Adopting Mechanistic Molecular Biology Approaches in Exposome Research for Causal Understanding.

Through investigating the combined impact of the environmental exposures experienced by an individual throughout their lifetime, exposome research provides opportunities to understand and mitigate negative health outcomes. While current exposome research is driven by epidemiological studies that identify associations between exposures and effects, new frameworks integrating more substantial population-level metadata, including electronic health and administrative records, will shed further light on characterizing environmental exposure risks. Molecular biology offers methods and concepts to study the biological and health impacts of exposomes in experimental and computational systems. Of particular importance is the growing use of omics readouts in epidemiological and clinical studies. This paper calls for the adoption of mechanistic molecular biology approaches in exposome research as an essential step in understanding the genotype and exposure interactions underlying human phenotypes. A series of recommendations are presented to make the necessary and appropriate steps to move from exposure association to causation, with a huge potential to inform precision medicine and population health. This includes establishing hypothesis-driven laboratory testing within the exposome field, supported by appropriate methods to read across from model systems research to human.

Innovative analytical methodologies for characterizing chemical exposure with a view to next-generation risk assessment.

The chemical burden on the environment and human population is increasing. Consequently, regulatory risk assessment must keep pace to manage, reduce, and prevent adverse impacts on human and environmental health associated with hazardous chemicals. Surveillance of chemicals of known, emerging, or potential future concern, entering the environment-food-human continuum is needed to document the reality of risks posed by chemicals on ecosystem and human health from a one health perspective, feed into early warning systems and support public policies for exposure mitigation provisions and safe and sustainable by design strategies. The use of less-conventional sampling strategies and integration of full-scan, high-resolution mass spectrometry and effect-directed analysis in environmental and human monitoring programmes have the potential to enhance the screening and identification of a wider range of chemicals of known, emerging or potential future concern. Here, we outline the key needs and recommendations identified within the European Partnership for Assessment of Risks from Chemicals (PARC) project for leveraging these innovative methodologies to support the development of next-generation chemical risk assessment.

Ketocarotenoid production in tomato triggers metabolic reprogramming and cellular adaptation: The quest for homeostasis.

Plants are sessile and therefore have developed an extraordinary capacity to adapt to external signals. Here, the focus is on the plasticity of the plant cell to respond to new intracellular cues. Ketocarotenoids are high-value natural red pigments with potent antioxidant activity. In the present study, system-level analyses have revealed that the heterologous biosynthesis of ketocarotenoids in tomato initiated a series of cellular and metabolic mechanisms to cope with the formation of metabolites that are non-endogenous to the plant. The broad multilevel changes were linked to, among others, (i) the remodelling of the plastidial membrane, where the synthesis and storage of ketocarotenoids occurs; (ii) the recruiting of core metabolic pathways for the generation of metabolite precursors and energy; and (iii) redox control. The involvement of the metabolites as regulators of cellular processes shown here reinforces their pivotal role suggested in the remodelled 'central dogma' concept. Furthermore, the role of metabolic reprogramming to ensure cellular homeostasis is proposed.

A Critical Review on the Opportunity to Use Placenta and Innovative Biomonitoring Methods to Characterize the Prenatal Chemical Exposome.

Adverse effects associated with chemical exposures during pregnancy include several developmental and reproductive disorders. However, considering the tens of thousands of chemicals present on the market, the effects of chemical mixtures on the developing fetus is still likely underestimated. In this critical review, we discuss the potential to apply innovative biomonitoring methods using high-resolution mass spectrometry (HRMS) on placenta to improve the monitoring of chemical exposure during pregnancy. The physiology of the placenta and its relevance as a matrix for monitoring chemical exposures and their effects on fetal health is first outlined. We then identify several key parameters that require further investigations before placenta can be used for large-scale monitoring in a robust manner. Most critical is the need for standardization of placental sampling. Placenta is a highly heterogeneous organ, and knowledge of the intraplacenta variability of chemical composition is required to ensure unbiased and robust interindividual comparisons. Other important variables include the time of collection, the sex of the fetus, and mode of delivery. Finally, we discuss the first applications of HRMS methods on the placenta to decipher the chemical exposome and describe how the use of placenta can complement biofluids collected on the mother or the fetus.

