Metabolomics Simultaneously Derives Benchmark Dose Estimates and Discovers Metabolic Biotransformations in a Rat Bioassay.

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).

Treatment of wild-type mice with 2,3-butanediol, a urinary biomarker of Fmo5 -/- mice, decreases plasma cholesterol and epididymal fat deposition.

We previously showed that Fmo5 -/- mice exhibit a lean phenotype and slower metabolic ageing. Their characteristics include lower plasma glucose and cholesterol, greater glucose tolerance and insulin sensitivity, and a reduction in age-related weight gain and whole-body fat deposition. In this paper, nuclear magnetic resonance (NMR) spectroscopy-based metabolite analyses of the urine of Fmo5 -/- and wild-type mice identified two isomers of 2,3-butanediol as discriminating urinary biomarkers of Fmo5 -/- mice. Antibiotic-treatment of Fmo5 -/- mice increased plasma cholesterol concentration and substantially reduced urinary excretion of 2,3-butanediol isomers, indicating that the gut microbiome contributed to the lower plasma cholesterol of Fmo5 -/- mice, and that 2,3-butanediol is microbially derived. Short- and long-term treatment of wild-type mice with a 2,3-butanediol isomer mix decreased plasma cholesterol and epididymal fat deposition but had no effect on plasma concentrations of glucose or insulin, or on body weight. In the case of long-term treatment, the effects were maintained after withdrawal of 2,3-butanediol. Short-, but not long-term treatment, also decreased plasma concentrations of triglycerides and non-esterified fatty acids. Fecal transplant from Fmo5 -/- to wild-type mice had no effect on plasma cholesterol, and 2,3-butanediol was not detected in the urine of recipient mice, suggesting that the microbiota of the large intestine was not the source of 2,3-butanediol. However, 2,3-butanediol was detected in the stomach of Fmo5 -/- mice, which was enriched for Lactobacillus genera, known to produce 2,3-butanediol. Our results indicate a microbial contribution to the phenotypic characteristic of Fmo5 -/- mice of decreased plasma cholesterol and identify 2,3-butanediol as a potential agent for lowering plasma cholesterol.

HMDB 5.0: the Human Metabolome Database for 2022.

The Human Metabolome Database or HMDB (https://hmdb.ca) has been providing comprehensive reference information about human metabolites and their associated biological, physiological and chemical properties since 2007. Over the past 15 years, the HMDB has grown and evolved significantly to meet the needs of the metabolomics community and respond to continuing changes in internet and computing technology. This year's update, HMDB 5.0, brings a number of important improvements and upgrades to the database. These should make the HMDB more useful and more appealing to a larger cross-section of users. In particular, these improvements include: (i) a significant increase in the number of metabolite entries (from 114 100 to 217 920 compounds); (ii) enhancements to the quality and depth of metabolite descriptions; (iii) the addition of new structure, spectral and pathway visualization tools; (iv) the inclusion of many new and much more accurately predicted spectral data sets, including predicted NMR spectra, more accurately predicted MS spectra, predicted retention indices and predicted collision cross section data and (v) enhancements to the HMDB's search functions to facilitate better compound identification. Many other minor improvements and updates to the content, the interface, and general performance of the HMDB website have also been made. Overall, we believe these upgrades and updates should greatly enhance the HMDB's ease of use and its potential applications not only in human metabolomics but also in exposomics, lipidomics, nutritional science, biochemistry and clinical chemistry.

NP-MRD: the Natural Products Magnetic Resonance Database.

The Natural Products Magnetic Resonance Database (NP-MRD) is a comprehensive, freely available electronic resource for the deposition, distribution, searching and retrieval of nuclear magnetic resonance (NMR) data on natural products, metabolites and other biologically derived chemicals. NMR spectroscopy has long been viewed as the 'gold standard' for the structure determination of novel natural products and novel metabolites. NMR is also widely used in natural product dereplication and the characterization of biofluid mixtures (metabolomics). All of these NMR applications require large collections of high quality, well-annotated, referential NMR spectra of pure compounds. Unfortunately, referential NMR spectral collections for natural products are quite limited. It is because of the critical need for dedicated, open access natural product NMR resources that the NP-MRD was funded by the National Institute of Health (NIH). Since its launch in 2020, the NP-MRD has grown quickly to become the world's largest repository for NMR data on natural products and other biological substances. It currently contains both structural and NMR data for nearly 41,000 natural product compounds from >7400 different living species. All structural, spectroscopic and descriptive data in the NP-MRD is interactively viewable, searchable and fully downloadable in multiple formats. Extensive hyperlinks to other databases of relevance are also provided. The NP-MRD also supports community deposition of NMR assignments and NMR spectra (1D and 2D) of natural products and related meta-data. The deposition system performs extensive data enrichment, automated data format conversion and spectral/assignment evaluation. Details of these database features, how they are implemented and plans for future upgrades are also provided. The NP-MRD is available at https://np-mrd.org.

