Integrated biomarker responses as environmental status descriptors of a coastal zone (Sao Paulo, Brazil)

Sao Paulo state (Brazil) has one of the most overpopulated coastal zones in South America, where previous studies have already detected sediment and water contamination. However, biological-based monitoring considering signals of xenobiotic exposure and effects are scarce. The present study employed a battery of biomarkers under field conditions to assess the environmental quality of this coastal zone. For this purpose, the activity of CYP 450, antioxidant enzymes, DNA damage, lipid peroxidation and lysosomal membrane were analysed in caged mussels and integrated using Factorial Analysis. A representation of estimated factor scores was performed in order to confirm the factor descriptions characterizing the studied areas. Biomarker responses indicated signals of mussels` impaired health during the monitoring, which pointed to the impact of different sources of contaminants in the water quality and identified critical areas. This integrated approach produced a rapid, sensitive and cost-effective assessment, which could be incorporated as a descriptor of environmental status in future coastal zones biomonitoring. (C) 2011 Elsevier Inc. All rights reserved.

Immobilization of marine fungi on silica gel, silica xerogel and chitosan for biocatalytic reduction of ketones

The scanning electron microscopy (SEM) analysis showed that whole living hyphal of marine fungi Aspergillus sclerotiorum CBMAI 849 and Penicillium citrinum CBMAI 1186 were immobilized on support matrices of silica gel, silica xerogel and/or chitosan. P. citrinum immobilized on chitosan catalyzed the quantitative reduction of 1-(4-methoxyphenyl)-ethanone (1) to the enantiomer (S)-1-(4-methoxyphenyl)-ethanol (3b), with excellent enantioselectivity (ee > 99%, yield = 95%). Interestingly, ketone 1 was reduced with moderate selectivity and conversion to alcohol 3b (ee = 69%, c 40%) by the free mycelium of P. citrinum. This free mycelium of P. citrinum catalyzed the production of the (R)-alcohol 3a, the antipode of the alcohol produced by the immobilized cells. P. citrinum immobilized on chitosan also catalyzed the bioreduction of 2-chloro-1-phenylethanone (2) to 2-chloro-1-phenylethanol (4a,b), but in this case without optical selectivity. These results showed that biocatalytic reduction of ketones by immobilization hyphal of marine fungi depends on the xenobiotic substrate and the support matrix used. (c) 2012 Elsevier B.V. All rights reserved.

Mercury-induced autoimmunity : Genetics and immunoregulation

The existence of immune self-tolerance allows the immune system to mount responses against infectious agents, but not against self-molecular constitutes. Although self-tolerance is a robust phenomenon, in some individuals as well as in experimental models, the self-tolerance breaks down and as a result, a self-destructive autoimmune disease emerges. The underlying mechanisms for the development of autoimmune diseases are not known, but genetic, environmental and immunological factors are suggested to be involved. In this thesis, we used murine mercury-induced autoimmunity to test this suggestion. In susceptible mice mercuric chloride induces a systemic autoimmune disease characterized by increased serum levels of IgG1 and IgE, production of anti-nucleolar autoantibodies (ANolA) and formation of renal IgG deposits. In contrast, in resistant DBA/2 (H-2d) mice, none of these characteristics develop after exposure to mercury. By crossing and backcrossing mercury-resistant DBA/2 mice to mercury susceptible strains, we found that the resistance was inherited as a dominant trait in F1 hybrids and that one gene or a cluster of genes located in the H-2 loci determined the resistance to ANolA production, whereas resistance to the other characteristics was found to be controlled by two or three non-H-2 genes. We further put forward the “cryptic peptide hypothesis” to investigate whether mercury and another xenobiotic metal use similar pathway(s) to induce the H-2 linked production of ANolA. We found that while mercury stimulated ANolA synthesis in all H-2 susceptible (H-2s, H-2q and H-2f) mouse strains, silver induced only ANolA responses in H-2s and H-2q mice, but not in H-2f mice. Further studies showed that the resistance to silver-induced ANolA production in H-2f mice was inherited as a dominant trait. We next tested the proposition that mercury induces more adverse immunological effects in mouse strains, which are genetically prone to develop autoimmune diseases, using tight-skin 1 mice, an animal model for human Scleroderma. It was found that in this strain, mercury induced a strong immune activation with autoimmune characteristics, but did not accelerate the development of dermal fibrosis, a characteristic in Tsk/1 mice. Finally we addressed the Th1/Th2 cross-regulation paradigm by examining if a Th1-type of response could interact with a Th2-type of response if simultaneous induced in susceptible mice. Our findings demonstrated that mercury-induced autoimmunity (Th2-type) and collagen-induced arthritis (CIA) (Th1-type) can interact in a synergistic, antagonistic or additive fashion, depending on at which stage of CIA mercury is administered.

Messa a punto di processi biotecnologici per la produzione di biocombustibili da matrici organiche di rifiuto

The bioproduction of materials and energy from renewable sources (industrial biotechnology) is getting more and more interest in order to improve environmental sustainability of chemical industrial processes and to decrease their dependence from oil. Anaerobic digestion of organic waste matrices (agricultural and industrial wastes, organic fraction of municipal wastes, sewage sludges etc.) may play an important role in the implementation of industrial biotechnology being a well developed strategy in the valorization of complex matrices, as it can mineralize them while producing bioenergy in the form of a biogas rich in methane. In this research the potential of anaerobic digestion in the treatment of polluted sewage sludge was studied by developing three set of anaerobic microcosms with sludges differently contaminated by xenobiotic compounds. The effect of different incubating temperatures and of exogenous carbon and vitamine sources was investigated along with the role of the occurring microbial populations in the pollutant degradation activity. So, while confirming the potential of anaerobic digestion for the biomethanization of sewage sludges, this work proved the effectiveness of this technology in the removal of pollutants too. Moreover, since the degradation of lignocellulose appears to be a limiting step in the anaerobic treatment of a wide range of biomass, the possibility of optimizing anaerobic digestion of lignocellulosic substrates was also studied. To this aim a research was carried out at the BOKUUniversity of Natural Resources and Applied Life Sciences, Department for Agrobiotechnology, IFA - Tulln, where mixed cellulolytic cultures were isolated from biogas plants while assessing the metabolic pathway leading to cellulose degradation and verifying their capability to grow on lignocellulose too, proving that on the long term such bacterial cultures could be used as inoculum in order to improve the hydrolysis of lignocellulose in anaerobic digestion plants.