An actionable annotation scoring framework for gas chromatography-high-resolution mass spectrometry.

Omics-based technologies have enabled comprehensive characterization of our exposure to environmental chemicals (chemical exposome) as well as assessment of the corresponding biological responses at the molecular level (eg, metabolome, lipidome, proteome, and genome). By systematically measuring personal exposures and linking these stimuli to biological perturbations, researchers can determine specific chemical exposures of concern, identify mechanisms and biomarkers of toxicity, and design interventions to reduce exposures. However, further advancement of metabolomics and exposomics approaches is limited by a lack of standardization and approaches for assigning confidence to chemical annotations. While a wealth of chemical data is generated by gas chromatography high-resolution mass spectrometry (GC-HRMS), incorporating GC-HRMS data into an annotation framework and communicating confidence in these assignments is challenging. It is essential to be able to compare chemical data for exposomics studies across platforms to build upon prior knowledge and advance the technology. Here, we discuss the major pieces of evidence provided by common GC-HRMS workflows, including retention time and retention index, electron ionization, positive chemical ionization, electron capture negative ionization, and atmospheric pressure chemical ionization spectral matching, molecular ion, accurate mass, isotopic patterns, database occurrence, and occurrence in blanks. We then provide a qualitative framework for incorporating these various lines of evidence for communicating confidence in GC-HRMS data by adapting the Schymanski scoring schema developed for reporting confidence levels by liquid chromatography HRMS (LC-HRMS). Validation of our framework is presented using standards spiked in plasma, and confident annotations in outdoor and indoor air samples, showing a false-positive rate of 12% for suspect screening for chemical identifications assigned as Level 2 (when structurally similar isomers are not considered false positives). This framework is easily adaptable to various workflows and provides a concise means to communicate confidence in annotations. Further validation, refinements, and adoption of this framework will ideally lead to harmonization across the field, helping to improve the quality and interpretability of compound annotations obtained in GC-HRMS.

Merging the exposome into an integrated framework for "omics" sciences.

The exposome concept encourages holistic consideration of the non-genetic factors (environmental exposures including lifestyle) that influence an individual's health over their life course. However, disconnect between the concept and practical application has promoted divergent interpretations of the exposome across disciplines and reinforced separation of the environmental (emphasizing exposures) and biological (emphasizing responses) research communities. In particular, while knowledge of biological responses can help to distinguish actual (i.e. experienced) from potential exposures, the inclusion of endogenous processes has generated confusion about the position of the exposome in a multi-omics systems biology context. We propose a reattribution of "exposome" to exclusively represent the totality of contact with external factors that a biological entity experiences, and introduce the term "functional exposomics" to denote the systematic study of exposure-phenotype interaction. This reoriented definition of the exposome allows a more readily integrable dataset for multi-omics and systems biology research.

Datasets from harmonised metabolic phenotyping of root, tuber and banana crop.

Biochemical characterisation of germplasm collections and crop wild relatives (CWRs) facilitates the assessment of biological potential and the selection of breeding lines for crop improvement. Data from the biochemical characterisation of staple root, tuber and banana (RTB) crops, i.e. banana (Musa spp.), cassava (Manihot esculenta), potato (Solanum tuberosum), sweet potato (Ipomoea batatas) and yam (Dioscorea spp.), using a metabolomics approach is presented. The data support the previously published research article "Metabolite database for root, tuber, and banana crops to facilitate modern breeding in understudied crops" (Price et al., 2020) [1]. Diversity panels for each crop, which included a variety of species, accessions, landraces and CWRs, were characterised. The biochemical profile for potato was based on five elite lines under abiotic stress. Metabolites were extracted from the tissue of foliage and storage organs (tuber, root and banana pulp) via solvent partition. Extracts were analysed via a combination of liquid chromatography - mass spectrometry (LC-MS), gas chromatography (GC)-MS, high pressure liquid chromatography with photodiode array detector (HPLC-PDA) and ultra performance liquid chromatography (UPLC)-PDA. Metabolites were identified by mass spectral matching to in-house libraries comprised from authentic standards and comparison to databases or previously published literature.

