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Prateek Tripathi - One of the best experts on this subject based on the ideXlab platform.

  • a toolbox of genes proteins Metabolites and promoters for improving drought tolerance in soybean includes the metabolite coumestrol and stomatal development genes
    BMC Genomics, 2016
    Co-Authors: Prateek Tripathi, Roel C Rabara, Neil R Reese, Marissa A Miller, Jai S Rohila, Senthil Subramanian, Qingxi J Shen, Dominique Morandi
    Abstract:

    The purpose of this project was to identify Metabolites, proteins, genes, and promoters associated with water stress responses in soybean. A number of these may serve as new targets for the biotechnological improvement of drought responses in soybean (Glycine max). We identified Metabolites, proteins, and genes that are strongly up or down regulated during rapid water stress following removal from a hydroponics system. 163 Metabolites showed significant changes during water stress in roots and 93 in leaves. The largest change was a root-specific 160-fold increase in the coumestan coumestrol making it a potential biomarker for drought and a promising target for improving drought responses. Previous reports suggest that coumestrol stimulates mycorrhizal colonization and under certain conditions mycorrhizal plants have improved drought tolerance. This suggests that coumestrol may be part of a call for help to the rhizobiome during stress. About 3,000 genes were strongly up-regulated by drought and we identified regulators such as ERF, MYB, NAC, bHLH, and WRKY transcription factors, receptor-like kinases, and calcium signaling components as potential targets for soybean improvement as well as the jasmonate and abscisic acid biosynthetic genes JMT, LOX1, and ABA1. Drought stressed soybean leaves show reduced mRNA levels of stomatal development genes including FAMA-like, MUTE-like and SPEECHLESS-like bHLH transcription factors and leaves formed after drought stress had a reduction in stomatal density of 22.34 % and stomatal index of 17.56 %. This suggests that reducing stomatal density may improve drought tolerance. MEME analyses suggest that ABRE (CACGT/CG), CRT/DRE (CCGAC) and a novel GTGCnTGC/G element play roles in transcriptional activation and these could form components of synthetic promoters to drive expression of transgenes. Using transformed hairy roots, we validated the increase in promoter activity of GmWRKY17 and GmWRKY67 during dehydration and after 20 μM ABA treatment. Our toolbox provides new targets and strategies for improving soybean drought tolerance and includes the coumestan coumestrol, transcription factors that regulate stomatal density, water stress-responsive WRKY gene promoters and a novel DNA element that appears to be enriched in water stress responsive promoters.

  • A toolbox of genes, proteins, Metabolites and promoters for improving drought tolerance in soybean includes the metabolite coumestrol and stomatal development genes
    BMC Genomics, 2016
    Co-Authors: Prateek Tripathi, Roel C Rabara, Marissa A Miller, Jai S Rohila, Senthil Subramanian, Qingxi J Shen, Dominique Morandi, R. Neil Reese, Heike Bucking, Vladimir Shulaev
    Abstract:

    Background: The purpose of this project was to identify Metabolites, proteins, genes, and promoters associated with water stress responses in soybean. A number of these may serve as new targets for the biotechnological improvement of drought responses in soybean (Glycine max). Results: We identified Metabolites, proteins, and genes that are strongly up or down regulated during rapid water stress following removal from a hydroponics system. 163 Metabolites showed significant changes during water stress in roots and 93 in leaves. The largest change was a root-specific 160-fold increase in the coumestan coumestrol making it a potential biomarker for drought and a promising target for improving drought responses. Previous reports suggest that coumestrol stimulates mycorrhizal colonization and under certain conditions mycorrhizal plants have improved drought tolerance. This suggests that coumestrol may be part of a call for help to the rhizobiome during stress. About 3,000 genes were strongly up-regulated by drought and we identified regulators such as ERF, MYB, NAC, bHLH, and WRKY transcription factors, receptor-like kinases, and calcium signaling components as potential targets for soybean improvement as well as the jasmonate and abscisic acid biosynthetic genes JMT, LOX1, and ABA1. Drought stressed soybean leaves show reduced mRNA levels of stomatal development genes including FAMA-like, MUTE-like and SPEECHLESS-like bHLH transcription factors and leaves formed after drought stress had a reduction in stomatal density of 22.34 % and stomatal index of 17.56 %. This suggests that reducing stomatal density may improve drought tolerance. MEME analyses suggest that ABRE (CACGT/CG), CRT/DRE (CCGAC) and a novel GTGCnTGC/G element play roles in transcriptional activation and these could form components of synthetic promoters to drive expression of transgenes. Using transformed hairy roots, we validated the increase in promoter activity of GmWRKY17 and GmWRKY67 during dehydration and after 20 mu M ABA treatment. Conclusions: Our toolbox provides new targets and strategies for improving soybean drought tolerance and includes the coumestan coumestrol, transcription factors that regulate stomatal density, water stress-responsive WRKY gene promoters and a novel DNA element that appears to be enriched in water stress responsive promoters.