Ricerca, mediante tecniche di proteomica, di biomarcatori proteici utili nella valutazione della qualità degli alimenti di origine animale

In the last decades, the increase of industrial activities and of the request for the world food requirement, the intensification of natural resources exploitation, directly connected to pollution, have aroused an increasing interest of the public opinion towards initiatives linked to the regulation of food production, as well to the institution of a modern legislation for the consumer guardianship. This work was planned taking into account some important thematics related to marine environment, collecting and showing the data obtained from the studies made on different marine species of commercial interest (Chamelea gallina, Mytilus edulis, Ostrea edulis, Crassostrea gigas, Salmo salar, Gadus morhua). These studies have evaluated the effects of important physic and chemical parameters variations (temperature, xenobiotics like drugs, hydrocarbons and pesticides) on cells involved in the immune defence (haemocytes) and on some important enzymatic systems involved in xenobiotic biotransformation processes (cytochrome P450 complex) and in the related antioxidant defence processes (Superoxide dismutase, Catalase, Heat Shock Protein), from a biochemical and bimolecular point of view. Oxygen is essential in the biological answer of a living organism. Its consume in the normal cellular breathing physiological processes and foreign substances biotransformation, leads to reactive oxygen species (ROS) formation, potentially toxic and responsible of biological macromolecules damages with consequent pathologies worsening. Such processes can bring to a qualitative alteration of the derived products, but also to a general state of suffering that in the most serious cases can provoke the death of the organism, with important repercussions in economic field, in the output of the breedings, of fishing and of aquaculture. In this study it seemed interesting to apply also alternative methodologies currently in use in the medical field (cytofluorimetry) and in proteomic studies (bidimensional electrophoresis, mass spectrometry) with the aim of identify new biomarkers to place beside the traditional methods for the control of the animal origin food quality. From the results it’s possible to point out some relevant aspects from each experiment: 1. The cytofluorimetric techniques applied to O. edulis and C. gigas could bring to important developments in the search of alternative methods that quickly allows to identify with precision the origin of a specific sample, contributing to oppose possible alimentary frauds, in this case for example related to presence of a different species, also under a qualitative profile, but morpholgically similar. A concrete perspective for the application in the inspective field of this method has to be confirmed by further laboratory tests that take also in account in vivo experiments to evaluate the effect in the whole organism of the factors evaluated only on haemocytes in vitro. These elements suggest therefore the possibility to suit the cytofluorimetric methods for the study of animal organisms of food interest, still before these enter the phase of industrial working processes, giving useful information about the possible presence of contaminants sources that can induce an increase of the immune defence and an alteration of normal cellular parameter values. 2. C. gallina immune system has shown an interesting answer to benzo[a]pyrene (B[a]P) exposure, dose and time dependent, with a significant decrease of the expression and of the activity of one of the most important enzymes involved in the antioxidant defence in haemocytes and haemolymph. The data obtained are confirmed by several measurements of physiological parameters, that together with the decrease of the activity of 7-etossi-resourifine-O-deetilase (EROD linked to xenobiotic biotransformation processes) during exposure, underline the major effects of B[a]P action. The identification of basal levels of EROD supports the possible presence of CYP1A subfamily in the invertebrates, still today controversial, never identified previously in C. gallina and never isolated in the immune cells, as confirmed instead in this study with the identification of CYP1A-immunopositive protein (CYP1A-IPP). This protein could reveal a good biomarker at the base of a simple and quick method that could give clear information about specific pollutants presence, even at low concentrations in the environment where usually these organisms are fished before being commercialized. 3. In this experiment it has been evaluated the effect of the antibiotic chloramphenicol (CA) in an important species of commercial interest, Chamelea gallina. Chloramphenicol is a drug still used in some developing countries, also in veterinary field. Controls to evaluate its presence in the alimentary products of animal origin, can reveal ineffective whereas the concentration results to be below the limit of sensitivity of the instruments usually used in this type of analysis. Negative effects of CA towards the CYP1A- IPP proteins, underlined in this work, seem to be due to the attack of free radicals resultant from the action of the antibiotic. This brings to a meaningful alteration of the biotransformation mechanisms through the free radicals. It seems particularly interesting to pay attention to the narrow relationships in C. gallina, between SOD/CAT and CYP450 system, actively involved in detoxification mechanism, especially if compared with the few similar works today present about mollusc, a group that is composed by numerous species that enter in the food field and on which constant controls are necessary to evaluate in a rapid and effective way the presence of possible contaminations. 4. The investigations on fishes (Gadus morhua, and Salmo salar) and on a bivalve mollusc (Mytilus edulis) have allowed to evaluate different aspects related to the possibility to identify a biomarker for the evaluation of the health of organisms of food interest and consequently for the quality of the final product through 2DE methodologies. In the seafood field these techniques are currently used with a discreet success only for vertebrates (fishes), while in the study of the invertebrates (molluscs) there are a lot of difficulties. The results obtained in this work have underline several problems in the correct identification of the isolated proteins in animal organisms of which doesn’t currently exist a complete genomic sequence. This brings to attribute some identities on the base of the comparison with similar proteins in other animal groups, incurring in the possibility to obtain inaccurate data and above all discordant with those obtained on the same animals by other authors. Nevertheless the data obtained in this work after MALDI-ToF analysis, result however objective and the spectra collected could be again analyzed in the future after the update of genomic database related to the species studied. 4-A. The investigation about the presence of HSP70 isoforms directly induced by different phenomena of stress like B[a]P presence, has used bidimensional electrophoresis methods in C. gallina, that have allowed to isolate numerous protein on 2DE gels, allowing the collection of several spots currently in phase of analysis with MALDI-ToF-MS. The present preliminary work has allowed therefore to acquire and to improve important methodologies in the study of cellular parameters and in the proteomic field, that is not only revealed of great potentiality in the application in medical and veterinary field, but also in the field of the inspection of the foods with connections to the toxicology and the environmental pollution. Such study contributes therefore to the search of rapid and new methodologies, that can increase the inspective strategies, integrating themselves with those existing, but improving at the same time the general background of information related to the state of health of the considered animal organism, with the possibility, still hypothetical, to replace in particular cases the employment of the traditional techniques in the alimentary field.

Manipolazione del metabolismo degli xenobiotici da frutta convenzionale ed attività chemiopreventiva

