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Benchmark dose (BMD) modeling estimates the dose of a chemical that causes a perturbation from baseline. Transcriptional BMDs have been shown to be relatively consistent with apical end point BMDs, opening the door to using molecular BMDs to derive human health-based guidance values for chemical exposure. Metabolomics measures the responses of small-molecule endogenous metabolites to chemical exposure, complementing transcriptomics by characterizing downstream molecular phenotypes that are more closely associated with apical end points. The aim of this study was to apply BMD modeling to in vivo metabolomics data, to compare metabolic BMDs to both transcriptional and apical end point BMDs. This builds upon our previous application of transcriptomics and BMD modeling to a 5-day rat study of triphenyl phosphate (TPhP), applying metabolomics to the same archived tissues. Specifically, liver from rats exposed to five doses of TPhP was investigated using liquid chromatography-mass spectrometry and 1H nuclear magnetic resonance spectroscopy-based metabolomics. Following the application of BMDExpress2 software, 2903 endogenous metabolic features yielded viable dose-response models, confirming a perturbation to the liver metabolome. Metabolic BMD estimates were similarly sensitive to transcriptional BMDs, and more sensitive than both clinical chemistry and apical end point BMDs. Pathway analysis of the multiomics data sets revealed a major effect of TPhP exposure on cholesterol (and downstream) pathways, consistent with clinical chemistry measurements. Additionally, the transcriptomics data indicated that TPhP activated xenobiotic metabolism pathways, which was confirmed by using the underexploited capability of metabolomics to detect xenobiotic-related compounds. Eleven biotransformation products of TPhP were discovered, and their levels were highly correlated with multiple xenobiotic metabolism genes. This work provides a case study showing how metabolomics and transcriptomics can estimate mechanistically anchored points-of-departure. Furthermore, the study demonstrates how metabolomics can also discover biotransformation products, which could be of value within a regulatory setting, for example, as an enhancement of OECD Test Guideline 417 (toxicokinetics).
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Biotransformação , Fígado , Metabolômica , Animais , Ratos , Fígado/metabolismo , Fígado/efeitos dos fármacos , Masculino , Relação Dose-Resposta a Droga , Benchmarking , Organofosfatos/toxicidade , Organofosfatos/metabolismo , Ratos Sprague-DawleyRESUMO
Lipid functions can be influenced by genetics, age, disease states, and lifestyle factors, particularly dietary patterns, which are crucial in diabetes management. Lipidomics is an expanding field involving the comprehensive exploration of lipids from biological samples. In this cross-sectional study, 396 participants from a Mediterranean region, including individuals with type 1 diabetes (T1D), type 2 diabetes (T2D), and non-diabetic individuals, underwent lipidomic profiling and dietary assessment. Participants completed validated food frequency questionnaires, and lipid analysis was conducted using ultra-high-performance liquid chromatography coupled with mass spectrometry (UHPLC/MS). Multiple linear regression models were used to determine the association between lipid features and dietary patterns. Across all subjects, acylcarnitines (AcCa) and triglycerides (TG) displayed negative associations with the alternate Healthy Eating Index (aHEI), indicating a link between lipidomic profiles and dietary habits. Various lipid species (LS) showed positive and negative associations with dietary carbohydrates, fats, and proteins. Notably, in the interaction analysis between diabetes and the aHEI, we found some lysophosphatidylcholines (LPC) that showed a similar direction with respect to aHEI in non-diabetic individuals and T2D subjects, while an opposite direction was observed in T1D subjects. The study highlights the significant association between lipidomic profiles and dietary habits in people with and without diabetes, particularly emphasizing the role of healthy dietary choices, as reflected by the aHEI, in modulating lipid concentrations. These findings underscore the importance of dietary interventions to improve metabolic health outcomes, especially in the context of diabetes management.
