Establishing relationships between particle-induced in vitro and in vivo inflammation endpoints to better extrapolate between in vitro markers and in vivo fibrosis.

BACKGROUND: Toxicity assessment for regulatory purposes is starting to move away from traditional in vivo methods and towards new approach methodologies (NAM) such as high-throughput in vitro models and computational tools. For materials with limited hazard information, utilising quantitative Adverse Outcome Pathways (AOPs) in a testing strategy involving NAM can produce information relevant for risk assessment. The aim of this work was to determine the feasibility of linking in vitro endpoints to in vivo events, and moreover to key events associated with the onset of a chosen adverse outcome to aid in the development of NAM testing strategies. To do this, we focussed on the adverse outcome pathway (AOP) relating to the onset of pulmonary fibrosis. RESULTS: We extracted in vivo and in vitro dose-response information for particles known to induce this pulmonary fibrosis (crystalline silica, specifically α-quartz). To test the in vivo-in vitro extrapolation (IVIVE) determined for crystalline silica, cerium dioxide nanoparticles (nano-CeO2) were used as a case study allowing us to evaluate our findings with a less studied substance. The IVIVE methodology outlined in this paper is formed of five steps, which can be more generally summarised into two categories (i) aligning the in vivo and in vitro dosimetry, (ii) comparing the dose-response curves and derivation of conversion factors. CONCLUSION: Our analysis shows promising results with regards to correlation of in vitro cytokine secretion to in vivo acute pulmonary inflammation assessed by polymorphonuclear leukocyte influx, most notable is the potential of using IL-6 and IL-1ß cytokine secretion from simple in vitro submerged models as a screening tool to assess the likelihood of lung inflammation at an early stage in product development, hence allowing a more targeted investigation using either a smaller, more targeted in vivo study or in the future a more complex in vitro protocol. This paper also highlights the strengths and limitations as well as the current difficulties in performing IVIVE assessment and suggestions for overcoming these issues.

The Road to Achieving the European Commission's Chemicals Strategy for Nanomaterial Sustainability-A PATROLS Perspective on New Approach Methodologies.

The European Green Deal outlines ambitions to build a more sustainable, climate neutral, and circular economy by 2050. To achieve this, the European Commission has published the Chemicals Strategy for Sustainability: Towards a Toxic-Free Environment, which provides targets for innovation to better protect human and environmental health, including challenges posed by hazardous chemicals and animal testing. The European project PATROLS (Physiologically Anchored Tools for Realistic nanOmateriaL hazard aSsessment) has addressed multiple aspects of the Chemicals Strategy for Sustainability by establishing a battery of new approach methodologies, including physiologically anchored human and environmental hazard assessment tools to evaluate the safety of engineered nanomaterials. PATROLS has delivered and improved innovative tools to support regulatory decision-making processes. These tools also support the need for reducing regulated vertebrate animal testing; when used at an early stage of the innovation pipeline, the PATROLS tools facilitate the safe and sustainable development of new nano-enabled products before they reach the market.

Functionalized Surface-Charged SiO2 Nanoparticles Induce Pro-Inflammatory Responses, but Are Not Lethal to Caco-2 Cells.

Nanoparticles (NPs) are widely used in food, and analysis of their potential gastrointestinal toxicity is necessary. The present study was designed to determine the effects of silica dioxide (SiO2), titanium dioxide (TiO2), and zinc oxide (ZnO) NPs on cultured THP-1 monocyte-derived macrophages and human epithelial colorectal adenocarcinoma (Caco-2) cells. Exposure to ZnO NPs for 24 h increased the production of redox response species (ROS) and reduced cell viability in a dose-dependent manner in THP-1 macrophages and Caco-2 cells. Although TiO2 and SiO2 NPs induced oxidative stress, they showed no apparent cytotoxicity against both cell types. The effects of functionalized SiO2 NPs on undifferentiated and differentiated Caco-2 cells were investigated using fluorescently labeled SiO2 NPs with neutral, positive, or negative surface charge. Exposure of both types of cells to the three kinds of SiO2 NPs significantly increased their interaction in a dose-dependent manner. The largest interaction with both types of cells was noted with exposure to more negatively surface-charged SiO2 NPs. Exposure to either positively or negatively, but not neutrally, surface-charged SiO2 NPs increased NO levels in differentiated Caco-2 cells. Exposure of differentiated Caco-2 cells to positively or negatively surface-charged SiO2 NPs also upregulated interleukin-8 expression. We conclude that functionalized surface-charged SiO2 NPs can induce pro-inflammatory responses but are noncytotoxic.