Marilyn A Huestis - One of the best experts on this subject based on the ideXlab platform.

  • pentylindole pentylindazole synthetic cannabinoids and their 5 fluoro analogs produce different primary Metabolites metabolite profiling for ab pinaca and 5f ab pinaca
    Aaps Journal, 2015
    Co-Authors: Ariane Wohlfarth, Mingshe Zhu, Karl B Scheidweiler, Marisol S. Castaneto, Shaokun Pang, Robert Kronstrand, Marilyn A Huestis
    Abstract:

    Whereas non-fluoropentylindole/indazole synthetic cannabinoids appear to be metabolized preferably at the pentyl chain though without clear preference for one specific position, their 5-fluoro analogs’ major Metabolites usually are 5-hydroxypentyl and pentanoic acid Metabolites. We determined metabolic stability and Metabolites of N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA) and 5-fluoro-AB-PINACA (5F-AB-PINACA), two new synthetic cannabinoids, and investigated if results were similar. In silico prediction was performed with MetaSite (Molecular Discovery). For metabolic stability, 1 μmol/L of each compound was incubated with human liver microsomes for up to 1 h, and for metabolite profiling, 10 μmol/L was incubated with pooled human hepatocytes for up to 3 h. Also, authentic urine specimens from AB-PINACA cases were hydrolyzed and extracted. All samples were analyzed by liquid chromatography high-resolution mass spectrometry on a TripleTOF 5600+ (AB SCIEX) with gradient elution (0.1% formic acid in water and acetonitrile). High-resolution full-scan mass spectrometry (MS) and information-dependent acquisition MS/MS data were analyzed with MetabolitePilot (AB SCIEX) using different data processing algorithms. Both drugs had intermediate clearance. We identified 23 AB-PINACA Metabolites, generated by carboxamide hydrolysis, hydroxylation, ketone formation, carboxylation, epoxide formation with subsequent hydrolysis, or reaction combinations. We identified 18 5F-AB-PINACA Metabolites, generated by the same biotransformations and oxidative defluorination producing 5-hydroxypentyl and pentanoic acid Metabolites shared with AB-PINACA. Authentic urine specimens documented presence of these Metabolites. AB-PINACA and 5F-AB-PINACA produced suggested metabolite patterns. AB-PINACA was predominantly hydrolyzed to AB-PINACA carboxylic acid, carbonyl-AB-PINACA, and hydroxypentyl AB-PINACA, likely in 4-position. The most intense 5F-AB-PINACA Metabolites were AB-PINACA pentanoic acid and 5-hydroxypentyl-AB-PINACA.

Johänning Janina - One of the best experts on this subject based on the ideXlab platform.

  • Metabolismus, Pharmakodynamik und klinische Relevanz der östrogenen Tamoxifenmetaboliten bei der endokrinen Therapie des Mammakarzinoms
    Universität Tübingen, 2020
    Co-Authors: Johänning Janina
    Abstract:

