Identification of a Variable Number of Tandem Repeats Polymorphism and Characterization of LEF-1 Response Elements in the Promoter of the IDO1 Gene

Indoleamine 2,3-dioxygenase (IDO) catalyzes the first and rate-limiting step of the kynurenine pathway that is an important component of immunomodulatory and neuromodulatory processes. The IDO1 gene is highly inducible by IFN-γ and TNF-α through interaction with cis-acting regulatory elements of the promoter region. Accordingly, functional polymorphisms in the IDO1 promoter could partly explain the interindividual variability in IDO expression that has been previously documented.A PCR-sequencing strategy, applied to DNA samples from healthy Caucasians, allowed us to identify a VNTR polymorphism in the IDO1 promoter, which correlates significantly with serum tryptophan concentration, controlled partially by IDO activity, in female subjects, but not in males. Although this VNTR does not appear to affect basal or cytokine-induced promoter activity in gene reporter assays, it contains novel cis-acting elements. Three putative LEF-1 binding sites, one being located within the VNTR repeat motif, were predicted in silico and confirmed by chromatin immunoprecipitation. Overexpression of LEF-1 in luciferase assays confirmed an interaction between LEF-1 and the predicted transcription factor binding sites, and modification of the LEF-1 core sequence within the VNTR repeat motif, by site-directed mutagenesis, resulted in an increase in promoter activity.The identification of a VNTR in the IDO1 promoter revealed a cis-acting element interacting with the most downstream factor of the Wnt signaling pathway, suggesting novel mechanisms of regulation of IDO1 expression. These data offer new insights, and suggest further studies, into the role of IDO in various pathological conditions, particularly in cancer where IDO and the Wnt pathway are strongly dysregulated

Variabilité génétique du métabolisme du tryptophane et troubles du comportement sous alcool

Interindividual variability in ethanol-induced behavioural disorders, such as blackouts and violent impulsive behaviours (BOVIB) following binge drinking could partly be explained by polymorphisms in genes encoding enzymes and transporters of the tryptophan (Trp) catabolic pathway. Indeed, Trp is the precursor of serotonin, a neurotransmitter that modulates mood, cognition and impulsivity, and is also transformed into various kynurenine metabolites, most of them displaying neuroactive properties and being involved in cognitive and memory dysfunctions. The aims of our work were (1) to test the BOVIB hypothesis in relation with a dysregulation of Trp metabolism in alcohol-dependent (AD) patients with or without BOVIB history, (2) to analyse the genetic variability of the kynurenine pathway (KP) leading to variations in expression and/or activity of key enzymes, (3) to explore interactions between the KP and the main biological neuroendocrine systems involved in ethanol addiction, using a rodent model of prenatal stress. The main results of our study showed (1) a significant difference in the rate-limiting enzyme tryptophan 2,3-dioxygenase (TDO) activity between AD patients with or without BOVIB history, (2) the existence of polymorphisms in the promoter region of two key genes of the KP, TDO2 and IDO1, (3) the presence of functional Glucocorticoid Responsive Elements (GRE) in the promoter of TDO2 which might be affected by some of the polymorphisms identified, and (4) the identification of LEF-1 response elements in the IDO1 promoter. These findings support the genetic variability of the Trp metabolism, as well as its potential role in individual vulnerability to ethanol-induced behavioural disorders.Les différences interindividuelles observées dans les troubles du comportement sous alcool, notamment au cours d’épisodes impulsifs associés à des troubles de la mémorisation (ivresses pathologiques ou IP), pourraient s’expliquer en partie par l’existence de polymorphismes affectant des gènes-clé du métabolisme du tryptophane (Trp), celui-ci étant en effet le précurseur, non seulement de la sérotonine, neurotransmetteur impliqué dans le contrôle de l’impulsivité, mais aussi de la kynurénine et de ses dérivés, dont certains présentent des propriétés neuromodulatrices impliquées dans les phénomènes de mémorisation. Nos travaux ont ainsi consisté à (1) étudier l’hypothèse mécanistique des IP en relation avec un dysfonctionnement du métabolisme du Trp chez des patients alcoolodépendants (AD) présentant ou non une susceptibilité aux IP, (2) étudier les variabilités d’origine génétique de la voie des kynurénines, à l’origine de variations d’expression et/ou d’activité, (3) explorer les interactions entre la voie des kynurénines et les axes biologiques impliqués dans l’addiction à l’alcool dans un modèle animal de stress prénatal. Les principaux résultats de ces travaux ont permis de montrer (1) une différence significative de l’activité de la tryptophane-2,3-dioxygénase (TDO), enzyme catalysant la 1ère étape de la voie des kynurénines, entre les patients AD IP+ et IP-, (2) l’existence d’un polymorphisme génétique affectant les régions promotrices de TDO2 et d’IDO1, gènes-clé de la voie des kynurénines, (3) le rôle des éléments de réponse aux glucocorticoides (GRE) dans l’expression de TDO2, ainsi que l’impact de certains polymorphismes affectant ces GRE, et enfin (4) l’existence d’un élément de réponse de type LEF1, non précédemment décrit, dans le promoteur d’IDO1. L’ensemble de ces résultats confirme l’existence d’une variabilité d’origine génétique du métabolisme du Trp, potentiellement à l’origine d’une vulnérabilité individuelle aux troubles du comportement sous alcool

