Validation of a set of reference genes to study response to herbicide stress in grasses

Abstract Background Non-target-site based resistance to herbicides is a major threat to the chemical control of agronomically noxious weeds. This adaptive trait is endowed by differences in the expression of a number of genes in plants that are resistant or sensitive to herbicides. Quantification of the expression of such genes requires normalising qPCR data using reference genes with stable expression in the system studied as internal standards. The aim of this study was to validate reference genes in Alopecurus myosuroides, a grass (Poaceae) weed of economic and agronomic importance with no genomic resources. Results The stability of 11 candidate reference genes was assessed in plants res…

Flavour from the molecules to the behaviour

Characterization of rat glutathione transferases in olfactory epithelium and mucus

International audience; The olfactory epithelium is continuously exposed to exogenous chemicals, including odorants. During the past decade, the enzymes surrounding the olfactory receptors have been shown to make an important contribution to the process of olfaction. Mammalian xenobiotic metabolizing enzymes, such as cytochrome P450, esterases and glutathione transferases (GSTs), have been shown to participate in odorant clearance from the olfactory receptor environment, consequently contributing to the maintenance of sensitivity toward odorants. GSTs have previously been shown to be involved in numerous physiological processes, including detoxification, steroid hormone biosynthesis, and am…

Oral enzymatic detoxification system: Insights obtained from proteome analysis to understand its potential impact on aroma metabolization

The oral cavity is an entry path into the body, enabling the intake of nutrients but also leading to the ingestion of harmful substances. Thus, saliva and oral tissues contain enzyme systems that enable the early neutralization of xenobiotics as soon as they enter the body. Based on recently published oral proteomic data from several research groups, this review identifies and compiles the primary detoxification enzymes (also known as xenobiotic-metabolizing enzymes) present in saliva and the oral epithelium. The functions and the metabolic activity of these enzymes are presented. Then, the activity of these enzymes in saliva, which is an extracellular fluid, is discussed with regard to the…

[Thu-P1-027] Pheromone-induced odour learning and outstanding detection abilities in the newborn rabbit

Perception of the wide, complex and changing odour environment requires that the olfactory system engages processing mechanisms ensuring efficient detection and discrimination of stimuli ending in specific motor actions and in adaptation. In newborn rabbits detecting and responding to the mammary pheromone (MP) emitted by the mother is crucial for survival, since the MP is a strong releaser of typical head-searching/oral grasping behaviour allowing to locate and orally grasp the maternal nipples during the daily nursing. Strikingly, the MP also functions as a natural reinforcer, i.e. as an unconditioned stimulus able to promote appetitive conditioning to a new odorant (CS) in a single and v…

Olfactory xenobiotic metabolizing enzymes have an impact on the stimulating properties of some odorants

International audience

Vitamin A modulates the effects of thyroid hormone on UDP-glucuronosyltransferase expression and activity in rat liver.

We studied the influence of thyroid hormones and vitamin A status on the regulation of UDP-glucuronosyltransferase (UGT) expression and the glucuronidation of thyroid hormones by UGTs. For this, we used an original model of rats fed with different vitamin A diets and implanted subcutaneously by osmotic minipumps delivering vehicle or thyroid hormones, which permitted the control of plasma thyroid hormone concentrations. The activity and expression of family 1 UGTs are correlated and were significantly modified by both thyroid status and amounts of retinol in the diet. Dietary vitamin A did not perturbe the UGT1A expression in thyroidectomized animals. Thyroid hormones and dietary vitamin A …

Activity and expression of drug metabolizing enzymes in olfactory mucosa of rats treated by hepatic inducers

International audience; Several drug-metabolizing enzymes (DME), such as cytochrome P450- dependent monooxygenases (CYP) and transferases have been characterized in the olfactory epithelium. Some of them are preferentially expressed in this tissue, while others are similar to those present in the liver. The role of these enzymes remains unclear. Since the olfactory mucosa is in direct contact with the external environment, these enzymes can contribute to the detoxification of chemical compounds. In addition, these enzymes could be involved in the olfaction process, especially in the biotransformation of odorants. Indeed, the rapid inactivation and clearance of odorants is a prerequisite for…