Application of 96-well plate SPE method for analysis of persistent organic pollutants in low volume blood serum samples.

Though many persistent organic pollutants (POPs) are closely regulated the human population is still exposed to these ubiquitous chemicals from the environment and diet. Safe management and human biomonitoring of POPs is necessary to understand the risk of exposure. Within human biomonitoring the mass of sample is often limited, therefore robust methods using smaller sample amounts are necessary. This study developed a 96-well plate solid phase extraction (SPE) method for determination of selected POPs: polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), polybrominated diphenyl ethers (PBDEs), hexabromocyclododecane (HBCD) and non-persistent novel flame retardants (NFRs) in low volume blood serum. Non-destructive clean-up coupling Oasis HLB extraction plate with Phree phospholipid removal plate was employed. Extraction efficiency was determined at low and high concentrations in certified reference materials NIST SRM 1957 and 1958, respectively. Target compounds deviated from certified values on average by 15% and 21% for SRM 1957 and SRM 1958, respectively. Observed limit of detections (LODs) ranged from 0.36 pg/mL (PCB 180) to 66.07 pg/mL (δ-HCH). The applicability for real samples is demonstrated on 48 samples from pregnant women enrolled in the pilot phase of the CELSPAC: TNG study. In total, 30 target compounds were detected in at least one sample. The method developed here provides a fast and reliable analysis of human blood serum with possibility to introduce automation for the sample preparation procedure.

The Exposome and Toxicology: A Win-Win Collaboration.

The development of the exposome concept has been one of the hallmarks of environmental and health research for the last decade. The exposome encompasses the life course environmental exposures including lifestyle factors from the prenatal period onwards. It has inspired many research programs and is expected to influence environmental and health research, practices, and policies. Yet, the links bridging toxicology and the exposome concept have not been well developed. In this review, we describe how the exposome framework can interface with and influence the field of toxicology, as well as how the field of toxicology can help advance the exposome field by providing the needed mechanistic understanding of the exposome impacts on health. Indeed, exposome-informed toxicology is expected to emphasize several orientations including (1) developing approaches integrating multiple stressors, in particular chemical mixtures, as well as the interaction of chemicals with other stressors, (2) using mechanistic frameworks such as the adverse outcome pathways to link the different stressors with toxicity outcomes, (3) characterizing the mechanistic basis of long-term effects by distinguishing different patterns of exposures and further exploring the environment-DNA interface through genetic and epigenetic studies, and (4) improving the links between environmental and human health, in particular through a stronger connection between alterations in our ecosystems and human toxicology. The exposome concept provides the linkage between the complex environment and contemporary mechanistic toxicology. What toxicology can bring to exposome characterization is a needed framework for mechanistic understanding and regulatory outcomes in risk assessment.

Towards a comprehensive characterisation of the human internal chemical exposome: Challenges and perspectives.

