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Environmental exposures during early life play a critical role in life-course health, yet the molecular phenotypes underlying environmental effects on health are poorly understood. In the Human Early Life Exposome (HELIX) project, a multi-centre cohort of 1301 mother-child pairs, we associate individual exposomes consisting of >100 chemical, outdoor, social and lifestyle exposures assessed in pregnancy and childhood, with multi-omics profiles (methylome, transcriptome, proteins and metabolites) in childhood. We identify 1170 associations, 249 in pregnancy and 921 in childhood, which reveal potential biological responses and sources of exposure. Pregnancy exposures, including maternal smoking, cadmium and molybdenum, are predominantly associated with child DNA methylation changes. In contrast, childhood exposures are associated with features across all omics layers, most frequently the serum metabolome, revealing signatures for diet, toxic chemical compounds, essential trace elements, and weather conditions, among others. Our comprehensive and unique resource of all associations ( https://helixomics.isglobal.org/ ) will serve to guide future investigation into the biological imprints of the early life exposome. Environmental exposures in early life can have lasting health effects, but the molecular mechanisms are not well understood. Here, the authors discover >1000 associations between exposure factors and child multi-omics profiles, revealing signatures for diet, toxic chemical compounds, essential trace elements, and weather conditions.

ObjectiveAcute liver failure (ALF) is characterised by overwhelming hepatocyte death and liver inflammation with massive infiltration of myeloid cells in necrotic areas. The mechanisms underlying resolution of acute hepatic inflammation are largely unknown. Here, we aimed to investigate the impact of Mer tyrosine kinase (MerTK) during ALF and also examine how the microenvironmental mediator, secretory leucocyte protease inhibitor (SLPI), governs this response.DesignFlow cytometry, immunohistochemistry, confocal imaging and gene expression analyses determined the phenotype, functional/transcriptomic profile and tissue topography of MerTK+ monocytes/macrophages in ALF, healthy and disease controls. The temporal evolution of macrophage MerTK expression and its impact on resolution was examined in APAP-induced acute liver injury using wild-type (WT) and Mer-deficient (Mer−/−) mice. SLPI effects on hepatic myeloid cells were determined in vitro and in vivo using APAP-treated WT mice.ResultsWe demonstrate a significant expansion of resolution-like MerTK+HLA-DRhigh cells in circulatory and tissue compartments of patients with ALF. Compared with WT mice which show an increase of MerTK+MHCIIhigh macrophages during the resolution phase in ALF, APAP-treated Mer−/− mice exhibit persistent liver injury and inflammation, characterised by a decreased proportion of resident Kupffer cells and increased number of neutrophils. Both in vitro and in APAP-treated mice, SLPI reprogrammes myeloid cells towards resolution responses through induction of a MerTK+HLA-DRhigh phenotype which promotes neutrophil apoptosis and their subsequent clearance.ConclusionsWe identify a hepatoprotective, MerTK+, macrophage phenotype that evolves during the resolution phase following ALF and represents a novel immunotherapeutic target to promote resolution responses following acute liver injury.

Parkinson's disease (PD), an age-dependent neurodegenerative disease, is characterised by the selective loss of dopaminergic neurons in the substantia nigra (SN). Mitochondrial dysfunction is a hallmark of PD, and mutations in PINK1, a gene necessary for mitochondrial fitness, cause PD. Drosophila melanogaster flies with pink1 mutations exhibit mitochondrial defects and dopaminergic cell loss and are used as a PD model. To gain an integrated view of the cellular changes caused by defects in the PINK1 pathway of mitochondrial quality control, we combined metabolomics and transcriptomics analysis in pink1-mutant flies with human induced pluripotent stem cell (iPSC)-derived neural precursor cells (NPCs) with a PINK1 mutation. We observed alterations in cysteine metabolism in both the fly and human PD models. Mitochondrial dysfunction in the NPCs resulted in changes in several metabolites that are linked to cysteine synthesis and increased glutathione levels. We conclude that alterations in cysteine metabolism may compensate for increased oxidative stress in PD, revealing a unifying mechanism of early-stage PD pathology that may be targeted for drug development. This article has an associated First Person interview with the first author of the paper.

Metabolic profiling, metabolomic and metabonomic studies require robust study protocols for any large-scale comparisons and evaluations. Detailed methods for solution-state NMR spectroscopy have been summarized in an earlier protocol. This protocol details the analysis of intact tissue samples by means of high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy and we provide a detailed description of sample collection, preparation and analysis. Described here are 1 H NMR spectroscopic techniques such as the standard one-dimensional, relaxation-edited, diffusion-edited and two-dimensional J-resolved pulse experiments, as well as one-dimensional 31 P NMR spectroscopy. These are used to monitor different groups of metabolites, e.g., sugars, amino acids and osmolytes as well as larger molecules such as lipids, non-invasively. Through the use of NMR-based diffusion coefficient and relaxation times measurements, information on molecular compartmentation and mobility can be gleaned. The NMR methods are often combined with statistical analysis for further metabonomics analysis and biomarker identification. The standard acquisition time per sample is 8–10 min for a simple one-dimensional 1 H NMR spectrum, giving access to metabolite information while retaining tissue integrity and hence allowing direct comparison with histopathology and MRI/MRS findings or the evaluation together with biofluid metabolic-profiling data.