Metabonomics study of the effects of single copy mutant KRAS in the presence or absence of WT allele using human HCT116 isogenic cell lines.

INTRODUCTION: KRAS was one of the earliest human oncogenes to be described and is one of the most commonly mutated genes in different human cancers, including colorectal cancer. Despite KRAS mutants being known driver mutations, KRAS has proved difficult to target therapeutically, necessitating a comprehensive understanding of the molecular mechanisms underlying KRAS-driven cellular transformation. OBJECTIVES: To investigate the metabolic signatures associated with single copy mutant KRAS in isogenic human colorectal cancer cells and to determine what metabolic pathways are affected. METHODS: Using NMR-based metabonomics, we compared wildtype (WT)-KRAS and mutant KRAS effects on cancer cell metabolism using metabolic profiling of the parental KRAS G13D/+ HCT116 cell line and its isogenic, derivative cell lines KRAS +/- and KRAS G13D/-. RESULTS: Mutation in the KRAS oncogene leads to a general metabolic remodelling to sustain growth and counter stress, including alterations in the metabolism of amino acids and enhanced glutathione biosynthesis. Additionally, we show that KRASG13D/+ and KRASG13D/- cells have a distinct metabolic profile characterized by dysregulation of TCA cycle, up-regulation of glycolysis and glutathione metabolism pathway as well as increased glutamine uptake and acetate utilization. CONCLUSIONS: Our study showed the effect of a single point mutation in one KRAS allele and KRAS allele loss in an isogenic genetic background, hence avoiding confounding genetic factors. Metabolic differences among different KRAS mutations might play a role in their different responses to anticancer treatments and hence could be exploited as novel metabolic vulnerabilities to develop more effective therapies against oncogenic KRAS.

Metabolic characterization of colorectal cancer cells harbouring different KRAS mutations in codon 12, 13, 61 and 146 using human SW48 isogenic cell lines.

INTRODUCTION: Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) mutations occur in approximately one-third of colorectal (CRC) tumours and have been associated with poor prognosis and resistance to some therapeutics. In addition to the well-documented pro-tumorigenic role of mutant Ras alleles, there is some evidence suggesting that not all KRAS mutations are equal and the position and type of amino acid substitutions regulate biochemical activity and transforming capacity of KRAS mutations. OBJECTIVES: To investigate the metabolic signatures associated with different KRAS mutations in codons 12, 13, 61 and 146 and to determine what metabolic pathways are affected by different KRAS mutations. METHODS: We applied an NMR-based metabonomics approach to compare the metabolic profiles of the intracellular extracts and the extracellular media from isogenic human SW48 CRC cell lines with different KRAS mutations in codons 12 (G12D, G12A, G12C, G12S, G12R, G12V), 13 (G13D), 61 (Q61H) and 146 (A146T) with their wild-type counterpart. We used false discovery rate (FDR)-corrected analysis of variance (ANOVA) to determine metabolites that were statistically significantly different in concentration between the different mutants. RESULTS: CRC cells carrying distinct KRAS mutations exhibited differential metabolic remodelling, including differences in glycolysis, glutamine utilization and in amino acid, nucleotide and hexosamine metabolism. CONCLUSIONS: Metabolic differences among different KRAS mutations might play a role in their different responses to anticancer treatments and hence could be exploited as novel metabolic vulnerabilities to develop more effective therapies against oncogenic KRAS.

Flavin-Containing Monooxygenase 1 Catalyzes the Production of Taurine from Hypotaurine.

