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2014;9(7): e100910
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P266
Liu Q - Van Spitsbergen J - Cariou R - Huang CY - Jiang N - Goetz G - Hutz RJ - Tonellato PJ - Carvan MJ
Histopathologic Alterations Associated with Global Gene Expression Due to Chronic Dietary TCDD Exposure in Juvenile Zebrafish
Plos-One
The goal of this project was to investigate the effects and possible developmental disease implication of chronic dietary TCDD exposure on global gene expression anchored to histopathologic analysis in juvenile zebrafish by functional genomic, histopathologic and analytic chemistry methods. Specifically, juvenile zebrafish were fed Biodiet starter with TCDD added at 0, 0.1, 1, 10 and 100 ppb, and fish were sampled following 0, 7, 14, 28 and 42 d after initiation of the exposure. TCDD accumulated in a dose- and time-dependent manner and 100 ppb TCDD caused TCDD accumulation in female (15.49 ppb) and male (18.04 ppb) fish at 28 d post exposure. Dietary TCDD caused multiple lesions in liver, kidney, intestine and ovary of zebrafish and functional dysregulation such as depletion of glycogen in liver, retrobulbar edema, degeneration of nasal neurosensory epithelium, underdevelopment of intestine, and diminution in the fraction of ovarian follicles containing vitellogenic oocytes. Importantly, lesions in nasal epithelium and evidence of endocrine disruption based on alternatively spliced vasa transcripts are two novel and significant results of this study. Microarray gene expression analysis comparing vehicle control to dietary TCDD revealed dysregulated genes involved in pathways associated with cardiac necrosis/cell death, cardiac fibrosis, renal necrosis/cell death and liver necrosis/cell death. These baseline toxicological effects provide evidence for the potential mechanisms of developmental dysfunctions induced by TCDD and vasa as a biomarker for ovarian developmental disruption.

2014;44:237-249
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P265
Couderc M - Gandar A - Kamari A - Allain Y - Zalouk-Vergnoux A - Herrenknecht C - Le Bizec B - Mouneyrac C - Poirier L
Neurodevelopmental and behavioral effects of nonylphenol exposure during gestational and breastfeeding period on F1 rats
Neurotoxicology
Nonylphenols (NP) are endocrine-disruptors known to be widely present in our environment. This study evaluated the effects of 4-n-NP on neurobehavioral development and memory capacity after perinatal exposure on the offspring rats. Dams were gavaged with 4-n-NP (0, 50 and 200 mg/kg/day) from gestational day 5 to postnatal day (PND) 21. Dams exposed to the higher dose lost weight during gestation and had a longer gestational duration. Juvenile female pups of the 200 mg 4-n-NP/kg/day group were lighter. Their thyroid somatic index (TSI) was also affected. For male pups, a decrease of TSI at weaning for the 200 mg 4-n-NP/kg/day group and an increase of GSI for the 50 mg 4-n-NP/kg/day group were observed. Physical maturation (incisives and eyes) were likewise affected. In open field (OF) tests, females were more active than males. In the first OF (PND 36), a treatment effect was observed only for males, particularly for the high dose group, which became as active as females. The second OF (PND 71) showed few differences between groups (treated vs control), the gender difference whatever the dose was not abolished. In the Morris Water Maze test, the study of the first 30 s showed that females (200 mg/kg/day) were mainly affected. Their performances were improved by 4-n-NP. These effects were particularly important for the first short-term memory test and observed to a lesser extent in the second evaluation of the long-term memory (PND 69). These data showed that perinatal 4-n-NP exposure induced behavioral and neuro-developmental impairments from 50 mg/kg/day. ©2014 Elsevier Inc. All rights reserved.

