Derivation of metabolic point of departure using high-throughput in vitro metabolomics: investigating the importance of sampling time points on benchmark concentration values in the HepaRG cell line.

Amongst omics technologies, metabolomics should have particular value in regulatory toxicology as the measurement of the molecular phenotype is the closest to traditional apical endpoints, whilst offering mechanistic insights into the biological perturbations. Despite this, the application of untargeted metabolomics for point-of-departure (POD) derivation via benchmark concentration (BMC) modelling is still a relatively unexplored area. In this study, a high-throughput workflow was applied to derive PODs associated with a chemical exposure by measuring the intracellular metabolome of the HepaRG cell line following treatment with one of four chemicals (aflatoxin B1, benzo[a]pyrene, cyclosporin A, or rotenone), each at seven concentrations (aflatoxin B1, benzo[a]pyrene, cyclosporin A: from 0.2048 µM to 50 µM; rotenone: from 0.04096 to 10 µM) and five sampling time points (2, 6, 12, 24 and 48 h). The study explored three approaches to derive PODs using benchmark concentration modelling applied to single features in the metabolomics datasets or annotated metabolites or lipids: (1) the 1st rank-ordered unannotated feature, (2) the 1st rank-ordered putatively annotated feature (using a recently developed HepaRG-specific library of polar metabolites and lipids), and (3) 25th rank-ordered feature, demonstrating that for three out of four chemical datasets all of these approaches led to relatively consistent BMC values, varying less than tenfold across the methods. In addition, using the 1st rank-ordered unannotated feature it was possible to investigate temporal trends in the datasets, which were shown to be chemical specific. Furthermore, a possible integration of metabolomics-driven POD derivation with the liver steatosis adverse outcome pathway (AOP) was demonstrated. The study highlights that advances in technologies enable application of in vitro metabolomics at scale; however, greater confidence in metabolite identification is required to ensure PODs are mechanistically anchored.

Automated Sample Preparation and Data Collection Workflow for High-Throughput In Vitro Metabolomics.

Regulatory bodies have started to recognise the value of in vitro screening and metabolomics as two types of new approach methodologies (NAMs) for chemical risk assessments, yet few high-throughput in vitro toxicometabolomics studies have been reported. A significant challenge is to implement automated sample preparation of the low biomass samples typically used for in vitro screening. Building on previous work, we have developed, characterised and demonstrated an automated sample preparation and analysis workflow for in vitro metabolomics of HepaRG cells in 96-well microplates using a Biomek i7 Hybrid Workstation (Beckman Coulter) and Orbitrap Elite (Thermo Scientific) high-resolution nanoelectrospray direct infusion mass spectrometry (nESI-DIMS), across polar metabolites and lipids. The experimental conditions evaluated included the day of metabolite extraction, order of extraction of samples in 96-well microplates, position of the 96-well microplate on the instrument's deck and well location within a microplate. By using the median relative standard deviation (mRSD (%)) of spectral features, we have demonstrated good repeatability of the workflow (final mRSD < 30%) with a low percentage of features outside the threshold applied for statistical analysis. To improve the quality of the automated workflow further, small method modifications were made and then applied to a large cohort study (4860 sample infusions across three nESI-DIMS assays), which confirmed very high repeatability of the whole workflow from cell culturing to metabolite measurements, whilst providing a significant improvement in sample throughput. It is envisioned that the automated in vitro metabolomics workflow will help to advance the application of metabolomics (as a part of NAMs) in chemical safety, primarily as an approach for high throughput screening and prioritisation.

Integrating in vitro metabolomics with a 96-well high-throughput screening platform.