Real time monitoring of the metabolic capacity of ex-vivo rat olfactory mucosa by on-line PTR-MS

Real time monitoring of the metabolic capacity of ex-vivo rat olfactory mucosa by on-line PTR-MS. Congrès français de Spectrométrie de Masse et d'Analyse Protéomique (SMAP 2015)

Function of nasal odorant metabolism in mammalian olfaction

Induction of cytochrome P450 and/or detoxication enzymes by various extracts or rosemary: description of specific patterns

The ability of rosemary to modulate cytochrome P450 (CYP) and detoxication enzymes in rat liver was evaluated by comparing the effects of dried leaves and leaf extracts with different chemical compositions: essential oil (EO) containing monoterpenes, a dichloromethane extract (DCME) containing phenolic diterpenes and a water-soluble extract (WSE) containing phenolic compounds such as rosmarinic acid and flavonoids. Chemical analyses were done in order to characterize the composition of extracts. Male Wistar rats received the leaves or extracts of rosemary in their diet at 0.5% (w/w) for 2 weeks. The effects of such treatments were evaluated for CYP (1A, 2B, 2E1), glutathione S-transferase (…

Effects of endocrine disruptors on genes associated with 17 beta-estradiol metabolism and excretion

International audience; In order to provide a global analysis of the effects of endocrine disruptors on the hormone cellular bioavailability, we combined 17 beta-estradiol (E2) cellular flow studies with real-time PCR and Western blot expression measurements of genes involved in the hormone metabolism and excretion. Three endocrine disruptors commonly found in food were chosen for this study, which was conducted in the estrogen receptor (ER) negative hepatoblastoma HepG2 cell line: bisphenol A (BPA), genistein (GEN) and resveratrol (RES). We showed that 24h after a single dose treatment with genistein, resveratrol or bisphenol A, the expression of ATP-binding cassette transporters (the mult…

Glutathione conjugation of the rabbit mammary pheromone 2-methylbut-2-enal

International audience; In the process of smell, the olfactory signal is initiated by the binding of odorous molecules to olfactory receptors. In the receptor environment,associated events are supposed to modulate this signal.Thus, the xenobiotic metabolizing enzymes, potentially involved in the clearance of the odorous molecules, could modulate the availability of these molecules for the olfactory receptors, and consequently could participate indirectly in the olfactory signal termination. A mammary pheromone, which is an odorous aldehyde (2-methylbut-2-enal or 2MB2) has been recently characterized in the rabbit by our group. The aim of this work was to elucidate the metabolism of 2MB2 in …

Odorant metabolizing enzymes in the peripheral olfactory process

Odorant metabolizing enzymes in the peripheral olfactory process

Effect of retinoids on UDP-glucuronosyltransferase 2B7 mRNA expression in Caco-2 cells.

Human UDP-glucuronosyltransferase 2B7 (UGT2B7) is one of the major isoforms involved in the glucuronidation of endogenous compounds and xenobiotics. This isoform is the only human UGT shown to glucuronidate retinoids and their oxidized derivatives. In this study, the effects of all-trans retinoic acid (atRA), 9-cis RA, and the RAR agonist TTNPB, on UGT2B7 and UGT2B15 mRNA expression in Caco-2 cells have been examined. Each of these retinoids significantly suppressed UGT2B7 mRNA expression in a concentration-dependent manner with IC50 values of 3.5, 0.3, and 0.2 microM, respectively. However, no inhibition was observed when two other UGTs, UGT2B15 or -1A6, were exposed to atRA, 9-cis RA, or …

Les systèmes de détoxification dans le signal olfactif

Impact of aldehyde dehydrogenases and aldo-keto reductases on human olfactory peri-receptor events

International audience

Rat olfactory bulb and epithelium UDP-glucuronosyltransferase 2A1 (UGT2A1) expression: in situ mRNA localization and quantitative analysis.

