Das Schilddrüsenhormon Triiodthyronin (T3) ist ein essenzieller Regulator physiologischer Prozesse der Entwicklung, des Wachstums und im Intermediärstoffwechsel. Täglich werden zahlreiche natürliche und synthetische Stoffe aufgenommen, die mit dem endokrinen System interferieren und deshalb als Endokrine Disruptoren (ED) bezeichnet werden. Zur Untersuchung einer direkten Interferenz von ED mit den Schilddrüsenhormonrezeptoren (TR) und ihrer transkriptionellen Aktivität wurde im Rahmen dieser Arbeit ein neues Luziferase-basiertes T3 Reportergensystem mit TRalpha1-transfizierten humanen Leberzellen aufgebaut. Durch Validierung mit dem synthetischen TR-Agonisten GC-1 und dem Antagonisten NH-3 konnte nachgewiesen werden, dass dieser Assay ein hoch-sensitives System zur Analyse von agonistischen sowie antagonistischen Effekten von Testsubtanzen darstellt. Zur Bestimmung der endokrinen Aktivitäten einiger humanrelevanter Vertreter aus den Stoffkategorien der Nahrungsmittel, Kosmetika, Pestizide und Industriechemikalien wurden Dosis-Wirkungskurven in Aktivierungs- und T3-Kompetitionsexperimenten ermittelt. In mikromolaren Konzentrationen wirkten von insgesamt 21 Testsubstanzen einige als reine Agonisten oder Antagonisten während andere gemischt agonistische/antagonistische Effekte hatten. Aufgrund ihrer hier beobachteten Effekte und der gegebenen Humanexposition wird eine eingehendere Analyse von 4-Methylbenzyliden Campher, 4-Nonylphenol, Acetochlor, Benzophenon 2, Benzophenon 3, Bisphenol A, Genistein, Octylmethoxycinnamat, Tetrabromobisphenol A und Xanthohumol empfohlen. Außerdem erwiesen sich einige Metaboliten von Schilddrüsenhormonen als potente Agonisten im T3-Reportergenassay und bedürfen weiterer Aufmerksamkeit. Für die molekulare Charakterisierung der Einflüsse solcher Substanzen auf die T3-regulierte Transaktivierung konnte mit dem hier etablierten Bioassay ein zuverlässiges neues Testsystem für reproduzierbare Screeningserien geschaffen werden. / Triiodothyronine (T3) is a crucial regulator of many physiological processes during development, growth and metabolism. A variety of natural and synthetic substances, which are collectively termed endocrine disrupters (ED) due to their interference with the endocrine system, is taken up on a daily base. A novel luciferase-based T3-responsive reporter gene system employing a human liver cell line transfected with thyroid hormone receptor (TR) alpha1 was established in this work to elucidate the potential molecular interference of certain ED with TR and their transcriptional activity. This assay was validated to be a highly sensitive and reliable tool for analyzing agonistic and antagonistic effects of test compounds using the synthetic TR agonist GC-1 and the antagonist NH-3. Dose-response data of test compounds contained in food, cosmetics, pesticides, plasticizers and other industrial chemicals were obtained after applying the substances alone in activation assays or in combination with T3 in competition assays. In total 21 test compounds were screened of which some acted as pure agonists or antagonists while others were mixed agonists/antagonists in the micromolar concentration range and only one was without effect. Follow-up studies are recommended for some of these substances with regard to their effects as determined in this bioassay and in light of information known on human exposure, i.e., 4-methylbenzyliden camphor, 4-nonylphenol, acetochlor, benzophenone 2, benzophenone 3, bisphenol A, genistein, octylmethoxycinnamate, tetrabromobisphenol A and xanthohumol. In addition some endogenous metabolites of thyroid hormones were surprisingly potent agonists in the T3 reporter gene assay and merit further attention. The novel bioassay established here represents a reliable tool for the screening and molecular characterization of substances interfering with T3-mediated transactivation of gene expression.