Toxicological Evaluation of SiO2 Nanoparticles by Zebrafish Embryo Toxicity Test.

As the use of nanoparticles (NPs) is increasing, the potential toxicity and behavior of NPs in living systems need to be better understood. Our goal was to evaluate the developmental toxicity and bio-distribution of two different sizes of fluorescently-labeled SiO2 NPs, 25 and 115 nm, with neutral surface charge or with different surface functionalization, rendering them positively or negatively charged, in order to predict the effect of NPs in humans. We performed a zebrafish embryo toxicity test (ZFET) by exposing the embryos to SiO2 NPs starting from six hours post fertilization (hpf). Survival rate, hatching time, and gross morphological changes were assessed at 12, 24, 36, 48, 60, and 72 hpf. We evaluated the effect of NPs on angiogenesis by counting the number of sub-intestinal vessels between the second and seventh intersegmental vessels and gene expression analysis of vascular endothelial growth factor (VEGF) and VEGF receptors at 72 hpf. SiO2 NPs did not show any adverse effects on survival rate, hatching time, gross morphology, or physiological angiogenesis. We found that SiO2 NPs were trapped by the chorion up until to the hatching stage. After chemical removal of the chorion (dechorionation), positively surface-charged SiO2 NPs (25 nm) significantly reduced the survival rate of the fish compared to the control group. These results indicate that zebrafish chorion acts as a physical barrier against SiO2 NPs, and removing the chorions in ZFET might be necessary for evaluation of toxicity of NPs.

Adoption of in vitro systems and zebrafish embryos as alternative models for reducing rodent use in assessments of immunological and oxidative stress responses to nanomaterials.

Assessing the safety of engineered nanomaterials (NMs) is paramount to the responsible and sustainable development of nanotechnology, which provides huge societal benefits. Currently, there is no evidence that engineered NMs cause detrimental health effects in humans. However, investigation of NM toxicity using in vivo, in vitro, in chemico, and in silico models has demonstrated that some NMs stimulate oxidative stress and inflammation, which may lead to adverse health effects. Accordingly, investigation of these responses currently dominates NM safety assessments. There is a need to reduce reliance on rodent testing in nanotoxicology for ethical, financial and legislative reasons, and due to evidence that rodent models do not always predict the human response. We advocate that in vitro models and zebrafish embryos should have greater prominence in screening for NM safety, to better align nanotoxicology with the 3Rs principles. Zebrafish are accepted for use by regulatory agencies in chemical safety assessments (e.g. developmental biology) and there is growing acceptance of their use in biomedical research, providing strong foundations for their use in nanotoxicology. We suggest that investigation of the response of phagocytic cells (e.g. neutrophils, macrophages) in vitro should also form a key part of NM safety assessments, due to their prominent role in the first line of defense. The development of a tiered testing strategy for NM hazard assessment that promotes the more widespread adoption of non-rodent, alternative models and focuses on investigation of inflammation and oxidative stress could make nanotoxicology testing more ethical, relevant, and cost and time efficient.

The Essential Elements of a Risk Governance Framework for Current and Future Nanotechnologies.