    Dissertation ist gesperrt bis 11. Dezember 2020 !Tamoxifen ist die Standardtherapie für Hormonrezeptor-positiven Brustkrebs bei prämenopausalen Frauen und eine Alternative zur Therapie mit Aromataseinhibitoren bei postmenopausalen Frauen. Dabei wird Tamoxifen von Cytochrom P450 (CYP-) Enzymen zu über 60 Metaboliten verstoffwechselt, welche zum Teil anti-östrogene wie auch östrogene Eigenschaften aufweisen. Die anti-östrogenen Metaboliten 4-Hydroxytamoxifen (4-OH-Tamoxifen) und N-Desmethyl-4-hydroxytamoxifen (Endoxifen) haben eine 100-fach stärkere Affinität zum Östrogenrezeptor (ER) und sind hauptsächlich für den antiproliferativen Effekt im Brustgewebe verantwortlich. Zu den östrogenen Metaboliten gehören Tamoxifen Bisphenol (Bisphenol) und beide Isomere von Metabolit E, welche in ER-positiven Brustkrebs-Zellen die Expression von Östrogen-regulierten Genen induzieren und somit die Zellproliferation beeinflussen. Darüber hinaus wurden diese Metaboliten in Xenotransplantaten eines Mausmodells für die Tamoxifenresistenz sowie in Mammakarzinomgewebe von Patientinnen mit klinischer Resistenz identifiziert. Sowohl die Metabolismuswege zu Metabolit E und Bisphenol als auch der Einfluss dieser Metaboliten auf den Therapieverlauf sind bisher weitestgehend unerforscht. Das Ziel dieser Arbeit war es demnach die Enzyme, welche für die Bildung von Bisphenol und Metabolit E verantwortlich sind, zu identifizieren und die pharmakodynamischen Effekte auf den ER und Auswirkungen auf den Fremdstoffmetabolismus in vitro zu untersuchen. Darüber hinaus wurden die Plasmaspiegel der östrogenen Metaboliten in Blutproben von prä- und postmenopausalen Frauen unter Tamoxifentherapie quantifiziert, deren Abhängigkeit von genetischen Varianten der beteiligten CYP-Enzyme analysiert sowie der Einfluss auf den Therapieverlauf untersucht. Mit Hilfe von humanen Lebermikrosomen konnte gezeigt werden, dass Metabolit E wie auch Bisphenol in NADPH-abhängigen mikrosomalen Reaktionen gebildet werden. Weitere Analysen mit Supersomes™ zeigten, dass Metabolit E hauptsächlich durch die Enzyme CYP2C19, 3A, 2D6 und 1A2 aus Tamoxifen, N-Desmethyltamoxifen (DM-Tamoxifen) und N-Didesmethyltamoxifen (DDM-Tamoxifen) metabolisiert wird. Kinetische Untersuchungen ergaben die höchste Clearance für Tamoxifen mit 0,112 µl mg-1 min-1. Für die Bildung von Bisphenol aus den anti-östrogenen Tamoxifenmetaboliten 4-OH-Tamoxifen, Endoxifen und N-Didesmethyl-4-hydroxytamoxifen (Norendoxifen) konnte kein spezifisches CYP-Isoenzym identifiziert werden. Im Gegensatz dazu zeigte die stereospezifische Hydroxylierung von Metabolit E zu Bisphenol durch CYP2C19 ((Z)-Isomer) und CYP2B6 ((E)-Isomer) eine bis zu 45-fach höhere Clearance verglichen mit den anti-östrogenen Tamoxifenmetaboliten. Bezüglich des Phase II-Metabolismus zeigten die UGT-Enzyme 1A8 und 2B7 höchste Aktivitäten für die Glucuronidierung von Metabolit E und Bisphenol. In beiden prä- und postmenopausalen Patientenkohorten war die genetische Variante CYP2C19*2 mit einer erniedrigten metabolischen Ratio (MR) von Bisphenol zu (Z)-Metabolit E assoziiert. Darüber hinaus führte das CYP3A4*22 Allel zu signifikant niedrigeren MRs von (Z)-Metabolit E zu Tamoxifen bzw. zu DM-Tamoxifen. Die stärkste östrogene Wirkung in einem ER-abhängigen Luciferase-Reportergen-Assay in MCF7-Zellen wurde für (E)-Metabolit E beobachtet. Darüber hinaus aktivierten alle drei östrogenen Metaboliten in Transkriptomanalysen von MCF7-Zellen in gleicher Weise wie Östradiol die Expression von Genen, die mit Zellzyklus, Transkription, DNA-Replikation, Mitose, Proliferation und Nukleotidmetabolismus assoziiert sind. Es konnte gezeigt werden, dass Bisphenol im Gegensatz zu den anti-östrogenen Tamoxifenmetaboliten die Aktivität aller untersuchten CYP-Enzyme (CYP1A2, 2B6, 2C8, 2C9, 2C19, 3A4) stark induziert, wobei niedrigste EC50-Werte für CYP2C19 (EC50=30,5 nM) gefunden wurden. Die in vitro beobachteten EC50-Konzentrationen waren allerdings höher als die gemessenen Patientenplasmaspiegel und setzen eine Gewebeanreicherung für eine biologische Wirkung in vivo voraus. In prämenopausalen Frauen konnte das Verhältnis von (E)-Metabolit E zu Tamoxifen als Risikofaktor für eine kürzere rezidivfreie Zeit (time to relapse; Hazard Ratio [HR] 2,813; 95 % Konfidenzintervall [KI] 1,372-5,767; p=0,005) bzw. ein kürzeres rezidivfreies Überleben (HR 2,022; 95 % KI 1,079-3,791; p=0,028) assoziiert werden. Dies impliziert, dass (E)-Metabolit E als stärkster Agonist am ER einen negativen Einfluss auf das Therapieansprechen von Tamoxifen hat und dass hohe Konzentrationen dieses Metaboliten ein möglicher Risikofaktor für das Therapieversagen von Tamoxifen sind. Die vorliegende Dissertation schafft die Basis für zukünftige Arbeiten pharmakogenetische Biomarker