Fipronil na bioenergética de mitocôndrias isoladas de figado de rato

Pós-graduação em Ciência Animal - FMVA

Genotoxicity evaluation of environmental pollutants using analysis of nucleolar alterations

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Protective effect of bixin on carbon tetrachloride-induced hepatotoxicity in rats

Background: The liver is an important organ for its ability to transform xenobiotics, making the liver tissue a prime target for toxic substances. The carotenoid bixin present in annatto is an antioxidant that can protect cells and tissues against the deleterious effects of free radicals. In this study, we evaluated the protective effect of bixin on liver damage induced by carbon tetrachloride (CCl4) in rats.Results: The animals were divided into four groups with six rats in each group. CCl4 (0.125 mL kg(-1) body wt.) was injected intraperitoneally, and bixin (5.0 mg kg(-1) body wt.) was given by gavage 7 days before the CCl4 injection. Bixin prevented the liver damage caused by CCl4, as noted by the significant decrease in serum aminotransferases release. Bixin protected the liver against the oxidizing effects of CCl4 by preventing a decrease in glutathione reductase activity and the levels of reduced glutathione and NADPH. The peroxidation of membrane lipids and histopathological damage of the liver was significantly prevented by bixin treatment.Conclusion: Therefore, we can conclude that the protective effect of bixin against hepatotoxicity induced by CCl4 is related to the antioxidant activity of the compound.

Histopathological changes in the perivisceral fat body of Rhinocricus padbergi (Diplopoda, Spirobolida) triggered by biosolids

Human activities generate a great amount of sewage daily, which is dumped into the sewer system. After sewage-treatment processes, sewage sludge is generated. Such byproduct can be treated by different methods; the result of treatment is a stabilized compost of reduced pathogenicity that has a similar inorganic chemical composition to the raw sewage sludge. After such pretreatment, sewage sludge is called a biosolids, and it can be used in agriculture. In this contest, the present study evaluated the effects of a sample of biosolids on the perivisceral fat body of a diplopod. These invertebrates are soil organisms that play an important role in the dynamics of terrestrial ecosystems, and as a consequence, they are in contact with xenobiotics present in this environmental compartment. Special emphasis is given on the interpretation of the effects of complex mixtures in target organs of diplopods. A semiquantitative analysis for the evaluation of histopathological changes in the perivisceral fat body was proposed. The sample-induced histopathological and ultrastructural changes in individuals exposed to it, and the severity of the effects was positively related to the exposure time, resulting in the deaths of exposed individuals after 90 days. Thus, the results indicate the need for caution in the use of biosolids as well as the need for improving waste management techniques, so they will produce environmentally innocuous final products.

Biomarkers of exposure to metal contamination and lipid peroxidation in the benthic fish Cathorops spixii from two estuaries in South America, Brazil

Biomarkers as lipid peroxidation, metallothionein and delta-aminolevulinic acid dehydratase were determined in Cathorops spixii to compare the biological responses of this fish from estuaries with distinct anthropogenic influence. Three areas were selected in two estuaries in accordance with the levels of contamination for the polluted (Santos/So Vicente) and with the hydrodynamic characteristics for the non-polluted (Canan,ia) estuary. Water characteristics and mercury levels in C. spixii confirmed a high human influence in the polluted system. In general, the biomarkers showed differences between the estuaries, suggesting disturbances in the specific cell mechanisms due to the presence of multiple xenobiotics in the contaminated system. Therefore, these biomarkers are recommended to promote more accurate information about the exposure to pollutants. Additionally, the study of the effect of the multiple xenobiotics on resident species such as the benthic fish C. spixii can favor a better assessment of the environmental quality of these systems.

Biomonitoring of coastal regions of So Paulo State, Brazil, using mussels Perna perna

The marine environment is constantly affected by anthropic actions, with causes consequent degradation of the waters and marine biota by various discharges of xenobiotics. In the present study, the focus was the study of a region of the marine coast of the State of So Paulo (city of Santos), which is one of the most industrialized parts of Brazil and suffers also from a strong impact of domestic effluents. The mussel Perna perna, very abundant in the coast of the State of So Paulo, Brazil, was selected as the biomonitoring organism for the determination of inorganic elements and a passive biomonitoring was performed. The organisms were collected at two sites in So Paulo State coast: Cocanha beach in Caraguatatuba (mussel farm) and Santos Bay (Itaipu and Palmas). Seasonally, the Perna perna were collected between September/08 and July/09 in each study sites. After removal and sample preparation, the elements As, Co, Cr, Fe, Se and Zn were determined by instrumental neutron activation analysis (INAA) and Cd, Pb and Hg were determined by atomic absorption spectroscopy (AAS) in this organism.