UDP-glucuronosyltransferases (UGTs) form a multigenic family of enzymes involved in the biotransformation and elimination of numerous endo- and xenobiotic compounds. Beside the diverse UGT isoforms present in the liver as well as in other tissues, the UGT2A1 isoform, also called olfactory UGT, was initially thought to be expressed in the nasal epithelium only. In this work, we demonstrate the UGT2A1 mRNA expression in the olfactory bulb, using in situ hybridization and quantitative reverse transcription-polymerase chain reaction (RT-PCR) techniques. Within the epithelium, UGT2A1 mRNA is mainly found in the sustentacular cells and to a lesser extent in Bowman's gland cells. Moreover, in situ…

Characterization of rat glutathione transferases in olfactory mucus and epithelium

International audience

Effects of typical inducers on olfactory xenobiotic-metabolizing enzyme, transporter, and transcription factor expression in rats.

International audience; Several xenobiotic-metabolizing enzymes (XMEs) have been identified in the olfactory mucosa (OM) of mammals. However, the molecular mechanisms underlying the regulation of these enzymes have been little explored. In particular, information on the expression of the transcriptional factors in this tissue is quite limited. The aim of the present study was to examine the impact of five typical inducers, Aroclor 1254, 3-methylcholanthrene, dexamethasone, phenobarbital, and ethoxyquin, on the activities and mRNA expression of several XMEs in the OM and in the liver of rats. We also evaluated the effects of these treatments on the mRNA expression of transcription factors an…

A self-inducible heterologous protein expression system in Escherichia coli

AbstractEscherichia coli is an important experimental, medical and industrial cell factory for recombinant protein production. The inducible lac promoter is one of the most commonly used promoters for heterologous protein expression in E. coli. Isopropyl-β-D-thiogalactoside (IPTG) is currently the most efficient molecular inducer for regulating this promoter’s transcriptional activity. However, limitations have been observed in large-scale and microplate production, including toxicity, cost and culture monitoring. Here, we report the novel SILEX (Self-InducibLe Expression) system, which is a convenient, cost-effective alternative that does not require cell density monitoring or IPTG inducti…

Odorant metabolism in olfactory mucus: characterization and impact on olfactory perception

Biotransformation of odorants modifies the olfactory signal

International audience

New insights in vertebrate >

International audience

Inhibition of the mammary pheromone catabolism by a second odorant present in the same mixture

National audience

Implication des enzymes dans la perception chimio-sensorielle : une piste dans les troubles olfacto-gustatifs induits par des xénobiotiques ?

International audience

Périréception et protéines porteuses

ISBN-10 : 978-2-7592-1770-0 ; issn : 1777-4624

Aroma volatile metabolites at olfactory mucosa level evidenced by in vitro PTR-Tof-MS studies

International audience; Olfactory mucosa can metabolize odorants through various enzymatic mechanisms participating in their clearance and therefore in the termination of the olfactory signal. Preliminary ex-vivo studies using headspace-GC revealed the formation of volatile metabolites when odorant molecules were injected above a fresh explant of rat olfactory mucosa. However, this method did not allow accessing the data during the first five minutes of contact between the odorant and the mucosa, thus limiting the olfactory biological significance. Using a direct-injection mass spectrometry technique (PTR-MS) we have been able for the first time to investigate the first moments of the enzym…

The NAOMI (Nasal Odorant Metabolites) consortium: an international, multidisciplinary consortium investigating how metabolites resulting from nasal enzymatic odorant metabolism can shape odor perception

International audience; A functional sense of smell is essential for evaluating and enjoying food, receiving information about conspecifics, and detecting threats via airborne chemical cues. Olfactory dysfunction has been associated with an impaired quality of life, reflected by a higher incidence of undesirable mental states such as depression and social insecurity. Addressing and overcoming related olfactory dysfunction issues require a detailed understanding of peripheral and central processes involved in olfaction. Consequently, there is a growing body of research unravelling molecular and cellular interactions that shape olfactory sensation. Recent studies have demonstrated that in the…

Odorant Metabolism Analysis by an Automated Ex Vivo Headspace Gas-Chromatography Method

International audience; In the olfactory epithelium (OE), odorant metabolizing enzymes have the dual function of volatile component detoxification and active clearance of odorants from the perireceptor environment to respectively maintain the integrity of the tissues and the sensitivity of the detection. Although emphasized by recent studies, this enzymatic mechanism is poorly documented in mammals. Thus, olfactory metabolism has been characterized mainly in vitro and for a limited number of odorants. The automated ex vivo headspace gas-chromatography method that was developed here was validated to account for odorant olfactory metabolism. This method easily permits the measurement of the f…

Molecules of the flavor

UDP-glucuronosyltransferases (UGTs) in neuro-olfactory tissues: expression, regulation, and function.

