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

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.     

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.     

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.     

Interactions of engineered nanomaterials in physiological media and implications for in vitro dosimetry

Abstract

In vitro toxicity assays are efficient and inexpensive tools for screening the increasing number of engineered nanomaterials (ENMs) entering the consumer market. However, the data produced by in vitro studies often vary substantially among different studies and from in vivo data. In part, these discrepancies may be attributable to lack of standardisation in dispersion protocols and inadequate characterisation of particle–media interactions which may affect the particle kinetics and the dose delivered to cells. In this study, a novel approach for preparation of monodisperse, stabilised liquid suspensions is presented and coupled with a numerical model which estimates delivered dose values. Empirically derived material- and media-specific functions are presented for each media–ENM system that can be used to convert administered doses to delivered doses. The interactions of ENMs with a variety of physiologic media were investigated and the importance of this approach was demonstrated by in vitro cytotoxicity assays using THP-1 macrophages.     

Epigenetic screening in product safety assessment: are we there yet?

There has been a growing concern that epigenetic events, that is, heritable changes in gene expression superimposed on DNA nucleotide sequences, may be involved in chemically and/or nutritionally mediated adverse health outcomes, such as reproductive toxicity and cancer. This concern has been driven by an increasing number of studies reporting toxicant-induced alterations to the epigenome in the form of changes in DNA methylation, histone/chromatin remodeling, and altered expression of non-coding RNAs. These three major mechanisms of epigenetic modifications may have coordinated, independent, or potentially antagonistic influences on gene expression. Complicating this understanding is the incomplete understanding of the normal state and dynamic variation of the epigenome, which differs widely between cells, tissues, developmental state, age, strain, and species. This review serves as a framework to outline characteristics composing an ideal epigenetic screen(s) for hazard identification in product safety assessment. In order to implement such a screen, first there needs to be a better understanding of adaptive versus adverse changes in the epigenome, which includes identification of robust and reproducible causal links between epigenetic changes and adverse apical end points, and second development of improved reporter assay tools to monitor such changes. An ideal screen would be in vitro-based, medium- to high-throughput, and assess all three branches of epigenome control (i.e. methylation, histone modifications, non-coding RNAs), although also being quantitative, objective, portable (i.e. lab to lab), and relevant to humans.     

DNA methylation changes in human lung epithelia cells exposed to multi-walled carbon nanotubes

Humans are increasingly exposed to nanoparticles and, although many of their physiological effects have been described, the molecular mechanisms underlying them are still largely unknown. The present study aimed to determine the possible role of certain epigenetic mechanisms in the cellular response of human lung epithelial cells that are triggered by long-term exposure to titanium dioxide nanoparticles (TiO₂NPs) and multi-walled carbon nanotubes (MWCNTs). The results showed that exposure to TiO₂NPs had only minor effects on genome-wide DNA methylation. However, we identified 755 CpG sites showing consistent DNA hypomethylation in cells exposed to MWCNTs. These sites were mainly located at low density CpG regions and enhancers, and very frequently on the X chromosome. Our results thus suggest that long-term MWCNT exposure may have important effects on the epigenome.     

Nanotoxicity of engineered nanomaterials (ENMs) to environmentally relevant beneficial soil bacteria – a critical review

