Direct Stimulation Of Human Fibroblasts By nCeO2 In Vitro Is Attenuated With An Amorphous Silica Coating

Background: Nano-scaled cerium oxide (nCeO2) is used in a variety of applications, including use as a fuel additive, catalyst, and polishing agent, yet potential adverse health effects associated with nCeO2 exposure remain incompletely understood. Given the increasing utility and demand for engineered nanomaterials (ENMs) such as nCeO2, “safety-bydesign” approaches are currently being sought, meaning that the physicochemical properties (e.g., size and surface chemistry) of the ENMs are altered in an effort to maximize functionality while minimizing potential toxicity. In vivo studies have shown in a rat model that inhaled nCeO2 deposited deep in the lung and induced fibrosis. However, little is known about how the physicochemical properties of nCeO2, or the coating of the particles with a material such as amorphous silica (aSiO2), may affect the bio-activity of these particles. Thus, we hypothesized that the physicochemical properties of nCeO2 may explain its potential to induce fibrogenesis, and that a nano-thin aSiO2 coating on nCeO2 may counteract that effect. Results: Primary normal human lung fibroblasts were treated at occupationally relevant doses with nCeO2 that was either left uncoated or was coated with aSiO2 (amsCeO2). Subsequently, fibroblasts were analyzed for known hallmarks of fibrogenesis, including cell proliferation and collagen production, as well as the formation of fibroblastic nodules. The results of this study are consistent with this hypothesis, as we found that nCeO2 directly induced significant production of collagen I and increased cell proliferation in vitro, while amsCeO2 did not. Furthermore, treatment of fibroblasts with nCeO2, but not amsCeO2, significantly induced the formation of fibroblastic nodules, a clear indicator of fibrogenicity. Such in vitro data is consistent with recent in vivo observations using the same nCeO2 nanoparticles and relevant doses. This effect appeared to be mediated through TGFβ signaling since chemical inhibition of the TGFβ receptor abolished these responses. Conclusions: These results indicate that differences in the physicochemical properties of nCeO2 may alter the fibrogenicity of this material, thus highlighting the potential benefits of “safety-by-design” strategies. In addition, this study provides an efficient in vitro method for testing the fibrogenicity of ENMs that strongly correlates with in vivo finding

Advanced computational modeling for in vitro nanomaterial dosimetry

Background: Accurate and meaningful dose metrics are a basic requirement for in vitro screening to assess potential health risks of engineered nanomaterials (ENMs). Correctly and consistently quantifying what cells “see,” during an in vitro exposure requires standardized preparation of stable ENM suspensions, accurate characterizatoin of agglomerate sizes and effective densities, and predictive modeling of mass transport. Earlier transport models provided a marked improvement over administered concentration or total mass, but included assumptions that could produce sizable inaccuracies, most notably that all particles at the bottom of the well are adsorbed or taken up by cells, which would drive transport downward, resulting in overestimation of deposition. Methods: Here we present development, validation and results of two robust computational transport models. Both three-dimensional computational fluid dynamics (CFD) and a newly-developed one-dimensional Distorted Grid (DG) model were used to estimate delivered dose metrics for industry-relevant metal oxide ENMs suspended in culture media. Both models allow simultaneous modeling of full size distributions for polydisperse ENM suspensions, and provide deposition metrics as well as concentration metrics over the extent of the well. The DG model also emulates the biokinetics at the particle-cell interface using a Langmuir isotherm, governed by a user-defined dissociation constant, KD, and allows modeling of ENM dissolution over time. Results: Dose metrics predicted by the two models were in remarkably close agreement. The DG model was also validated by quantitative analysis of flash-frozen, cryosectioned columns of ENM suspensions. Results of simulations based on agglomerate size distributions differed substantially from those obtained using mean sizes. The effect of cellular adsorption on delivered dose was negligible for KD values consistent with non-specific binding (> 1 nM), whereas smaller values (≤ 1 nM) typical of specific high-affinity binding resulted in faster and eventual complete deposition of material. Conclusions: The advanced models presented provide practical and robust tools for obtaining accurate dose metrics and concentration profiles across the well, for high-throughput screening of ENMs. The DG model allows rapid modeling that accommodates polydispersity, dissolution, and adsorption. Result of adsorption studies suggest that a reflective lower boundary condition is appropriate for modeling most in vitro ENM exposures. Electronic supplementary material The online version of this article (doi:10.1186/s12989-015-0109-1) contains supplementary material, which is available to authorized users