A reduced cancer risk associated with fruit and vegetable phytochemicals initially dictated chemopreventive approaches focused on specific green variety consumption or even single nutrient supplementations. However, these strategies not only failed to provide any health benefits but gave rise to detrimental effects. In parallel, public-health chemoprevention programmes were developed in the USA and Europe to increase whole vegetable consumption. Among these, the National Cancer Institute (NCI) sponsored plan “5 to 9 a day for a better health” was one of the most popular. This campaign promoted wide food choice through the consumption of at least 5 to 9 servings a day of colourful fruits and vegetables. In this study the effects of the diet suggested by NCI on transcription, translation and catalytic activity of both xenobiotic metabolizing (XME) and antioxidant enzymes were studied in the animal model. In fact, the boost of both antioxidant defences and “good” phase-II together with down-regulation of “bad” phase-I XMEs is still considered one of the most widely-used strategies of cancer control. Six male Sprague Dawley rats for each treatment group were used. According to the Italian Society of Human Nutrition, a serving of fruit, vegetables and leafy greens corresponds to 150, 250 and 50 g, respectively, in a 70 kg man. Proportionally, rats received one or five servings of lyophilized onion, tomato, peach, black grape or lettuce – for white, red, yellow, violet or green diet, respectively - or five servings of each green (“5 a day” diet) by oral gavage daily for 10 consecutive days. Liver subcellular fractions were tested for various cytochrome P450 (CYP) linked-monooxygenases, phase-II supported XMEs such as glutathione S-transferase (GST) and UDP-glucuronosyl transferase (UDPGT) as well as for some antioxidant enzymes. Hepatic transcriptional and translational effects were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. dROMs test was used to measure plasmatic oxidative stress. Routine haematochemical parameters were also monitored. While the five servings administration didn’t significantly vary XME catalytic activity, the lower dose caused a complex pattern of CYP inactivation with lettuce exerting particularly strong effects (a loss of up to 43% and 45% for CYP content and CYP2B1/2-linked XME, respectively; P<0.01). “5 a day” supplementation produced the most pronounced modulations (a loss of up to 60% for CYP2E1-linked XME and a reduction of CYP content of 54%; P<0.01). Testosterone hydroxylase activity confirmed these results. RT-PCR and Western blot analysis revealed that the “5 a day” diet XMEs inactivations were a result of both a transcriptional and a translational effect while lettuce didn’t exert such effects. All administrations brought out none or fewer modulation of phase-II supported XMEs. Apart from “5 a day” supplementation and the single serving of lettuce, which strongly induced DT- diaphorase (an increase of up to 141 and 171%, respectively; P<0.01), antioxidant enzymes were not significantly changed. RT-PCR analysis confirmed DT-diaphorase induction brought about by the administration of both “5 a day” diet and a single serving of lettuce. Furthermore, it unmasked a similar result for heme-oxygenase. dROMs test provided insight into a condition of high systemic oxidative stress as a consequence of animal diet supplementation with “5 a day” diet and a single serving of lettuce (an increase of up to 600% and 900%, respectively; P<0.01). Haematochemical parameters were mildly affected by such dietary manipulations. According to the classical chemopreventive theory, these results could be of particular relevance. In fact, even if antioxidant enzymes were only mildly affected, the phase-I inactivating ability of these vegetables would be a worthy strategy to cancer control. However, the recorded systemic considerable amount of reactive oxygen species and the complexity of these enzymes and their functions suggest caution in the widespread use of vegan/vegetarian diets as human chemopreventive strategies. In fact, recent literature rather suggests that only diets rich in fruits and vegetables and poor in certain types of fat, together with moderate caloric intake, could be associated with reduced cancer risk.

Effetto dei modulatori del sistema serotoninergico sulla trasduzione del segnale AMPc-dipendente nel mitilo

Lo sviluppo della medicina ha determinato un utilizzo sempre crescente di sostanze farmacologiche, le quali una volta escrete dagli organismi raggiungono le acque dei fiumi e dei laghi, per arrivare poi all’ambiente marino costiero, che ne risulta sempre maggiormente contaminato. Negli organismi non bersaglio esposti ai residui dei farmaci in ambiente, queste sostanze potrebbero indurre effetti simili a quelli specifici nel caso i bersagli molecolari siano stati conservati durante l’evoluzione, oppure avere effetti inattesi se i bersagli molecolari sono conservati ma hanno una differente funzione. Questo lavoro di tesi è volto a studiare i potenziali effetti indotti dalla fluoxetina (FX, farmaco antidepressivo inibitore dell’uptake della serotonina), e dal propranololo (PROP, farmaco bloccante sia dei recettori β-adrenergici che serotoninergici nell’uomo) nei mitili Mytilus galloprovincialis, esposti a tali sostanze, valutandone l’interazione con i meccanismi di trasduzione del segnale AMPc-dipendente. Sono stati valutati in particolare i livelli di AMPc e l’attività dell’enzima PKA, inoltre si è studiato se i farmaci influiscano con i meccanismi di regolazione del gene ABCB1, che codifica per la P-glicoproteina (Pgp), che ha il compito di estrudere all’esterno della cellula gli xenobiotici che vi sono entrati. Gli studi sono stati condotti dopo esposizione dei mitili in vivo ai due farmaci ed alla loro miscela per 7 giorni in acquario. I risultati hanno indicato che la FX causa una diminuzione statisticamente significativa dei livelli di AMPc, dell’attività della PKA e anche dell’espressione del gene ABCB1 rispetto al controllo, sia nel mantello che nella ghiandola digestiva. Nella ghiandola digestiva il PROP provoca una significativa riduzione dei livelli di AMPc, dell’attività della PKA e dell’espressione del gene ABCB1 rispetto ai valori di controllo. Nel mantello, invece, il PROP aumenta i livelli di AMPc e l’espressione del gene ABCB1, anche se non ha effetti significativi sull’attività della PKA. Per caratterizzare i recettori per la serotonina (5HT), e il possibile ruolo di antagonista giocato dal PROP, abbiamo inoltre trattato in vitro emociti di mitilo con la 5HT l’agonista fisiologico del recettore, usata da sola ed in presenza del PROP. I dati ottenuti dimostrano che negli emociti di mitilo sono espressi recettori 5HT1 accoppiati a proteine G inibitrici, e che il PROP blocca l’effetto della 5HT, agendo come antagonista dei recettori 5HT1. Nell’insieme i dati dimostrano che i farmaci possono avere effetti sugli organismi acquatici anche a concentrazioni molto basse come quelle ambientali. I dati della tesi non dimostrano che PROP e FX hanno effetti deleteri sulle popolazioni o le comunità dei molluschi, ma debbono essere considerati come indicatori della vulnerabilità degli animali a questi composti. Si è dimostrato per la prima volta che gli emociti di mitilo possiedono recettori di tipo 5HT1 correlati alla riduzione dei livelli intracellulari di AMPc, e soprattutto che il sistema AMPc/PKA è deputato alla regolazione dell’espressione dei geni ABCB1 codificanti per proteine del complesso Multi Xenobiotic Resistance.

