Use of reconstituted metabolic networks to assist in metabolomic data visualization and mining

Metabolomics experiments seldom achieve their aim of comprehensively covering the entire metabolome. However, important information can be gleaned even from sparse datasets, which can be facilitated by placing the results within the context of known metabolic networks. Here we present a method that allows the automatic assignment of identified metabolites to positions within known metabolic networks, and, furthermore, allows automated extraction of sub-networks of biological significance. This latter feature is possible by use of a gap-filling algorithm. The utility of the algorithm in reconstructing and mining of metabolomics data is shown on two independent datasets generated with LC–MS LTQ-Orbitrap mass spectrometry. Biologically relevant metabolic sub-networks were extracted from both datasets. Moreover, a number of metabolites, whose presence eluded automatic selection within mass spectra, could be identified retrospectively by virtue of their inferred presence through gap filling

Comparison of the in vivo biotransformation of two emerging estrogenic contaminants, BP2 and BPS, in zebrafish embryos and adults

Zebrafish embryo assays are increasingly used in the toxicological assessment of endocrine disruptors. Among other advantages, these models are 3R-compliant and are fit for screening purposes. Biotransformation processes are well-recognized as a critical factor influencing toxic response, but major gaps of knowledge exist regarding the characterization of functional metabolic capacities expressed in zebrafish. Comparative metabolic studies between embryos and adults are even scarcer. Using ³H-labeled chemicals, we examined the fate of two estrogenic emerging contaminants, benzophenone-2 (BP2) and bisphenol S (BPS), in 4-day embryos and adult zebrafish. BPS and BP2 were exclusively metabolized through phase II pathways, with no major qualitative difference between larvae and adults except the occurrence of a BP2-di-glucuronide in adults. Quantitatively, the biotransformation of both molecules was more extensive in adults. For BPS, glucuronidation was the predominant pathway in adults and larvae. For BP2, glucuronidation was the major pathway in larvae, but sulfation predominated in adults, with ca. 40% conversion of parent BP2 and an extensive release of several conjugates into water. Further larvae/adults quantitative differences were demonstrated for both molecules, with higher residue concentrations measured in larvae. The study contributes novel data regarding the metabolism of BPS and BP2 in a fish model and shows that phase II conjugation pathways are already functional in 4-dpf-old zebrafish. Comparative analysis of BP2 and BPS metabolic profiles in zebrafish larvae and adults further supports the use of zebrafish embryo as a relevant model in which toxicity and estrogenic activity can be assessed, while taking into account the absorption and fate of tested substances

Characterization of Novel Ligands of ER alpha, Er beta, and PPAR gamma: The Case of Halogenated Bisphenol A and Their Conjugated Metabolites

International audienceThe capability of the flame retardants tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA) to activate peroxysome proliferator-activated receptors (PPARs) alpha, beta, and gamma and estrogen receptors (ERs) alpha and beta has been recently investigated, but the activity of their biotransformation products and of their lower molecular weight analogues formed in the environment remains unexplored. The aim of this study was to investigate the relationship between the degree of halogenation of BPA analogues and their affinity and activity towards human PPAR gamma and ERs and to characterize active metabolites of major marketed halogenated bisphenols. The biological activity of all compounds was studied using reporter cell lines expressing these nuclear receptors (NRs). We used NR-based affinity columns to rapidly evaluate the binding affinity of halogenated bisphenols for PPAR gamma and ERs and to trap active metabolites of TBBPA and TCBPA formed in HepG2 cells. The agonistic potential of BPA analogs highly depends on their halogenation degree: the bulkier halogenated BPA analogs, the greater their capability to activate PPAR gamma. In addition, PPAR gamma-based affinity column, HGELN-PPAR gamma reporter cell line and crystallographic analysis clearly demonstrate that the sulfation pathway, usually considered as a detoxification process, leads for TBBPA and TCBPA, to the formation of sulfate conjugates which possess a residual PPAR gamma-binding activity. Our results highlight the effectiveness NR-based affinity columns to trap and characterize biologically active compounds from complex matrices. Polyhalogenated bisphenols, but also some of their metabolites, are potential disrupters of PPAR gamma activity