The holistic characterisation of the human internal chemical exposome using high-resolution mass spectrometry (HRMS) would be a step forward to investigate the environmental ætiology of chronic diseases with an unprecedented precision. HRMS-based methods are currently operational to reproducibly profile thousands of endogenous metabolites as well as externally-derived chemicals and their biotransformation products in a large number of biological samples from human cohorts. These approaches provide a solid ground for the discovery of unrecognised biomarkers of exposure and metabolic effects associated with many chronic diseases. Nevertheless, some limitations remain and have to be overcome so that chemical exposomics can provide unbiased detection of chemical exposures affecting disease susceptibility in epidemiological studies. Some of these limitations include (i) the lack of versatility of analytical techniques to capture the wide diversity of chemicals; (ii) the lack of analytical sensitivity that prevents the detection of exogenous (and endogenous) chemicals occurring at (ultra) trace levels from restricted sample amounts, and (iii) the lack of automation of the annotation/identification process. In this article, we discuss a number of technological and methodological limitations hindering applications of HRMS-based methods and propose initial steps to push towards a more comprehensive characterisation of the internal chemical exposome. We also discuss other challenges including the need for harmonisation and the difficulty inherent in assessing the dynamic nature of the internal chemical exposome, as well as the need for establishing a strong international collaboration, high level networking, and sustainable research infrastructure. A great amount of research, technological development and innovative bio-informatics tools are still needed to profile and characterise the "invisible" (not profiled), "hidden" (not detected) and "dark" (not annotated) components of the internal chemical exposome and concerted efforts across numerous research fields are paramount.

Open, High-Resolution EI+ Spectral Library of Anthropogenic Compounds.

To address the lack of high-resolution electron ionisation mass spectral libraries (HR-[EI+]-MS) for environmental chemicals, a retention-indexed HR-[EI+]-MS library has been constructed following analysis of authentic compounds via GC-Orbitrap MS. The library is freely provided alongside a compound database of predicted physicochemical properties. Currently, the library contains over 350 compounds from 56 compound classes and includes a range of legacy and emerging contaminants. The RECETOX Exposome HR-[EI+]-MS library expands the number of freely available resources for use in full-scan chemical exposure studies and is available at: https://doi.org/10.5281/zenodo.4471217.

The effect of ß-cyclocitral treatment on the carotenoid content of transgenic Marsh grapefruit (Citrus paradisi Macf.) suspension-cultured cells.

This work reports the development of suspension culture system of transgenic Marsh grapefruit (Citrus paradisi Macf., Rutaceae) callus overexpressing bacterial phytoene synthase; and the use of this suspension culture to investigate the effects of ß-cyclocitral on carotenoid content and composition. At a ß-cyclocitral concentration of 0.5 mM and after ten days cultivation, analysis of the carotenoids showed a significant increase in the content of ß-, α-carotene, and phytoene predominantly. The maximal increase in total provitamin A carotenoids content following ß-cyclocitral application was ~2-fold higher than the control, reaching 245.8 µg/g DW. The trend for increased transcript levels of biosynthetic genes PSY and ZDS correlated with the enhancement of the content of these carotenes following ß-cyclocitral treatment and GC-MS based metabolite profiling showed significant changes of metabolite levels across intermediary metabolism. These findings suggest that ß-cyclocitral can act as a chemical elicitor, to enhance the formation of carotenes in citrus suspension-cultured cells (SCC), which could be utilized in studying the regulation of carotenoid biosynthesis and biotechnological application to the renewable production of nutritional carotenoids.

Suspect and non-targeted screening of chemicals of emerging concern for human biomonitoring, environmental health studies and support to risk assessment: From promises to challenges and harmonisation issues.

Large-scale suspect and non-targeted screening approaches based on high-resolution mass spectrometry (HRMS) are today available for chemical profiling and holistic characterisation of biological samples. These advanced techniques allow the simultaneous detection of a large number of chemical features, including markers of human chemical exposure. Such markers are of interest for biomonitoring, environmental health studies and support to risk assessment. Furthermore, these screening approaches have the promising capability to detect chemicals of emerging concern (CECs), document the extent of human chemical exposure, generate new research hypotheses and provide early warning support to policy. Whilst of growing importance in the environment and food safety areas, respectively, CECs remain poorly addressed in the field of human biomonitoring. This shortfall is due to several scientific and methodological reasons, including a global lack of harmonisation. In this context, the main aim of this paper is to present an overview of the basic principles, promises and challenges of suspect and non-targeted screening approaches applied to human samples as this specific field introduce major specificities compared to other fields. Focused on liquid chromatography coupled to HRMS-based data acquisition methods, this overview addresses all steps of these new analytical workflows. Beyond this general picture, the main activities carried out on this topic within the particular framework of the European Human Biomonitoring initiative (project HBM4EU, 2017-2021) are described, with an emphasis on harmonisation measures.