Assessment of the sublethal toxicity of organochlorine pesticide endosulfan in juvenile common carp (Cyprinus carpio)

This study is aimed at evaluating the sublethal effects of endosulfan (EDS) in juvenile common carp (Cyprinus carpio). For this purpose, fish were exposed for 15 days to the technical EDS (95% pure) diluted in dimethyl sulfoxide (DMSO) 0.1% of the total volume in water solution in a semi-static system at sublethal concentration (1 mu g/L). Subsequently, the liver somatic index (LSI) and factor condition (K) were determined. The total cytocrome P450 (CYP), CYP1A isoform, and the ethoxyresorufin-O-deethylase (EROD) activity were determined from the hepatic microsomal fraction as well as the activity of the oxidative stress enzyme system such as superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GP(X)), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH). Among the parameters assessed, EDS at the sublethal concentration in subchronic exposure caused significant changes in liver somatic indices as well as induction of the phase I biotransformation system and oxidative stress in juvenile common carp (Cyprinus carpio). Thus, it is seen that the use of biochemical biomarkers of environmental contamination in this study proved to be an extremely important tool for detecting the adverse effects of xenobiotics in the aquatic environment, even at low concentration.

Caratterizzazione del legame di molecole di interesse farmaceutico alla sieroalbumina umana mediante biocromatografia e dicroismo circolare