Deposition of engineered nanomaterials (ENMs) in various environmental compartments is projected to continue rising exponentially. Terrestrial environments are expected to be the largest repository for environmentally released ENMs. Because ENMs are enriched in biosolids during wastewater treatment, agriculturally applied biosolids facilitate ENM exposure of key soil micro-organisms, such as plant growth-promoting rhizobacteria (PGPR). The ecological ramifications of increasing levels of ENM exposure of terrestrial micro-organisms are not clearly understood, but a growing body of research has investigated the toxicity of ENMs to various soil bacteria using a myriad of toxicity end-points and experimental procedures. This review explores what is known regarding ENM toxicity to important soil bacteria, with a focus on ENMs which are expected to accumulate in terrestrial ecosystems at the highest concentrations and pose the greatest potential threat to soil micro-organisms having potential indirect detrimental effects on plant growth. Knowledge gaps in the fundamental understanding of nanotoxicity to bacteria are identified, including the role of physicochemical properties of ENMs in toxicity responses, particularly in agriculturally relevant micro-organisms. Strategies for improving the impact of future research through the implementation of in-depth ENM characterization and use of necessary experimental controls are proposed. The future of nanotoxicological research employing microbial ecoreceptors is also explored, highlighting the need for continued research utilizing bacterial isolates while concurrently expanding efforts to study ENM–bacteria interactions in more complex environmentally relevant media, e.g. soil. Additionally, the particular importance of future work to extensively examine nanotoxicity in the context of bacterial ecosystem function, especially of plant growth-promoting agents, is proposed.     

Quantum dot induced acute changes in lung mechanics are mouse strain dependent

Abstract

Introduction: Concerns have been raised regarding occupational exposure to engineered nanomaterials (ENMs). Potential impacts on lung function from inhalation exposures are of concern as the lung is a sensitive ENM target in animals. Epidemiological data suggest that occupational exposure to ENMs may impact respiratory and cardiovascular health. Quantum dots (QDs) are ENMs with outstanding semiconductor and fluorescent properties with uses in biomedicine and electronics. QDs are known to induce inflammation and cytotoxicity in rodents and high dose exposures impact lung function 2 weeks after exposure. However, effects of mouse strain and the temporality of QD effects on lung function at more occupationally relevant doses have not been well-established.

Objective: We evaluated the impact of QD exposure on respiratory mechanics in C57BL/6J and A/J mice. Previous work found a greater initial inflammatory response to QD exposure in A/J mice compared to C57BL/6J mice. Thus, we hypothesized that A/J mice would be more sensitive to QD-induced effects on lung mechanics.

Methods: C57BL/6J and A/J mice were exposed to 6?μg/kg Cd equivalents of amphiphilic polymer-coated Cd/Se core, ZnS shell QDs via oropharyngeal aspiration. Lung mechanics were measured using forced oscillation, and inflammation was characterized by neutrophils and cytokines in bronchoalveolar lavage fluid.

Results: Both strains showed signs of QD-induced acute lung inflammation. However, lung mechanics were impacted by QD exposure in A/J mice only.

Conclusions: Our findings suggest that susceptibility to QDs and similar ENM-induced changes in lung function may depend at least in part on genetic background.     

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.     

Using a holistic approach to assess the impact of engineered nanomaterials inducing toxicity in aquatic systems

In this report, we critically reviewed selected intrinsic physicochemical properties of engineered nanomaterials (ENMs) and their role in the interaction of the ENMs with the immediate surroundings in representative aquatic environments. The behavior of ENMs with respect to dynamic microenvironments at the nano–bio–eco interface level, and the resulting impact on their toxicity, fate, and exposure potential are elaborated. Based on this literature review, we conclude that a holistic approach is urgently needed to fulfill our knowledge gap regarding the safety of discharged ENMs. This comparative approach affords the capability to recognize and understand the potential hazards of ENMs and their toxicity mechanisms, and ultimately to establish a quantitative and reliable system to predict such outcomes.     

Assessing nanomaterial exposures in aquatic ecotoxicological testing: Framework and case studies based on dispersion and dissolution