Societies worldwide are investing considerable resources into the safe development and use of nanomaterials. Although each of these protective efforts is crucial for governing the risks of nanomaterials, they are insufficient in isolation. What is missing is a more integrative governance approach that goes beyond legislation. Development of this approach must be evidence based and involve key stakeholders to ensure acceptance by end users. The challenge is to develop a framework that coordinates the variety of actors involved in nanotechnology and civil society to facilitate consideration of the complex issues that occur in this rapidly evolving research and development area. Here, we propose three sets of essential elements required to generate an effective risk governance framework for nanomaterials. (1) Advanced tools to facilitate risk-based decision making, including an assessment of the needs of users regarding risk assessment, mitigation, and transfer. (2) An integrated model of predicted human behavior and decision making concerning nanomaterial risks. (3) Legal and other (nano-specific and general) regulatory requirements to ensure compliance and to stimulate proactive approaches to safety. The implementation of such an approach should facilitate and motivate good practice for the various stakeholders to allow the safe and sustainable future development of nanotechnology.

Impact of serum as a dispersion agent for in vitro and in vivo toxicological assessments of TiO2 nanoparticles.

Nanoparticles (NP) have a tendency to agglomerate after dispersion in physiological media, which can be prevented by the addition of serum. This may however result in modification of the toxic potential of particles due to the formation of protein corona. Our study aimed to analyze the role of serum that is added to improve the dispersion of 10 nm TiO2 NPs on in vitro and in vivo effects following the exposure via the respiratory route. We characterized NP size, surface charge, sedimentation rate, the presence of protein corona and the oxidant-generating capacity after NP dispersion in the presence/absence of serum. The effect of serum on NP internalization, cytotoxicity and pro-inflammatory responses was assessed in a human pulmonary cell line, NCI-H292. Serum in the dispersion medium led to a slower sedimentation, but an enhanced cellular uptake of TiO2 NPs. Despite this greater uptake, the pro-inflammatory response in NCI-H292 cells was lower after serum supplementation (used either as a dispersant or as a cell culture additive), which may be due to a reduced intrinsic oxidative potential of TiO2 NPs. Interestingly, serum could be added 2 h after the NP treatment without affecting the pro-inflammatory response. We also determined the acute pulmonary and hepatic toxicity in vivo 24 h after intratracheal instillation of TiO2 NPs in C57BL/6N mice. The use of serum resulted in an underestimation of the local acute inflammatory response in the lung, while a systemic response on glutathione reduction remained unaffected. In conclusion, serum as a dispersion agent for TiO2 NPs can lead to an underestimation of the acute pro-inflammatory response in vitro and in vivo. To avoid potential unwanted effects of dispersants and medium components, we recommend that the protocol of NM preparation should be thoroughly tested, and reflect as close as possible realistic exposure conditions.

A novel semi-supervised algorithm for the taxonomic assignment of metagenomic reads.

BACKGROUND: Taxonomic assignment is a crucial step in a metagenomic project which aims to identify the origin of sequences in an environmental sample. Among the existing methods, since composition-based algorithms are not sufficient for classifying short reads, recent algorithms use only the feature of similarity, or similarity-based combined features. However, those algorithms suffer from the computational expense because the task of similarity search is very time-consuming. Besides, the lack of similarity information between reads and reference sequences due to the length of short reads reduces significantly the classification quality. RESULTS: This paper presents a novel taxonomic assignment algorithm, called SeMeta, which is based on semi-supervised learning to produce a fast and highly accurate classification of short-length reads with sufficient mutual overlap. The proposed algorithm firstly separates reads into clusters using their composition feature. It then labels the clusters with the support of an efficient filtering technique on results of the similarity search between their reads and reference databases. Furthermore, instead of performing the similarity search for all reads in the clusters, SeMeta only does for reads in their subgroups by utilizing the information of sequence overlapping. The experimental results demonstrate that SeMeta outperforms two other similarity-based algorithms on different aspects. CONCLUSIONS: By using a semi-supervised method as well as taking the advantages of various features, the proposed algorithm is able not only to achieve high classification quality, but also to reduce much computational cost. The source codes of the algorithm can be downloaded at http://it.hcmute.edu.vn/bioinfo/metapro/SeMeta.html.

A Multilaboratory Toxicological Assessment of a Panel of 10 Engineered Nanomaterials to Human Health--ENPRA Project--The Highlights, Limitations, and Current and Future Challenges.