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Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Lipidômica , Humanos , Masculino , Feminino , Diabetes Mellitus Tipo 2/dietoterapia , Adulto , Estudos Transversais , Pessoa de Meia-Idade , Diabetes Mellitus Tipo 1/dietoterapia , Comportamento Alimentar , Região do Mediterrâneo , Lipídeos/sangue , Dieta Saudável , Dieta , Triglicerídeos/sangue , Cromatografia Líquida de Alta Pressão , Dieta Mediterrânea , Padrões Dietéticos , Carnitina/análogos & derivadosRESUMO
Objective: The study objective was to determine whether adequately delivered bilateral remote ischemic preconditioning is cardioprotective in young children undergoing surgery for 2 common congenital heart defects with or without cyanosis. Methods: We performed a prospective, double-blind, randomized controlled trial at 2 centers in the United Kingdom. Children aged 3 to 36 months undergoing tetralogy of Fallot repair or ventricular septal defect closure were randomized 1:1 to receive bilateral preconditioning or sham intervention. Participants were followed up until hospital discharge or 30 days. The primary outcome was area under the curve for high-sensitivity troponin-T in the first 24 hours after surgery, analyzed by intention-to-treat. Right atrial biopsies were obtained in selected participants. Results: Between October 2016 and December 2020, 120 eligible children were randomized to receive bilateral preconditioning (n = 60) or sham intervention (n = 60). The primary outcome, area under the curve for high-sensitivity troponin-T, was higher in the preconditioning group (mean: 70.0 ± 50.9 µg/L/h, n = 56) than in controls (mean: 55.6 ± 30.1 µg/L/h, n = 58) (mean difference, 13.2 µg/L/h; 95% CI, 0.5-25.8; P = .04). Subgroup analyses did not show a differential treatment effect by oxygen saturations (pinteraction = .25), but there was evidence of a differential effect by underlying defect (pinteraction = .04). Secondary outcomes and myocardial metabolism, quantified in atrial biopsies, were not different between randomized groups. Conclusions: Bilateral remote ischemic preconditioning does not attenuate myocardial injury in children undergoing surgical repair for congenital heart defects, and there was evidence of potential harm in unstented tetralogy of Fallot. The routine use of remote ischemic preconditioning cannot be recommended for myocardial protection during pediatric cardiac surgery.
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Grouping/read-across is widely used for predicting the toxicity of data-poor target substance(s) using data-rich source substance(s). While the chemical industry and the regulators recognise its benefits, registration dossiers are often rejected due to weak analogue/category justifications based largely on the structural similarity of source and target substances. Here we demonstrate how multi-omics measurements can improve confidence in grouping via a statistical assessment of the similarity of molecular effects. Six azo dyes provided a pool of potential source substances to predict long-term toxicity to aquatic invertebrates (Daphnia magna) for the dye Disperse Yellow 3 (DY3) as the target substance. First, we assessed the structural similarities of the dyes, generating a grouping hypothesis with DY3 and two Sudan dyes within one group. Daphnia magna were exposed acutely to equi-effective doses of all seven dyes (each at 3 doses and 3 time points), transcriptomics and metabolomics data were generated from 760 samples. Multi-omics bioactivity profile-based grouping uniquely revealed that Sudan 1 (S1) is the most suitable analogue for read-across to DY3. Mapping ToxPrint structural fingerprints of the dyes onto the bioactivity profile-based grouping indicated an aromatic alcohol moiety could be responsible for this bioactivity similarity. The long-term reproductive toxicity to aquatic invertebrates of DY3 was predicted from S1 (21-day NOEC, 40 µg/L). This prediction was confirmed experimentally by measuring the toxicity of DY3 in D. magna. While limitations of this 'omics approach are identified, the study illustrates an effective statistical approach for building chemical groups.
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Compostos Azo , Corantes , Daphnia , Poluentes Químicos da Água , Daphnia/efeitos dos fármacos , Animais , Compostos Azo/toxicidade , Compostos Azo/química , Corantes/toxicidade , Poluentes Químicos da Água/toxicidade , Metabolômica , Testes de Toxicidade/métodos , Transcriptoma/efeitos dos fármacos , Daphnia magna , MultiômicaRESUMO
BACKGROUND: In this study, we evaluated the lipidome alterations caused by type 1 diabetes (T1D) and type 2 diabetes (T2D), by determining lipids significantly associated with diabetes overall and in both sexes, and lipids associated with the glycaemic state. METHODS: An untargeted lipidomic analysis was performed to measure the lipid profiles of 360 subjects (91 T1D, 91 T2D, 74 with prediabetes and 104 controls (CT)) without cardiovascular and/or chronic kidney disease. Ultra-high performance liquid chromatography-electrospray ionization mass spectrometry (UHPLC-ESI-MS) was conducted in two ion modes (positive and negative). We used multiple linear regression models to (1) assess the association between each lipid feature and each condition, (2) determine sex-specific differences related to diabetes, and (3) identify lipids associated with the glycaemic state by considering the prediabetes stage. The models were adjusted by sex, age, hypertension, dyslipidaemia, body mass index, glucose, smoking, systolic blood pressure, triglycerides, HDL cholesterol, LDL cholesterol, alternate Mediterranean diet score (aMED) and estimated glomerular filtration rate (eGFR); diabetes duration and glycated haemoglobin (HbA1c) were also included in the comparison between T1D and T2D. RESULTS: A total of 54 unique lipid subspecies from 15 unique lipid classes were annotated. Lysophosphatidylcholines (LPC) and ceramides (Cer) showed opposite effects in subjects with T1D and subjects with T2D, LPCs being mainly up-regulated in T1D and down-regulated in T2D, and Cer being up-regulated in T2D and down-regulated in T1D. Also, Phosphatidylcholines were clearly down-regulated in subjects with T1D. Regarding sex-specific differences, ceramides and phosphatidylcholines exhibited important diabetes-associated differences due to sex. Concerning the glycaemic state, we found a gradual increase of a panel of 1-deoxyceramides from normoglycemia to prediabetes to T2D. CONCLUSIONS: Our findings revealed an extensive disruption of lipid metabolism in both T1D and T2D. Additionally, we found sex-specific lipidome changes associated with diabetes, and lipids associated with the glycaemic state that can be linked to previously described molecular mechanisms in diabetes.