INTRODUCTION: High-throughput screening (HTS) is emerging as an approach to support decision-making in chemical safety assessments. In parallel, in vitro metabolomics is a promising approach that can help accelerate the transition from animal models to high-throughput cell-based models in toxicity testing. OBJECTIVE: In this study we establish and evaluate a high-throughput metabolomics workflow that is compatible with a 96-well HTS platform employing 50,000 hepatocytes of HepaRG per well. METHODS: Low biomass cell samples were extracted for metabolomics analyses using a newly established semi-automated protocol, and the intracellular metabolites were analysed using a high-resolution spectral-stitching nanoelectrospray direct infusion mass spectrometry (nESI-DIMS) method that was modified for low sample biomass. RESULTS: The method was assessed with respect to sensitivity and repeatability of the entire workflow from cell culturing and sampling to measurement of the metabolic phenotype, demonstrating sufficient sensitivity (> 3000 features in hepatocyte extracts) and intra- and inter-plate repeatability for polar nESI-DIMS assays (median relative standard deviation < 30%). The assays were employed for a proof-of-principle toxicological study with a model toxicant, cadmium chloride, revealing changes in the metabolome across five sampling times in the 48-h exposure period. To allow the option for lipidomics analyses, the solvent system was extended by establishing separate extraction methods for polar metabolites and lipids. CONCLUSIONS: Experimental, analytical and informatics workflows reported here met pre-defined criteria in terms of sensitivity, repeatability and ability to detect metabolome changes induced by a toxicant and are ready for application in metabolomics-driven toxicity testing to complement HTS assays.

Systematic Organization of COVID-19 Data Supported by the Adverse Outcome Pathway Framework.

Adverse Outcome Pathways (AOP) provide structured frameworks for the systematic organization of research data and knowledge. The AOP framework follows a set of key principles that allow for broad application across diverse disciplines related to human health, including toxicology, pharmacology, virology and medical research. The COVID-19 pandemic engages a great number of scientists world-wide and data is increasing with exponential speed. Diligent data management strategies are employed but approaches for systematically organizing the data-derived information and knowledge are lacking. We believe AOPs can play an important role in improving interpretation and efficient application of scientific understanding of COVID-19. Here, we outline a newly initiated effort, the CIAO project (https://www.ciao-covid.net/), to streamline collaboration between scientists across the world toward development of AOPs for COVID-19, and describe the overarching aims of the effort, as well as the expected outcomes and research support that they will provide.

Understanding COVID-19 through adverse outcome pathways - 2nd CIAO AOP Design Workshop.

The CIAO project (Modelling the Pathogenesis of COVID-19 using the Adverse Outcome Pathway framework) aims at a holistic assembly of knowledge to deliver a truly transdisciplinary description of the entire COVID-19 physiopathology starting with the initial contact with the SARS-CoV-2 virus and ending with one or several adverse outcomes, e.g., respiratory failure. On 27-28 January 2021, a group of 50+ scientists from numerous organizations around the world met in the 2nd CIAO AOP Design Workshop to discuss the depiction of the COVID-19 disease process as a series of key events (KEs) in a network of AOPs. During the workshop, 74 such KEs forming 13 AOPs were identified, covering COVID-19 manifestations that affect the respiratory, neurological, liver, cardiovascular, kidney and gastrointestinal systems. Modulating factors influencing the course and severity of the disease were also addressed, as was a possible extension of the investigations beyond purely biological phenomena. The workshop ended with the creation of seven working groups, which will further elaborate on the AOPs to be presented and discussed in the 3rd CIAO workshop on 28-29 April 2021.

Carbon Nanomaterials Promote M1/M2 Macrophage Activation.

Toxic effects of certain carbon nanomaterials (CNM) have been observed in several exposure scenarios both in vivo and in vitro. However, most of the data currently available has been generated in a high-dose/acute exposure setup, limiting the understanding of their immunomodulatory mechanisms. Here, macrophage-like THP-1 cells, exposed to ten different CNM for 48 h in low-cytotoxic concentration of 10 µg mL-1 , are characterized by secretion of different cytokines and global transcriptional changes. Subsequently, the relationships between cytokine secretion and transcriptional patterns are modeled, highlighting specific pathways related to alternative macrophage activation. Finally, time- and dose-dependent activation of transcription and secretion of M1 marker genes IL-1ß and tumor necrosis factor, and M2 marker genes IL-10 and CSF1 is confirmed among the three most responsive CNM, with concentrations of 5, 10, and 20 µg mL-1 at 24, 48, and 72 h of exposure. These results underline CNM effects on the formation of cell microenvironment and gene expression leading to specific patterns of macrophage polarization. Taken together, these findings imply that, instead of a high and toxic CNM dose, a sub-lethal dose in controlled exposure setup can be utilized to alter the cell microenvironment and program antigen presenting cells, with fascinating implications for novel therapeutic strategies.