International audience; This work aims to review uridine diphosphate (UDP)-glucuronosyltransferase (UGT) expression and activities along different neuronal structures involved in the common physiological process of olfaction: olfactory epithelium, olfactory bulb, and olfactory cortex. For the first time, using high-throughput in situ hybridization data generated by the Allen Brain Atlas (ABA), we present quantitative analysis of spatial distribution of UGT genes in the mouse brain. The olfactory area is a central nervous system site with the highest expression of UGTs, including UGT isoforms not previously identified in the brain. Since there is evidence of the transfer of xenobiotics to th…

CHARACTERIZATION OF MICROSOMAL CYTOCHROME P450-DEPENDENT MONOOXYGENASES IN THE RAT OLFACTORY MUCOSA

Nasal administration of a drug ensures therapeutic action by rapid systemic absorption and/or the entry of some molecules into the brain through different routes. Many recent studies have pointed out the presence of xenobiotic-metabolizing enzymes in rat olfactory mucosa (OM). Nevertheless, very little is known about the precise identity of isoforms of cytochrome P450 (P450)-dependent monooxygenases (P450) and their metabolic function in this tissue. Therefore, we evaluated mRNA expression of 19 P450 isoforms by semiquantitative reverse transcriptase-polymerase chain reaction and measured their microsomal activity toward six model substrates. For purposes of comparison, studies were conduct…

Cytochrome P450-dependent metabolism of caffeine in [i]Drosophila melanogaster[/i]

Caffeine (1, 3, 7-trimethylxanthine), an alkaloid produced by plants, has antioxidant and insecticide properties that can affect metabolism and cognition. In vertebrates, the metabolites derived from caffeine have been identified, and their functions have been characterized. However, the metabolites of caffeine in insects remain unknown. Thus, using radiolabelled caffeine, we have identified some of the primary caffeine metabolites produced in the body of Drosophila melanogaster males, including theobromine, paraxanthine and theophylline. In contrast to mammals, theobromine was the predominant metabolite (paraxanthine in humans; theophylline in monkeys; 1, 3, 7-trimethyluric acid in rodents…

Major sulfation pathway for resveratrol metabolism in human hepatic derived cells and resveratrol –dependent inducibility of conjugating enzymes. Perspective in nutritional prevention

Odorant-odorant metabolic competitions: ex vivo inhibition of the mammary pheromone catabolism

International audience

Carboxyl nonsteroidal anti-inflammatory drugs are efficiently glucuronidated by microsomes of the human gastrointestinal tract.

Limited studies have been carried out on the biotransformation of carboxyl nonsteroidal anti-inflammatory drugs (NSAIDs) in the liver. However, the role of the intestine in NSAID metabolism has not been investigated. In this report, the contribution of UDP-glucuronosyltransferases (UGTs) in the human gastrointestinal (GI) tract from five donors to the glucuronidation of the NSAIDs, RS-ketoprofen, S-naproxen, RS- and S-etodolac, was investigated. UGT activity and, for some donors, mRNA levels were evaluated. All NSAIDs were glucuronidated throughout the GI tract; however, glucuronidation was low in stomach and duodenum as compared to the remainder of the intestine. RT-PCR analysis demonstrat…

New insights in vertebrate 'biotransfolfaction'

International audience

Odorant metabolism catalyzed by olfactory mucosal enzymes influences peripheral olfactory responses in rats.

International audience; A large set of xenobiotic-metabolizing enzymes (XMEs), such as the cytochrome P450 monooxygenases (CYPs), esterases and transferases, are highly expressed in mammalian olfactory mucosa (OM). These enzymes are known to catalyze the biotransformation of exogenous compounds to facilitate elimination. However, the functions of these enzymes in the olfactory epithelium are not clearly understood. In addition to protecting against inhaled toxic compounds, these enzymes could also metabolize odorant molecules, and thus modify their stimulating properties or inactivate them. In the present study, we investigated the in vitro biotransformation of odorant molecules in the rat …

How odorant metabolizing enzymes shape odor perception?