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Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Estado Pré-Diabético , Masculino , Feminino , Humanos , Lipidômica , Estado Pré-Diabético/diagnóstico , Estado Pré-Diabético/complicações , HDL-Colesterol , Ceramidas , FosfatidilcolinasRESUMO
Untargeted metabolomics is an established approach in toxicology for characterising endogenous metabolic responses to xenobiotic exposure. Detecting the xenobiotic and its biotransformation products as part of the metabolomics analysis provides an opportunity to simultaneously gain deep insights into its fate and metabolism, and to associate the internal relative dose directly with endogenous metabolic responses. This integration of untargeted exposure and response measurements into a single assay has yet to be fully demonstrated. Here we assemble a workflow to discover and analyse pharmaceutical-related measurements from routine untargeted UHPLC-MS metabolomics datasets, derived from in vivo (rat plasma and cardiac tissue, and human plasma) and in vitro (human cardiomyocytes) studies that were principally designed to investigate endogenous metabolic responses to drug exposure. Our findings clearly demonstrate how untargeted metabolomics can discover extensive biotransformation maps, temporally-changing relative systemic exposure, and direct associations of endogenous biochemical responses to the internal dose.
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Metabolômica , Xenobióticos , Humanos , Ratos , Animais , Metaboloma , BiotransformaçãoRESUMO
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.
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Benchmarking , Benzo(a)pireno , Aflatoxina B1 , Ciclosporina , Rotenona , Metabolômica , Linhagem Celular , LipídeosRESUMO
Biomarker discovery using biobank samples collected from veterinary clinics would deliver insights into the diverse population of pets and accelerate diagnostic development. The acquisition, preparation, processing, and storage of biofluid samples in sufficient volumes and at a quality suitable for later analysis with most suitable discovery methods remain challenging. Metabolomics analysis is a valuable approach to detect health/disease phenotypes. Pre-processing changes during preparation of plasma/serum samples may induce variability that may be overcome using dried blood spots (DBSs). We report a proof of principle study by metabolite fingerprinting applying UHPLC-MS of plasma and DBSs acquired from healthy adult dogs and cats (age range 1-9 years), representing each of 4 dog breeds (Labrador retriever, Beagle, Petit Basset Griffon Vendeen, and Norfolk terrier) and the British domestic shorthair cat (n = 10 per group). Blood samples (20 and 40 µL) for DBSs were loaded onto filter paper, air-dried at room temperature (3 h), and sealed and stored (4°C for ~72 h) prior to storage at -80°C. Plasma from the same blood draw (250 µL) was prepared and stored at -80°C within 1 h of sampling. Metabolite fingerprinting of the DBSs and plasma produced similar numbers of metabolite features that had similar abilities to discriminate between biological classes and correctly assign blinded samples. These provide evidence that DBSs, sampled in a manner amenable to application in in-clinic/in-field processing, are a suitable sample for biomarker discovery using UHPLC-MS metabolomics. Further, given appropriate owner consent, the volumes tested (20-40 µL) make the acquisition of remnant blood from blood samples drawn for other reasons available for biobanking and other research activities. Together, this makes possible large-scale biobanking of veterinary samples, gaining sufficient material sooner and enabling quicker identification of biomarkers of interest.
