Sandra V. Pirela

Advanced computational modeling for in vitro nanomaterial dosimetry.

BackgroundAccurate 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.MethodsHere 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.ResultsDose 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.ConclusionsThe 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.

Short-term exposure to engineered nanomaterials affects cellular epigenome

Abstract 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 in vitro 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 Alu/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.

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

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

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.1409582

Consumer exposures to laser printer-emitted engineered nanoparticles: A case study of life-cycle implications from nano-enabled products

Abstract It is well established that printers emit nanoparticles during their operation. To-date, however, the physicochemical and toxicological characterization of “real world” printer-emitted nanoparticles (PEPs) remains incomplete, hampering proper risk assessment efforts. Here, we investigate our earlier hypothesis that engineered nanomaterials (ENMs) are used in toners and ENMs are released during printing (consumer use). Furthermore, we conduct a detailed physicochemical and morphological characterization of PEPs in support of ongoing toxicological assessment. A comprehensive suite of state of the art analytical methods and tools was employed for the physicochemical and morphological characterization of 11 toners widely utilized in printers from major printer manufacturers and their PEPs. We confirmed that a number of ENMs incorporated into toner formulations (e.g. silica, alumina, titania, iron oxide, zinc oxide, copper oxide, cerium oxide, carbon black among others) and released into the air during printing. All evaluated toners contained large amounts of organic carbon (OC, 42–89%), metals/metal oxides (1–33%), and some elemental carbon (EC, 0.33–12%). The PEPs possess a composition similar to that of toner and contained 50–90% OC, 0.001–0.5% EC and 1–3% metals. While the chemistry of the PEPs generally reflected that of their toners, considerable differences are documented indicative of potential transformations taking place during consumer use (printing). We conclude that: (i) Routine incorporation of ENMs in toners classifies them as nano-enabled products (NEPs); (ii) These ENMs become airborne during printing; (iii) The chemistry of PEPs is complex and it reflects that of the toner and paper. This work highlights the importance of understanding life-cycle (LC) nano-EHS implications of NEPs and assessing real world exposures and associated toxicological properties rather than focusing on “raw” materials used in the synthesis of an NEP.

Development and characterization of an exposure platform suitable for physico-chemical, morphological and toxicological characterization of printer-emitted particles (PEPs).

Abstract An association between laser printer use and emissions of particulate matter (PM), ozone and volatile organic compounds has been reported in recent studies. However, the detailed physico-chemical, morphological and toxicological characterization of these printer-emitted particles (PEPs) and possible incorporation of engineered nanomaterials into toner formulations remain largely unknown. In this study, a printer exposure generation system suitable for the physico-chemical, morphological, and toxicological characterization of PEPs was developed and used to assess the properties of PEPs 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 the size fractionation and sampling of PEPs and an exposure chamber for inhalation toxicological studies. Eleven commonly used laser printers were evaluated and ranked based on their PM emission profiles. Results show PM peak emissions are brand independent and varied between 3000 to 1 300 000 particles/cm3, with modal diameters ranging from 49 to 208 nm, with the majority of PEPs in the nanoscale (<100 nm) size. Furthermore, it was shown that PEPs can be affected by certain operational parameters and printing conditions. The release of nanoscale particles from a nano-enabled product (printer toner) raises questions about health implications to users. The presented PEGS platform will help in assessing the toxicological profile of PEPs and the link to the physico-chemical and morphological properties of emitted PM and toner formulations.

Small airway epithelial cells exposure to printer-emitted engineered nanoparticles induces cellular effects on human microvascular endothelial cells in an alveolar-capillary co-culture model

Abstract The printer is one of the most common office equipment. Recently, it was reported that toner formulations for printing equipment constitute nano-enabled products (NEPs) and contain engineered nanomaterials (ENMs) that become airborne during printing. To date, insufficient research has been performed to understand the potential toxicological properties of printer-emitted particles (PEPs) with several studies using bulk toner particles as test particles. These studies demonstrated the ability of toner particles to cause chronic inflammation and fibrosis in animal models. However, the toxicological implications of inhalation exposures to ENMs emitted from laser printing equipment remain largely unknown. The present study investigates the toxicological effects of PEPs using an in vitro alveolar-capillary co-culture model with Human Small Airway Epithelial Cells (SAEC) and Human Microvascular Endothelial Cells (HMVEC). Our data demonstrate that direct exposure of SAEC to low concentrations of PEPs (0.5 and 1.0 µg/mL) caused morphological changes of actin remodeling and gap formations within the endothelial monolayer. Furthermore, increased production of reactive oxygen species (ROS) and angiogenesis were observed in the HMVEC. Analysis of cytokine and chemokine levels demonstrates that interleukin (IL)-6 and MCP-1 may play a major role in the cellular communication observed between SAEC and HMVEC and the resultant responses in HMVEC. These data indicate that PEPs at low, non-cytotoxic exposure levels are bioactive and affect cellular responses in an alveolar-capillary co-culture model, which raises concerns for potential adverse health effects.