Endogene DNA-Schädigung als Ursache genetischer Instabilität

Zusammenfassung Diese Arbeit beschreibt Untersuchungen über die zellulären Mechanismen, die zur Bildung dieser DNA-Schäden führen, sowie über die biologischen Auswirkungen dieser Schäden. Die Untersuchungen zu Uracil in der DNA wurden in ung-knockout-MEFs und Mäusen durchgeführt, die es erlauben, die Konsequenzen eines Ausfalls der wichtigsten Reparaturglykosylase für Uracil zu beleuchten. Die Ergebnisse zeigen eine deutliche Akkumulation von Uracil in den ung-/--Mausfibroblasten im Vergleich zum Wildtyp. In frisch isolierten Leber- und Milzzellen der Mäuse konnte dieser genotypspezifische Unterschied, wenn auch weniger ausgeprägt, ebenso beobachtet werden, nicht jedoch in reifen Spermien. Dieser gewebespezifische Unterschied und die quantitativ stärker ausgeprägte Akkumulation in ung-/--Mausfibroblasten im Vergleich zu den Mäusegeweben gab Anlass zur Vermutung, dass die Proliferation der Zellen für den Haupteintrag an Uracil in die DNA verantwortlich ist. Erstmals konnte in Versuche mit konfluenten (nicht mehr proliferierenden) ung-/--Mausfibroblasten gezeigt werden, dass nicht die spontane hydrolytische Desaminierung von Cytosin, sondern der Fehleinbau von dUMP während der DNA-Replikation die Hauptquelle für Uracil in der DNA von Säugerzellen darstellt. Da der Uracilmetabolismus ein wichtiges Target in der Chemotherapie ist, lag es nahe, das zur Verfügung stehende ung-knockout-Modell der MEFs zur Untersuchung mit Fluorpyrimidinen, die als Zytostatika verwendet werden, einzusetzen. Da bisher die Ursachen der beobachteten Apoptose der Tumorzellen und aller anderen metabolisch hochaktiven Zellen eines behandelten Organismus noch nicht vollständig verstanden ist, wurden diese Zellen mit verschiedenen Fluorpyrimidinen behandelt, die als Thymidylatsynthasehemmer die de novo Synthese von Thymidin unterbinden. Es konnte gezeigt werden, dass ung-/- Mausfibroblasten, im Gegensatz zu ung+/+ Mausfibroblasten, verstärkt Uracil in der DNA akkumulieren. Obwohl die ung+/+ Mausfibroblasten keine erhöhten Uracil-Spiegel in der DNA aufwiesen, zeigten sie bei Inkubation mit einem der beiden Thymidylatsynthasehemmern, 5-Fluoruracil (5-FU), die gleiche Sensitivität in einem nachfolgenden Proliferationsversuch wie die ung-/- Mausfibroblasten. Dies lässt darauf schließen, dass weder Reparatur noch Einbau von Uracil in die DNA für die beobachtete Toxizität dieser Zytostatika notwendig sind. Ein weiterer Schwerpunkt dieser Arbeit war die Untersuchung des DNA-schädigenden Potenzials endogener ROS, die aus dem Fremdstoffmetabolismus stammen. Dazu wurden V79-Zellen verwendet, die mit dem humanen Enzym Cytochrom 2E1 (CYP2E1) transfiziert wurden (V79 CYP2E1) sowie Zellen, die ebenfalls durch Transfektion das humane Enzym Cytochromreduktase (auch Oxidoreduktase genannt) überexprimieren (V79 hOR). Beide Enzyme sind zusammen an der Hydroxylierung von Fremdstoffen beteiligt, bei der die Reduktion von molekularem Sauerstoff durch Übertragung von zwei Elektronen notwendig ist. Wird anstatt zweier Elektronen in Folge nur eines auf den Sauerstoff übertragen, so führt dieser von der Substratoxygenierung enkoppelte Vorgang zur Bildung von Superoxid. Daher galt es zu klären, ob das so erzeugte Superoxid und daraus gebildete ROS in der Lage sind, die DNA zu schädigen. Es konnte gezeigt werden, dass die Überexpression von CYP2E1 nicht zu einem erhöhten basalen Gleichgewichtsspiegel oxidativer DNA-Schäden führt und die Metabolisierung von Ethanol durch dieses Enzym ebenfalls keine DNA-Modifikationen verursacht. Die Überexpression der Cytochromreduktase hingegen führte gegenüber dem Wildtyp zu einem erhöhten basalen Gleichgewichtsspiegel oxidativer Basenmodifikationen nach Depletion von Glutathion, einem wichtigen zellulären Antioxidans. Im Mikrokerntest, der gentoxische Ereignisse wie Chromosomenbrüche in Zellen aufzeigt, zeigte sich schon ohne Glutathion-Depletion eine doppelt so hohe Mikrokernrate im Vergleich zum Wildtyp. In weiteren Versuchen wurden die V79-hOR-Zellen mit dem chinoiden Redoxcycler Durochinon inkubiert, um zu untersuchen, ob das vermutlich durch die Reduktase vermittelte Redoxcycling über Generierung von ROS in der Lage ist, einen oxidativen DNA-Schaden und Toxizität zu verursachen. Hier zeigte sich, dass die Überexpression der Reduktase Voraussetzung für Toxizität und den beobachteten DNA-Schaden ist. Die Wildtyp-Zellen zeigten weder einen DNA-Schaden noch Zytotoxizität, auch eine zusätzliche Glutathion-Depletion änderte nichts an dem Befund. Die V79-hOR-Zellen hingegen reagierten auf die Inkubation mit Durochinon mit einer konzentrationsabhängigen Zunahme der Einzelstrangbrüche und oxidativen Basenmodifikationen, wobei sich der DNA-Schaden durch vorherige Glutathion-Depletion verdoppeln ließ.