zur Vorhersage der (E)-Metabolit E Plasmakonzentration zu identifizieren und damit einen Beitrag für eine personalisierte Tamoxifentherapie bei Brustkrebs zu leisten.Tamoxifen is the standard therapy for hormone receptor positive breast cancer in premenopausal women and the major option to aromatase inhibitors in postmenopausal women. It is extensively metabolized and bioactivated by enzymes of the cytochrome P450 (CYP) family to more than 60 compounds with anti-estrogenic and estrogenic properties. The anti-estrogenic Metabolites 4-hydroxytamoxifen (4-OH-tamoxifen) and N-desmethyl-4-hydroxytamoxifen (endoxifen) have a more than 100-fold higher affinity to the estrogen receptor (ER) than tamoxifen itself and are therefore believed to mediate the anti-proliferative effect of the drug. The estrogenic Metabolites Tamoxifen bisphenol (bisphenol) and both isomers of metabolite E were shown to induce the expression of certain estrogen-regulated mRNA and therefore impact cell proliferation in ER positive breast cancer cell lines. In addition, these Metabolites were identified in a xenograft mouse model of tamoxifen resistance as well as in tumour tissue of tamoxifen treated breast cancer patients with clinical resistance. However, the metabolic pathways leading to Bisphenol and metabolite E as well as their impact on tamoxifen therapy are still unknown. Hence, the purpose of this work was to identify the enzymes involved in the formation of the estrogenic tamoxifen Metabolites and to analyse the pharmacodynamic effects regarding their ER-activating potency and the xenobiotic metabolism in vitro. Furthermore, the plasma concentrations of metabolite E and bisphenol were quantified in blood samples of pre- and postmenopausal women under tamoxifen therapy and the dependency on genetic variants of the involved CYP enzymes as well as to clinical outcome were analyzed. Human liver microsome experiments showed that both metabolite E and bisphenol are formed by NADPH-dependent microsomal reactions. In particular, it could be shown that metabolite E formation is mediated by CYP2C19, 3A, 2D6 and 1A2 from tamoxifen, N-desmethyl tamoxifen (DM-tamoxifen) and N-didesmethyl tamoxifen (DDM-tamoxifen) with tamoxifen having the highest metabolic clearance of 0.112 µl mg-1 min-1. Regarding bisphenol formation from the anti-estrogenic tamoxifen metabolite precursors 4-OH-tamoxifen, endoxifen and N-didesmethyl-4-hydroxy tamoxifen (norendoxifen), no specific CYP-isoenzyme could be identified. In contrast, the stereospecific hydroxylation of metabolite E to bisphenol was catalysed by CYP2C19 ((Z)-isomer) and CYP2B6 ((E)-isomer) with an up to 45-fold higher metabolic clearance compared to anti-estrogenic tamoxifen Metabolites. With regard to phase II metabolism, UGT1A8 and 2B7 were identified as main enzymes for the glucuronidation of metabolite E and bisphenol. In both pre- and postmenopausal breast cancer patients, CYP2C19*2 variants significantly lowered the metabolic ratio (MR) of bisphenol to (Z)-metabolite E. Furthermore CYP3A4*22 significantly decreased the MR of (Z)-metabolite E to both tamoxifen and DM-tamoxifen, respectively. The estrogenic effects on the ER based on an ER-dependent luciferase-reportergene-assay in MCF7-cells was strongest for (E)-metabolite E. Moreover, whole transcriptome analysis in MCF7-cells showed that all three estrogenic Metabolites displayed almost identical activated pathways associated with cell cycle, transcription, DNA-replication, mitosis, proliferation and nucleotid metabolism when compared to estradiol. Regarding the pharmacodynamic effects on the xenobiotic metabolism, bisphenol strongly induced the activity of all tested CYP-enzymes (CYP1A2, 2B6, 2C8, 2C9, 2C19, 3A4) with lowest EC50-values for CYP2C19 (EC50=30,5 nM). However, given that the observed EC50-concentrations in vitro are higher than those found in patient plasma a biological impact would require a tissue enrichment of estrogenic tamoxifen Metabolites in vivo. In premenopausal women the ratio of (E)-metabolite E to tamoxifen was identified as a risk factor of reduced time to relapse (hazard ratio [HR] 2.813; 95 % confidence interval [CI]: 1.372-5.767; p=0.005) and relapse-free survival (HR 2.022; 95 % CI: 1.079-3.791; p=0.028). This suggests that (E)-metabolite E due to its property as the strongest ER-agonist among the investigated estrogenic Metabolites, negatively influences the response to tamoxifen therapy and is a risk factor for therapeutic failure in the clinic. This dissertation provides a basis for future works to identify of pharmacogenetic biomarkers as predictors for (E)-metabolite E plasma concentration which contributes to further personalise tamoxifen treatment of hormone receptor positive breast cancer patients