Active metabolism of thyroid hormone during metamorphosis of amphioxus

International audienceThyroid hormones (THs), and more precisely the 3,3′,5-triiodo-l-thyronine (T3) acetic derivative 3,3′,5-triiodothyroacetic acid (TRIAC), have been shown to activate metamorphosis in amphioxus. However, it remains unknown whether TRIAC is endogenously synthesized in amphioxus and more generally whether an active TH metabolism is regulating metamorphosis. Here we show that amphioxus naturally produces TRIAC from its precursors T3 and l-thyroxine (T4), supporting its possible role as the active TH in amphioxus larvae. In addition, we show that blocking TH production inhibits metamorphosis and that this effect is compensated by exogenous T3, suggesting that a peak of TH production is important for advancement of proper metamorphosis. Moreover, several amphioxus genes encoding proteins previously proposed to be involved in the TH signaling pathway display expression profiles correlated with metamorphosis. In particular, thyroid hormone receptor (TR) and deiodinases gene expressions are either up- or down-regulated during metamorphosis and by TH treatments. Overall, these results suggest that an active TH metabolism controls metamorphosis in amphioxus, and that endogenous TH production and metabolism as well as TH-regulated metamorphosis are ancestral in the chordate lineage

Preliminary data on the disposition of Benzo[c]fluorene in rat

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants and food contaminants. Part of them is mutagenic/genotoxic in vitro and in vivo and has shown clear carcinogenic effects in mammals. Therefore, these chemicals are of public health concern and require appropriate hazard characterization. The genotoxic process resulting in the carcinogenicity of PAHs is due to the formation of highly reactive metabolites, produced through different metabolic pathways. Among the PAH found in food samples, benzo[c]fluorene (B[c]F) was demonstrated to be carcinogenic in rodents 1,2 and the occurrence of corresponding pulmonary adducts has been demonstrated3. JECFA4 and EFSA5 recently highlighted the need for additional occurrence and toxicological data regarding this compound. In this context, we started to study B[c]F distribution and biotransformation in Sprague Dawley rats exposed to a single oral dose of radiolabelled B[c]F (20 µg/kg body weight). Extraction protocols of B[c]F residues from rats tissues, fluids, and excreta were developed with the aim to analyze both parental B[c]F and metabolites. Metabolic profiles were determined by radio-HPLC. Analysis of 0-24h urine samples showed an extensive renal excretion and the presence of many metabolites (>20). Liquid-solid extractions from liver samples excised 24h post-dose resulted in more than 50% unextractable radioactivity suggesting the presence of bound residues. Radio-HPLC profiles of extracts indicated the presence of approximately 30% of unmetabolized B[c]F, as well as several polar metabolites. Experimental developments and analyses of additional tissues and fluids are in progress, with the aim to better understand the metabolic mechanisms that trigger the carcinogenicity of this molecule. Our data will be used to develop a PBPK model in order to describe the toxicokinetics of B[c]F in rats and ultimately to extrapolate the toxicokinetics to humans

Comparison of the in vivo biotransformation of two emerging estrogenic contaminants, BP2 and BPS, in zebrafish embryos and adults

Zebrafish embryo assays are increasingly used in the toxicological assessment of endocrine disruptors. Among other advantages, these models are 3R-compliant and are fit for screening purposes. Biotransformation processes are well-recognized as a critical factor influencing toxic response, but major gaps of knowledge exist regarding the characterization of functional metabolic capacities expressed in zebrafish. Comparative metabolic studies between embryos and adults are even scarcer. Using ³H-labeled chemicals, we examined the fate of two estrogenic emerging contaminants, benzophenone-2 (BP2) and bisphenol S (BPS), in 4-day embryos and adult zebrafish. BPS and BP2 were exclusively metabolized through phase II pathways, with no major qualitative difference between larvae and adults except the occurrence of a BP2-di-glucuronide in adults. Quantitatively, the biotransformation of both molecules was more extensive in adults. For BPS, glucuronidation was the predominant pathway in adults and larvae. For BP2, glucuronidation was the major pathway in larvae, but sulfation predominated in adults, with ca. 40% conversion of parent BP2 and an extensive release of several conjugates into water. Further larvae/adults quantitative differences were demonstrated for both molecules, with higher residue concentrations measured in larvae. The study contributes novel data regarding the metabolism of BPS and BP2 in a fish model and shows that phase II conjugation pathways are already functional in 4-dpf-old zebrafish. Comparative analysis of BP2 and BPS metabolic profiles in zebrafish larvae and adults further supports the use of zebrafish embryo as a relevant model in which toxicity and estrogenic activity can be assessed, while taking into account the absorption and fate of tested substances