Differential cell type-specific transcriptional regulation of the CYP1A1 gene

Cytochrome P450 1A1 (CYP1A1) monooxygenase plays an important role in the metabolism of environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) and halogenated polycyclic aromatic hydrocarbons (HAHs). Oxidation of these compounds converts them to the metabolites that subsequently can be conjugated to hydrophilic endogenous entities e.g. glutathione. Derivates generated in this way are water soluble and can be excreted in bile or urine, which is a defense mechanism. Besides detoxification, metabolism by CYP1A1 may lead to deleterious effects since the highly reactive intermediate metabolites are able to react with DNA and thus cause mutagenic effects, as it is in the case of benzo(a) pyrene (B[a]P). CYP1A1 is normally not expressed or expressed at a very low level in the cells but it is inducible by many PAHs and HAHs e.g. by B[a]P or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Transcriptional activation of the CYP1A1 gene is mediated by aryl hydrocarbon receptor (AHR), a basic-helix-loop-helix (bHLH) transcription factor. In the absence of a ligand AHR stays predominantly in the cytoplasm. Ligand binding causes translocation of AHR to the nuclear compartment, its heterodimerization with another bHLH protein, the aryl hydrocarbon nuclear translocator (ARNT) and binding of the AHR/ARNT heterodimer to a DNA motif designated dioxin responsive element (DRE). This process leads to the transcriptional activation of the responsive genes containing DREs in their regulatory regions, e.g. that coding for CYP1A1. TCDD is the most potent known agonist of AHR. Since it is not metabolized by the activated enzymes, exposure to this compound leads to a persisting activation of AHR resulting in diverse toxic effects in the organism. To enlighten the molecular mechanisms that mediate the toxicity of xenobiotics like TCDD and related compounds, the AHR-dependent regulation of the CYP1A1 gene was investigated in two cell lines: human cervix carcinoma (HeLa) and mouse hepatoma (Hepa). Study of AHR activation and its consequence concerning expression of the CYP1A1 enzyme confirmed the TCDD-dependent formation of the AHR/ARNT complex on DRE leading to an increase of the CYP1A1 transcription in Hepa cells. In contrast, in HeLa cells formation of the AHR/ARNT heterodimer and binding of a protein complex containing AHR and ARNT to DRE occurred naturally in the absence of TCDD. Moreover, treatment with TCDD did not affect the AHR/ARNT dimer formation and binding of these proteins to DRE in these cells. Even though the constitutive complex on DRE exists in HeLa, transcription of the CYP1A1 gene was not increased. Furthermore, the CYP1A1 level in HeLa cells remained unchanged in the presence of TCDD suggesting repressional mechanism of the AHR complex function which may hinder the TCDD-dependent mechanisms in these cells. Similar to the native, the mouse CYP1A1-driven reporter constructs containing different regulatory elements were not inducible by TCDD in HeLa cells, which supported a presence of cell type specific trans-acting factor in HeLa cells able to repress both the native CYP1A1 and CYP1A1-driven reporter genes rather than species specific differences between CYP1A1 genes of human and rodent origin. The different regulation of the AHR-mediated transcription of CYP1A1 gene in Hepa and HeLa cells was further explored in order to elucidate two aspects of the AHR function: (I) mechanism involved in the activation of AHR in the absence of exogenous ligand and (II) factor that repress function of the exogenous ligand-independent AHR/ARNT complex. Since preliminary studies revealed that the activation of PKA causes an activation of AHR in Hepa cells in the absence of TCDD, the PKA-dependent signalling pathway was the proposed endogenous mechanism leading to the TCDD-independent activation of AHR in HeLa cells. Activation of PKA by forskolin or db-cAMP as well as inhibition of the kinase by H89 in both HeLa and Hepa cells did not lead to alterations in the AHR interaction with ARNT in the absence of TCDD and had no effect on binding of these proteins to DRE. Moreover, the modulators of PKA did not influence the CYP1A1 activity in these cells in the presence and in the absence of TCDD. Thus, an involvement of PKA in the regulation of the CYP1A1 Gen in HeLa cells was not evaluated in the course of this study. Repression of genes by transcription factors bound to their responsive elements in the absence of ligands has been described for nuclear receptors. These receptors interact with protein complex containing histone deacetylase (HDAC), enzyme responsible for the repressional effect. Thus, a participation of histone deacetylase in the transcriptional modulation of CYP1A1 gene by the constitutively DNA-bound AHR/ARNT complex was supposed. Inhibition of the HDAC activity by trichostatin A (TSA) or sodium butyrate (NaBu) led to an increase of the CYP1A1 transcription in the presence but not in the absence of TCDD in Hepa and HeLa cells. Since amount of the AHR and ARNT proteins remained unchanged upon treatment of the cells with TSA or NaBu, the transcriptional upregulation of CYP1A1 gene was not due to an increased expression of the regulatory proteins. These findings strongly suggest an involvement of HDAC in the repression of the CYP1A1 gene. Similar to the native human CYP1A1 also the mouse CYP1A1-driven reporter gene transfected into HeLa cells was repressed by histone deacetylase since the presence of TSA or NaBu led to an increase in the reporter activity. Induction of reporter gene did not require a presence of the promoter or negative regulatory regions of the CYP1A1 gene. A promoter-distal fragment containing three DREs together with surrounding sequences was sufficient to mediate the effects of the HDAC inhibitors suggesting that the AHR/ARNT binding to its specific DNA recognition site may be important for the CYP1A1 repression. Histone deacetylase is recruited to the specific genes by corepressors, proteins that bind to the transcription factors and interact with other members of the HDAC complex. Western blot analyses revealed a presence of HDAC1 and the corepressors mSin3A (mammalian homolog of yeast Sin3) and SMRT (silencing mediator for retinoid and thyroid hormone receptor) in both cell types, while the corepressor NCoR (nuclear receptor corepressor) was expressed exclusively in HeLa cells. Thus the high inducibility of CYP1A1 in Hepa cells may be due to the absence of NCoR in these cells in contrast to the non-responsive HeLa cells, where the presence of NCoR would support repression of the gene by histone deacetylase. This hypothesis was verified in reporter gene experiments where expression constructs coding for the particular members of the HDAC complex were cotransfected in Hepa cells together with the TCDD-inducible reporter constructs containing the CYP1A1 regulatory sequences. An overexpression of NCoR however did not decrease but instead led to a slight increase of the reporter gene activity in the cells. The expected inhibition was observed solely in the case of SMRT that slightly reduced constitutive and TCDD-induced reporter gene activity. A simultaneous expression of NCoR and SMRT shown no further effects and coexpression of HDAC1 with the two corepressors did not alter this situation. Thus, additional factors that are likely involved in the repression of CYP1A1 gene by HDAC complex remained to be identified. Taking together, characterisation of an exogenous ligand independent AHR/ARNT complex on DRE in HeLa cells that repress transcription of the CYP1A1 gene creates a model system enabling investigation of endogenous processes involved in the regulation of AHR function. This study implicates HDAC-mediated repression of CYP1A1 gene that contributes to the xenobiotic-induced expression in a tissue specific manner. Elucidation of these processes gains an insight into mechanisms leading to deleterious effects of TCDD and related compounds.

Alterazioni del sistema di trasduzione AMPc-dipendente nel mitilo mediterraneo Mytilus galloprovincialis esposto a fluoxetina

Negli ultimi decenni le nuove scoperte mediche e il miglioramento dello stile di vita nei paesi occidentali hanno determinato un aumento del consumo di sostanze terapeutiche, nonché della gamma di prodotti farmaceutici e parafarmaceutici comunemente utilizzati. Gli studi di cinetica dei farmaci dimostrano con certezza che essi, seppur metabolizzati, mantengono inalterato il loro potere d’azione, rimanendo biodisponibili anche una volta escreti dall’organismo. A causa della loro eccessiva polarità tali molecole sono inoltre difficilmente trattenute dai convenzionali impianti di depurazione dei reflui urbani, dai quali confluiscono inevitabilmente verso le acque fluviali e/o costiere le quali risultano. In ragione di ciò, la valutazione degli effetti che la loro presenza può provocare sulla qualità dei sistemi di approvigionamento idrico e sulla biologia delle specie tipiche degli ecosistemi acquatici, ha classificato tali composti come una nuova classe di inquinanti emergenti, il cui impatto ambientale non risulta ancora del tutto arginato attraverso adeguate contromisure legislative. I farmaci sono sostanze bioattive progettate per avere effetti specifici a bassissime concentrazioni negli organismi target attraverso specifici meccanismi d’azione. Nel caso in cui i bersagli cellulari su cui agiscono siano evolutivamente conservati negli organismi non target, essi possono esercitare le proprie funzioni attraverso i medesimi meccanismi di regolazione fisiologica attivati nelle specie target, dando origine a effetti specifici, o anche aspecifici, nel caso in cui tali bersagli siano deputati alla regolazioni di funzioni differenti. Pertanto lo scopo del presente lavoro di tesi è stato quello di analizzare le possibili alterazioni di carattere fisiologico in individui di mitilo mediterraneo (Mytilus galloprovincialis) esposti a concentrazioni ambientali di fluoxetina, farmaco antidepressivo appartenente alla classe degli inibitori selettivi del riassorbimento presinaptico della serotonina (SSRI). Più nel dettaglio, a seguito di un’esposizione per 7 giorni a dosaggi compresi tra 0.03 e 300 ng/L di FX, sono stati analizzati i livelli intracellulari di AMPc e l’attività dell’enzima PKA nei diversi trattamenti sperimentali effettuati. Inoltre sono stati valutati i livelli di espressione genica del recettore serotoninergico 5HTmyt1 e della la P-glicoproteina (Pgp, gene ABCB1), trasportatore di membrana responsabile del sistema di detossificazione noto come Multi-xenobiotic resistance (MXR). Nella ghiandola digestiva, la FX causa una diminuzione statisticamente significativa dei livelli di AMPc, dell’attività della PKA e dell’espressione del gene ABCB1 rispetto al controllo. Al contrario nel mantello il farmaco non produce alterazioni dei livelli intracellulari di AMPc e dell’attività della PKA mentre si apprezza una sottoespressione del gene ABCB1 nei trattamenti a dosaggi intermedi. In entrambi i tessuti si nota un aumento dell’espressione genica di 5HTmyt1 alle minori concentrazioni di FX, mentre ai dosaggi più alti non si registrano alterazioni significative rispetto al controllo. Nel loro insieme i risultati indicano che nei mitili, concentrazioni ambientali di FX producono significative alterazioni di diversi parametri fisiologici attraverso una modulazione specifica dei medesimi bersagli molecolari coinvolti nella terapia umana. La riduzione dei livelli di AMPc/PKA apprezzata nella ghiandola digestiva risulta in linea con la funzione inibitoria svolta dal recettore 5HTmyt1 su tale via di trasduzione, mentre l’assenza di variazioni significative registrata nel mantello supporta l’ipotesi di un’interazione tra il sistema serotoninergico e catecolaminergico nella regolazione dei processi legati al ciclo riproduttivo che si verificano in tale tessuto. In conclusione, i dati dimostrano che l’espressione del gene codificante la proteina Pgp è regolata dalla FX attraverso uno specifico meccanismo d'azione AMPc-dipendente modulato dalla serotonina; tuttavia, è ipotizzabile anche un effetto non specifico indotto dalla FX stessa, per esempio attraverso l’induzione di stress ossidativo. Inoltre essi evidenziano la presenza di un meccanismo di regolazione retroattivo sulla espressione dei recettori 5HTmyt1 in funzione delle concentrazioni extracellulari di serotonina modulate dall’azione della FX.