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The expanding therapeutic use of thalidomide (TD) remains limited by its species-specific teratogenicity in humans and rabbits, but not rodents. The R and S isomers of TD may be selectively responsible for its respective therapeutic and teratogenic effects, but rapid in vivo racemization makes this impossible to confirm. Fluorothalidomide (FTD), a fluorinated TD analogue with stable, non-racemizing isomers, may serve as a model compound for determining stereoselective effects. In vivo, FTD was undetectable in plasma, suggesting rapid breakdown, as confirmed in vitro, where FTD hydrolyzed up to 22-fold faster than TD. Unlike TD, FTD in pregnant rabbits and mice was highly toxic and lethal to both dams and fetuses. In rabbit embryo culture, FTD initiated optic (eye) vesicle and hindbrain but not classic limb bud embryopathies. Chemical instability, potent general toxicity and absence of limb bud embryopathies make FTD an unsuitable stereoselective model for TD teratogenesis. TD teratogenesis may involve its bioactivation by embryonic prostaglandin H synthases (PHSs) to a free radical intermediate that increases embryopathic reactive oxygen species (ROS) formation. However, the teratogenic potential of rapidly formed TD hydrolysis products and the determinants of species-specific teratogenesis are unclear. For some teratogens, mouse strains that are resistant in vivo are susceptible in embryo culture, suggesting maternal and/or placental determinants of risk. However, TD and two hydrolysis products, 2-phthalimidoglutaramic acid (PGMA) and 2-phthalimidoglutaraic acid (PGA), were non-embryopathic in CD-1 mouse embryo culture. Also, mice deficient in oxoguanine glycosylase 1 (OGG1), which repairs oxidatively damaged DNA, were resistant to TD embryopathies in culture and in vivo. Therefore, murine resistance to TD teratogenesis is dependent on embryonic factors, rather than maternal/placental determinants or increased DNA repair. In contrast, rabbit embryos exposed in culture to TD, PGMA and PGA exhibited head/brain, otic (ear) vesicle and classic limb bud embryopathies, validating the first mammalian embryo culture model for TD teratogenesis and providing the first evidence of a teratogenic role for TD hydrolysis products. Pretreatment with eicosatetraynoic acid (ETYA), a dual PHS/lipoxygenase inhibitor, or phenylbutylnitrone (PBN), a free radical spin trapping agent, completely blocked TD, PGMA and PGA-initiated embryopathies, implicating a PHS-dependent, ROS-mediated embryopathic mechanism.

The expanding therapeutic use of thalidomide (TD) remains limited by its species-specific teratogenicity in humans and rabbits, but not rodents. The R and S isomers of TD may be selectively responsible for its respective therapeutic and teratogenic effects, but rapid in vivo racemization makes this impossible to confirm. Fluorothalidomide (FTD), a fluorinated TD analogue with stable, non-racemizing isomers, may serve as a model compound for determining stereoselective effects. In vivo, FTD was undetectable in plasma, suggesting rapid breakdown, as confirmed in vitro, where FTD hydrolyzed up to 22-fold faster than TD. Unlike TD, FTD in pregnant rabbits and mice was highly toxic and lethal to both dams and fetuses. In rabbit embryo culture, FTD initiated optic (eye) vesicle and hindbrain but not classic limb bud embryopathies. Chemical instability, potent general toxicity and absence of limb bud embryopathies make FTD an unsuitable stereoselective model for TD teratogenesis. TD teratogenesis may involve its bioactivation by embryonic prostaglandin H synthases (PHSs) to a free radical intermediate that increases embryopathic reactive oxygen species (ROS) formation. However, the teratogenic potential of rapidly formed TD hydrolysis products and the determinants of species-specific teratogenesis are unclear. For some teratogens, mouse strains that are resistant in vivo are susceptible in embryo culture, suggesting maternal and/or placental determinants of risk. However, TD and two hydrolysis products, 2-phthalimidoglutaramic acid (PGMA) and 2-phthalimidoglutaraic acid (PGA), were non-embryopathic in CD-1 mouse embryo culture. Also, mice deficient in oxoguanine glycosylase 1 (OGG1), which repairs oxidatively damaged DNA, were resistant to TD embryopathies in culture and in vivo. Therefore, murine resistance to TD teratogenesis is dependent on embryonic factors, rather than maternal/placental determinants or increased DNA repair. In contrast, rabbit embryos exposed in culture to TD, PGMA and PGA exhibited head/brain, otic (ear) vesicle and classic limb bud embryopathies, validating the first mammalian embryo culture model for TD teratogenesis and providing the first evidence of a teratogenic role for TD hydrolysis products. Pretreatment with eicosatetraynoic acid (ETYA), a dual PHS/lipoxygenase inhibitor, or phenylbutylnitrone (PBN), a free radical spin trapping agent, completely blocked TD, PGMA and PGA-initiated embryopathies, implicating a PHS-dependent, ROS-mediated embryopathic mechanism.

Background & Aims: Oxidative stress and apoptosis are proposed mechanisms of cellular injury in studies of xenobiotic hepatotoxicity. The aim of this work is to find early signal markers of drug-induced injury of the liver by focusing on select antioxidant/oxidant and apoptotic genes. As well, to address the relationship between conventional liver dysfunction markers and the measured mRNA and protein expressions in the D-galactosamine/lipopolysaccharide and tert-butylhydroperoxide hepatotoxicity models. Furthermore, potential hepatoprotective capabilities of antioxidant polyphenols quercetin and curcumin were evaluated in relation to its modulation of the oxidative stress and apoptotic parameters in the given xenobiotic hepatotoxicity models. Methods: Biochemical markers testing the hepatic function included aminotransferases (ALT, AST) and bilirubin. Measurements of TBARS and conjugated dienes were used to assess lipoperoxidation. Plasma levels of catalase and reduced glutathione were used as indicators of the oxidative status of the cell. Real time PCR was used to analyse the mRNA expressions of the inducible nitric oxide synthase (NOS-2), heme oxygenase-1 (HO-1), superoxide dismutase (SOD-1), glutathione peroxidase (Gpx-1), caspase 3 (Casp3), BH3 interacting domain death agonist (Bid) and Bcl-2...