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Untargeted lipidomics has previously been applied to the study of daphnids and the discovery of biomarkers that are indicative of toxicity. Typically, liquid chromatography-mass spectrometry is used to measure the changes in lipid abundance in whole-body homogenates of daphnids, each only ca. 3 mm in length which limits any biochemical interpretation of site-specific toxicity. Here, we applied mass spectrometry imaging of Daphnia magna to combine untargeted lipidomics with spatial resolution to map the molecular perturbations to defined anatomical regions. A desorption electrospray ionization-mass spectrometry (DESI-MS) method was optimized and applied to tissue sections of daphnids exposed to bisphenol-A (BPA) compared to unexposed controls, generating an untargeted mass spectrum at each pixel (35 µm2/pixel) within each section. First, unique lipid profiles from distinct tissue types were identified in whole-body daphnids using principal component analysis, specifically distinguishing appendages, eggs, eye, and gut. Second, changes in the lipidome were mapped over four stages of normal egg development and then the effect of BPA exposure on the egg lipidome was characterized. The primary perturbations to the lipidome were annotated as triacylglycerides and phosphatidylcholine, and the distributions of the individual lipid species within these classes were visualized in whole-body D. magna sections as ion images. Using an optimized DESI-MS workflow, the first ion images of D. magna tissue sections were generated, mapping both their baseline and BPA-perturbed lipidomes.
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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.
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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.
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Ensaios de Triagem em Larga Escala , Metabolômica , Animais , Espectrometria de Massas/métodos , Metaboloma , Metabolômica/métodos , Manejo de EspécimesRESUMO
Endogenous metabolite levels describe the molecular phenotype that is most downstream from chemical exposure. Consequently, quantitative changes in metabolite levels have the potential to predict mode-of-action and adversity, with regulatory toxicology predicated on the latter. However, toxicity-related metabolic biomarker resources remain highly fragmented and incomplete. Although development of the S1500+ gene biomarker panel has accelerated the application of transcriptomics to toxicology, a similar initiative for metabolic biomarkers is lacking. Our aim was to define a publicly available metabolic biomarker panel, equivalent to S1500+, capable of predicting pathway perturbations and/or adverse outcomes. We conducted a systematic review of multiple toxicological resources, yielding 189 proposed metabolic biomarkers from existing assays (BASF, Bowes-44, and Tox21), 342 biomarkers from databases (Adverse Outcome Pathway Wiki, Comparative Toxicogenomics Database, QIAGEN Ingenuity Pathway Analysis, and Toxin and Toxin-Target Database), and 435 biomarkers from the literature. Evidence mapping across all 8 resources generated a panel of 722 metabolic biomarkers for toxicology (MTox700+), of which 462 (64%) are associated with molecular pathways and 575 (80%) with adverse outcomes. Comparing MTox700+ and S1500+ revealed that 418 (58%) metabolic biomarkers associate with pathways shared across both panels, with further metabolites mapping to unique pathways. Metabolite reference standards are commercially available for 646 (90%) of the panel metabolites, and assays exist for 578 (80%) of these biomarkers. This study has generated a publicly available metabolic biomarker panel for toxicology, which through its future laboratory deployment, is intended to help build foundational knowledge to support the generation of molecular mechanistic data for chemical hazard assessment.
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Transcriptoma , Biomarcadores , Bases de Dados Factuais , FenótipoRESUMO
The gut microbiome produces vitamins, nutrients, and neurotransmitters, and helps to modulate the host immune system-and also plays a major role in the metabolism of many exogenous compounds, including drugs and chemical toxicants. However, the extent to which specific microbial species or communities modulate hazard upon exposure to chemicals remains largely opaque. Focusing on the effects of collateral dietary exposure to the widely used herbicide atrazine, we applied integrated omics and phenotypic screening to assess the role of the gut microbiome in modulating host resilience in Drosophila melanogaster. Transcriptional and metabolic responses to these compounds are sex-specific and depend strongly on the presence of the commensal microbiome. Sequencing the genomes of all abundant microbes in the fly gut revealed an enzymatic pathway responsible for atrazine detoxification unique to Acetobacter tropicalis. We find that Acetobacter tropicalis alone, in gnotobiotic animals, is sufficient to rescue increased atrazine toxicity to wild-type, conventionally reared levels. This work points toward the derivation of biotic strategies to improve host resilience to environmental chemical exposures, and illustrates the power of integrative omics to identify pathways responsible for adverse health outcomes.
