NMR metabolomic approaches for plants, toxicology and medicine

NMR metabolomic approaches for plants, toxicology and medicine This article describes the principles as well as the analytical and chemometric tools that support the development of NMR metabolomic approaches. These analytical methods are illustrated through a range of selected examples in various domains of biology and medicine, concerning the study of genetically modified organisms, plant/environment interactions, environmental toxicology, pharmacology, cancerology, or molecular epidemiology

Approche métabolomique pour la recherche de marqueurs métaboliques de la qualité technologique des foies gras

La maîtrise de la fonte lipidique des foies gras à la cuisson représente aujourd’hui un enjeu majeur pour les acteurs de la filière. L’amélioration des différentes étapes : élevage-gavage, abattage et transformation des foies a permis de réduire les pertes lipidiques à la cuisson. Cependant une part de la variabilité de ce paramètre reste mal contrôlée. Pour mieux comprendre cette variabilité, les caractéristiques intrinsèques de deux groupes de foies gras avec des valeurs extrêmes de rendement technologique ont été comparées. Leurs métabolites hydrophiles ont été analysés en RMN du proton, puis comparés. Ces résultats ont renforcé l’idée que i) le catabolisme protéique est plus élevé dans les foies avec des rendements technologiques faibles que dans ceux avec des rendements élevés et ii) que le stress oxydatif semble moindre dans les foies avec des rendements technologiques élevés. Les membranes plasmiques pourraient avoir une meilleure intégrité ce qui expliquerait la plus grande rétention de lipides lors de la cuisson dans ce groupe de foie gras

The Dividing Line between Federal and State Promotion of Aeronautics

<p>The model xeno-estrogen bisphenol A (BPA) has been extensively studied over the past two decades, contributing to major advances in the field of endocrine disrupting chemicals research. Besides its well documented adverse effects on reproduction and development observed in rodents, latest studies strongly suggest that BPA disrupts several endogenous metabolic pathways, with suspected steatogenic and obesogenic effects. BPA's adverse effects on reproduction are attributed to its ability to activate estrogen receptors (ERs), but its effects on metabolism and its mechanism(s) of action at low doses are so far only marginally understood. Metabolomics based approaches are increasingly used in toxicology to investigate the biological changes induced by model toxicants and chemical mixtures, to identify markers of toxicity and biological effects. In this study, we used proton nuclear magnetic resonance (¹H-NMR) based untargeted metabolite profiling, followed by multivariate statistics and computational analysis of metabolic networks to examine the metabolic modulation induced in human hepatic cells (HepG2) by an exposure to low and very low doses of BPA (10⁻⁶M, 10⁻⁹M, and 10⁻¹²M), vs. the female reference hormone 17β-estradiol (E2, 10⁻⁹M, 10⁻¹²M, and 10⁻¹⁵M). Metabolomic analysis combined to metabolic network reconstruction highlighted different mechanisms at lower doses of exposure. At the highest dose, our results evidence that BPA shares with E2 the capability to modulate several major metabolic routes that ensure cellular functions and detoxification processes, although the effects of the model xeno-estrogen and of the natural hormone can still be distinguished.</p

Metabolic Fate of 2,4-Dichlorophenol and Related Plant Residues in Rats

This study compared the metabolic fate of [14C]-DCP, [14C]-residues from radish plants, and purified [14C]-DCP-(acetyl)glucose following oral administration in rats. A rapid excretion of radioactivity in urine occurred for [14C]-DCP, [14C]-DCP-(acetyl)glucose, and soluble residues, 69, 85, and 69% within 48 h, respectively. Radio-HPLC profiles of 0−24 h urine from rats fed [14C]-DCP and [14C]-DCP-(acetyl)glucose were close and qualitatively similar to those obtained from plant residues. No trace of native plant residues was detected under the study conditions. The structures of the two major peaks were identified by MS as the glucuronide and the sulfate conjugates of DCP. The characterization of a dehydrated glucuronide conjugate by MS and NMR of DCP was unusual. In contrast to soluble residues, bound residues were mainly excreted in feces, 90% within 48 h, whereas total residues were eliminated in both urine and feces. For total residues, the radioactivity in feces was higher than expected from the percentage of soluble and bound residues in radish plants. This result highlighted that less absorption took place when residues were present in the plant matrix as compared to plant-free residues and DCP

Metabolic Fate of [14C]Diuron and [14C]Linuron in Wheat (Triticum aestivum) and Radish (Raphanus sativus)