ENPRA was one of the earlier multidisciplinary European Commission FP7-funded projects aiming to evaluate the risks associated with nanomaterial (NM) exposure on human health across pulmonary, cardiovascular, hepatic, renal, and developmental systems. The outputs from this project have formed the basis of this review. A retrospective interpretation of the findings across a wide range of in vitro and in vivo studies was performed to identify the main highlights from the project. In particular, focus was placed on informing what advances were made in the hazard assessment of NM, as well as offering some suggestions on the future of "nanotoxicology research" based on these observations, shortcomings, and lessons learned from the project. A number of issues related to the hazard assessment of NM are discussed in detail and include use of appropriate NM for nanotoxicology investigations; characterization and dispersion of NM; use of appropriate doses for all related investigations; need for the correct choice of experimental models for risk assessment purposes; and full understanding of the test systems and correct interpretation of data generated from in vitro and in vivo systems. It is hoped that this review may assist in providing information in the implementation of guidelines, model systems, validation of assessment methodology, and integrated testing approaches for risk assessment of NM. It is vital to learn from ongoing and/or completed studies to avoid unnecessary duplication and offer suggestions that might improve different aspects of experimental design.

Comparing the CORAL and Random Forest approaches for modelling the in vitro cytotoxicity of silica nanomaterials.

Nanotechnology is one of the most important technological developments of the 21st century. In silico methods to predict toxicity, such as quantitative structure-activity relationships (QSARs), promote the safe-by-design approach for the development of new materials, including nanomaterials. In this study, a set of cytotoxicity experimental data corresponding to 19 data points for silica nanomaterials were investigated, to compare the widely employed CORAL and Random Forest approaches in terms of their usefulness for developing so-called 'nano-QSAR' models. 'External' leave-one-out cross-validation (LOO) analysis was performed, to validate the two different approaches. An analysis of variable importance measures and signed feature contributions for both algorithms was undertaken, in order to interpret the models developed. CORAL showed a more pronounced difference between the average coefficient of determination (R²) for training and for LOO (0.83 and 0.65 for training and LOO, respectively), compared to Random Forest (0.87 and 0.78 without bootstrap sampling, 0.90 and 0.78 with bootstrap sampling), which may be due to overfitting. With regard to the physicochemical properties of the nanomaterials, the aspect ratio and zeta potential were found to be the two most important variables for Random Forest, and the average feature contributions calculated for the corresponding descriptors were consistent with the clear trends observed in the data set: less negative zeta potential values and lower aspect ratio values were associated with higher cytotoxicity. In contrast, CORAL failed to capture these trends.

The biologically effective dose in inhalation nanotoxicology.

In all branches of toxicology, the biologically effective dose (BED) is the fraction of the total dose of a toxin that actually drives any toxic effect. Knowledge of the BED has a number of applications including in building structure-activity relationships, the selection of metrics, the design of safe particles, and the determination of when a nanoparticle (NP) can be considered to be "new" for regulatory purposes. In particle toxicology, we define the BED as "the entity within any dose of particles in tissue that drives a critical pathophysiogically relevant form of toxicity (e.g., oxidative stress, inflammation, genotoxicity, or proliferation) or a process that leads to it." In conventional chemical toxicology, researchers generally use the mass as the metric to describe dose (such as mass per unit tissue or cells in culture) because of its convenience. Concentration, calculated from mass, may also figure in any description of dose. In the case of a nanoparticle dose, researchers use either the mass or the surface area. The mass of nanoparticles is not the only driver of their activity: the surfaces of insoluble particles interact with biological systems, and soluble nanoparticles can release factors that interact with these systems. Nanoparticle shape can modify activity. In this Account, we describe the current knowledge of the BED as it pertains to different NP types. Soluble toxins released by NPs represent one potential indicator of BED for wholly or partially soluble NPs composed of copper or zinc. Rapid dissolution of these NPs into their toxic ions in the acidic environment of the macrophage phagolysosome causes those ions to accumulate, which leads to lysosome destabilization and inflammation. In contrast, soluble NPs that release low toxicity ions, such as magnesium oxide NPs, are not inflammogenic. For insoluble NPs, ζ potential can serve as a BED measurement because the exposure of the particle surface to the acidic milieu of the phagolysosome and interactions with the lysosomal membrane can compromise the integrity of the NPs. Researchers have explored oxidative potential of NPs most extensively as an indicator of the BED: the ability of an NP to cause oxidative stress in cells is a key factor in determining cell toxicity, inflammogenicity, and oxidative DNA adduct formation. Finally we discuss BEDs for high aspect ratio nanoparticles because long fibers or nanoplatelets can cause inflammation and further effects. These consequences arise from the paradoxically small aerodynamic diameter of fibers or thin platelets. As a result, these NPs can deposit beyond the ciliated airways where their extended dimensions prevent them from being fully phagocytosed by macrophages, leading to frustrated phagocytosis. Although knowledge is accumulating on the BED for NPs, many questions and challenges remain in understanding and utilizing this important nanotoxicological parameter.