2015;224:171-182
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P264
Dellschaft NS - Alexandre-Gouabau MC - Gardner DS - Antignac JP - Keisler DH - Budge H - Symonds ME - Sebert SP
Maternal caloric restriction during late gestation followed by exposure to a low activity obesogenic environment after weaning promotes insulin resistance in young adult offspring, an adaptation dependent on accelerated postnatal growth
Journal of Endocrinology
Maternal caloric restriction during late gestation reduces birth weight, but whether long-term adverse metabolic outcomes of intra-uterine growth retardation (IUGR) are dependent on either accelerated postnatal growth or exposure to an obesogenic environment after weaning is not established. We induced IUGR in twin-pregnant sheep using a 40% maternal caloric restriction commencing from 110 days of gestation until term (w147 days), compared with mothers fed to 100% of requirements. Offspring were reared either as singletons to accelerate postnatal growth or as twins to achieve standard growth. To promote an adverse phenotype in young adulthood, after weaning, offspring were reared under a low-activity obesogenic environment with the exception of a subgroup of IUGR offspring, reared astwins, maintained in a standard activity environment. We assessed glucose tolerance together with leptin and cortisol responses to feeding in young adulthood when the hypothalamus was sampled for assessment of genes regulating appetite control, energy and endocrine sensitivity. Caloric restriction reduced maternal plasma glucose, raised non-esterified fatty acids, and changed the metabolomic profile, but had no effect on insulin, leptin, or cortisol. IUGR offspring whose postnatal growth was enhanced and were obese showed insulin and leptin resistance plus raised cortisol. This was accompanied by increased hypothalamic gene expression for energy and glucocorticoid sensitivity. These long-term adaptations were reduced but not normalized in IUGR offspring whose postnatal growth was not accelerated and remained lean in a standard post-weaning environment. IUGR results in an adverse metabolic phenotype, especially when postnatal growth is enhanced and offspring progress to juvenile-onset obesity. ©2015 Society for Endocrinology.

2014;14:2369-2388
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P263
Courant F - Antignac JP - Dervilly-Pinel G - Le Bizec B
Basics of mass spectrometry-based metabolomics
Journal of Proteome Research
The emerging field of metabolomics, aiming to characterize small molecule metabolites present in biological systems, promises immense potential for different areas such as medicine, environmental sciences, agronomy, etc. The purpose of this article is to guide the reader through the history of the field, then through the main steps of the metabolomics workflow, from study design to structure elucidation, and help the reader to understand the key phases of a metabolomics investigation and the rationale underlying the protocols and techniques used. This article is not intended to give standard operating procedures as several papers related to this topic were already provided, but is designed as a tutorial aiming to help beginners understand the concept and challenges of MS-based metabolomics. A real case example is taken from the literature to illustrate the application of the metabolomics approach in the field of doping analysis. Challenges and limitations of the approach are then discussed along with future directions in research to cope with these limitations. This tutorial is part of the International Proteomics Tutorial Programme (IPTP18). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

2014;1362: 241-249
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P262
Deceuninck Y - Bichon E - Durand S - Bemrah-Aouachria N - Zendong Z - Morvan ML - Marchand P - Dervilly-Pinel G - Antignac JP - Leblanc JC - Le Bizec B
Development and validation of a MIP/GC-MS/MS method for the determination of bisphenol A residues in wide range of food items
Journal of Chromatography A
BPA-containing products are widely used in foodstuffs packaging as authorized within the European Union (UE no. 10/2011). Therefore, foods and beverages are in contact with BPA which can migrate from food contact material to foodstuffs. An accurate assessment of the exposure of the consumers to BPA is crucial for a non-ambiguous risk characterization. In this context, an efficient analytical method using gas chromatography coupled to tandem mass spectrometry (GC–MS/MS), in the selected reaction monitoring(SRM) mode, was developed for the quantification of BPA in foodstuffs at very low levels (<0.5 µg.kg−1). A standard operating procedure, based on the combination of two successive solid phase extractions (SPE), was developed for various liquid and solid foodstuffs. The use of 13C12-BPA as internal standard allowed accurate quantification of BPA by isotopic dilution. Control charts based on both blank and certified materials have been implemented to ensure analytical data quality. The developed analytical method has been validated according to in-house validation requirements. R2 was better than 0.9990 within the range [0-100 µg.kg−1], the trueness was 4.2%. Repeatability and within-laboratory reproducibility ranged from 7.5% to 19.0% and 2.5% to 12.2%, respectively, at 0.5 and 5.0 µg.kg−1 depending on the matrices tested for. The detection and quantification limits were 0.03 and 0.10 µg.kg−1, respectively. The reporting limit was 0.35 µg.kg−1, taking into account the mean of the laboratory background contamination. The global uncertainty was 22.2% at 95% confidence interval.© 2014 Elsevier B.V. All rights reserved.