Analysis of Tomato Post-Harvest Properties: Fruit Color, Shelf Life, and Fungal Susceptibility.

A wide variety of fresh market and processing tomatoes (Solanum lycopersicum) is grown and consumed worldwide. Post-harvest losses are a major contributing factor to losses in crop productivity and can account for up to 50% of the harvest. To select and breed elite tomato varieties, it is important to characterize fruit quality and evaluate the post-harvest properties of tomato fruits. This includes the analysis of shelf life (the period during which a fruit remains suitable for consumption without qualitative deterioration), color, and pathogen susceptibility. Tomato shelf life depends upon the rate of fruit softening which accompanies fruit ripening and exacerbates damage during transport and handling. Furthermore, the susceptibility of tomatoes to fruit pathogens is also often linked to fruit ripening, especially for necrotrophic fungi such as Botrytis cinerea, also known as gray mold. The methods described here are critical for determining fruit quality and fungal susceptibility during storage. © 2020 The Authors. Basic Protocol 1: Fruit color as a determinant of fruit quality Basic Protocol 2: Shelf life test of tomato fruits Basic Protocol 3: Botrytis cinerea pathogen test of tomato fruits Support Protocol: Preparation of Botrytis spore inoculum.

Metabolite database for root, tuber, and banana crops to facilitate modern breeding in understudied crops.

Roots, tubers, and bananas (RTB) are vital staples for food security in the world's poorest nations. A major constraint to current RTB breeding programmes is limited knowledge on the available diversity due to lack of efficient germplasm characterization and structure. In recent years large-scale efforts have begun to elucidate the genetic and phenotypic diversity of germplasm collections and populations and, yet, biochemical measurements have often been overlooked despite metabolite composition being directly associated with agronomic and consumer traits. Here we present a compound database and concentration range for metabolites detected in the major RTB crops: banana (Musa spp.), cassava (Manihot esculenta), potato (Solanum tuberosum), sweet potato (Ipomoea batatas), and yam (Dioscorea spp.), following metabolomics-based diversity screening of global collections held within the CGIAR institutes. The dataset including 711 chemical features provides a valuable resource regarding the comparative biochemical composition of each RTB crop and highlights the potential diversity available for incorporation into crop improvement programmes. Particularly, the tropical crops cassava, sweet potato and banana displayed more complex compositional metabolite profiles with representations of up to 22 chemical classes (unknowns excluded) than that of potato, for which only metabolites from 10 chemical classes were detected. Additionally, over 20% of biochemical signatures remained unidentified for every crop analyzed. Integration of metabolomics with the on-going genomic and phenotypic studies will enhance 'omics-wide associations of molecular signatures with agronomic and consumer traits via easily quantifiable biochemical markers to aid gene discovery and functional characterization.

Carotenoid profiling of yams: Clarity, comparisons and diversity.

Screening carotenoids of elite accessions of yam (Dioscorea spp.) used in the global yam breeding program has been conducted to quantitatively determine the carotenoid composition of the crop. Comparisons to previous data reporting cerotenoid levels in yam has been made, in order to deduce greater perspectives across multiple studies. Characterisation of complex species and accession -specific profiles have shown a rich base of diversity that can inform breeding strategies. Key findings include; (i) the identification of accessions rich in ß-carotene which can aid provitamin A biofortification, (ii) Data disputing the commonly held belief that yellow Guinea yam (D. cayennensis) has higher ß-carotene content than that of white Guinea yam (D. rotundata), and (iii) the tentative identification of C25-epoxy-apocarotenoid persicaxanthin with potential implications for tuber dormancy.