  • Metabolismus, Pharmakodynamik und klinische Relevanz der östrogenen Tamoxifenmetaboliten bei der endokrinen Therapie des Mammakarzinoms
    Universität Tübingen, 2020
    Co-Authors: Johänning Janina
    Abstract:

    Tamoxifen ist die Standardtherapie für Hormonrezeptor-positiven Brustkrebs bei prämenopausalen Frauen und eine Alternative zur Therapie mit Aromataseinhibitoren bei postmenopausalen Frauen. Dabei wird Tamoxifen von Cytochrom P450 (CYP-) Enzymen zu über 60 Metaboliten verstoffwechselt, welche zum Teil anti-östrogene wie auch östrogene Eigenschaften aufweisen. Die anti-östrogenen Metaboliten 4-Hydroxytamoxifen (4-OH-Tamoxifen) und N-Desmethyl-4-hydroxytamoxifen (Endoxifen) haben eine 100-fach stärkere Affinität zum Östrogenrezeptor (ER) und sind hauptsächlich für den antiproliferativen Effekt im Brustgewebe verantwortlich. Zu den östrogenen Metaboliten gehören Tamoxifen Bisphenol (Bisphenol) und beide Isomere von Metabolit E, welche in ER-positiven Brustkrebs-Zellen die Expression von Östrogen-regulierten Genen induzieren und somit die Zellproliferation beeinflussen. Darüber hinaus wurden diese Metaboliten in Xenotransplantaten eines Mausmodells für die Tamoxifenresistenz sowie in Mammakarzinomgewebe von Patientinnen mit klinischer Resistenz identifiziert. Sowohl die Metabolismuswege zu Metabolit E und Bisphenol als auch der Einfluss dieser Metaboliten auf den Therapieverlauf sind bisher weitestgehend unerforscht. Das Ziel dieser Arbeit war es demnach die Enzyme, welche für die Bildung von Bisphenol und Metabolit E verantwortlich sind, zu identifizieren und die pharmakodynamischen Effekte auf den ER und Auswirkungen auf den Fremdstoffmetabolismus in vitro zu untersuchen. Darüber hinaus wurden die Plasmaspiegel der östrogenen Metaboliten in Blutproben von prä- und postmenopausalen Frauen unter Tamoxifentherapie quantifiziert, deren Abhängigkeit von genetischen Varianten der beteiligten CYP-Enzyme analysiert sowie der Einfluss auf den Therapieverlauf untersucht. Mit Hilfe von humanen Lebermikrosomen konnte gezeigt werden, dass Metabolit E wie auch Bisphenol in NADPH-abhängigen mikrosomalen Reaktionen gebildet werden. Weitere Analysen mit Supersomes™ zeigten, dass Metabolit E hauptsächlich durch die Enzyme CYP2C19, 3A, 2D6 und 1A2 aus Tamoxifen, N-Desmethyltamoxifen (DM-Tamoxifen) und N-Didesmethyltamoxifen (DDM-Tamoxifen) metabolisiert wird. Kinetische Untersuchungen ergaben die höchste Clearance für Tamoxifen mit 0,112 µl mg-1 min-1. Für die Bildung von Bisphenol aus den anti-östrogenen Tamoxifenmetaboliten 4-OH-Tamoxifen, Endoxifen und N-Didesmethyl-4-hydroxytamoxifen (Norendoxifen) konnte kein spezifisches CYP-Isoenzym identifiziert werden. Im Gegensatz dazu zeigte die stereospezifische Hydroxylierung von Metabolit E zu Bisphenol durch CYP2C19 ((Z)-Isomer) und CYP2B6 ((E)-Isomer) eine bis zu 45-fach höhere Clearance verglichen mit den anti-östrogenen Tamoxifenmetaboliten. Bezüglich des Phase II-Metabolismus zeigten die UGT-Enzyme 1A8 und 2B7 höchste Aktivitäten für die Glucuronidierung von Metabolit E und Bisphenol. In beiden prä- und postmenopausalen Patientenkohorten war die genetische Variante CYP2C19*2 mit einer erniedrigten metabolischen Ratio (MR) von Bisphenol zu (Z)-Metabolit E assoziiert. Darüber hinaus führte das CYP3A4*22 Allel zu signifikant niedrigeren MRs von (Z)-Metabolit E zu Tamoxifen bzw. zu DM-Tamoxifen. Die stärkste östrogene Wirkung in einem ER-abhängigen Luciferase-Reportergen-Assay in MCF7-Zellen wurde für (E)-Metabolit E beobachtet. Darüber hinaus aktivierten alle drei östrogenen Metaboliten in Transkriptomanalysen von MCF7-Zellen in gleicher Weise wie Östradiol die Expression von Genen, die mit Zellzyklus, Transkription, DNA-Replikation, Mitose, Proliferation und Nukleotidmetabolismus assoziiert sind. Es konnte gezeigt werden, dass Bisphenol im Gegensatz zu den anti-östrogenen Tamoxifenmetaboliten die Aktivität aller untersuchten CYP-Enzyme (CYP1A2, 2B6, 2C8, 2C9, 2C19, 3A4) stark induziert, wobei niedrigste EC50-Werte für CYP2C19 (EC50=30,5 nM) gefunden wurden. Die in vitro beobachteten EC50-Konzentrationen waren allerdings höher als die gemessenen Patientenplasmaspiegel und setzen eine Gewebeanreicherung für eine biologische Wirkung in vivo voraus. In prämenopausalen Frauen konnte das Verhältnis von (E)-Metabolit E zu Tamoxifen als Risikofaktor für eine kürzere rezidivfreie Zeit (time to relapse; Hazard Ratio [HR] 2,813; 95 % Konfidenzintervall [KI] 1,372-5,767; p=0,005) bzw. ein kürzeres rezidivfreies Überleben (HR 2,022; 95 % KI 1,079-3,791; p=0,028) assoziiert werden. Dies impliziert, dass (E)-Metabolit E als stärkster Agonist am ER einen negativen Einfluss auf das Therapieansprechen von Tamoxifen hat und dass hohe Konzentrationen dieses Metaboliten ein möglicher Risikofaktor für das Therapieversagen von Tamoxifen sind. Die vorliegende