In vivo epigenetic effects induced by engineered nanomaterials: A case study of copper oxide and laser printer-emitted engineered nanoparticles

Abstract Evidence continues to grow on potential environmental health hazards associated with engineered nanomaterials (ENMs). While the geno- and cytotoxic effects of ENMs have been investigated, their potential to target the epigenome remains largely unknown. The aim of this study is two-fold: 1) determining whether or not industry relevant ENMs can affect the epigenome in vivo and 2) validating a recently developed in vitro epigenetic screening platform for inhaled ENMs. Laser printer-emitted engineered nanoparticles (PEPs) released from nano-enabled toners during consumer use and copper oxide (CuO) were chosen since these particles induced significant epigenetic changes in a recent in vitro companion study. In this study, the epigenetic alterations in lung tissue, alveolar macrophages and peripheral blood from intratracheally instilled mice were evaluated. The methylation of global DNA and transposable elements (TEs), the expression of the DNA methylation machinery and TEs, in addition to general toxicological effects in the lung were assessed. CuO exhibited higher cell-damaging potential to the lung, while PEPs showed a greater ability to target the epigenome. Alterations in the methylation status of global DNA and TEs, and expression of TEs and DNA machinery in mouse lung were observed after exposure to CuO and PEPs. Additionally, epigenetic changes were detected in the peripheral blood after PEPs exposure. Altogether, CuO and PEPs can induce epigenetic alterations in a mouse experimental model, which in turn confirms that the recently developed in vitro epigenetic platform using macrophage and epithelial cell lines can be successfully utilized in the epigenetic screening of ENMs.

Nanoparticle exposures from nano-enabled toner-based printing equipment and human health: state of science and future research needs

Abstract Toner formulations used by laser printers (LP) and photocopiers (PC), collectively called “toner-based printing equipment” (TPE), are nano-enabled products (NEP) because they contain several engineered nanomaterials (ENM) that improve toner performance. It has been shown that during consumer use (printing), these ENM are released in the air, together with other semi-volatile organic nanoparticles, and newly formed gaseous co-pollutants such as volatile organic compounds (VOC). The aim of this review is to detail and analyze physico-chemical and morphological (PCM), as well as the toxicological properties of particulate matter (PM) emissions from TPE. The review covers evolution of science since the early 2000, when this printing technology first became a subject of public interest, as well as the lagging regulatory framework around it. Important studies that have significantly changed our understanding of these exposures are also highlighted. The review continues with a critical appraisal of the most up-to-date cellular, animal and human toxicological evidence on the potential adverse human health effects of PM emitted from TPE. We highlight several limitations of existing studies, including (i) use of high and often unrealistic doses in vitro or in vivo; (ii) unrealistically high-dose rates in intratracheal instillation studies; (iii) improper use of toners as surrogate for emitted nanoparticles; (iv) lack of or inadequate PCM characterization of exposures; and (v) lack of dosimetry considerations in in vitro studies. Presently, there is compelling evidence that the PM0.1 from TPE are biologically active and capable of inducing oxidative stress in vitro and in vivo, respiratory tract inflammation in vivo (in rats) and in humans, several endpoints of cellular injury in monocultures and co-cultures, including moderate epigenetic modifications in vitro. In humans, limited epidemiological studies report typically 2–3 times higher prevalence of chronic cough, wheezing, nasal blockage, excessive sputum production, breathing difficulties, and shortness of breath, in copier operators relative to controls. Such symptoms can be exacerbated during chronic exposures, and in individuals susceptible to inhaled pollutants. Thus respiratory, immunological, cardiovascular, and other disorders may be developed following such exposures; however, further toxicological and larger scale molecular epidemiological studies must be done to fully understand the mechanism of action of these TPE emitted nanoparticles. Major research gaps have also been identified. Among them, a methodical risk assessment based on “real world” exposures rather than on the toner particles alone needs to be performed to provide the much-needed data to establish regulatory guidelines protective of individuals exposed to TPE emissions at both the occupational and consumer level. Industry-wide molecular epidemiology as well as mechanistic animal and human studies are also urgently needed.

Toxicological Assessment of CoO and La2O3 Metal Oxide Nanoparticles in Human Small Airway Epithelial Cells

Cobalt monoxide (CoO) and lanthanum oxide (La2O3) nanoparticles are 2 metal oxide nanoparticles with different redox potentials according to their semiconductor properties. By utilizing these two nanoparticles, this study sought to determine how metal oxide nanoparticles mode of toxicological action is related to their physio-chemical properties in human small airway epithelial cells (SAEC). We investigated cellular toxicity, production of superoxide radicals and alterations in gene expression related to oxidative stress, and cellular death at 6 and 24 h following exposure to CoO and La2O3(administered doses: 0, 5, 25, and 50 µg/ml) nanoparticles. CoO nanoparticles induced gene expression related to oxidative stress at 6 h. After characterizing the nanoparticles, transmission electron microscope analysis showed SAEC engulfed CoO and La2O3nanoparticles. CoO nanoparticles were toxic after 6 and 24 h of exposure to 25.0 and 50.0 µg/ml administered doses, whereas, La2O3nanoparticles were toxic only after 24 h using the same administered doses. Based upon the Volumetric Centrifugation Methodin vivoSedimentation, Diffusion, and Dosimetry, the dose of CoO and La2O3nanoparticles delivered at 6 and 24 h were determined to be: CoO: 1.25, 6.25, and 12.5 µg/ml; La2O3: 5, 25, and 50 µg/ml and CoO: 4, 20, and 40 µg/ml; and La2O3: 5, 25, 50 µg/ml, respectively. CoO nanoparticles produced more superoxide radicals and caused greater stimulation of total tyrosine and threonine phosphorylation at both 6 and 24 h when compared with La2O3nanoparticles. Taken together, these data provide evidence that different toxicological modes of action were involved in CoO and La2O3metal oxide nanoparticle-induced cellular toxicity.