Integration of genome-wide mRNA and miRNA expression, and DNA methylation data of three cell lines exposed to ten carbon nanomaterials.

We present data derived from an exposure experiment in which three cell-lines representative of cell types of the respiratory tissue (epithelial type-I A549, epithelial type-II BEAS-2B, and macrophage THP-1) have been exposed to ten different carbon-based nanomaterials for 48 h. In particular, we provide: genome-wide mRNA and miRNA expression, and DNA methylation; gene tables, containing information on the aberrations induced in these three genomic data layers at the gene level; mechanism of action (MOA) maps representing the comparative functional alteration induced in each cell line and each exposure.

Elucidating differential nano-bio interactions of multi-walled andsingle-walled carbon nanotubes using subcellular proteomics.

Understanding the relationship between adverse exposure events and specific material properties will facilitate predictive classification of carbon nanotubes (CNTs) according to their mechanisms of action, and a safe-by-design approach for the next generation of CNTs. Mass-spectrometry-based proteomics is a reliable tool to uncover the molecular dynamics of hazardous exposures, yet challenges persist with regards to its limited dynamic range when sampling whole organisms, tissues or cell lysates. Here, the simplicity of the sub-cellular proteome was harnessed to unravel distinctive adverse exposure outcomes at the molecular level, between two CNT subtypes. A549, MRC9 and human macrophage cells, were exposed for 24h to non-cytotoxic doses of single-walled or multi-walled CNTs (swCNTs or mwCNTs). Label-free proteomics on enriched cytoplasmic fractions was complemented with analyses of reactive oxygen species (ROS) production and mitochondrial integrity. The extent/number of modulated proteoforms indicated the single-walled variant was more bioactive. Greater enrichment of pathways corresponding to oxido-reductive activity was consistent with greater intracellular ROS induction and mitochondrial dysfunction capacities of swCNTs. Other compromised cellular functions, as revealed by pathway analysis were; ribosome, spliceosome and DNA repair. Highly upregulated proteins (fold change in abundance >6) such as, APOC3, RBP4 and INS are also highlighted as potential markers of hazardous CNT exposure. We conclude that, changes in cytosolic proteome abundance resulting from nano-bio interactions, elucidate adverse response pathways and their distinctive molecular components. Our results indicate that CNT-protein interactions might have a thus far unappreciated significance for protein trafficking, and this warrants further investigation.

Multi-omics analysis of ten carbon nanomaterials effects highlights cell type specific patterns of molecular regulation and adaptation.

New strategies to characterize the effects of engineered nanomaterials (ENMs) based on omics technologies are emerging. However, given the intricate interplay of multiple regulatory layers, the study of a single molecular species in exposed biological systems might not allow the needed granularity to successfully identify the pathways of toxicity (PoT) and, hence, portraying adverse outcome pathways (AOPs). Moreover, the intrinsic diversity of different cell types composing the exposed organs and tissues in living organisms poses a problem when transferring in vivo experimentation into cell-based in vitro systems. To overcome these limitations, we have profiled genome-wide DNA methylation, mRNA and microRNA expression in three human cell lines representative of relevant cell types of the respiratory system, A549, BEAS-2B and THP-1, exposed to a low dose of ten carbon nanomaterials (CNMs) for 48 h. We applied advanced data integration and modelling techniques in order to build comprehensive regulatory and functional maps of the CNM effects in each cell type. We observed that different cell types respond differently to the same CNM exposure even at concentrations exerting similar phenotypic effects. Furthermore, we linked patterns of genomic and epigenomic regulation to intrinsic properties of CNM. Interestingly, DNA methylation and microRNA expression only partially explain the mechanism of action (MOA) of CNMs. Taken together, our results strongly support the implementation of approaches based on multi-omics screenings on multiple tissues/cell types, along with systems biology-based multi-variate data modelling, in order to build more accurate AOPs.