International audience; Lingering in the background of every odorant-receptor interaction is an odor metabolizing enzyme. Over the last several years, a fascinating set of studies in rodents, rabbits, insects and humans has demonstrated that these enzymes participate in the active biotransformation of odorous molecules within the perireceptor environment. These mechanisms may exert two unique effects: first, to maintain odor sensitivity by reducing receptor saturation, and second, to transform the local milieu of the odor components giving rise to unique odor percepts. This symposium will focus on the impact of odorant metabolizing enzymes (OMEs) on functional and behavioral aspects of olfa…

Odorants' metabolism in Human: a critical role in odor perception revealed

When the nose must remain responsive: glutathione conjugation of the mammary pheromone in the newborn rabbit

In insects, xenobiotic-metabolizing enzymes were demonstrated to regulate pheromones inactivation, clearing them from the olfactory periphery and keeping receptors ready for stimulation renewal. Here, we investigate whether similar processes could occur in mammals, focusing on the pheromonal communication between female rabbits and their newborns. Lactating rabbits emit in their milk a volatile aldehyde, 2-methylbut-2-enal, that elicits searching-grasping in neonates; called the mammary pheromone (MP), it is critical for pups which are constrained to find nipples within the 5 min of daily nursing. For newborns, it is thus essential to remain sensitive to this odorant during the whole nursin…

Expression and differential localization of xenobiotic transporters in the rat olfactory neuro-epithelium.

International audience; Transporters, such as multidrug resistance P-glycoproteins (MDR), multidrug resistance-related proteins (MRP) and organic anion transporters (OATs), are involved in xenobiotic metabolism, particularly the cellular uptake or efflux of xenobiotics (and endobiotics) or their metabolites. The olfactory epithelium is exposed to both inhaled xenobiotics and those coming from systemic circulation. This tissue has been described as a pathway for xenobiotics to the brain via olfactory perineural space. Thereby, olfactory transporters and xenobiotic metabolizing enzymes, dedicated to the inactivation and the elimination of xenobiotics, have been involved in the toxicological p…

Role of CYTOCHROME p450 in the perception of caffeine in Drosophila melanogaster

Poster

Rôle des enzymes du métabolisme des xénobiotiques dans la perception de la caféine chez la drosophile

National audience; Les animaux ont développé des systèmes de communication avec leur environnement indispensables à leur survie et leur reproduction. Les systèmes chimiosensoriels gustatifs et olfactifs détectent et transmettent au niveau central les informations chimiques de l’environnement (xénobiotique) afin que l’individu distingue les sources alimentaires des substances toxiques et reconnaisse ses proies de ses congénères. Ces systèmes, pour être efficaces, doivent être très sensibles et discriminants, et éviter la saturation des récepteurs. Dans l’espace péri-récepteur, les enzymes du métabolisme des xénobiotiques (EMX), impliquées normalement dans la détoxication, pourraient égalemen…

Métabolisme périphérique des odorants : arrêt ou modulation du signal olfactif ?

Métabolisme périphérique des odorants : arrêt ou modulation du signal olfactif ?. 1. réunion du GDR (Groupe De Réflexion) Odorant Odeur Olfaction

Endocrine disruptors found in food modulate estradiol cellular metabolism

Major sulfation pathway for resveratrol metabolism in human hepatic derived cells and resveratrol –dependent inducibility of conjugating enzymes. Perspective in nutritional prevention

ARET : colloque 2018 au CSGA Dijon. 28 et 29 mai 2018 - CSGA, 9E boulevard Jeanne d'Arc, Dijon

L'Association pour la REcherche en Toxicologie (ARET) organise un colloque sur le thème "Substances odorantes et sapides : du plaisir des sens aux cibles biologiques", lundi 28 et mardi 29 mai 2018 au CSGA. Ce colloque bénéficie du soutien du département AlimH. Invité d'honneur de la table ronde: Roland Salesse.