Metabolism of xenobiotics in plants usually occurs in three phases, phase I (primary metabolism), phase II (conjugation processes), and phase III (storage). The uptake and metabolism of [14C]diuron and [14C]linuron were investigated in wheat and radish. Seeds were sown in quartz sand and irrigated with a nutrient solution of either radioactive herbicide. Plants were harvested after two weeks, and metabolites were extracted and then analyzed by radio-reverse-high-performance liquid chromatography (HPLC). Uptake of the two molecules was higher in radish compared to wheat. Translocation of parent compounds and related metabolites from roots to aerial plant parts was important, especially for radish. A large proportion of extractable residues were found in radish whereas nonextractable residues amounted to 30% in wheat, mainly associated with roots. Chemical structure of metabolites was thereafter identified by acid, alkaline, and enzymatic hydrolyses followed by electrospray ionization mass spectrometry (ESI-MS) and proton nuclear magnetic resonance spectroscopy (1H NMR). This study highlighted the presence of diuron and linuron metabolites conjugated to sugars in addition to N-demethylation and N-demethoxylation products

Phenotypic prediction based on metabolomic data : lasso vs Bolasso, primary data vs wavelet transformation

International audienceUnderstanding the relations between various 'omics data (such as metabolomics or genomics data) and phenotypes of interest is one of the current major challenges in biology. This question can be addressed by trying to learn a way to predict the phenotype value from the omic from joint observations of the omic and of the phenotype. In this paper, we focus on the prediction of a phenotype related to the quality of the meat from metabolomic data. As metabolomic data are high dimensional data and as, conjointly, the number of observations is often restricted, model selection methods are a way both to obtain a relevant solution to the prediction problem but also to select the most important metabolomes related to the phenotype under study. During the past years, model selection has know a growing interest in the statistical community: the first - and also probably the most known - selection method has been introducted by \citep{Tibshirani:1996} under the name of LASSO. Several variants of this original approach has then been proposed such as, recently, a bootstraped LASSO, named BOLASSO, introduced in (Bach, 2009). The proposal of this paper is to combine a wavelet representation of the metabolome spectra (see (Mallat, 1999) and (Antonini, 1992) for a complete introduction to wavelets) with the BOLASSO approach. We compare this methodology to more classical methods using either the original spectra as predictors (instead of the wavelet representation) or the original LASSO to select the model. The following section deals with the methodological description of the approach whereas the next one details the experiments and results

Specific Metabolic Fingerprint of a Dietary Exposure to a Very Low Dose of Endosulfan

Like other persistent organochlorine pesticides, endosulfan residues have been detected in foods including fruit, vegetables, and fish. The aim of our study was to assess the impact of a dietary exposure to low doses of endosulfan from foetal development until adult age on metabolic homeostasis in mice and to identify biomarkers of exposure using an 1H-NMR-based metabonomic approach in various tissues and biofluids. We report in both genders an increase in plasma glucose as well as changes in levels of factors involved in the regulation of liver oxidative stress, confirming the prooxidant activities of this compound. Some metabolic changes were distinct in males and females. For example in plasma, a decrease in lipid LDL and choline content was only observed in female. Lactate levels in males were significantly increased. In conclusion, our results show that metabolic changes in liver could be linked to the onset of pathologies like diabetes and insulin resistance. Moreover from our results it appears that the NMR-based metabonomic approach could be useful for the characterization in plasma of a dietary exposure to low dose of pesticide in human

Immunological and Metabolomic Impacts of Administration of Cry1Ab Protein and MON 810 Maize in Mouse

We have investigated the immunological and metabolomic impacts of Cry1Ab administration to mice, either as a purified protein or as the Cry1Ab-expressing genetically modified (GM) MON810 maize. Humoral and cellular specific immune responses induced in BALB/cJ mice after intra-gastric (i.g.) or intra-peritoneal (i.p.) administration of purified Cry1Ab were analyzed and compared with those induced by proteins of various immunogenic and allergic potencies. Possible unintended effects of the genetic modification on the pattern of expression of maize natural allergens were studied using IgE-immunoblot and sera from maize-allergic patients. Mice were experimentally sensitized (i.g. or i.p. route) with protein extracts from GM or non-GM maize, and then anti-maize proteins and anti-Cry1Ab–induced immune responses were analyzed. In parallel, longitudinal metabolomic studies were performed on the urine of mice treated via the i.g. route. Weak immune responses were observed after i.g. administration of the different proteins. Using the i.p. route, a clear Th2 response was observed with the known allergenic proteins, whereas a mixed Th1/Th2 immune response was observed with immunogenic protein not known to be allergenic and with Cry1Ab. This then reflects protein immunogenicity in the BALB/c Th2-biased mouse strain rather than allergenicity. No difference in natural maize allergen profiles was evidenced between MON810 and its non-GM comparator. Immune responses against maize proteins were quantitatively equivalent in mice treated with MON810 vs the non-GM counterpart and no anti-Cry1Ab–specific immune response was detected in mice that received MON810. Metabolomic studies showed a slight “cultivar” effect, which represented less than 1% of the initial metabolic information. Our results confirm the immunogenicity of purified Cry1Ab without evidence of allergenic potential. Immunological and metabolomic studies revealed slight differences in mouse metabolic profiles after i.g. administration of MON810 vs its non-GM counterpart, but no significant unintended effect of the genetic modification on immune responses was seen