Linking the Exposure to Engineered Nanoparticles Released From Nano-Enabled Products to Toxicology: a Case Study of Laser Printers

A research gap in the fields of exposure assessment and toxicology that remains unaddressed is the assimilation of experimental conditions to those of the real world human exposure. Currently, there is a lack of understanding of the properties of particles released by nano-enabled products (NEPs). Thus, we designed a multi-tiered methodology to physico-chemically, morphologically and toxicologically characterize engineered nanomaterials (ENMs) released from NEPs (i.e., toner powder). It is well established that printers emit nanoparticles during their operation; however, the physico-chemical and toxicological characterization of real world printer-emitted nanoparticles (PEPs) remains incomplete, hampering proper risk assessment efforts. For example, a number of studies estimating the potential adverse effects of PEPs used bulk toner particles as test particles rather than the actual particulate matter released by laser printers. Thus, the public health implications of exposure to PEPs remain largely unknown. For this project, a printer exposure generation system suitable for the subsequent physico-chemical, morphological, and toxicological characterization of PEPs was developed and used to assess the properties of particulate matter released from the use of commercially available laser printers. The system consists of a glovebox type environmental chamber for uninterrupted printer operation, real-time and time-integrated particle sampling instrumentation for size fractionation and sampling of PEPs and an exposure chamber for inhalation toxicological studies. Results from our extensive analysis show that laser printers emit up to 1,300,000 particles/cm3, most of which are nanoparticles. Further, we confirmed that a number of ENMs incorporated into toner formulations (e.g., silica, alumina, titania, ceria,) become airborne during printing. Both in vitro and in vivo toxicological evaluation showed PEPs are biologically reactive and may cause significant cytotoxicity, membrane integrity damage, reactive oxygen species production, pro-inflammatory cytokine release, angiogenesis, cytoskeletal and epigenetic changes as well as lung inflammation. This work highlights the importance of understanding life-cycle nano environmental health and safety implications of NEPs and assessing real world exposures and their associated toxicological properties rather than focusing on ‘‘raw’’ materials used in the synthesis of an NEP. Such analysis can be achieved for pollutants emitted by any NEP by employing the multi-tiered methodology described in this dissertation.Environmental Healt

Effects of Laser Printer–Emitted Engineered Nanoparticles on Cytotoxicity, Chemokine Expression, Reactive Oxygen Species, DNA Methylation, and DNA Damage: A Comprehensive in Vitro Analysis in Human Small Airway Epithelial Cells, Macrophages, and Lymphoblasts

Background: Engineered nanomaterials (ENMs) incorporated into toner formulations of printing equipment become airborne during consumer use. Although information on the complex physicochemical and toxicological properties of both toner powders and printer-emitted particles (PEPs) continues to grow, most toxicological studies have not used the actual PEPs but rather have primarily used raw toner powders, which are not representative of current exposures experienced at the consumer level during printing. Objectives: We assessed the biological responses of a panel of human cell lines to PEPs. Methods: Three physiologically relevant cell lines—small airway epithelial cells (SAECs), macrophages (THP-1 cells), and lymphoblasts (TK6 cells)—were exposed to PEPs at a wide range of doses (0.5–100 μg/mL) corresponding to human inhalation exposure durations at the consumer level of 8 hr or more. Following treatment, toxicological parameters reflecting distinct mechanisms were evaluated. Results: PEPs caused significant membrane integrity damage, an increase in reactive oxygen species (ROS) production, and an increase in pro-inflammatory cytokine release in different cell lines at doses equivalent to exposure durations from 7.8 to 1,500 hr. Furthermore, there were differences in methylation patterns that, although not statistically significant, demonstrate the potential effects of PEPs on the overall epigenome following exposure. Conclusions: The in vitro findings obtained in this study suggest that laser printer–emitted engineered nanoparticles may be deleterious to lung cells and provide preliminary evidence of epigenetic modifications that might translate to pulmonary disorders. Citation Pirela SV, Miousse IR, Lu X, Castranova V, Thomas T, Qian Y, Bello D, Kobzik L, Koturbash I, Demokritou P. 2016. Effects of laser printer–emitted engineered nanoparticles on cytotoxicity, chemokine expression, reactive oxygen species, DNA methylation, and DNA damage: a comprehensive in vitro analysis in human small airway epithelial cells, macrophages, and lymphoblasts. Environ Health Perspect 124:210–219; http://dx.doi.org/10.1289/ehp.140958