The unique behavior of engineered nanomaterials (ENM) in aqueous media and dynamic changes in particle settling, agglomeration and dissolution rates is a challenge to the consistency, reliability and interpretation of standard aquatic hazard bioassay results. While the toxicological endpoints (e.g., survival, growth, reproduction, etc.) in ecotoxicity bioassays are largely applicable to ENMs, the standard methods as written for dissolved substances are confounded by the dynamic settling, agglomeration and dissolution of particulate ENMs during the bioassay. A testing framework was designed to serve as a starting point to identify approaches for the consistent conduct of aquatic hazard tests that account for the behavior of ENMs in test media and suitable data collection to support representative exposure metrology. The framework was demonstrated by conducting three case studies testing ENMs with functionally distinct characteristics and behaviors. Pretests with a temporal sampling of particle concentration, agglomeration and dissolution were conducted on each ENM in test media. Results indicated that a silver nanoparticle (AgNP) powder was not dispersible, a nano-TiO₂ powder was dispersible but unstable, and a polyvinylpyrrolidinone-coated AgNP was relatively stable in test media. Based on these functional results, Ceriodaphnia dubia bioassays were conducted to compare different exposure summary methods (nominal, arithmetic average, geometric average, time-weighted average) for calculating and expressing toxicity endpoints. Results indicated that while arithmetic means were effective for expressing the toxicity of more stable materials, time-weighted averaged concentrations were appropriate for the unstable nano-TiO₂.     

Workshop report: Strategies for setting occupational exposure limits for engineered nanomaterials

Occupational exposure limits (OELs) are important tools for managing worker exposures to chemicals; however, hazard data for many engineered nanomaterials (ENMs) are insufficient for deriving OELs by traditional methods. Technical challenges and questions about how best to measure worker exposures to ENMs also pose barriers to implementing OELs. New varieties of ENMs are being developed and introduced into commerce at a rapid pace, further compounding the issue of OEL development for ENMs. A Workshop on Strategies for Setting Occupational Exposure Limits for Engineered Nanomaterials, held in September 2012, provided an opportunity for occupational health experts from various stakeholder groups to discuss possible alternative approaches for setting OELs for ENMs and issues related to their implementation. This report summarizes the workshop proceedings and findings, identifies areas for additional research, and suggests potential avenues for further progress on this important topic.     

Assessing the protection of the nanomaterial workforce

Responsible development of any technology, including nanotechnology, requires protecting workers, the first people to be exposed to the products of the technology. In the case of nanotechnology, this is difficult to achieve because in spite of early evidence raising health and safety concerns, there are uncertainties about hazards and risks. The global response to these concerns has been the issuance by authoritative agencies of precautionary guidance to strictly control exposures to engineered nanomaterials (ENMs). This commentary summarizes discussions at the “Symposium on the Health Protection of Nanomaterial Workers” held in Rome (25 and 26 February 2015). There scientists and practitioners from 11 countries took stock of what is known about hazards and risks resulting from exposure to ENMs, confirmed that uncertainties still exist, and deliberated on what it would take to conduct a global assessment of how well workers are being protected from potentially harmful exposures.     

Genotoxicity of engineered nanomaterials: A critical review

In view of the fast-growing industrial applications of engineered nanomaterials (ENMs), the evaluation of their genotoxic potential and of their mode of action is a necessity to conduct adequate hazard/risk assessment and to produce safer and sustainable ENMs. This review aims at: (i) Providing an evaluation of in vitro and in vivo genotoxicity data available for ENM, and (ii) proposing minimal criteria for conducting nano-genotoxicity assays. The possible modes of action of ENM (i.e., generation of reactive oxygen species (ROS) and mechanical interference with cellular components) and the potential cellular targets are discussed. The available studies are evaluated on the basis of specific quality criteria after categorisation according to ENMs type/size investigated. No definitive conclusion can be drawn concerning the genotoxic activity of ENMs, essentially because of the limited number of data, incomplete physico-chemical characterization of ENMs examined and shortcomings in experimental approaches. This evaluation revealed gaps to be considered in future studies (e.g., one-sided approach focusing mainly on ROS as mode of action) and the need to develop adequate positive controls for genotoxicity assays when conducted with nanomaterials.     