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Atrazina/toxicidade , Drosophila melanogaster/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Inseticidas/toxicidade , Acetobacter/genética , Acetobacter/metabolismo , Animais , Drosophila melanogaster/microbiologia , Feminino , Inativação Metabólica , MasculinoRESUMO
BACKGROUND: The Investigation/Study/Assay (ISA) Metadata Framework is an established and widely used set of open source community specifications and software tools for enabling discovery, exchange, and publication of metadata from experiments in the life sciences. The original ISA software suite provided a set of user-facing Java tools for creating and manipulating the information structured in ISA-Tab-a now widely used tabular format. To make the ISA framework more accessible to machines and enable programmatic manipulation of experiment metadata, the JSON serialization ISA-JSON was developed. RESULTS: In this work, we present the ISA API, a Python library for the creation, editing, parsing, and validating of ISA-Tab and ISA-JSON formats by using a common data model engineered as Python object classes. We describe the ISA API feature set, early adopters, and its growing user community. CONCLUSIONS: The ISA API provides users with rich programmatic metadata-handling functionality to support automation, a common interface, and an interoperable medium between the 2 ISA formats, as well as with other life science data formats required for depositing data in public databases.
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Disciplinas das Ciências Biológicas , Metadados , Bases de Dados Factuais , SoftwareRESUMO
Discovering modes of action and predictive biomarkers of drug-induced structural cardiotoxicity offers the potential to improve cardiac safety assessment of lead compounds and enhance preclinical to clinical translation during drug development. Cardiac microtissues are a promising, physiologically relevant, in vitro model, each composed of ca. 500 cells. While untargeted metabolomics is capable of generating hypotheses on toxicological modes of action and discovering metabolic biomarkers, applying this technology to low-biomass microtissues in suspension is experimentally challenging. Thus, we first evaluated a filtration-based approach for harvesting microtissues and assessed the sensitivity and reproducibility of nanoelectrospray direct infusion mass spectrometry (nESI-DIMS) measurements of intracellular extracts, revealing samples consisting of 28 pooled microtissues, harvested by filtration, are suitable for profiling the intracellular metabolome and lipidome. Subsequently, an extensive workflow combining nESI-DIMS untargeted metabolomics and lipidomics of intracellular extracts with ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) analysis of spent culture medium, to profile the metabolic footprint and quantify drug exposure concentrations, was implemented. Using the synthetic drug and model cardiotoxin sunitinib, time-resolved metabolic and lipid perturbations in cardiac microtissues were investigated, providing valuable data for generating hypotheses on toxicological modes of action and identifying putative biomarkers such as disruption of purine metabolism and perturbation of polyunsaturated fatty acid levels.
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Omics methodologies are widely used in toxicological research to understand modes and mechanisms of toxicity. Increasingly, these methodologies are being applied to questions of regulatory interest such as molecular point-of-departure derivation and chemical grouping/read-across. Despite its value, widespread regulatory acceptance of omics data has not yet occurred. Barriers to the routine application of omics data in regulatory decision making have been: 1) lack of transparency for data processing methods used to convert raw data into an interpretable list of observations; and 2) lack of standardization in reporting to ensure that omics data, associated metadata and the methodologies used to generate results are available for review by stakeholders, including regulators. Thus, in 2017, the Organisation for Economic Co-operation and Development (OECD) Extended Advisory Group on Molecular Screening and Toxicogenomics (EAGMST) launched a project to develop guidance for the reporting of omics data aimed at fostering further regulatory use. Here, we report on the ongoing development of the first formal reporting framework describing the processing and analysis of both transcriptomic and metabolomic data for regulatory toxicology. We introduce the modular structure, content, harmonization and strategy for trialling this reporting framework prior to its publication by the OECD.