Dissertation schafft die Basis für zukünftige Arbeiten pharmakogenetische Biomarker zur Vorhersage der (E)-Metabolit E Plasmakonzentration zu identifizieren und damit einen Beitrag für eine personalisierte Tamoxifentherapie bei Brustkrebs zu leisten.Tamoxifen is the standard therapy for hormone receptor positive breast cancer in premenopausal women and the major option to aromatase inhibitors in postmenopausal women. It is extensively metabolized and bioactivated by enzymes of the cytochrome P450 (CYP) family to more than 60 compounds with anti-estrogenic and estrogenic properties. The anti-estrogenic Metabolites 4-hydroxytamoxifen (4-OH-tamoxifen) and N-desmethyl-4-hydroxytamoxifen (endoxifen) have a more than 100-fold higher affinity to the estrogen receptor (ER) than tamoxifen itself and are therefore believed to mediate the anti-proliferative effect of the drug. The estrogenic Metabolites Tamoxifen bisphenol (bisphenol) and both isomers of metabolite E were shown to induce the expression of certain estrogen-regulated mRNA and therefore impact cell proliferation in ER positive breast cancer cell lines. In addition, these Metabolites were identified in a xenograft mouse model of tamoxifen resistance as well as in tumour tissue of tamoxifen treated breast cancer patients with clinical resistance. However, the metabolic pathways leading to Bisphenol and metabolite E as well as their impact on tamoxifen therapy are still unknown. Hence, the purpose of this work was to identify the enzymes involved in the formation of the estrogenic tamoxifen Metabolites and to analyse the pharmacodynamic effects regarding their ER-activating potency and the xenobiotic metabolism in vitro. Furthermore, the plasma concentrations of metabolite E and bisphenol were quantified in blood samples of pre- and postmenopausal women under tamoxifen therapy and the dependency on genetic variants of the involved CYP enzymes as well as to clinical outcome were analyzed. Human liver microsome experiments showed that both metabolite E and bisphenol are formed by NADPH-dependent microsomal reactions. In particular, it could be shown that metabolite E formation is mediated by CYP2C19, 3A, 2D6 and 1A2 from tamoxifen, N-desmethyl tamoxifen (DM-tamoxifen) and N-didesmethyl tamoxifen (DDM-tamoxifen) with tamoxifen having the highest metabolic clearance of 0.112 µl mg-1 min-1. Regarding bisphenol formation from the anti-estrogenic tamoxifen metabolite precursors 4-OH-tamoxifen, endoxifen and N-didesmethyl-4-hydroxy tamoxifen (norendoxifen), no specific CYP-isoenzyme could be identified. In contrast, the stereospecific hydroxylation of metabolite E to bisphenol was catalysed by CYP2C19 ((Z)-isomer) and CYP2B6 ((E)-isomer) with an up to 45-fold higher metabolic clearance compared to anti-estrogenic tamoxifen Metabolites. With regard to phase II metabolism, UGT1A8 and 2B7 were identified as main enzymes for the glucuronidation of metabolite E and bisphenol. In both pre- and postmenopausal breast cancer patients, CYP2C19*2 variants significantly lowered the metabolic ratio (MR) of bisphenol to (Z)-metabolite E. Furthermore CYP3A4*22 significantly decreased the MR of (Z)-metabolite E to both tamoxifen and DM-tamoxifen, respectively. The estrogenic effects on the ER based on an ER-dependent luciferase-reportergene-assay in MCF7-cells was strongest for (E)-metabolite E. Moreover, whole transcriptome analysis in MCF7-cells showed that all three estrogenic Metabolites displayed almost identical activated pathways associated with cell cycle, transcription, DNA-replication, mitosis, proliferation and nucleotid metabolism when compared to estradiol. Regarding the pharmacodynamic effects on the xenobiotic metabolism, bisphenol strongly induced the activity of all tested CYP-enzymes (CYP1A2, 2B6, 2C8, 2C9, 2C19, 3A4) with lowest EC50-values for CYP2C19 (EC50=30,5 nM). However, given that the observed EC50-concentrations in vitro are higher than those found in patient plasma a biological impact would require a tissue enrichment of estrogenic tamoxifen Metabolites in vivo. In premenopausal women the ratio of (E)-metabolite E to tamoxifen was identified as a risk factor of reduced time to relapse (hazard ratio [HR] 2.813; 95 % confidence interval [CI]: 1.372-5.767; p=0.005) and relapse-free survival (HR 2.022; 95 % CI: 1.079-3.791; p=0.028). This suggests that (E)-metabolite E due to its property as the strongest ER-agonist among the investigated estrogenic Metabolites, negatively influences the response to tamoxifen therapy and is a risk factor for therapeutic failure in the clinic. This dissertation provides a basis for future works to identify of pharmacogenetic biomarkers as predictors for (E)-metabolite E plasma concentration which contributes to further personalise tamoxifen treatment of hormone receptor positive breast cancer patients