Background Environment and diet in early life can affect development and health throughout the life course. Metabolic phenotyping of urine and serum represents a complementary systems-wide approach to elucidate environment–health interactions. However, large-scale metabolome studies in children combining analyses of these biological fluids are lacking. Here, we sought to characterise the major determinants of the child metabolome and to define metabolite associations with age, sex, BMI and dietary habits in European children, by exploiting a unique biobank established as part of the Human Early-Life Exposome project ( http://www.projecthelix.eu ). Methods Metabolic phenotypes of matched urine and serum samples from 1192 children (aged 6–11) recruited from birth cohorts in six European countries were measured using high-throughput 1 H nuclear magnetic resonance (NMR) spectroscopy and a targeted LC-MS/MS metabolomic assay (Biocrates Absolute IDQ p180 kit). Results We identified both urinary and serum creatinine to be positively associated with age. Metabolic associations to BMI z-score included a novel association with urinary 4-deoxyerythronic acid in addition to valine, serum carnitine, short-chain acylcarnitines (C3, C5), glutamate, BCAAs, lysophosphatidylcholines (lysoPC a C14:0, lysoPC a C16:1, lysoPC a C18:1, lysoPC a C18:2) and sphingolipids (SM C16:0, SM C16:1, SM C18:1). Dietary-metabolite associations included urinary creatine and serum phosphatidylcholines (4) with meat intake, serum phosphatidylcholines (12) with fish, urinary hippurate with vegetables, and urinary proline betaine and hippurate with fruit intake. Population-specific variance (age, sex, BMI, ethnicity, dietary and country of origin) was better captured in the serum than in the urine profile; these factors explained a median of 9.0% variance amongst serum metabolites versus a median of 5.1% amongst urinary metabolites. Metabolic pathway correlations were identified, and concentrations of corresponding metabolites were significantly correlated ( r  > 0.18) between urine and serum. Conclusions We have established a pan-European reference metabolome for urine and serum of healthy children and gathered critical resources not previously available for future investigations into the influence of the metabolome on child health. The six European cohort populations studied share common metabolic associations with age, sex, BMI z-score and main dietary habits. Furthermore, we have identified a novel metabolic association between threonine catabolism and BMI of children.

The receptor tyrosine kinase, MERTK, plays an essential role in homeostasis of the retina via efferocytosis of shed outer nuclear segments of photoreceptors. The Royal College of Surgeons rat model of retinal degeneration has been linked to loss-of-function of MERTK, and together with the MERTK knock-out mouse, phenocopy retinitis pigmentosa in humans with MERTK mutations. Given recent efforts and interest in MERTK as a potential immuno-oncology target, development of a strategy to assess ocular safety at an early pre-clinical stage is critical. We have applied a state-of-the-art, multi-modal imaging platform to assess the in vivo effects of pharmacological inhibition of MERTK in mice. This involved the application of mass spectrometry imaging (MSI) to characterize the ocular spatial distribution of our highly selective MERTK inhibitor; AZ14145845, together with histopathology and transmission electron microscopy to characterize pathological and ultra-structural change in response to MERTK inhibition. In addition, we assessed the utility of a human retinal in vitro cell model to identify perturbation of phagocytosis post MERTK inhibition. We identified high localized total compound concentrations in the retinal pigment epithelium (RPE) and retinal lesions following 28 days of treatment with AZ14145845. These lesions were present in 4 of 8 treated animals, and were characterized by a thinning of the outer nuclear layer, loss of photoreceptors (PR) and accumulation of photoreceptor outer segments at the interface of the RPE and PRs. Furthermore, the lesions were very similar to that shown in the RCS rat and MERTK knock-out mouse, suggesting a MERTK-induced mechanism of PR cell death. This was further supported by the observation of reduced phagocytosis in the human retinal cell model following treatment with AZ14145845. Our study provides a viable, translational strategy to investigate the pre-clinical toxicity of MERTK inhibitors but is equally transferrable to novel chemotypes.

Background The adverse health effects of early life exposure to tobacco smoking have been widely reported. In spite of this, the underlying molecular mechanisms of in utero and postnatal exposure to tobacco smoke are only partially understood. Here, we aimed to identify multi-layer molecular signatures associated with exposure to tobacco smoke in these two exposure windows. Methods We investigated the associations of maternal smoking during pregnancy and childhood secondhand smoke (SHS) exposure with molecular features measured in 1203 European children (mean age 8.1 years) from the Human Early Life Exposome (HELIX) project. Molecular features, covering 4 layers, included blood DNA methylation and gene and miRNA transcription, plasma proteins, and sera and urinary metabolites. Results Maternal smoking during pregnancy was associated with DNA methylation changes at 18 loci in child blood. DNA methylation at 5 of these loci was related to expression of the nearby genes. However, the expression of these genes themselves was only weakly associated with maternal smoking. Conversely, childhood SHS was not associated with blood DNA methylation or transcription patterns, but with reduced levels of several serum metabolites and with increased plasma PAI1 (plasminogen activator inhibitor-1), a protein that inhibits fibrinolysis. Some of the in utero and childhood smoking-related molecular marks showed dose-response trends, with stronger effects with higher dose or longer duration of the exposure. Conclusion In this first study covering multi-layer molecular features, pregnancy and childhood exposure to tobacco smoke were associated with distinct molecular phenotypes in children. The persistent and dose-dependent changes in the methylome make CpGs good candidates to develop biomarkers of past exposure. Moreover, compared to methylation, the weak association of maternal smoking in pregnancy with gene expression suggests different reversal rates and a methylation-based memory to past exposures. Finally, certain metabolites and protein markers evidenced potential early biological effects of postnatal SHS, such as fibrinolysis.