Regulation der Stress-aktivierten Proteinkinasen durch den gentoxischen Benzo[a]pyren-Metaboliten BPDE

Polyzyklische aromatische Kohlenwasserstoffe (PAK) sind ubiquitäre Verschmutzungen der Umwelt und entstehen während der unvollständigen Verbrennung organischen Materials wie Holz, Kohle und Erdöl. Werden diese chemisch nicht reaktiven PAK in den Körper aufgenommen, durchlaufen sie eine Reihe von enzymatischen Umsetzungen, die unter der Bezeichnung Fremdstoffmetabolismus zusammengefasst werden. Die chemische Umsetzung des PAK und Prokarzinogens Benzo[a]pyren (B[a]P) führt u.a. zur Bildung des reaktiven Metaboliten B[a]P-7,8-dihydrodiol-9,10-epoxid (BPDE). BPDE ist stark elektrophil und kann auf Grund dieser Eigenschaft an nukleophile Makromoleküle wie Proteine und DNA binden. Die Bildung von BPDE-DNA-Addukten resultiert in der Entstehung von Mutationen und kann zur Tumorbildung führen. Im Rahmen der vorliegenden Arbeit sollte die Wirkung von BPDE als Modellsubstanz für gentoxische Agenzien auf intrazelluläre Signalkaskaden und die Konsequenzen der BPDE-Exposition bezüglich der Zellaktivität untersucht werden. Es konnte gezeigt werden, dass BPDE-Behandlung von Mausfibroblasten eine intrazelluläre Signalkaskade induziert, welche zur Aktivierung der Stressaktivierten Proteinkinasen (SAPK) JNK und p38 führt. An dieser Signalkaskade sind Src-ähnliche Kinasen beteiligt. BPDE-Behandlung führt in den untersuchten Mausfibroblasten zur Induktion von DNA-Einzelstrangbrüchen, deren Auftreten zeitlich mit der SAPK-Aktivierung korreliert. Die BPDEinduzierten DNA-Strangbrüche sind die Folge der Entfernung dieser Läsionen aus dem Genom durch die Nukleotidexzisionsreparatur (NER). Erkannt werden BPDE-DNA-Addukte durch die NERProteine XPA und XPC (Xeroderma Pigmentosum Komplementationsgruppe A und C). Nach der Erkennung von BPDE-DNA-Addukten kommt es zur Rekrutierung von Nukleasen, welche die vorliegende Läsion und umliegende Nukleotide aus dem Genom entfernen. In XPA- und XPCdefizienten Mausfibroblasten induziert BPDE daher keine DNA-Strangbrüche. Jedoch ist nur in XPCdefizienten Zellen, aber nicht in XPA-defizienten Zellen, die SAPK-Aktivierung drastisch reduziert. Behandlung von Mausfibroblasten mit Benzo[c]phenanthren-3,4-Diol-1,2-Epoxid, einem PAK, dessen DNA-Addukte schlecht durch NER-Faktoren erkannt und repariert werden, führt zu keiner SAPKAktivierung. Die Aktivierung von p38 und JNK scheint demnach abhängig zu sein von der Erkennung des primären DNA-Schadens. Die XPC-abhängige SAPK-Aktivierung schützt die Zellen vor BPDEabhängiger Toxizität, da sowohl XPC- als auch p38-defiziente Mausfibroblasten eine höhere Sensitivität gegenüber BPDE zeigen als korrespondierende Wildtypzellen. Zusamenfassend konnte in dieser Arbeit ein neuer Signalweg beschrieben werden, in dem DNASchäden, verursacht durch BPDE, über die XPC-abhängige DNA-Schadenserkennung, die Aktivierung der SAPK induziert. Diese Aktivierung der SAPK schützt vor BPDE-induzierter Toxizität.

Studio degli effetti funzionali e tossici di derivati di Brassicaceae in modelli sperimentali

I vegetali appartenenti alla famiglia delle Brassicaceae, sono ricchi di molecole biologicamente attive note per le numerose proprietà salutari. L’effetto di un estratto di germogli di cavolo nero toscano (TBCSE) è stato investigato, in termini chemiopreventivi, sugli enzimi epatici del metabolismo degli xenobiotici e antiossidanti, in ratti trattati con TBCSE. I risultati hanno mostrato un complesso pattern di modulazione, con una prevalente inibizione, del sistema citocromo P450-dipendente, e induzioni significative degli enzimi di fase II (glutatione transferasi e glucuronosiltransferasi) e antiossidanti (catalasi, NAD(P)H:chinone reduttasi, glutatione reduttasi e perossidasi). Successivamente, l’effetto di TBCSE è stato studiato nei confronti delle alterazioni provocate da un’alimentazione iperlipidica nel ratto. Il trattamento si è dimostrato efficace nel contrastare gli effetti deleteri dei grassi presenti nella dieta, come l’iperlipidemia, l’aumento del peso corporeo e del fegato, l’indebolimento delle attività degli enzimi antiossidanti e del potenziale detossificante a livello epatico. Complessivamente, TBCSE emerge essere un promettente prodotto nutraceutico con potenziali effetti chemiopreventivi, e da impiegare come strategia alimentare per contrastare gli effetti correlati ad una dieta iperlipidica. Il consumo di dosi sovralimentari di molecole isolate dalle Brassicaceae, tramite per esempio integratori dietetici, come strategia alimentare preventiva, potrebbe tuttavia rappresentare un rischio per la salute. La potenziale tossicità del sulforafane, glucorafanina, indolo-3-carbinolo, e 3,3'-diindolimetano, è stata valutata in epatociti primari di ratto. La citotossicità e l’induzione di stress ossidativo, osservate a concentrazioni non lontane da quelle che potrebbero essere raggiunte in vivo, insieme ad una forte modulazione dell’espressione genica, riguardante principalmente il metabolismo degli xenobiotici, risposte ad alterazioni dello stato ossidoredutivo, eventi di riparazione del DNA e di proteine, induzione dell’apoptosi, e meccanismi (co)cancerogeni, sottolineano la potenzialità di queste molecole di determinare un rischio tossicologico, in seguito ad un’assunzione prolungata e ad alte dosi.