ITS-NANO--prioritising nanosafety research to develop a stakeholder driven intelligent testing strategy.

BACKGROUND: To assess the risk of all nanomaterials (NMs) on a case-by-case basis is challenging in terms of financial, ethical and time resources. Instead a more intelligent approach to knowledge gain and risk assessment is required. METHODS: A framework of future research priorities was developed from the accorded opinion of experts covering all major stake holder groups (government, industry, academia, funders and NGOs). It recognises and stresses the major topics of physicochemical characterisation, exposure identification, hazard identification and modelling approaches as key components of the current and future risk assessment of NMs. RESULTS: The framework for future research has been developed from the opinions of over 80 stakeholders, that describes the research priorities for effective development of an intelligent testing strategy (ITS) to allow risk evaluation of NMs. In this context, an ITS is a process that allows the risks of NMs to be assessed accurately, effectively and efficiently, thereby reducing the need to test NMs on a case-by-case basis.For each of the major topics of physicochemical characterisation, exposure identification, hazard identification and modelling, key-priority research areas are described via a series of stepping stones, or hexagon diagrams structured into a time perspective. Importantly, this framework is flexible, allowing individual stakeholders to identify where their own activities and expertise are positioned within the prioritisation pathway and furthermore to identify how they can effectively contribute and structure their work accordingly. In other words, the prioritisation hexagon diagrams provide a tool that individual stakeholders can adapt to meet their own particular needs and to deliver an ITS for NMs risk assessment. Such an approach would, over time, reduce the need for testing by increasing the reliability and sophistication of in silico approaches.The manuscript includes an appraisal of how this framework relates to the current risk assessment approaches and how future risk assessment could adapt to accommodate these new approaches. A full report is available in electronic format (pdf) at http://www.nano.hw.ac.uk/research-projects/itsnano.html. CONCLUSION: ITS-NANO has delivered a detailed, stakeholder driven and flexible research prioritisation (or strategy) tool, which identifies specific research needs, suggests connections between areas, and frames this in a time-perspective.

Engineered nanomaterial risk. Lessons learnt from completed nanotoxicology studies: potential solutions to current and future challenges.

PARTICLE_RISK was one of the first multidisciplinary projects funded by the European Commission's Framework Programme that was responsible for evaluating the implications of nanomaterial (NM) exposure on human health. This project was the basis for this review which identifies the challenges that exist within the assessment of NM risk. We have retrospectively reflected on the findings of completed nanotoxicology studies to consider what progress and advances have been made within the risk assessment of NMs, as well as discussing the direction that nanotoxicology research is taking and identifying the limitations and failings of existing research. We have reflected on what commonly encountered challenges exist and explored how these issues may be resolved. In particular, the following is discussed (i) NM selection (ii) NM physico-chemical characterisation; (iii) NM dispersion; (iv) selection of relevant doses and concentrations; (v) identification of relevant models, target sites and endpoints; (vi) development of alternatives to animal testing; and (vii) NM risk assessment. These knowledge gaps are relatively well recognised by the scientific community and recommendations as to how they may be overcome in the future are provided. It is hoped that this will help develop better defined hypothesis driven research in the future that will enable comprehensive risk assessments to be conducted for NMs. Importantly, the nanotoxicology community has responded and adapted to advances in knowledge over recent years to improve the approaches used to assess NM hazard, exposure and risk. It is vital to learn from existing information provided by ongoing or completed studies to avoid unnecessary duplication of effort, and to offer guidance on aspects of the experimental design that should be carefully considered prior to the start of a new study.