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Metabolômica/normas , Organização para a Cooperação e Desenvolvimento Econômico/normas , Toxicogenética/normas , Toxicologia/normas , Transcriptoma/fisiologia , Documentação/normas , HumanosRESUMO
Incorporating safety data early in the drug discovery pipeline is key to reducing costly lead candidate failures. For a single drug development project, we estimate that several thousand samples per day require screening (<10 s per acquisition). While chromatography-based metabolomics has proven value at predicting toxicity from metabolic biomarker profiles, it lacks sufficiently high sample throughput. Acoustic mist ionization mass spectrometry (AMI-MS) is an atmospheric pressure ionization approach that can measure metabolites directly from 384-well plates with unparalleled speed. We sought to implement a signal processing and data analysis workflow to produce high-quality AMI-MS metabolomics data and to demonstrate its application to drug safety screening. An existing direct infusion mass spectrometry workflow was adapted, extended, optimized, and tested, utilizing three AMI-MS data sets acquired from technical and biological replicates of metabolite standards and HepG2 cell lysates and a toxicity study. Driven by criteria to minimize variance and maximize feature counts, an algorithm to extract the pulsed scan data was designed; parameters for signal-to-noise-ratio, replicate filter, sample filter, missing value filter, and RSD filter were all optimized; normalization and batch correction strategies were adapted; and cell phenotype filtering was implemented to exclude high cytotoxicity samples. The workflow was demonstrated using a highly replicated HepG2 toxicity data set, comprising 2772 samples from exposures to 16 drugs across 9 concentrations and generated in under 5 h, revealing metabolic phenotypes and individual metabolite changes that characterize specific modes of action. This AMI-MS workflow opens the door to ultrahigh-throughput metabolomics screening, increasing the rate of sample analysis by approximately 2 orders of magnitude.
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Metaboloma , Metabolômica , Acústica , Descoberta de Drogas , Espectrometria de MassasRESUMO
Iron is typically the dominant metal in the ultrafine fraction of airborne particulate matter. Various studies have investigated the toxicity of inhaled nano-sized iron oxide particles (FeOxNPs) but their results have been contradictory, with some indicating no or minor effects and others finding effects including oxidative stress and inflammation. Most studies, however, did not use materials reflecting the characteristics of FeOxNPs present in the environment. We, therefore, analysed the potential toxicity of FeOxNPs of different forms (Fe3O4, α-Fe2O3 and γ-Fe2O3) reflecting the characteristics of high iron content nano-sized particles sampled from the environment, both individually and in a mixture (FeOx-mix). A preliminary in vitro study indicated Fe3O4 and FeOx-mix were more cytotoxic than either form of Fe2O3 in human bronchial epithelial cells (BEAS-2B). Follow-up in vitro (0.003, 0.03, 0.3 µg/mL, 24 h) and in vivo (Sprague-Dawley rats, nose-only exposure, 50 µg/m3 and 500 µg/m3, 3 h/d × 3 d) studies therefore focused on these materials. Experiments in vitro explored responses at the molecular level via multi-omics analyses at concentrations below those at which significant cytotoxicity was evident to avoid detection of responses secondary to toxicity. Inhalation experiments used aerosol concentrations chosen to produce similar levels of particle deposition on the airway surface as were delivered in vitro. These were markedly higher than environmental concentrations. No clinical signs of toxicity were seen nor effects on BALF cell counts or LDH levels. There were also no significant changes in transcriptomic or metabolomic responses in lung or BEAS-2B cells to suggest adverse effects.
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Lesão Pulmonar Aguda/fisiopatologia , Inflamação/fisiopatologia , Pulmão/efeitos dos fármacos , Nanopartículas Magnéticas de Óxido de Ferro/toxicidade , Lesão Pulmonar Aguda/induzido quimicamente , Aerossóis/química , Aerossóis/toxicidade , Poluentes Atmosféricos/toxicidade , Animais , Linhagem Celular , Humanos , Inflamação/induzido quimicamente , Exposição por Inalação , Pulmão/patologia , Material Particulado/toxicidade , Ratos , Ratos Sprague-DawleyRESUMO
SUMMARY: Implementing and combining methods from a diverse range of R/Bioconductor packages into 'omics' data analysis workflows represents a significant challenge in terms of standardization, readability and reproducibility. Here, we present an R/Bioconductor package, named struct (Statistics in R using Class-based Templates), which defines a suite of class-based templates that allows users to develop and implement highly standardized and readable statistical analysis workflows. Struct integrates with the STATistics Ontology to ensure consistent reporting and maximizes semantic interoperability. We also present a toolbox, named structToolbox, which includes an extensive set of commonly used data analysis methods that have been implemented using struct. This toolbox can be used to build data-analysis workflows for metabolomics and other omics technologies. AVAILABILITY AND IMPLEMENTATION: struct and structToolbox are implemented in R, and are freely available from Bioconductor (http://bioconductor.org/packages/struct and http://bioconductor.org/packages/structToolbox), including documentation and vignettes. Source code is available and maintained at https://github.com/computational-metabolomics.