Genetic variability of tryptophan metabolism and its implication in ethanol-induced behavioural disorders

Les différences interindividuelles observées dans les troubles du comportement sous alcool, notamment au cours d’épisodes impulsifs associés à des troubles de la mémorisation (ivresses pathologiques ou IP), pourraient s’expliquer en partie par l’existence de polymorphismes affectant des gènes-clé du métabolisme du tryptophane (Trp), celui-ci étant en effet le précurseur, non seulement de la sérotonine, neurotransmetteur impliqué dans le contrôle de l’impulsivité, mais aussi de la kynurénine et de ses dérivés, dont certains présentent des propriétés neuromodulatrices impliquées dans les phénomènes de mémorisation. Nos travaux ont ainsi consisté à (1) étudier l’hypothèse mécanistique des IP en relation avec un dysfonctionnement du métabolisme du Trp chez des patients alcoolodépendants (AD) présentant ou non une susceptibilité aux IP, (2) étudier les variabilités d’origine génétique de la voie des kynurénines, à l’origine de variations d’expression et/ou d’activité, (3) explorer les interactions entre la voie des kynurénines et les axes biologiques impliqués dans l’addiction à l’alcool dans un modèle animal de stress prénatal. Les principaux résultats de ces travaux ont permis de montrer (1) une différence significative de l’activité de la tryptophane-2,3-dioxygénase (TDO), enzyme catalysant la 1ère étape de la voie des kynurénines, entre les patients AD IP+ et IP-, (2) l’existence d’un polymorphisme génétique affectant les régions promotrices de TDO2 et d’IDO1, gènes-clé de la voie des kynurénines, (3) le rôle des éléments de réponse aux glucocorticoides (GRE) dans l’expression de TDO2, ainsi que l’impact de certains polymorphismes affectant ces GRE, et enfin (4) l’existence d’un élément de réponse de type LEF1, non précédemment décrit, dans le promoteur d’IDO1. L’ensemble de ces résultats confirme l’existence d’une variabilité d’origine génétique du métabolisme du Trp, potentiellement à l’origine d’une vulnérabilité individuelle aux troubles du comportement sous alcool.Interindividual variability in ethanol-induced behavioural disorders, such as blackouts and violent impulsive behaviours (BOVIB) following binge drinking could partly be explained by polymorphisms in genes encoding enzymes and transporters of the tryptophan (Trp) catabolic pathway. Indeed, Trp is the precursor of serotonin, a neurotransmitter that modulates mood, cognition and impulsivity, and is also transformed into various kynurenine metabolites, most of them displaying neuroactive properties and being involved in cognitive and memory dysfunctions. The aims of our work were (1) to test the BOVIB hypothesis in relation with a dysregulation of Trp metabolism in alcohol-dependent (AD) patients with or without BOVIB history, (2) to analyse the genetic variability of the kynurenine pathway (KP) leading to variations in expression and/or activity of key enzymes, (3) to explore interactions between the KP and the main biological neuroendocrine systems involved in ethanol addiction, using a rodent model of prenatal stress. The main results of our study showed (1) a significant difference in the rate-limiting enzyme tryptophan 2,3-dioxygenase (TDO) activity between AD patients with or without BOVIB history, (2) the existence of polymorphisms in the promoter region of two key genes of the KP, TDO2 and IDO1, (3) the presence of functional Glucocorticoid Responsive Elements (GRE) in the promoter of TDO2 which might be affected by some of the polymorphisms identified, and (4) the identification of LEF-1 response elements in the IDO1 promoter. These findings support the genetic variability of the Trp metabolism, as well as its potential role in individual vulnerability to ethanol-induced behavioural disorders

Identification par LC-MS-MS de métabolites de médicaments produits in vitro (application au diagnostic d intoxications médicamenteuses aiguës)

LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

False positive amphetamines and 3,4-methylenedioxymethamphetamine immunoassays in the presence of metoprolol—two cases reported in clinical toxicology