2015; 11:403-411
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P261
Dervilly-Pinel G - Chéreau S - Cesbron N - Monteau F - Le Bizec B
LC-HRMS based metabolomics screening model to detect various b-agonists treatments in bovines
Metabolomics
A study was performed to determine if untargeted metabolomics of urine could be used to establish a predictive tool for identifying β-agonists misuse in cattle. Although prohibited for more than 20 years within the EU, growth promoting practices for livestock fattening purposes are still suspected. Methods based on gas- or liquid chromatography coupled to (tandem) mass spectrometry are today considered as the state-of-the-art to monitor, in a targeted approach, residues of known drugs. To overcome the detection of anabolic practices, new synthetic xenobiotic growth promoters have been designed and new ways of applications, such as the administration of low dose cocktails, have been developed. In this context, innovative screening strategies are urgently needed to enable efficient control of such practices. Omic technologies have recently shown their relevance in highlighting physiological response resulting from anabolic compounds administration. LC-HRMS based metabolomics is one of the approaches allowing profiling biological matrices to reveal biological effects of a drug. In the present work, a metabolomics study performed on urine samples collected in the frame of several independent experiments involving different animals, different β-agonists treatments and different parameters (doses, compounds mixture, treatment length), allowed highlighting biomarkers of interest and implementing a robust statistical model to predict for β-agonists treated bovines. Performances of the proposed model fit with EU requirements for screening methods. © Springer Science+Business Media New York 2014.

2014;86(12):5649-5655
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P260
Doué M - Dervilly-Pinel G - Gicquiau A - Pouponneau K - Monteau F - Le Bizec B
High throughput identification and quantification of anabolic steroid esters by atmospheric solids analysis probe (ASAP) mass spectrometry for efficient screening of drug preparations
Analytical Chemistry
Recent developments in ambient mass spectrometry (AMS), such as atmospheric solids analysis probe (ASAP) mass spectrometry, open a whole new range of possibilities to screen for drug preparations. In this study, the potential of ASAP for the rapid identification and quantification of anabolic steroid esters has been evaluated. These compounds are known to be used both in human and in food producing animals to enhance performances and to improve the rate of growth, respectively. Using a triple quadrupole (QqQ) MS instrument, mechanism of ionization and fragmentation in both positive and negative mode were studied for a range of 21 selected steroid esters (based on testosterone, estradiol, nandrolone, and boldenone) which highlighted common neutral mass loss of 96.1, thus allowing rapid screening in minutes to reveal steroid ester presence with minimal sample preparation. Ester identification is further achieved through an efficient 2 min workflow on a QqQ MS instrument. Moreover, the use of isotope labeled internal standards permitted the quantification of the corresponding steroid esters in selected reaction monitoring (SRM) mode, for the first time in ASAP. This approach was successfully applied for characterization of oily commercial preparations. These results open new perspectives in hormone (and drug) rapid analysis by ASAP-MS in the near future. © 2014 American Chemical Society.