Einfluss der Modulation von P-Glykoprotein (P-gp) auf die Verfügbarkeit und Wirksamkeit der pharmakologischen Behandlung psychiatrischer Erkrankungen im Tiermodell

P-Glykoprotein (P-gp) ist ein ATP-verbrauchender Transporter, der in Organschranken exprimiert wird, um Fremdstoffe auszuschleusen, darunter auch Psychopharmaka. Im Rahmen dieser Arbeit wurde im Tiermodell der Maus untersucht, welche pharmakokinetischen und pharmakodynamischen Konsequenzen sich bei Verabreichung von Risperidon als P-gp Modellsubstrat ergeben, wenn die Expression von P-gp induziert wird. Als potenzielle Induktoren wurden Dexamethason, Rifampicin, Quercetin, 5-Pregnen-3ß-ol-20-on-16α-Carbonitril (PCN) und Acitretin geprüft. Es konnte gezeigt werden, dass alle Substanzen die Verteilung von Risperidon und seinem aktiven Metaboliten 9-Hydroxyrisperidon beeinflussten. Während sich für Quercetin und Acitretin leichte P-gp inhibitorische Eigenschaften ergaben, die an Hand von erhöhten Konzentrationen von Risperidon und 9-Hydroxyrisperidon gezeigt werden konnten, führten die bekannten P-gp Induktoren Rifampicin, Dexamethason und PCN zu verringerten Konzentrationen im Vergleich zur Kontrollgruppe. Durch Western Blot Untersuchungen wurde bestätigt, dass die Induktoren die P-gp Expression im Hirngewebe tendenziell steigerten. Dies sprach dafür, dass bei Verabreichung einer Komedikation, die P-gp induziert, mit einer veränderten Verteilung von P-gp Substraten zu rechnen ist. Darüber hinaus konnte nachgewiesen werden, dass durch eine Hemmung bzw. Induktion von P-gp nicht nur die Pharmakokinetik, sondern auch die Pharmakodynamik von Risperidon und 9-Hydroxyrisperidon verändert wird. Dies wurde durch verhaltenspharmakologische Untersuchungen gezeigt. Durch Risperidon induzierte motorische Effekte auf dem RotaRod waren nach Induktion von P-gp abgeschwächt. Dies zeigte sich auch für Haloperidol, welches kein Substrat ist. Da P-gp abhängige Effekte in diesem Fall keine bedeutende Rolle spielen, ist davon auszugehen, dass neben der Induktion von P-gp an der Blut-Hirn Schranke auch andere Mechanismen wie z.B. eine Induktion von Enzymen der CYP-Familie an den beobachteten Effekten beteiligt sind. Bei Untersuchungen von kognitiven Leistungen in der Barnes Maze konnte gezeigt werden, dass Haloperidol im Gegensatz zu Risperidon das Lernverhalten negativ beeinflussen kann. Eine P-gp Induktion schien jedoch keinen deutlichen Einfluss auf das Lernverhalten unter Antipsychotika-Gabe zu haben und sprach vielmehr für substanzabhängige Effekte der einzelnen Antipsychotika bzw. P-gp Modulatoren. Zusatzuntersuchungen zur Hirngängigkeit von Acitretin, einem synthetischen Retinoid, welches derzeit als potenzielles Antidementivum geprüft wird, konnten belegen, dass es die Blut-Hirn Schranke überwindet. Bereits 1h nach Injektion war Acitretin in hoher Konzentration im Gehirn nachweisbar. Durch die Analyse zur Verteilung von Acitretin in Hirngewebe und Serum von P-gp Wildtyp und P-gp doppel knockout Mäusen konnte belegt werden, dass Acitretin nicht P-gp abhängig transportiert wird. Die Daten insgesamt betrachtet, lassen den Schluss zu, dass durch Verabreichung von Medikamenten, die P-gp Modulatoren sind, bei Antipsychotika mit pharmakokinetischen Interaktionen zu rechnen ist, welche die Wirksamkeit der Medikamente einschränken können.

In vivo and in vitro investigation of the tissue-specific activity of the human CYP3A4 and CYP3A5 promoters

The human cytochrome P450 3A4 (CYP3A4), the predominant but variably expressed cytochrome P450 in adult liver and small intestine is involved in the metabolism of over 50% of currently used drugs. Its paralog CYP3A5 plays a crucial role in the disposition of several drugs with low therapeutic index, including tacrolimus. Limited information is available for the CYP3A5 transcriptional regulation and its induction by xenobiotics remains controversial. In the first part of this study, we analysed the CYP3A5 transcriptional regulation and its induction by xenobiotics in vivo using transgenic mice. To this end, two transgenic strains were established by pronuclear injection of a plasmid, expressing firefly luciferase driven by a 6.2 kb of the human CYP3A5 promoter. A detailed analysis of both strains shows a tissue distribution largely reflecting that of CYP3A5 transcripts in humans. Thus, the highest luciferase activity was detected in the small intestine, followed by oesophagus, testis, lung, adrenal gland, ovary, prostate and kidney. However, no activity was observed in the liver. CYP3A5-luc transgenic mice were similarly induced in both sexes with either PCN or TCPOBOP in small intestine in a dose-dependent manner. Thus, the 6.2 kb upstream promoter of CYP3A5 mediates the broad tissue activity in transgenic mice. CYP3A5 promoter is inducible in the small intestine in vivo, which may contribute to the variable expression of CYP3A in this organ. rnThe hepato-intestinal level of the detoxifying oxidases CYP3A4 and CYP3A5 is adjusted to the xenobiotic exposure mainly via the xenosensor and transcriptional factor PXR. CYP3A5 is additionally expressed in several other organs lacking PXR, including kidney. In the second part of this study, we investigated the mechanism of the differential expression of CYP3A5 and CYP3A4 and its evolutionary origin using renal and intestinal cells, and comparative genomics. For this examination, we established a two-cell line models reflecting the expression relationships of CYP3A4 and CYP3A5 in the kidney and small intestine in vivo. Our data demonstrate that the CYP3A5 expression in renal cells was enabled by the loss of a suppressing Yin Yang 1 (YY1)-binding site from the CYP3A5 promoter. This allowed for a renal CYP3A5 expression in a PXR-independent manner. The YY1 element is retained in the CYP3A4 gene, leading to its suppression, perhaps via interference with the NF1 activity in renal cells. In intestinal cells, the inhibition of CYP3A4 expression by YY1 is abrogated by a combined activating effect of PXR and NF1 acting on their respective response elements located adjacent to the YY1-binding site on CYP3A4 proximal promoter. CYP3A4 expression is further facilitated by a point mutation attenuating the suppressing effect of YY1 binding site. The differential expression of CYP3A4 and CYP3A5 in these organs results from the loss of the YY1 binding element from the CYP3A5 promoter, acting in concert with the differential organ expression of PXR, and with the higher accumulation of PXR response elements in CYP3A4. rn