Negli ultimi anni, un crescente numero di studiosi ha focalizzato la propria attenzione sullo sviluppo di strategie che permettessero di caratterizzare le proprietà ADMET dei farmaci in via di sviluppo, il più rapidamente possibile. Questa tendenza origina dalla consapevolezza che circa la metà dei farmaci in via di sviluppo non viene commercializzato perché ha carenze nelle caratteristiche ADME, e che almeno la metà delle molecole che riescono ad essere commercializzate, hanno comunque qualche problema tossicologico o ADME [1]. Infatti, poco importa quanto una molecola possa essere attiva o specifica: perché possa diventare farmaco è necessario che venga ben assorbita, distribuita nell’organismo, metabolizzata non troppo rapidamente, ne troppo lentamente e completamente eliminata. Inoltre la molecola e i suoi metaboliti non dovrebbero essere tossici per l’organismo. Quindi è chiaro come una rapida determinazione dei parametri ADMET in fasi precoci dello sviluppo del farmaco, consenta di risparmiare tempo e denaro, permettendo di selezionare da subito i composti più promettenti e di lasciar perdere quelli con caratteristiche negative. Questa tesi si colloca in questo contesto, e mostra l’applicazione di una tecnica semplice, la biocromatografia, per caratterizzare rapidamente il legame di librerie di composti alla sieroalbumina umana (HSA). Inoltre mostra l’utilizzo di un’altra tecnica indipendente, il dicroismo circolare, che permette di studiare gli stessi sistemi farmaco-proteina, in soluzione, dando informazioni supplementari riguardo alla stereochimica del processo di legame. La HSA è la proteina più abbondante presente nel sangue. Questa proteina funziona da carrier per un gran numero di molecole, sia endogene, come ad esempio bilirubina, tiroxina, ormoni steroidei, acidi grassi, che xenobiotici. Inoltre aumenta la solubilità di molecole lipofile poco solubili in ambiente acquoso, come ad esempio i tassani. Il legame alla HSA è generalmente stereoselettivo e ad avviene a livello di siti di legame ad alta affinità. Inoltre è ben noto che la competizione tra farmaci o tra un farmaco e metaboliti endogeni, possa variare in maniera significativa la loro frazione libera, modificandone l’attività e la tossicità. Per queste sue proprietà la HSA può influenzare sia le proprietà farmacocinetiche che farmacodinamiche dei farmaci. Non è inusuale che un intero progetto di sviluppo di un farmaco possa venire abbandonato a causa di un’affinità troppo elevata alla HSA, o a un tempo di emivita troppo corto, o a una scarsa distribuzione dovuta ad un debole legame alla HSA. Dal punto di vista farmacocinetico, quindi, la HSA è la proteina di trasporto del plasma più importante. Un gran numero di pubblicazioni dimostra l’affidabilità della tecnica biocromatografica nello studio dei fenomeni di bioriconoscimento tra proteine e piccole molecole [2-6]. Il mio lavoro si è focalizzato principalmente sull’uso della biocromatografia come metodo per valutare le caratteristiche di legame di alcune serie di composti di interesse farmaceutico alla HSA, e sul miglioramento di tale tecnica. Per ottenere una miglior comprensione dei meccanismi di legame delle molecole studiate, gli stessi sistemi farmaco-HSA sono stati studiati anche con il dicroismo circolare (CD). Inizialmente, la HSA è stata immobilizzata su una colonna di silice epossidica impaccata 50 x 4.6 mm di diametro interno, utilizzando una procedura precedentemente riportata in letteratura [7], con alcune piccole modifiche. In breve, l’immobilizzazione è stata effettuata ponendo a ricircolo, attraverso una colonna precedentemente impaccata, una soluzione di HSA in determinate condizioni di pH e forza ionica. La colonna è stata quindi caratterizzata per quanto riguarda la quantità di proteina correttamente immobilizzata, attraverso l’analisi frontale di L-triptofano [8]. Di seguito, sono stati iniettati in colonna alcune soluzioni raceme di molecole note legare la HSA in maniera enantioselettiva, per controllare che la procedura di immobilizzazione non avesse modificato le proprietà di legame della proteina. Dopo essere stata caratterizzata, la colonna è stata utilizzata per determinare la percentuale di legame di una piccola serie di inibitori della proteasi HIV (IPs), e per individuarne il sito(i) di legame. La percentuale di legame è stata calcolata attraverso il fattore di capacità (k) dei campioni. Questo parametro in fase acquosa è stato estrapolato linearmente dal grafico log k contro la percentuale (v/v) di 1-propanolo presente nella fase mobile. Solamente per due dei cinque composti analizzati è stato possibile misurare direttamente il valore di k in assenza di solvente organico. Tutti gli IPs analizzati hanno mostrato un’elevata percentuale di legame alla HSA: in particolare, il valore per ritonavir, lopinavir e saquinavir è risultato maggiore del 95%. Questi risultati sono in accordo con dati presenti