Dominique Morandi - One of the best experts on this subject based on the ideXlab platform.

  • a toolbox of genes proteins Metabolites and promoters for improving drought tolerance in soybean includes the metabolite coumestrol and stomatal development genes
    BMC Genomics, 2016
    Co-Authors: Prateek Tripathi, Roel C Rabara, Neil R Reese, Marissa A Miller, Jai S Rohila, Senthil Subramanian, Qingxi J Shen, Dominique Morandi
    Abstract:

    The purpose of this project was to identify Metabolites, proteins, genes, and promoters associated with water stress responses in soybean. A number of these may serve as new targets for the biotechnological improvement of drought responses in soybean (Glycine max). We identified Metabolites, proteins, and genes that are strongly up or down regulated during rapid water stress following removal from a hydroponics system. 163 Metabolites showed significant changes during water stress in roots and 93 in leaves. The largest change was a root-specific 160-fold increase in the coumestan coumestrol making it a potential biomarker for drought and a promising target for improving drought responses. Previous reports suggest that coumestrol stimulates mycorrhizal colonization and under certain conditions mycorrhizal plants have improved drought tolerance. This suggests that coumestrol may be part of a call for help to the rhizobiome during stress. About 3,000 genes were strongly up-regulated by drought and we identified regulators such as ERF, MYB, NAC, bHLH, and WRKY transcription factors, receptor-like kinases, and calcium signaling components as potential targets for soybean improvement as well as the jasmonate and abscisic acid biosynthetic genes JMT, LOX1, and ABA1. Drought stressed soybean leaves show reduced mRNA levels of stomatal development genes including FAMA-like, MUTE-like and SPEECHLESS-like bHLH transcription factors and leaves formed after drought stress had a reduction in stomatal density of 22.34 % and stomatal index of 17.56 %. This suggests that reducing stomatal density may improve drought tolerance. MEME analyses suggest that ABRE (CACGT/CG), CRT/DRE (CCGAC) and a novel GTGCnTGC/G element play roles in transcriptional activation and these could form components of synthetic promoters to drive expression of transgenes. Using transformed hairy roots, we validated the increase in promoter activity of GmWRKY17 and GmWRKY67 during dehydration and after 20 μM ABA treatment. Our toolbox provides new targets and strategies for improving soybean drought tolerance and includes the coumestan coumestrol, transcription factors that regulate stomatal density, water stress-responsive WRKY gene promoters and a novel DNA element that appears to be enriched in water stress responsive promoters.

  • A toolbox of genes, proteins, Metabolites and promoters for improving drought tolerance in soybean includes the metabolite coumestrol and stomatal development genes
    BMC Genomics, 2016
    Co-Authors: Prateek Tripathi, Roel C Rabara, Marissa A Miller, Jai S Rohila, Senthil Subramanian, Qingxi J Shen, Dominique Morandi, R. Neil Reese, Heike Bucking, Vladimir Shulaev
    Abstract:

    Background: The purpose of this project was to identify Metabolites, proteins, genes, and promoters associated with water stress responses in soybean. A number of these may serve as new targets for the biotechnological improvement of drought responses in soybean (Glycine max). Results: We identified Metabolites, proteins, and genes that are strongly up or down regulated during rapid water stress following removal from a hydroponics system. 163 Metabolites showed significant changes during water stress in roots and 93 in leaves. The largest change was a root-specific 160-fold increase in the coumestan coumestrol making it a potential biomarker for drought and a promising target for improving drought responses. Previous reports suggest that coumestrol stimulates mycorrhizal colonization and under certain conditions mycorrhizal plants have improved drought tolerance. This suggests that coumestrol may be part of a call for help to the rhizobiome during stress. About 3,000 genes were strongly up-regulated by drought and we identified regulators such as ERF, MYB, NAC, bHLH, and WRKY transcription factors, receptor-like kinases, and calcium signaling components as potential targets for soybean improvement as well as the jasmonate and abscisic acid biosynthetic genes JMT, LOX1, and ABA1. Drought stressed soybean leaves show reduced mRNA levels of stomatal development genes including FAMA-like, MUTE-like and SPEECHLESS-like bHLH transcription factors and leaves formed after drought stress had a reduction in stomatal density of 22.34 % and stomatal index of 17.56 %. This suggests that reducing stomatal density may improve drought tolerance. MEME analyses suggest that ABRE (CACGT/CG), CRT/DRE (CCGAC) and a novel GTGCnTGC/G element play roles in transcriptional activation and these could form components of synthetic promoters to drive expression of transgenes. Using transformed hairy roots, we validated the increase in promoter activity of GmWRKY17 and GmWRKY67 during dehydration and after 20 mu M ABA treatment. Conclusions: Our toolbox provides new targets and strategies for improving soybean drought tolerance and includes the coumestan coumestrol, transcription factors that regulate stomatal density, water stress-responsive WRKY gene promoters and a novel DNA element that appears to be enriched in water stress responsive promoters.