Nanomaterial genotoxicity evaluation using the high-throughput p53-binding protein 1 (53BP1) assay.

Toxicity evaluation of engineered nanomaterials is challenging due to the ever increasing number of materials and because nanomaterials (NMs) frequently interfere with commonly used assays. Hence, there is a need for robust, high-throughput assays with which to assess their hazard potential. The present study aimed at evaluating the applicability of a genotoxicity assay based on the immunostaining and foci counting of the DNA repair protein 53BP1 (p53-binding protein 1), in a high-throughput format, for NM genotoxicity assessment. For benchmarking purposes, we first applied the assay to a set of eight known genotoxic agents, as well as X-ray irradiation (1 Gy). Then, a panel of NMs and nanobiomaterials (NBMs) was evaluated with respect to their impact on cell viability and genotoxicity, and to their potential to induce reactive oxygen species (ROS) production. The genotoxicity recorded using the 53BP1 assay was confirmed using the micronucleus assay, also scored via automated (high-throughput) microscopy. The 53BP1 assay successfully identified genotoxic compounds on the HCT116 human intestinal cell line. None of the tested NMs showed any genotoxicity using the 53BP1 assay, except the positive control consisting in (CoO)(NiO) NMs, while only TiO2 NMs showed positive outcome in the micronucleus assay. Only Fe3O4 NMs caused significant elevation of ROS, not correlated to DNA damage. Therefore, owing to its adequate predictivity of the genotoxicity of most of the tested benchmark substance and its ease of implementation in a high throughput format, the 53BP1 assay could be proposed as a complementary high-throughput screening genotoxicity assay, in the context of the development of New Approach Methodologies.

Probabilistic human health risk assessment of 1,3-butadiene and styrene exposure using Monte Carlo simulation technique in the carpet production industry.

Chemicals containing Volatile Organic Compounds (VOCs) are commonly used in the machine carpet production. 1,3-butadiene and styrene are main components of the carpenter's glue used in carpet factories. Exposition to these chemicals can lead to a number of adverse health effects. This is the first study of the human health risk assessment due to inhalational exposure to 1,3-butadiene (BD) and styrene (ST) performed among workers in the carpet factories in Kashan city, Iran. The importance of the study was related with the fact of high popularity of carpet production in the South Asia countries. Inhalation exposure to BD and ST were measured based on the National Institute for Occupational Safety and Health (NIOSH) 1024 and 1501 methods, respectively. The cancerogenic risk (CR) and non-cancerogenic risk described as Hazard Quotient (HQ) values were calculated based on the United States Environmental Protection Agency (USEPA) method. The sensitivity and uncertainty analysis were performed by the Monte Carlo simulation (MCS) technique. The average concentration measured of BD and ST during work shifts of employees were 0.039 mg m-3 (0.017 ppm) and 12.108 mg m-3 (2.84 ppm), respectively. The mean ± SD value of estimated cancerogenic risk in inhalation exposure to BD and ST were equal to 5.13 × 10-3 ± 3.85 × 10-4 and 1.44 × 10-3 ± 2.36 × 10-4, respectively exceeding the acceptable risk level of 10-6 defined by USEPA. The average non-carcinogenic risk (HQ) values of BD and ST were equal to 8.50 × 100 and 5.13 × 100, respectively exceeding the acceptable risk level of 1. As the results of our studies exceeded both cancerogenic and non-carcinogenic risk values it indicates that adverse health effects due to inhalational exposure to BD and ST for workers in the machine carpet industry are very likely. To avoid negative health effects protective measures for employees in the factories should be introduced immediately and furher detailed research are recommended.