in letteratura, ottenuti attraverso il biosensore ottico [9]. Inoltre, questi risultati sono coerenti con la significativa riduzione di attività inibitoria di questi composti osservata in presenza di HSA. Questa riduzione sembra essere maggiore per i composti che legano maggiormente la proteina [10]. Successivamente sono stati eseguiti degli studi di competizione tramite cromatografia zonale. Questo metodo prevede di utilizzare una soluzione a concentrazione nota di un competitore come fase mobile, mentre piccole quantità di analita vengono iniettate nella colonna funzionalizzata con HSA. I competitori sono stati selezionati in base al loro legame selettivo ad uno dei principali siti di legame sulla proteina. In particolare, sono stati utilizzati salicilato di sodio, ibuprofene e valproato di sodio come marker dei siti I, II e sito della bilirubina, rispettivamente. Questi studi hanno mostrato un legame indipendente dei PIs ai siti I e II, mentre è stata osservata una debole anticooperatività per il sito della bilirubina. Lo stesso sistema farmaco-proteina è stato infine investigato in soluzione attraverso l’uso del dicroismo circolare. In particolare, è stato monitorata la variazione del segnale CD indotto di un complesso equimolare [HSA]/[bilirubina], a seguito dell’aggiunta di aliquote di ritonavir, scelto come rappresentante della serie. I risultati confermano la lieve anticooperatività per il sito della bilirubina osservato precedentemente negli studi biocromatografici. Successivamente, lo stesso protocollo descritto precedentemente è stato applicato a una colonna di silice epossidica monolitica 50 x 4.6 mm, per valutare l’affidabilità del supporto monolitico per applicazioni biocromatografiche. Il supporto monolitico monolitico ha mostrato buone caratteristiche cromatografiche in termini di contropressione, efficienza e stabilità, oltre che affidabilità nella determinazione dei parametri di legame alla HSA. Questa colonna è stata utilizzata per la determinazione della percentuale di legame alla HSA di una serie di poliamminochinoni sviluppati nell’ambito di una ricerca sulla malattia di Alzheimer. Tutti i composti hanno mostrato una percentuale di legame superiore al 95%. Inoltre, è stata osservata una correlazione tra percentuale di legame è caratteristiche della catena laterale (lunghezza e numero di gruppi amminici). Successivamente sono stati effettuati studi di competizione dei composti in esame tramite il dicroismo circolare in cui è stato evidenziato un effetto anticooperativo dei poliamminochinoni ai siti I e II, mentre rispetto al sito della bilirubina il legame si è dimostrato indipendente. Le conoscenze acquisite con il supporto monolitico precedentemente descritto, sono state applicate a una colonna di silice epossidica più corta (10 x 4.6 mm). Il metodo di determinazione della percentuale di legame utilizzato negli studi precedenti si basa su dati ottenuti con più esperimenti, quindi è necessario molto tempo prima di ottenere il dato finale. L’uso di una colonna più corta permette di ridurre i tempi di ritenzione degli analiti, per cui la determinazione della percentuale di legame alla HSA diventa molto più rapida. Si passa quindi da una analisi a medio rendimento a una analisi di screening ad alto rendimento (highthroughput- screening, HTS). Inoltre, la riduzione dei tempi di analisi, permette di evitare l’uso di soventi organici nella fase mobile. Dopo aver caratterizzato la colonna da 10 mm con lo stesso metodo precedentemente descritto per le altre colonne, sono stati iniettati una serie di standard variando il flusso della fase mobile, per valutare la possibilità di utilizzare flussi elevati. La colonna è stata quindi impiegata per stimare la percentuale di legame di una serie di molecole con differenti caratteristiche chimiche. Successivamente è stata valutata la possibilità di utilizzare una colonna così corta, anche per studi di competizione, ed è stata indagato il legame di una serie di composti al sito I. Infine è stata effettuata una valutazione della stabilità della colonna in seguito ad un uso estensivo. L’uso di supporti cromatografici funzionalizzati con albumine di diversa origine (ratto, cane, guinea pig, hamster, topo, coniglio), può essere proposto come applicazione futura di queste colonne HTS. Infatti, la possibilità di ottenere informazioni del legame dei farmaci in via di sviluppo alle diverse albumine, permetterebbe un migliore paragone tra i dati ottenuti tramite esperimenti in vitro e i dati ottenuti con esperimenti sull’animale, facilitando la successiva estrapolazione all’uomo, con la velocità di un metodo HTS. Inoltre, verrebbe ridotto anche il numero di animali utilizzati nelle sperimentazioni. Alcuni lavori presenti in letteratura dimostrano l’affidabilita di colonne funzionalizzate con albumine di diversa origine [11-13]: l’utilizzo di colonne più corte potrebbe aumentarne le applicazioni.