Effect of silver nanoparticles (AgNPs) exposure on microRNA expression and global DNA methylation in endothelial cells EA.hy926

The aim of this study was to determine the effect of AgNPs on the epigenome of endothelial cells EA.hy926, including the levels of expression of microRNAs (miRNAs) and global DNA methylation patterns. In addition, evaluation of the expression of inflammatory genes and the levels of VCAM-1 protein (miRNA-126 target) was performed. The expression levels of analyzed miRNAs (microRNAs-126, 155 and 146) were reduced significantly and there were not observed changes in inflammatory gene expression. Regarding the levels of protein vascular cell adhesion molecule 1 (VCAM-1), they increase significantly to 0.5 μM AgNPs at 24 h of exposure. As far as DNA methylation is concerned, we found that AgNPs induce a state of global hyper-methylation. In conclusion, it was demonstrated that direct contact between AgNPs and endothelial cells resulted in the dysregulation of highly enriched and vastly functional miRNAs and DNA hypermethylation, that may have multiple effects on endothelium function and integrity.     

Inhaled silica-coated TiO2 nanoparticles induced airway irritation,airflow limitation and inflammation in mice

Abstract

The wide use of nanotechnology is here to stay. However, the knowledge on the health effects of different engineered nanomaterials (ENMs) is lacking. In this study, irritation and inflammation potential of commercially available silica-coated TiO₂ ENMs (10?×?40?nm, rutile) were studied. Single exposure (30?min) at mass concentrations 5, 10, 20 and 30?mg/m³, and repeated exposure (altogether 16?h, 1?h/day, 4 days/week for 4 weeks) at mass concentration of 30?mg/m³ to silica-coated TiO₂ induced first phase of pulmonary irritation (P1), which was seen as rapid, shallow breathing. During repeated exposures, P1 effect was partly evolved into more intense pulmonary irritation. Also sensory irritation was observed at the beginning of both single and repeated exposure periods, and the effect intensified during repeated exposures. Airflow limitation started to develop during repeated exposures. Repeated exposure to silica-coated TiO₂ ENMs induced also pulmonary inflammation: inflammatory cells infiltrated in peribronchial and perivascular areas of the lungs, neutrophils were found in BAL fluids, and the number of CD3 and CD4 positive T cells increased significantly. In line with these results, pulmonary mRNA expression of chemokines CXCL1, CXCL5 and CXCL9 was enhanced. Also expression of mRNA levels of proinflammatory cytokines TNF-α and IL-6 was elevated after repeated exposures. Taken together, these results indicated that silica-coated TiO₂ ENMs induce pulmonary and sensory irritation after single and repeated exposure, and airflow limitation and pulmonary inflammation after repeated exposure.     

Alterations in DNA methylation and airway hyperreactivity in response to in utero exposure to environmental tobacco smoke

Abstract

Growing evidence indicates that prenatal exposure to maternal smoking is a risk factor for the development of asthma in children. However, the effects of prenatal environmental tobacco smoke (ETS) exposure on the genome and lung immune cells are unclear. This study aims to determine whether in utero ETS exposure alters DNA methylation patterns and increases airway hyperreactivity (AHR) and inflammation. Pregnant C57BL/6 mice were exposed daily to a concentration of 1.0?mg/m³ ETS. AHR was determined in the 6-week-old offspring by measurement of airway resistance. Global and gene promoter methylation levels in lung DNA from offspring were analyzed by luminometric methylation and pyrosequencing assays, respectively. Offspring exposed to ETS showed a marked increase in the number of alveolar macrophages in the bronchoalveolar lavage fluid and level of IL-13 in the airways compared with offspring of filtered-air exposed dams (controls). ETS exposure significantly augmented AHR compared with controls. In the methylation analysis, ETS-exposed offspring had a significantly lower level of global DNA methylation than the controls. We observed a significant increase in IFN-γ, and significant decrease in IL-13 methylation levels in the ETS group compared with controls. Collectively, these data suggest that in utero ETS exposure increases the risk of pulmonary inflammation and AHR through altered DNA methylation, but additional studies are needed to fully determine the causal link between changes in methylation and cytokines levels, as well as AHR.