Obesity promotes fumonisin B1 hepatotoxicity

Obesity, which is a worldwide public health issue, is associated with chronic inflammation that contribute to long-term complications, including insulin resistance, type 2 diabetes and non-alcoholic fatty liver disease. We hypothesized that obesity may also influence the sensitivity to food contaminants, such as fumonisin B1 (FB1), a mycotoxin produced mainly by the Fusarium verticillioides. FB1, a common contaminant of corn, is the most abundant and best characterized member of the fumonisins family. We investigated whether diet-induced obesity could modulate the sensitivity to oral FB1 exposure, with emphasis on gut health and hepatotoxicity. Thus, metabolic effects of FB1 were assessed in obese and non-obese male C57BL/6J mice. Mice received a high-fat diet (HFD) or normal chow diet (CHOW) for 15 weeks. Then, during the last three weeks, mice were exposed to these diets in combination or not with FB1 (10 mg/kg body weight/day) through drinking water. As expected, HFD feeding induced significant body weight gain, increased fasting glycemia, and hepatic steatosis. Combined exposure to HFD and FB1 resulted in body weight loss and a decrease in fasting blood glucose level. This co-exposition also induces gut dysbiosis, an increase in plasma FB1 level, a decrease in liver weight and hepatic steatosis. Moreover, plasma transaminase levels were significantly increased and associated with liver inflammation in HFD/FB1-treated mice. Liver gene expression analysis revealed that the combined exposure to HFD and FB1 was associated with reduced expression of genes involved in lipogenesis and increased expression of immune response and cell cycle-associated genes. These results suggest that, in the context of obesity, FB1 exposure promotes gut dysbiosis and severe liver inflammation. To our knowledge, this study provides the first example of obesity-induced hepatitis in response to a food contaminant.L.D. PhD was supported by the INRAE Animal Health department. This work was also supported by grants from the French National Research Agency (ANR) Fumolip (ANR-16-CE21-0003) and the Hepatomics FEDER program of Région Occitanie. We thank Prof Wentzel C. Gelderblom for generously providing the FB1 and for his interest and support in our project. B.C. laboratory is supported by a Starting Grant from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No. ERC-2018-StG- 804135), a Chaire d'Excellence from IdEx Université de Paris - ANR-18-IDEX-0001, an Innovator Award from the Kenneth Rainin Foundation, an ANR grant EMULBIONT ANR-21-CE15-0042-01 and the national program “Microbiote” from INSERM. We thank Anexplo (Genotoul, Toulouse) for their excellent work on plasma biochemistry. Neutral Lipids MS and NMR experiments were performed with instruments in the Metatoul-AXIOM platform. Sphingolipid MS analysis were performed with instruments in the RUBAM platform. The FB1 plasma levels were determined using an UPLC-MS/MS instrument part of the Ghent University MSsmall expertise centre for advanced mass spectrometry analysis of small organic molecules. We thank Elodie Rousseau-Bacquié and all members of the EZOP staff for their assistance in the animal facility. We are very grateful to Talal al Saati for histology analyses and review, and we thank all members of the US006/CREFRE staff at the histology facility and the Genom'IC platforms (INSERM U1016, Paris, France) for their expertise.Peer reviewe

Trypacidin, a Spore-Borne Toxin from Aspergillus fumigatus, Is Cytotoxic to Lung Cells

Inhalation of Aspergillus fumigatus conidia can cause severe aspergillosis in immunosuppressed people. A. fumigatus produces a large number of secondary metabolites, some of which are airborne by conidia and whose toxicity to the respiratory tract has not been investigated. We found that spores of A. fumigatus contain five main compounds, tryptoquivaline F, fumiquinazoline C, questin, monomethylsulochrin and trypacidin. Fractionation of culture extracts using RP-HPLC and LC-MS showed that samples containing questin, monomethylsulochrin and trypacidin were toxic to the human A549 lung cell line. These compounds were purified and their structure verified using NMR in order to compare their toxicity against A549 cells. Trypacidin was the most toxic, decreasing cell viability and triggering cell lysis, both effects occurring at an IC50 close to 7 µM. Trypacidin toxicity was also observed in the same concentration range on human bronchial epithelial cells. In the first hour of exposure, trypacidin initiates the intracellular formation of nitric oxide (NO) and hydrogen peroxide (H2O2). This oxidative stress triggers necrotic cell death in the following 24 h. The apoptosis pathway, moreover, was not involved in the cell death process as trypacidin did not induce apoptotic bodies or a decrease in mitochondrial membrane potential. This is the first time that the toxicity of trypacidin to lung cells has been reported