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.

Emerging contaminants in agricultural ecosystems: impact of selected pharmaceutical on water and soil ecology and pratical implications

Pharmaceuticals are useful tools to prevent and treat human and animal diseases. Following administration, a significant fraction of pharmaceuticals is excreted unaltered into faeces and urine and may enter the aquatic ecosystem and agricultural soil through irrigation with recycled water, constituting a significant source of emerging contaminants into the environment. Understanding major factors influencing their environmental fate is consequently needed to value the risk, reduce contamination, and set up bioremediation technologies. The antiviral drug Tamiflu (oseltamivir carboxylate, OC) has received recent attention due to the potential use as a first line defence against H5N1 and H1N1 influenza viruses. Research has shown that OC is not removed during conventional wastewater treatments, thus having the potential to enter surface water bodies. A series of laboratory experiments investigated the fate and the removal of OC in surface water systems in Italy and Japan and in a municipal wastewater treatment plant. A preliminary laboratory study investigated the persistence of the active antiviral drug in water samples from an irrigation canal in northern Italy (Canale Emiliano Romagnolo). After an initial rapid decrease, OC concentration slowly decreased during the remaining incubation period. Approximately 65% of the initial OC amount remained in water at the end of the 36-day incubation period. A negligible amount of OC was lost both from sterilized water and from sterilized water/sediment samples, suggesting a significant role of microbial degradation. Stimulating microbial processes by the addition of sediments resulted in reduced OC persistence. Presence of OC (1.5 μg mL-1) did not significantly affect the metabolic potential of the water microbial population, that was estimated by glyphosate and metolachlor mineralization. In contrast, OC caused an initial transient decrease in the size of the indigenous microbial population of water samples. A second laboratory study focused on basic processes governing the environmental fate of OC in surface water from two contrasting aquatic ecosystems of northern Italy, the River Po and the Venice Lagoon. Results of this study confirmed the potential of OC to persist in surface water. However, the addition of 5% of sediments resulted in rapid OC degradation. The estimated half-life of OC in water/sediment of the River Po was 15 days. After three weeks of incubation at 20 °C, more than 8% of 14C-OC evolved as 14CO2 from water/sediment samples of the River Po and Venice Lagoon. OC was moderately retained onto coarse sediments from the two sites. In water/sediment samples of the River Po and Venice Lagoon treated with 14C-OC, more than 30% of the 14C-residues remained water-extractable after three weeks of incubation. The low affinity of OC to sediments suggests that the presence of sediments would not reduce its bioavailability to microbial degradation. Another series of laboratory experiments investigated the fate and the removal of OC in two surface water ecosystems of Japan and in the municipal wastewater treatment plant of the city of Bologna, in Northern Italy. The persistence of OC in surface water ranged from non-detectable degradation to a half-life of 53 days. After 40 days, less than 3% of radiolabeled OC evolved as 14CO2. The presence of sediments (5%) led to a significant increase of OC degradation and of mineralization rates. A more intense mineralization was observed in samples of the wastewater treatment plant when applying a long incubation period (40 days). More precisely, 76% and 37% of the initial radioactivity applied as 14C-OC was recovered as 14CO2 from samples of the biological tank and effluent water, respectively. Two bacterial strains growing on OC as sole carbon source were isolated and used for its removal from synthetic medium and environmental samples, including surface water and wastewater. Inoculation of water and wastewater samples with the two OC-degrading strains showed that mineralization of OC was significantly higher in both inoculated water and wastewater, than in uninoculated controls. Denaturing gradient gel electrophoresis and quantitative PCR analysis showed that OC would not affect the microbial population of surface water and wastewater. The capacity of the ligninolytic fungus Phanerochaete chrysosporium to degrade a wide variety of environmentally persistent xenobiotics has been largely reported in literature. In a series of laboratory experiments, the efficiency of a formulation using P. chrysosporium was evaluated for the removal of selected pharmaceuticals from wastewater samples. Addition of the fungus to samples of the wastewater treatment plant of Bologna significantly increased (P < 0.05) the removal of OC and three antibiotics, erythromycin, sulfamethoxazole, and ciprofloxacin. Similar effects were also observed in effluent water. OC was the most persistent of the four pharmaceuticals. After 30 days of incubation, approximately two times more OC was removed in bioremediated samples than in controls. The highest removal efficiency of the formulation was observed with the antibiotic ciprofloxacin. The studies included environmental aspects of soil contamination with two emerging veterinary contaminants, such as doramectin and oxibendazole, wich are common parasitic treatments in cattle farms.

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.

Serumfreie Collagen-Monolayer- und Sandwich-Kulturen primärer Hepatozyten als ein wertvolles Modell zur Detektion von Toxizität und Arzneimittelwechselwirkungen in vitro

