Garfunkel E, Strickland PA, Mainelis G, Lioy PJ, Tetley TD, Chung KF, Zhang J, Ryan M, Porter A, Schwander S. Variability in bioreactivity linked to changes in size and zeta potential of diesel exhaust particles in human immune cells. PLoS ONE 2014;9(5):e97304 (12 pp.). |
R834693 (2014) R834693 (2015) R834693 (Final) |
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Seiffert J, Hussain F, Wiegman C, Li F, Bey L, Baker W, Porter A, Ryan MP, Chang Y, Gow A, Zhang J, Zhu J, Tetley TD, Chung KF. Pulmonary toxicity of instilled silver nanoparticles: influence of size, coating and rat strain. PLOS ONE 2015;10(3):e0119726 (17 pp.). |
R834693 (2013) R834693 (2014) R834693 (2015) R834693 (Final) |
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Seiffert J, Buckley A, Leo B, Martin NG, Zhu J, Dai R, Hussain F, Guo C, Warren J, Hodgson A, Gong J, Ryan MP, Zhang JJ, Porter A, Tetley TD, Gow A, Smith R, Chung KF. Pulmonary effects of inhalation of spark-generated silver nanoparticles in Brown-Norway and Sprague-Dawley rats. Respiratory Research 2016;17(1):85 (15 pp.). |
R834693 (2014) R834693 (2015) R834693 (Final) |
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Subramaniam P, Lee SJ, Shah S, Patel S, Starovoytov V, Lee K-B. Generation of a library of non-toxic quantum dots for cellular imaging and siRNA delivery. Advanced Materials 2012;24(29):4014-4019. |
R834693 (2011) R834693 (2012) R834693 (2013) R834693 (2014) R834693 (2015) R834693 (Final) |
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Zhang J, Nazarenko Y, Zhang L, Calderon L, Lee KB, Garfunkel E, Schwander S, Tetley TD, Chung KF, Porter AE, Ryan M, Kipen H, Lioy PJ, Mainelis G. Impacts of a nanosized ceria additive on diesel engine emissions of particulate and gaseous pollutants. Environmental Science & Technology 2013;47(22):13077-13085. |
R834693 (2013) R834693 (2014) R834693 (2015) R834693 (Final) |
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Supplemental Keywords:
Consumer products, manufactured nanoparticles, diesel exhaust particles, inhalation exposureProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
- Final Report
- 2015 Progress Report
- 2013 Progress Report
- 2012 Progress Report
- 2011 Progress Report
- Original Abstract
28 journal articles for this project