Taurine is one of the most abundant amino acids in mammalian tissues. It is obtained from the diet and by de novo synthesis from cysteic acid or hypotaurine. Despite the discovery in 1954 that the oxygenation of hypotaurine produces taurine, the identification of an enzyme catalyzing this reaction has remained elusive. In large part, this is due to the incorrect assignment, in 1962, of the enzyme as an NAD-dependent hypotaurine dehydrogenase. For more than 55 years, the literature has continued to refer to this enzyme as such. Here we show, both in vivo and in vitro, that the enzyme that oxygenates hypotaurine to produce taurine is flavin-containing monooxygenase (FMO) 1. Metabolite analysis of the urine of Fmo1-null mice by 1H NMR spectroscopy revealed a buildup of hypotaurine and a deficit of taurine in comparison with the concentrations of these compounds in the urine of wild-type mice. In vitro assays confirmed that human FMO1 catalyzes the conversion of hypotaurine to taurine, utilizing either NADPH or NADH as cofactor. FMO1 has a wide substrate range and is best known as a xenobiotic- or drug-metabolizing enzyme. The identification that the endogenous molecule hypotaurine is a substrate for the FMO1-catalyzed production of taurine resolves a long-standing mystery. This finding should help establish the role FMO1 plays in a range of biologic processes in which taurine or its deficiency is implicated, including conjugation of bile acids, neurotransmitter, antioxidant and anti-inflammatory functions, and the pathogenesis of obesity and skeletal muscle disorders. SIGNIFICANCE STATEMENT: The identity of the enzyme that catalyzes the biosynthesis of taurine from hypotaurine has remained elusive. Here we show, both in vivo and in vitro, that flavin-containing monooxygenase 1 catalyzes the oxygenation of hypotaurine to produce taurine.

Metabolic Biomarkers of Ageing in C57BL/6J Wild-Type and Flavin-Containing Monooxygenase 5 (FMO5)-Knockout Mice.

It was recently demonstrated in mice that knockout of the flavin-containing monooxygenase 5 gene, Fmo5, slows metabolic ageing via pleiotropic effects. We have now used an NMR-based metabonomics approach to study the effects of ageing directly on the metabolic profiles of urine and plasma from male, wild-type C57BL/6J and Fmo5-/- (FMO5 KO) mice back-crossed onto the C57BL/6J background. The aim of this study was to identify metabolic signatures that are associated with ageing in both these mouse lines and to characterize the age-related differences in the metabolite profiles between the FMO5 KO mice and their wild-type counterparts at equivalent time points. We identified a range of age-related biomarkers in both urine and plasma. Some metabolites, including urinary 6-hydroxy-6-methylheptan-3-one (6H6MH3O), a mouse sex pheromone, showed similar patterns of changes with age, regardless of genetic background. Others, however, were altered only in the FMO5 KO, or only in the wild-type mice, indicating the impact of genetic modifications on mouse ageing. Elevated concentrations of urinary taurine represent a distinctive, ageing-related change observed only in wild-type mice.

A guide to the identification of metabolites in NMR-based metabonomics/metabolomics experiments.

Metabonomics/metabolomics is an important science for the understanding of biological systems and the prediction of their behaviour, through the profiling of metabolites. Two technologies are routinely used in order to analyse metabolite profiles in biological fluids: nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), the latter typically with hyphenation to a chromatography system such as liquid chromatography (LC), in a configuration known as LC-MS. With both NMR and MS-based detection technologies, the identification of the metabolites in the biological sample remains a significant obstacle and bottleneck. This article provides guidance on methods for metabolite identification in biological fluids using NMR spectroscopy, and is illustrated with examples from recent studies on mice.

The phenotype of a knockout mouse identifies flavin-containing monooxygenase 5 (FMO5) as a regulator of metabolic ageing.

We report the production and metabolic phenotype of a mouse line in which the Fmo5 gene is disrupted. In comparison with wild-type (WT) mice, Fmo5(-/-) mice exhibit a lean phenotype, which is age-related, becoming apparent after 20 weeks of age. Despite greater food intake, Fmo5(-/-) mice weigh less, store less fat in white adipose tissue (WAT), have lower plasma glucose and cholesterol concentrations and enhanced whole-body energy expenditure, due mostly to increased resting energy expenditure, with no increase in physical activity. An increase in respiratory exchange ratio during the dark phase, the period in which the mice are active, indicates a switch from fat to carbohydrate oxidation. In comparison with WT mice, the rate of fatty acid oxidation in Fmo5(-/-) mice is higher in WAT, which would contribute to depletion of lipid stores in this tissue, and lower in skeletal muscle. Five proteins were down regulated in the liver of Fmo5(-/-) mice: aldolase B, ketohexokinase and cytosolic glycerol 3-phosphate dehydrogenase (GPD1) are involved in glucose or fructose metabolism and GPD1 also in production of glycerol 3-phosphate, a precursor of triglyceride biosynthesis; HMG-CoA synthase 1 is involved in cholesterol biosynthesis; and malic enzyme 1 catalyzes the oxidative decarboxylation of malate to pyruvate, in the process producing NADPH for use in lipid and cholesterol biosynthesis. Down regulation of these proteins provides a potential explanation for the reduced fat deposits and lower plasma cholesterol characteristic of Fmo5(-/-) mice. Our results indicate that disruption of the Fmo5 gene slows metabolic ageing via pleiotropic effects.