A secretomics analysis reveals major differences in the macrophage responses towards different types of carbon nanotubes.

Certain types of carbon nanotubes (CNT) can evoke inflammation, fibrosis and mesothelioma in vivo, raising concerns about their potential health effects. It has been recently postulated that NLRP3 inflammasome activation is important in the CNT-induced toxicity. However, more comprehensive studies of the protein secretion induced by CNT can provide new information about their possible pathogenic mechanisms. Here, we studied protein secretion from human macrophages with a proteomic approach in an unbiased way. Human monocyte-derived macrophages (MDM) were exposed to tangled or rigid, long multi-walled CNT (MWCNT) or crocidolite asbestos for 6 h. The growth media was concentrated and secreted proteins were analyzed using 2D-DIGE and DeCyder software. Subsequently, significantly up- or down-regulated protein spots were in-gel digested and identified with an LC-MS/MS approach. Bioinformatics analysis was performed to reveal the different patterns of protein secretion induced by these materials. The results show that both long rigid MWCNT and asbestos elicited ample and highly similar protein secretion. In contrast, exposure to long tangled MWCNT induced weaker protein secretion with a more distinct profile. Secretion of lysosomal proteins followed the exposure to all materials, suggesting lysosomal damage. However, only long rigid MWCNT was associated with apoptosis. This analysis suggests that the CNT toxicity in human MDM is mediated via vigorous secretion of inflammation-related proteins and apoptosis. This study provides new insights into the mechanisms of toxicity of high aspect ratio nanomaterials and indicates that not all types of CNT are as hazardous as asbestos fibers.

Phagocytosis of nano-sized titanium dioxide triggers changes in protein acetylation.

Nano-sized titanium dioxide (nTiO2) is one of the most produced engineered nanomaterials and therefore carries a high risk for workplace exposure. In several nanosafety studies, exposure to nTiO2 has been shown to trigger inflammation in mice lung and to cause oxidative stress. Here, cytoplasmic proteome changes in human monocyte derived macrophages were investigated with two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry to evaluate the adverse cellular effects after exposure to different types of TiO2 nanoparticles (NPs). Both studied TiO2 NPs (rutile TiO2 with or without silica coating) evoked similar proteome alterations. The identified proteins were linked to metabolic homeostasis, cytoskeleton remodeling and oxidative stress. The abundances of chloride intracellular channel protein 1 and cathepsin D changed only after exposure to nTiO2 as compared to a coarse particle analog. Enrichment analysis revealed that 70% of the proteins with changed intensities contained known acetylation sites, and it was possible to confirm a significant induction of cytoplasmic protein acetylation after nTiO2 exposure. The course of the events during phagocytosis could account for the observed membrane maintenance, metabolic and cytoskeletal protein expression changes. Lysine acetylation of cytoplasmic proteins in macrophages is emerging as a major cell regulation mechanism after nTiO2 exposure. BIOLOGICAL SIGNIFICANCE: While the amount of nanosafety research conducted in recent years has been constantly increasing, proteomics has not yet been utilized widely in this field. In addition, reversible protein post-translational modifications (PTMs) such as acetylation and phosphorylation have not been investigated in-depth in nanomaterial exposed cells. Proteome changes observed in nanomaterial exposed macrophages revealed active phagocytosis of the particles and provided new insights into underlying mechanisms of biological responses to nTiO2 exposures. Moreover, reversible protein acetylation might be a major cellular regulation event occurring in nanomaterial exposed cells.

Long, needle-like carbon nanotubes and asbestos activate the NLRP3 inflammasome through a similar mechanism.