Roel C Rabara - One of the best experts on this subject based on the ideXlab platform.

  • a toolbox of genes proteins Metabolites and promoters for improving drought tolerance in soybean includes the metabolite coumestrol and stomatal development genes
    BMC Genomics, 2016
    Co-Authors: Prateek Tripathi, Roel C Rabara, Neil R Reese, Marissa A Miller, Jai S Rohila, Senthil Subramanian, Qingxi J Shen, Dominique Morandi
    Abstract:

    The purpose of this project was to identify Metabolites, proteins, genes, and promoters associated with water stress responses in soybean. A number of these may serve as new targets for the biotechnological improvement of drought responses in soybean (Glycine max). We identified Metabolites, proteins, and genes that are strongly up or down regulated during rapid water stress following removal from a hydroponics system. 163 Metabolites showed significant changes during water stress in roots and 93 in leaves. The largest change was a root-specific 160-fold increase in the coumestan coumestrol making it a potential biomarker for drought and a promising target for improving drought responses. Previous reports suggest that coumestrol stimulates mycorrhizal colonization and under certain conditions mycorrhizal plants have improved drought tolerance. This suggests that coumestrol may be part of a call for help to the rhizobiome during stress. About 3,000 genes were strongly up-regulated by drought and we identified regulators such as ERF, MYB, NAC, bHLH, and WRKY transcription factors, receptor-like kinases, and calcium signaling components as potential targets for soybean improvement as well as the jasmonate and abscisic acid biosynthetic genes JMT, LOX1, and ABA1. Drought stressed soybean leaves show reduced mRNA levels of stomatal development genes including FAMA-like, MUTE-like and SPEECHLESS-like bHLH transcription factors and leaves formed after drought stress had a reduction in stomatal density of 22.34 % and stomatal index of 17.56 %. This suggests that reducing stomatal density may improve drought tolerance. MEME analyses suggest that ABRE (CACGT/CG), CRT/DRE (CCGAC) and a novel GTGCnTGC/G element play roles in transcriptional activation and these could form components of synthetic promoters to drive expression of transgenes. Using transformed hairy roots, we validated the increase in promoter activity of GmWRKY17 and GmWRKY67 during dehydration and after 20 μM ABA treatment. Our toolbox provides new targets and strategies for improving soybean drought tolerance and includes the coumestan coumestrol, transcription factors that regulate stomatal density, water stress-responsive WRKY gene promoters and a novel DNA element that appears to be enriched in water stress responsive promoters.

  • A toolbox of genes, proteins, Metabolites and promoters for improving drought tolerance in soybean includes the metabolite coumestrol and stomatal development genes
    BMC Genomics, 2016
    Co-Authors: Prateek Tripathi, Roel C Rabara, Marissa A Miller, Jai S Rohila, Senthil Subramanian, Qingxi J Shen, Dominique Morandi, R. Neil Reese, Heike Bucking, Vladimir Shulaev
    Abstract:

    Background: The purpose of this project was to identify Metabolites, proteins, genes, and promoters associated with water stress responses in soybean. A number of these may serve as new targets for the biotechnological improvement of drought responses in soybean (Glycine max). Results: We identified Metabolites, proteins, and genes that are strongly up or down regulated during rapid water stress following removal from a hydroponics system. 163 Metabolites showed significant changes during water stress in roots and 93 in leaves. The largest change was a root-specific 160-fold increase in the coumestan coumestrol making it a potential biomarker for drought and a promising target for improving drought responses. Previous reports suggest that coumestrol stimulates mycorrhizal colonization and under certain conditions mycorrhizal plants have improved drought tolerance. This suggests that coumestrol may be part of a call for help to the rhizobiome during stress. About 3,000 genes were strongly up-regulated by drought and we identified regulators such as ERF, MYB, NAC, bHLH, and WRKY transcription factors, receptor-like kinases, and calcium signaling components as potential targets for soybean improvement as well as the jasmonate and abscisic acid biosynthetic genes JMT, LOX1, and ABA1. Drought stressed soybean leaves show reduced mRNA levels of stomatal development genes including FAMA-like, MUTE-like and SPEECHLESS-like bHLH transcription factors and leaves formed after drought stress had a reduction in stomatal density of 22.34 % and stomatal index of 17.56 %. This suggests that reducing stomatal density may improve drought tolerance. MEME analyses suggest that ABRE (CACGT/CG), CRT/DRE (CCGAC) and a novel GTGCnTGC/G element play roles in transcriptional activation and these could form components of synthetic promoters to drive expression of transgenes. Using transformed hairy roots, we validated the increase in promoter activity of GmWRKY17 and GmWRKY67 during dehydration and after 20 mu M ABA treatment. Conclusions: Our toolbox provides new targets and strategies for improving soybean drought tolerance and includes the coumestan coumestrol, transcription factors that regulate stomatal density, water stress-responsive WRKY gene promoters and a novel DNA element that appears to be enriched in water stress responsive promoters.