In dieser Arbeit wurden Zellkulturen primärer Hepatozyten von Ratte und Mensch hinsichtlich ihrer Eignung untersucht Speziesunterschiede der toxischen Wirkung und des Metabolismus von Substanzen darzustellen und inwieweit die in vitro-Ergebnisse in vivo vergleichbar bzw. übertragbar sind. Des Weiteren wurde ein Zellkulturmodell entwickelt, das eine Kultivierung von primären Hepatozyten aus Ratte, Mensch und Maus über einen Zeitraum von mindestens einer bis zwei Wochen erlaubt.rnrnDie Zellkulturen primärer Hepatozyten von Ratte und Mensch zeigten deutliche Unterschiede in der substanzinduzierten Veränderung der Genexpression nach Behandlung mit den, vor allem für den Menschen, lebertoxischen Substanzen Diclofenac und Troglitazon. Diese Unterschiede traten hauptsächlich in der Induktion fremdstoffmetabolisierender Enzyme sowie deren transkriptionsregulierenden Kernrezeptoren in den humanen Hepatozyten auf. Ebenso war eine verstärkte Stressantwort zu beobachten.rnDeutliche Speziesunterschiede konnten ebenso in der Wirkung der Arzneimittelentwicklungssubstanz EMD 392949 auf die Aktivität bzw. Genexpression von Cytochrom P450 Enzymen sowie deren Regulatoren nachgewiesen werden. Des Weiteren konnte hier eine sehr gute Übereinstimmung der Ergebnisse aus den Zellkulturen primärer Ratten- bzw. Humanhepatozyten mit jenen aus in vivo-Experimenten mit Ratten bzw. Affen (Macaca fascicularis) beobachtet werden, was die Aussagekraft der Primärkulturen verdeutlichte.rnDie große Übereinstimmung zwischen Enzymaktivität und Genexpression in der Induktion fremdstoffmetabolisierender Enzyme konnte durch die Behandlung mit einer Reihe speziesspezifischer Induktoren in Zellkulturen primärer Ratten- bzw. Humanhepatozyten bestätigt werden; vor allem nach dem von der amerikanischen Arzneimittelzulassungsbehörde (FDA, Food and Drug Administartion) vorgeschlagenen Bewertungsschema zur Untersuchung der CYP-Induktion.rnrnDie Lebensdauer sowie der Differenzierungsgrad von primären Hepatozyten in Kultur sind stark abhängig von den Zellkulturbedingungen. Durch diese Arbeit konnte gezeigt werden, dass spezifische Eigenschaften von Rattenleberzellen durch Kultivierung in einem Sandwich aus zwei hydratisierten Collagengelschichten und unter serumfreien Bedingungen für einen Zeitraum von mindestens zwei Wochen aufrechterhalten werden können. Dieses Kulturmodel konnte auf Primärhepatozyten von Mensch und Maus übertragen werden und erweitert die möglichen Anwendungen hin zu einer Behandlung über einen längeren Zeitraum und der Untersuchung von subchronischen Effekten.rn

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

Untersuchungen zum Einfluss des HMG-CoA-Reduktase-Inhibitors Lovastatin auf die Toxizität ionisierender Strahlung sowie des Anthrazyklinderivats Doxorubicin im Mausmodell