Les herbicides sont des polluants suscitant de grandes inquiétudes en raison de leur ubiquité environnementale résultant de leur usage intensif dans l’agriculture moderne et de leur persistance dans les sols et les eaux. Les herbicides peuvent être dégradés par des microorganismes, des plantes ou d’autres processus naturels, produisant alors une vaste gamme de métabolites dont l’impact sur les écosystèmes reste méconnu. Dans un contexte d’évaluation des risques environnementaux, l’étude de la réponse des plantes à des mélanges complexes de xénobiotiques est importante pour estimer les effets des contaminations, notamment dans le cas de pollution résiduelle. Afin d’étudier l’impact de cette diversité de polluants, les mécanismes de réponse et les cibles impliquées, la plante modèle Arabidopsis thaliana a été confrontée à des doses variables de molécules de la famille des triazines constituant une série chimique cohérente : atrazine, herbicide encore largement utilisé au niveau mondial, déséthylatrazine, métabolite chloré de l’atrazine, et hydroxyatrazine, métabolite de déchloration de l’atrazine. Ce travail montre que l’exposition de courte durée à des doses variables d’atrazine, de déséthylatrazine et d’hydroxyatrazine, au niveau racinaire, affecte de manière spécifique et dose-dépendante la croissance précoce et le développement de la plante. La caractérisation d’effets directs et multiples sur la respiration et la croissance racinaire a permis de révéler des mécanismes d’action non-canoniques, distincts de l’action classiquement décrite des triazines sur le photosystème II. Afin d’identifier ces mécanismes, activés en absence de dommages cellulaires, une analyse transcriptomique au niveau du génome entier a été effectuée. Les trois triazines induisent des changements coordonnés et spécifiques dans l’expression des gènes. L’analyse fonctionnelle des gènes différentiellement exprimés et de leur promoteur révèle que les voies de signalisation liées à la fois aux hormones végétales, à la perception de faibles niveaux d’énergie, aux stress environnementaux ainsi qu’aux interactions biotiques sont impliquées dans la réponse aux faibles doses de triazines. Les triazines affectent, en particulier, l’expression de gènes connus pour être régulés par les cytokinines. De manière intéressante, cette famille d’hormones végétales montre des caractéristiques chimiques similaires à celles des triazines. Des études développementales utilisant différentes modalités d’exposition aux triazines et aux cytokinines ont alors été effectuées sur des génotypes sauvages et sur des mutants de la voie de signalisation des cytokinines. L’identification d’interactions spécifiques entre les triazines et les composants de la signalisation des cytokinines a alors mis en évidence des mécanismes potentiels de compétition et/ou d’antagonisme. La caractérisation de ces perturbations au niveau de la transduction du signal pourra permettre à terme d’évaluer l’efficacité des herbicides sur les cultures ainsi que l’impact des contaminations xénobiotiques sur les communautés végétales naturelles. Enfin, l’identification des interactions entre stress xénobiotique, biotique et abiotique approfondira les connaissances sur les effets croisés de la pollution chimique et des stress liés au changement climatique. / Herbicides are pollutants of high concern due to their environmental ubiquity resulting from extensive use in modern agriculture and persistence in soil and water. Degradation events on active molecules mediated by microorganisms, by plants and by natural processes give rise to a plethora of herbicide metabolites of unknown impact on ecosystems. Study of plant behavior toward such complex mixtures of xenobiotic structures is important to evaluate the effects of contaminations, especially in the context of residual pollution. In order to understand the mechanisms underlying the action of this diversity of compounds, the model plant Arabidopsis thaliana was confronted to variable doses of the widely-used triazine herbicide atrazine, and of two of its metabolites, desethylatrazine and hydroxyatrazine. Short exposure to varying concentrations of atrazine, desethylatrazine and hydroxyatrazine was found to affect early growth and development in various dose-dependent and distinct manners. These differential effects pointed out to the multiple involvement of non-canonical mechanisms, directly affecting respiration and root development. In order to identify these mechanisms, which are activated in the absence of major adverse physiological effects, a genome-wide transcriptomic analysis was carried out. All of the triazines under study induced coordinated and specific changes in gene expression. Functional analysis of differentially expressed genes and of their promoters revealed that signaling pathways related to plant hormones, low energy sensing, environmental stresses and biotic interactions were involved in low-dose triazine responses. In particular, triazines affected the expression of genes known to be regulated by cytokinins. Interestingly, this family of plant hormones shares similar chemical features with triazine compounds. Developmental studies on plants bearing mutations in cytokinin sensing and signaling pathways were then carried out under variable triazine exposures. The identification of specific interactions between triazine compounds and cytokinin-signaling components highlighted potential mechanisms of competition and/or antagonism. The characterization of such signal transduction modifications and perturbations will be useful to assess herbicide efficiency in crop systems and xenobiotic contamination impact on natural plant communities. Finally, the identification of crosstalk processes between xenobiotic, abiotic and biotic stress signaling gives novel insights into the interplay between chemical pollution and climate change stressors.