Short-term exposure to engineered nanomaterials affects cellular epigenome

<p>Extensive incorporation of engineered nanomaterials (ENMs) into industrial and biomedical applications increases the risks of exposure to these potentially hazardous materials. While the geno- and cytotoxic effects of ENMs have been investigated, the potential of ENMs to target the cellular epigenome remains largely unknown. Our goal was to determine whether industry relevant ENMs can affect the epigenome at low cytotoxic doses. A panel of cells relevant to inhalation exposures such as human and murine macrophages (THP-1 and RAW264.7, respectively) and human small airway epithelial cells (SAEC) were exposed to printer-emitted engineered nanoparticles (PEPs), mild steel welding fumes (MS-WF), copper oxide (CuO) and titanium dioxide nanoparticles. Toxicological effects, including cytotoxicity, oxidative stress and inflammatory responses were assessed, taking into consideration <i>in vitro</i> dosimetry. The effects of ENMs on cellular epigenome were determined by addressing the global and transposable elements (TEs)-associated DNA methylation and expression of DNA methylation machinery and TEs. The percentage of ENMs-induced cytotoxicity for all cell lines was in the range of 0–15%. Oxidative stress was evident in SAEC after exposure to PEPs and in THP-1 when exposed to CuO. In addition, exposure to ENMs resulted in modest alterations in DNA methylation of two most abundant TEs in mammalian genomes, LINE-1 and <i>Alu</i>/SINE, their transcriptional reactivation, and decreased expression of DNA methylation machinery in a cell-, dose- and ENM-dependent manner. These results indicate that exposure to ENMs at environmentally relevant concentrations, aside from the geno- and cytotoxic effects, can also affect the epigenome of target cells.</p

Synergistic effects of engineered nanoparticles and organics released from laser printers using nano-enabled toners: Potential health implications from exposures to the emitted organic aerosol

Recent studies have shown that engineered nanoparticles (ENPs) are incorporated into toner powder used in printing equipment and released during their use. Thus, understanding the functional and structural composition and the potential synergistic effects of this complex aerosol and released gaseous co-pollutants is critical in assessing their potential toxicological implications and risks. In this study, toner powder and PEPs were thoroughly examined for the functional and molecular composition of the organic fraction and the concentration profile of 16 Environmental Protection Agency (EPA)-priority polycyclic aromatic hydrocarbons (PAH) using state-of-the-art analytical methods. Results show significant differences in abundance of the non-exchangeable organic hydrogen of toner powder and PEPs, with a stronger aromatic spectral signature in PEPs. Changes in the structural composition of PEPs are indicative of radical additions and free-radical polymerization favored by catalytic reactions, resulting in formation of functionalized organic species. Particularly, accumulation of aromatic carbons with strong styrene-like molecular signatures on PEPs is associated with formation of semi-volatile heavier aromatic species (i.e., PAHs). Further, the transformation of low molecular weight PAHs in the toner powder to high molecular weight PAHs in PEPs was documented and quantified. This may be a result of synergistic effects from catalytic metal/metal oxide ENPs incorporated into the toner and the presence/release of semi-volatile organic species (SVOCs). The presence of known carcinogenic PAHs on PEPs raises public health concerns and warrants further toxicological assessment