Hintergund: HMG-CoA-Reduktase-Inhibitoren (Statine) sind klinisch etablierte Cholesterinsenker. Über die Inhibition der intrinsischen Cholesterinbiosynthese hinaus zeigen sie sogenannte pleiotrope biologische Effekte. Ein Großteil dieser Wirkungen wird auf die Inhibition kleiner Ras homologer GTPasen (Rho GTPasen) zurückgeführt. In vitro schützt das Statinderivat Lovastatin (Lova) primäre humane Endothelzellen vor der Zytotoxizität von ionisierender Strahlung (IR) und dem Krebsmedikament Doxorubicin (Doxo). Zielsetzung: Die Relevanz dieser Befunde für ein in vivo Mausmodell sollte in der vorliegenden Arbeit überprüft werden. Dafür wurden BALB/c-Mäuse mit IR oder Doxo behandelt und der Einfluss einer Kobehandlung mit Lova auf verschiedene Toxizitätsendpunkte untersucht (24 h nach einer einzelnen hohen Dosis IR (i), 14 Tage nach zwei geringen Dosen IR (ii), 48 h nach einer einzelnen hohen Dosis Doxo (iii), sowie 8 Tage nach drei niedrigen Dosen Doxo (iv)). Eine mögliche gleichzeitige Protektion von Tumorzellen durch die Statingabe wurde in einem Xenotransplantationsexperiment überprüft (v), in dem das gleiche Behandlungsschema wie bei iv angewendet wurde. Ergebnisse: Es konnte gezeigt werden, dass eine Statinbehandlung Normalgewebe vor Doxo- und IR-induzierter Toxizität schützt, ohne gleichzeitig protektiv auf transformierte Zellen zu wirken. Dieser Effekt ist wahrscheinlich von einer Inhibition der kleinen GTPasen Rac1 und RhoA abhängig und einer daraus folgenden Modifizierung der DNA-Schadensantwort. i: Die Statinvorbehandlung der Mäuse hatte keinen Einfluss auf die Bildung von initialen IR-induzierten DNA-Doppelstrangbrüchen (DSB) in der Leber. Die Lova-Behandlung wirkte sich jedoch auf IR-induzierte Stressantworten aus, was sich in einer Minderung der Expression von Inflammations- und Fibrosesurrogatmarkern in Leber und Darm widerspiegelte. ii: In der Lunge der Tiere wurde ein Anstieg von molekularen Inflammations- und Fibrosesurrogatmarkern detektiert, der bei Statinkobehandlung ausblieb. Zudem verhinderte die Kobehandlung mit Lova eine IR-induzierte Abnahme der Thrombozytenzahl, ohne sich auf die durch IR verringerte Leukozytenzahl im Blut auszuwirken. iii: Die Verabreichung einer hohen Dosis Doxo induzierte DSB-Formation in der Leber. Die Statinvorbehandlung reduzierte deren Menge um ca. 50 %. Dieser genoprotektive Effekt war unabhängig von der Entstehung reaktiver Sauerstoffspezies sowie einer Änderung des Doxo-Imports oder Exports. Die Expression von proinflammatorischen und profibrotischen Genen fiel besonders in der Leber und im Herzen durch die Lova-Kobehandlung geringer aus, als in der nur mit Doxo behandelten Gruppe. Zudem verringerte Lova die durch Doxo induzierte Hochregulation von für den AP1-Komplex kodierenden Genen sowie von Zellzykluskontrollfaktoren. Die Lova-Vorbehandlung führte darüber hinaus im Herzen zu einem reduzierten mRNA-Spiegel der Topoisomerasen II α und β. iv: Es konnten schwere Herz- und Leberschäden detektiert werden (gemessen an Gldh-, Gpt- sowie cTn-I-Serumkonzentrationen), die bei einer Kobehandlung mit dem Statin nicht auftraten. Die Lova-Kobehandlung verhinderte außerdem eine durch die Doxo-Behandlung verringerte Leukozytenzahl. Molekulare Marker für frühe fibrotische Ereignisse, sowie für Inflammation und Hypertrophie waren in der Leber und im Herzen nach der Doxo-Behandlung erhöht. Das Statin war auch hier in der Lage, diese toxischen Wirkungen des Anthrazyklins zu mindern. Auch die Doxo-induzierte Expression von Surrogatmarkern für Zellantworten auf oxidativen Stress wurde in der Leber abgeschwächt. In der Leber und im Herzen wiesen die mit Doxo behandelten Tiere höhere mRNA Spiegel von an Zellzykluskontrolle beteiligten Faktoren sowie von DNA-Reparatur und Fremdstoffmetabolismus assoziierten Genen auf. Am stärksten wurde die Expression von Topoisomerase II alpha - ein molekularer Marker für Zellproliferation und bedeutsame Zielstruktur von Doxo - in der Leber hochreguliert. Die Statin-Kobehandlung verhinderte all diese Doxo-induzierten Expressionsänderungen. Im Gegensatz zur Leber wurde die Top2a-mRNA Menge im Herzen durch die Doxo-Applikation reduziert. Auch hier bewirkte die Kobehandlung mit dem Statin, dass die Expression nahe dem Kontrollniveau blieb. v: Die Kobehandlung mit Lova führte zu keinem Schutz der Tumorzellen vor Doxo, sondern erhöhte sogar dessen antineoplastisches Potential.rnFazit: Die Erkenntnisse aus vorhergegangenen in vitro Versuchen konnten zum großen Teil auf die in vivo Situation im Mausmodell übertragen werden. Sie stehen im Einklang mit Ergebnissen anderer Gruppen, welche die Inhibition kleiner GTPasen mit einer geringeren, durch zytotoxische Substanzen induzierten, Inflammation und Fibrose korrelieren konnten. Eine Kobehandlung mit Lova während einer Krebstherapie erscheint somit als vielversprechende Möglichkeit Doxo- oder IR-induzierte Nebenwirkungen auf Normalgewebe zu mildern.

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.