The influence of exposure approaches to in vitro lung epithelial barrier models to assess engineered nanomaterial hazard.

Exposure to engineered nanomaterials (ENM) poses a potential health risk to humans through long-term, repetitive low-dose exposures. Currently, this is not commonplace within in vitro lung cell cultures. Therefore, the purpose of this study was to consider the optimal exposure approach toward determining the stability, sensitivity and validity of using in vitro lung cell mono- and co-cultures to determine ENM hazard. A range of exposure scenarios were conducted with DQ12 (previously established as a positive particle control) (historic and re-activated), TiO2 (JRC NM-105) and BaSO4 (JRC NM-220) on both monocultures of A549 cells as well as co-cultures of A549 cells and differentiated THP-1 cells. Cell cultures were exposed to either a single, or a repeated exposure over 24, 48- or 72-hours at in vivo extrapolated concentrations of 0-5.2 µg/cm2, 0-6 µg/cm2 and 0-1µg/cm2. The focus of this study was the pro-inflammatory, cytotoxic and genotoxic response elicited by these ENMs. Exposure to DQ12 caused pro-inflammatory responses after 48 hours repeat exposures, as well as increases in micronucleus frequency. Neither TiO2 nor BaSO4 elicited a pro-inflammatory response at this time point. However, there was induction of IL-6 after 24 hours TiO2 exposure. In conclusion, it is important to consider the appropriateness of the positive control implemented, the cell culture model, the time of exposure as well as the type of exposure (bolus or fractionated) before establishing if an in vitro model is appropriate to determine the level of response to the specific ENM of interest.

Understanding the Role and Impact of Poly (Ethylene Glycol) (PEG) on Nanoparticle Formulation: Implications for COVID-19 Vaccines.

Poly (ethylene glycol) (PEG) is a widely used polymer in a variety of consumer products and in medicine. PEGylation refers to the conjugation of PEG to drugs or nanoparticles to increase circulation time and reduce unwanted host responses. PEG is viewed as being well-tolerated, but previous studies have identified anti-PEG antibodies and so-called pseudoallergic reactions in certain individuals. The increased use of nanoparticles as contrast agents or in drug delivery, along with the introduction of mRNA vaccines encapsulated in PEGylated lipid nanoparticles has brought this issue to the fore. Thus, while these vaccines have proven to be remarkably effective, rare cases of anaphylaxis have been reported, and this has been tentatively ascribed to the PEGylated carriers, which may trigger complement activation in susceptible individuals. Here, we provide a general overview of the use of PEGylated nanoparticles for pharmaceutical applications, and we discuss the activation of the complement cascade that might be caused by PEGylated nanomedicines for a better understanding of these immunological adverse reactions.

Transgenic zebrafish larvae as a non-rodent alternative model to assess pro-inflammatory (neutrophil) responses to nanomaterials.

Hazard studies for nanomaterials (NMs) commonly assess whether they activate an inflammatory response. Such assessments often rely on rodents, but alternative models are needed to support the implementation of the 3Rs principles. Zebrafish (Danio rerio) offer a viable alternative for screening NM toxicity by investigating inflammatory responses. Here, we used non-protected life stages of transgenic zebrafish (Tg(mpx:GFP)i114) with fluorescently-labeled neutrophils to assess inflammatory responses to silver (Ag) and zinc oxide (ZnO) NMs using two approaches. Zebrafish were exposed to NMs via water following a tail fin injury, or NMs were microinjected into the otic vesicle. Zebrafish were exposed to NMs at 3 days post-fertilization (dpf) and neutrophil accumulation at the injury or injection site was quantified at 0, 4, 6, 8, 24, and 48 h post-exposure. Zebrafish larvae were also exposed to fMLF, LTB4, CXCL-8, C5a, and LPS to identify a suitable positive control for inflammation induction. Aqueous exposure to Ag and ZnO NMs stimulated an enhanced and sustained neutrophilic inflammatory response in injured zebrafish larvae, with a greater response observed for Ag NMs. Following microinjection, Ag NMs stimulated a time-dependent neutrophil accumulation in the otic vesicle which peaked at 48 h. LTB4 was identified as a positive control for studies investigating inflammatory responses in injured zebrafish following aqueous exposure, and CXCL-8 for microinjection studies that assess responses in the otic vesicle. Our findings support the use of transgenic zebrafish to rapidly screen the pro-inflammatory effects of NMs, with potential for wider application in assessing chemical safety (e.g. pharmaceuticals).