Unmittelbare, dauerhafte sowie evolutionäre Anpassung an Fremdstoffexpositionen

Eine der Hauptursachen für unerwünschte oder reduzierte Wirkungen von Medikamenten ist die Induktion von Enzymen und Transportern des Medikamentenstoffwechsels. Diese Induktion stellt ursprünglich eine physiologische Reaktion auf die Aufnahme von potentiell schädlichen Fremdstoffen aus der Umwelt dar und sichert so die Gesundheit und Fortpflanzungsfähigkeit von Lebewesen. Beim Menschen sowie anderen Säugetieren werden Fremdstoffe hauptsächlich von den nukleären Rezeptoren PXR und CAR in der Leber und im Dünndarm detektiert. Zu den Medikamenten, welche über PXR und CAR wirken, gehören unter anderem Antikonvulsiva, Statine, antiretrovirale Medikamente, Glucocorticoide sowie Antimykotika. Die durch Fremdstoffe aktivierten Transkriptionsfaktoren PXR und CAR steigern die Menge der Enzyme und Transporter des Fremdstoffmetabolismus. Hierzu zählen vor allem die Cytochrom P450-Enzyme (Cyp-Enzyme) mit breitem Substratspektrum oder der Transporter MDR1, welcher eine Vielzahl von Substraten über Membranen transportiert. Durch die Biotransformation werden die induzierenden, lipophilen Substanzen so modifiziert, dass sie leichter über den Urin oder die Galle ausgeschieden werden können. \r\nDie Dauer der Induktion sollte auf die Zeit der Fremdstoffexposition beschränkt sein, um Störungen des endogenen Stoffwechsels zu vermindern. In dieser Arbeit werden jedoch Hinweise auf dauerhafte und sogar generationsübergreifende Effekte von Medikamenten in Mäusen geliefert. Nachkommen von Müttern, welche bereits vor ihrer Verpaarung einmalig mit TCPOBOP, einem Liganden des murinen CAR, injiziert wurden, hatten eine ungefähr 100-fach gesteigerte Genexpression von Cyp2b10. Auch gab es Expressionsänderungen von Genen, deren Produkte eine Rolle im Lipidstoffwechsel sowie bei Immunkrankheiten spielen. Eine Hochdurchsatz-RNA-Sequenzierung der injizierten Elterngeneration ergab außerdem dauerhafte Expressionsveränderungen anderer Gene des Medikamentenstoffwechsels sowie von Genen mit Verbindung zum Energiemetabolismus. \r\nBerücksichtigt man die enge evolutionäre Verwandtschaft der nukleären Rezeptoren CAR und PXR, sind Langzeitveränderungen auch für PXR möglich und wurden im Verlauf dieser Arbeit ebenfalls untersucht. Eine Hochdurchsatz-Sequenzierung ergab für Mäuse, welche mit dem PXR-Aktivator PCN induziert wurden, dass selbst noch drei Monate nach der Exposition Gene verändert exprimiert waren, welche im Zusammenhang mit Lebernekrosen stehen. Bei Nachkommen von PCN-injizierten Müttern wurden Gene unterschiedlich exprimiert, welche eine Rolle bei der Energiehomöostase sowie im Glukosestoffwechsel spielen. Im Erwachsenenalter sind bei diesen Nachkommen darüber hinaus noch Gene unterschiedlich exprimiert, deren Produkte eine Funktion in der Immunantwort haben. \r\nDa Erwachsene aufgrund ihrer Lebensdauer sowie der absoluten Krankheitshäufigkeit wesentlich öfter Kontakt mit Fremdstoffen haben, war medizinisch von besonderem Interesse, ob anhaltende Genexpressionsänderungen auch bei Erwachsenen zu beobachten sind. So konnte im Rahmen dieser Arbeit gezeigt werden, dass auch einmalig exponierte Adulttiere Gene dauerhaft verändert exprimieren und die Veränderungen im Medikamentenstoffwechsel an die nächste Generation übertrugen. \r\n\r\nBisher sind klinische Studien zur Risikobewertung von Medikamenten (Pharmakovigilanz) nicht generationsübergreifend angelegt. Diese Arbeit gibt Anstöße dafür, dass dies in Zukunft für viel mehr Medikamente notwendig werden könnte. Neben Veränderungen im Medikamentenstoffwechsel ergeben sich Nebenwirkungen von PXR- und CAR-Liganden vor allem aus ihrer Beteiligung an endogenen Stoffwechselwegen. Nach Aktivierung von CAR, welcher viele metabolische Stoffwechselwege steuert, treten beispielsweise Störungen des Energiestoffwechsels auf. Ein tieferes Verständnis der Rezeptoraktivität von CAR samt einer gezielten Modulierung seiner Aktivität würde wichtige Beiträge zum Verständnis der Regulation des Fremdstoffmetabolismus sowie der Entstehung von Nebenwirkungen durch eine Behandlung mit CAR-Liganden leisten. Dauerhafte Veränderungen endogener Stoffwechselwege könnten dann möglicherweise über eine pharmakologische Modulierung der CAR-Aktivität reduziert werden. \r\nZu diesem Zweck wurden im Verlauf dieser Arbeit die CAR-Rezeptoren der Amphibien (Xenopus tropicalis, Xenopus laevis) und Reptilien (Anolis carolinensis) erstmals kloniert, als Proteine exprimiert und charakterisiert. Vergleiche zwischen Tierarten ermöglichen ein besseres Verständnis von humanen Proteinen. Funktionelle Analysen ergaben Ähnlichkeiten des Xenopus laevis-CAR mit dem PXR der Säugetiere: eine niedrige basale Aktivität sowie eine starke Induzierbarkeit durch Liganden. In weiteren funktionellen Analysen wurden die Determinanten der basalen Aktivität des Xenopus laevis-CAR untersucht. Die basale Aktivität war nicht abhängig von der subzellulären Lokalisation, sondern ergab sich aus der Proteinstruktur, welche nur beim CAR der Landvertebraten in einer aktiven Konformation fixiert ist. Ähnlich dem PXR der Säugetiere besitzt CAR der Amphibien eine Aktivierungsdomäne, welche erst durch Ligandenbindung in eine aktive Konformation gebracht wird. Mutationen einzelner Aminosäuren zum jeweils humanen Homolog erhöhten die basale Aktivität des Xenopus laevis-CAR auf die des humanen Rezeptors. Diese Mutanten mit erhöhter basalen Aktivität zeigten eine verstärkte Interaktion mit dem Kofaktor PGC-1a, einem Regulator des Energiestoffwechsels bei Säugetieren. Die hepatischen Zielgene des CAR der Amphibien überlappen zum Teil mit den humanen Zielgenen und spielen ebenfalls eine Rolle im Energiestoffwechsel.