International audienceAbstract Amphetamines, frequently used recreational drugs with high risk of toxicity, are commonly included in urine drug screens. This screening is based on enzyme immunoassay, which is a quick and easy-to-perform technique, but may lack specificity resulting from cross-reactivity with other compounds, causing false positive results. We present two cases of presumed false positive MULTIGENT® amphetamine/methamphetamine and MULTIGENT® ecstasy (Abbott®) immunoassays with the beta-blocker metoprolol. Both metoprolol-poisoned patients presented positive urine screening despite no history of drug abuse. No confirmation for amphetamine molecular structures was found with gas chromatography–mass spectrometry. The cross-reactivity was further investigated by doping urine samples with metoprolol and its two major phase-I metabolites. Metoprolol showed positive results for both amphetamine and MDMA tests at low concentrations (200 and 150 μg/mL, respectively). Metoprolol metabolites cross-reacted with the amphetamines immunoassay only, but at higher concentrations (i.e., 2000 μg/mL for α-hydroxymetoprolol and 750 μg/mL for O-demethylmetoprolol). In conclusion, false positive results in amphetamines and MDMA immunoassays are possible in the presence of metoprolol. Toxicologists should be aware of frequent analytical interferences with immunoassays and a detailed medication history should be taken into consideration for interpretation. In vitro investigation of suspected cross-reactivity should include not only the parent drug but also its related metabolites

Characterization of functional polymorphisms and glucocorticoid-responsive elements in the promoter of TDO2, a candidate gene for ethanol-induced behavioural disorders

Aims: In response to acute ethanol consumption, tryptophan 2,3-dioxygenase (TDO) induces the kynurenine pathway (KP) through a glucocorticoid-mediated mechanism, which could lead to a dramatic accumulation of neurotoxic metabolites in association with serotonin depletion. As a result, interindividual variability in ethanol-induced behavioural disorders, such as black-outs and violent impulsive behaviours (BOVIBs) following binge drinking, could be partly explained by genetic polymorphisms affecting the KP. The aim of this study was to identify polymorphisms on the promoter of the TDO2 gene that could affect expression and/or activity of TDO through glucocorticoid inductio.Methods: Polymorphisms were screened using a PCR-sequencing strategy applied to 31 alcohol-dependent patients and 49 unrelated healthy volunteers, and functionally analysed with bioinformatic prediction tools and gene reporter assays in HepG2 and A549 cell lines. Results: We identified 12 polymorphisms in the human TDO2 promoter region, 2 of them corresponding to previously unknown single-nucleotide polymorphisms (SNPs) and 3 of them located in putative glucocorticoid-responsive elements (GREs). Gene reporter assays using HepG2 and A549 cell lines confirmed the presence of several functional GREs in the promoter region of TDO2 and revealed that some of the identified polymorphisms affect the promoter activity under glucocorticoid receptor over-expression and dexamethasone exposure conditions. Conclusions: Correlational studies in larger samples could help to determine whether these polymorphisms are responsible for variations of expression and/or activity of TDO, inparticular under conditions where release of glucocorticoids is increased, such as acute ethanol intake. If confirmed, such results would be of major interest in explaining part of the interindividual variability observed in behavioural responses to acute ethanol consumption.11 page(s

Is R(+)-Baclofen the best option for the future of Baclofen in alcohol dependence pharmacotherapy? Insights from the preclinical side

International audienceFor several decades, studies conducted to evaluate the efficacy of RS(+/-)-Baclofen in the treatment of alcohol dependence yielded contrasting results. Human and animal studies recently questioned the use of the racemic drug in patients since a potential important role of the different enantiomers has been revealed with an efficacy thought to reside with the active R(+)-enantiomer. Here we conducted experiments in the postdependent rat model of alcohol dependence to compare the efficacy of R(+)-Baclofen or S(-)-Baclofen to that of RS(+/-)-Baclofen on ethanol intake, seeking, and relapse. R(+)-Baclofen was more effective than RS(+/-)-Baclofen in reducing ethanol intake and seeking during acute withdrawal and during relapse after abstinence. We also used an original population approach in order to identify drug responders. We found a significant proportion of responders to S(-)-Baclofen and RS(+/-)-Baclofen, displaying an increase in ethanol intake, and this increasing effect on alcohol intake was not seen in the R(+)-Baclofen group. At an intermediate dose of R(+)-Baclofen, devoid of any motor side effects, we identified a very large proportion of responders (75%) with a large decrease in ethanol intake (90% decrease). Finally, the response to RS(+/-)-Baclofen on ethanol intake was correlated to plasma level of Baclofen. R(+)-Baclofen and RS(+/-)-Baclofen were effective in reducing sucrose intake. Our study has important clinical implication since it suggests that the wide variability in the therapeutic responses of patients to RS(+/-)-Baclofen may come from the sensitivity to the R(+)-Baclofen but also to the one of the S(-)-Baclofen that can promote an increase in ethanol intake