Metabolite profiling of yam (Dioscorea spp.) accessions for use in crop improvement programmes.

INTRODUCTION: Ninety-seven percent of yam (Dioscorea spp.) production takes place in low income food deficit countries (LIFDCs) and the crop provides 200 calories a day to approximately 300 million people. Therefore, yams are vital for food security. Yams have high-yield potential and high market value potential yet current breeding of yam is hindered by a lack of genomic information and genetic resources. New tools are needed to modernise breeding strategies and unlock the potential of yam to improve livelihood in LIFDCs. OBJECTIVES: Metabolomic screening has been undertaken on a diverse panel of Dioscorea accessions to assess the utility of the approach for advancing breeding strategies in this understudied crop. METHODS: Polar and lipophilic extracts from tubers of accessions from the global yam breeding program have been comprehensively profiled via gas chromatography-mass spectrometry. RESULTS: A visual pathway representation of the measured yam tuber metabolome has been delivered as a resource for biochemical evaluation of yam germplasm. Over 200 compounds were routinely measured in tubers, providing a major advance for the chemo-typing of this crop. Core biochemical redundancy concealed trends that were only elucidated following detailed mining of global metabolomics data. Combined analysis on leaf and tuber material identified a subset of metabolites which allow accurate species classification and highlighted the potential of predicting tuber composition from leaf profiles. Metabolic variation was accession-specific and often localised to compound classes, which will aid trait-targeting for metabolite markers. CONCLUSIONS: Metabolomics provides a standalone platform with potential to deliver near-future crop gains for yam. The approach compliments the genetic advancements currently underway and integration with other '-omics' studies will deliver a significant advancement to yam breeding strategies.

Metabolite profiling of Dioscorea (yam) species reveals underutilised biodiversity and renewable sources for high-value compounds.

Yams (Dioscorea spp.) are a multispecies crop with production in over 50 countries generating ~50 MT of edible tubers annually. The long-term storage potential of these tubers is vital for food security in developing countries. Furthermore, many species are important sources of pharmaceutical precursors. Despite these attributes as staple food crops and sources of high-value chemicals, Dioscorea spp. remain largely neglected in comparison to other staple tuber crops of tropical agricultural systems such as cassava (Manihot esculenta) and sweet potato (Ipomoea batatas). To date, studies have focussed on the tubers or rhizomes of Dioscorea, neglecting the foliage as waste. In the present study metabolite profiling procedures, using GC-MS approaches, have been established to assess biochemical diversity across species. The robustness of the procedures was shown using material from the phylogenetic clades. The resultant data allowed separation of the genotypes into clades, species and morphological traits with a putative geographical origin. Additionally, we show the potential of foliage material as a renewable source of high-value compounds.

Study of the influence of yeast inoculum concentration (Yarrowia lipolytica and Kluyveromyces lactis) on blue cheese aroma development using microbiological models.

Yarrowia lipolytica and Kluyveromyces lactis occur as part of Stilton cheese microflora yet are not controlled during production. This study investigated the influence of their inoculum concentration on aroma production. Models of Y. lipolytica and K. lactis, with Penicillium roqueforti, were analysed using instrumental and sensory analysis. Different concentrations of Y. lipolytica produced important changes in the aroma profiles of microbiological models, analysed by solid-phase microextraction (SPME GC-MS). Sensory analysis with discrimination tests showed differences were detectable via human perception but did not concern the similarity to blue cheese odour. Increasing the inoculum concentration of K. lactis resulted in decreased variation between replicates. Partial least squares (PLS) regression on Flash profile data showed models inoculated with low concentrations of K. lactis exhibited blue cheese-related attributes, associated with increased ketone production. Results suggest that controlling the amount of Y. lipolytica and K. lactis during production offers potential to manipulate blue cheese aroma development.