Background Multiple omics technologies are increasingly applied to detect early, subtle molecular responses to environmental stressors for future disease risk prevention. However, there is an urgent need for further evaluation of stability and variability of omics profiles in healthy individuals, especially during childhood. Methods We aimed to estimate intra-, inter-individual and cohort variability of multi-omics profiles (blood DNA methylation, gene expression, miRNA, proteins and serum and urine metabolites) measured 6 months apart in 156 healthy children from five European countries. We further performed a multi-omics network analysis to establish clusters of co-varying omics features and assessed the contribution of key variables (including biological traits and sample collection parameters) to omics variability. Results All omics displayed a large range of intra- and inter-individual variability depending on each omics feature, although all presented a highest median intra-individual variability. DNA methylation was the most stable profile (median 37.6% inter-individual variability) while gene expression was the least stable (6.6%). Among the least stable features, we identified 1% cross-omics co-variation between CpGs and metabolites (e.g. glucose and CpGs related to obesity and type 2 diabetes). Explanatory variables, including age and body mass index (BMI), explained up to 9% of serum metabolite variability. Conclusions Methylation and targeted serum metabolomics are the most reliable omics to implement in single time-point measurements in large cross-sectional studies. In the case of metabolomics, sample collection and individual traits (e.g. BMI) are important parameters to control for improved comparability, at the study design or analysis stage. This study will be valuable for the design and interpretation of epidemiological studies that aim to link omics signatures to disease, environmental exposures, or both.

Urinary metabolic profiling is a promising powerful tool to reflect dietary intake and can help understand metabolic alterations in response to diet quality. Here, we used 1 H NMR spectroscopy in a multicountry study in European children (1147 children from 6 different cohorts) and identified a common panel of 4 urinary metabolites (hippurate, N -methylnicotinic acid, urea, and sucrose) that was predictive of Mediterranean diet adherence (KIDMED) and ultra-processed food consumption and also had higher capacity in discriminating children’s diet quality than that of established sociodemographic determinants. Further, we showed that the identified metabolite panel also reflected the associations of these diet quality indicators with C-peptide, a stable and accurate marker of insulin resistance and future risk of metabolic disease. This methodology enables objective assessment of dietary patterns in European child populations, complementary to traditional questionary methods, and can be used in future studies to evaluate diet quality. Moreover, this knowledge can provide mechanistic evidence of common biological pathways that characterize healthy and unhealthy dietary patterns, and diet-related molecular alterations that could associate to metabolic disease.

The leading cause of drug-induced liver injury in the developed world is overdose with N -acetyl- p -aminophenol (APAP). A comparative metabonomic approach was applied to the study of both xenobiotic and endogenous metabolic profiles reflective of in vivo exposure to APAP (300 mg/kg) and its structural isomer N -acetyl- m -aminophenol (AMAP; 300 mg/kg) in C57BL/6J mice, which was anchored with histopathology. Liver and urine samples were collected at 1 h, 3 h and 6 h post-treatment and analyzed by 1 H nuclear magnetic resonance (NMR) spectroscopy and gas chromatography–mass spectrometry (liver only). Histopathology revealed the presence of centrilobular necrosis from 3 h post-APAP treatment, while an AMAP-mediated necrotic endpoint was not observed within the timescale of this study, yet two of five treated mice showed minimal centrilobular eosinophilia. The 1 H-NMR xenobiotic metabolic profile of APAP-treated animals comprised of mercapturate (urine and liver) and glutathionyl (liver) conjugates detected at 1 h post-treatment. This finding corroborated the hepatic endogenous metabolic profile which showed depletion of glutathione from 1 h onwards. In contrast, AMAP glutathionyl conjugates were not detected, nor was AMAP-induced depletion of hepatic glutathione observed. APAP administration induced significant endogenous hepatic metabolic perturbations, primarily linked to oxidative and energetic stress, and perturbation of amino acid metabolism. Early depletion of glutathione was followed by depletion of additional sulfur-containing metabolites, while altered levels of mitochondrial and glycolytic metabolites indicated a disruption of energy homeostasis. In contrast, AMAP administration caused minimal, transient, distinct metabolic perturbations and by 6 h the metabolic profiles of AMAP-treated mice were indistinguishable from those of controls.