2014;491–492:176–183
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P259
Rivière G - Sirot V - Marchand P - Veyrand B - Tard A - Le Bizec B - Leblanc JC
Food risk assessment to perfluoroalkyls acids and brominated flame retardants in the french population, results from the second french total diet study
Science of the Total Environment
To determine the exposure of the French population to toxic compounds contaminating the food chain, a total diet study was performed in France between 2007 and 2009. This study was designed to reflect the consumption habits of the French population and covered the most important foods in terms of consumption, selected nutrients and contribution to contamination. Based on French consumption data, the present study reports the dietary exposure to perfluoroalkyl acids (16 congeners) and brominated flame retardants (polybrominated diphenyl ethers, hexabromocyclododecane and polybrominated biphenyls). Comparison of the calculated dietary exposures with the generally accepted health-based guidance values revealed that most compounds do not pose any risk. There are however knowledge gaps for some congeners in these large chemical classes. © 2014 Elsevier B.V. All rights reserved.

2015;11(1):184-197
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P257
Jacob C - Dervilly-Pinel G - Biancotto G - Monteau F - Le Bizec B
Global urine fingerprinting by LC-ESI(+)-HRMS for better characterization of metabolic pathway disruption upon anabolic practices in bovine
Metabolomics
The use of anabolic agents in meat producing animals is forbidden in Europe since 1988. Nevertheless, the possibility of widespread abuse of hormonal substances still exists, mainly due to economical benefits. The use of ‘‘omics’’ approach is a promising strategy to highlight anabolic hormone abuse by indirectly detecting their physiological action. In this context, the aim of this work was to set up a liquid chromatography – high resolution mass spectrometry based metabolomics workflow for the screening of a combined trenbolone acetate/estradiol implant abuse in cattle urine. Therefore, an untargeted metabolomics approach combining the information provided by reverse-phase liquid chromatography and hydrophilic interaction chromatography both coupled to high resolution mass spectrometry was developed and applied to characterize and compare cattle urinary metabolic profiles from non-implanted and implanted animals. The combination of both separation modes improved the metabolite information richness. Discrimination between treated and untreated animals was observed by application of multivariate statistical analysis. Moreover, OPLS models permitted to highlight the candidate biomarkers appearing as the ions which contribute the most in the observed discrimination. From the results obtained, metabolomics approaches can be considered as a powerful strategy for the detection of fraudulent anabolic treatments in cattle since global urinary metabolic response provides helpful discrimination. © Springer Science+Business Media New York 2014

2015;11:1117–1130
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P256
Le Boucher C - Courant F - Royer AL - Jeanson S - Lortal S - Dervilly-Pinel G - Thierry A - Le Bizec B
LC-HRMS fingerprinting as an efficient approach to highlight fine differences in cheese metabolome during ripening
Metabolomics
New approaches, mainly based on mass spectrometry techniques, are being developed and appear as a must in the modern food science and microbiology research to investigate food quality and safety. To date, the investigation of cheese ripening mechanisms has mostly used targeted approaches. The aims of the present project were to assess the use of untargeted metabolomics as an approach to investigate the influence of altering one ripening parameter to generate fine differences in the microbial metabolism within cheese. Two cheeses were made which varied with respect to the spatial distribution of bacterial colonies, leading to cheeses with only big or only small colonies. Liquid chromatography high resolution mass spectrometry metabolic fingerprints were acquired on cheese extracts collected after 2, 13 and 27 days of ripening using two different extraction methods (water or acetonitrile) and analyzed using two different simultaneous ionization modes (positive and negative electrospray). Data processing involving XCMS and multivariate statistical analysis highlighted significant discriminant profiles of the cheese metabolomes according to the two different spatial distributions compared. The different fractions investigated (water and acetonitrile extractions in two ionization modes) were complementary and resulted in a view as global as possible of the cheese metabolome which had been modulated by the spatial distribution of bacterial colonies. Some of the metabolites were then identified using an in-house database. These results show the relevance of cheese LC–HRMS fingerprinting to understand the influence of a ripening parameter generating fine differences on microbial metabolism within cheese. © Springer Science+Business Media New York 2015.

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