Developing metabolomics approaches for chemical risk assessment

Chemical use is highly dynamic and the chemical industry is projected to grow four-fold over the next four decades, according to the Organisation for Economic Co-operation and Development (OECD)1. However, complete toxicity data are often lacking for many chemicals and their potential harmful impacts on human and environmental health remain poorly characterised. It is widely recognised that traditional toxicity testing methods are no longer fit for purpose to address the huge backlog of untested substances worldwide. Therefore, there is a rapid demand for novel mechanistic approaches to fill information gaps for more robust chemical safety assessment. Among all omics, metabolomics is particularly well placed to address this knowledge gap as it captures the dynamics of molecular perturbations in a biological system, in response to xenobiotic exposure. The overarching aim of this thesis was to develop and implement untargeted metabolomics approaches suited for safety assessment of chemicals in regulatory toxicology, exploring both toxicodynamics (TD; i.e. endogenous biochemistry) and toxicokinetics (TK; primarily xenobiotic metabolism). Specifically, two applications of toxicometabolomics were explored: 1) a regulatory scenario of chemical grouping for subsequent read-across (G/RAx), and 2) characterisation of xenobiotic metabolism through untargeted metabolomics. Firstly, a novel G/RAx workflow was developed to conduct biology-based grouping of seven data-poor azo dyes, whereby mechanistic multi-omics data (i.e. metabolic, lipid and transcriptional responses in Daphnia magna) were considered along conventional in silico structure-based approaches. These proof-of-principle investigations demonstrated the potential of metabolomics for more robust chemical grouping to facilitate future toxicity prediction (i.e. read-across), simultaneously addressing the relative lack of similar regulatory-relevant case studies. Additionally, untargeted (specifically, nESI-DIMS and UHPLC-MS/MS) metabolomics, principally designed to probe the endogenous toxicity responses, unravelled promising insights into the metabolism of xenobiotics, thus demonstrating the ‘added value’ of this approach. Specifically, the TK/ADME (absorption, distribution, metabolism, elimination) information was successfully extracted from untargeted MS$^1$ nESI-DIMS metabolomics datasets for the seven azo dyes from the multi-omics grouping study. Building upon this work, untargeted UHPLC-MS/MS metabolomics was applied to characterise ADME/TK properties of five industrial chemicals in rat plasma, revealing rich biotransformation patterns for these xenobiotics. Although further research is warranted to address the existing knowledge gaps (e.g. annotation and identification of xenobiotic biotransformation products), this power to simultaneously characterise TK and TD properties (all within a single toxicometabolomics assay) holds a promising potential for deeper characterisation of chemical toxicity (i.e. via discovery of mode(s) of action, MoAs), ultimately empowering future chemical safety assessment

Multi-omics bioactivity profile-based chemical grouping and read-across:a case study with Daphnia magna and azo dyes

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

Myopathy-Sensitive G-Actin Segment 227-235 Is Involved in Salt-Induced Stabilization of Contacts within the Actin Filament

Formation of stable actin filaments, critically important for actin functions, is determined by the ionic strength of the solution. However, not much is known about the elements of the actin fold involved in ionic-strength-dependent filament stabilization. In this work, F-actin was destabilized by Cu2+ binding to Cys374, and the effects of solvent conditions on the dynamic properties of F-actin were correlated with the involvement of Segment 227-235 in filament stabilization. The results of our work show that the presence of Mg2+ at the high-affinity cation binding site of Cu-modified actin polymerized with MgCl2 strongly enhances the rate of filament subunit exchange and promotes the filament instability. In the presence of 0.1 M KCl, the filament subunit exchange was 2–3-fold lower than that in the MgCl2-polymerized F-actin. This effect correlates with the reduced accessibility of the D-loop and Segment 227-235 on opposite filament strands, consistent with an ionic-strength-dependent conformational change that modulates involvement of Segment 227-235 in stabilization of the intermonomer interface. KCl may restrict the mobility of the α-helix encompassing part of Segment 227-235 and/or be bound to Asp236 at the boundary of Segment 227-235. These results provide experimental evidence for the involvement of Segment 227-235 in salt-induced stabilization of contacts within the actin filament and suggest that they can be weakened by mutations characteristic of actin-associated myopathies