Drosophila melanogaster glutathione transferase overexpressed in the sensory organs after exposure to bitter molecules in food

International audience

Caffeine metabolism in Drosophila melanogaster

International audience

Oxidative stress and xenobiotic detoxification enzymes in rat astrocytes: Correlations with brain aging

Meeting Abstract; International audience

P45 - Drosophila melanogaster glutathione transferase overexpressed in the sensory organs after exposure to bitter molecules in food

International audience

Influence of vitamin A status on the regulation of uridine (5′-)diphosphate-glucuronosyltransferase (UGT) 1A1 and UGT1A6 expression by L-triiodothyronine

The uridine (5′-)diphosphate-glucuronosyltransferases (UGT) are involved in the phase II of various xenobiotics and endogenous compounds. They are responsible for glucuronidation of many substrates, especially including bilirubin (UGT1A1) and phenolic compounds (UGT1A6). We previously showed that the expression of both isoforms is regulated at the transcriptional level by thyroid hormone in rat liver. In this present study, effects of vitamin A dietary intake (0, 1.72, 69 ug retinol acetate/g food) on the regulation of UGT1A1 and UGT1A6 activity and expression by 3,5,3′ triiodo-L-THYRONINE (l-T3) were examined in the same organ. Activities were determined toward bilirubin and 4-nitrophenol.…

Structural and enzymatic characterization of a Drosophila melanogaster glutathione transferase overexpressed in the sensory organs after exposure to bitter molecules in food

Glutathione transferases (GSTs) are ubiquitous key detoxification enzymes that catalyze the conjugation of glutathione to a large variety of xenobiotic chemical including odorants and sapid molecules. A previous study in Drosophila melanogaster demonstrated the existence of a GST specifically expressed in the antenna and involved in signal termination by catalyzing biotransformation of odorant molecules. Genomic analysis allows identifying forty GSTs in Drosophila melanogaster but their biochemistry is poorly documented. Here we demonstrated that among the GSTs, dmelGSTD2-2 was strongly and preferentially overexpressed in the sensory organs (antennae, proboscis, legs, wings) after an exposu…

Role of xenobiotic metabolizing enzymes in the perception of caffeine in Drosophila melanogaster

Poster

Role of human metabolizing enzymes in food perception

Flavor is the main factor determining food acceptability. Flavor corresponds to the combination of the signals from the gustatory system, the olfactory system and the trigeminal system. Interactions between proteins and flavor molecules in oral/nasal cavities are proposed to modulate flavor perception through a series of events knowns as perireceptor events. Among these proteins involved in this modulation are found enzymes mainly known for their role in the detoxification process. These enzymes are able to recognize a large panel of molecules. We proposed to explore the role of these enzymes in food perception. We demonstrated that enzymatic activities present in the oral epithelium and sa…

Effect of oxidative stress on UDP-glucuronosyltransferases in rat astrocytes.

WOS:000309170300003; International audience; The present work reports data regarding effects of an induced oxidative stress on the mainly expressed isoforms of UDP-glucuronosyltransferases (UGTs) in the brain. UGT1A6 and UGT1A7 expression and enzymatic activities toward the 1-naphthol were analyzed in rat cultured astrocytes following the exposure for 48 h to redox-cycling xenobiotic compounds such as quinones and bipyridinium ions. The expression of NADPH:cytochrome P450 reductase and NAD(P)H:quinone oxidoreductase 1 (NQO1) was also investigated. Oxidative stress induced significant deleterious changes in astrocyte morphology, decreased cell viability and inhibited catalytic function of UG…

Function of nasal odorant metabolism in vertebrate olfaction

National audience

Role of CYP6d5 in the metabolism and the sensory perception of caffeine in Drosophila melanogaster

National audience

Interactions between odorants and glutathione transferases in the human olfactory cleft

AbstractXenobiotic metabolizing enzymes and other proteins, including odorant-binding proteins located in the nasal epithelium and mucus, participate in a series of processes modulating the concentration of odorants in the environment of olfactory receptors (ORs) and finely impact odor perception. These enzymes and transporters are thought to participate in odorant degradation or transport. Odorant biotransformation results in 1) changes in the odorant quantity up to their clearance and the termination of signaling and 2) the formation of new odorant stimuli (metabolites). Enzymes, such as cytochrome P450 and glutathione transferases (GSTs), have been proposed to participate in odorant clea…