Carbon nanomaterials (CNM) are targets of great interest because they have multiple applications in industry but also because of the fear of possible harmful heath effects of certain types of CNM. The high aspect ratio of carbon nanotubes (CNT), a feature they share with asbestos, is likely the key factor for reported toxicity of certain CNT. However, the mechanism to explain this toxicity is unclear. Here we investigated whether different CNM induce a pro-inflammatory response in human primary macrophages. Carbon black, short CNT, long, tangled CNT, long, needle-like CNT, and crocidolite asbestos were used to compare the effect of size and shape on the potency of the materials to induce secretion of interleukin (IL) 1-family cytokines. Our results demonstrated that long, needle-like CNT and asbestos activated secretion of IL-1ß from LPS-primed macrophages but only long, needle-like CNT induced IL-1α secretion. SiRNA experiments demonstrated that the NLRP3 inflammasome was essential for long, needle-like CNT and asbestos-induced IL-1ß secretion. Moreover, it was noted that CNT-induced NLRP3 inflammasome activation depended on reactive oxygen species (ROS) production, cathepsin B activity, P2X(7) receptor, and Src and Syk tyrosine kinases. These results provide new information about the mechanisms by which long, needle-like materials may cause their harmful health effects. Furthermore, the techniques used here may be of use in future risk assessments of nanomaterials.

Proteomic characterization of engineered nanomaterial-protein interactions in relation to surface reactivity.

Adsorption of proteins onto an engineered nanoparticle surface happens immediately after particles come in contact with a biological fluid. However, at the moment very little is known about the mechanisms of interactions between biomolecules and nanomaterials. In this study, eleven thoroughly characterized materials were first investigated in vitro for their ability to enter human lung epithelial cells and human monocyte-derived macrophages. All tested materials were taken up by primary macrophages and epithelial cells. Some of the engineered nanomaterials (ENM) were found in the cytoplasm. Large quantitative and qualitative variation in the binding efficiencies to cellular proteins was observed between different tested nanoparticles. Pulmonary surfactant components significantly reduced the overall protein adsorption on the surface of ENMs. Fibrinogen chains were attached to all materials after exposure to plasma proteins. Common ENM-bound cytoplasmic protein identifications were peroxiredoxin 1, annexin A2, and several ribosomal and cytoskeletal proteins. The underlying mechanism of the ENM-plasma protein interaction may diverge from that of cell lysate proteins, as the binding efficiency to cell lysate proteins appears to depend on the characteristics of the ENM surface, whereas the adsorbed plasma proteins are involved in particle phagocytosis and seem to cover ENMs independently of the their surface properties. Identification of the composition of the nanomaterial-protein complex is crucial for understanding of the uptake mechanisms, biodistribution, and clearance of ENMs, knowledge which is required for safety evaluation and biomedical applications of these materials.

The single-stranded DNA genome of novel archaeal virus halorubrum pleomorphic virus 1 is enclosed in the envelope decorated with glycoprotein spikes.

Only a few archaeal viruses have been subjected to detailed structural analyses. Major obstacles have been the extreme conditions such as high salinity or temperature needed for the propagation of these viruses. In addition, unusual morphotypes of many archaeal viruses have made it difficult to obtain further information on virion architectures. We used controlled virion dissociation to reveal the structural organization of Halorubrum pleomorphic virus 1 (HRPV-1) infecting an extremely halophilic archaeal host. The single-stranded DNA genome is enclosed in a pleomorphic membrane vesicle without detected nucleoproteins. VP4, the larger major structural protein of HRPV-1, forms glycosylated spikes on the virion surface and VP3, the smaller major structural protein, resides on the inner surface of the membrane vesicle. Together, these proteins organize the structure of the membrane vesicle. Quantitative lipid comparison of HRPV-1 and its host Halorubrum sp. revealed that HRPV-1 acquires lipids nonselectively from the host cell membrane, which is typical of pleomorphic enveloped viruses.