Biomarkers of nanomaterials hazard from multi-layer data.

There is an urgent need to apply effective, data-driven approaches to reliably predict engineered nanomaterial (ENM) toxicity. Here we introduce a predictive computational framework based on the molecular and phenotypic effects of a large panel of ENMs across multiple in vitro and in vivo models. Our methodology allows for the grouping of ENMs based on multi-omics approaches combined with robust toxicity tests. Importantly, we identify mRNA-based toxicity markers and extensively replicate them in multiple independent datasets. We find that models based on combinations of omics-derived features and material intrinsic properties display significantly improved predictive accuracy as compared to physicochemical properties alone.

The carcinogenic action of crystalline silica: a review of the evidence supporting secondary inflammation-driven genotoxicity as a principal mechanism.

In 1987 the International Agency for Research on Cancer (IARC) classified crystalline silica (CS) as a probable carcinogen and in 1997 reclassified it as a Group 1 carcinogen, i.e., that there was sufficient evidence for carcinogenicity in experimental animals and sufficient evidence for carcinogenicity in humans. The Working Group noted that "carcinogenicity in humans was not detected in all industrial circumstances studied, carcinogenicity may be dependent on inherent characteristics of the crystalline silica or on external factors affecting its biological activity or distribution of its polymorphs." This unusual statement that the physicochemical form of the CS influences its carcinogenicity is well understood at the toxicological level and arises as a consequence of the fact that CS activity depends on the reactivity of the CS surface, which can be blocked by a number of agents. We reviewed the literature on CS genotoxicity that has been published since the 1997 monograph, with special reference to the mechanism of CS genotoxicity. The mechanism of CS genotoxicity can be primary, a result of direct interaction of CS with target cells, or indirect, as a consequence of inflammation elicited by quartz, where the inflammatory cell-derived oxidants cause the genotoxicity. The review revealed a number of papers supporting the hypothesis that the CS genotoxic and inflammatory hazard is a variable one. In an attempt to attain a quantitative basis for the potential mechanism, we carried out analysis of published data and noted a 5-fold greater dose required to reach a threshold for genotoxic effects than for proinflammatory effects in the same cell line in vitro. When we related the calculated threshold dose at the proximal alveolar region for inflammation in a published study with the threshold dose for genotoxicity in vitro, we noted that a 60-120-fold greater dose was required for direct genotoxic effects in vitro. These data strongly suggests that inflammation is the driving force for genotoxicity and that primary genotoxicity of deposited CS would play a role only at very high, possibly implausible, exposures and deposited doses. Although based on rat studies and in vitro studies, and therefore with caveats, the analysis supports the hypothesis that the mechanism of CS genotoxicity is via inflammation-driven secondary genotoxicity. This may have implications for setting of the CS standard in workplaces. During the writing of this review (in May 2009), IARC undertook a review of carcinogenic substances, including CS. The Working Group met to reassess 10 separate agents including CS. This was not a normal monograph working group published as a large single monograph, but was published as a two-page report. This review group reaffirmed the carcinogenicity of "silica dust, crystalline in the form of quartz or cristobalite" as a Group 1 agent, with the lung as the sole tumor site. Of special relevance to the present review is that the cited "established mechanism events" for CS are restricted to the words "impaired particle clearance leading to macrophage activation and persistent inflammation." The lack of mention of direct genotoxicity is in line with the conclusions reached in the present review.